Method for screening antigen-specific binding polypeptides

Abstract

The present application relates to a method for screening antigen-specific binding polypeptides. The screening method comprises: providing a first phage library displaying antigen-specific binding polypeptides and a second phage library displaying antigen-specific binding polypeptides.

Description

A method for screening antigen-specific binding polypeptides Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a method for screening antigen-specific binding polypeptides. Background Art

[0002] Cell membrane proteins are widely distributed on the cell membrane and include various types such as receptors, channels, and transporters. They play key roles in physiological processes such as cell signaling, substance transport, and cell adhesion, making them ideal targets for regulating physiological and pathological states.

[0003] For the screening of cell membrane protein antibodies, the step of obtaining active membrane proteins that can be used for screening is very challenging. Membrane proteins are usually folded and assembled in a complex way in the physiological environment, which makes it complicated to replicate the antigens of their natural structure in vitro. In addition, membrane proteins may become unstable after leaving the physiological environment and easily lose their natural conformation. Some important membrane proteins have low abundance in cells, and it is difficult to obtain sufficient antigens. Therefore, the method of using isolated and purified antigens for specific antibody screening is often difficult to achieve for membrane protein targets.

[0004] One approach to addressing this issue is to use whole cells expressing membrane proteins as antigens for antibody screening. The lipid environment of the cell membrane allows membrane proteins to be presented in their native conformation and with appropriate post-translational modifications, making it easier to obtain membrane protein antibodies with therapeutic functions. However, in whole cell expression systems, in addition to the target membrane protein, a large number of other cellular proteins are present. Antibodies may recognize and bind to other proteins in the cell rather than the target membrane protein. This greatly complicates the screening process. Screening for antibodies that specifically bind to the target membrane protein often requires repeated rounds of screening, which is time-consuming and results in low screening efficiency and success rates.

[0005] Some methods for improving the efficiency of screening cells expressing membrane proteins as antigens have been proposed in the prior art (see Antibodies 2017, 6(3), 10; Strategies for Selecting Membrane Protein-Specific Antibodies using Phage Display with Cell-Based Panning). For example, the use of blocking agents, such as bovine serum albumin (BSA), milk, and casein, is a common practice for blocking nonspecific sites on the cell membrane to prevent nonspecific binding events, but it does not prevent non-target protein-specific binding events. For example, before contacting the phage library with the positive cell line, the library is exposed to a similar cell line that is negative for the desired antigen. For example, alternating between cell lines expressing the same target antigen can help eliminate any irrelevant binding molecules enriched in the first round. These methods can, to some extent, increase the probability that positive clones in the phage display library contact the target membrane protein. However, the eluted phage library may still contain a large number of clones that specifically bind to non-target proteins and clones that bind nonspecifically. These methods cannot effectively increase the probability of screening positive clones in the phage library, and further screening and verification still require a lot of work and time.

[0006] Therefore, it is urgent to develop new screening methods for membrane protein antibodies to improve the efficiency of screening membrane protein antibodies. Summary of the Invention

[0007] The present application provides a method for screening antigen-specific binding polypeptides with broad practicality and applicability, high screening efficiency and high success rate, which includes using a first phage library to "block" cells expressing the antigen to increase the possibility of positive clones in the second phage library binding to the target membrane protein antigen, and utilizing the difference in screening markers between the first phage library and the second phage library, and using different culture conditions after elution to distinguish the clones in the first phage library from the second phage library, thereby greatly reducing the workload of screening positive clones and reducing the probability of screening noise clones.

[0008] In one aspect, the present application provides a method for screening antigen-specific binding polypeptides, the screening method comprising the following steps:

[0009] a) providing a first phage library displaying an antigen-specific binding polypeptide and a second phage library displaying an antigen-specific binding polypeptide, wherein the first phage library and the second phage library have screening markers, the first phage library has a first screening marker, and the second phage library has a second screening marker different from the first screening marker;

[0010] b) after contacting the antigen with the first phage library, contacting the antigen with a second phage library, thereby obtaining antigen-binding phage; and

[0011] c) under conditions in which the first screening marker is not displayed and the second screening marker is displayed, screening the second phage library for antigen-specific binding phages from the antigen-binding phages, wherein the antigen-specific binding phages display the antigen-specific binding polypeptide.

[0012] In certain embodiments, the steps for preparing the first phage library are the same as the steps for preparing the second phage library.

[0013] In certain embodiments, the antigen-binding polypeptide sequences displayed by the first phage library and the second phage library are of the same type.

[0014] In certain embodiments, the antigen-binding polypeptides displayed by the first phage library and the second phage library are both selected from the group consisting of Fab, scFv, VHH, full-length antibody, fusion protein and polypeptide.

[0015] In certain embodiments, the antigen-binding polypeptide sequences displayed by the first phage library and the second phage library are obtained in the same manner.

[0016] In certain embodiments, the first phage library and the second phage library are both selected from one of the following phage libraries: a natural library, an immune library, a semisynthetic library, and a synthetic library.

[0017] In certain embodiments, the library capacity of the first phage library and the second phage library is the same or different.

[0018] In certain embodiments, the library capacity of the first phage library is smaller or larger than the library capacity of the second phage library.

[0019] In certain embodiments, the library capacity of the first phage library is 1 / 100 to 1 / 1000 of that of the second phage library.

[0020] In certain embodiments, the selection marker comprises a resistance gene.

[0021] In certain embodiments, the resistance gene comprises an antibiotic resistance gene.

[0022] In certain embodiments, the first screening marker is selected from one of the kanamycin resistance gene, the puromycin resistance gene, the hygromycin resistance gene and the ampicillin resistance gene, and the second screening marker includes one selected from the kanamycin resistance gene, the puromycin resistance gene, the hygromycin resistance gene and the ampicillin resistance gene and is different from the first screening marker.

[0023] In certain embodiments, the antigen comprises a membrane protein and / or a membrane protein fragment.

[0024] In certain embodiments, the antigen comprises a G protein coupled receptor (GPCR) and / or a fragment thereof.

[0025] In certain embodiments, the antigen comprises glucagon receptor (GCGR) and / or a fragment thereof.

[0026] In certain embodiments, the antigen comprises a cell expressing the antigen.

[0027] In certain embodiments, the cells in which the antigen is expressed include natural cells and / or engineered cells.

[0028] In certain embodiments, the antigen comprises cells expressing the antigen via an integrated expression vector and / or cells expressing the antigen via an episomal expression vector.

[0029] In certain embodiments, the integrating expression vector comprises a single-site integrating expression vector.

[0030] In certain embodiments, the antigen comprises a cell expressing the antigen via the Flp-In system.

[0031] In certain embodiments, the cells comprise mammalian cells.

[0032] In certain embodiments, the cells comprise CHO cells.

[0033] In certain embodiments, the method for screening an antigen-specific binding polypeptide described herein further comprises the step of providing the antigen, wherein the step of providing the antigen comprises:

[0034] a) constructing a vector for expressing the antigen; and

[0035] b) transducing the vector expressing the antigen into cells, so that the cells express the antigen.

[0036] In certain embodiments, the vector for expressing the antigen comprises a Flp-In system vector.

[0037] In certain embodiments, the cells comprise mammalian cells.

[0038] In certain embodiments, the cells comprise CHO cells.

[0039] In certain embodiments, the antigen comprises a G protein coupled receptor (GPCR) and / or a fragment thereof.

[0040] In certain embodiments, the antigen comprises glucagon receptor (GCGR) and / or a fragment thereof.

[0041] In certain embodiments, screening the second phage library for antigen-specific binding phage comprises the step of obtaining the antigen-specific binding phage.

[0042] In certain embodiments, obtaining antigen-binding phage comprises the following steps:

[0043] a) dissociating the antigen-binding phage from the antigen;

[0044] b) collecting antigen-binding phage that have dissociated from the antigen; and / or

[0045] c) allowing the antigen-binding phage to infect bacteria.

[0046] In certain embodiments, the antigen-binding phage comprises a phage particle, a phage vector and / or bacteria infected by the phage.

[0047] In certain embodiments, screening the antigen-specific binding phage in the second phage library from the antigen-binding phage comprises allowing the antigen-binding phage to infect bacteria, and then culturing the bacteria under conditions in which the bacteria infected by the second phage library can grow but the bacteria infected by the first phage library cannot grow.

[0048] In certain embodiments, wherein the selection marker is an antibiotic resistance gene, culturing the bacteria comprises using a culture medium containing an antibiotic corresponding to the second selection marker and not containing an antibiotic corresponding to the first selection marker.

[0049] In certain embodiments, it further comprises the step of obtaining the antigen-specific binding polypeptide based on the antigen-specific binding phage in the second phage library.

[0050] In certain embodiments, the step of obtaining the antigen-specific binding polypeptide comprises expressing and / or isolating the antigen-specific binding polypeptide displayed by phage.

[0051] In certain embodiments, the step of obtaining the antigen-specific binding polypeptide comprises the step of converting the antigen-specific binding polypeptide into IgG form.

[0052] In certain embodiments, the step of obtaining an antigen-specific binding polypeptide comprises evaluating the expression level, binding affinity to the antigen, and / or biological function of the antigen-specific binding polypeptide.

[0053] In certain embodiments, the step of obtaining antigen-specific binding polypeptides comprises evaluating by FACS analysis.

[0054] In certain embodiments, the method of screening for antigen-specific binding polypeptides described herein further comprises: obtaining antigen-specific binding phages in the first phage library from the antigen-binding phages.

[0055] In certain embodiments, the step of contacting the antigen with the first phage library further comprises contacting the antigen with a polypeptide known to specifically bind to the antigen.

[0056] In certain embodiments, the method of screening for antigen-specific binding polypeptides described herein is repeated one or more times.

[0057] On the other hand, the present application provides a phage library, comprising a first phage library displaying an antigen-specific binding polypeptide and a second phage library displaying an antigen-specific binding polypeptide, wherein the first phage library and the second phage library have screening markers, the first phage library has a first screening marker, and the second phage library has a second screening marker different from the first screening marker.

[0058] On the other hand, the present application provides a vector comprising a first phage library antigen-binding polypeptide gene display vector and a second phage library antigen-binding polypeptide gene display vector, wherein the screening marker of the first phage library antigen-binding polypeptide gene display vector is different from the screening marker of the second phage library antigen-binding polypeptide gene display vector.

[0059] On the other hand, the present application provides a kit, wherein the kit comprises the phage library described in the present application and / or the vector described in the present application.

[0060] On the other hand, the present application provides a use of the phage library described in the present application, the vector described in the present application and / or the kit described in the present application in screening antigen-specific binding polypeptides.

[0061] On the other hand, the present application provides an antigen-specific binding phage obtained by the method described in the present application.

[0062] On the other hand, the present application provides an antigen-specific binding polypeptide obtained by the method described in the present application.

[0063] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0065] FIG1 shows the vector constructed in the present application for efficiently expressing the native structure hGCGR on the surface of FCHO cells.

[0066] FIG2 shows the antigen-binding polypeptide gene display vector used to construct the ZM-Fab library in this application.

[0067] FIG3 shows the antigen-binding polypeptide gene display vector used to construct the Kana-Fab library in this application.

[0068] FIG4 shows the results of FACS analysis of the specific binding of Volagidemab full-length antibody and Fab to hGCGR on the surface of F-hGCGR cells.

[0069] FIG5 shows FACS images of negative clones and positive clones obtained by the screening method described in the present application, which can clearly distinguish positive clones (cells located in the upper left quadrant) from negative clones (cells located in the lower left quadrant).

[0070] FIG6A-FIG6B show the dose-dependent binding of the positive clones obtained by the screening method described in the present application to the natural structure of hGCGR on the cell surface after conversion into full-length antibodies.

[0071] FIG7 shows that the positive clones obtained by the screening method described in this application have biological functions after being converted into full-length antibodies. DETAILED DESCRIPTION

[0072] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0073] Definition of terms

[0074] In this application, the term "screening" may include selection, screening, or any appropriate combination of selection and / or screening techniques. When using a phage library for screening, "screening" generally refers to the process of selecting phage clones with specific properties or functions from the entire library. In some cases, "screening" may also include the process of isolating, purifying, and / or amplifying the phage clones with specific properties or functions.

[0075] In this application, the term "bacteriophage" generally refers to a parasitic virus that targets bacteria rather than eukaryotic organisms. In this application, the term "bacteriophage" may include bacteriophages at any stage of their life cycle. For example, the bacteriophages described herein may include bacteriophage particles and / or bacteriophage vectors. For example, the bacteriophages described herein may also include bacteria infected by the bacteriophage, cultures of the bacteria, and / or mixtures thereof. For example, the bacteriophages described herein may also include cells containing bacteriophage vectors and / or bacteriophage genomes. Phage particles generally refer to structures formed by bacteriophages inside bacterial hosts that contain genetic material and proteins.

[0076] Those skilled in the art will have a clear and unambiguous understanding of the meaning of bacteriophage based on the scenario or context of the description. For example, when describing the binding of a bacteriophage to an antigen, those skilled in the art will understand that this generally refers to the binding of an antigen-binding protein displayed on the surface of the phage particle to the antigen. For example, when describing the growth of a phage bearing a second selection marker, those skilled in the art will understand that this generally includes the growth of bacteria containing the phage vector.

[0077] In this application, the term "phage vector" generally refers to a nucleic acid vector used for phage display or gene cloning. A phage vector can include a nucleic acid molecule comprising an antigen-binding polypeptide encoding the phage, and when the phage comprises the phage vector, the antigen-binding polypeptide can be displayed on its surface. The phage vector can be DNA or RNA, and can be linear or circular, depending on the type of phage. The phage vector can be double-stranded DNA, double-stranded RNA, or single-stranded RNA. For example, the phage vector can be a plasmid. In some cases, the phage vector carries exogenous DNA and can replicate autonomously in bacteria. The backbone of the phage vector can include a plasmid origin of replication, a phage origin of replication, a multiple cloning site, a screening marker (e.g., an antibiotic resistance gene), a phage packaging signal, and / or an expression control element (e.g., a promoter, a terminator, a ribosome binding site, etc.). In some cases, the phage vector can be amplified in bacteria (e.g., Escherichia coli) and cannot be assembled into a phage by itself. It needs to be packaged into phage particles that display the antigen-binding polypeptide with the help of a helper phage. Phage particles can carry phage vectors and allow the phage vectors to enter new host cells. For example, phage vectors include antigen-binding polypeptide gene display vectors.

[0078] In this application, the terms "phage library" and "phage display library" can be used interchangeably and generally refer to a library comprising polynucleotides encoding different recombinant polypeptides capable of being displayed on phage. For example, the recombinant polypeptides include antigen-binding polypeptides. In some cases, the phage library may also include a collection of phages containing the polynucleotides. For example, the phage library displaying recombinant polypeptides generally refers to a collection of phages expressing different recombinant polypeptides on the surface of phages. In some cases, the phage library may also include a collection of bacteria containing the polynucleotides. In some cases, the phage library may also include a collection of bacteria transfected with a vector containing the polynucleotides. In some cases, the phage library may also include a collection of vectors containing the polynucleotides, wherein each vector contains a polynucleotide encoding at least one recombinant polypeptide capable of being displayed on a phage.

[0079] In this application, the library may refer to a diverse collection or mixture of polynucleotides, which may contain polynucleotides encoding different antigen-binding polypeptides. For example, the polynucleotide library collection may contain at least 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 1015 or more different polynucleotides. The polynucleotides in the phage library can be derived from a single species (e.g., a mammal, such as a human), its tissues, organs and / or cells. The library can contain polynucleotides of a common genus. For example, the genus can be a polynucleotide encoding a certain type or class of immunoglobulin subunit polypeptides. For example, the polynucleotide can encode a Fab, scFv, VHH, full-length antibody, fusion protein and / or polypeptide.

[0080] Phage libraries displaying antigen-binding peptides can be categorized into human, mouse, and rabbit libraries, depending on the species of origin of the displayed antigen-specific binding peptide sequences. Choosing the appropriate phage library depends on the researcher's experimental objectives and the species of interest.

[0081] Phage libraries displaying antigen-binding peptides can be categorized into Fab libraries, scFv libraries, VHH libraries, full-length antibody libraries, fusion protein libraries, and peptide libraries, depending on the type of antigen-specific binding peptide sequences displayed. Choosing the appropriate phage library type depends on the researcher's specific experimental objectives and research needs.

[0082] Phage libraries displaying antigen-binding polypeptides can be categorized into natural, immune, semisynthetic, and synthetic libraries, depending on how the displayed antigen-specific binding polypeptide sequences are obtained. For example, within the classification of phage antibody libraries, natural libraries typically refer to antibody libraries constructed using unimmunized lymphocytes, immune libraries typically refer to antibody libraries constructed using B cells or tissues from animals or humans immunized with antigens, semisynthetic libraries typically refer to antibody libraries constructed from a combination of artificially synthesized sequences and natural sequences, and synthetic libraries typically refer to antibody libraries in which all variable region sequences are artificially synthesized. Choosing the appropriate type of phage library depends on the researcher's specific experimental objectives and research needs.

[0083] The "capacity" of a phage library generally refers to the number of phage clones contained in the phage library. Phage library capacity is typically expressed as the number of clones or phage particles. A larger library capacity indicates a greater number of phage clones contained in the library. The capacity of a phage library can be determined by methods known in the art. For example, the capacity of a phage library can be estimated by titration, where a serial dilution of a phage library sample is co-cultured with infectable bacteria and the number of phage particles is determined by observing or counting the infected bacterial clones.

[0084] The "diversity" of a phage library generally refers to the diversity of the polynucleotides contained in the phage library. The diversity of a phage library can be measured using methods known in the art. For example, by sequencing a portion of clones in a phage library and then counting the different sequences, a certain degree of diversity in the library can be obtained.

[0085] In this application, "display" in phage display technology generally refers to the process of making foreign proteins or peptides visible on the surface of phage particles. In this application, "display" in antigen display technology generally refers to the process of displaying target antigens (usually proteins or peptides) on a surface for identification, detection or screening, for example, it can include displaying the target antigen in some way on cells, particles or other surfaces.

[0086] In this application, the term "antigen-binding phage" generally refers to a phage that displays an antigen-binding polypeptide. In some cases, an antigen-binding phage may refer to a phage vector used to display the antigen-binding polypeptide. In some cases, an antigen-binding phage may refer to a bacterium that contains the phage vector of the antigen-binding polypeptide.

[0087] In this application, the term "antigen-specific binding phage" generally refers to a phage that displays an antigen-specific binding polypeptide. In some cases, an antigen-specific binding phage may refer to a phage vector used to display the antigen-specific binding polypeptide. In some cases, an antigen-specific binding phage may refer to a bacterium that contains a phage vector that displays the antigen-specific binding polypeptide.

[0088] In this application, the term "antigen-binding polypeptide gene display vector" generally refers to a phage vector for displaying antigen-binding polypeptide genes. The antigen-binding polypeptide gene display vector is designed so that the antigen-binding polypeptide gene can be inserted into a specific site in the phage vector. This usually involves the use of appropriate promoters and terminators to ensure that the gene can be expressed in the phage particle. The antigen-binding polypeptide gene is usually fused with the structural protein gene of the phage so that the antigen-binding polypeptide can be stably displayed on the surface of the phage particle. The antigen-binding polypeptide gene display vector also generally includes expression control elements, such as promoters and regulatory sequences, to ensure that the antigen-binding polypeptide is properly expressed on the surface of the phage particle. The antigen-binding polypeptide gene display vector may also include a selection marker so that the particles displayed on the surface of the phage particle can be easily screened and separated.

[0089] In this application, the term "selectable marker" generally refers to a marker that can be used to identify, separate or mark a specific property or state of a specific organism, molecule or cell.

[0090] In the present application, the term "resistance gene" generally refers to a gene that a cell or organism has resistance to a specific environmental pressure or external influence. In vitro culture, resistance genes are generally used for selective screening of target cells or organisms. This resistance can be resistance to diseases, drugs, harmful substances, environmental conditions, etc. For example, a target cell that contains a resistance gene for a certain substance (for example, an antibiotic) and can express can grow in an environment containing the substance (for example, an antibiotic), while a target cell that does not contain or does not express the resistance gene cannot grow. The resistance gene can include drug resistance genes, poison resistance genes and / or environmental resistance genes.

[0091] In this application, the terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably and generally include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded.

[0092] In this application, the terms "polypeptide" or "protein" are used interchangeably to refer generally to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers.

[0093] In this application, the term "antigen-binding polypeptide" generally includes polypeptides that can specifically bind to the antigen and polypeptides that can non-specifically bind to the antigen. In this application, the "specific binding" generally refers to highly selective binding. Highly selective binding can mean that among many possible antigens, the polypeptide can accurately recognize and bind to a specific antigen. In some cases, "specific binding" can also include high-affinity binding. High-affinity binding can refer to a strong interaction between the antigen and the polypeptide. For example, the high-affinity binding can mean that the polypeptide has at least about 10 -4 M or less, or about 10 -5 M or less, or about 10 -6 M or less, or about 10 - 7 M or less, or about 10 -8 M or less, or about 10 -9 M or less, or about 10 -10 M or less, or about 10 - 11 M or less, or about 10 -12 M or less, or less K dIn some cases, "specific binding" may also include the exclusion of binding to non-target antigens.

[0094] In this application, "non-specific binding" is used in contrast to "specific binding" and generally refers to intermolecular binding that is not restricted by a specific structure or sequence and lacks high selectivity. In some cases, non-specific binding can include binding to multiple different targets. For example, a non-specific polypeptide can refer to a polypeptide that can bind to multiple different antigens.

[0095] In the present application, the binding of the polypeptide to the antigen can be detected by commonly used means in the art, for example, enzyme-linked immunosorbent assay (ELISA), immunoblotting, flow cytometry, biosensor technology, etc.

[0096] In this application, the term "antibody" generally refers to an immunoglobulin that is reactive to a specified protein or peptide or a fragment thereof. The antibody can be an antibody from any class, including but not limited to IgG, IgA, IgM, IgD and IgE, and an antibody from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). The antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibody of the present application can be derived from any species. In this application, the antigen-specific binding polypeptide can include an antibody.

[0097] As used herein, the term "antigen-binding fragment of an antibody" generally refers to the amino acid residues of an antibody that are responsible for antigen binding. The antigen-binding portion of an antibody comprises amino acid residues from a "complementarity determining region" or "CDR." As used herein, the antigen-specific binding polypeptide may include an antigen-binding fragment of an antibody.

[0098] In this application, the term "Fab" generally refers to two identical antigen-binding fragments produced by papain digestion of an antibody with a complete structure (e.g., removing the Fc region and hinge region). Fab can be composed of a complete light chain, a heavy chain variable region (VH), and the first constant domain (CH1) of the heavy chain. Each Fab can have a single antigen-binding site.

[0099] In this application, the term "scFv" generally refers to a monovalent molecule formed by covalently linking a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker.

[0100] In this application, the term "VHH" generally refers to an antibody comprising a variable antigen-binding domain of a heavy chain antibody (see Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH may also be referred to as a nanobody (Nb) and / or a single domain antibody.

[0101] In this application, the term "full-length antibody" generally refers to an antibody that is structurally similar to most naturally occurring antibodies (e.g., IgG antibodies). For example, a full-length antibody can include a molecule containing two complete heavy chains and two complete light chains. A "full-length antibody" can also include an antibody that is structurally similar to a naturally occurring dromedary antibody that contains only two complete heavy chains (often with an abnormally long CDR3 region) and no light chains.

[0102] In this application, the term "fusion protein" generally refers to a polypeptide or protein that contains the amino acid sequence of a first polypeptide or protein, or a fragment, analog or derivative thereof, and the amino acid sequence of a heterologous polypeptide or protein (i.e., a second polypeptide or protein, or a fragment, analog or derivative thereof, that is different from the first polypeptide or protein, or a fragment, analog or derivative thereof, or that is not generally a part of the first polypeptide or protein, or a fragment, analog or derivative thereof). In some cases, a fusion protein may comprise a prophylactic or therapeutic drug fused to a heterologous protein, polypeptide or peptide. The heterologous protein, polypeptide or peptide may or may not be a different type of prophylactic or therapeutic drug. For example, two different proteins, polypeptides or peptides with immunomodulatory activity may be fused together to form a fusion protein. In some cases, the fusion protein retains or increases the activity of the original polypeptide or protein before fusion with the heterologous protein, polypeptide or protein. For example, a fusion protein may include a peptibody. The term "peptibody" generally refers to a molecule comprising a peptide fused directly or indirectly to another molecule (e.g., an antibody Fc region), wherein the peptide portion specifically binds to a desired target.

[0103] In this application, the terms "cell membrane protein" and "membrane protein" can be used interchangeably and generally refer to proteins anchored in the lipid bilayer of a cell or organelle. In this application, the membrane protein includes a membrane protein that can serve as a therapeutic target.

[0104] In this application, the term "engineered" or "genetically engineered" generally refers to a method of modifying the genome of a cell by artificial means, including but not limited to deleting coding or non-coding regions or portions thereof, or inserting coding regions or portions thereof.

[0105] In this application, the term "integrative expression vector" generally refers to a vector that carries exogenous genes and can be stably integrated into the host cell genome, and the term "free expression vector" generally refers to a DNA vector that carries exogenous genes and is characterized by being able to exist in the host cell without having to be integrated into the host cell genome.

[0106] In this application, the term "Flp-In system" generally refers to a system for the stable integration of exogenous genes into the genome of mammalian cells. This system is implemented through recombination technology mediated by Flp recombinase (Flippase) in Escherichia coli. Specifically, the Flp-In system utilizes the specific recognition and recombination of FRT (Flippase Recognition Target) sequences by Flp recombinase.

[0107] In this application, the term "dissociation" generally refers to the weakening or interruption of the binding force between the antigen and the antigen-binding polypeptide, resulting in the separation of the two. The term "elution" generally refers to the steps including dissociation and collection of the dissociated antigen-binding polypeptide.

[0108] In this application, the term "infection" generally refers to the process by which a bacteriophage binds to a specific receptor on the surface of a target bacterium, then invades and injects at least a portion of its genome into the host bacterium. For example, the bacteriophage contains a phage vector, which, when infecting a bacterium, enables the phage vector to enter the bacterial cell (e.g., E. coli).

[0109] In this application, the term "test kit" generally refers to a combination of reagents and other materials. The test kit is expected to include reagents, such as buffers, protein stabilizing reagents, signal generating systems (e.g., fluorescent signal generating systems), antibodies, control proteins, and test containers (e.g., microtiter plates, etc.). The term "test kit" is not intended to be limited to a specific combination of reagents and / or other materials. For example, the test kit also includes instructions for using the reagents. The test kit can be packaged in any suitable manner, typically having components in a single container or (if necessary) in multiple containers and an instruction sheet for performing the test. The test kit can be prepared by a variety of methods known in the art.

[0110] In this application, the term "comprising" and its variations, including "containing", "including", and other forms, generally refer to the inclusion of other components, elements, values, steps, etc. In some cases, "comprising" encompasses "including" and "consisting of". The term "consisting of" does not include other components, elements, integers, steps, etc.

[0111] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0112] Detailed Description of the Invention

[0113] In one aspect, the present application provides a method for screening an antigen-specific binding polypeptide, the screening method comprising the following steps:

[0114] a) providing a first phage library displaying an antigen-specific binding polypeptide and a second phage library displaying an antigen-specific binding polypeptide, wherein the first phage library and the second phage library have screening markers, the first phage library has a first screening marker, and the second phage library has a second screening marker different from the first screening marker;

[0115] b) after contacting the antigen with the first phage library, contacting the antigen with a second phage library, thereby obtaining antigen-binding phage; and

[0116] c) under conditions in which the first screening marker is not displayed and the second screening marker is displayed, screening the second phage library for antigen-specific binding phages from the antigen-binding phages, wherein the antigen-specific binding phages display the antigen-specific binding polypeptide.

[0117] In certain embodiments, the first phage library and the second phage library are "identical." Because the first phage library and the second phage library are "identical," the antigen-nonspecific binding phage in the first phage library and the antigen-nonspecific binding phage in the second phage library may also be similar. Therefore, the first phage library can be used to block non-target antigens, thereby increasing the probability that antigen-specific binding polypeptides displayed by antigen-specific binding phage in the second phage library will bind to the target antigen, thereby improving screening efficiency.

[0118] The sameness of the first phage library and the second phage library described in this application generally refers to being similar or consistent in many aspects. The sameness of the first phage library and the second phage library generally refers to overall consistency, rather than the antigen-binding polypeptides displayed by the two phage libraries being identical in sequence one to one. For example, the sameness includes partial duplication of the antigen-binding polypeptides displayed by the two phage libraries. Even if the two phage libraries are identical, they may differ in tiny details. Based on the randomness of the phage library, the exogenous DNA fragments inserted in the library may be random, resulting in the genomic fragments represented by each clone being possibly not exactly the same. Therefore, although described as "same" as a whole, differences can be allowed, particularly in terms of the randomness and diversity of the phage library. For example, "same" described in this application does not mean that the antigen-binding polypeptide sequences displayed in the two phage libraries are completely identical.

[0119] In certain embodiments, the steps for preparing the first phage library are the same as the steps for preparing the second phage library.

[0120] In the present application, "the same preparation steps" means that in the process of providing the first phage library and the second phage library, the preparation steps of the two are similar or identical. This means that the key steps, operating procedures or experimental methods used in preparing the two phage libraries are relatively consistent. This does not mean that the preparation steps for providing the first phage library and the second phage library are exactly the same. There may be some differences, such as the use of DNA fragments encoding different antigen-binding polypeptides, different screening markers, different screening conditions or fine-tuning of other experimental parameters. Overall, the preparation processes of the two phage libraries are similar in key steps and may be based on the same basic principles or methods.

[0121] The steps for preparing a phage library are known to those skilled in the art, and the specific preparation steps may vary depending on the purpose of the experiment, the type of phage used, and the properties of the exogenous polynucleotide fragment. When preparing a phage library, those skilled in the art will typically adjust and optimize according to the experimental requirements. For example, the preparation of the phage library may include the following steps: constructing a phage display vector, transferring the phage display vector into cells, phage packaging, phage enrichment or screening, and / or phage library storage. The steps for constructing a phage display vector may include preparing exogenous polynucleotide fragments, inserting polynucleotide fragments into a phage display vector backbone, screening and / or amplification of the phage display vector, etc. The exogenous polynucleotide fragment encodes an antigen-binding polypeptide or a fragment thereof, and the phage display vector contains a gene encoding an antigen-binding polypeptide.

[0122] The preparation of exogenous polynucleotide fragments can include obtaining DNA of a gene of interest, cDNA (complementary DNA) or other exogenous polynucleotide fragments, which can be obtained by methods such as PCR amplification, enzyme digestion, and chemical synthesis. Transforming the phage display vector into cells can include introducing the phage display vector into the host Escherichia coli, which can be achieved by methods such as heat shock transformation and electroporation; and using helper phage to infect Escherichia coli to package the phage vector. The preparation of the phage library can also include selectively culturing the transformed or transfected Escherichia coli. Selective culture can include culturing the transformed Escherichia coli on a culture medium containing appropriate antibiotics to select phage display vectors with inserted DNA fragments. Phage library storage can include preserving the obtained phage display vector, preserving the Escherichia coli cells transformed with the phage display vector, or preserving the phage particles to form a phage library, from which the DNA fragments inserted in different clones can be obtained again.

[0123] The screening method for antigen-specific binding polypeptides described herein can be applied to phage libraries prepared using different preparation steps. For example, phage libraries prepared using any method can be screened using the screening method described herein, provided that the conditions for the first phage library and the second phage library are met.

[0124] The preparation steps of the first phage library and the second phage library of the present application may include the phage library preparation steps disclosed in PCT patent application No. PCT / CN2020 / 085706 (published on April 20, 2020, publication number WO2020216191A1), PCT patent application No. PCT / CN2022 / 084229 (published on October 6, 2022, publication number WO2022206868A1), and the disclosures of the above PCT patent applications are expressly incorporated herein by reference. For example, the first phage library and the second phage library of the present application can be phage libraries displaying antibodies or antibody fragments described in PCT patent application No. PCT / CN2020 / 085706. For example, the first phage library and the second phage library of the present application can be a phage library containing one or more first vectors to be screened, a phage library containing one or more second vectors to be screened, or a phage library containing one or more double-substituted vectors to be screened as described in PCT patent application No. PCT / CN2022 / 084229.

[0125] In certain embodiments, the screening marker of the antigen-binding polypeptide gene display vector used to provide the first phage library is different from the screening marker of the antigen-binding polypeptide gene display vector used to provide the second phage library.

[0126] In certain embodiments, the antigen-binding polypeptide sequence types displayed by the first phage library and the second phage library are the same. For example, the antigen-binding polypeptide sequences displayed by the first phage library and the second phage library are the same antibody type or antibody antigen-binding fragment type. In certain embodiments, the antigen-binding polypeptide sequence types displayed by the first phage library and the second phage library are both selected from the group consisting of: Fab, scFv, VHH, full-length antibody, fusion protein, and polypeptide. Those skilled in the art are aware of methods for controlling the antigen-binding polypeptide sequence types displayed by the first phage library and the second phage library when preparing phage libraries. For example, antigen-binding polypeptides of the same polypeptide sequence type can be obtained by using respectively identical upstream primers and downstream primers when preparing exogenous polynucleotide fragments.

[0127] In certain embodiments, the antigen-binding polypeptide sequences displayed by the first phage library and the second phage library are obtained in the same manner. The acquisition method generally refers to the method for obtaining exogenous polynucleotide molecules encoding the antigen-binding polypeptide sequences. The same acquisition method may include the same source and / or the same preparation method for the exogenous polynucleotide molecules.

[0128] For example, the same acquisition method can refer to the same template material, the same primer pair and the same PCR step, and the exogenous polynucleotide molecule is obtained by amplification by PCR. In this case, due to the non-specificity of PCR and the diversity of template materials, the product obtained by PCR can be a mixture of different DNA fragments. Therefore, even if the same template material, the same primer and the same PCR step are used, each PCR reaction may obtain different product mixtures. The same sequence acquisition method described herein can include the situation that this type of product mixture is obtained differently.

[0129] In certain embodiments, the first phage library and the second phage library are one selected from the following phage libraries: a natural library, an immune library, a semisynthetic library, and a synthetic library.

[0130] In certain embodiments, the first phage library and the second phage library have the same or different library capacities. In certain embodiments, the first phage library has a smaller or larger library capacity than the second phage library.

[0131] The steps for regulating and determining phage library capacity are well known to those skilled in the art. The specific methods may vary depending on the experimental objectives, the type of phage used, and the properties of the exogenous polynucleotide fragment. For example, when preparing a phage library, the library capacity can be controlled by adjusting the experimental parameters for phage transfection into E. coli. Adjusting the amount of phage vector used during the transformation process can affect the copy number of the phage vector in the E. coli. Increasing the amount of phage vector may result in more transformation events, thereby increasing the library capacity. Adjusting the number of E. coli colonies is also a key factor affecting library capacity. Using more E. coli may increase the number of transformation events, thereby increasing the library capacity. Similarly, reducing the amount of phage vector may result in fewer transformation events, thereby reducing the library capacity; using fewer E. coli may reduce the transformation time, thereby reducing the library capacity. Common methods for determining library capacity typically include counting transformed colonies or analyzing the insert fragments in the library to estimate the library size.

[0132] In the present application, the storage capacity and relative storage capacity difference of the first phage library and the second phage library can be adjusted according to various factors to achieve specific experimental objectives and screening effects. For example, the storage capacity of the first phage library and the second phage library can be adjusted according to the desired screening effect, the type of antigen, the diversity of the phage library, the type of displayed antigen-binding polypeptides, and other purposes. For example, when the antigen is a cell expressing a membrane protein antigen, the storage capacity of the first phage library and the second phage library can be adjusted according to factors such as the type of membrane protein, the expression of the membrane protein, and the type of cell. The storage capacity of the first phage library can be greater than, less than, or equal to the storage capacity of the second phage library.

[0133] Since the first phage library and the second phage library are "identical," the antigen-specific binding phage in the first phage library and the antigen-specific binding phage in the second phage library may also be similar. Therefore, in certain embodiments, if the second phage library has a larger capacity than the first phage library, the probability of containing antigen-specific binding phage different from those in the first phage library will theoretically be greater, and the probability of screening antigen-specific binding phage may be higher. In certain embodiments, the capacity of the first phage library may be smaller than that of the second phage library.

[0134] In certain embodiments, if the constructed specific cell membrane antigen-expressing cells have significantly different cell membrane antigen expression compared to the corresponding negative cells, for example, if the expression level of the cell membrane antigen is significantly higher than that of the negative cells, the library capacity of the first phage library can be larger than that of the second phage library. Even so, the antigen-specific binding phages in the first phage library will not saturate the specific antigen, and the antigen-specific binding phages in the second phage library will still have the opportunity to bind to the specific antigen on the cell surface. Therefore, in some cases, the library capacity of the first phage library can be larger than that of the second phage library to improve the blocking effect of the first phage library on non-specific binding and the blocking effect of specific binding to non-target antigens.

[0135] In the present application, the conditions that can cause the first screening marker to not be displayed and the second screening marker to be displayed generally refer to conditions that can distinguish phages having the first screening marker from phages having the second screening marker.

[0136] After contacting the antigen with the first phage library, the antigen is then contacted with the second phage library to obtain antigen-binding phage. The antigen-binding phage obtained at this point includes phage from both the first and second phage libraries. Therefore, the different first and second screening markers allow the antigen-binding phage from the first phage library to be distinguished from those from the second phage library. Because the first phage library is contacted with the antigen first, theoretically, the proportion of nonspecific binding phage in the phage obtained from the first phage library is much higher than that from the phage obtained from the second phage library. Furthermore, after the first phage library blocks non-target antigens, the proportion of antigen-specific binding phage in the second phage library is higher than that from the phage obtained from the first phage library. Therefore, under conditions that prevent the first screening marker from being displayed and the second screening marker from being displayed, the probability of obtaining antigen-specific binding phage is increased.

[0137] In certain embodiments, the differentiation can be demonstrated by whether or not the bacteria can grow normally. For example, the conditions under which the first screening marker is not displayed and the second screening marker is displayed can be conditions under which the phage in the first phage library cannot grow normally, while the phage in the second phage library can grow normally. For example, the conditions under which the first screening marker is not displayed and the second screening marker is displayed can be conditions under which bacteria containing the phage vector contained in the first phage library cannot grow normally, while bacteria containing the phage vector contained in the second phage library can grow normally.

[0138] In some embodiments, the phage library has a screening marker, which may refer to phages in the phage library having a screening marker on their genome. In some embodiments, the phage library has a screening marker, which may refer to phages in the phage library comprising a phage vector having a screening marker.

[0139] In certain embodiments, the screening marker may be a resistance gene, and the first screening marker and the second screening marker are different resistance genes.

[0140] In certain embodiments, the first screening marker can be selected from the group consisting of kanamycin resistance gene, puromycin resistance gene, hygromycin resistance gene and ampicillin resistance gene, and the second screening marker can be selected from the group consisting of kanamycin resistance gene, puromycin resistance gene, hygromycin resistance gene and ampicillin resistance gene that is different from the first screening marker.

[0141] For example, the first screening marker may be a kanamycin resistance gene, the second screening marker may be an ampicillin resistance gene, and the condition for not displaying the first screening marker and displaying the second screening marker may be using a culture medium containing ampicillin but not kanamycin. In a culture medium containing ampicillin, phages carrying the ampicillin resistance gene can grow normally, while phages carrying the kanamycin resistance gene cannot grow normally.

[0142] In certain embodiments, providing the first phage library and / or the second phage library requires a helper phage to package the phage vector, and the helper phage also has a corresponding screening marker (e.g., a resistance gene). In this case, regardless of whether the screening marker of the helper phage is the same as or different from the first screening marker of the first phage library, the first phage can achieve a certain degree of "blocking" effect.

[0143] The first screening marker and the second screening marker may also be different types of screening markers, as long as they can distinguish between antigen-binding phages in the first phage library and the second phage library. Different differentiation methods can be used according to specific needs. Those skilled in the art can select the most suitable screening marker type based on the purpose and conditions of the experiment to achieve accurate screening and differentiation of phages.

[0144] The screening method described in the present application can be used to screen polypeptides that specifically bind to a variety of different antigens and / or different forms of antigens.

[0145] In certain embodiments, the antigen may include a membrane protein and / or a membrane protein fragment. For example, the membrane protein includes, but is not limited to, a variety of membrane proteins such as G protein-coupled receptors (GPCRs), ion channel proteins, and transporters. For example, the antigen may include G protein-coupled receptors (GPCRs). GPCRs are a class of membrane proteins widely present on cell membranes that mediate cellular responses to many signaling molecules and are targets for many drugs, including those that regulate neurotransmitters, hormones, and medications. For example, the antigen may include the glucagon receptor (GCGR) and / or its fragments. The glucagon receptor (GCGR) is a G protein-coupled receptor that belongs to the seven-transmembrane protein family and is primarily expressed in tissues such as the liver, kidney, and heart. It is the receptor for glucagon, a hormone secreted by the pancreas whose primary function is to increase blood glucose levels. Due to the key role of GCGR in regulating blood glucose balance, it has become a target of some research and drug development. Several anti-GCGR drugs have been studied and developed to regulate blood sugar levels, which is potentially important in the treatment of metabolic diseases such as diabetes. Antibody drugs targeting GCGR may also become a strategy for regulating blood sugar balance.

[0146] In certain embodiments, the antigen may include a cell that expresses the antigen.

[0147] In certain embodiments, the cells expressing the antigen include natural cells and / or engineered cells. In certain embodiments, the cells expressing the antigen include cells expressing the antigen through an integrative expression vector and / or cells expressing the antigen through an episomal expression vector. In certain embodiments, the integrative expression vector includes a single-site integrative expression vector. In certain embodiments, the antigen may include cells expressing the antigen through the Flp-In system. For example, the Flp-In system can be used to integrate the antigen gene into a specific site of the target cell, which helps to ensure stable antigen expression and helps to avoid uncontrolled gene expression. In certain embodiments, the cells expressing the antigen may include mammalian cells. In certain embodiments, the cells expressing the antigen may include CHO cells. The basis for selecting cells expressing the antigen may depend on the specific requirements of the target antigen and the experimental system used.

[0148] The antigen-expressing cells described herein may also include cells engineered to improve the efficiency of antibody screening corresponding to the antigen. For example, the antigen-expressing cells described herein may also include glycosylphosphatidylinositol-anchored antigen-binding specific polypeptide display and β-arrestin reporter cells (see Ren, H., Li, J., Zhang, N. et al. Function-based high-throughput screening for antibody antagonists and agonists against G protein-coupled receptors. Commun Biol 3, 146 (2020).).

[0149] In certain embodiments, the antigen may include cells expressing membrane proteins and / or membrane protein fragments. For example, the antigen may include cells expressing G protein coupled receptors (GPCRs) and / or fragments thereof.

[0150] For example, the antigen may include cells expressing glucagon receptor (GCGR) and / or its fragments. In certain embodiments, the method for screening antigen-specific binding polypeptides provided herein further comprises the step of providing the antigen. For example, when the antigen is an engineered cell, the step of providing the antigen may include:

[0151] a) constructing a vector for expressing the antigen; and

[0152] b) transducing the vector expressing the antigen into cells, so that the cells express the antigen.

[0153] For example, the vector for expressing the antigen includes a Flp-In system vector. For example, the cell includes a mammalian cell. For example, the cell includes a CHO cell. For example, the antigen includes a G protein coupled receptor (GPCR) and / or a fragment thereof. For example, the antigen includes a cell expressing a G protein coupled receptor (GPCR) and / or a fragment thereof. For example, the antigen includes a glucagon receptor (GCGR) and / or a fragment thereof. For example, the antigen includes a cell expressing a glucagon receptor (GCGR) and / or a fragment thereof. For example, the antigen includes a cell expressing a glucagon receptor (GCGR) and / or a fragment thereof.

[0154] In certain embodiments, obtaining antigen-binding phage may comprise the following steps:

[0155] a) dissociating the antigen-binding phage from the antigen;

[0156] b) collecting antigen-binding phage that have dissociated from the antigen; and / or

[0157] c) allowing the antigen-binding phage to infect bacteria.

[0158] In the present application, the antigen-binding phage can include phages in various forms or states, for example, phages can include phage particles, phage vectors, bacteria containing the phage vectors and / or bacteria infected by the phage. For example, the antigen-binding phage is dissociated from the antigen, and those skilled in the art will understand that it generally refers to the dissociation of the antigen-binding polypeptide displayed on the surface of the phage particle from the antigen. For example, collecting the antigen-binding phage that is dissociated from the antigen generally refers to collecting the culture after dissociation, which may contain a mixture of phage particles, phage vectors, bacteria infected by the phage and / or bacteria containing the phage vector. For example, causing the antigen-binding phage to infect bacteria generally refers to transforming bacteria with the antigen-binding phage vector.

[0159] In certain embodiments, the methods for screening for antigen-specific binding polypeptides described herein may further comprise amplifying and / or preserving the antigen-specific binding phage obtained by screening. For example, the amplification and / or preservation may be achieved by transforming and / or culturing bacteria infected with the antigen-specific binding phage. For example, amplification may be achieved by infecting Escherichia coli with the aid of a helper phage.

[0160] For example, screening the antigen-specific binding phage in the second phage library from the antigen-binding phage includes allowing the antigen-binding phage to infect bacteria, and then culturing the bacteria under conditions where bacteria infected with the second phage library can grow but bacteria infected with the first phage library cannot grow. For example, when the selection marker is an antibiotic resistance gene, culturing the bacteria includes using a culture medium containing an antibiotic corresponding to the second selection marker and not containing an antibiotic corresponding to the first selection marker.

[0161] In certain embodiments, the method for screening antigen-specific binding polypeptides described in the present application may further include a step of further screening, a step of improving screening efficiency, a step of obtaining the antigen-specific binding polypeptide and / or a step of verifying the function and / or activity of the antigen-specific binding polypeptide.

[0162] In certain embodiments, the method for screening antigen-specific binding polypeptides described herein may further comprise the step of obtaining the antigen-specific binding polypeptide based on the antigen-specific binding phages in the second phage library.

[0163] In certain embodiments, the step of obtaining the antigen-specific binding polypeptide may include the following steps:

[0164] a) extracting phage DNA: extracting phage DNA from the antigen-specific binding phage, or extracting phage DNA from bacteria infected by the antigen-specific binding phage, which can be achieved by conventional DNA extraction methods in the art;

[0165] b) PCR amplification: Using PCR technology, specific primers are used to amplify the antigen-specific binding polypeptide gene. The primer design is usually based on the known phage vector and antibody framework region. PCR amplification can ensure the selective amplification of the target antibody gene.

[0166] c) Sequencing: Sequencing the antibody gene fragments amplified by PCR. Sequencing can be performed using Sanger sequencing or other high-throughput sequencing technologies. The sequencing results will provide the DNA sequence information of the antibody.

[0167] d) Sequence analysis: Analyze the sequenced DNA sequence and / or the amino acid sequence encoded by the DNA sequence to obtain information about the structure and function of the antigen-specific binding polypeptide;

[0168] e) Constructing an expression vector: Based on the sequencing results, design and construct an appropriate expression vector to contain the complete antigen-specific binding polypeptide gene. This may involve inserting the antigen-specific binding polypeptide gene into an appropriate expression vector to ensure that the antigen-specific binding polypeptide can be effectively expressed in the host cell;

[0169] f) Cell transfection and expression: introducing the constructed expression vector into host cells, and expressing the antigen-specific binding polypeptide gene by cell transfection or other appropriate methods; and / or

[0170] g) Protein expression detection: Ensure that the antigen-specific binding peptide is expressed in the cells. This can be done by detecting the protein expression level of the antibody through methods such as Western blot and ELISA.

[0171] In certain embodiments, the step of obtaining the antigen-specific binding polypeptide may further include the step of isolating and / or purifying the antigen-specific binding polypeptide. The steps of isolating and / or purifying the antigen-specific binding polypeptide may be known to those skilled in the art. For example, the antigen-specific binding polypeptide may be isolated and / or purified by ion exchange chromatography, affinity chromatography, liquid chromatography, or the like.

[0172] In certain embodiments, the step of obtaining the antigen-specific binding polypeptide may further comprise obtaining amino acid sequence information of the antigen-specific binding polypeptide obtained by separation and / or purification, and / or analyzing the amino acid sequence to obtain information about the structure and function of the antigen-specific binding polypeptide. The amino acid sequence information may be obtained by amino acid sequencing. Amino acid sequencing methods are known to those skilled in the art. For example, amino acid sequence information may be obtained by amino acid sequencing methods such as mass spectrometry, Edman degradation, gas chromatography-mass spectrometry, and enzymatic hydrolysis.

[0173] In certain embodiments, when the antigen-specific binding fragment is an antibody fragment, the step of obtaining the antigen-specific binding polypeptide may further include converting it into a full-length antibody. In certain embodiments, the step of obtaining the antigen-specific binding polypeptide may further include converting the antigen-specific binding polypeptide into an IgG form. The step of converting into an IgG form may be known to those skilled in the art. For example, it may be achieved through genetic engineering or biosynthesis.

[0174] In certain embodiments, the step of the antigen-specific binding polypeptide may also include assessing the expression of the antigen-specific binding polypeptide, its binding affinity to the antigen and / or its biological function. The method and specific steps for assessing the expression of the antigen-specific binding polypeptide, its binding affinity to the antigen and / or its biological function may be known to those skilled in the art, and different techniques may be selected according to specific experimental requirements. For example, the method for assessing the expression of the antigen-specific binding polypeptide, its binding affinity to the antigen and / or its biological function may include immunoblotting, enzyme-linked immunosorbent assay, flow cytometry, biosensor technology, cell function experiments, bioactivity analysis, surface plasma mass spectrometry, Raman spectroscopy and other methods.

[0175] In certain embodiments, the method of screening an antigen-specific binding polypeptide described herein may further comprise evaluating the binding of the antigen-specific binding polypeptide to the antigen by FACS (fluorescence activated cell sorting) analysis.

[0176] For example, the phage displaying the antigen-specific binding polypeptide can be directly used in FACS analysis to assess the combination of the antigen-specific binding polypeptide and the antigen. For example, the culture after the phage displaying the antigen-specific binding polypeptide infects bacteria can be directly used in FACS analysis to assess the combination of the antigen-specific binding polypeptide and the antigen. For example, the antigen-specific binding polypeptide after the separation and / or purification can be used in FACS analysis to assess the combination of the antigen-specific binding polypeptide and the antigen. The specific steps of FACS analysis and assessment can be well known to those skilled in the art, and can include steps such as labeled antibodies or antigen-specific binding polypeptides, processing cell samples, flow cytometer analysis and data analysis.

[0177] In certain embodiments, the method for screening antigen-specific binding polypeptides described herein may further include screening the antigen-specific binding polypeptides displayed in the first phage library. Based on the fact that the first phage library and the second phage library are "the same," there are theoretically phages displaying antigen-specific binding polypeptides in the first phage library. However, under conditions where the first screening marker is not displayed and the second screening marker is displayed, phages displaying antigen-specific binding polypeptides in the first phage library cannot be obtained. Therefore, before placing the obtained antigen-binding phages under conditions where the first screening marker is not displayed and the second screening marker is displayed, the method described herein may also obtain antigen-binding phages in the first phage library under conditions where the first screening marker is displayed, and screen for antigen-specific binding polypeptides therein.

[0178] In certain embodiments, the method for screening antigen-specific binding polypeptides described herein may further include amplifying and / or preserving the antigen-binding phage obtained before placing the obtained antigen-binding phage under conditions that cause the first screening marker to be non-displayed and the second screening marker to be displayed. For example, if no antigen-specific binding polypeptide is obtained in the second phage library, the antigen-specific binding polypeptides displayed in the first phage library may be screened again. Alternatively, in order to screen for different antigen-specific binding polypeptides, the antigen-specific binding polypeptides displayed in the first phage library may be screened again.

[0179] In certain embodiments, in order to screen for a diverse array of antigen-specific binding polypeptides, the methods for screening for antigen-specific binding polypeptides described herein may further include, during the step of contacting the antigen with the first phage library, contacting a polypeptide known to specifically bind to the antigen with the antigen. For example, in this case, the probability of obtaining a novel antigen-specific binding polypeptide having a different antigen-binding epitope than the polypeptide known to specifically bind to the antigen can be increased.

[0180] In certain embodiments, the method for screening antigen-specific binding polypeptides described herein may further include a further screening step based on the affinity and / or biological function of the antigen-specific binding polypeptides to the target antigen. For example, the method for screening antigen-specific binding polypeptides described herein may further include quantitative or qualitative determination of affinity of the antigen-specific binding polypeptides to select polypeptides with higher binding affinity. For example, the method for screening antigen-specific binding polypeptides described herein may further include quantitative or qualitative determination of the effect of the antigen-specific binding polypeptides on the function of the target antigen to select polypeptides with better biological function. For example, the method for screening antigen-specific polypeptides described herein may further include displaying the obtained antigen-binding specific polypeptides on cells for a further screening step.

[0181] The method for screening antigen-specific binding polypeptides described herein can be combined with methods known in the prior art for improving screening efficiency. For example, for the purpose of further improving screening efficiency or screening different antigen-specific binding proteins. In certain embodiments, the method for screening antigen-specific binding polypeptides described herein can include the step of using a blocking agent. For example, the blocking agent can include bovine serum albumin (BSA), milk and / or casein. In certain embodiments, the method for screening antigen-specific binding polypeptides described herein can include the step of exposing the library to a similar cell line that is negative for the desired antigen before contacting the phage library with the positive cell line. In certain embodiments, the method for screening antigen-specific binding polypeptides described herein can include the step of alternating cell lines expressing the same target antigen. In addition, the method for screening antigen-specific binding polypeptides described herein can also include further optimization of screening and / or culture conditions. For example, further optimization of the screening and / or culture conditions can include optimizing the solution formulation used for dissociation when dissociating the antigen-binding phage from the antigen. For example, further optimization of the screening and / or culture conditions can include adjusting the pH of the eluent. For example, further optimization of the screening and / or culture conditions may include the use of an eluent comprising a competitive binder. For example, further optimization of the screening and / or culture conditions may include the adjustment of the culture temperature and culture time.

[0182] In certain embodiments, the method for screening antigen-specific binding polypeptides described herein can be repeated one or more times. In certain embodiments, the repetition can include using the obtained antigen-binding phage as the "second phage library" for the next round, contacting the antigen with the first phage library, and then contacting the antigen with the second phage library. In certain embodiments, the repetition can include using the antigen-binding phage in the obtained second phage library as the "second phage library" for the next round, contacting the antigen with the first phage library, and then contacting the antigen with the second phage library. Alternatively, in certain embodiments, the repetition can include continuing to screen the antigen-binding phage in the obtained second phage library in other ways.

[0183] In some embodiments, using the screening method of the antigen-specific polypeptide described in the present application, it is possible to obtain an antigen-specific binding polypeptide that specifically binds to a target antigen, a phage that displays the antigen-specific polypeptide, a cell that includes a phage that displays the antigen-specific polypeptide, a nucleic acid molecule that encodes the antigen-specific binding polypeptide, and / or a vector that includes a nucleic acid molecule that encodes the antigen-specific binding polypeptide. In some embodiments, using the screening method of the antigen-specific polypeptide described in the present application, it is possible to improve the probability and / or efficiency of obtaining an antigen-specific binding polypeptide that specifically binds to a target antigen, a phage that displays the antigen-specific polypeptide, a cell that includes a phage that displays the antigen-specific polypeptide, a nucleic acid molecule that encodes the antigen-specific binding polypeptide, and / or a vector that includes a nucleic acid molecule that encodes the antigen-specific binding polypeptide. In some embodiments, using the screening method of the antigen-specific polypeptide described in the present application, it is possible to reduce the workload of obtaining an antigen-specific binding polypeptide that specifically binds to a target antigen, a phage that displays the antigen-specific polypeptide, a cell that includes a phage that displays the antigen-specific polypeptide, a nucleic acid molecule that encodes the antigen-specific binding polypeptide, and / or a nucleic acid molecule that encodes the antigen-specific binding polypeptide compared to a case where the method is not used.

[0184] In certain embodiments, the antigen-specific polypeptide screening methods described herein can be used to determine whether a phage library contains phage that specifically binds to a target antigen. In certain embodiments, the antigen-specific polypeptide screening methods described herein can be used to determine whether a phage library contains no or substantially no phage that specifically binds to a target antigen. The screening results of the methods described herein may depend on a variety of factors, including the quality of the phage library, the nature of the antigen, the screening conditions, and the like.

[0185] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0186] Example

[0187] Example 1 Construction of an autoimmune disease donor-specific Fab phage display library

[0188] Referring to the method for displaying a phage library of antibodies or antibody fragments in WO2020216191A1, a Fab phage library was constructed.

[0189] Using PBMCs from more than 100 autoimmune disease donors as raw materials, we extracted total RNA, amplified antibody genes, and constructed autoimmune disease donor-specific VH sublibrary (the library capacity reached 5.6x10 8 ), KLC sub-library (library capacity reaches 3.6x10 8 ), and LLC sub-library (library capacity reaches 1.2x10 8 The vector fragment, VH sublibrary fragment, KLC sublibrary fragment, LLC sublibrary fragment, and linker fragment were prepared and purified by ligation of the four fragments to obtain 213 μg of ligation product (the vector structure is shown in Figure 2). 337 strains of TG1 electroporated bacteria (Lucigen) were transformed to construct a Fab phage display bacterial library. The ligation transformation efficiency (LTE) reached 1.47×10 9 / ug connection product, the storage capacity reaches 3.14x10 11 , the diversity reaches 2x10 11 Fab expression analysis of bacterial clones showed that the expression capacity was 1.96x10 11 M13KO7 helper phage was used to infect the ZM-Fab library, and the ZM-Fab phage library (second phage library: ZM-Fab library) was constructed by packaging and amplification. The total amount of the phage library was 1.58x10 16 , ZM-Fab phage library 3.4x10 13 / mL / tube, which is 100 times the capacity of the bacterial library, and frozen at -80 degrees for subsequent screening of antigen-specific Fab.

[0190] The Amp resistance gene of the original vector pComb-3X-Fab-Amp used to construct the ZM-Fab library was replaced with the Kana resistance gene to obtain the pComb-3X-Fab-Kana vector. The pComb-3X-Fab-Kana vector was digested to prepare a vector fragment containing the Kana resistance gene. Referring to the method for displaying a phage library of antibodies or antibody fragments in WO2020216191A1, the same raw materials as those used for the preparation of ZM-Fab (more than 100 PBMCs from autoimmune disease donors as raw materials) were used to prepare VH sublibrary fragments, KLC sublibrary fragments, LLC sublibrary fragments, and linker fragments. The Kana-resistant Fab library was constructed by connecting the four fragments, with a library capacity of 2x10 8The Kana-Fab library (first phage library: Kana-Fab library, vector structure as shown in Figure 3) was packaged and amplified for freezing. The phage concentration was 2x10 13 / mL.

[0191] Example 2 Construction of FCHO cell line expressing hGCGR (F-hGCGR)

[0192] 1) Two expression cassettes containing the eGFP gene and the hGCGR gene (SEQ ID NO: 1) were inserted into the Flp-In vector pcDNA5 / FRT to obtain pDGB4-eGFP-hGCGR. The vector structure is shown in Figure 1;

[0193] 2) The vector pDGB4-eGFP-hGCGR was transformed into FCHO cells and selected by hygromycin pressure to obtain single-site stably integrated cells (F-hGCGR) that efficiently expressed the native hGCGR structure on the FCHO cell surface. The F-hGCGR cells were cultured, expanded, and frozen.

[0194] Example 3 Expression and purification of hGCGR-specific positive control antibody Volagidemab and phage displaying Volagidemab-Fab

[0195] Volagidemab, which has entered clinical trials, was selected as a positive control. Volagidemab is a monoclonal antibody that antagonizes the glucagon receptor (GCGR). The antibody amino acid sequences (Volagidemab HC - SEQ ID NO: 3, Volagidemab LC - SEQ ID NO: 4) were obtained through patent searches. After expression and purification, the full-length Volagidemab antibody was obtained.

[0196] With reference to the method for displaying antibody or antibody fragment phage libraries in WO2020216191A1, the volagidemab display vector pComb-3X-volagidemab-Fab was constructed and transformed into TG1 bacteria. Expression was induced with IPTG to obtain a bacterial supernatant containing volagidemab-Fab.

[0197] Example 4 FACS analysis of the specific binding of Volagidemab full-length antibody and Fab to hGCGR on the surface of F-hGCGR cells

[0198] Volagidemab full-length antibody and Fab were used to stain F-hGCGR cells and primary FCHO cells, respectively. PE-labeled mouse anti-human kappa light chain antibody was used as a secondary antibody to stain both cells. As shown in Figure 4, neither the full-length Volagidemab antibody nor the Fab bound to FCHO cells, but both did bind to F-hGCGR cells with strong signals. This indicates that both the full-length Volagidemab antibody and the Fab specifically bind to hGCGR on the surface of F-hGCGR cells.

[0199] Example 5 Screening of hGCGR-specific Fab from the ZM-Fab phage library

[0200] 1) Thaw a tube of frozen F-hGCGR cells (T175, 90% confluence), wash twice with 1.5 mL of PBS (centrifugation at 500g for 3 minutes each time), suspend the cells in 1 mL of F12 medium + 10% FBS, and keep on ice until ready to use.

[0201] 2) Add 0.5 mL of Kana-Fab library (first phage library, 2x10 13 / mL) to the cell suspension, mix well, shake at 4 degrees for 60 minutes (20 rpm), and block the cells with Kana-Fab library.

[0202] 3) Centrifuge and discard the supernatant, suspend the cells with 1 mL of F12 culture medium + 10% FBS, add 0.5 mL of ZM-Fab library (second phage library), and shake at 4°C for 120 minutes (20 rpm).

[0203] 4) Centrifuge and discard the supernatant, then wash 4 times with PBS (1.8 mL / time).

[0204] 5) Add 300 μL of pH 2.2 glycine solution (0.2 M), pipette several times to suspend the cell pellet, and let it stand at room temperature for 10 minutes (shake gently several times every 2-3 minutes) to elute the phage.

[0205] 6) Centrifuge at 1000g for 3 minutes. Transfer the supernatant to a new 1.5mL centrifuge tube and neutralize the phage eluate with 110µL of Tris solution (pH 8.0-1M). Store at 4°C or proceed to the next step.

[0206] Example 6 Phage eluate infects TG1 bacteria and is titrated on a plate

[0207] 1) Prepare TG1 bacteria and adjust the bacteria to the logarithmic growth phase, OD280 = 0.2-0.3.

[0208] 2) Mix 200 μL of phage eluate with 2 mL of TG1 bacterial solution and incubate at 37°C for 30 minutes.

[0209] 3) Take 10uL of phage-infected TG1 bacterial solution and dilute it 10-fold six times with culture medium. Take 5uL of each dilution and spread it on Amp-resistant plates for titration. Spread the remaining bacterial solution on 3 large square plates (225cm 2 The plates were incubated at 30°C overnight.

[0210] 4) The next day, observe, analyze, and calculate the screening titer. Design the next step based on the screening results: Collect the plate colonies from this round and use M13KO7 helper phage to package and amplify the phage library for the next round of screening. The specific operation process is described above; alternatively, inoculate the colonies, induce Fab expression with IPTG, and screen and analyze hGCGR-positive clones by FACS.

[0211] Example 7 FACS screening and analysis of hGCGR positive clones

[0212] After 2-5 rounds of screening, an appropriate number of colonies were inoculated from the plates of the appropriate round, and IPTG was used to induce the expression of soluble Fab. The binding of Fab to F-hGCGR was analyzed by FACS.

[0213] 1) Take a 96-well deep-well plate and add 400 μL / well of 2YT-Amp-0.2% glucose culture medium.

[0214] 2) Inoculate an appropriate number of colonies into a 96-well deep-well plate and incubate at 37°C, 250 rpm for 2-3 hours.

[0215] 3) Add 2YT-Amp-IPTG culture medium (400 μL / well) to a final IPTG concentration of 1 μM. Incubate overnight at 250 rpm and 30°C.

[0216] 4) The next day, transfer 100 μL / well of the bacterial solution from the 96-well deep-well plate to a new deep-well plate, add DMSO (10 μL / well) to the new deep-well plate, mix well, and store at -80°C.

[0217] 5) Centrifuge the original deep-well plate at 3000G for 10 minutes, transfer the supernatant containing the induced expressed Fab (400uL / well) to a new deep-well plate and store at 4°C.

[0218] 6) Thaw F-hGCGR cells. To analyze clones in a 96-well deep-well plate, cells should be at 1 / 2-T175-90% confluence. Wash twice with staining buffer (PBS-1% FBS).

[0219] 7) Resuspend the cells in 10 mL of staining buffer and aliquot into a 96-well round-bottom ELISA plate at 100 μL / well.

[0220] 8) Transfer 30 μL / well of the Fab supernatant after overnight induction to the cell suspension plate, mix well, and incubate on ice for 30 minutes.

[0221] 9) Add staining buffer (200 μL / well), centrifuge the plate at 1000 × g for 5 minutes, and discard the supernatant.

[0222] 10) Add 30uL of secondary antibody solution (40uL-PEK + 40uL PEL + 2920uL staining buffer), suspend the cells, and incubate on ice for 30 minutes.

[0223] 11) Add staining buffer, 100 uL / well, and perform FACS analysis.

[0224] 12) Inoculate PE-positive clones for FACS analysis, send samples for sequencing, and identify positive Fab clones with hGCGR-specific unique amino acid sequences.

[0225] Comparison of FACS images of positive clones and negative clones:

[0226] 1. Positive clones (cells located in the upper left quadrant) and negative clones (cells located in the lower left quadrant) can be clearly distinguished;

[0227] 2. The characteristics of the positive clones (the expression intensity of the antibody and the binding ability to the cell surface antigen) can be preliminarily judged by the proportion of positive cells and their positioning in the image.

[0228] Example 8 Construction of Volagidemab light chain replacement library, heavy chain replacement library, light and heavy chain double replacement library, and screening of hGCGR-specific Fab

[0229] With reference to the preparation method of the chain replacement phage library in WO2022206868A1 and the process of direct screening of functional antibody patents, the Volagidemab light chain replacement library (LC-ZH) and the Volagidemab heavy chain replacement library (HC-ZH) were constructed, and a group of hGCGR-specific positive Fab clones containing new light chains and a group of hGCGR-specific positive Fab clones containing new heavy chains were screened. Then, a group of new light chain genes and a group of new heavy chain genes were combined to construct a light and heavy chain double Fab replacement library (DZH), and a group of light and heavy chain double replacement hGCGR-specific positive Fab clones were screened. FACS-positive clones were inoculated, sent for sequencing, and sequence analysis was performed on the sequencing results to obtain unique sequence-positive clones from 4 libraries.

[0230] Example 9 Screening of hGCGR-specific Fab clones and data analysis

[0231] This example statistically analyzes the screening of hGCGR-specific Fabs from the natural original library (ZM-Fab), light chain replacement library (LC-ZH), heavy chain replacement library (VH-ZH), and light and heavy chain double replacement library (DZH).

[0232] Each library was screened for 1 to 5 rounds, depending on the needs. An appropriate number of colonies were inoculated from each round of screening, and the culture supernatants of the clones from three to four 96-well deep-well plates were analyzed by FACS to screen for hGCGR-specific binding Fabs. A total of 14 96-well deep-well plates and 1,344 clones were analyzed.

[0233] The ZM-Fab library was screened for three rounds, and four 96-well plates were analyzed by FACS, yielding 223 FACS-positive clones with a total positive rate of 58% (24-93%). Sequencing analysis of 215 FACS-positive clones yielded six unique sequence Fabs.

[0234] The heavy chain replacement library (VH-ZH library) was screened for 5 rounds, and FACS analysis was performed on 4 96-well plates, yielding 283 FACS-positive clones with a total positive rate of 73% (28-96%). Sequencing analysis was performed on 75 FACS-positive clones, yielding 29 unique Fab sequences.

[0235] The light chain replacement library (LC-ZH library) was screened for three rounds, and three 96-well plates were analyzed by FACS, yielding 107 FACS-positive clones with a total positive rate of 37% (2-85%). Sequencing analysis of 75 FACS-positive clones yielded 19 unique sequence Fabs.

[0236] The double-permuted light and heavy chain library (VH-LC-DZH library) was screened for one round, and three 96-well plates were analyzed by FACS, yielding 198 FACS-positive clones with a total positive rate of 69% (42-82%). Sequencing analysis of 139 FACS-positive clones yielded 29 unique sequence Fabs.

[0237] The following table shows the screening data statistics for hGCGR-specific Fabs screened from the natural original library (ZM-Fab), light chain permuted library (LC-ZH), heavy chain permuted library (VH-ZH), and light and heavy chain double permuted library (DZH):

[0238] The statistics of sequencing results of hGCGR-specific Fabs screened from the natural original library (ZM-Fab), light chain permuted library (LC-ZH), heavy chain permuted library (VH-ZH), and light and heavy chain double permuted library (DZH) are shown in the following table:

[0239] The results show that the screening method of the present application has a very high screening efficiency, and high-affinity specific antibodies can be screened in 2-3 rounds. For double-permutation phage libraries, positive clones can be obtained in the first round of screening. For natural original libraries, light chain permutation libraries, and heavy chain permutation libraries, positive clones can be obtained in the second round of screening, and the positive clone rate is high. In addition, multiple clones with unique sequences can be screened efficiently.

[0240] Example 10: Conversion of Fab into full-length antibody, expression and analysis of the dose-dependent binding of the full-length antibody to the native cell surface structure hGCGR

[0241] Volagidemab was used as a positive control, and an antibody numbered DDBKB001-15 (obtained during the screening process) was randomly selected as a negative control.

[0242] Based on the results of FACS analysis and sequence analysis, 10 positive Fab clones with high FACS signal intensities that were significantly different from Volagidemab in CDR region sequences were selected and numbered GR01-GR10. The corresponding full-length antibodies (IgG1 subtype) were expressed and purified. GR01 and GR02 were from the natural human Fab library (ZM-Fab); GR03 and GR04 were from the light chain replacement library; GR05 and GR06 were from the heavy chain replacement library; and GR07, GR08, and GR09 were from the light and heavy chain double replacement library. Ultimately, with the exception of GR08 (due to the different preferred codons for bacterial and cell-synthesized proteins, individual positive Fab clones could not be expressed in cells when converted into full-length antibodies), 9 full-length antibodies were expressed and purified.

[0243] FACS analysis showed a dose-dependent binding of 11 antibodies (including positive and negative controls) to cell surface hGCGR (the final concentrations of the reaction system antibodies were 5nM, 50nM, 500nM, 5uM, 50uM, and 500uM, respectively, from low to high). Preliminary results showed that GR05 had the strongest signal. To confirm the reliability of the analysis results, GR05 was selected to repeat the dose-dependent binding experiment (GR05B). The results are shown in Figures 6A and 6B. The binding curves of the GR06 and GR10 full-length antibodies overlapped with the negative control and showed no specific binding ability to hGCGR. The other seven antibodies all showed specific binding ability to hGCGR, comparable to or even stronger than Volagidemab, with GR02 and GR05, in particular, significantly higher than the positive control.

[0244] Because bacteria lack post-transcriptional modifications, full-length antibodies undergo post-transcriptional modifications and structural changes during cellular expression, leading to the loss of specific binding capacity in some Fab-positive clones. This example demonstrates that the screening method described herein can efficiently identify antibodies that exhibit antigen-specific binding capacity after conversion to full-length antibodies.

[0245] Example 11: Converting Fab into full-length antibodies and expressing and analyzing the biological functions of full-length antibodies at the cellular level

[0246] This example illustrates that the screening method described in this application can efficiently screen for antibodies that have biological functions after conversion into full-length antibodies.

[0247] In this example, a cAMP kit was used to detect the effect of the antibody to be tested or the control substance in inhibiting the production of cAMP by glucagon in HEK293 / GCGR / Gα15 cells.

[0248] The materials and reagents used in this example are shown in the table below:

[0249] Experimental methods:

[0250] 11.1 Solution preparation

[0251] 11.1.1 Preparation of test medium

[0252] Mix 98% cell culture medium and 2% FBS (for example, 49 ml cell culture medium and 1 ml FBS). Store at 2-8°C and the shelf life is 6 months.

[0253] 11.1.2 Preparation of stimulation buffer

[0254] 0.5 M IBMX was added to 2% FBS assay medium to dilute it to a concentration of 2.5 mM in stimulation buffer.

[0255] 11.1.3 Preparation of Agonist, Test Antibody, and Control Standard Working Solutions

[0256] Except for the agonist Glucagon, all test antibodies and control standards are solutions. 271 μl of DMSO is added to 1 mg of glucagon to prepare a 1 mM stock solution.

[0257] The stock solution of Glucagon was diluted to 10 nM with stimulation buffer to obtain solution SD1, which was then serially diluted 4-fold starting from SD1, for a total of 10 concentration gradients.

[0258] The stock solutions of the test antibody and the control standard were diluted to 666.7 nM to obtain solution SD1, which was then serially diluted 2-fold starting from SD1 for a total of 10 concentration gradients.

[0259] The stock solution of Glucagon was prepared into a 50 pM working solution using stimulation buffer.

[0260] 11.2 Cell Treatment

[0261] 11.2.1 Cell Preparation

[0262] The cells were passaged and expanded in advance. When the cell density reached 80%, the cells were washed 1-2 times with PBS and digested with 0.25% trypsin. The digestion was terminated when the cells became round under microscope. The cells were stained with trypan blue to determine if the cell viability was greater than 95%. The cells were prepared into a density of 8×10 5cells / ml, and 5 μl of cell suspension was inoculated into each well of a 384-well plate.

[0263] 11.2.2 Treatment of test antibodies and control standards

[0264] Then, 3 μl of the antibody to be tested, control standard, and assay medium were added to each well. The 384-well plate was sealed with a membrane and placed in a microplate constant temperature shaker at 500 rpm for 3 min at room temperature. The plate was then transferred to a 37°C cell culture incubator and incubated for 30 min.

[0265] Remove the well plate, add 50pM Glucagon working solution to the test antibody and control standard wells, 2μl / well, final concentration 10pM; add gradient diluted Glucagon working solution to the agonist wells, 2μl / well.

[0266] The 384-well plate was sealed with a membrane and placed in a microplate constant temperature shaker at 500 rpm for 3 min at room temperature, and then transferred to a 37°C cell culture incubator for further incubation for 30 min.

[0267] 11.2.3 Cell Lysis and Detection Solution Treatment

[0268] Use the lysis detection buffer in the cAMP detection kit to dilute 20× cAMP Eu Cryptate and 20× cAMP d2 reagent to 1×. Then mix the two dilutions 1:1 and add 10 μl / well to the cell wells. Seal the 384-well plate with a membrane and place it in a microplate constant temperature shaker at 350 rpm at room temperature for 3 minutes. Then let it stand at room temperature for 1 hour before detection.

[0269] 11.3 Detection and Data Processing

[0270] Use a SpectraMax Paradigm microplate reader with excitation at 340 nm, collect signal values ​​under emission light conditions of 616 nm and 665 nm, calculate the ratio of acceptor and donor emission signals for each individual well, plot the relationship between HTRF ratio and compound concentration, determine the concentration of cAMP by standard curve analysis, plot the relationship between compound concentration and cAMP concentration, and calculate the specific activity of the compound.

[0271] The results are shown in FIG7 and the table below, indicating that the screening method described in this application successfully screened out compounds with similar or even lower EC values ​​than the control standard Volagidemab. 50Value-added, biologically functional antibodies (wherein, DDBAB03 and DDBAB04 are full-length antibodies converted from Fab obtained by screening using the screening method described in this application, DDBAB11 is an antibody obtained based on the Volagidemab sequence using the same purification method as DDBAB03 and DDBAB04, and Volagidemab is a commercially available antibody).

[0272] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to fall within the scope of the appended claims and their equivalents.

Claims

1. A method for screening an antigen-specific binding polypeptide, the screening method comprising the following steps: a) providing a first phage library displaying an antigen-specific binding polypeptide and a second phage library displaying an antigen-specific binding polypeptide, wherein the first phage library and the second phage library have screening markers, the first phage library has a first screening marker, and the second phage library has a second screening marker different from the first screening marker; b) contacting the antigen with the first phage library, and then contacting the antigen with the second phage library, thereby obtaining antigen-binding phage; and c) under conditions in which the first screening marker is not displayed and the second screening marker is displayed, screening the second phage library for antigen-specific binding phages from the antigen-binding phages, wherein the antigen-specific binding phages display the antigen-specific binding polypeptide.

2. The method according to claim 1, wherein the preparation step of providing the first phage library is the same as the preparation step of providing the second phage library.

3. The method according to any one of claims 1-2, wherein the antigen-binding polypeptide sequences displayed by the first phage library and the second phage library are of the same type.

4. The method according to any one of claims 1 to 3, wherein the antigen-binding polypeptides displayed by the first phage library and the second phage library are each selected from the group consisting of Fab, scFv, VHH, full-length antibody, fusion protein, and polypeptide.

5. The method according to any one of claims 1 to 4, wherein the antigen-binding polypeptide sequences displayed by the first phage library and the second phage library are obtained in the same manner.

6. The method according to any one of claims 1 to 5, wherein the first phage library and the second phage library are both selected from one of the following phage libraries: a natural library, an immune library, a semisynthetic library, and a synthetic library.

7. The method according to any one of claims 1 to 6, wherein the library capacity of the first phage library is smaller than, larger than or equal to the library capacity of the second phage library.

8. The method of any one of claims 1 to 7, wherein the selection marker comprises a resistance gene.

9. The method of any one of claims 1 to 8, wherein the resistance gene comprises an antibiotic resistance gene.

10. The method according to any one of claims 1 to 9, wherein the first screening marker is selected from one of a kanamycin resistance gene, a puromycin resistance gene, a hygromycin resistance gene, and an ampicillin resistance gene, and the second screening marker is selected from one of a kanamycin resistance gene, a puromycin resistance gene, a hygromycin resistance gene, and an ampicillin resistance gene and is different from the first screening marker.

11. The method according to any one of claims 1 to 10, wherein the antigen comprises a membrane protein and / or a membrane protein fragment.

12. The method of any one of claims 1-11, wherein the antigen comprises a G protein coupled receptor (GPCR) and / or a fragment thereof.

13. The method of any one of claims 1-12, wherein the antigen comprises glucagon receptor (GCGR) and / or a fragment thereof.

14. The method of any one of claims 1-13, wherein the antigen comprises a cell expressing the antigen.

15. The method of claim 14, wherein the cells expressing the antigen comprise natural cells and / or engineered cells.

16. The method according to any one of claims 1 to 15, wherein the antigen comprises a cell expressing the antigen via an integrated expression vector and / or a cell expressing the antigen via an episomal expression vector. The method of claim 16 , wherein the integrating expression vector comprises a single-site integrating expression vector.

18. The method of any one of claims 1-17, wherein the antigen comprises a cell expressing the antigen via the Flp-In system.

19. The method of any one of claims 14-18, wherein the cells comprise mammalian cells.

20. The method of any one of claims 14-19, wherein the cells comprise CHO cells.

21. The method according to any one of claims 1 to 20, further comprising the step of providing the antigen, the step of providing the antigen comprising: a) constructing a vector for expressing the antigen; and b) transducing the vector expressing the antigen into cells, so that the cells express the antigen.

22. The method according to claim 21, wherein the vector for expressing the antigen comprises a Flp-In system vector.

23. The method of any one of claims 21-22, wherein the cells comprise mammalian cells.

24. The method of any one of claims 21-23, wherein the cells comprise CHO cells.

25. The method of any one of claims 21-24, wherein the antigen comprises a G protein coupled receptor (GPCR) and / or a fragment thereof.

26. The method of any one of claims 21-25, wherein the antigen comprises glucagon receptor (GCGR) and / or a fragment thereof.

27. The method according to any one of claims 1 to 26, wherein the screening of the second phage library for antigen-specific binding phage comprises the step of obtaining the antigen-specific binding phage.

28. The method according to any one of claims 1 to 27, wherein obtaining antigen-binding phage comprises the following steps: a) dissociating the antigen-binding phage from the antigen; b) collecting antigen-binding phage that have dissociated from the antigen; and / or c) allowing the antigen-binding phage to infect bacteria.

29. The method of any one of claims 1-28, wherein the antigen-binding phage comprises a phage particle, a phage vector, a bacterium comprising the phage vector, and / or a bacterium infected by the phage.

30. The method according to any one of claims 1 to 29, wherein screening the antigen-specific binding phage in the second phage library from the antigen-binding phage comprises allowing the antigen-binding phage to infect bacteria, and then culturing the bacteria under conditions in which bacteria infected by the second phage library can grow but bacteria infected by the first phage library cannot grow.

31. The method according to claim 30, wherein the selection marker is an antibiotic resistance gene, and culturing the bacteria comprises using a culture medium containing an antibiotic corresponding to the second selection marker and not containing an antibiotic corresponding to the first selection marker.

32. The method according to any one of claims 1 to 31, further comprising the step of obtaining the antigen-specific binding polypeptide based on the antigen-specific binding phage in the second phage library.

33. The method of claim 32, wherein the step of obtaining the antigen-specific binding polypeptide comprises expressing and / or isolating the antigen-specific binding phage-displayed antigen-specific binding polypeptide.

34. The method according to any one of claims 32-33, wherein the step of obtaining the antigen-specific binding polypeptide comprises the step of converting the antigen-specific binding polypeptide into an IgG form.

35. The method according to any one of claims 32-34, wherein the step of obtaining an antigen-specific binding polypeptide comprises evaluating the expression level, binding affinity to an antigen, and / or biological function of the antigen-specific binding polypeptide.

36. The method of any one of claims 32-35, wherein the step of obtaining antigen-specific binding polypeptides comprises evaluating by FACS analysis.

37. The method of any one of claims 1 to 36, further comprising: The first phage library is screened for antigen-specific binding phages from the antigen-binding phages.

38. The method according to any one of claims 1 to 37, wherein the step of contacting the antigen with the first phage library further comprises contacting the antigen with a polypeptide known to be able to specifically bind to the antigen.

39. The method of any one of claims 1-38, wherein the screening method is repeated one or more times.

40. A phage library comprising a first phage library displaying an antigen-specific binding polypeptide and a second phage library displaying an antigen-specific binding polypeptide, wherein the first phage library and the second phage library have screening markers, the first phage library has a first screening marker, and the second phage library has a second screening marker different from the first screening marker.

41. A vector comprising a first phage library antigen-binding polypeptide gene display vector and a second phage library antigen-binding polypeptide gene display vector, wherein the screening marker of the first phage library antigen-binding polypeptide gene display vector is different from the screening marker of the second phage library antigen-binding polypeptide gene display vector.

42. A kit, wherein the kit comprises the phage library of claim 40 and / or the vector of claim 41.

43. Use of the phage library according to claim 40, the vector according to claim 41 and / or the kit according to claim 42 in screening for antigen-specific binding polypeptides.

44. An antigen-specific binding phage obtained by the method of any one of claims 1-39.

45. An antigen-specific binding polypeptide obtained by the method of any one of claims 1-39.

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