Method for screening for agonist antibodies that induce receptor dimerization

Abstract

The present invention relates to a method for screening for agonist antibodies that induce receptor dimerization. The screening method according to one embodiment enables the identification of agonist antibodies that mimic the signaling mechanism of natural signaling molecules and, in particular, enables complex screening for heterodimerization of single receptors or receptors that function by inducing homodimerization, and also of various types of receptors.

Description

Method for screening agonist antibodies that induce receptor dimerization

[0001] It relates to a novel agent antibody screening method.

[0002] Antibody screening is the process of selecting antibodies with high affinity and specificity for a specific antigen. To develop therapeutic or diagnostic antibodies, highly purified antibodies through antibody screening are required. Major screening technologies include hybridoma technology and phage display technology. Hybridoma fusion technology involves fusing B cells from an antigen-exposed animal (usually a mouse) with cancer cells to create hybridomas, and then selecting cells that secrete the desired antibody. Phage display technology is a widely used screening method that selects phages that bind to an antigen from a large-scale phage library created by expressing antibody fragments (scFv or Fab) on the surface of bacteriophages.

[0003] Existing affinity-based antibody screening methods, such as hybridoma technology or phage display, have limitations in selectively identifying agonists. Agonist antibodies are antibodies that bind to receptors present on the cell membrane or cytoplasm and induce a biological response. Unlike antagonist antibodies, which function by blocking or neutralizing their target, agonist antibodies mimic the actions of natural signaling molecules, activating signal transduction pathways, the immune system, or cell death or proliferation. Affinity-based screening methods, which simply select antibodies with high binding affinity, are suitable for developing antibodies that block activity, such as antagonist antibodies. However, they are not suitable for screening agonist antibodies, which require specific structural changes to activate receptors.

[0004] Against this backdrop, the present inventors have conducted research efforts to develop a screening technology capable of selecting agonist antibodies that regulate a desired cellular response, and have completed the present invention by confirming that it is possible to screen agonist antibodies capable of inducing homo- or heterodimerization of various receptors by expressing a combinatorial library of potential agonists inside eukaryotic cells using dual antibodies.

[0005] One aspect provides a method for screening for agonist antibodies or antigen-binding fragments capable of inducing receptor-to-receptor dimerization.

[0006] One aspect provides a method for screening for an agonist antibody or antigen-binding fragment capable of inducing receptor-to-receptor dimerization. The antibody or antigen-binding fragment screened using the method of the present disclosure is capable of inducing receptor-to-receptor dimerization.

[0007] Specifically, the method may include the following steps:

[0008] (a) producing a first DNA library comprising a gene encoding an antibody or antigen-binding fragment that binds to a first receptor; and (ii) a second DNA library comprising a gene encoding an antibody or antigen-binding fragment that binds to a second receptor;

[0009] (b) a step of producing a first vector library comprising the first DNA library and the transmembrane domain and a second vector library comprising the second DNA library and the transmembrane domain;

[0010] (c) a step of transfecting the first vector library and the second vector library into a group of reporter cells whose intracellular fluorescence changes due to receptor dimerization; and

[0011] (d) A step of measuring the fluorescence of reporter cells included in the reporter cell group and selecting an antibody or antigen-binding fragment that changes cell fluorescence expression compared to control cells.

[0012]

[0013] (a) A detailed description is given of the steps for producing a DNA library. Step (a) is a step for producing a DNA library containing a gene encoding an antibody or antigen-binding fragment that binds to a receptor.

[0014] The term "receptor" as used herein refers to a protein structure that plays a crucial role in allowing cells to detect and respond to signals via specific ligands. A receptor may comprise "subunits," which are individual protein components that constitute the receptor protein as its functional unit. A single subunit or a complex composed of multiple subunits may be collectively referred to as a receptor.

[0015] In one specific example, the first or second receptor may be a subunit of a receptor capable of forming a dimer. That is, the receptor of the present invention may be one in which a ligand binds, receptor subunits combine to form a dimer, and thus function.

[0016] The above receptor may be a receptor in which receptor subunits form homodimers and / or heterodimers.

[0017] The above subunits may belong to a homologous receptor family, or each may belong to a heterologous receptor family.

[0018] In one specific example, the first or second receptor may be at least one selected from the group consisting of IL-18 α, IL-18 β, IL-2, EGFR (HER1), HER2, HER3, HER4, PDGFR-α, PDGFR-β, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IL-10Rα, IL-10Rβ, IL-22R1, IL-10R2, IL-28Rα, IL-12Rβ1, IL-12Rβ2, IL-12Rβ1, IL-23R, IL-1R1, IL-1R2, IL-1RAcP, FGFR1, FGFR2, FGFR3, FGFR4, HGF, GHR, PRLR, EpoR and LEPR, and may include all receptors that act by forming dimers, without being limited thereto.

[0019] The term "antibody," "antigen-binding region or site," or "antigen-binding polypeptide" as used in the present invention means a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.

[0020] The antibody or antigen-binding fragment thereof may be a whole antibody, or any antigen-binding fragment or single chain thereof. The term "whole antibody" refers to a structure in which two full-length light chains and two full-length heavy chains are linked by disulfide bonds. The whole antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG is a subtype that includes IgG1, IgG2, IgG3, and IgG4. The term "antigen-binding fragment" refers to a fragment of the whole antibody structure, which includes a portion of the antibody that can bind to an antigen.

[0021] The antibody or antigen-binding fragment thereof may comprise any protein or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule having biological activity that binds to an antigen among the whole antibody. Examples thereof include, but are not limited to, a complementarity determining region (CDR) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0022] The antibody or antigen-binding fragment thereof may include at least one selected from the group consisting of a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a scFab fragment, an Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), an scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, an antibody variant, a multimeric antibody, a minibody, a diabody, a triabody, a tetrabody, a Bis-scFv, a nanobody, a bispecific antibody, and a multispecific antibody.

[0023] The term "ScFv (single-chain Fv, single-chain fragment antibody or antibody fragment)" used in the present invention refers to an antibody in which variable regions of light and heavy chains are linked within a single polypeptide chain. ScFv may include a linker (linker, linking portion) composed of a peptide chain of about 15 amino acids, and may have a structure of, for example, light chain variable region-linking portion-heavy chain variable region, or heavy chain variable region-linking portion-light chain variable region, and in this case, has an antigen specificity identical or similar to that of the original antibody.

[0024] The term "library" as used in the present invention refers to a collection of genes having different sequences. The library is used to mean a library composed of a single gene.

[0025] (a) The DNA library of step means any one of the following DNA libraries:

[0026] (i) a first DNA library comprising a gene encoding an antibody or antigen-binding fragment that binds to a first receptor; and

[0027] (ii) a second DNA library comprising a gene encoding an antibody or antigen-binding fragment that binds to a second receptor.

[0028] The first receptor and the second receptor may be the same or different. That is, the first DNA library and the second DNA library may be the same or different.

[0029] The above DNA library may be an scFv DNA library.

[0030] The above DNA library may contain one or more DNAs.

[0031] In one specific example, the DNA library can be produced by eluting phages that bind to a receptor from an antibody phage library and extracting DNA from the eluted phages.

[0032] The above phage elution may be performed by repeating panning one or more times. "Panning" refers to a technique for amplifying the number of phages by binding phages to a specific antigen, removing unbound phages, recovering the bound phages, and infecting host cells.

[0033] The above panning may be performed using beads mixed with a receptor.

[0034] The above panning may be performed one or more times, two or more times, three or more times, four or more times, or five or more times, and may be performed repeatedly as many times as necessary to achieve the purpose, without being limited thereto.

[0035]

[0036] (b) Step is described in detail. Step (b) is a step of producing a vector library including the above gene library.

[0037] The term "vector" as used herein refers to a genetic construct containing a base sequence encoding a target protein operably linked to suitable regulatory sequences so as to enable expression of the target protein in a suitable host. The regulatory sequences may include, but are not limited to, a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After being introduced into a suitable host cell, the vector may replicate or function independently of the host genome, or may be integrated into the genome itself.

[0038] The vector used in the present invention is not particularly limited as long as it is capable of expression within a host cell, and any vector known in the art may be introduced into the host cell. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages, either in a natural or recombinant state.

[0039] (b) The vector library of step means any one of the following vector libraries:

[0040] (i) a first vector library comprising the first DNA library and the transmembrane domain; and

[0041] (ii) a second vector library comprising the second DNA library and the transmembrane domain.

[0042] The protein expressed within the cell through the above vector library contains a transmembrane domain, so that it is expressed in a form attached to the cell membrane rather than being secreted outside the cell.

[0043] The above-mentioned transmembrane domain comprises at least one transmembrane domain selected from the group consisting of platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), hepatocyte growth factor receptor (HGFR), tropomyosin receptor kinase (Trk), insulin receptor (IR), Leukocyte receptor tyrosine kinase (LTK), angiopoietin receptor, cholecystokinin (CCK) receptor, neurotrophic factor (NGF) receptor, receptor tyrosine kinase-like orphan receptors (ROR), discoidin domain receptor (DDR), rearranged during transfection receptor (RETR), tyrosine-protein kinase-like (PTK), related to receptor tyrosine kinase (RYK), muscle-specific kinase (MuSK), CD63, CD9, and CD81. It may be. Preferably, the transmembrane domain may comprise a transmembrane domain of PDGFR.

[0044] The above vector may be a vector capable of acting in an autocrine manner.

[0045] In one specific example, the vector library may encode a dual antibody or an antigen-binding fragment thereof. The dual antibody or antigen-binding fragment thereof refers to a domain that induces heavy chain binding. For example, the vector library may include a gene encoding an Fc domain that induces heavy chain binding. That is, the first vector library may include a gene encoding a first Fc domain that induces heavy chain binding; and the second vector library may include a gene encoding a second Fc domain that induces heavy chain binding.

[0046] The bispecific antibody may have any one structure selected from the group consisting of Knobs-into-Holes (KiH), Triomab quadroma, Asymmetric Reengineering Technology Immunoglobulin (ART-Ig), CrossMAb, BiMAb, FcΔAdp, XmAb, DuoBody, Azymetric, Bispecific Engagement by Antibodies based on the T-cell receptor (BEAT), Biclonics, Dock-aNd-Lock (DNL), Adaptir (previously SCORPION), Tandem diAbody (TandAb), Payload-packaged EDV (EngeneIC Delivery Vehicle), nanocells actively targeted with tandem scFvS, Bispecific T-cell Engager (BiTE), Nanobody-based, Dual-Affinity-ReTargeting (DART), and Immune-mobilizing monoclonal T-cell receptors Against Cancer (ImmTAC). Preferably, it may be a Knobs-into-Holes (KiH) structure.

[0047] Specifically, the first vector may comprise a gene encoding an antibody or antigen-binding fragment that binds to a first receptor; a gene encoding a first Fc domain that induces heavy chain binding; and a gene encoding a transmembrane domain;

[0048] The second vector may comprise a gene encoding an antibody or antigen-binding fragment that binds to a second receptor; a gene encoding a second Fc domain that induces heavy chain binding; and a gene encoding a transmembrane domain.

[0049] More specifically, the first vector may comprise a gene encoding an antibody or antigen-binding fragment that binds to a first receptor; a gene encoding an Fc comprising a Knob mutation; and a gene encoding a transmembrane domain;

[0050] The second vector may comprise a gene encoding an antibody or antigen-binding fragment that binds to a second receptor; a gene encoding an Fc comprising a Hole mutation; and a gene encoding a transmembrane domain.

[0051] The first vector library and the second vector library may be the same or different.

[0052] The above vector library may include one or more vectors.

[0053] The domains that induce the above heavy chain binding and the transmembrane domains may be connected directly or via a linker. The linker may be a flexible linker. Linkers may be applied without limitation as long as they perform additional functions, such as increasing or decreasing water solubility, increasing the distance between the two components being connected to provide flexibility, or enhancing stability, while not inducing immunogenicity or affecting activity.

[0054] The linker may be a peptide linker and may be 1 to 10, or 2 to 10 amino acids long, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (e.g., up to 20) amino acids long, such as, but not limited to, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids long. The peptide linker may be composed of neutral amino acids (more specifically, Gly, Ser, Ala, Thr or a combination of these four amino acids). For example, (GS) n , (G2S) n , (G3S) n , (G4S) n , G n , LE, SSGG or GGGGSGGGGG.

[0055]

[0056] (c) Step is described in detail. Step (c) is the step of transfecting the vector library into a group of reporter cells.

[0057] The term "reporter cell" as used herein refers to a cell that expresses a fluorescent gene through intracellular signaling when a specific substance binds to a cellular receptor. The reporter cell may express fluorescence by a fusion protein comprising a protein and a fluorescent protein that translocates into and / or out of the nucleus in response to a stimulus of interest. For example, any suitable fluorescent protein may be used, including but not limited to green fluorescent protein, red fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, orange fluorescent protein, near-infrared fluorescent protein, and any derivatives thereof, as deemed suitable for the intended use.

[0058] The above reporter cells may be monoclonal reporter cells.

[0059] The above reporter cells may be monoclonal reporter cells that express a fluorescent protein when a receptor activation signal is induced by receptor-to-receptor dimerization.

[0060] The method of introducing a vector into a cell in the present application includes any method of introducing a nucleic acid into a cell (e.g., transfection or transformation), and depending on the cell, a suitable standard technique known in the art can be selected and performed. Examples include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0061]

[0062] (d) Step is described in detail. Step (d) is a step of measuring the fluorescence of reporter cells included in the reporter cell group and selecting an antibody or antigen-binding fragment that changes the cellular fluorescence expression compared to the control cells.

[0063] Specifically, the selection step includes a step of selecting cells with increased cell fluorescence expression compared to a control group using the fluorescence intensity of fluorescence expressed from the reporter cells as an indicator, and selecting an antibody or antigen-binding fragment expressed by the reporter cells with increased fluorescence expression as the final antibody or antigen-binding fragment.

[0064] The above step (d) may additionally include the following step (e):

[0065] (e) A step of extracting DNA from a group of reporter cells in which fluorescence expression has increased compared to the control cells in step (d) above, and performing steps (a) to (d) again on the extracted DNA.

[0066] Specifically, the method of the present invention may perform a first screening to select cells with increased cellular fluorescence expression compared to a control group using the fluorescence intensity of fluorescence expressed from reporter cells as an indicator, and then perform a second screening to extract a DNA library from the cells screened in the first screening, create a new vector library containing the same, re-transfect the same into a group of reporter cells, and then select cells with increased cellular fluorescence expression compared to the control group. The screening may be repeated one or more times, two or more times, three or more times, four or more times, or five or more times, and may be performed in a number of times to improve the degree of increase in GFP expression compared to the control group, without being limited thereto.

[0067]

[0068] The present method identifies the presence and extent of fluorescent protein expression when an activation signal is induced by receptor-to-receptor dimerization in reporter cells. This allows for intuitive identification of reporter cells in which receptor activation has been induced without any additional processing. Furthermore, any antibody or antigen-binding fragment capable of inducing receptor-to-receptor dimerization can be screened without limitation. The present method is characterized by its ability to screen for antibodies or antigen-binding fragments that induce homodimerization and / or heterodimerization.

[0069]

[0070] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0071] The screening method according to the aspect of the day allows the discovery of agonist antibodies that mimic the signaling mode of natural signaling molecules, and in particular, allows for complex screening of not only single receptors or receptors that operate by inducing homodimerization, but also heterodimerization of various receptors.

[0072] Figure 1 is a schematic diagram of the process of obtaining phage particles through solution panning.

[0073] Figure 2 shows the polyclonal phage ELISA results for phage pools according to the number of panning rounds.

[0074] Figure 3 is a diagram comparing GFP reactivity between cells to select monoclonal reporter cell candidates.

[0075] Figure 4 shows the results of flow cytometry analysis performed to select the final monoclonal reporter cells.

[0076] Figure 5 is a schematic diagram of the screening process.

[0077] Figure 6 is an image showing the results of flow cytometry analysis performed after the first screening.

[0078] Figure 7 is an image showing the results of flow cytometry analysis performed after the second screening.

[0079] Figures 8 and 9 are graphs confirming the efficacy of the final screened antibodies.

[0080] The following examples are provided for more detailed description. However, these examples are provided solely to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0081]

[0082] <Example>

[0083]

[0084] 1. Obtaining phage particles that bind to receptors

[0085] (1) Securing phage particles

[0086] First, phages binding to each IL-18 receptor (IL-18R) subunit were obtained. Solution-phase panning was performed using recombinant human IL-18Rα extracellular domain (11102-H08H, Sino Biological) and IL-18Rβ extracellular domain (10176-H08H) and Dynabeads M-270 Epoxy beads (14301; Invitrogen; Thermo Fisher Scientific). The beads were mixed with the IL-18R extracellular domain and incubated on a rotator at 37°C for more than 12 h, and were used for each round of panning.

[0087] IL-18R-conjugated beads were incubated with a human combinatorial antibody phage library for 2 h at room temperature while rotating on a rotator. The beads were then washed with PBST (phosphate-buffered saline, 0.05% Tween 20 in PBS), and residual phages were eluted with a solution containing 0.2 M glycine-HCl and 0.1% bovine serum albumin (BSA), pH 2.2.

[0088] The eluted phage was infected with Escherichia coli XL1-Blue (200228, Agilent) and amplified. Carbenicillin (100 mg / mL) was added, and the culture was incubated at 37°C for 2 h. The culture was then diluted with super broth (SB). M13KO7 helper phage (N0.15S; New England Biolabs) was added, and the culture was incubated for an additional 2 h at 37°C. After completion of the incubation, kanamycin sulfate (70 μg / mL) was added, and the culture was incubated at 37°C for more than 12 h.

[0089] The next day, the generated phage particles were concentrated using the polyethylene glycol / NaCl precipitation method, and resuspended in a solution containing 1% BSA in Tris-buffered saline to obtain purified phage particles.

[0090] Panning was performed in a total of three rounds, and its schematic diagram is shown in Figure 1.

[0091]

[0092] (2) Confirmation of phage amplification

[0093] The amplification of phages binding to each IL-18R subunit was confirmed using a polyclonal phage ELISA technique.

[0094] Specifically, recombinant human IL-18Rα extracellular domain, IL-18Rβ extracellular domain, and BSA were coated on 96-well half-area plates (3690; Corning) by reacting with carbonate coating buffer (421701, BioLegend) at 4°C for more than 12 hours. The plates were washed once with PBS and blocked with PBS containing 5% skim milk at 37°C for 1 hour. Afterwards, phage supernatant was added without washing and reacted at 37°C for 2 hours. Each well was washed five times with PBS, and HRP-conjugated anti-M13 bacteriophage antibody (1:2000, 11973-MM05T-H) was added and reacted at 37°C for 1 hour. Finally, Substrate Reagent A (51-2606KZ, BD Biosciences) and Substrate Reagent B (51-2607KZ, BD Biosciences) were mixed to form a tetramethylbenzidine (TMB) substrate, which was then added to the plate and reacted for 10 minutes. The reaction was terminated with 2 N sulfuric acid. The absorbance was measured at 450 nm using a microplate reader, and the results are shown in Figure 2.

[0095] As shown in Figure 2, it was confirmed that the number of phages binding to each receptor was amplified as the number of rounds increased.

[0096]

[0097] 2. Creation of DNA libraries and vector libraries

[0098] The following process was followed to obtain an scFv DNA library from the phage pool obtained in the second round of panning for each IL-18R subunit in Example 1.

[0099] Specifically, 50 μL of XL-1 Blue at an OD of 1.0 was added to 50 mL of SB medium supplemented with 100 μL of 5 mg / mL tetracycline, and the mixture was cultured in a shaking incubator at 37°C for 1 hour in a 250 mL flask. 200 μL of phage binding to each IL-18R subunit was added to each 2 mL of the cultured XL-1 blue, and the infection was performed at room temperature for 15 minutes. 10 mL of SB medium and 12.2 μL of 100 mg / mL carbenicillin were added, and the mixture was cultured in a shaking incubator at 37°C for 16 hours. The following day, the scFv DNA library was obtained using a Plasmid Mini Extraction kit (K-3030, BIONEER).

[0100] Next, in the screening step, the scFv DNA library binding to each receptor subunit was transferred to an MTA-type scFv-Fc (knob or hole) vector capable of expressing bispecific antibodies on the cell membrane surface to induce dimerization of IL-18R subunits. The IL-18Rα-binding scFv DNA library was transferred to an MTA-knob vector containing knob mutations in Fc, and the IL-18Rβ-binding scFv DNA library was transferred to an MTA-hole vector containing hole mutations in Fc. First, the MTA vector (MTA-knob, MTA-hole) and the scFv DNA library binding to each subunit were cleaved using SfiI restriction enzyme (R0123S; NEW ENGLAND Biolabs) by incubation at 50°C for 4 hours. Afterwards, the truncated vector and scFv DNA libraries were recovered through gel electrophoresis and ligated using T4 DNA ligase (M202S; NEW ENGLAND Biolabs) at 16°C for 16 h. The completed libraries were electroporated into XL-1 Blue, and colonies were recovered to obtain MTA-knob and MTA-hole vector libraries containing scFv DNA libraries that bind to each IL-18R subunit.

[0101]

[0102] 3. Production of reporter cells

[0103] To generate monoclonal reporter cells expressing GFP fluorescence by IL-18 signaling, HEKBlue-IL18 reporter cells were seeded at 6 x 10 in a 12-well plate. 5After seeding at 10 cells / well, pGreenFire1-NF-κB (EF1α-neo) Lentivector (TR012PA-N; System Biosciences) was transfected into HEKBlueIL-18 reporter cells using Lipofectamine 3000 (L3000075; Invivogen; Thermo Fisher Scientific). Three days after transfection, cells were treated with G418 antibiotics (300 ug / ml) and cultured for 24 h at 37°C and 5% CO2 to select only cells containing the vector.

[0104] After treating the selected cells with 10 ng / mL of recombinant human IL-18 (rhIL-18), cells showing GFP reactivity were separated into 96-well plates at 1 cell / well using flow cytometry. After a certain period of time, among the cell groups with a confluency of 70% or more in the well, the cell group showing a fluorescence value more than three times that of the PBS-treated group in the rhIL-18-treated group was selected from the candidate group using the Phospha-Light™ SEAP Reporter Gene Assay System (T1017; Invitrogen; Thermo Fisher Scientific) to select monoclonal reporter cells with excellent IL-18 reactivity (Fig. 3).

[0105] Afterwards, among the selected groups, the clone (9C7) with the least noise and the greatest increase in GFP expression following IL-18 treatment was selected as the final reporter cell through flow cytometry analysis (Fig. 4).

[0106]

[0107] 4. Screening progress

[0108] (1) Transfection of vector library into reporter cells

[0109] In Example 3, monoclonal reporter cells were constructed to detect IL-18 signals based on GFP expression, and the MTA-knob and MTA-hole vector libraries containing scFv libraries binding to each IL-18R subunit obtained in Example 2 were transfected using Lipofectamine 3000. Various combinations of scFvs binding to each receptor expressed through a flexible linker on the membrane induce dimerization of IL-18R to transmit IL-18 signals, and GFP is expressed in activated cells.

[0110] Cells with confirmed GFP expression were selected through flow cytometry, and the scFv DNA sequence was confirmed from the GFP-positive cells extracted through two screening processes as described below. A schematic diagram of the screening process is shown in Figure 5.

[0111]

[0112] (2) Primary screening and scFv DNA library extraction

[0113] After extracting scFv DNA from GFP-positive cells in the first screening, a scFv DNA library was amplified from this to construct new MTA-knob and MTA-hole vector libraries.

[0114] First, C32 MTA vector was used as a negative control, and membrane-tethered rhIL-18 was used as a positive control. First, 5 X 10 GFP-based monoclonal cells were seeded in a 100-pi dish. 6After seeding cells / dish, they were cultured overnight. Each IL-18R subunit-specific MTA scFv DNA library (7.5 µg each) and the negative and positive controls (15 µg each) were transfected with Lipofectamine 3000, and cultured for 24 h at 37°C and 5% CO2. The culture medium was then replaced, and flow cytometry sampling was performed 48 h later. As a result of the first screening experiment, a cell group with increased GFP expression was identified in the experimental group (Panning 2R output combination) compared to the negative control group (Fig. 6).

[0115] These were separated into e-tubes containing 500 μl of genomic DNA extraction buffer (GuSCN, 0.2 M EDTA, Triton X-100 in 0.1 M Tris-HCl). The separated cells were resuspended and centrifuged (13,000 xg, 10 min). Only the supernatant was taken and placed in a silica-based column and centrifuged (12,000 rpm, 30 sec). After discarding the through-out solution, 500 μl of binding buffer (GuSCN in 0.1 M Tris-HCl) was added to the column and centrifuged (12,000 rpm, 30 sec). Afterwards, 600 μl of wash buffer (10 mM NaCl in 70% EtOH) was added to the column and centrifuged (12,000 rpm, 30 sec). This process was repeated once more, and centrifugation was performed under the conditions of 12,000 rpm and 2 min of idle rotation. Finally, 25 ul of DW was added to the column, left at 37°C for 10 min, and centrifugation was performed (12,000 rpm, 1 min) to extract the scFv DNA library.

[0116]

[0117] (3) Secondary screening and scFv DNA library extraction

[0118] The newly constructed library from the first screening was retransfected into reporter cells to conduct a second screening round.

[0119] The extracted scFv DNA was amplified by PCR and then amplified using the Sfi1 restriction enzyme at 50°C for 1 hour. The primers used were as follows:

[0120] - FW: 5'-ATGTACAGGATGCAACTCCTG-3' (SEQ ID NO: 1)

[0121] - RV(knob): 5'-CTTATCGTCGTCATCCTTGTAATC-3' (SEQ ID NO: 2)

[0122] - RV (hole): 5'-GTGATGGTGGTGATGGTG-3' (SEQ ID NO: 3)

[0123] After PCR, cuts were made in each of the scFv DNA, MTA-knob, and MTA-hole vectors, and then ligation of the Sfi1-cut insert and vector was performed overnight at 16°C using T4 DNA Ligase.

[0124] After constructing a new MTA-knob and MTA-hole vector library, a secondary screening was conducted. The experimental process was identical to the primary screening experiment, and the secondary screening results confirmed a cell group with increased GFP expression in the experimental group (Panning 2R output combination) compared to the negative control group, and the degree of GFP expression increase was confirmed to be approximately twofold higher than in the primary screening (Fig. 7).

[0125] Afterwards, the cell group with increased GFP expression was selected using the same method as the first screening, and scFv DNA was extracted from this.

[0126]

[0127] 5. Antibody screening

[0128] The extracted scFv DNA was amplified to create new MTA-knob and MTA-hole vector libraries, and each library was electroporated into XL-1 blue, and approximately 10 colonies were recovered from each.

[0129] As a result, three types of scFv (α2, α3, α5) were identified that bind to IL-18Rα, and six types (β1, β3, β4, β5, β6, β10) were identified that bind to IL-18Rβ.

[0130]

[0131] <Experimental Example> Evaluation of the Efficacy of Combinations of scFv Sequences

[0132] The scFv binding to IL-18Rα was cloned using an expression vector (pFuse expression vector) with a knob mutation applied to the Fc, and the scFv binding to IL-18Rβ was cloned using an expression vector with a hole mutation applied to the Fc using the above-mentioned method. Afterwards, they were co-transfected into Expi293F (A14527; Gibco™; Thermo Fisher Scientific) cells using the ExpiFectamine™ 293 Transfection Kit (A14524; Gibco™; Thermo Fisher Scientific). A total of 18 types of combinations were expressed in the form of bispecific antibodies, and purified using a HiTrap Protein G HP column (17-0404-01; Cytiva) equipped with an AKTA Prime Plus protein purification system (Cytiva, Marlborough, MA, USA). Among these, nine purified clones were obtained and treated on HEKBLUE-IL18 cells, and the efficacy of the agent was evaluated using QUANTI-Blue™ (rep-qbs; InvivoGen), and the results are shown in Fig. 8. Additionally, clones 2-6, which showed the highest efficacy, were treated at different concentrations to evaluate the efficacy of the agent, and the results are shown in Fig. 9.

[0133]

[0134] As shown in Figures 8 and 9, antibodies exhibiting high agent efficacy could be selected through the screening method of the present invention.

[0135]

[0136] In summary, the screening method of the present invention enables the discovery of agonistic antibodies that mimic the signaling mechanisms of natural signaling molecules. The screening method of the present invention enables complex screening of not only single receptors or receptors that function through homodimerization, but also heterodimerization of various receptors, making it a useful tool for the discovery of agonistic antibodies.

[0137]

[0138] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A method for screening an agonist antibody or antigen-binding fragment capable of inducing dimerization between receptors, (a) producing a first DNA library comprising a gene encoding an antibody or antigen-binding fragment that binds to a first receptor; and a second DNA library comprising a gene encoding an antibody or antigen-binding fragment that binds to a second receptor; (b) a step of producing a first vector library comprising the first DNA library and the transmembrane domain and a second vector library comprising the second DNA library and the transmembrane domain; (c) a step of transfecting the first vector library and the second vector library into a group of reporter cells whose intracellular fluorescence changes due to receptor dimerization; and (d) a step of measuring the fluorescence of reporter cells included in the reporter cell group and selecting an antibody or antigen binding fragment that changes cell fluorescence expression compared to control cells; A screening method comprising:

2. A screening method according to claim 1, wherein the first vector library and the second vector library encode a dual antibody or an antigen-binding fragment thereof.

3. In claim 1, the first vector of step (b) comprises a gene encoding an antibody or antigen-binding fragment that binds to a first receptor; a gene encoding an Fc containing a Knob mutation; and a gene encoding a transmembrane domain. A screening method, wherein the second vector comprises a gene encoding an antibody or antigen-binding fragment that binds to a second receptor; a gene encoding an Fc containing a Hole mutation; and a gene encoding a transmembrane domain.

4. A screening method according to claim 3, wherein the Fc and transmembrane domains are connected directly or via a linker.

5. A screening method according to claim 1, wherein the vector of step (b) is a vector capable of acting in an autocrine manner.

6. A screening method according to claim 1, wherein the first or second DNA library of step (a) is produced by eluting phages that bind to the first or second receptor from an antibody phage library and extracting DNA from the eluted phages.

7. A screening method according to claim 6, wherein the elution of the phage is performed by repeating panning at least once.

8. A screening method according to claim 1, wherein the following steps are additionally repeated one or more times: (e) A step of extracting DNA from a group of reporter cells in which fluorescence expression has increased compared to the control cells in step (d) above, and performing steps (a) to (d) again on the extracted DNA.

9. A screening method according to claim 1, wherein the agent antibody or antigen-binding fragment capable of inducing inter-receptor dimerization is an antibody or antigen-binding fragment that induces inter-receptor heterodimerization.

10. A screening method according to claim 1, wherein the first or second receptor is a subunit of a receptor that can act by forming a dimer.

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