pH-sensitive antibodies

Abstract

A major challenge in the field of antibody-based therapeutics is the development of antibodies with enhanced duration of action, extended half-lives, and the ability to be delivered to patients at lower doses. Current approaches to achieving this rely on manipulating antibodies to impose cumbersome pH-dependent antigen-binding properties, typically heavily relying on the introduction of histidine mutations into specific antibody complementarity-determining regions (CDRs), thus necessitating further optimization of pH-sensitive interactions in a low-throughput, case-by-case manner. In this invention, the inventors provide antibodies and other antigen-binding proteins that possess mutations outside of a paratope that confer universal pH-sensitive binding properties to the antibody. The invention also relates to methods for isolating and preparing such antigen-binding proteins, compositions, and their medical use.

Description

[Technical Field]

[0001] The present invention relates to an antigen-binding protein, such as an antibody, which includes an amino acid mutation that confers pH-dependent antigen-binding properties to the antigen-binding protein, a method for obtaining the same, and its medical applications. [Background technology]

[0002] Antigen-binding proteins, such as antibodies, are an attractive class of therapeutic agents due to the typically high specificity and affinity of their variable domains for their targets, as well as their ability to perform effector functions.

[0003] Antibody-mediated targeted degradation is an attractive strategy for neutralizing antigens due to its potential to achieve therapeutic effects at very low doses and to modulate target function without binding to inhibitory epitopes. Non-stoichiometric drugs: The classic antibody-based approach to degrading extracellular proteins using target ratios is via neonatal Fc receptors (FcRn) that recycle antibodies. FcRn, which binds to the antibody Fc domain in a pH-dependent manner, has been used to degrade plasma antigens (Reference 1). Antibodies with low affinity for the antigen at endosomal acidic pH selectively release targets for lysosomal degradation during recycling, while the antibody itself is returned to the plasma unbound. This allows the antibody to neutralize multiple target molecules (non-stoichiometric neutralization), theoretically enabling administration at lower doses than conventional antibodies (Reference 1).

[0004] While Fc variants with different pH dependencies have been developed for FcRn, the mechanisms behind how antibodies can respond to pH-dependent antigen binding are not well understood. Current approaches to manipulating pH-dependent antigen binding properties are cumbersome and typically rely on introducing histidine mutations into specific antibody complementarity-determining regions (CDRs), thus necessitating further optimization of pH-sensitive interactions in a low-throughput, case-by-case manner.

[0005] The lack of methods and determinants for conferring universal pH-sensitive antigen-binding properties to antibodies limits the development of therapeutic antibodies with improved pharmacokinetic characteristics. [Overview of the project]

[0006] The present invention is defined in the appended claims.

[0007] In the field of antibody engineering, approaches utilizing FcRn recycling to increase the circulating half-life of antibodies have been described in this technology (Reference 2). FcRn receptors naturally have high affinity for the Fc domain of antibodies at acidic pH and low affinity at neutral pH. This allows antibodies to bind to FcRn in acidic endosomes and be rescued from lysosomal transfer and degradation. Upon returning to the cell surface, the antibody is subsequently released from FcRn into the plasma due to its lower affinity at neutral pH.

[0008] This principle is used in specific applications where monoclonal antibodies are engineered to have weaker antigen binding at acidic pH levels and recycled into a cycle that can target more toxic antigens.

[0009] By manipulating the antibody Fc domain to interact with FcRn with higher affinity at neutral pH, the rate of internalization and, consequently, the rate of antigen degradation of recycling antibodies is increased. More specifically, recycling IgG antibodies typically bind to congener antigens with high affinity at near-neutral pH (7.4) and with low affinity at acidic pH (<6.0), enabling antigen release within endosomes and subsequent lysosomal degradation of the antigen. On the other hand, antibodies are rescued via the FcRn-mediated pathway (Reference 1).

[0010] On the other hand, processes for manipulating antibodies with pH-dependent antigen-binding properties typically require multiple discovery steps, multiple manipulation steps, cumbersome design, and analysis of individual variants to selectively adjust antibody affinity at both neutral and acidic pH levels.

[0011] Many of these approaches focus on the use of this amino acid residue due to the alteration of the charge state of histidine at the acidic pH of endosomes. Histidine residues can be introduced into antibody CDRs in a low-throughput manner by creating a rationally designed panel of antibody variants that can be tested one by one (Reference 1), or they can be introduced on a large scale semi-randomly into the CDRs of synthetic antibody libraries, for example, via phage display selection (Reference 4) (Reference 3). More specifically, manipulation of pH-sensitive binding in antibody variable domains is typically achieved by either sequentially introducing histidine mutations into the CDR loop (histidine walking) or by a combinatorial histidine scanning approach using in vitro display techniques (References 1, 5).

[0012] Histidine residues exhibit charge changes within the physiological pH range of the antibody recycling pathway; therefore, they have been successfully introduced into the complementarity-determining region (CDR) of antibodies to manipulate the pH switch to non-pH-dependent antibodies based on the computational design or in vitro display techniques described above (Reference 6). In attempts to a priori discover antibodies with pH-dependent antigen-binding properties using phage display, histidine-doped libraries have been created in which histidine residues have been increased in the antibody CDR.

[0013] However, this approach suffers from the limitation of introducing mutations in the region of antigen-binding proteins that are involved in antigen recognition and therefore affect antibody function. Furthermore, because of the very high variability in the CDR region of antibodies, such mutations are specific to each specific antibody.

[0014] Understanding how antibody binding can be affected by pH changes expands the possibilities for manipulating pH-dependent antibodies, particularly pH-determining factors located outside the antibody paratope, which is beneficial, for example, when pH-dependent interactions cannot be absorbed into the antibody paratope without significantly impairing the antibody's function.

[0015] In this invention, the inventors provide antibodies and other antigen-binding proteins that possess mutations outside of a paratope having pH-sensitive binding properties. Such antibodies were identified, for example, in a panel of light-chain shuffle cross-reactive antibodies specific to long-chain α-neurotoxins by investigating the molecular basis for antibody neutralization and the relationships between antibody paratopes, epitopes, and paratope-independent factors regarding pH-dependent antigen binding. Examples of antibodies with pH-sensitive binding properties include antibodies that bind to different targets, such as antibodies specific to long-chain α-neurotoxins in snake venom, antibodies specific to tumor necrosis factor alpha (TNF-alpha), such as adalimumab, and antibodies specific to vascular endothelial growth factor (VEGF), such as bevacizumab, which supports the universality of the principle provided herein, because these mutations are located outside the paratope and can therefore be introduced into substantially any antibody.

[0016] Therefore, light chains optimized for these antibodies can be given pH-dependent antigen binding. The antibody pool was screened for pH-dependent binding properties to different long-chain α-neurotoxins using biolayer interferometry, which identified one antibody capable of pH-dependent binding to all long-chain α-neurotoxins tested. To investigate the mechanism of this antibody's pH-dependent binding properties to antigens, X-ray crystallography was chosen to determine whether pH-dependent binding was facilitated by the antibody paratope or epitope.

[0017] Structural studies revealed that cross-reactivity is achieved by utilizing conserved functional constraints in long-chain α-neurotoxins required for nAChR inhibition, as determined by the antibody heavy-chain paratope. In addition to cross-reactivity, one light-chain shuffle antibody, 2555_01_A01, was able to bind to all long-chain α-neurotoxins tested in a pH-dependent manner. Structural characterization of 2555_01_A01 bound to α-cbtx at different pH levels suggests that broad pH-dependent binding was conferred at the light-heavy-chain interface, away from the paratope-epitope interface. Furthermore, the inventors determined the crystal structure of this antibody bound to long-chain α-neurotoxins at 1.6 Å and elucidated the basis of the neutralization mechanism of this antibody lineage. Through antibody heavy-chain complementarity determination region 3, these antibodies neutralized long-chain α-neurotoxins and achieved broad cross-reactivity by mimicking the conserved interaction between long-chain α-neurotoxins and acetylcholine receptors. This antibody also binds to all long-chain α-neurotoxins tested in a pH-dependent manner, initiating further structural studies to investigate the pH-dependent binding mechanism.

[0018] Importantly, by determining the structure of antibodies that bind to long-chain α-neurotoxins at different pH levels, a network of residues located at the interface between the heavy and light chains of the antibody, which respond in a coordinated manner to low pH levels in the antibody structure, was identified.

[0019] Furthermore, the analyzed antibody paratopes and epitopes were shared with other antibodies that were not pH-dependent. This indicates that the pH-dependent binding mechanism of this antibody resides outside the paratopes and epitopes, anticipating further structural studies.

[0020] Based on these findings, the inventors have discovered that pH-dependent binding conferred by the antibody framework itself, specifically mediated by amino acid residues located at the light-heavy chain interface, can provide a universal method for introducing pH-dependent antigen-binding properties into antibodies.

[0021] A key advantage of this approach is that pH-dependent antigen binding may be less dependent on CDR, thereby dramatically expanding the range of suitable paratopes for antibody recycling. Secondly, this strategy could improve the speed and accuracy of discovery activities seeking pH-dependent antibodies against different targets. While not constrained by theory, the inventors believe that the framework interface facilitates antibody-antigen binding by modulating the orientation of the heavy and light chains. Since the heavy-light chain orientation can be manipulated to change its conformation in a pH-dependent manner, this could serve as a universal approach to reduce binding affinity at a given pH, enabling the design of in vitro display libraries consisting of pH-dependent antibodies with this synthetic framework.

[0022] The present invention also provides a method for improving the discovery of pH-dependent antibodies using in vitro display technology. The inventors observed that pH-dependent antigen binding can be encoded only at positions far from the paratope, possibly at the heavy-light-chain interface within the antibody framework, by using a minimal set of mutations that do not affect antigen binding. Therefore, the inventors propose an approach to manipulate a comprehensive pH switch in the antibody variable domain using a library of variable light and heavy chains of antigen-binding proteins having one or more mutations from an identified set.

[0023] Therefore, to introduce a comprehensive pH switch into the antibody variable domain, we designed and validated a phage display library targeting the antibody heavy-light chain interface framework region. Through pH-dependent binding manipulated to the antibody as a predetermined feature, the present invention provides a consistent, high-throughput approach to discovering and / or manipulating pH-dependent antibodies against different targets by a priori using in vitro display technology in a single discovery activity.

[0024] Therefore, generally speaking, the inventors describe a pH-dependent antigen-binding protein that includes a minimal set of mutations outside the paratope that confers pH-dependent antigen-binding properties to the pH-dependent antigen-binding protein, and an approach to manipulate it by targeting the antibody framework region (FWR).

[0025] Furthermore, using point mutations, the inventors identified further framework residue mutations associated with increased pH sensitivity of antigen-binding proteins.

[0026] The inventors also identified further framework residues related to the pH sensitivity of antigen-binding proteins based on in silico analysis of interaction sites at the heavy-chain and light-chain interfaces with amino acid conservation determined by alignment of 328 therapeutic antibodies.

[0027] The present invention may also have the potential to further improve the pharmacology of antibodies that suffer from target-mediated clearance, improving their cost-effectiveness by having a longer duration of action and lower doses, and resulting in therapies with better patient compliance (by administering them less frequently). Another example involves designing specialized in vitro display antibody libraries with pre-established pH-dependent binding properties that can be used in a comprehensive manner to discover recycling antibodies and / or antibodies that bind to target antigens at the correct time and precise anatomical location.

[0028] In one embodiment, the present invention relates to a method for isolating an antigen-binding protein having a pH-dependent scaffold, wherein the scaffold consists of a variable region that is not part of a paratope, and the method: - A step of providing a library comprising antigen-binding proteins, each containing an antibody light chain variable region (VL), wherein the VL contains one or more mutations located outside the paratope of the antigen-binding protein, and the library comprises a plurality of antigen-binding proteins, each containing different mutations in the VL. -The process includes the step of selecting an antigen-binding protein from the library that exhibits a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, This relates to a method for isolating antigen-binding proteins having a pH-dependent scaffold.

[0029] In a second aspect, the present invention relates to a method for producing a pH-dependent antigen-binding protein that binds to a specific epitope, wherein the method is: -The step of isolating an antigen-binding protein having a pH-dependent scaffold according to the method described herein, - A step of providing an antigen-binding protein that binds to the specific epitope, -The step of replacing a paratope of the antigen-binding protein having a pH-dependent scaffold with a paratope of the antigen-binding protein bound to the epitope, This relates to a method for creating a pH-dependent antigen-binding protein that binds to a specific epitope.

[0030] A third aspect of the present invention relates to a pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parent FWR1, FWR2, FWR2', FWR3, and FWR4, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, and the pH-dependent antigen-binding protein does not contain heavy chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, respectively, and light chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 4, 5, and 6, respectively.

[0031] A fourth aspect of the present invention is a pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, and the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and one or more mutations, compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, according to Kabat, result in VH39, VH44, VH89, VH105 The present invention relates to a pH-dependent antigen-binding protein that is introduced at a residue position selected from the group consisting of VL38, VL43, VL50, VL85, and VL100, and the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, and the pH-dependent antigen-binding protein does not contain the heavy chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, respectively, or the light chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 4, 5, and 6, respectively.

[0032] A fifth aspect of the present invention is a method for generating a pH-dependent antigen-binding protein directed toward an antigen of interest, wherein the method is: - A step of providing an antigen-binding protein that binds to an antigen of interest; - The step of introducing one or more mutations in the antigen-binding protein at a residue position selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and VL100, according to Kabat, This relates to a method for generating a pH-dependent antigen-binding protein targeted to an antigen of interest.

[0033] As described herein, in some embodiments of the antigen-binding proteins, pH-dependent antigen-binding proteins, and methods described herein, one or more mutations are introduced into the antigen-binding protein to confer pH-dependent binding properties to the antigen binding. In some embodiments, the pH-dependent binding properties are conferred to the antigen-binding protein by mutations in specific amino acid residues at specific residue positions in the VH and VL of the antigen-binding protein.

[0034] Therefore, an aspect of the present invention is an antigen-binding protein comprising an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein one or more mutations are introduced at residue positions selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98 and VL100 according to Kabat numbering. The VH contains one or more of the following amino acid residues, for example, two, three, or four: - S, E, R, T, or A, ranked 39th - P, R, N, S, K, Q, A, Y, or T, ranked 44th - T, N, I, Q, A, L, Y, D, F, S, or K, and / or - 105th place T, R, K, P, D, I, S, or T, and / or The VL contains one or more of the following amino acid residues, for example, two, three, four, five, six, or seven: - N, ranked 36th. - 38th place: L, Y, S, I, T, A, R, F, or V - 43rd place: P, T, A, R, Q, K, or V, - 46th place A or S, - 49th place A, H, or S, - S, N, T, F, V, L, S, A, H, or R in the 85th position and / or - S, Y, W, or L in 100th place, In the formula, all positions are indicated according to Kabat numbering, relating to the antigen-binding protein.

[0035] A sixth aspect of the present invention relates to a composition comprising a pH-dependent antigen-binding protein described herein and a pharmaceutically acceptable excipient.

[0036] A seventh aspect of the present invention relates to a pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL) for use in a method of treatment for patients requiring treatment for cancer, autoimmune diseases, metabolic diseases, or hematological diseases, wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, or to the composition described herein.

[0037] An eighth aspect of the present invention relates to a method for treating cancer, autoimmune diseases, metabolic diseases, or hematological diseases, comprising administering to a patient in need of treatment for cancer, autoimmune diseases, metabolic diseases, or hematological diseases a pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, or a composition described herein.

[0038] A ninth aspect of the present invention relates to the use of a pH-dependent antigen-binding protein, or a composition described herein, in the manufacture of a pharmaceutical for the treatment of cancer, autoimmune diseases, metabolic diseases, or hematological diseases, wherein the pH-dependent antigen-binding protein, or a composition described herein, has a pH-dependent antigen-binding protein having a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH.

[0039] A tenth aspect of the present invention relates to the use of the pH-dependent antigen-binding protein, or the composition described herein, in an in vitro method for the detection and / or diagnosis of cancer, wherein the pH-dependent antigen-binding protein, or the composition described herein, has a pH-dependent antigen-binding protein, or a composition described herein, having a pH-dependent antigen-binding protein, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH.

[0040] An eleventh aspect of the present invention is: - To provide the pH-dependent antigen-binding protein described herein, - The pH-dependent antigen-binding protein is brought into contact with the antigen to which it binds, - Includes detecting contact between the pH-dependent antigen-binding protein and the antigen, This relates to an in vitro antigen detection method for detecting the antigen.

[0041] A twelfth aspect of the present invention is: - To provide the pH-dependent antigen-binding protein described herein, - The pH-dependent antigen-binding protein is brought into contact with the antigen it binds to in a complex mixture. - This includes separating the pH-dependent antigen-binding protein / antigen complex from the complex mixture. This relates to an in vitro antigen purification method for purifying the antigen. [Brief explanation of the drawing]

[0042] [Figure 1]Characterization of binding and in vivo neutralization of long-chain α-neurotoxins. (A) Light chain complementarity-determining region loop sequences of the antibodies used in this study, numbered according to the Kabat scheme (CDRL1 extends from residues 22-34, CDRL2 from residues 50-56, and CDRL3 from residues 89-97, including positions 95, 95A, and 95B). The characteristics of amino acid residues are indicated by different gray shading below the sequence (charge: R, E, D, H; polarity: T, S, Q, N; nonpolar: G, I, A, Y, V, W, P). (B) Affinity of anti-long-chain neurotoxin clones against α-bgtx at pH 7.4. Data points are shaped according to the antibody light chain germline. (C-E) Differences in pH-sensitive binding ratios of anti-long-chain neurotoxin clones against α-cbtx, α-eptx, and α-bgtx. The difference in affinity between IGVLλ bound to α-bgtx and the parent clone was measured as the product of the difference in their affinity under steady-state conditions at pH 5.5 and pH 7.4, while all other difference in affinity was measured as the change in dissociation rate between pH 5.5 and 7.4. [Figure 2] (A) 2555_01_A01 antibody bound to α-cbtx. The antibody heavy chain (HC, dark gray) recognizes finger II (finger II, light gray) of α-cbtx via CDRH3, which is structurally stabilized by an intramolecular disulfide bridge (indicated by the black arrow). The antibody light chain (LC) is stained a medium gray. (B) Overlay of the SEC chromatogram of 2555_01_A01 that forms a complex with α-cbtx when unbound. [Figure 3]Neutralization of α-cbtx by 2555_01_A01 antibody via receptor mimicry. (A) From left to right: Structure of Torpedo nAChR bound to α-bgtx (6uwz). Finger II of α-bgtx (structure above, Bgtx) is bound to the interface between the gamma (structure on the left, light gray, gamma) domain and the alpha (structure on the right, medium gray, alpha) domain of nAChR, with R33 on finger II shown in stick format. The important response element loop C (dark gray, dotted circle on the left) is illustrated on the alpha domain of nAChR. Overlays of tyrosine residues on loop C and CDRH3 (dark gray cyclic structure, dotted circle in the center), coordinating to R33 and R36 on the long-chain α-neurotoxin, show a mimicry of the interaction between nAChR and 2555_01_A01. Right: 2555_01_A01 bound to α-cbtx. (B) Core interaction between CDRH3 and finger II of α-cbtx, targeting conserved residues (R33, R36, D26) in long-chain α-neurotoxin. (C) Reverse view of the interaction between the CDRH3 and CDRL3 loops to the α-cbtx skeleton and C-terminus. [Figure 4] Effect of light chain on pH-sensitive binding of 2555_01_A01 antibody. (A) Back view of the interface between α-cbtx (light gray, R70) and CDRL3 (dark gray) at pH 6.0. Hydrogen bonds are indicated by dotted lines. (B) Remodeled H95b hydrogen bonds after rotational isomerization at pH 4.5, indicated by black arrows. (C) Effect of D95a and H95b on pH-dependent binding due to double mutations to the non-pH-dependent HE motif seen in the non-pH-dependent 2554_01_D11 antibody. (D) Proposed residue network involved in pH-dependent binding of 2555_01_A01, as seen in the pH 4.5 model. (E) Fo-Fo subtraction of proposed residues using refined pH 5.5 and pH 4.5 datasets. pH 4.5 model shown, with electron density on the phenyl ring highlighted at pH 5.5. The heavy chain and related residues are shown in dark gray (e.g., D95, Y100l), the light chain in medium gray (e.g., H34, S50), and α-cbtx in light gray (e.g., R33). The Fourier map is outlined using 3σ. [Figure 5] Characterization of 2554_01_D11 antibody binding by SPR and framework library design. (A) Sensograms of binding of monovalent 2554_01_D11 Fab to immobilized α-cobra toxin, measured at pH 7.4 and (B) pH 5.1. The order of the curves from top to bottom in each graph corresponds to the decrease in antibody concentration tested, as shown in the right panel. (C) Highlighted sites of the library design selected for library creation, numbered using the Kabat scheme (VH: Q39, G44, V89, Q105, VL: Q38, S43, D85, G100). (D) Residue positions are shown on the variable domain of the 2555_01_A01 antibody (bottom of VH and VL, stick format), which is a light chain variant of the 2554_01_D11 (2555_01_A01) clone. [Figure 6] Phage enrichment from two selections using a binding framework interface library. Enrichment was calculated by the factor difference between the test selection and the non-antigen control selection. [Figure 7] Upscaling of insertion fragments after golden gate assembly. (A) Upscaling of individual heavy chain (HC) and light chain (LC). Two PCRs were performed on the heavy chain because the reverse primer contained the mutation site. (B) PCR optimization of the scFv library assembled after gel extraction. Different input volumes of extracted scFv and concentrations of each primer were tested. The scFv reference was prepared from the purchased scFv vector used to construct the library. A primer concentration of 1 ng scFv + 0.1 μM showed the purest product and proceeded to electroporation. [Figure 8]Cloning efficiency of scFv libraries to pSANG4 phagemide. (A) Agarose gel analysis of colony PCR amplicons from individual transformants using pSANG5th_For and gpiIII primers. Reference amplicons were prepared from the template pSANG4 phagemide used to create the library. (B) Restriction digestion of amplicons from six library transformants and the reference using BpiI endonuclease. The reference scFv, which has a BpiI restriction enzyme recognition site, was included as a control. [Figure 9] The monoclonal DELFIA (dissociation-enhanced lantanide fluorescence immunoassay) binding signals of 92 framework-shifted scFv randomly selected from a second antibody-phage display selection were measured as time-resolved fluorescence (TRF) at excitation wavelengths of 320 nm and emission wavelengths of 615 nm using a Victor Nivo Multimode Microplate reader. The x-axis represents the baseline binding affinity of the clone to the target antigen, with higher signals indicating higher binding. The y-axis represents the ratio of DELFIA signals obtained at pH 7.4 and pH 5.8 for each clone, also known as pH sensitivity. "Parent antibody" refers to the parent antibody used to construct the chain-interface library. "pH-sensitive antibody" refers to a positive control antigen-binding protein that exhibits very high pH sensitivity in the range of 10–11. [Figure 10] Amino acid sequence alignment of scFv (heavy chain) with a pH-sensitive DELFIA signal greater than 1.25 (pH 7.4 DELFIA / pH 5.8 DELFIA). The gray areas highlight mutated residues in the framework mutation library used for phage display discovery. The dots correspond to the same amino acids as clone 2554_01_D11, and "*" indicates deleted residues compared to clone 2554_01_D11. [Figure 11]Amino acid sequence alignment of scFv (light chain) with a pH-sensitive DELFIA signal greater than 1.25 (pH 7.4 DELFIA / pH 5.8 DELFIA). The gray areas highlight the mutated residues in the framework mutation library. The dots correspond to the same amino acids as clone 2554_01_D11. [Figure 12] Point mutations and associated pH sensitivity: This figure shows different point mutations and the levels of pH sensitivity they result in. pH sensitivity was calculated by dividing the mean DELFIA signal at pH 7.4 by the mean signal at pH 5.8 (pH 7.4 / pH 5.8). AVR: mean, Pos CTR: positive control. [Figure 13] Antibody sequence alignment from the Therapeutic Structural Antibody database (Thera-SAbDab) (https: / / opig.stats.ox.ac.uk / webapps / sabdab-sabpred / therasabdab / search / ). The consensus sequences of the full heavy chain, lambda light chain, and kappa light chain are each split across two horizontal rows labeled "1 / 2" and "2 / 2". Split sequences 1 / 2 and 2 / 2 are directly contiguous to each chain starting from sequence "1 / 2". The "+" symbol can be any amino acid. The hash symbol (#) indicates a position in the framework mutation library where the inventors mutated and observed an increase in pH sensitivity compared to the parent antibody. The asterisk (*) highlights an amino acid (AA) shown as an interfacial interaction AA calculated as the embedded solvent-contactable surface area using the ChimeraX function:interface. Triangles (▲) highlight additional residues identified by point mutations and associated with increased pH sensitivity of scFv. Circles (●) highlight additional residues associated with pH sensitivity of antigen-binding proteins, identified based on interfacial interactions and conservation. [Figure 14]Structural information-based manipulation of the D11 light chain improves pH-dependent binding to α-cobra toxin. DELFIA screening of the effect of light chain mutations on the ability to bind to cobra toxin in a pH-dependent manner. Binding was evaluated in pH shift groups of pH 7.4 (dark bar) or pH 5.8 (gray bar). [Figure 15] The identified framework mutations are identical in the blockbuster antibodies. A. Results of a DELFIA immunoassay showing that mutagenesis of eight strategic amino acid residues located at the heavy-light chain interface increases the pH dependence of the antibody-antigen interaction between the model antigen (α-cobra toxin) and the antibody (2554_01_D11 - shown as gray dots in the graph), although the binding signal is preserved, thus demonstrating the usefulness of the versatile pH switch of the present invention. B. Display of conserved similarities in four key amino acid residues identified in the mutant D11 (2554_01_D11) antibody (circled dots in panel A) compared to two blockbuster mAbs, bevacizumab (Avastin) and adalimumab (Humira). Dark ribbon: heavy chain, gray ribbon: light chain, dark spheres: four positions where the mutation induces pH-dependent antigen binding. These models demonstrate that the frame mutation that gives pH-dependent antigen-binding properties to 2554_01_D11 is conserved in other (blockbuster) mAbs. [Figure 16] Site-directed mutagenesis of bevacizumab and adalimumab in scFv format induces increased pH-dependent antigen binding. Site-directed mutagenesis of amino acid residues located at the heavy-light chain interface (lower and upper panels, respectively) of adalimumab and bevacizumab in scFv format (black bars) induces increased pH-dependent antigen binding when measured by DELFIA immunoassay. Parent antibodies (unmutated bevacizumab, upper panel; unmutated adalimumab, lower panel) are shown in gray. [Figure 17]Sequence alignment of the top five bevacizumab and adalimumab candidates from Figure 16. A. Sequences of the top five bevacizumab candidates (sequences 1, 2, 3, 4, and 5 shaded in gray) show mutations at four identified residue positions. B. Sequences of the top five adalimumab candidates (sequences 1, 2, 3, 4, and 5 shaded in gray) show mutations at four identified residue positions. Lightly colored residue positions highlight mutated residues, which are labeled "X" in the parental antibody consensus sequence above the five mutated sequences. [Figure 18] Site-directed mutagenesis of antibody 2554_01_D11 in scFv format induces increased pH-dependent antigen binding. DELFIA immunoassay shows that mutagenesis of strategic amino acid residues VH45, VH47, VH91, VL36, VL44, VL46, VL49, VL87, and / or VL98 located at the heavy-light chain interface increases the pH dependence of antibody-antigen interaction between the model antigen (α-cobra toxin) and the antibody (2554_01_D11). D11 scFv clones labeled with an asterisk "*" are those of sequence number 254. [Modes for carrying out the invention]

[0043] Detailed explanation definition As used herein, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include multiple references. Therefore, for example, a reference to "antigen-binding protein" includes multiple such antigen-binding proteins.

[0044] As used herein, the term “antigen-binding protein” includes antibodies, antibody fragments, and other antigen-binding protein constructs. The term encompasses intact antibodies comprising at least two full-length heavy chains and two full-length light chains, as well as their derivatives, variants, fragments, and variants, examples of which include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, domain antibodies, single-chain antibodies, scFv, or bispecific or multispecific antigen-binding proteins. Preferably, the antigen-binding protein is an antibody or its antigen-binding fragment. The antibodies according to the present invention are generally human monoclonal antibodies.

[0045] As used herein, the term “amino acid” means any of the 20 naturally occurring amino acids commonly used in polypeptide formation, or analogs or derivatives of those amino acids, or any non-naturally occurring amino acid; preferably, the term “amino acid” means any of the 20 naturally occurring amino acids commonly used in polypeptide formation.

[0046] Unless otherwise indicated, amino acids are abbreviated and referred to by conventional nomenclature known to those skilled in the art, such as the conventional single-letter amino acid codes in accordance with the International Union of Pure and Applied Chemistry (IUPAC) (www.iupac.org) "Nomenclature and Symbolism of Amino Acids and Peptides".

[0047] As used herein, the term “complementarity-determining regions” (CDRs) refers to three hypervariable regions of the light chain (LCDR1 or CDRL1, LCDR2 or CDRL2, and LCDR3 or CDRL3) and / or three hypervariable regions of the heavy chain (HCDR1 or CDRH1, HCDR2 or CDRH2, and HCDR3 or CDRH3) located within four more highly conserved framework regions of an antigen-binding protein. These hypervariable regions are primarily responsible for antigen recognition. In antigen-binding proteins, the CDRs are arranged relative to each other in three-dimensional space to form an antigen-binding surface.

[0048] As used herein, the terms “pH-dependent antigen-binding protein” or “pH-sensitive antigen-binding protein” refer to an antigen-binding protein that can bind to an antigen under a first pH condition, for example, a neutral pH including the near-neutral pH of the human body (7.35–7.45) as used herein, e.g., physiological plasma pH (7.4), and can be dissociated from the antigen under another pH condition, for example, an acidic pH defined as pH < 7, preferably pH < 6, e.g., the acidic conditions encountered in cellular endosomes, more preferably a pH in the range of pH 5–pH 6, e.g., pH 5.8, e.g., pH 5.5. The reverse is also included in this term. Preferably, a difference of at least one order of magnitude in binding affinity is observed between the two pH conditions, for example, as measured by Kd. Unless otherwise indicated herein, “higher binding affinity” or “lower binding affinity” refers to the binding affinity of an antigen-binding protein to that antigen. In some embodiments of this specification, pH dependence or pH sensitivity is measured as the ratio of the DELFIA TRF signal (dissociation-enhanced lantanide fluorescence immunoassay time-resolved fluorescence) obtained from the binding of an antigen-binding protein to its antigen at two pH conditions, for example, the ratio of the DELFIA signal obtained from the binding of an antigen-binding protein to its antigen at a neutral pH, e.g., pH 7.4, to the DELFIA signal obtained from the binding of an antigen-binding protein to its antigen at an acidic pH, e.g., pH 5.8 or pH 5.5.

[0049] As used herein, "paratope" refers to the antigen-binding site(s) of an antigen-binding protein that is involved in binding to an antigen epitope.

[0050] As used herein, the term Framework Region (FWR) refers to a region of the variable region of an antibody or other fragment of an antibody, where three hypervariable regions (complementarity-determining regions (CDRs)) are interspersed on each of the variable light and / or heavy chains. For example, for a variable region of full-length IgG containing three CDRs and four FWRs in each variable region, the numbering order of the FWRs as used herein, from the N-terminus to the C-terminus of the variable region, is as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. Furthermore, as used herein, FWRX' refers to the residue position directly adjacent to the C-terminus of the last residue of the FWRX, where X may be 1, 2, 3, or 4.

[0051] As used herein, the term “scaffold” refers to a variable region of an antigen-binding protein that is not part of the paratope. Therefore, the term scaffold includes antibody framework regions (FWRs) and / or complementarity-determining regions (CDRs) that are not involved in epitope binding.

[0052] As used herein, “Kabat numbering” refers to the Kabat antibody amino acid residue numbering system. This scheme defines specific locations in the CDR and FWR where insertions and gaps may occur. In this system, additional amino acid insertions are annotated with letters as described in Kabat EA, Te Wu T, Foeller C, Perry HM, Gottesman KS. Sequences of Proteins of Immunological Interest. (1991). USD Department of Health and Human Services, Public Health Service, National Institutes of Health.

[0053] As used herein, "VH FWRX" and "VL FWRX" refer to the framework region numbers X of the variable region (VH) of the heavy chain and the variable region (VL) of the light chain, respectively, of the antigen-binding protein, where X may be 1, 2, 3, or 4.

[0054] As used herein, "VH XX" or "VL XX" refers to the residue at position "XX" of the sequence in the variable region (VH) of the heavy chain or the variable region (VL) of the light chain, respectively. For example, VH39 refers to the residue at position 39 of the antibody heavy chain variable region (VH). In preferred embodiments, the residue numbering follows the Kabat standard described above.

[0055] As used herein, a specific amino acid at a particular residue position in a sequence is indicated by a conventional single-letter abbreviation of that amino acid, placed at the residue position where the amino acid is found. For example, "L45" refers to the amino acid leucine found at residue position 45. To specify the type of chain (variable region of the light chain or variable region of the heavy chain) in which the specific amino acid is found at that particular position, "VL" or "VH" may be added. For example, "VH L45" refers to the amino acid leucine found at residue position 45 in the variable region of the heavy chain.

[0056] As used herein, the term “variant” refers to either a naturally occurring variation of a given peptide, or a modification of a given peptide or protein prepared by recombinant technology, in which one or more amino acid residues are altered by amino acid substitution, addition, or deletion. The term “variant” may also define either a naturally occurring gene variant of a DNA sequence or the RNA or protein product encoded therein, or a modification of a DNA sequence or the RNA or protein product prepared by recombinant technology.

[0057] When used herein, "K d " or "KD" refers to the equilibrium dissociation constant of the ligand-receptor complex. d The value is expressed in molar concentration units (M) and the dissociation rate (koff ) and meeting speed (k on It is obtained by dividing by ). The association rate, dissociation rate, and equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. The binding affinity may be compared between two different conditions, such as two different pH conditions, to compare the binding affinity of an antibody to that antigen under two conditions.

[0058] When used herein, the "k" of the binding protein (e.g., antibody) from the association complex (e.g., antibody / antigen complex) off " or "K Dis The "dissociation rate constant" or "dissociation rate constant" is known in the art. This value indicates the rate of antibody dissociation from the target antigen, or the separation of the Ab-Ag complex into free antibody and antigen over time.

[0059] As used herein, the term “developability” refers to the suitability of an antigen-binding protein clone for future antibody development. This can be investigated by characterizing the biophysical properties that indicate how well the antibody can be developed and manufactured on a large scale without aggregation, precipitation, denaturation, or suboptimal pharmacokinetics in vivo.

[0060] Method for isolating and / or creating antigen-binding proteins with pH-dependent scaffolds. The present invention enables the direct discovery of pH-dependent antibodies from mutant libraries, such as phage display libraries, by manipulating pH-sensitive antigen-binding protein scaffolds, and enables the design of phage display libraries with predetermined pH-dependent binding properties to antigen-binding proteins. This is achieved, for example, by selectively selecting and / or introducing pH-dependent binding in non-pH-dependent antibodies using variable regions that are not part of the paratope.

[0061] In one embodiment, the present invention relates to a method for isolating an antigen-binding protein having a pH-dependent scaffold, wherein the scaffold consists of a variable region that is not part of the paratope, and the method: - A step of providing a library comprising antigen-binding proteins, each containing an antibody light chain variable region (VL), wherein the VL contains one or more mutations located outside the paratope of the antigen-binding protein, and the library comprises a plurality of antigen-binding proteins, each containing different mutations in the VL. -The process includes the step of selecting an antigen-binding protein from the library that exhibits a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, This relates to a method for isolating antigen-binding proteins having a pH-dependent scaffold.

[0062] In a second aspect, the present invention relates to a method for producing a pH-dependent antigen-binding protein that binds to a specific epitope, wherein the method is: The steps described herein for isolating an antigen-binding protein having a pH-dependent scaffold, - A step of providing an antigen-binding protein that binds to the specific epitope, -The step of replacing a paratope of the antigen-binding protein having a pH-dependent scaffold with a paratope of the antigen-binding protein bound to the epitope, This relates to a method for creating a pH-dependent antigen-binding protein that binds to a specific epitope.

[0063] In some embodiments of the present invention, one or more antigen-binding proteins in the library further comprise a heavy chain containing a variable region (VH), the VH comprising one or more mutations located outside the paratope of the antigen-binding protein.

[0064] In other embodiments, one or more mutations are located at residue positions at the interface between the VL and VH of the antigen-binding protein.

[0065] In further embodiments, the parent sequence of VL and / or the parent sequence of VH is a sequence of an antigen-binding protein that does not have pH-dependent antigen binding. In yet further embodiments, the parent sequence of VL and / or the parent sequence of VH is a sequence of an antigen-binding protein that has low pH-dependent antigen binding to an antigen-binding protein containing one or more mutations described herein.

[0066] In other embodiments, one or more mutations are not located within the complementarity-determining region (CDR).

[0067] Those skilled in the art will understand that a classic approach to modulating the pH dependence of antigen-binding proteins involves manipulating the CDR region, particularly by mutations to and / or from histidine residues. In this specification, we provide an approach comprising manipulating one or more antibody framework regions (FWRs) within the antigen-binding protein variable region, which are located directly beneath the CDR and consist of a beta sheet and a hairpin loop.

[0068] Therefore, in a preferred embodiment of the present invention, one or more mutations are located within one or more framework regions (FWRs).

[0069] In yet another embodiment, the antigen-binding protein of the library includes an antibody light chain variable region (VL), which includes three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, and at least one of FWR1, FWR2, FWR3, and FWR4 contains one or more mutations compared to the parental FWR1, FWR2, FWR3, and FWR4.

[0070] FWR1, FWR2, FWR3, and FWR4 may be framework regions derived from any antibody, for example, any type of antibody described herein.

[0071] In some embodiments, the antigen-binding protein in the library includes VL FWR1 of SEQ ID NO: 26, except that it has one or two amino acid mutations.

[0072] In some embodiments, the antigen-binding protein in the library includes VL FWR2 of SEQ ID NO: 27, except that it has one or two amino acid mutations.

[0073] In other embodiments, the antigen-binding protein in the library includes VL FWR3 of SEQ ID NO: 28, except that it has one or two amino acid mutations.

[0074] In yet another embodiment, the antigen-binding protein of the library includes VL FWR4 of SEQ ID NO: 29, except that it has one or two amino acid mutations.

[0075] In some embodiments, the antigen-binding proteins in the library include VL FWR1 of SEQ ID NO: 26, SEQ ID NO: 151, SEQ ID NO: 157, or SEQ ID NO: 163, except that they have one or two amino acid mutations.

[0076] In some embodiments, the antigen-binding proteins in the library include VL FWR2 of SEQ ID NO: 27, SEQ ID NO: 152, SEQ ID NO: 158, or SEQ ID NO: 164, except that they have one or two amino acid mutations.

[0077] In some embodiments, the antigen-binding proteins in the library include VL FWR3 of SEQ ID NO: 28, SEQ ID NO: 153, SEQ ID NO: 159, or SEQ ID NO: 165, except that they have one or two amino acid mutations.

[0078] In some embodiments, the antigen-binding protein in the library includes VL FWR4 of SEQ ID NO: 29 or SEQ ID NO: 154, except that it has one or two amino acid mutations.

[0079] In some embodiments, the antigen-binding protein in the library has the VL sequence of SEQ ID NO: 35. In further embodiments, the VL sequence is encoded by the sequence of SEQ ID NO: 37.

[0080] In some embodiments of the present invention, the antigen-binding protein of the library comprises an antibody heavy chain variable region (VH), the VH comprising three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, and at least one of the FWR1, FWR2, FWR3, and FWR4 contains one or more mutations compared to the parental FWR1, FWR2, FWR3, and FWR4.

[0081] In other embodiments, the antigen-binding protein in the library includes VH FWR1 of SEQ ID NO: 30, except that it has one or two amino acid mutations.

[0082] In other embodiments, the antigen-binding protein in the library includes VH FWR2 of SEQ ID NO: 31, except that it has one or two amino acid mutations.

[0083] In a further embodiment, the antigen-binding protein of the library includes VH FWR3 of SEQ ID NO: 32, except that it has one or two amino acid mutations.

[0084] In yet another embodiment, the antigen-binding protein of the library includes VH FWR4 of SEQ ID NO: 33, except that it has one or two amino acid mutations.

[0085] In some embodiments, the antigen-binding proteins in the library include VH FWR1 of SEQ ID NO: 30, SEQ ID NO: 155, SEQ ID NO: 160, or SEQ ID NO: 166, except that they have mutations in one or two amino acids.

[0086] In some embodiments, the antigen-binding proteins in the library include VH FWR2 of SEQ ID NO: 31, SEQ ID NO: 156, SEQ ID NO: 161, or SEQ ID NO: 167, except that they have one or two amino acid mutations.

[0087] In some embodiments, the antigen-binding proteins in the library include VH FWR3 of SEQ ID NO: 32, SEQ ID NO: 157, SEQ ID NO: 162, or SEQ ID NO: 168, except that they have one or two amino acid mutations.

[0088] In some embodiments, the antigen-binding protein in the library includes VH FWR4 of SEQ ID NO: 33, except that it has one or two amino acid mutations.

[0089] In some embodiments, the antigen-binding protein of the library has the VH sequence of SEQ ID NO: 34. In further embodiments, the VH sequence is encoded by the sequence of SEQ ID NO: 36.

[0090] In a preferred embodiment, one or more mutations are not located at residue positions occupied by histidine residues.

[0091] In other preferred embodiments, one or more mutations are not mutations in histidine residues.

[0092] In yet another embodiment, one or more mutations are located at a residue position at least one amino acid away from a histidine residue, for example, at a residue position at least two amino acids away from a histidine residue, for example, at least five amino acids, for example, at least eight amino acids, for example, at least ten amino acids, for example, at least fifteen amino acids, for example, at least 20 amino acids, for example, at least 25 amino acids, for example, at least 50 amino acids away.

[0093] The inventors have identified specific FWR residue positions that are particularly useful for conferring pH-dependent binding to antigen-binding proteins. In some embodiments, these positions are independent of the FWR sequence itself.

[0094] In some embodiments, one or more mutations are located in VH residues selected from the group consisting of 39, 44, 89, and 105 according to Kabat numbering.

[0095] In other embodiments, one or more mutations are located at VL residue positions selected from the group consisting of 38, 43, 85, and 100, according to Kabat numbering.

[0096] In some embodiments, the antigen-binding protein in the library is: a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4, sequence number 39 This includes one or more mutations introduced at the residue positions of VH39, VH44, VH89, VH105, VL38, VL43, VL85 and / or VL100, according to Kabat numbering.

[0097] In some applications, it may be beneficial for an antigen-binding protein to exhibit a specific combination of framework regions, and therefore, in some embodiments of the antigen-binding proteins described herein, one or more mutations may be as follows: i. FWR1 of sequence number 26 ii. FWR2 of sequence number 27 iii. FWR3 of sequence number 28, and iv. FWR4 of sequence number 29 or i. FWR1 of sequence number 151 ii. FWR2 of sequence number 152 iii. FWR3 of sequence number 153, and iv. FWR4 of sequence number 154 or i. FWR1 of sequence number 151 ii. FWR2 of sequence number 158 iii. FWR3 of sequence number 159, and iv. FWR4 of sequence number 154 or i. FWR1 of sequence number 163 ii. FWR2 of sequence number 164 iii. FWR3 of sequence number 165, and iv. FWR4 of sequence number 154 Within the VL region of the antigen-binding protein, including the framework region, And the following: i. FWR1 of sequence number 30 ii. FWR2 of Sequence ID 31 iii. FWR3 of sequence number 32, and iv. FWR4 of sequence number 33 or i. FWR1 of sequence number 155 ii. FWR2 of sequence number 156 iii. FWR3 of sequence number 157, and iv. FWR4 of sequence number 33 or i. FWR1 of sequence number 160 ii. FWR2 of sequence number 161 iii. FWR3 of sequence number 162, and iv. FWR4 of sequence number 33 or i. FWR1 of sequence number 166 ii. FWR2 of sequence number 167 iii. FWR3 of sequence number 168, and iv. It is introduced into the VH region of the antigen-binding protein containing the framework region of FWR4 in SEQ ID NO: 33.

[0098] In other embodiments, the antigen-binding protein in the library is: a) VH FWR2 of sequence number 156 b) VH FWR3 of sequence number 157 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 152 e) VL FWR3 with sequence number 153 f) VL FWR4, sequence number 154 The system includes one or more mutations introduced at the residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably one or more mutations introduced at the residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering.

[0099] In further embodiments, the antigen-binding proteins in the library are: a) VH FWR2 of sequence number 161 b) VH FWR3 of sequence number 162 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 158 e) VL FWR3, sequence number 159 f) VL FWR4, sequence number 154 The system includes one or more mutations introduced at the residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably one or more mutations introduced at the residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering.

[0100] In some embodiments, the antigen-binding protein in the library is: a) VH FWR2 of sequence number 164 b) VH FWR3 of sequence number 165 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 167 e) VL FWR3, sequence number 168 f) VL FWR4, sequence number 154 The system includes one or more mutations introduced at the residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably one or more mutations introduced at the residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering.

[0101] In a preferred embodiment, the step of selecting antigen-binding proteins from the library that exhibit a lower KD at acidic pH than neutral pH, or a higher KD at acidic pH than neutral pH, includes using an in vitro display technique.

[0102] The selection step may be carried out as described in Example 6, for example.

[0103] In other preferred embodiments, the in vitro display technology is selected from the group consisting of phage displays, ribosome displays, yeast displays, bacterial displays, mammalian displays, and CIS displays.

[0104] In some embodiments of the methods described herein, the antigen-binding protein of the library includes or comprises the pH-dependent antigen-binding protein described herein or the antigen-binding protein described herein.

[0105] pH-dependent antigen-binding protein A third aspect of the present invention relates to a pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parent FWR1, FWR2, FWR2', FWR3, and FWR4, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, and the pH-dependent antigen-binding protein does not contain heavy chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, respectively, and light chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 4, 5, and 6, respectively.

[0106] Another aspect of the present invention is a pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, and the pH-dependent antigen-binding protein Sequence numbers 1, 2, and 3, respectively Sequence numbers 178, 179, and 180, respectively. The numbers 181, 182, 183, and Each of the heavy chain complementarity determination regions 1, 2, and 3 of sequence numbers 184, 185, and 186, respectively, and Sequence numbers 4, 5, and 6, respectively These are sequence numbers 169, 170, and 171 respectively. Sequence numbers 172, 173, 174, and This relates to a pH-dependent antigen-binding protein that does not contain light chain complementarity-determining regions 1, 2, and 3 of any of sequence numbers 175, 176, and 177, respectively.

[0107] In a preferred embodiment, the pH-dependent antigen-binding protein is as defined above.

[0108] Residual mutation - location In some embodiments, one or more mutations in a pH-dependent antigen-binding protein are located at residue positions at the interface between the VL and VH of the pH-dependent antigen-binding protein.

[0109] In other embodiments, one or more mutations in the pH-dependent antigen-binding protein are not located at residue positions occupied by histidine residues.

[0110] In further embodiments, one or more mutations in the pH-dependent antigen-binding protein are not mutations in histidine residues.

[0111] In other embodiments, one or more mutations in the pH-dependent antigen-binding protein are located at a residue position at least one amino acid away from a histidine residue, for example, at a residue position at least two amino acids away from a histidine residue, for example, at least five amino acids, for example, at least eight amino acids, for example, at least ten amino acids, for example, at least fifteen amino acids, for example, at least 20 amino acids, for example, at least 25 amino acids, for example, at least 50 amino acids away.

[0112] In some embodiments, one or more mutations in the pH-dependent antigen-binding protein are located in VH residues selected from the group consisting of 39, 44, 89, and 105 according to Kabat numbering, preferably in VH residues selected from the group consisting of 44, 89, and 105.

[0113] In some embodiments, one or more mutations are located in VH residues selected from the group consisting of 45, 47, 91, and 103 according to Kabat numbering.

[0114] In some embodiments, one or more mutations are located in VH residues selected from the group consisting of L45, W47, Y91, and W103.

[0115] In some embodiments, one or more mutations in the pH-dependent antigen-binding protein are located at a VL residue position selected from the group consisting of 38, 43, 85, and 100 according to Kabat numbering, preferably at the VL residue position of 38.

[0116] In some embodiments, one or more mutations are located at VL residue positions selected from the group consisting of 32, 46, and 49 according to Kabat numbering.

[0117] In other embodiments, one or more mutations are located at VL residue positions selected from the group consisting of 46 and 49 according to Kabat numbering.

[0118] In some embodiments, one or more mutations are located at VL residue positions selected from the group consisting of 36, 44, 87, and 98 according to Kabat numbering.

[0119] In some embodiments, one or more mutations are located in VL residues selected from the group consisting of Y32, T46, and Y49 according to Kabat numbering.

[0120] In other embodiments, one or more mutations are located in VL residues selected from the group consisting of T46 and Y49 according to Kabat numbering.

[0121] In some embodiments, one or more mutations are located in VL residues selected from the group consisting of Y36, P44, Y87, and F98 according to Kabat numbering.

[0122] In other embodiments, one or more mutations are located in VH residues selected from the group consisting of 39, 44, 45, 47, 89, 91, 103, and 105 according to Kabat numbering.

[0123] In some embodiments, one or more mutations are located in VL residues selected from the group consisting of 32, 36, 38, 43, 44, 46, 49, 85, 87, 98, and 100, according to Kabat numbering.

[0124] In some embodiments, one or more mutations are located in VL residues selected from the group consisting of 36, 38, 43, 44, 46, 49, 85, 87, 98, and 100, according to Kabat numbering.

[0125] In some embodiments, one or more mutations are located in VH residues selected from the group consisting of Q39, G44, L45, W47, V89, Y91, W103, and Q105, according to Kabat numbering.

[0126] In some embodiments, one or more mutations are located at VL residue positions selected from the group consisting of Y32, Y36, Q38, D43, P44, T46, Y49, D85, Y87, F98, and G100, according to Kabat numbering.

[0127] In other embodiments, one or more mutations are located in VL residues selected from the group consisting of Y36, Q38, D43, P44, T46, Y49, D85, Y87, F98, and G100, according to Kabat numbering.

[0128] In a preferred embodiment, the antigen-binding protein is: a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4, sequence number 39 It includes one or more mutations introduced at residue positions selected from the group consisting of VH39, VH44, VH89, VH105, VL38, VL43, VL85, and VL100, according to Kabat numbering.

[0129] In other embodiments, the antigen-binding protein further comprises VH FWR1 of SEQ ID NO: 30 and VL FWR1 of SEQ ID NO: 26.

[0130] In other embodiments, the antigen-binding protein is: a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) It includes one or more mutations introduced at residue positions selected from the group consisting of VH45, VH47, VH91, VH103, VL36, VL44, VL87, and VL98, according to Kabat numbering.

[0131] In yet another embodiment, the antigen-binding protein is: a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) It includes one or more mutations introduced at residue positions selected from the group consisting of VL32, VL46, and VL49 according to Kabat numbering.

[0132] In some embodiments, the antigen-binding protein is: a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) It includes one or more mutations introduced at residue positions selected from the group consisting of VL46 and VL49 according to Kabat numbering.

[0133] In other embodiments, the antigen-binding protein is: a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) The mutations include one or more mutations introduced at residue positions selected from the group consisting of VH39, VH44, VH45, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL46, VL49, VL85, VL87, VL98, and VL100, according to Kabat numbering.

[0134] In some embodiments, the antigen-binding protein is: a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) The mutations include one or more mutations introduced at residue positions selected from the group consisting of VH39, VH44, VH45, VH89, VH91, VH103, VH105, VL36, VL38, VL43, VL46, VL49, VL85, VL87, VL98, and VL100, according to Kabat numbering.

[0135] In other embodiments, the antigen-binding protein is: a) VH FWR2 of sequence number 156 b) VH FWR3 of sequence number 157 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 152 e) VL FWR3 with sequence number 153 f) VL FWR4, sequence number 154 The system includes one or more mutations introduced at the residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably one or more mutations introduced at the residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering.

[0136] In some embodiments, the antigen-binding protein is: a) VH FWR2 of sequence number 161 b) VH FWR3 of sequence number 162 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 158 e) VL FWR3, sequence number 159 f) VL FWR4, sequence number 154 The system includes one or more mutations introduced at the residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably one or more mutations introduced at the residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering.

[0137] In further embodiments, the antigen-binding protein is: a) VH FWR2 of sequence number 164 b) VH FWR3 of sequence number 165 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 167 e) VL FWR3, sequence number 168 f) VL FWR4, sequence number 154 The system includes one or more mutations introduced at the residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably one or more mutations introduced at the residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering.

[0138] As described herein, in some embodiments of the antigen-binding proteins, pH-dependent antigen-binding proteins, and methods described herein, one or more mutations are introduced into the antigen-binding protein to confer pH-dependent binding properties to the antigen binding. In some embodiments, the pH-dependent binding properties are conferred to the antigen-binding protein by mutations in specific amino acid residues at specific residue positions in the VH and VL of the antigen-binding protein.

[0139] Therefore, an aspect of the present invention is an antigen-binding protein comprising an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein one or more mutations are introduced at residue positions selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98 and VL100 according to Kabat numbering. The VH contains one or more of the following amino acid residues, for example, two, three, or four: - S, E, R, T, or A, ranked 39th - P, R, N, S, K, Q, A, Y, or T, ranked 44th - T, N, I, Q, A, L, Y, D, F, S, or K, and / or - 105th place T, R, K, P, D, I, S, or T, and / or The VL contains one or more of the following amino acid residues, for example, two, three, four, five, six, or seven: - N, ranked 36th. - 38th place: L, Y, S, I, T, A, R, F, or V - 43rd place: P, T, A, R, Q, K, or V, - 46th place A or S, - 49th place A, H, or S, - S, N, T, F, V, L, S, A, H, or R in the 85th position and / or - S, Y, W, or L in 100th place, In the formula, all positions are indicated according to Kabat numbering, relating to the antigen-binding protein.

[0140] In some embodiments, VH includes one or more of the following amino acid residues, for example, two, three, or four: 39th place S, E, or A 44th place: P, R, N, S, K, Q, A, or T 89 - position T, N, I, Q, A, L, Y, D, F, S, or K, and / or 105 - position T, R, K, P, D, I, S, or T, wherein all positions are shown according to Kabat numbering.

[0141] In some embodiments, VL comprises one or more, such as 2, such as 3, such as 4, such as 5, such as 6, such as 7 of the following amino acid residues, N at position 36, L, Y, S, I, T, A, R, F, or V at position 38, P, T, A, or V at position 43, A or S at position 46, A, H, or S at position 49, S, N, T, F, V, L, S, A, or R at position 85, and / or S, Y, or L at position 100, wherein all positions are shown according to Kabat numbering.

[0142] In preferred embodiments, the antigen - binding protein comprises: a) VH FWR2 of WVRX1APGQX2X3EX4MG (SEQ ID NO: 98) b) VH FWR3 of RVTITADX5STSTAYMX6LX7SLRSDDTAX8YX9CAR (SEQ ID NO: 99) c) X 10 GX 11 VH FWR4 of GTLVTVSS (SEQ ID NO: 100), wherein X1, X2, X3, X4, X5, X6,X7, X8, X9, X 10 , X 11 each may be any amino acid, provided that at least one of X1, X2, X8, X 11 is selected from: X1 is any amino acid other than Q, preferably X1 is S, E, or A, and / or X2 is any amino acid other than G, preferably X2 is P, R, N, S, K, Q, A, or T, and / or X8 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, or K, and / or X 11 is any amino acid other than Q, more preferably X 11 is T, R, K, P, D, I, or T, and d) The light chain CDR1 of TRSX1GSIGSDX2VH (SEQ ID NO: 104) e) The VL FWR2 of WX3QX4RPGSX5X6TX7VIX8 (SEQ ID NO: 101) f) GVPDRFSGSIDSSSNSASLTISGLKTEDEAX9YX 10 C (SEQ ID NO: 102) of VL FWR3 g) X 11 GX 12 GTKLTVX 13 (SEQ ID NO: 103) of VL FWR4, wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 each may be any amino acid, provided that at least one of X2, X4, X5, X7, X8, X9, X 12 is selected from the following: X2 is any amino acid other than Y, preferably X2 is A, S, T, or H, and / or X4 is any amino acid other than Q, preferably X4 is L, Y, S, I, T, A, R, or V, and / or X5 is any amino acid other than S, preferably X5 is P, T, A, or V, and / or X7 is any amino acid other than T, preferably X7 is A, or S, and / or X8 is any amino acid other than Y, preferably X8 is A, H, or S, and / or X9 is any amino acid other than D, preferably X9 is S, N, T, F, V, L, S, A, or R, and / or X 12 is any amino acid other than G, preferably X 12 It is S, Y, or L.

[0143] In further embodiments, the antigen-binding protein includes: a) VH FWR2 of WVRX1APGQX2X3EX4MG (Sequence ID 98) b) VH FWR3 of RVTITADX5STSTAYMX6LX7SLRSDDTAX8YX9CAR (Sequence ID 99) c)X 10 GX 11 GTLVTVSS (Sequence ID 100) VH FWR4, In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X8, X 11 At least one of the following is selected: X1 is any amino acid other than Q, preferably X1 is S, E, or A, and / or X2 is any amino acid other than G, preferably X2 is P, R, N, S, K, Q, A, or T, and / or X8 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, or K, and / or X 11 is any amino acid other than Q, preferably X 11 is T, R, K, P, D, I, or T. Furthermore d) VL FWR2 of WX1QX2RPGSX3X4TX5VIX6 (Sequence ID 101) e) VL FWR3 of GVPDRFSGSIDSSSNSASLTISGLKTEDEAX7YX8C (Sequence ID 102) f)X9GX 10 GTKLTVX 11 VL FWR4 (Sequence ID 103), In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X10 , X 11 Each of these can be any amino acid, however X2, X3, X5, X6, X7, X 10 At least one of the following is selected: X2 is any amino acid other than Q, preferably X2 is L, Y, S, I, T, A, R, or V, and / or X3 is any amino acid other than S, preferably X3 is P, T, A, or V, and / or X5 is any amino acid other than T, preferably X5 is A or S, and / or X6 is any amino acid other than Y, preferably X6 is A, H, or S, and / or X7 is any amino acid other than D, preferably X7 is S, N, T, F, V, L, S, A, or R, and / or X 10 is any amino acid other than G, preferably X 10 It is S, Y, or L.

[0144] In other embodiments, the antigen-binding protein includes: a) VH FWR2 of WVRX1APGKX2X3EX4VS (Sequence ID 187) b) VH FWR3 of RFTISRDX5AKNSLYLX6MX7SLRAEDTAX8YX9CAK (Sequence ID 188) c)X 10 GX 11 GTLVTVSS (Sequence ID 100) VH FWR4, In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X8, X 11 At least one of the following is selected: X1 is any amino acid other than Q, preferably X 1は , S, E, or A, and / or X2 is any amino acid other than G, preferably X2 is P, R, N, S, K, Q, A, Y, or T, more preferably X2 is Y, R, or T, and / or X8 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, Y, D, F, S, or K, more preferably X8 is Y, D, F, or S, and / or X 11 is any amino acid other than Q, preferably X 11 is T, R, K, P, D, I, S, or T, and more preferably X 11 is T, S, or P, Furthermore d) VL FWR2 of WX1QX2KPGKX3X4KX5LIX6 (Sequence ID 190) e) VL FWR3 of GVPSRFSGSGSGTDFTLTISSLQPEDVAX7YX8C (Sequence ID 191) f)X9GX 10 GTKVEIX 11 VL FWR4 (Sequence ID 192), In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X3, X5, X6, X7, X 10 At least one of the following is selected: X1 is any amino acid other than Y, preferably X1 is N, and / or X2 is any amino acid other than Q, preferably X2 is L, Y, S, I, T, A, R, F, or V, more preferably X2 is S, R, or L, and / or X3 is any amino acid other than A, preferably X3 is P, T, or V, and / or X5 is any amino acid other than L, preferably X5 is A or S, and / or X6 is any amino acid other than Y, preferably X6 is A, H, or S, and / or X7 is any amino acid other than T, preferably X7 is N, F, V, L, S, A, or R, and / or X 10 is any amino acid other than Q, preferably X 10 It is S, Y, or L.

[0145] In some embodiments, the antigen-binding protein includes: a) VH FWR2 of WVRQAPGKX1LEWVS (Sequence ID 193) b) VH FWR3 of RFTISRDNAKNSLYLQMNSLRAEDTAX2YYCAK (SEQ ID NO: 194) c) VH FWR4 of WGX3GTLVTVSS (Sequence ID 195) Furthermore d) VL FWR2 of WYQX4KPGKAPKLLIY (Sequence ID 196), In the formula, each of X1, X2, X3, and X4 can be any amino acid, provided that at least one of X1, X2, X3, and X4 is selected from the following: X1 is any amino acid other than G, preferably X1 is P, R, N, S, K, Q, A, Y, or T, more preferably X1 is Y, R, or T, and / or X2 is any amino acid other than V, preferably X2 is T, N, I, Q, A, L, Y, D, F, S, or K, more preferably X2 is Y, D, F, or S, and / or X3 is any amino acid other than Q, preferably X3 is T, R, K, P, D, I, S, or T, more preferably X3 is S, T, or P, and / or X4 is any amino acid other than Q, preferably X4 is L, Y, S, I, T, A, R, F, or V, and more preferably X4 is R, S, or L.

[0146] In a further embodiment, the antigen-binding protein comprises: a) the VH FWR2 of WVRX1APGKX2X3EX4VG (SEQ ID NO: 197) b) RFTFSLDX5SKSTAYLX6MX7SLRX8EDTAX9YX 10 the VH FWR3 of CAK (SEQ ID NO: 198) c) X 11 GX 12 the VH FWR4 of GTLVTVSS (SEQ ID NO: 189), wherein each of X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 can be any amino acid, provided that at least one of X1, X2, X8, X 11 is selected from: X1 is any amino acid other than Q, preferably X1 is S, E, or A, and / or X2 is any amino acid other than G, preferably X2 is P, R, N, S, K, Q, A, or T, even more preferably X2 is A, P, S, or T, and / or X8 is any amino acid other than A, preferably X8 is T, and / or X9 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, or K, even more preferably X8 is A, T, I, or L, and / or X 12 is any amino acid other than Q, preferably X 11 is T, R, K, P, D, I, S, or T, even more preferably X 11 is T, D, S, or P, and d) the VL FWR2 of WX1QX2KPGKX3X4KX5LIX6 (SEQ ID NO: 190) e) the VL FWR3 of GVPSRFSGSGSGTDFTLTISSLQPEDFAX7YX8C (SEQ ID NO: 199) f) X9GX 10 GTKVEIX11 VL FWR4 (Sequence ID 192), In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X3, X5, X6, X7, X 10 At least one of the following is selected: X1 is any amino acid other than Y, preferably X1 is N, and / or X2 is any amino acid other than Q, preferably X2 is L, Y, S, I, T, A, R, F, or V, more preferably X2 is F, L, S, T, or I, and / or X3 is any amino acid other than A, preferably X3 is P, T, or V, and / or X5 is any amino acid other than V, preferably X5 is A or S, and / or X6 is any amino acid other than Y, preferably X6 is A, H, or S, and / or X7 is any amino acid other than T, preferably X7 is S, N, F, V, L, A, or R, and / or X 10 is any amino acid other than Q, preferably X 10 It is S, Y, or L.

[0147] In some embodiments, the antigen-binding protein includes: a) VH FWR2 of WVRQAPGKX1LEWVG (Sequence ID 200) b) VH FWR3 of RFTFSLDTSKSTAYLQMNSLRX2EDTAX3YYCAK (Sequence ID 201) c) VH FWR4 of WGX4GTLVTVSS (Sequence ID 195) Furthermore d) VL FWR2 of WYQX5KPGKAPKVLIY (Sequence ID 202), In the formula, each of X1, X2, X3, and X4 can be any amino acid, provided that at least one of X1, X2, X3, and X4 is selected from the following: X1 is any amino acid other than G, preferably X1 is P, R, N, S, K, Q, A, Y, or T, more preferably X1 is A, P, S, or T, and / or X2 is any amino acid other than A, preferably X8 is T, and / or X3 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, Y, D, F, S, or K, preferably X2 is A, T, I, or L, and / or X4 is any amino acid other than Q, preferably X3 is T, R, K, P, D, I, S, or T, preferably X3 is T, D, S, or P, and / or X5 is any amino acid other than Q, preferably X4 is L, Y, S, I, T, A, R, F, or V, and more preferably X4 is F, L, S, T, or I.

[0148] In other embodiments, the antigen-binding protein includes: a) VH FWR2 of WVRX1APGKX2X3EX4MG (Sequence ID 203) b) VH FWR3 of RVTMTEDX5STDTAYMX6LX7SLRSEDTAX8YX9CST (Sequence ID 204) c)X 10 GX 11 GTLVTVSS (Sequence ID 189) VH FWR4, In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X8, X 11 At least one of the following is selected: X1 is any amino acid other than Q, preferably X1 is S, E, or A, and / or X2 is any amino acid other than G, preferably X2 is P, R, N, S, K, Q, A, or T, and / or X8 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, or K, and / or X 11 is any amino acid other than Q, preferably X 11 is T, R, K, P, D, I, S, or T. Furthermore d) VL FWR2 of WX1QX2KPGKX3X4KX5LIX6 (Sequence ID 190) e) VL FWR3 of GVPSRFSGSGSGTEFTLTISSLQPEDLAX7YX8C (Sequence ID 205) f)X9GX 10 GTKVEIX 11 VL FWR4 (Sequence ID 192), In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X3, X5, X6, X7, X 10 At least one of the following is selected: X1 is any amino acid other than Y, preferably X1 is N, and / or X2 is any amino acid other than Q, preferably X2 is L, Y, S, I, T, A, R, F, or V, and / or X3 is any amino acid other than A, preferably X3 is P, T, or V, and / or X5 is any amino acid other than R, preferably X5 is A or S, and / or X6 is any amino acid other than Y, preferably X6 is A, H, or S, and / or X7 is any amino acid other than S, preferably X7 is N, F, V, L, A, or R, and / or X 10 is any amino acid other than Q, preferably X10 It is S, Y, or L.

[0149] In a preferred embodiment of the antigen-binding protein of the present invention, the variable light chain framework region (VL FWR) contains three light chain complementarity-determining regions (CDRL), and the heavy chain framework region (VH FWR) contains three heavy chain complementarity-determining regions (CDRH). In a more preferred embodiment of the antigen-binding protein of the present invention, the four variable light chain framework regions (VL FWR) contain three consecutive light chain complementarity-determining regions (VL CDR) in the format VL FWR1-CDRL1-VL FWR2-CDRL2-VL FWR3-CDRL3-VL FWR4, and the four variable heavy chain framework regions (VH FWR) contain three consecutive heavy chain complementarity-determining regions (CDRH) in the format VH FWR1-CDRH1-VH FWR2-CDRH2-VH FWR3-CDRH3-VH FWR4.

[0150] In other embodiments, the antigen-binding protein includes: a) VH FWR2 of sequence number 39 b) VH FWR3 of sequence number 40 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 43 e) FWR2' is S50 according to Kabat numbering. f) VL FWR3, sequence number 45 g) VL FWR4 of sequence number 47.

[0151] In other embodiments, the antigen-binding protein further comprises VH FWR1 of SEQ ID NO: 38 and VL FWR1 of SEQ ID NO: 41.

[0152] In some embodiments, VL FWR1 of the 2555_01_A01 antibody is the one specified by SEQ ID NO: 41. In other embodiments, VL FWR2 of the 2555_01_A01 antibody is the one specified by SEQ ID NO: 42.

[0153] In some embodiments, the VL FWR2 of the 2555_01_A01 antibody is that of SEQ ID NO: 43. In other embodiments, the VL FWR2 of the 2555_01_A01 antibody is that of SEQ ID NO: 44.

[0154] In some embodiments, the VL FWR3 of the 2555_01_A01 antibody is that of SEQ ID NO: 45. In other embodiments, the VL FWR3 of the 2555_01_A01 antibody is that of SEQ ID NO: 46.

[0155] In further embodiments, the antigen-binding protein includes a VH sequence selected from the group consisting of SEQ ID NOs: 77-87. In other embodiments, the antigen-binding protein includes a VL sequence selected from the group consisting of SEQ ID NOs: 35 and 88-94.

[0156] In some embodiments, the antigen-binding protein includes a VH sequence selected from the group consisting of SEQ ID NOs: 105-124 and a VL sequence selected from the group consisting of SEQ ID NOs: 125-144.

[0157] In yet another embodiment, the antigen-binding protein includes a VH sequence selected from the group consisting of SEQ ID NOs: 77-87 and 105-124, and a VL sequence selected from SEQ ID NOs: 35, 88-94, and 125-144.

[0158] A fourth aspect of the present invention is a pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, and the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and one or more mutations, compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, according to Kabat, are VH39, VH44, VH45, VH89, VH91, VH103, VH105, VL32, VL This invention relates to a pH-dependent antigen-binding protein introduced at a residue position selected from the group consisting of 36, VL38, VL43, VL46, VL49, VL50, VL85, VL87, VL98, and VL100, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, and the pH-dependent antigen-binding protein does not contain the heavy chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, respectively, or the light chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 4, 5, and 6, respectively.

[0159] A key advantage of the present invention is that, by the method described herein, a novel synthetic library of antibodies having pH-dependent binding predetermined by the framework region can be created by using the pH-dependent antibodies subsequently discovered from the library as a comprehensive scaffold.

[0160] Importantly, the "predetermined" pH-dependent antigen binding in each clone reduces the need for histidine doping or other low-throughput manipulation approaches to discover recyclable antibodies. Furthermore, using such libraries allows for the identification of amino acid substitutions at the heavy / light chain interface, which robustly confer pH sensitivity to the antibody, leading to the possibility of routinely introducing these substitutions into monoclonal antibodies on a case-by-case basis through rational design.

[0161] Therefore, a fifth aspect of the present invention is a method for generating a pH-dependent antigen-binding protein directed toward an antigen of interest, wherein the method is: - A step of providing an antigen-binding protein that binds to an antigen of interest; - The step of introducing one or more mutations in the antigen-binding protein at residue positions selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and VL100, according to Kabat numbering, This relates to a method for generating a pH-dependent antigen-binding protein targeted to an antigen of interest.

[0162] In some embodiments of the method, one or more mutations are introduced at residue positions selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and VL100, according to Kabat numbering.

[0163] In other embodiments of the method, one or more mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH L45, VH W47, VH V89, VH Y91, VH W103, VH Q105, VL Y32, VL Y36, VL Q38, VL S43, VL P44, VL T46, VL Y49, VL E50, VL D85, VL Y87, VL F98, and VL G100, according to the Kabat numbering of another amino acid.

[0164] In a further embodiment of the method, one or more mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH L45, VH W47, VH V89, VH Y91, VH W103, VH Q105, VL Y36, VL Q38, VL S43, VL P44, VL T46, VL Y49, VL E50, VL D85, VL Y87, VL F98, and VL G100, according to the Kabat numbering of another amino acid.

[0165] In yet another embodiment of the method, one or more mutations are introduced at residue positions from the group consisting of VH39, VH44, VH89, VH105, VL38, VL43, VL50, VL85 and VL100, according to the Kabat numbering of another amino acid.

[0166] In some embodiments of the method, one or more mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH V89, VH Q105, VL Q38, VL S43, VL E50, VL D85, and VL G100, according to the Kabat numbering of another amino acid.

[0167] In some embodiments of the method, one or more mutations are introduced at residue positions selected from the group consisting of VH45, VH47, VH91, and VH103 according to Kabat numbering.

[0168] In other embodiments of the method, one or more mutations are introduced at residue positions selected from the group consisting of VH L45, VH W47, VH Y91, and VH W103 according to Kabat numbering.

[0169] In other embodiments of the method, one or more mutations are introduced at residue positions selected from the group consisting of VL32, VL46, and VL49 according to Kabat numbering.

[0170] In a further embodiment of the method, one or more mutations are introduced at residue positions selected from the group consisting of VL46 and VL49 according to Kabat numbering.

[0171] In a preferred embodiment of the method, one or more mutations are introduced at residue positions selected from the group consisting of VL Y32, VL T46, and VL Y49 according to Kabat numbering.

[0172] In other embodiments of the method, one or more mutations are introduced at residue positions selected from the group consisting of VL T46 and VL Y49 according to Kabat numbering.

[0173] In other embodiments of the method, one or more mutations are introduced at residue positions selected from the group consisting of VL36, VL44, VL87, and VL98 according to Kabat numbering.

[0174] In some embodiments of the method, one or more mutations are introduced at residue positions selected from the group consisting of VL Y36, VL P44, VL Y87, and VL F98, according to Kabat numbering.

[0175] In a preferred embodiment, the step of introducing one or more mutations further includes testing whether the antigen-binding protein containing one or more mutations has a higher binding affinity at an acidic pH than at a neutral pH, or a lower binding affinity at an acidic pH than at a neutral pH.

[0176] It may be beneficial to ensure that antigen-binding proteins containing one or more mutations have comparable or greater development potential than antibodies containing the parent residues, and in particular, it may be beneficial to select constructed or isolated antibodies that exhibit maximum thermal stability.

[0177] Therefore, in some embodiments, the method further includes the step of selecting an antibody that exhibits the best thermal stability, for example, the best Fab fragment melting temperature.

[0178] Residual mutation - Characteristics of mutant amino acids or mutations to said amino acids Those skilled in the art will understand that certain amino acid residues have side chains that can be charged. At a neutral pH (7), aspartic acid and glutamic acid, for example, are negatively charged (acidic side chains), while lysine, arginine, and histidine are positively charged (basic side chains).

[0179] In a preferred embodiment, one or more mutations are located at residue positions occupied by charged residues.

[0180] In further embodiments, one or more mutations are mutations in charged residues.

[0181] In other embodiments, one or more mutations are located at residue positions occupied by residues that can participate in hydrogen bonding.

[0182] In yet another embodiment, one or more mutations are mutations in residues that can participate in hydrogen bonding.

[0183] The amino acid (AA) residues that mutate in the method or pH-dependent antigen-binding protein of the present invention are preferably AAs having a hydrogen donor or acceptor atom in their side chain that is involved in hydrogen bonding. Those skilled in the art will understand that hydrogen bonding involves the interaction of a hydrogen atom located between a pair of other atoms that have a high affinity for electrons. The donor atom of the pair is typically nitrogen, oxygen, or fluorine covalently bonded to the hydrogen atom as -NH, -OH, or -FH. The acceptor atom is typically nitrogen, oxygen, or fluorine having a lone pair of electrons. Typically, polar amino acids can form hydrogen bonds through their side chains.

[0184] Therefore, in some embodiments, one or more mutations are located at residue positions occupied by residues selected from the group consisting of arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, threonine, tryptophan, and tyrosine.

[0185] In other embodiments, one or more mutations are mutations in residues selected from the group consisting of arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, threonine, tryptophan, and tyrosine.

[0186] In further embodiments, one or more mutations are located at residue positions occupied by residues selected from the group consisting of arginine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, serine, threonine, tryptophan, and tyrosine.

[0187] In yet further embodiments, one or more mutations are mutations in residues selected from the group consisting of arginine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, serine, threonine, tryptophan, and tyrosine.

[0188] The mutated AA of the present invention may also preferably be charged AA.

[0189] Therefore, in some embodiments, one or more mutations are located at residue positions occupied by residues selected from the group consisting of arginine, aspartic acid, glutamic acid, histidine, and lysine.

[0190] In other embodiments, one or more mutations are mutations in residues selected from the group consisting of arginine, aspartic acid, glutamic acid, histidine, and lysine.

[0191] In further embodiments, one or more mutations are located at residue positions occupied by residues selected from the group consisting of arginine, aspartic acid, glutamic acid, histidine, and lysine.

[0192] In yet further embodiments, one or more mutations are mutations in residues selected from the group consisting of arginine, aspartic acid, glutamic acid, histidine, and lysine.

[0193] pH dependence-Kd In some embodiments, pH-dependent antigen-binding proteins have a lower Kd value for their antigen at acidic pH compared to their Kd value at neutral pH.

[0194] In a preferred embodiment, the Kd value at an acidic pH decreases by 2 times, for example, 5 times, for example, 10 times, for example, 25 times, for example, 50 times, for example, 75 times, for example, 100 times, for example, 125 times, for example, 250 times, for example, 500 times, for example, 750 times, for example, 1000 times, compared to a neutral pH.

[0195] In other embodiments, the pH-dependent antigen-binding protein has a higher Kd value for its antigen at acidic pH compared to its Kd value at neutral pH.

[0196] In a preferred embodiment, the Kd value at an acidic pH increases by 2 times, for example, 5 times, for example, 10 times, for example, 25 times, for example, 50 times, for example, 75 times, for example, 100 times, for example, 125 times, for example, 250 times, for example, 500 times, for example, 750 times, for example, 1000 times compared to a neutral pH.

[0197] Antibody types and possible modifications In some embodiments, the pH-dependent antigen-binding protein is selected from the group consisting of full-length antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, scFv, diabodies, and triabodies.

[0198] In other embodiments, the pH-dependent antigen-binding protein includes an immunoglobulin constant region.

[0199] In further embodiments, the constant region of pH-dependent antigen-binding protein immunoglobulin is selected from the group consisting of IgG, IgM, IgA, IgD, and IgE.

[0200] In yet another embodiment, the constant region of pH-dependent antigen-binding protein immunoglobulin is selected from the group consisting of IgG and IgA.

[0201] In some embodiments, the constant region of pH-dependent antigen-binding protein immunoglobulin is selected from the group consisting of IgG1, IgA1, and IgA2.

[0202] In other embodiments, the constant region of pH-dependent antigen-binding protein immunoglobulin is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0203] In preferred embodiments, the pH-dependent antigen-binding protein is a monoclonal antibody. In further embodiments, the pH-dependent antigen-binding protein is a human antibody or a chimeric antibody.

[0204] Chimeric antibodies retain the CDR from the original species, which is incorporated into an antibody derived from another species, such as human. Other chimeric antibodies retain the variable region from the original species, which is fused to the constant region of another species, such as human. The advantages of chimeric antibodies are their reduced immunogenicity in humans compared to mouse antibodies and their lower production cost compared to what is required to create fully humanized antibodies.

[0205] The antibody may be a multispecific antibody (e.g., a bispecific antibody) formed from at least two different antibodies, and / or an antibody fragment.

[0206] In some embodiments, the antigen-binding protein is a human antibody or an antibody based on a human scaffold. The antibody may also be a humanized antibody containing a CDR region (and possibly some other residues) transferred from another species having the desired specificity, affinity, and capability. The humanized antibody may also contain a synthetic CDR region, for example, from a synthetic antibody library.

[0207] Antibody production can be achieved by any standard method in the art for generating antibodies.

[0208] Recombinant antibodies can be isolated from a library of genes encoding antibody fragments. Antibody fragments may be the aforementioned antibody fragments, e.g., Fab, Fv fragments, single-chain fragments of heavy and light chain variable domains, or single-domain antibodies, e.g., polypeptides containing or consisting of VH or VL domains. Antigen-binding proteins may be single-chain antibodies containing linked heavy and light chain variable domains; for example, an antigen-binding protein may be scFv. Gene libraries may be obtained from natural sources, as in the case of naive or immunized libraries, or they may be created by synthetic means. Isolation of specific antibodies from a library can be mediated by panning a phage-display antibody library against a specific antigen or complex mixture. Libraries can be obtained by panning a phage-display antibody library over several specific antigens of several origins. Phage-display antibody libraries can be screened by sequential selection against human antigens or fragments thereof, followed by panning of the phage-display antibody library against mouse antigens, or fragments of mouse antigens corresponding to fragments of the human antigen. These panning processes can be performed in either order. Alternatively, methods such as yeast display, bacterial display, and ribosome display can be applied to the selection of monoclonal recombinant antibodies.

[0209] Antibodies can be human single-domain antibodies or single-domain antibodies based on human sequences, where diversity is artificially created. Several different libraries of useful human single-domain antibodies are available.

[0210] Those skilled in the art will understand that antibodies can be usefully labeled for specific applications. In some embodiments of the present invention, pH-dependent antigen-binding proteins include detection labels.

[0211] In further embodiments, the detection label is selected from the group consisting of colorimetric, fluorescent, luminescent, magnetic, and paramagnetic labels.

[0212] In other embodiments, the detection label is biotin.

[0213] In yet another embodiment, the detection label is gold nanoparticles.

[0214] composition A sixth aspect of the present invention relates to a composition comprising a pH-dependent antigen-binding protein described herein and a pharmaceutically acceptable excipient.

[0215] Medical use and treatment methods A seventh aspect of the present invention relates to a pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL) for use in a method of treatment for patients requiring treatment for cancer, autoimmune diseases, metabolic diseases, or hematological diseases, wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, or to the composition described herein.

[0216] In a preferred embodiment, the cancer is a solid tumor carcinoma.

[0217] In a more preferred embodiment, the pH-dependent antigen-binding protein is a pH-dependent antigen-binding protein described herein.

[0218] An eighth aspect of the present invention relates to a method for treating cancer, autoimmune diseases, metabolic diseases, or hematological diseases, comprising administering to a patient in need of treatment for cancer, autoimmune diseases, metabolic diseases, or hematological diseases a pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, or a composition described herein.

[0219] A ninth aspect of the present invention relates to the use of a pH-dependent antigen-binding protein, or a composition described herein, in the manufacture of a pharmaceutical for the treatment of cancer, autoimmune diseases, metabolic diseases, or hematological diseases, wherein the pH-dependent antigen-binding protein, or a composition described herein, has a pH-dependent antigen-binding protein having a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH.

[0220] in vitro method A tenth aspect of the present invention relates to the use of the pH-dependent antigen-binding protein, or the composition described herein, in an in vitro method for the detection and / or diagnosis of cancer, wherein the pH-dependent antigen-binding protein, or the composition described herein, has a pH-dependent antigen-binding protein, or a composition described herein, having a pH-dependent antigen-binding protein, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH.

[0221] pH-sensitive antigen-binding properties can vary; typically, they bind to antigens at physiological pH while releasing them at lower pH levels (e.g., within endosomes), enabling the recycling of antibodies into the antibody cycle. This approach may be useful in different applications requiring the clearance of circulating proteins (e.g., toxins, cytokines). Conversely, pH-sensitive antigen-binding properties can enable binding at low pH and release of antigens in high pH environments. This property may be useful in targeted therapies in acidic environments such as the tumor microenvironment.

[0222] In preferred embodiments, plasma recycling of pH-dependent antigen-binding proteins is improved compared to antigen-binding proteins including parental FWR1, FWR2, FWR2', FWR3, and FWR4.

[0223] In other embodiments, pH-dependent antigen binding improves the clearance of the antigen from plasma compared to antigen-binding proteins including parental FWR1, FWR2, FWR2', FWR3, and FWR4.

[0224] In yet another embodiment, pH-dependent antigen binding improves the release of the antigen from the endosome compared to antigen-binding proteins including parental FWR1, FWR2, FWR2', FWR3, and FWR4.

[0225] In further embodiments, intracellular uptake of pH-dependent antigen-binding proteins is improved in an acidic microenvironment compared to antigen-binding proteins including parental FWR1, FWR2, FWR2', FWR3, and FWR4.

[0226] An eleventh aspect of the present invention is: - To provide the pH-dependent antigen-binding protein described herein, - The pH-dependent antigen-binding protein is brought into contact with the antigen to which it binds, - Includes detecting contact between the pH-dependent antigen-binding protein and the antigen, This relates to an in vitro antigen detection method for detecting the antigen.

[0227] A twelfth aspect of the present invention is: - To provide the pH-dependent antigen-binding protein described herein, - The pH-dependent antigen-binding protein is brought into contact with the antigen it binds to in a complex mixture. - Separating the pH-dependent antigen-binding protein / antigen complex from the complex mixture, This relates to an in vitro antigen purification method for purifying the antigen.

[0228] In preferred embodiments of the antigen-binding proteins described herein, VL and VH each comprise three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are referred to as FWR2', and at least one of FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, and the pH-dependent antigen-binding proteins do not comprise the heavy chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 4, 5, and 6, respectively.

[0229] In a more preferred embodiment, the three complementarity-determining regions CDR1, CDR2, and CDR3, the four framework regions FWR1, FWR2, FWR3, and FWR4, and FWR2' of the antigen-binding protein described herein are as described for the pH-dependent antigen-binding protein described herein.

[0230] In some embodiments of the methods described herein, one or more mutations are introduced at residue positions selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL36, VL38, VL43, VL44, VL46, VL49, VL85, VL87, VL98, and VL100, according to Kabat numbering.

[0231] In other embodiments of the method described herein, one or more mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH L45, VH W47, VH V89, VH Y91, VH W103, VH Q105, VL Y32, VL Y36, VL Q38, VL S43, VL P44, VL T46, VL Y49, VL D85, VL Y87, VL F98, and VL G100, according to the Kabat numbering of another amino acid.

[0232] In yet another embodiment of the method described herein, one or more mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH L45, VH W47, VH V89, VH Y91, VH W103, VH Q105, VL Y36, VL Q38, VL S43, VL P44, VL T46, VL Y49, VL D85, VL Y87, VL F98, and VL G100, according to the Kabat numbering of another amino acid.

[0233] In other embodiments of the method described herein, one or more mutations are introduced at residue positions from the group consisting of VH39, VH44, VH89, VH105, VL38, VL43, VL85 and VL100, according to the Kabat numbering of another amino acid.

[0234] In preferred embodiments of the methods, pH-dependent antigen-binding proteins, antigen-binding proteins, compositions, or uses described herein, the binding affinity that is higher at acidic pH than neutral pH, or lower at acidic pH than neutral pH, corresponds to a lower KD or a higher KD, respectively.

[0235] In some embodiments of the methods described herein, pH-dependent antigen-binding proteins, antigen-binding proteins, compositions, or uses, the binding affinity that is higher at acidic pH than neutral pH, or lower at acidic pH than neutral pH, is measured as the ratio of DELFIA signals at acidic pH and neutral pH.

[0236] In preferred embodiments of the methods described herein, pH-dependent antigen-binding proteins, antigen-binding proteins, compositions, or uses, the binding affinity that is higher at an acidic pH than neutral pH, or lower at an acidic pH than neutral pH, is at least 1.25 times, for example, at least 1.3 times, for example, at least 1.5 times, for example, at least 1.6 times, for example, at least 1.7 times, for example, at least 1.8 times, at least 1.9 times, for example, at least 2 times, for example, at least 2.2 times, for example, at least 2.5 times, for example, at least 5 times, for example, at least 6 times, for example, at least 10 times, for example, at least 10.5 times. [Examples]

[0237] Example 1: Profiling of cross-reactivity and pH-dependent binding of light chain shuffle antibodies Materials and methods SuperTEV expression SuperTEV endoprotease was expressed using the pET39-mCherry-SuperTEV expression vector in BL21(DE3) cells (New England Biolabs, NEB-C2527H), as previously described (Reference 11).

[0238] Antibody format expression Full-length human IgG was generated using mammalian expression, as previously reported (Reference 7). The Fab was generated in two batches, the first from human fetal kidney cells and the second from Chinese hamster ovary cells (Reference 8).

[0239] To generate scFv for crystallographic analysis, expression was increased using a Tunair® shaking flask, as previously described.18 C-terminal TEV-His tagged scFv was generated in BL21 (DE3) cells (New England Biolabs, NEB-C2527H) and purified by nickel affinity purification. After nickel affinity purification, scFv was buffer-exchanged using a PD-10 column (Merck, GE17-0851-01) in 20 mM Tris, 50 mM NaCl, 5 mM EDTA, pH 8.0 buffer and concentrated to 10 mg / mL using a 10 kDa MWCO membrane (Fisher Scientific, 10781543). The C-terminal tag was removed by incubation with superTEV endoprotease overnight at 4°C using superTEV:scFv in a molar ratio of 1:20.

[0240] After the C-terminal tag was removed, monomeric scFv was purified by size exclusion chromatography using an NGC Quest® 10 Plus chromatography system and a Superdex 75 10 / 60 HiLoad column (Cytiva, 28989333) with 5 mM Tris, 20 mM NaCl, pH 8.0 buffer as the eluent at 4°C. The concentration of scFv was estimated based on the predicted A280 absorbance of 1 mg / mL of protein using the Expasy ProtParam tool.

[0241] Octet screening for pH-sensitive antibodies The affinity and pH sensitivity of antibody Fab fragments were characterized using the Octet RED96 system (ForteBio). Before starting the assay, all reagents were transferred to a black 96-well plate (Greiner Bio-One, 655209), and the assay was performed at 24°C with a shaking rate of 1000 rpm. The streptavidin biosensor tip (Sartorius, 18-5136) was equilibrated in kinetic buffer (Sartorius, 18-1105) prepared in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4.2H2O, 1.4 mM KH2PO4, pH 7.4) in the dark for 10 minutes before starting the assay. The long-chain α-neurotoxin was biotinylated as previously described (Reference 8) and diluted to a concentration of 0.4 μg / mL in kinetic buffer. Equilibrated biosensors were immersed in wells of biotinylated long-chain α-neurotoxin for 120 seconds to allow sufficient loading, and biosensors without toxin coating were used as a reference. The biosensors were then transferred to HEPES-MES (10 mM HEPES, 50 mM MES, NaCl 0.05% P20, pH 7.4) running buffer, equilibrated for 30 seconds, and then primed for 3 cycles in regeneration buffer of 10 mM glycine, 2 M NaCl, pH 2.0. Each cycle consisted of 10 seconds of regeneration and 10 seconds of neutralization in kinetic buffer. After transferring the primed biosensors to running buffer for 60 seconds to obtain a stable baseline, they were transferred to Fab-containing wells for 120 seconds. Antibody Fab fragments were prepared in running buffer in a 3x titration series at concentrations ranging from 10x lower to 10x higher than the expected KD. Dissociation of bound Fab in running buffer was performed for 1000 seconds to ensure sufficient dissociation for high-affinity interactions. For affinity and dissociation rates measured at acidic pH, the running buffer was adjusted to pH 5.5. Data were processed using Octet evaluation software (version 12.2.2.4). All curves were fitted using a 1:1 coupling model with global fit, after subtracting the reference from the coupling curve. KD values ​​were determined either by the product of kinetic rates (kd / ka) or by steady-state analysis.The flow cell was regenerated by a regeneration solution in a 5 x 10-second cycle, followed by neutralization.

[0242] result To determine whether cross-reactivity and pH sensitivity could be linked, the inventors screened a panel of antibodies for pH-sensitive binding to three long-chain α-neurotoxins: N. kaouthia (α-cbtx), D. polylepis (α-eptx), and B. multicinctus (α-bgtx) using biolayer interferometry (BLI). Parental antibodies were discovered from a naive antibody phage display library against α-cbtx and affinity-matured by light chain shuffling using α-cbtx and α-eptx antigens to improve cross-reactivity.1 A total of seven affinity-matured light chain shuffle clones were selected for further characterization based on their binding signals to α-cbtx and α-eptx (Table 2) and the diversity of their light chain CDR sequences. Clones were classified into two light germlines, IGVL3-23:2558_02_G09,2555_01_A01,2555_01_A04,2551_01_B11 and IGLV6-57:2554_01_D11,2554_01_E01,2551_01_A12, and the parental antibody. Clones from the same germline had similar CDR sequences, and all clones had the same CDRL3 and CDRL2 loop lengths. The LCDR1 loop showed the most variation in both length and sequence diversity between germlines (Figure 1A).

[0243] The antibody affinities to α-cbtx and α-eptx were consistent with previously reported SPR values. The values ​​ranged from 33.8 to 2.9 nM for α-cbtx, and the clones showed slightly higher affinity for α-eptx, ranging from 0.89 to 0.44 nM (Tables 1 and 2). To evaluate whether light chain shuffling would improve cross-reactivity, α-bgtx was included because it was not used in the discovery process and has low sequence identity with α-cbtx (58%). All clones bound to α-bgtx with considerably low affinity, and the KD values ​​ranged from 3 μM to 122 nM (Figure 1C, Tables 3 and 4). The effect of light chains on cross-reactivity was most pronounced in the light chain shuffled clones derived from the IGLV6-57 germline, which showed an order of magnitude higher binding affinity to α-bgtx compared to both the parental antibody and the clones derived from the IGLV3-23 germline (Figure 1B). This suggests that affinity-mature clones carrying light chains derived from the IGLV6-57 germline exhibit improved interaction with the conserved region of the long-chain α-neurotoxin, possibly the conserved finger II region, which is central to the inhibition of nAChRs.

[0244] Antibodies classified as pH-dependent typically exhibit at least a four-fold difference in affinity or dissociation rate between pH 7.4 and pH < 6.0.1,2 Based on this, one antibody derived from the IGVL3-23 germline, 2555_01_A01, showed pH-dependent binding to all three long-chain α-neurotoxins (Figures 1C-E). 2555_01_A01 was the most pH-sensitive antibody for α-cbtx, with an average 19-fold difference in dissociation rate between pH 7.4 and pH 5.5, followed by α-bgtx (11.7-fold) and α-eptx (7.90-fold). In contrast, the parental antibody and the remaining affinity-mature clones exhibited moderate pH sensitivity to each of the long-chain α-neurotoxins, with pH sensitivity ranging from 1.82 to 4.94-fold. The antibody with the lowest pH dependence was derived from the IGVL6-57 germline. While it also exhibited higher cross-reactivity and a pH-dependent binding ratio difference of 1.82–3.35 between pH 5.5 and pH 7.4, the 2554_01_D11 antibody, which showed the highest affinity for α-bgtx and broadly neutralized the neurotoxic agent, was the most clearly highlighted. The most pH-sensitive antibody, 2555_01_A01, was also able to neutralize long-chain α-neurotoxins in preliminary incubation in in vivo experiments (data not shown).

[0245] To investigate the basis of the broad cross-reactivity of this lineage of antibodies and the role of the light chain in promoting pH-dependent binding, 2555_01_A01 was selected for structural characterization by X-ray crystallography.

[0246] JPEG2026519648000002.jpg161159

[0247] JPEG2026519648000003.jpg57159

[0248] JPEG2026519648000004.jpg76159

[0249] JPEG2026519648000005.jpg115159

[0250] conclusion In this example, the inventors analyzed how cross-reactive antibodies against long-chain α-neurotoxins can accept both antibody function and pH-dependent binding properties. The inventors report on the neutralization mechanism of a lineage of cross-reactive light-chain shuffled human monoclonal antibodies targeting long-chain α-neurotoxins, discovered by phage display technology. Insights into the pH-dependent binding mechanism of one neutralizing antibody, 2555_01_A01, are also reported, which exhibits both pH-dependent binding and cross-reactivity, and thus demonstrate that these two therapeutically appropriate properties are not mutually exclusive.

[0251] In addition to the potential for development, the inventors propose another parameter that may influence the ability of these antibodies to neutralize long-chain α-neurotoxins: namely, the dissociation rate of the antibodies, as opposed to overall affinity. This hypothesis is supported by a comparison of the dissociation rates of three antibodies against α-cbtx. In contrast to the slow dissociation rate of 2554_01_D11 from α-cbtx (2.18 × 10⁻⁴ / s), both antibodies 2555_01_A01 and 2552_02_B02 have faster dissociation rates of 4.86 × 10⁻⁴ / s, 6.26 × 10⁻⁴ / s (2555_01_A01, two measurements), and 5.5 × 10⁻⁴ / s (2552_02_B02, one measurement), respectively (Table 1). These faster dissociation rates allow α-cbtx to be released from the α-cbtx-antibody complex over time and accumulate at the neuromuscular junction, even when affinity is higher, as determined for 2552_02_B02. Accumulation of α-cbtx at the neuromuscular junction may lead to strong binding to nAChR, preventing dissociation and causing complete neuromuscular blockade, resulting in the observed delayed lethality. These observations and this hypothesis highlight the appropriateness of extending the observation period of monoclonal IgG during in vivo evaluation to differentiate between antibody candidates.

[0252] Example 2: Overall Structure Materials and methods The antibody format was expressed and prepared as described in Example 1.

[0253] Crystallography: Sample preparation, data acquisition, and model construction Lyophilized long-chain α-neurotoxin α-cbtx (Latoxan) was resuspended in Tris-NaCl (5mM Tris, 20mM NaCl, pH 8.0) buffer at a concentration of 5–10 mg / mL. Freshly prepared scFv was added in a molar ratio of scFv:α-cbtx of 1:3 and incubated overnight at 4°C to conjugate. The complex was purified using an NGC Quest® 10 Plus chromatography system and a Superdex 75 10 / 300GL column (Cytiva) with Tris-NaCl (5mM Tris, 20mM NaCl, pH 8.0) buffer as the eluent at 4°C. The scFv-α-cbtx complex was concentrated to 14 mg / mL using a 3.0 kDa MWCO ultracentrifuge unit (Fisher Scientific) and then plated.

[0254] Crystallization was performed at 21°C using the sitting drop vapor diffusion method. Drops were set up on a SWISSCI MRC 2-well crystallization plate (JENA) in a 96-well drop format with a total volume of 0.3 μL, using a 1:1 or 1:2 molar ratio of protein to reservoir. The wells were sealed with crystal-clear tape and equilibrated at 21°C with 50 μL of reservoir solution. Under the 1:2 condition (0.1 M Bis-Tris, 0.2 M ammonium sulfate, 25% PEG3350, pH 6.5), small crystals appeared in less than one week. Optimization screening was performed around this condition with 1.2 μL drops and 100 μL reservoirs. Crystals formed after two weeks and were collected using onboard CryoLoops (Hampton Research). Freeze-protection was performed by adding glycerol to adjacent drops without crystals until the final concentration reached 20%. Several crystals were fished out using a 300 μm loop. After equilibrating the loop edge by contacting it with the freezing solution for approximately 5 seconds, the crystals were rapidly frozen with liquid nitrogen and then transported to the beamline for remote data acquisition.

[0255] Diffraction data was collected at the P13 beamline (PETRAIII, EMBL, Germany) (PMID: 28009574). This beamline is equipped with a 6M PILATUS detector. Data was collected at 100K, with a full 360° sweep, an oscillation of 0.1°, an exposure time of 0.050 seconds, and 12700 eV. The complete dataset was processed from 360° (3600 images) with an X-ray beam reduced to 50% intensity.

[0256] The structure of 2555_01_A01 bound to α-cbtx was determined by molecular substitution using Phaser-MR (PMID:17164524), using the AlphaFold2 (PMID:34265844) model of the expected scFv structure and the toxin PDB ID 4AEA as search models. Model construction and refinement were performed using phenix.refine (PMID:20124702) and Coot (PMID:20383002). Data acquisition and refinement statistics are summarized in Table 1. Molecular graphics were displayed using the PyMOL molecular graphics system (version 2.2r7pre, Schroedinger, LLC).

[0257] result First, to understand the structural basis of the broad recognition of long-chain α-neurotoxins by 2555_01_A01 and related clones, we determined the crystal structure of the 2555_01_A01 antibody in scFv format bound to α-cbtx at a resolution of 1.6 Å (Figure 2A, Table 5). Two scFv molecules and two α-cbtx molecules existed in an asymmetric unit and exhibited P212121 space group symmetry. Each α-cbtx molecule was bound to two scFv molecules, forming an interface with the heavy chain from one scFv and the light chain from the other in a 1:2 (α-cbtx:scFv) stoichiometry. During the preparation of the complex by gel filtration, a 1:1 stoichiometry was clearly observed to be preferable (Figure 2B), indicating that one of these interfaces was the crystallographically driven interface. To assess whether either the heavy-chain or light-chain interface is a crystallographically driven interface, the complex formation significance scores (CSS) of the heavy-chain and light-chain-α-cbtx interfaces were compared using PISA. The CSS score for light-chain α-cbtx was 0, meaning this interface is not important for complex formation, while the heavy-chain interface had a score of 0.41. Therefore, the α-cbtx molecule forming the interface with the heavy chain was treated as the major molecule involved in its scFv interaction and selected to characterize this interaction.

[0258] JPEG2026519648000006.jpg170159

[0259] conclusion The inventors were able to observe the paratope-epitope interaction between 2555_01_A01 scFv and α-cbtx at high resolution, revealing the recognition of a highly conserved epitope by the antibody CDRH3 loop. At a length of 24 residues, CDRH3 was stabilized by an intramolecular disulfide bridge and extended between fingers I and II of α-cbtx, recognizing the conformational epitope in the three-finger neurotoxin fold. The complex exhibited a dense interface with a total embedding surface area of ​​952.4 Å2, 864 Å2 due to the contribution of CDRH3, and 87.8 Å2 due to the CDRL3 loop.

[0260] Example 3: Heavy chain CDR3 contains a determinant of cross-reactivity. Materials and methods The antibody format was expressed and prepared as described in Example 1.

[0261] The crystal structure analysis was performed as described in Example 2 above.

[0262] result Long-chain α-neurotoxins induce paralysis by inhibiting nicotinic acetylcholine receptor (nAChR)-acetylcholine interactions. Loop C, a loop of nAChR, is a crucial response element required for neurotransmitter binding and is targeted by R33 and R36 residues at the tip of finger II on the long-chain α-neurotoxin. Both arginine residues bind to aromatic residues on loop C and coordinate with the acetylcholine molecule. Structural alignment of electric ray AChR and the bound 2555_01_A01 structure revealed that the aromatic Y190nAChR and Y198nAChR residues on loop C can be superimposed on Y99HC and Y100eHC on CDRH3 of 2555_01_A01, which bind to R33cbtx and R36cbtx on α-cbtx (Figure 3A). The CDRH3 loop forms numerous hydrophobic interactions and hydrogen bonds with the α-cbtx backbone, as well as 11 hydrogen bonds and salt bridges to the R33cbtx, R36cbtx, and D26cbtx residues (Figure 3B). The hydroxyl group of Y100eHC on CDRH3 forms a hydrogen bond to D26cbtx and is oriented between the R36cbtx and R33cbtx residues, forming a cation-π interaction with both R33cbtx and R36cbtx. The guanidinium group of R33cbtx is involved in a further cation-π interaction with Y99HC and a salt bridge to D95HC on CDRH3, forming the core of the interaction. The interaction with R36cbtx is supported by two hydrogen bonds from the S100cHC residue on CDRH3. Since R36 is valine in α-bgtx, the reduced ability of α-bgtx to form hydrogen bonds and cation-π interactions with Y100eHC and S100cHC may explain the low affinity of all antibodies for α-bgtx.

[0263] Looking at the C-terminal side of α-cbtx (Figure 3C), the contribution of the light chain to the interaction becomes clear. D95aLC on CDRL3 forms a salt bridge to R70cbtx and a hydrogen bond with Y100fHC, enhancing the cation-π interaction between Y100fHC and R70cbtx. The 95aLC position on CDRL3 is variant in all affinity-matured light chains, which may explain the increased affinity of 2555_01_A01 and related antibodies to α-cbtx compared to the parental antibody with glycine at this position. Overall, the CDRH3 loop of 2555_01_A01 and related clones achieves cross-reactivity by binding to long-chain α-neurotoxins via receptor mimicry, and consequently neutralizes the long-chain α-neurotoxins by inhibiting residues crucial for nAChR inhibition. These results explain the cross-reactivity of 2555_01_A01 and related clones via CDRH3 interaction, but do not explain the broad pH-dependent binding of 2555_01_A01, since the paratope of 2555_01_A01 is shared with clones that bind in a pH-independent manner. Therefore, the pH sensitivity of 2555_01_A01 must lie outside the paratope-epitope interface.

[0264] conclusion By determining the binding structure of 2555_01_A01 to α-cbtx, the inventors found that the antibody utilizes the conserved functional constraints in long-chain α-neurotoxins necessary for inhibiting nAChRs by mimicking the conserved interactions that long-chain α-neurotoxins form with nAChRs via receptor mimicry. Therefore, the inventors showed that specificity is primarily determined by the paratope that interacts with the neurotoxin to mimic the acetylcholine receptor-neurotoxin interface. This resulted in broad reactivity to multiple antigens through binding via the antibody's CDRH3 loop. This neutralization mechanism may be useful for optimizing this antibody and other antibodies for improved cross-reactivity, for example, through structure-based manipulations that present residues on the antibody paratope to further mimic nAChRs and form interactions with other conserved residues on the long-chain α-neurotoxin that are important for its function.

[0265] Example 4: Broad pH sensitivity determinants located at the light chain interface Materials and methods The antibody format was expressed and prepared as described in Example 1.

[0266] The crystal structure analysis was performed as described in Example 2 above.

[0267] result Since the light chain is a variable component between each antibody, we investigated the role of the light chain in the observed pH-dependent binding of 2555_01_A01. Crystallographic optimization screening allowed us to create crystals and determine the structures of 2555_01_A01 bound to α-cbtx at different pH levels (Table 5). The space group and asymmetric units were the same across all three structures, and there were no obvious differences in crystal packing, allowing us to identify potential determinants of pH-dependent binding. First, we focused on the charged residues in the 2555_01_A01 light chain and identified two charged residues, G95aLCD and S95bLCH, introduced into the CDRL3 loop after light chain shuffling. By analyzing their roles in bonding, the inventors observed that H95bLC forms a hydrogen bond with S95LC located near the apex of the β-hairpin loop of CDRL3, thereby positioning D95aLC to interact with R70cbtx on α-cbtx (Figure 4A). By comparing the stereochemistry of H95bLC in the structures determined at each pH, ​​it was found that H95bLC is pH-responsive, showing changes in rotational isomer positions and hydrogen bond network at pH 4.5 (Figure 4B), where the indole ring of H95bLC switches hydrogen bonding from the S95LC backbone to the D95aLC backbone, destabilizing the interaction between D95aLC and R70cbtx and effectively reducing its affinity for α-cbtx. The effects of H95bLC and D95aLC (which may be protonated) on interaction with α-cbtx were tested using double mutants D95aLCH and H95bLCE (2555_01_A01-HE), which were residues observed at equivalent positions in the non-pH-dependent 2554_01_D11 antibody. This resulted in a 2-3 fold reduction in pH-dependent binding to α-cbtx (Figure 4C), explaining the increased level of pH-dependent binding of 2555_01_A01 to α-cbtx compared to the long-chain α-neurotoxins α-bgtx and α-eptx. Unlike α-bgtx and α-eptx, the long-chain α-neurotoxins are characterized by P70, in contrast to R70 found in α-cbtx, and cannot form salt crosslinks with D95aLC, thus failing to reduce the affinity of the 2555_01_A01 antibody to these long-chain α-neurotoxins via the CDRL3 loop.However, this does not fully explain the pH-dependent binding of 2555_01_A01, and 2555_01_A01-HE was still 10 times more pH-sensitive than the non-pH-dependent 2554_01_D11 antibody (Figure 4C). 2555_01_A01 was also more pH-sensitive than antibodies containing both D95ALC and H95bLC residues (Figure 1A). Therefore, H95bLC and D95aLC residues alone cannot explain the broad pH-dependent binding behavior of 2555_01_A01.

[0268] The 2555_01_A01 antibody contains another histidine residue, H34LC, located in the CDRL1 loop, which is conserved across all antibodies (Figure 1A).

[0269] The inventors hypothesized that, despite being conserved, the amino acid residue environment surrounding this histidine is unique to 2555_01_A01 and may contribute to its broad pH-dependent binding.

[0270] To investigate this, the inventors compared the structures of 2555_01_A01 bound to α-cbtx at pH 5.5 and pH 4.5, where the resolution limits and statistics are nearly identical, to identify differences in the environment of H34LC. Analysis of the structure of 2555_01_A01 bound to α-cbtx at pH 5.5 revealed that H34LC, when exposed to the solvent, forms side-chain-mediated hydrogen bonds with water molecules and the main chain of S50LC on CDRL2. H34LC is located at the interface between the heavy and light chains, packed directly beneath Y100lHC on CDRH3, where it forms a π-π interaction (Figure 4D). At pH 5.5, Y100lHC occupies a pocket at the upper interface of H34LC, with the phenol ring of Y100lHC positioned toward the LCDR2 loop and the main chain of Y100lHC toward the CDRH3 loop. The Y100lHC backbone forms a hydrogen bond with the carboxyl group of the D95HC side chain in CDRH3, stabilizing the interaction between D95HC on α-cbtx and R33cbtx (Figure 4D). At pH 4.5, the Y100lHC side chain forms a new hydrogen bond with S50LC, which then forms weaker intramolecular and intermolecular hydrogen bonds with itself and H34LC, accepting the new hydrogen bond to Y100lHC (Figure 4E). Errors related to these hydrogen bonds were checked on a web server (http: / / cluster.physics.iisc.ernet.in / dpi / ), confirming that the difference in hydrogen bonds between pH 5.5 and pH 4.5 was greater than the associated error, indicating a change in length between the two pH values. Next, the difference in electron density of these residues was compared by subtracting the X-ray crystal diffraction patterns between the pH 5.5 and pH 4.5 datasets. This allows for the detection of changes in atomic coordinate positions with higher sensitivity than removing model bias using a refined model. This reveals the positive electron density on the upper plane of the phenol ring of Y100lHC, which means that the phenol ring moved downward toward H34LC at pH 4.5 due to a more favorable cation-π interaction with the positively charged H34LC (Figure 4F).Positive electron density was observed along the main chain of Y100lHC and above the carboxyl group of the side chain of D95HC, indicating that the change in the position of Y100lHC led to the interaction between D95HC and R33cbtx. The electron density of S50 also changed, which was expected given the changes in the hydrogen bond network already observed at this residue. Differences in other electron density maps were mainly observed at the cysteine ​​bridge, which is likely due to radiation damage during data acquisition. In summary, these findings indicate that S50, Y100lHC, and D95HC all respond to the pH change between pH 5.5 and pH 4.5 as a result of protonation of H34, which shifts the position of the Y100lHC residue, and that Y100lHC influences the interaction between D95HC and R33cbtx by weakening the hydrogen bond that stabilizes D95HC. Since R33cbtx is highly conserved, this explains why pH-dependent binding is observed for all long-chain α-neurotoxins.

[0271] In particular, all non-pH-dependent antibodies contain D95HC, Y100lHC, and H34LC residues, but they differ at the S50LC position, containing either D, E, or H (Figure 1A). S50LC does not form a hydrogen bond with Y100lHC at pH 5.5 (or pH 6.0, data not shown), but D, E, and H residues have hydrogen-bonding ability and have longer side chains than S50LC, which allows Y100lHC to be stabilized at the chain interface by forming a more stable hydrogen bond than S50 at neutral pH. The goal is to prevent the influence of H34LC protonation on the position and binding of Y100lHC.

[0272] conclusion As illustrated in previous examples, in contrast to the specificity primarily determined by paratopes, we have shown that the determinants of pH-dependent binding are located away from the toxin interface. The pH-dependent antibody is functional and can neutralize long-chain alpha-neurotoxins in vivo.

[0273] Specifically, in relation to the origin of the pH-dependent antigen-binding properties observed in 2555_01_A01, the inventors found that the light chain plays a role in giving the antibody these properties, independently of the antibody paratope.

[0274] The inventors describe a model in which, in contrast to the protonation of H34LC itself, the hydrogen bonding network surrounding H34LC allows H34LC to influence binding by modulating the positions of residues on CDRH3 that are important for interaction with conserved residues on long-chain α-neurotoxins.

[0275] A complete explanation of the fundamentals of paratope-independent pH-dependent antigen-binding mechanisms is useful for engineering purposes that currently rely heavily on the single use of histidine residues. Furthermore, in antibodies, many amino acid residues exist outside the paratope (i.e., the framework region and any CDRs that do not interact with the antigen), and these constitute a larger sequence space for manipulating pH-dependent binding properties than those in the paratope. Therefore, manipulating pH-dependent antigen-binding properties outside the antibody paratope is advantageous at several antibody-antigen interfaces where manipulating the paratope can adversely affect the specificity and function of the antibody.

[0276] In this study, the inventors found that the paratope-independent mechanism is robust in promoting pH-dependent binding to a series of long-chain α-neurotoxins, consistently yielding nearly an order of magnitude difference in affinity between pH 5.5 and pH 7.4. In particular, the difference in the pH-dependent binding ratio allows for the release of long-chain α-neurotoxins within the duration of the antibody recycling pathway.

[0277] The inventors propose that the chain interface environment is the stage for this paratope-independent pH-dependent binding mechanism of the 2555_01_A01 antibody, specifically conferred by histidine residues within the antibody's CDRL1 loop. In a similar setting, Kolmar et al. (Reference 9) identified key histidine residues in the CDRL1 and CDRL3 loops that confer paratope-independent pH-dependent antigen-binding properties to five carcinoembryonic antigen-associated cell adhesion molecules. In their approach, light chains were selected because they were excluded from binding interactions to allow pairing with two different heavy chains in a bispecific antibody format without affecting their binding, a technique called the "common light chain technique." They identified histidine mutations that confer pH-dependent antigen-binding properties by common light chains accumulated at anchoring positions in the CDR1 and CDR3 loops, at sites comparable to the histidine residues located in the 2555_01_A01 light chain. The light chain used in the bispecific antibody developed by Kolmar et al. does not participate in interaction with the antigen; therefore, the introduction of pH-dependent antigen-binding properties by the common light chain was also paratope-independent, as in 2555_01_A01. This observation suggests that a similar pH-dependent binding mechanism was introduced into the antibody paratope by both sets of antibody light chains, and this was then conferred to the heavy chain, which has significantly different target specificities.

[0278] However, in the study by Kolmar et al., they did not explain the mechanism of light chain involvement in conferring pH-dependent binding. In this study, we observed structural changes in residues located at the chain interface surrounding the histidine residue located in the CDRL1 loop of 2555_01_A01 between structures determined at pH 5.5 and pH 4.5. The authors hypothesized that these residues are important in promoting the pH-dependent antigen-binding properties of 2555_01_A01 by influencing the loop structure and paratope of CDRH3.

[0279] Surprisingly, however, this histidine residue was conserved across all antibodies included in this study. While not bound by theory, we hypothesize that the hydrogen bond network at 2555_01_A01 allows this antibody to bind in a pH-dependent manner by enabling histidine to influence the loop structure and binding of the antibody's CDRH3.

[0280] This invention provides a paratope-independent approach to conferring pH-dependent antigen-binding properties. Antibodies capable of releasing antigens for lysosomal degradation during in vivo antibody recycling are characterized by a dissociation rate of 10⁻² to 10⁻³ / s at pH ≤ 6.0 and a slow dissociation rate at neutral pH, typically ≤ 10⁻⁴ / s. Here, 2555_01_A01 was verified to be able to release α-cbtx in a human FcRn cell antibody recycling assay during the recycling pathway because the bound 2555_01_A01 antibody was not detected after recycling (data not shown). This is in contrast to the non-pH-dependent 2554_01_D11 antibody, which remained complexed with α-cbtx during recycling, indicating that 2555_01_A01 may have kinetics that can promote antibody-mediated lysosomal degradation of α-cbtx. In this assay, a multiplier difference in dissociation rate between pH 7.4 and pH 5.5 was sufficient for 2555_01_A01 to release α-cbtx. However, antibodies with slower dissociation rates than 2555_01_A01 required a larger multiplier difference, potentially necessitating the introduction of a pH-dependent determinant at the paratope-epitope interface. Nevertheless, we demonstrated that a pH-dependent antigen-binding mechanism, driven independently of the antibody paratope, was accepted by cross-neutralizing antibodies against long-chain α-neurotoxins. pH-dependent antigen-binding via this mechanism does not adversely affect any parameters important to antibody function and therefore may be a useful approach for manipulating this property in antibodies without impairing antibody specificity.

[0281] Overall, the structures presented herein in previous examples outline the broad cross-reactivity and neutralization mechanisms of antibodies discovered using phage display from the naive antibody repertoire, leading to manipulations for improving neutralizing efficacy. For example, antibodies may be manipulated to further mimic the conserved interaction between function-critical nAChRs and long-chain α-neurotoxins. Finally, evidence of an allosteric pH-sensitive binding mechanism is presented for the broad pH-dependent binding of 2555_01_A01, which is conferred by the antibody light chain and allows for the linking of pH sensitivity with cross-reactivity.

[0282] Example 5: Framework Domain - Library Design and Verification Materials and methods Fab and IgG expression Both antibody formats were generated using previously described mammalian expression (Reference 10). Briefly, the VH and VL domains were subcloned from the scFv-pSANG10-3F vector into the pINT12 vector for Fab expression and the pINT3 vector for IgG expression. The individual VH and VL domains were PCR amplified using pSang10_pelB (CGCTGCCCAGCCGGCCATGG, SEQ ID NO: 48) and HLINK3_R (CTGAACCGCCTCCACCACTCGA, SEQ ID NO: 49) primers for the VH domain, and LLINK2_F (CTCTGGCGGTGGCGCTAGC, SEQ ID NO: 50) and 2097_R (GATGGTGATGATGATGTGCGGATGCG, SEQ ID NO: 51) primers for the VL domain. Subsequently, VH was cleaved using NcoI and XhoI endonucleases, and VL was cleaved with NheI and NotI. Each domain was incorporated into either a pINT3 or pINT12 vector using four-component ligation with T4 DNA ligase (Roche, 10481220001). This vector contained the respective heavy chain constant domains of Fab and IgG, in addition to a stuffer region containing CL and CMV promoters, which are also cleaved at NcoI and NotI. The resulting Fab and IgG formats were generated using transient mammalian expression with Expi293® cells along with ExpiFectamine® 293 (ThermoFisher, A14525), according to the manufacturer's guidelines. The cells were harvested, and the resulting supernatant containing IgG was purified using Protein A resin (Neo Biotech, NB-45-00036-100), and Fab was purified using anti-CH1 resin (Thermo Scientific, 194320010). The purified antibodies were then desalted in PBS and rapidly frozen for long-term storage.

[0283] SPR Antibody affinity at variable pH was measured by immobilizing a long neurotoxin on a CM5 dextran tip by amine coupling, as previously described (Reference 7), and flowing the antibody Fab fragment in buffered HEPES-MES (10 mM HEPES, 50 mM MES-NaCl, 0.05% P20, pH 7.4) at either pH 7.4 or pH 5.1. Double background subtraction was performed using a protein-free reference flow cell and buffer-only injection. Affinity was determined as the product of koff / kon rates using a 1:1 model and global fit.

[0284] Creation of a phage display library using Golden Gate Assembly This framework library was derived from the codon-optimized 2554_01_D11 scFv sequence purchased from Eurofins in the pEX-K168 vector. The pSANG4 phagemide was sequence-validated and used to obtain the phagemide backbone. Individual insertion fragments containing the 2554_01_D11 framework and CDR region were PCR-amplified using either mutagenic or wild-type oligonucleotides (included to reduce the average number of mutations per clone). The phagemide backbone was amplified using pSANG4Myc_BbsI_For and pSANG4M13_BbsI_Rev primers. All primers (Table 6) were ordered from TAG Copenhagen and contained BpiI IIS type restriction sites for golden gate assembly. Overhang fidelity was checked with the NEB fidelity tool, and for mutagenic oligonucleotides, NNK codons were used to diversify the framework positions, one per oligo.

[0285] All PCRs were performed using Q5 Hot Start HF polymerase (NEB, M0494S) in a two-step PCR program, and unless otherwise specified, the following templates were used. Reagents were prepared in 25 μL volumes, consisting of 0.5 μM of each primer, a 1x dilution of Q5 high-fidelity master mix, 40 pg of DNA, and 25 μL of nuclease-free water (Thermo Scientific, 10977035). DNA was amplified using the following program: initial denaturation, 98°C, 30 sec, 30 cycles, 98°C, 10 sec, and 72°C, 15 sec amplification, final extension, 72°C, 5 min. Four PCRs were performed on each insertion fragment and purified using the Genejet PCR purification kit (Thermo Scientific, K0702). To prepare the phagemide skeleton, 1 ng of pSANG4 phagemide was used as a template, and 20 PCR cycles were performed with amplification times of 98°C for 10 seconds and 72°C for 110 seconds. The DNA template was removed from the pSANG4 skeleton amplicons by adding 0.5 μL of FastDigest DpnI (Thermo Scientific, FD1704) to each PCR and incubating at 37°C for 15 minutes. Finally, the phagemide amplicons were gel-purified using the GeneJET gel extraction kit (Thermo Scientific, K0692). The purity of the total amplicons was confirmed by agarose gel electrophoresis and quantified using Nanodrop (Thermo Scientific, NanoDropOne).

[0286] Libraries were constructed using a sequential Golden Gate assembly reaction. First, individual VH and VL domains were assembled from 2.4 μg of insertion fragments, which is equivalent to 2700 fmol (approximately 300 ng) each of the three mutagenic insertion fragments and their competing wild-type counterparts, added in equimolar ratios to reduce the average mutation frequency of each clone. To improve scalability, the amount of enzyme in each assembly was reduced below the manufacturer's guidelines, to 0.6 μL of BpiI (Fisher Scientific, FD1014), 90 U of T4 DNA ligase, and 2.8 μL of 10× T4 ligase buffer (NEB, M0202T) per 1000 fmol of DNA, and then to a final volume of 600 μL with nuclease-free water. The reaction mixture was left overnight in a 37°C water bath, and there was no apparent damage to the assembly efficiency (Figure 7). After extraction from a 1.2% agarose gel, 4 ng of each assembled domain (more than 100 times the theoretical diversity) was divided into four separate PCRs for amplification, cleaned using the GeneJET PCR purification kit (Thermo Scientific, K0702), and then scFv was assembled. This was done to scale up each domain while maintaining diversity. Using the same enzymes and incubation conditions as described for the VH and VL subassemblies, scFv was assembled using equimolar ratios of each VH and VL domain and extracted from a 1.2% agarose gel to obtain 21 ng (2 × 10¹⁰) of scFv molecules. Finally, 21 PCRs were performed on the extracted scFv to obtain a sufficient amount of DNA to construct a large library. Each PCR used 1 ng of extracted scFv and 0.1 μM of Insert_1_For and pSANG4_Myc_M13_Rev primers (Figure 7) as input. The number of cycles was reduced to 15 to lower the likelihood of off-site mutations, and the amplicons were purified using the GeneJET PCR purification kit to obtain 2.6 μg of DNA.

[0287] Finally, the golden gate integration of library DNA into the phagemid backbone was performed using 4.4 μg of phagemid backbone and 2.2 μg of scFv DNA in a 1:3 molar ratio of phagemid:scFv. To drive efficient assembly, the enzyme levels were increased to 1400 U of T4 DNA ligase, 12.5 μL of ligase buffer, and 2.8 μL of BpiI per 1000 fmol of DNA, and incubated overnight at 37°C with nuclease-free water to a final volume of 800 μL. The library was cleaned using the MiniElute PCR purification kit (Qiagen, 28006) and eluted using 40 μL of nuclease-free water preheated to 60°C to obtain a total of 3.2 μg of DNA. As previously described (reference 7), electroporation was performed a total of 20 times using a 2 μL library for each aliquot of electrocompetent TG1 cells (Lucigen, 60000-PQ763-F).

[0288] To confirm the presence of the inserted fragment and correct assembly, individual transformants were picked for colony PCR screening using phagemide-specific primers (pSANG_5th_For and pSANG_seq_Rev) and subjected to sequencing using gpII_Rev primers.

[0289] Creation of the scFv library and cloning into the pSANG4 phagemid vector. Golden Gate assembly of the scFv library was performed in two separate assemblies, as described in the section on the creation of phage display libraries by Golden Gate assembly. The first was by assembling individual light and heavy chains, and the second was by assembling full-length scFvs. To maintain diversity and create a large library, the amount of DNA was scaled up by PCR between each assembly step. When the assembled heavy and light chains were amplified by PCR, clear bands of the expected size were predominant around 400 bp in HC and around 500 bp in LC (Figure 7A). The initial scaling of the subsequently assembled scFvs showed some nonspecific products, which decreased as the amount of scFv DNA extracted with primers and gels was reduced (Figure 7B).

[0290] The assembled scFv was scaled up and electroporated into TG1 cells. Colony PCR was then performed on randomly selected transformants using pSANG4 skeleton-specific primers to assess the proportion of clones containing the scFv insertion fragment. The expected 2kb amplicon size was present in 56 / 57 clones (Figure 8A). Five colony PCR products were treated with BpiI enzyme to confirm that the insertion fragment originated from the golden gate assembly and not from the phagemid template DNA used to generate the phagemid skeleton. None of the transformants were cleaved, confirming that the scFv insertion fragment was from assembled DNA. The template phagemid scFv amplicon contained two BpiI restriction enzyme recognition sites, which resulted in multiple bands after incubation with BpiI (Figure 8B).

[0291] result To manipulate antibodies with conserved pH-dependent determinants, the inventors hypothesized that a structural change located at the center of the antibody's variable region within the light-heavy-chain framework interface could provide a robust means of reducing binding affinity at acidic pH, regardless of the antigen. To identify framework mutations capable of conferring such pH-sensitive antigen-binding properties, the inventors selected 2554_01_D11, a light-chain shuffled anti-long-chain α-neurotoxin IgG previously discovered using phage display selection (Reference 10). The affinity of 2554_01_D11 for α-cobra toxin was high at both pH 7.4 and pH 5.1, with dissociation constant (KD) values ​​of 2.3 nM and 10.4 nM, respectively (Figures 5A and B). The dissociation rate was two orders of magnitude lower than previously reported pH-sensitive clones discovered using in vitro display techniques (References 3, 9), and therefore these were selected for framework manipulation.

[0292] To create a binding framework interface library based on the single-chain variable fragment (scFv) format, the inventors selected residues located at homologous positions in the heavy and light chains because residues in this region introduce crucial structural changes for neutralization of antibodies created using in vivo discovery approaches (Figures 5C and D). The inventors also pre-selected residues located in the framework interface region that eliminate light chain pairing, such as those found in single-domain antibodies.

[0293] In total, eight residues were selected for randomization using NNK codons, which were four heavy chains: Q39, G44, V89, and Q105, and four light chains: Q38, S43, D85, and G100. The library size was estimated to have 4.5 × 10¹⁰ unique clonal diversity based on the number of individual transformants, approaching the theoretical size of the library. Colony PCR screening showed that 56 / 57 insertion fragments (98%) were full length (Figure 8). Sequencing of 27 colonies revealed that 19 (70%) had open reading frames, were unique, and contained the mutations observed at each position. A concise approach was used to maintain wild-type residues in each clone while generating the library using Golden Gate assembly. This was approached by spiking competing wild-type assembly fragments to reduce the mutation level of each clone. According to this approach, no clones contained mutations at all eight locations selected in the library design, and the number of mutations for each clone ranged from 2 to 7.

[0294] conclusion Therefore, the inventors verified the design and usefulness of a library created to introduce pH-dependent determinants and to be used as a basis for identifying pH-dependent antigen-binding scaffolds.

[0295] Example 6: Selection of framework mutations that provide universal pH-sensitive antigen-binding properties to scFv using phage display Materials and methods Phage display Phage display selection was performed in solution using 50 nM biotinylated α-cobra toxin. Phages were rescued from the library by seeding 200 mL of cells in 2TY glucose ampicillin (100 μg / mL) medium (2TYGA) at OD600=0.1; this cell count was equal to the theoretical size of the library. The cells were incubated at 37°C and 280 rpm to OD600=0.5, then infected with a 10-fold excess of proteolytically sensitive helper phage at 37°C and 150 rpm for 1 hour. The culture was spun at 3,200 rpm for 2 minutes, the supernatant was discarded, and the cells were resuspended in 2TYKA medium (2TYGA + 50 μg / mL kanamycin). The phages were then grown overnight at 25°C and 280 rpm. One TG1 colony was selected from a pre-prepared plate and inoculated into 5 mL of 2TY medium. The culture was incubated overnight at 30°C and 280 rpm. The following day, the culture was centrifuged at 10,500 × g at 4°C for 10 minutes to obtain the supernatant. 1 / 10 volume of PEG / NaCl (20% PEG6000 / 2.5M NaCl) was added to precipitate the phages. After incubation on ice for 1 hour, the phages were pelletized by centrifuging at 4,500 rpm at 4°C for 10 minutes. The phage pellet was resuspended in 1 mL of PBS, transferred to an Eppendorf tube, and centrifuged at 14,000 rpm at 4°C for 10 minutes to remove residual cells. The supernatant was transferred to an Eppendorf tube containing 250 μL of PEG / NaCl and spun at 14,000 rpm at 4°C for 10 minutes. The supernatant was discarded, and the phage pellet was resuspended in 1 mL of PBS. The solution was spun at 14,000 rpm for 10 minutes at 4°C until no more cell pellets were observed. The phages were immediately used for phage display selection, and for long-term storage, the phages were stored in 20% (w / v) glycerol at -80°C.

[0296] Two selections were performed in parallel using 212 phages, with and without α-cobra toxin. Phages, biotinylated α-cobra toxin (100 nM), and 2 × 80 μL streptavidin-coated Dynabeads (Fisher Scientific, M-280) were blocked in 3% MPBS (PBS + 3% milk: VWR, A0830.100) at room temperature for 1 hour with inversion. Equivolutes of blocked α-cobra toxin were mixed with phages, and the phages were allowed to bind for 1 hour with inversion. Blocked streptavidin beads were then added to the phage-α-cobra toxin solution for 5 minutes with inversion to capture phages bound to biotinylated α-cobra toxin. The captured streptavidin Dynabeads were washed three times with PBST (PBS + Tween20) and three times with PBS, and then phages were eluted by adding 100 μL of trypsin (1 mg / mL, Sigma-Aldrich, T9201-500MG) and incubating for 15 minutes with inversion. TG1 cells grown to OD600=0.5 were infected with this phage eluate and incubated at 37°C and 150 rpm for 1 hour. Cells for seeding were prepared by spinning at 2000 g at room temperature for 10 minutes. To determine the background and enrichment of antigen-specific phages, dilution plates were prepared on 2TYGA plates at dilutions ranging from 5 to 500,000 times the supernatant. The following day, colonies were scraped from the output plates, resuspended in 2TYGA medium containing 25% glycerol, and then homogenized for several hours with inversion. OD600 was measured, and cells were stored at -80°C for subsequent rescue and selection. Concentration was determined by dividing the number of colony-forming units on the test plate by the number of colony-forming units from the antigen-free selection.

[0297] result To validate the library's usefulness, two phage display selections were performed using 50 nM α-cobra toxin in both steps, and the selected clones were sent for DNA sequencing. The α-cobra toxin concentration was kept the same in both steps to minimize selection bias for KD with 2 nM parental antibody (Figure 5A), although this might have been preferable with stricter selection conditions (decreased α-cobra toxin concentration).

[0298] Phage enrichment was observed between both trials (Figure 6). Sequencing of 10 clones confirmed that these clones were full-length variable domains, unique, and that each position in the library was fully covered (Table 7). No wild-type sequences were found, and most clones had 2-3 mutations. Off-site mutations were also observed, and the binding ability of these individual clones has not yet been verified.

[0299] TIFF2026519648000007.tif97163TIFF2026519648000008.tif233162

[0300] TIFF2026519648000009.tif14165TIFF2026519648000010.tif248160TIFF2026519648000011.tif249160TIFF2026519648000012.tif50161VH: Variable heavy chain, VL: Variable light chain, Bolded region of WT sequence: Region selected for library creation, Bolded region of C sequence: Observed mutation

[0301] conclusion In this study, we created a framework chain interface library from non-pH-dependent antibodies and used it as a basis for discovering antigen-binding proteins based on the theoretical premise that pH-dependent binding, specifically mediated and conferred by amino acid residues located at the light-heavy chain interface by the antibody framework itself, can provide a universal method for introducing pH-dependent antigen-binding properties into antibodies.

[0302] Example 7: Discovery of pH-dependent antigen-binding proteins from a chain interface library Materials and methods Selecting an SCFv From the phage display selection output of Example 6, 92 monoclonal colonies expressing individual scFv were randomly obtained for testing by the DELFIA immunoassay. To express soluble scFv, the scFv gene from the third selection was subcloned from the phage display vector (pIONTAS1) to the pSANG10-3F expression vector using NcoI and NotI restriction endonucleases. The expression vector was then transformed into E. coli BL21 (DE3) cells (New England Biolabs) according to the protocol from Martin et al. (Reference 13).

[0303] Individual scFv-producing monoclonal colonies (276 colonies) were obtained and expressed in a 96-well format using self-induction medium.

[0304] DELFIA Immunoassay The DELFIA immunoassay was performed as described in Example 8 of this specification.

[0305] Array alignment The sequences were aligned, and mutant residues within the framework mutation library were highlighted.

[0306] result We confirmed that the 2554_01_D11 antibody, used as a parent for creating the chain-interface phage display library, is pH-insensitive.

[0307] scFVs exhibiting similar affinity to the parent to α-cobra toxin were identified, and furthermore, they showed higher pH sensitivity ranging from 2 to 2.5 times as measured by the DELFIA immunoassay described herein (Figure 9, Table 8). Variable heavy and light chain alignments of scFv clones obtained with pH sensitivity greater than 1.25, as measured by the ratio of the DELFIA signal at neutral pH 7.4 to the DELFIA signal at acidic pH 5.8, showed specific mutations in the framework's mutant residues within the variable heavy and light chains (Figures 10 and 11).

[0308] TIFF2026519648000013.tif175165

[0309] The clones are as shown in the alignments in Figures 10 and 11 (for example, clone F09_F09_R in Figures 10 and 11 is clone "F09" in Table 8 above), and are also included in the 92 clones plotted on Figure 9. pH sensitivity is calculated as described herein based on DELFIA values ​​measured at pH 7.4 and pH 5.8. Post CTR: positive control.

[0310] conclusion The chain interface libraries designed by introducing the mutations described herein successfully enabled the identification of pH-dependent antigen-binding proteins that exhibit similar affinity to the parent antigen-binding proteins used to construct the library and possess 2 to 2.5 times higher pH sensitivity.

[0311] Example 8: Further identification of residues involved in the pH dependence of antigen-binding proteins by point mutations Materials and methods Point mutation approach For point mutations, in vivo assembly (IVA) cloning technology was used, employing unique primers for introducing each mutation as described in Reference 12.

[0312] DELFIA assay Day 1 Glycerol stock was inoculated into 2YTGK medium and grown overnight at 30°C at 800 revolutions per minute (rpm).

[0313] Day 2 The O / N culture was inoculated into self-inducing medium and grown / expressed O / N at 800 rpm and 30°C.

[0314] Black Maxisorp plates were coated overnight at 4°C with 2.5 ug / mL of anti-FLAG M2 antibody (Sigma).

[0315] Day 3 The plate was washed three times with phosphate-buffered saline (PBS). The wells were blocked for 1 hour with shaking in 200 μL of PBS (MPBS) with 3% (w / v) skim milk. The scFv cultures were centrifuged at 3000 g for 10-20 minutes. The plate was washed with PBS. Next, 25 μL of 6% MPBS was added to each well, followed by 25 μL of supernatant to each well.

[0316] The plates were incubated at room temperature for 1 hour, and then washed three times with PBS pH 7.4 and three times with PBST pH 7.4.

[0317] Next, 50 μL of 50 nM biotinylated α-cobra toxin, prepared in 3% MPBS pH 7.4, was added and incubated for 1 hour.

[0318] The plates were washed three times with 200 μL of PBS and three times with 200 μL of PBST at either pH 7.4 or pH 5.8 to prime their wells for antigen release in the next step.

[0319] α-cobra toxin was dissociated by adding 200 μL of 3% MPBS at pH 7.4 or pH 5.8 for 1 hour, followed by washing with PBS at either pH 5.8 or pH 7.4 under different conditions, and then washing all wells with 200 μL of PBST at pH 7.4.

[0320] 50 μL of a 1 / 500 dilution of streptavidin-europium in DELFIA assay buffer was added to each well (= 10 ng of streptavidin-europium per well), and the mixture was incubated at room temperature for 30 minutes.

[0321] The DELFIA-enhanced solution was divided into equal volumes for detection on all plates, and the plates were left in the dark until they reached room temperature.

[0322] The plate was washed three times with PBS + 0.1% Tween and three times with PBS in a plate washer. Then, 50 μL of enhancement solution was added. Next, the plate was incubated for 15 minutes with shaking, and the top plate was covered.

[0323] Switch on the Victor Nivo plate reader at least 30 minutes in advance to allow the lamp to warm up. The plate was read using a Vector Nivo plate reader set to 25°C and equipped with the correct filter for DELFIA (excitation 320-340nm, emission 615nm).

[0324] result DELFIA immunoassay identified point mutations that increased pH sensitivity ranging from 1.25 to a maximum of 6 times.

[0325] conclusion By introducing a point mutation (2554_01_D11) into the parent antigen-binding protein used to create a chain interface library, we identified further residue positions that confer pH dependence to the antigen-binding protein, as measured by the DELFIA assay.

[0326] Example 9: Identification of further residues involved in the pH dependence of antigen-binding proteins by TheraSAb-Dab analysis Materials and methods Alignment of therapeutic antibodies Antibody sequences were downloaded from the Therapeutic Structural Antibody database (Thera-SAbDab) (https: / / opig.stats.ox.ac.uk / webapps / sabdab-sabpred / therasabdab / search / (accessed October 23, 2023)). All sequences were downloaded from Thera-SAbDab using the "get all therapeutics" button. From the downloaded sequences, only those with the "Genetically Human" trait in "Genetics (Bispecifics delimited with semicolon)" were retained for analysis. The heavy chains of the 328 obtained antibody sequences were aligned using Jalview (version 2.11.2.7) with the "muscle with defaults" alignment option. The light chains of the 328 antibody sequences were split into kappa and lambda and aligned using the same parameters as above.

[0327] Interface analysis: Five human antibody structures (8AHN, 8BSF, 8DCC, 7U8E, 3NPS) were randomly obtained and downloaded from the SAb-Dab website (https: / / opig.stats.ox.ac.uk / webapps / sabdab-sabpred / sabdab / search / ?all=true). The heavy-chain-light-chain interface was shown using the ChimeraX functional "interface," followed by clicking the network line between the heavy and light chains and selecting "Select contact residues of X and X." This interface command calculates the embedded solvent-exposed surface area (SASA) for each pair of chains in a specific set of atoms based on the chain ID and creates a network diagram of the interchain interface. This function was used with the default parameters used in CimeraX (https: / / www.rbvi.ucsf.edu / chimerax / docs / user / commands / interfaces.html).

[0328] result The five antibody structures were aligned using Jalview as described above, and the residue positions that were shown to interact with the counterchain in all five structures were transferred to the Thera-SAbDab alignment to create Figure 13.

[0329] conclusion By comparing the interaction points of the heavy-chain and light-chain interfaces, indicated using the Chimera-x function "interface," with the amino acid conservation (determined by alignment) of 328 therapeutic antibodies, several additional residue positions were selected, and it was found that these have the potential to introduce pH sensitivity.

[0330] Example 10: Site-directed mutagenesis of bevacizumab, adalimumab, and 2554_01_D11 Materials and methods Site-directed mutagenesis was performed using wild-type bevacizumab and adalimumab, and their sequences were found using https: / / opig.stats.ox.ac.uk / webapps / sabdab-sabpred / therasabdab / search / . The amino acid sequences of the heavy and light chains were found, back-translated, and ligated together using flexible linker peptides (Gly4 Ser, Gly4 Ser, Gly3 Ala Ser) to create scFv versions of the antibodies. The resulting DNA sequences were ordered from https: / / www.twistbioscience.com. Furthermore, the wild-type gene 2554_01_D11 was PCR-amplified using GTAACCACCACACCCGCCGCGCTTAATG (SEQ ID NO: 221) and CACCATACCCACGCCGAAACAAGCGC (SEQ ID NO: 222), and then subjected to site-directed mutagenesis.

[0331] Using this sequence as a template, site-directed mutagenesis was performed using primers containing the NNK / NNM motif (Table 11) along with the following materials (Table 9).

[0332] TIFF2026519648000014.tif73161

[0333] The following PCR settings (Table 10) were used.

[0334] TIFF2026519648000015.tif49165

[0335] After PCR mutagenesis and purification using the GeneJET PCR purification kit (K0701), the fragments were combined and digested with Eco31I (FD0294) at 37°C for 30 minutes. Digestion was performed in a 100 μL volume containing 35 μL of Ultrapure H2O, 10 μL of FastDigest buffer, 50 μL of DNA (6 μg), and 5 μL of FastDigest enzyme (Eco31I). After restriction, the restricted overlapping DNA sequence was purified and ligated in an 80 μL volume using T4-ligase (4 μL), 10× ligation buffer (8 μL), 900 μg of DNA fragment (10 μL), and up to 80 μL of Ultrapure H2O. Next, PCR was performed using the above materials and temperature, but only the outer primers were used to ensure that only the full-length scFv sequence was amplified. These full-length scFv samples were digested using 1 μL of FastDigest enzymes NcoI and NotI, 10 μL of PCR solution, 2 μL of 10×FD buffer, and 16 μL of ultrapure H2O. Additionally, 1 μg of pSANG10-3F expression vector was digested using the same setup.

[0336] The digested vector (50 ng) and scFv gene (111 ng) were purified using the GeneJET PCR purification kit (K0701). Ligation was performed using 50 ng of vector DNA, 111 ng of scFv gene (vector:insertion ratio of 1:15), 1 μL of T4 ligase, 2 μL of 10x T4 ligase buffer, and up to 20 μL of Ultrapure H2O. Ligation was performed at 37°C for 30–60 minutes.

[0337] The ligated vector + scFv was transformed into BL21(DE3) competent E. coli (NEB, C2527H) by first thawing the cells on ice for 10 minutes, then mixing 50 μL of cells with 5 μL of ligation product and incubating on ice for 30 minutes. Next, the cells were heat-shocked in a 42°C water bath for 10 seconds, incubated on ice for 5 minutes, and transferred to 950 μL of SOC medium. The cells were then incubated at 37°C for 1 hour with shaking at 250 rpm, and then 100 μL of cells were seeded into a 2YTGK plate (2YT + 2% glucose + 50 μg / mL kanamycin). The remaining 900 μL of cell culture was centrifuged at 2400 × g, resuspended in 100-200 μL volumes, and seeded into a second 2TYGK plate. The plate was incubated overnight at 37°C.

[0338] The following day, the plates were transferred to 96-well polypropylene plates containing 150 μL of 2TYGK medium, allowed to grow overnight, then 50 μL of 50% glycerol was added, and the plates were stored at -80°C.

[0339] TIFF2026519648000016.tif140165TIFF2026519648000017.tif245165

[0340] Sequencing of the scFv gene Sequencing was performed using the s10b primer (GGCTTTGTTAGCAGCCGGATCTCA) (SEQ ID NO: 253) with the eurofins Mix2Seq service, and the sequencing was analyzed using snapgene.

[0341] result Site-directed mutagenesis at four residue positions that were found to induce pH dependence in 2554_01_D11 (Figure 15.B) also induced pH dependence in other (blockbuster) mAbs, bevacizumab and adalimumab (Figure 16). The top five mutants exhibiting the highest pH dependence were sequenced (Figure 17).

[0342] conclusion The frame mutation that gives pH-dependent antigen-binding properties to 2554_01_D11 is conserved in other (blockbuster) mAbs.

[0343] Array Overview Sequence ID 1 Heavy chain CDR1 of antibody 2555_01_A01 GGTFSSYA

[0344] Sequence ID 2 Heavy chain CDR2 of antibody 2555_01_A01 IIPIFGTA

[0345] Sequence ID 3 Heavy chain CDR3 of antibody 2555_01_A01 DNLGYCSGGSCYSDYYYYYMDV

[0346] Sequence ID 4 Light chain CDR1 of antibody 2555_01_A01 NIGQQI

[0347] Sequence ID 5 Light chain CDR2 of antibody 2555_01_A01 SDS

[0348] Sequence ID 6 Light chain CDR3 of antibody 2555_01_A01 QVWDSGSDHVV

[0349] Sequence ID 7 Parent antibody light chain CDR1 TRSSGSIASTYVH

[0350] Sequence ID 8 Parental 2554_01_D11, 2551_01_A12 antibody light chain CDR2 EDNQRPS

[0351] Sequence ID 9 Parent antibody light chain CDR3 QSYDSSNGSVV

[0352] Sequence ID 10 Light chain CDR1 of antibodies 2554_01_D11 and 2554_01_E01 TRSSGSIGSDYVH

[0353] Sequence ID 11 Light chain CDR3 of the 2554_01_D11 antibody QSYDRSNHEVV

[0354] Sequence ID 12 Light chain CDR2 of the 2554_01_E01 antibody EDNRRPS

[0355] Sequence ID 13 Light chain CDR3 of the 2554_01_E01 antibody QSYDSTTNHVV

[0356] Sequence ID 14 Light chain CDR1 of the 2551_01_A12 antibody TRSSGRIVSDYVH

[0357] Sequence ID 15 Light chain CDR3 of the 2551_01_A12 antibody QSYDSSNAYVV

[0358] Sequence ID 16 Light chain CDR1 of antibodies 2558_02_G09 and 2555_01_A01 EGDNIGQQIVH

[0359] Sequence ID 17 Light chain CDR2 of the 2558_02_G09 antibody DGSRRPS

[0360] Sequence ID 18 Light chain CDR3 of the 2558_02_G09 antibody QVWDITSDHVV

[0361] Sequence ID 19 Light chain CDR2 of the 2555_01_A01 antibody SDSDRPS

[0362] Sequence ID 20 Light chain CDR1 of the 2555_01_A04 antibody GGDYIGGESVH

[0363] Sequence ID 21 Light chain CDR2 of the 2555_01_A04 antibody DDTHRPS

[0364] Sequence ID 22 Light chain CDR3 of the 2555_01_A04 antibody QVWDVSSDHVV

[0365] Sequence ID 23 Light chain CDR1 of the 2551_01_B11 antibody GGHNIGSNIVH

[0366] Sequence ID 24 Light chain CDR2 of the 2551_01_B11 antibody HNTNRPS

[0367] Sequence ID 25 Light chain CDR3 of the 2551_01_B11 antibody QVWDSSSEHVV

[0368] Sequence ID 26 Library parent VL FWR1 NFMLTQPRSVSESPGKTVTISC

[0369] Sequence ID 27 Library parent VL FWR2 WYQQRPGSSPTTVIY

[0370] Sequence ID 28 Library parent VL FWR3 GVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC

[0371] Sequence ID 29 Library parent VL FWR4 FGGGTKLTVL

[0372] Sequence ID 30 Library parent VH FWR1 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS

[0373] Sequence ID 31 Library parent VH FWR2 WVRQAPGQGLEWMG

[0374] Sequence ID 32 Library parent VH FWR3 RVTITADESTSTAYMELRSLRSDDTAVYYCAR

[0375] Sequence ID 33 The library contains parent VH FWR4, 2555_01_A01 antibody, adalimumab, bevacizumab, and facinumab heavy chain FWR4. WGQGTLVTVSS

[0376] Sequence ID 34 Library VH primary sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTVSS

[0377] Sequence ID 35 WT, as well as the light chain VL primary sequences and VLs of C1, C2, and C9 clones obtained after a second phage display selection from the framework chain interface library. NFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0378] Sequence ID 36 Library VH polynucleotide sequences CAGGTGCAGCTGGTGCAATCTGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGG GCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGACAACCTAGGATATTGTAGTGGTGGTAGCTGCTACTCTGACTACTACTACTACTACATGGACGTCTGGGGCCAGGGCACCCTGGTCACCGTCTCGAGT

[0379] Sequence ID 37 Library VL polynucleotide sequences AATTTTATGCTGACTCAGCCCCGCTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGGCAGCGACTATGTGCATTGGTACCAGCAGCGCCGGGCAGCTCCCCCACCACTGTCATCTATGAGGATAACCAAAGACCC TCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATCGCAGCAATCATGAAGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0380] Sequence ID 38 2555_01_A01 antibody heavy chain FWR1 QVQLVQSGAEVKKPGSSVKVSCKAS

[0381] Sequence ID 39 2555_01_A01 antibody heavy chain FWR2 ISWVRQAPGQGLEWMGG

[0382] Sequence ID 40 2555_01_A01 antibody heavy chain FWR3 NYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCAR

[0383] Sequence ID 41 Light chain FWR1 of the 2555_01_A01 antibody SYELTQPPSVSVAPGRTATITC

[0384] Sequence ID 42 2555_01_A01 antibody alternative light chain FWR1 SYELTQPPSVSVAPGRTATITCEGD

[0385] Sequence ID 43 Light chain FWR2 of the 2555_01_A01 antibody WYQQKPGQAPVAVIS

[0386] Sequence ID 44 Alternative light chain FWR2 for 2555_01_A01 antibody VHWYQQKPGQAPVAVIS

[0387] Sequence ID 45 Light chain FWR3 of the 2555_01_A01 antibody GIPERFSGSNSGNTATLTISRVEAGDEADYYC

[0388] Sequence ID 46 2555_01_A01 antibody alternative light chain FWR3 DRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYC

[0389] Sequence ID 47 Light chain FWR4 of the 2555_01_A01 antibody FGGGTKVTVL

[0390] Sequence ID 48 pSang10_pelB primer used for PCR amplification of the VH domain CGCTGCCCAGCCGGCCATGG

[0391] Sequence ID 49 HLINK3_R primer CTGAACCGCCTCCACCACTCGA used for PCR amplification of the VH domain

[0392] Sequence ID 50 LLINK2_F primer CTCTGGCGGTGGCGCTAGC used for PCR amplification of VL domain

[0393] Sequence ID 51 2097_R primer used for PCR amplification of VL domain GATGGTGATG ATGATGTGCGGATGCG

[0394] Sequence ID 52 Primer Insert 1_For GGCTAC GAAGAC ACCCCAGCCGGCCATGGCTC

[0395] Sequence ID 53 Primer H2_39Rev GGCTAC GAAGA CTACCATCCACTCCAAACCTTGGCCCGGTGCKNNACGAACCCAGCTAATCG

[0396] Sequence ID 54 Primer H2_42Rev GGCTAC GAAGAC TACCATCCACTCCAAACCKNNGCCCGGTGCCTGACGAACCCAGCTAATCG

[0397] Sequence ID 55 Primer H2_43Rev GGCTAC GAAGAC TACCATCCACTCCAAKNNTTGGCCCGGTGCCTGACGAACCCAGCTAATCG

[0398] Sequence ID 56 Primer H2_WTRev GGCTAC GAAGAC TACCATCCACTCCAAACCTTGGCCCGGTGCCTGACGAACCCAGCTAATCG

[0399] Sequence ID 57 Primer H2_45Rev GGCTAC GAAGAC TACCATCCACTCKNNACCTTGGCCCGGTGCCTGACGAACCCAGCTAATCG

[0400] Sequence ID 58 Primer H2_For GGCTAC GAAGAC TAATGGGTGGTATTATCCCGATTTTTGGTACTGCTAATTATGCGC

[0401] Sequence ID 59 Primer H3_89Rev GGCTAC GAAGAC TATCACGGGCGCAGTAATAKNNAGCGGTATCATCGCTACG

[0402] Sequence ID 60 Primer H3_WTRev GGCTAC GAAGAC TATCACGGGCGCAGTAATACACAGCGGTATCATCGCTACG

[0403] Sequence ID 61 Primer H3_For GGCTAC GAAGAC TAGTGATAATCTGGGTTATTGCAGCGGCGGCTCC

[0404] Sequence ID 62 Primer H4_105Rev GGCTAC GAAGAC TAAGACGGTGACTAAGGTACCKNNACCCCAAACATCC

[0405] Sequence ID 63 Primer H4_WTRev GGCTAC GAAGAC TAAGACGGTGACTAAGGTACCTTGACCCCAAACATCC

[0406] Sequence ID 64 Primer H4_For GGCTAC GAAGAC TAGTCTCGAGCGGTGGTGGCGGCTCCGG

[0407] Sequence ID 65 Primer L2_37Rev GGCTAC GAAGAC TAGACGGTCGTCGGTGACGAGCCCGGGCGKNNCTGATACCAATGCAC

[0408] Sequence ID 66 Primer L2_43Rev GGCTAC GAAGAC TAGACGGTCGTCGGKNNCGAGCCCGGGCGCTGCTGATACC

[0409] Sequence ID 67 PrimerL2_WTRev GGCTAC GAAGAC TAGACGGTCGTCGGTGACGAGCCCGGGCGCTGCTGATACCAATGCAC

[0410] Sequence ID 68 Primer L2_For GGCTAC GAAGAC TACGTCATCTATGAGGACAACCAGCGTCCGAGCGGGGTGC

[0411] Sequence ID 69 Primer L3_85Rev GGCTAC GAAGAC TAGCTTTGGCAGTAGTAKNNCGCCTCGTCCTCG

[0412] Sequence ID 70 primerL3_WTRev GGCTAC GAAGAC TAGCTTTGGCAGTAGTAGTCCGCCTCGTCCTCG

[0413] Sequence ID 71 Primer L4_100For GGCTAC GAAGAC TAAAGCTACGACCGCTCTAACCACGAAGTTGTTTTTGGCNNKGGTACGAAGCTGAC

[0414] Sequence ID 72 Primer pSANG4_Myc_M13_Rev v2 GGCTAC GAAGAC TACAACTTTCAACAGTTTCTGCGGCCCCATTCAGATCCTCTTC

[0415] Sequence ID 73 Primer pSANG4Myc_BbsI_For GGCTAC GAAGAC TAGTTGTTTAGCAAAACCTCATACAGAAAATTCATTTACTAACGTCTGG

[0416] Sequence ID 74 Primer pSANG4_Vector_Rev GGCTAC GAAGAC CATGGGCCGCATAGAAAGGAACAAC

[0417] Sequence ID 75 Primer pSANG5th_For TGGAAAAACGCCAGCAACGC

[0418] Sequence ID 76 Primer-96gIII CCCTCATAGTTGCGTAACG

[0419] Sequence ID 77 VH WT clones obtained after the second phage display selection from the framework chain interface library. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTV

[0420] Sequence ID 78 VH C1 clone obtained after the second phage display selection from the framework chain interface library. HVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQTLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTV

[0421] Sequence ID 79 VH C2 clone obtained after the second phage display selection from the framework chain interface library. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRSAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGVGTLVTV

[0422] Sequence ID 80 VH C3 clone obtained after the second phage display selection from the framework chain interface library. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFKGRVTITADESTSTAYMELRSLRSDDTADYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTV

[0423] Sequence ID 81 VH C4 clone obtained after the second phage display selection from the framework chain interface library. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQRLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAQYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTV

[0424] Sequence ID 82 VH C5 clone obtained after the second phage display selection from the framework chain interface library. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRRAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAQYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTV

[0425] Sequence ID 83 VH C6 clone obtained after the second phage display selection from the framework chain interface library. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQNLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTV

[0426] Sequence ID 84 VH C7 clone obtained after the second phage display selection from the framework chain interface library. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRTAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTATYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTV

[0427] Sequence ID 85 VH C8 clone obtained after the second phage display selection from the framework chain interface library. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQYLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTV

[0428] Sequence ID 86 VH C9 clone obtained after the second phage display selection from the framework chain interface library. QFQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTATYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTV

[0429] Sequence ID 87 VH C10 clone obtained after the second phage display selection from the framework chain interface library. QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVREAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAIYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTV

[0430] Sequence ID 88 VL C3 clone obtained after the second phage display selection from the framework chain interface library. NFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSRPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0431] Sequence ID 89 VL C4 clone obtained after the second phage display selection from the framework chain interface library. NFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSQPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEATYYCQSYDRSNHEVVFGVGTKLTVL

[0432] Sequence ID 90 VL C5 clone obtained after the second phage display selection from the framework chain interface library. NFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSKPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0433] Sequence ID 91 VL C6 clone obtained after the second phage display selection from the framework chain interface library. NFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGWGTKLTVL

[0434] Sequence ID 92 VL C7 clone obtained after the second phage display selection from the framework chain interface library. NFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQFRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0435] Sequence ID 93 VL C8 clone obtained after the second phage display selection from the framework chain interface library. NFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAAYYCQSYDRSNHEVVFGGGTKLTVL

[0436] Sequence ID 94 VL C10 clone obtained after the second phage display selection from the framework chain interface library. NFMLTQPRSVSESPGKTVTISCTRYSGSIGSDYVHWYQVRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAHYYCQSYDRSNHEVVFGHGTKLTVL

[0437] Sequence ID 95 The consensus VH sequence of scFV randomly obtained from the second trial onwards, when pH sensitivity increased. XVQLVQSXAEVKKPGSXVKVSCKASGGTFSSYAISWVRXAPGQXXEXMGGIIPIFGTANYAQKFQGRVTITADXSTSTAYMXLXSLRSDDTAXYXCARDNLGYCSGGSCYSDYYYYYMDVXGXGTLVTVSS

[0438] Sequence ID 96 The consensus VL sequence of scFV randomly obtained from the second trial onwards, when pH sensitivity increased. SSGGGGSGGGGSGGGASNFMLTQPRSVSESPGKTVTISCTRSXGSIGSDXVHWXQXRPGSXXTXVIXEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAXYXCQSYDRSNHEVVXGXGTKLTVX SSGGGGSGGGGSGGGASNFMLTQPRSVSESPGKTVTISCTRSXGSIGSDXVHWXQXRPGSXXTXVIXEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAXYXCQSYDRSNHEVVXGXGTKLTVX

[0439] Sequence ID 97 Consensus VH FWR1 of scFV randomly obtained from the second trial onwards, where pH sensitivity increased. XVQLVQSXAEVKKPGSXVKVSCKASGGTFSXVQLVQSXAEVKKPGSXVKVSCKASGGTFS

[0440] Sequence ID 98 Consensus VH FWR2 of scFV randomly obtained from the second trial onwards, where pH sensitivity increased. WVRXAPGQXXEXMG

[0441] Sequence ID 99 Consensus VH FWR3 of scFV randomly obtained from the second trial onwards, where pH sensitivity increased. RVTITADXSTSTAYMXLXSLRSDDTAXYXCAR

[0442] Sequence ID 100 scFv randomly obtained from the second trial onwards, when pH sensitivity increased, as well as consensus VH FWR4 for adalimumab, bevacizumab, and facinumab. XGXGTLVTVSS

[0443] Sequence ID 101 Consensus VL FWR2 scFv values ​​obtained randomly from the second trial onwards, showing increased pH sensitivity. WXQXRPGSXXTXVIX

[0444] Sequence ID 102 Consensus VL FWR3 scFv values ​​obtained randomly from the second trial onwards, showing increased pH sensitivity. GVPDRFSGSIDSSSNSASLTISGLKTEDEAXYXC

[0445] Sequence ID 103 Consensus VL FWR4 scFv randomly obtained from the second trial onwards, where pH sensitivity increased. XGXGTKLTVX

[0446] Sequence ID 104 Consensus light chain CDR1 from the point mutation approach TRSXGSIGSDXVH

[0447] Sequence ID 105 VH sequence of 2554_01_D11(+1), VH sequence of B04_F_B04_R(+1), VH sequence of B02_F_B02_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTVSSGG

[0448] Sequence ID 106 VH array of F09_F_09_R(+1) QVQLVQSGAEVKKPGSYVKVSCKASGGTFSSYAISWVRSAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTVSSGG

[0449] Sequence ID 107 VH array of F05_F_F05_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRSAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTVSSGG

[0450] Sequence ID 108 VH array of G02_F_G02_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMLRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYYMDVWGQGTLVTVSSGG

[0451] Sequence ID 109 VH array of D01_F_D01_R(+1), VH array of H03_F_H03_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGTGTLVTVSSGG

[0452] Sequence ID 110 VH array of A08_F_A08_R(+1) QVQLVQSAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYYMDVWGRGTLVTVSSGG

[0453] Sequence ID 111 VH array of D05_F_D05_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTATYYCARDNLGYCSGGSCYSDYYYYYMDVWGTGTLVTVSSGG

[0454] Sequence ID 112 VH array of E08_F_E08_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQPLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGRGTLVTVSSGG

[0455] Sequence ID 113 VH array of D12_F_D12_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQRLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTANYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTVSSGG

[0456] Sequence ID 114 VH array of F07_F_F07_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQNLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAIYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTVSSGG

[0457] Sequence ID 115 VH array of G05_F_G05_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQSLEWMGGIIPIFGTANYAQKFQGRVTITADSTSTAYMELRSLRSDDTAQYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTVSSGG

[0458] Sequence ID 116 VH sequence of B05_F_B05_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGKGTLVTVSSGG

[0459] Sequence ID 117 VH array of C07_F_C07_R(+1), VH array of C09_F_C09_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQKLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGPGTLVTVSSGG

[0460] Sequence ID 118 VH array of E05_F_E05_R(+1) HVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVREAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGDGTLVTVSSGG

[0461] Sequence ID 119 VH array of H02_F_H02_(+1) HVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQQLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTATYYCARDNLGYCSGGSCYSDYYYYYMDVWGPGTLVTVSSGG

[0462] Sequence ID 120 VH array of D08_F_D08_R(+1) HVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQQLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTALYYCARDNLGYCSGGSCYSDYYYYYMDVWGKGTLVTVSSGG

[0463] Sequence ID 121 VH array of C04_F_C04_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQALEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAAYYCARDNLGYCSGGSCYSDYYYYYMDVWGPGTLVTVSSGG

[0464] Sequence ID 122 VH array of F03_F_F03_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQKLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAKYYCARDNLGYCSGGSCYSDYYYYYMDVWGIGTLVTVSSGG

[0465] Sequence ID 123 VH sequence of B03_F_B03_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQTLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGTGTLVTVSSGG

[0466] Sequence ID 124 VH array of B10_F_B10_R(+1) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRAAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELKSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYYMDVWGPGTLVTVSSGG

[0467] Sequence ID 125 VL sequence of 2554_01_D11(+1), VL sequence of F05_F_F05_R(+1), VL sequence of G02_F_G02_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0468] Sequence ID 126 VL sequence of F09_F_09_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQLRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0469] Sequence ID 127 VL sequence of B04_F_B04_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQYRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0470] Sequence ID 128 VL sequence of B02_F_B02_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQSRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEASYYCQSYDRSNHEVVFGGGTKLTVL

[0471] Sequence ID 129 VL array of D01_F_D01_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEANYYCQSYDRSNHEVVFGGGTKLTVL

[0472] Sequence ID 130 VL sequence of H03_F_H03_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQIRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEATYYCQSYDRSNHEVVFGGGTKLTVL

[0473] Sequence ID 131 VL sequence of A08_F_A08_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQTRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAFYYCQSYDRSNHEVVFGGGTKLTVL

[0474] Sequence ID 132 VL sequence of D05_F_D05_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGSGTKLTVL

[0475] Sequence ID 133 VL array of E08_F_E08_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVI

[0476] Sequence ID 134 VL array of D12_F_D12_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQARPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0477] Sequence ID 135 VL sequence of F07_F_F07_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQRRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0478] Sequence ID 136 VL sequence of G05_F_G05_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSPPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0479] Sequence ID 137 VL sequence of B05_F_B05_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSYGSIGSDYVHWYQQRPGSTPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0480] Sequence ID 138 VL sequence of C07_F_C07_R(+1), VL sequence of C09_F_C09_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSTPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGYGTKLTVL

[0481] Sequence ID 139 VL sequence of E05_F_E05_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAVYYCQSYDRSNHEVVFGGGTKLTVL

[0482] Sequence ID 140 VL array of H02_F_H02_(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEALYYCQSYDRSNHEVVFGGGTKLTVL

[0483] Sequence ID 141 VL array of D08_F_D08_R(+1), VL array of C04_F_C04_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEASYYCQSYDRSNHEVVFGGGTKLTVL

[0484] Sequence ID 142 VL sequence of F03_F_F03_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEASYYCQSYDRSNHEVVFGGGTKLTVL

[0485] Sequence ID 143 VL sequence of B03_F_B03_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQQRPGSVPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAAYYCQSYDRSNHEVVFGLGTKLTVL

[0486] Sequence ID 144 VL array of B10_F_B10_R(+1) ASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWYQVRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEARYYCQSYDRSNHEVVFGGGTKLTVL

[0487] Sequence ID 145 Heavy chain XXQVQLVESGGGLVQPGGSLRLSCAASGFTFSSGSYAMSWVRQAPGKGLEWVGGISPKTSGGWNTYYADSVKGRFTISRDNSKNFDTFTLYLQMNSLRAEDTAVYYCATXRRDQLYGXXPLXYGGGGGGGGYYYYGSGXEXLYDAFDYDPQYSWGQGTLVTVSS

[0488] Sequence ID 146 Lambda light chain QSVLTQPPSVSGAPGQTVTISCXGSSSNIGSYYDVSWYQQLPGTAPKLLIYDDSNRPSGVPDRFSGSXDXKSGNTASLTISGLQAEDEADYYCQSWDSSLSGXVVFGGGTKLTVL

[0489] Sequence ID 147 Kappa Light Chain DIVMTQSPSSLSASVGDRVTITCRASQSILYSSSSKSYLAWYQQKPGQAPKLLIYGASSRASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSWVPPLXFTFGQGTKVEIK

[0490] Sequence ID 148 Adalimumab MAEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSGGGGS GGGGSGGGASDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKAAASAHHHHHH

[0491] Sequence ID 149 bevacizumab MAEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSGGGG SGGGGSGGGASDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKAAAASAHHHHHH

[0492] Sequence ID 150 Facinumab MAQVQLVQSGAEVKKPGASVKVSCKVSGFTLTELSIHWVRQAPGKGLEWMGGFDPEDGETIYAQKFQGRVTMTEDTSTDTAYMELTSLRSEDTAVYYCSTIFGVVTNFDNWGQGTLVTVSSGGGGSG GGGSGGGASDIQMTQSPSSLSASAGDRVTITCRASQAIRNDLGWYQQKPGKAPKRLIYAAFNLQSGVPSRFSGSGSGTEFLTISSLQPEDLASYYCQQYNRYPWTFGQGTKVEIKAAASAHHHHHH

[0493] Sequence ID 151 Adalimumab and bevacizumab VL FWR1 DIQMTQSPSSLSASVGDRVTITC

[0494] Sequence ID 152 Adalimumab VL FWR2 WYQQKPGKAPKLLIY

[0495] Sequence ID 153 Adalimumab VL FWR3 GVPSRFSGSGSGTDFLTISSLQPEDVATYYC

[0496] Sequence ID 154 Adalimumab, bevacizumab, and facinumab VL FWR4 FGQGTKVEIK

[0497] Sequence ID 155 Adalimumab VH FWR1 EVQLVESGGGLVQPGRSLRLSCAASGFTFD

[0498] Sequence ID 156 Adalimumab VH FWR2 WVRQAPGKGLEWVS

[0499] Sequence ID 157 Adalimumab VH FWR3 RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAK

[0500] Sequence ID 158 Bevacizumab VL FWR2 WYQQKPGKAPKVLIY

[0501] Sequence ID 159 Bevacizumab VL FWR3 GVPSRFSGSGSGTDFLTISSLQPEDFATYYC

[0502] Sequence ID 160 Bevacizumab VH FWR1 EVQLVESGGGLVQPGGSLRLSCAASGYTFT

[0503] Sequence ID 161 Bevacizumab VH FWR2 WVRQAPGKGLEWVG

[0504] Sequence ID 162 Bevacizumab VH FWR3 RFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAK

[0505] Sequence ID 163 Facinumab VL FWR1 DIQMTQSPSSLSASAGDRVTITC

[0506] Sequence ID 164 Facinumab VL FWR2 WYQQKPGKAPKRLIY

[0507] Sequence ID 165 Facinumab VL FWR3 GVPSRFSGSGSGTEFTLTISSLQPEDLASYYC

[0508] Sequence ID 166 Facinumab VH FWR1 QVQLVQSGAEVKKPGASVKVSCKVSGFTLT

[0509] Sequence ID 167 Facinumab VH FWR2 WVRQAPGKGLEWMG

[0510] Sequence ID 168 Facinumab VH FWR3 RVTMTEDTSTDTAYMELTSLRSEDTAVYYCST

[0511] Sequence ID 169 Adalimumab light chain CDR1 RASQGIRNYLA

[0512] Sequence ID 170 Adalimumab light chain CDR2 AASTLQS

[0513] Sequence ID 171 Adalimumab light chain CDR3 QRYNRAPYT

[0514] Sequence ID 172 Bevacizumab light chain CDR1 SASQDISNYLN

[0515] Sequence ID 173 Bevacizumab light chain CDR2 FTSSLHS

[0516] Sequence ID 174 Bevacizumab light chain CDR3 QQYSTVPWT Sequence ID 175 Facinumab light chain CDR1 RASQAIRNDLG

[0517] Sequence ID 176 Facinumab light chain CDR2 AAFNLQS

[0518] Sequence ID 177 Facinumab light chain CDR3 QQYNRYPWT

[0519] Sequence ID 178 Adalimumab heavy chain CDR1 DYAMH

[0520] Sequence ID 179 Adalimumab heavy chain CDR2 AITWNSGHIDYADSVEG

[0521] Sequence ID 180 Adalimumab heavy chain CDR3 VSYLSTASSLDY

[0522] Sequence ID 181 bevacizumab heavy chain CDR1 NYGMN

[0523] Sequence ID 182 bevacizumab heavy chain CDR2 WINTYTGEPTYAADFKR

[0524] Sequence ID 183 Bevacizumab heavy chain CDR3 YPHYYGSSHWYFDV

[0525] Sequence ID 184 Facinumab heavy chain CDR1 ELSIH

[0526] Sequence ID 185 Facinumab heavy chain CDR2 GFDPEDGETIYAQKFQG

[0527] Sequence ID 186 Facinumab heavy chain CDR3 IFGVVTNFDN

[0528] Sequence ID 187 Adalimumab Consensus 1 heavy-chain FWR2 WVRXAPGKXXEXVS

[0529] Sequence ID 188 Adalimumab Consensus 1 heavy-chain FWR3 RFTISRDXAKNSLYLXMXSLRAEDTAXYXCAK

[0530] Sequence ID 189 Adalimumab, bevacizumab, and facinumab Consensus 1 heavy-chain FWR4 XGXGTLVTVSS

[0531] Sequence ID 190 Adalimumab, bevacizumab, and facinumab Consensus 1 light chain FWR2 WXQXKPGKXXKXLIX

[0532] Sequence ID 191 Adalimumab Consensus 1 light chain FWR3 GVPSRFSGSGSGTDFLTISSLQPEDVAXYXC

[0533] Sequence ID 192 Adalimumab, bevacizumab, and facinumab Consensus 1 light chain FWR4 XGXGTKVEIX

[0534] Sequence ID 193 Adalimumab Consensus 2 heavy-chain FWR2 WVRQAPGKXLEWVS

[0535] Sequence ID 194 Adalimumab Consensus 2 heavy-chain FWR3 RFTISRDNAKNSLYLQMNSLRAEDTAXYYCAK

[0536] Sequence ID 195 Adalimumab, bevacizumab, and facinumab Consensus 2 heavy-chain FWR4 WGXGTLVTVSS

[0537] Sequence ID 196 Adalimumab Consensus 2 light chain FWR2 WYQXKPGKAPKLLIY

[0538] Sequence ID 197 Bevacizumab Consensus 1 heavy-chain FWR2 WVRXAPGKXXEXVG

[0539] Sequence ID 198 Bevacizumab Consensus 1 heavy-chain FWR3 RFTFSLDXSKSTAYLXMXSLRXEDTAXYXCAK

[0540] Sequence ID 199 Bevacizumab Consensus 1 Light Chain FWR3 GVPSRFSGSGSGTDFTLTISSLQPEDFAXYXC

[0541] Sequence ID 200 Bevacizumab Consensus 2 heavy-chain FWR2 WVRQAPGKXLEWVG

[0542] Sequence ID 201 Bevacizumab Consensus 2 heavy-chain FWR3 RFTFSLDTSKSTAYLQMNSLRXEDTAXYYCAK

[0543] Sequence ID 202 Bevacizumab Consensus 2 Light Chain FWR2 WYQXKPGKAPKVLIY

[0544] Sequence ID 203 Fascinumab Consensus 1 heavy-chain FWR2 WVRXAPGKXXEXMG

[0545] Sequence ID 204 Fasinumab Consensus 1 heavy-chain FWR3 RVTMTEDXSTDTAYMXLXSLRSEDTAXYXCST

[0546] Sequence ID 205 Fascinumab Consensus 1 Light Chain FWR3 GVPSRFSGSGSGTEFTLTISSLQPEDLAXYXC

[0547] Sequence ID 206 Fascinumab Consensus 2 heavy-chain FWR2 WVRQAPGKXLEWMG

[0548] Sequence ID 207 Fascinumab Consensus 2 heavy-chain FWR3 RVTMTEDTSTDTAYMELTSLRSEDTAXYYCST

[0549] Sequence ID 208 Fascinumab Consensus 2 Light Chain FWR2 WYQXKPGKAPKRLIY

[0550] Sequence ID 209 Parental consensus sequence of bevacizumab MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKXLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAXYYCAKYPHYYGSSHWYFDVWGXGTLVTVSSGGGGSG GGGSGGGASDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQXKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0551] Sequence ID 210 Bevacizumab variant 1 MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKALEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAAYYCAKYPHYYGSSHWYFDVWGTGTLVTVSSGGGGSG GGGSGGGASDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQFKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0552] Sequence ID 211 Bevacizumab variant 2 MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKALEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTATYYCAKYPHYYGSSHWYFDVWGDGTLVTVSSGGGGSG GGGSGGGASDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQLKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0553] Sequence ID 212 Bevacizumab variant 3 MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKPLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAIYYCAKYPHYYGSSHWYFDVWGSGTLVTVSSGGGGSG GGGSGGGASDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQSKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0554] Sequence ID 213 Bevacizumab variant 4 MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKSLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRTEDTATYYCAKYPHYYGSSHWYFDVWGSGTLVTVSSGGGGSG GGGSGGGASDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQTKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0555] Sequence ID 214 Bevacizumab variant 5 MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKTLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTALYYCAKYPHYYGSSHWYFDVWGPGTLVTVSSGGGGSG GGGSGGGASDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQIKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0556] Sequence ID 215 Adalimumab parent consensus sequence MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKXLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAXYYCAKVSYLSTASSLDYWGXGTLVTVSSGGGGGSGG GGSGGGASDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQXKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0557] Sequence ID 216 Adalimumab mutant 1 MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKYLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAYYYCAKVSYLSTASSLDYWGSGTLVTVSSGGGGGSGG GGSGGGASDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQSKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0558] Sequence ID 217 Adalimumab mutant 2 MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTADYYCAKVSYLSTASSLDYWGQGTLVTVSSGGGGGSGG GGSGGGASDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQRKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0559] Sequence ID 218 Adalimumab mutant 3 MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKRLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAFYYCAKVSYLSTASSLDYWGTGTLVTVSSGGGGGSGG GGSGGGASDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQLKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0560] Sequence ID 219 Adalimumab variant 4 MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKRLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAFYYCAKVSYLSTASSLDYWGTGTLVTVSSGGGGGSGG GGSGGGASDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQLKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0561] Sequence ID 220 Adalimumab mutant 5 MKYLLPTAAAGLLLLAAQPAMAEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKTLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTASYYCAKVSYLSTASSLDYWGPGTLVTVSSGGGGGSGG GGSGGGASDIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKAAASAHHHHHHKLDYKDHDGDYKDHDIDYKDDDDK

[0562] Sequence ID 221 2554_01_D11 PCR primer1 GTAACCACCACACCCGCCGCGCTTAATG

[0563] Sequence ID 222 2554_01_D11 PCR Primer 2 and Vector(1).FOR CACCATACCCACGCCGAAACAAGCGC

[0564] Sequence ID 223 Ada_1_Forward and Bev_1_Forward GGTCTGCTGCTCCTCGCT

[0565] Sequence ID 224 Ada_1_Reverse TATGGTCTCACTAACCCATTCCAAKNNTTTACCTG

[0566] Sequence ID 225 Ada_2_Forward TATGGTCTCATTAGTGCAATTACCTGGAACTCTGG

[0567] Sequence ID 226 Ada_2_Reverse TATGGTCTCACCTTGGCACAGTAGTAKNNAGC

[0568] Sequence ID 227 Ada_3_Forward TATGGTCTCAAAGGTTTCTTACTTGTCCACCGC

[0569] Sequence ID 228 Ada_3_Reverse TATGGTCTCACAGTGACCAAAGTACCKNNACCC

[0570] Sequence ID 229 Ada_4_Forward TATGGTCTCAACTGTCAGCAGTGGTGGTGGTGGTAGC

[0571] Sequence ID 230 Ada_4_Reverse TATGGTCTCATGGAGCCTTACCCGGTTTKNNTTG

[0572] Sequence ID 231 Ada_5_Forward TATGGTCTCATCCAAAGCTGCTGATTTACGCT

[0573] Sequence ID 232 Ada_5_Reverse GTGATGGTGATGATGATGTGCGG

[0574] Sequence ID 233 Bev_1_Reverse TATGGTCTCAGACCCATTCCAAKNNCTTACCCG

[0575] Sequence ID 234 Bev_2_Forward TATGGTCTCAGGTCGGTTGGATTAATACCTACACCG

[0576] Sequence ID 235 Bev_2_Reverse TATGGTCTCAACTTAGCACAATAGTAKNNAGCAGTGT

[0577] Sequence ID 236 Bev_3_Forward TATGGTCTCAAAGTACCCACACTATTACGGTTCC

[0578] Sequence ID 237 Bev_3_Reverse TATGGTCTCACCAAAGTACCKNNACCCCAA

[0579] Sequence ID 238 Bev_4_Forward TATGGTCTCATTGGTTACTGTGAGTTCCGGT

[0580] Sequence ID 239 Bev_4_Reverse TATGGTCTCACTTTACCTGGCTTKNNTTGGTACCA

[0581] Sequence ID 240 Bev_5_Forward TATGGTCTCAAAAGCCCCGAAAGTTTTGATTTATTTCAC

[0582] Sequence ID 241 Bev_5_Reverse TGATGGTGATGATGATGTGCGG

[0583] Sequence ID 242 Fragment 1(1).FOR TATGGTCTCAGGGAGGGATCATCCCTATCTTTG

[0584] Sequence ID 243 Fragment 1(1).REV TATGGTCTCACTCTCGCACAMNNATACACGG

[0585] Sequence ID 244 Fragment 2(1).FOR TATGGTCTCAAGAGACAACCTAGGATATTGTAGTGGTG

[0586] Sequence ID 245 Fragment 2(1).REV TATGGTCTCACGCTGCCAATGCTGCCACTGCTGCG

[0587] Sequence ID 246 Fragment 3(1).FOR TATGGTCTAGCGACTATGTGCATTGGNNKCAGC

[0588] Sequence ID 247 Fragment 3(1).REV TATGGTCTCAGATGACMNNGGTMNNGGAGC

[0589] Sequence ID 248 Fragment 4(1).FOR TATGGTCTCACATCNNKGAGGATAACCAAAGACC

[0590] Sequence ID 249 Fragment 4(1).REV TATGGTCTCACTGCGATCATAAGACTGACAMNNGTAGTC

[0591] Sequence ID 250 Fragment 5(1).FOR TATGGTCTCAGCAGCAATCATGAAGTGGTGNNKGG

[0592] Sequence ID 251 Fragment 5(1).REV TGCGGATGCGGCCGCGGGCTGA

[0593] Sequence ID 252 Vector(1). REV TATGGTCTCATCCCATMNNCTCMNNCCCT

[0594] Sequence ID 253 s10b primer GGCTTTGTTAGCAGCCGGATCTCA

[0595] Sequence ID 254 Array of D11 scFv clones "*" from Figure 18 MAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELRSLRSDDTAVYYCARDNLGYCSGGSCYSDYYYYYMDVWGQGTLVTVSSGGGGSGGGGSGGGASNFMLTQPRSVSESPGKTVTISCTRSSGSIGSDYVHWNQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDRSNHEVVFGGGTKLTVL

[0596] References 1.M.L.Murtaugh,S.W.Fanning,T.M.Sharma,A.M.Terry,J.R.Horn,A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches:Engineering pH-Sensitive Protein Switches.Protein Science.20,1619-1631(2011). 2.B.C.Mackness,J.A.Jaworski,E.Boudanova,A.Park,D.Valente,C.Mauriac,O.Pasquier,T.Schmidt,M.Kabiri,A.Kandira,K.Radosevic,H.Qiu,Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life.mAbs.11,1276-1288(2019). 3.C.Schroeter,R.Guenther,L.Rhiel,S.Becker,L.Toleikis,A.Doerner,J.Becker,A.Schoenemann,D.Nasu,B.Neuteboom,H.Kolmar,B.Hock,A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display.mAbs.7,138-151(2015). 4.L.Ledsgaard,A.Ljungars,C.Rimbault,C.V.Sorensen,T.Tulika,J.Wade,Y.Wouters,J.McCafferty,A.H.Laustsen,Advances in antibody phage display technology.Drug Discovery Today.27,2151-2169(2022). 5.T.Igawa,S.Ishii,T.Tachibana,A.Maeda,Y.Higuchi,S.Shimaoka,C.Moriyama,T.Watanabe,R.Takubo,Y.Doi,T.Wakabayashi,A.Hayasaka,S.Kadono,T.Miyazaki,K.Haraya,Y.Sekimori,T.Kojima,Y.Nabuchi,Y.Aso,Y.Kawabe,K.Hattori,Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization.Nat Biotechnol.28,1203-1207(2010). 6.J.Chaparro-Riggers,H.Liang,R.M.DeVay,L.Bai,J.E.Sutton,W.Chen,T.Geng,K.Lindquist,M.G.Casas,L.M.Boustany,C.L.Brown,J.Chabot,B.Gomes,P.Garzone,A.Rossi,P.Strop,D.Shelton,J.Pons,A.Rajpal,Increasing Serum Half-life and Extending Cholesterol Lowering in Vivo by Engineering Antibody with pH-sensitive Binding to PCSK9.Journal of Biological Chemistry.287,11090-11097(2012). 7.L.Ledsgaard,A.H.Laustsen,U.Pus,J.Wade,P.Villar,K.Boddum,P.Slavny,E.W.Masters,A.S.Arias,S.Oscoz,D.T.Griffiths,A.M.Luther,M.Lindholm,R.A.Leah,M.S.Moller,H.Ali,J.McCafferty,B.Lomonte,J.M.Gutierrez,A.Karatt-Vellatt,In vitro discovery of a human monoclonal antibody that neutralizes lethality of cobra snake venom.mAbs.14,2085536(2022). 8.L.Ledsgaard,J.Wade,K.Boddum,I.Oganesyan,J.Harrison,T.P.Jenkins,P.Villar,R.A.Leah,R.Zenobi,J.McCafferty,B.Lomonte,J.M.Gutierrez,A.H.Laustsen,A.Karatt-Vellatt,“Discovery of a broadly-neutralizing human antibody that can rescue mice challenged with neurotoxin-rich snake venoms”(preprint,Bioengineering,2022),doi:10.1101 / 2022.06.17.496531. 9.J.P.Bogen,S.C.Hinz,J.Grzeschik,A.Ebenig,S.Krah,S.Zielonka,H.Kolmar,Dual Function pH Responsive Bispecific Antibodies for Tumor Targeting and Antigen Depletion in Plasma.Front.Immunol.10,1892(2019). 10.L.Ledsgaard,J.Wade,T.P.Jenkins,K.Boddum,I.Oganesyan,J.A.Harrison,P.Villar,R.A.Leah,R.Zenobi,S.Schoffelen,B.Voldborg,A.Ljungars,J.McCafferty,B.Lomonte,J.M.Gutierrez,A.H.Laustsen,A.Karatt-Vellatt,Discovery and optimization of a broadly-neutralizing human monoclonal antibody against long-chain α-neurotoxins from snakes.Nat Commun.14,682(2023). 11.C.Rimbault,PDKnudsen,A.Damsbo,K.Boddum,H.Ali,CMHackney,L.Ellgaard,M.-F.Bohn,AHLaustsen,A single-chain variable fragment selected against a conformational epitope of a recombinantly produced snake toxin using phage display.New Biotechnology.76,23-32(2023). 12. J. Garcia-Nafria, JFWatson, IHGreger, IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly. Sci Rep 6,27459(2016). 13. CD Martin, G. Rojas, JNMitchell, KJ Vincent, J. Wu, J. McCafferty, DJ Schofield, A simple vector system to improve performance and utilization of recombinant antibodies. BMC Biotechnol 6,46 (2006).

[0597] item The present invention may be further defined by any one of the following:

[0598] 1. A method for isolating an antigen-binding protein having a pH-dependent scaffold, wherein the scaffold consists of a variable region that is not part of a paratope, and the method is: - A step of providing a library comprising antigen-binding proteins, each containing an antibody light chain variable region (VL), wherein the VL contains one or more mutations located outside the paratope of the antigen-binding protein, and the library comprises a plurality of antigen-binding proteins, each containing different mutations in the VL, -The step of selecting an antigen-binding protein from the library that exhibits a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, The method for isolating an antigen-binding protein having a pH-dependent scaffold.

[0599] 2. A method for creating a pH-dependent antigen-binding protein that binds to a specific epitope, wherein the method is: - The step of isolating an antigen-binding protein having a pH-dependent scaffold according to the method of item 1, - The step of providing an antigen-binding protein that binds to the specific epitope; -The step of replacing a paratope of the antigen-binding protein having a pH-dependent scaffold with a paratope of the antigen-binding protein bound to the epitope, The method for creating a pH-dependent antigen-binding protein that binds to the specific epitope.

[0600] 3. The method according to any one of the preceding items, wherein one or more antigen-binding proteins in the library further comprises a heavy chain containing a variable region (VH), and the VH contains one or more mutations located outside the paratope of the antigen-binding protein.

[0601] 4. The method according to any one of the preceding items, wherein the one or more mutations are located at a residue position at the interface between the VL and VH of the antigen-binding protein.

[0602] 5. The method according to any one of the preceding items, wherein the parent sequence of VL and / or the parent sequence of VH are sequences of antigen-binding proteins that do not have pH-dependent antigen binding.

[0603] 6. The method according to any one of the preceding items, wherein one or more of the mutations are not located within a complementarity-determining region (CDR).

[0604] 7. The method according to any one of the preceding items, wherein the one or more mutations are located in one or more framework regions (FWRs).

[0605] 8. The method according to any one of the preceding items, wherein the antigen-binding protein of the library comprises an antibody light chain variable region (VL), the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, and at least one of the FWR1, FWR2, FWR3, and FWR4 contains one or more mutations compared to the parental FWR1, FWR2, FWR3, and FWR4.

[0606] 9. The method according to item 8, wherein the antigen-binding protein in the library comprises VL FWR1 of SEQ ID NO: 26, SEQ ID NO: 151, or SEQ ID NO: 163, except that it has one or two amino acid mutations.

[0607] 10. The method according to any one of items 8 to 9, wherein the antigen-binding protein in the library includes VL FWR2 of SEQ ID NO: 27, SEQ ID NO: 152, SEQ ID NO: 158, or SEQ ID NO: 164, except that it has one or two amino acid mutations.

[0608] 11. The method according to item 8, wherein the antigen-binding protein in the library includes VL FWR2 of SEQ ID NO: 27, except that it has one or two amino acid mutations.

[0609] 12. The method according to any one of items 8 to 11, wherein the antigen-binding protein in the library includes VL FWR3 of SEQ ID NO: 28, SEQ ID NO: 153, SEQ ID NO: 159, or SEQ ID NO: 165, except that it has one or two amino acid mutations.

[0610] 13. The method according to item 8 or 11, wherein the antigen-binding protein in the library includes VL FWR3 of SEQ ID NO: 28, except that it has one or two amino acid mutations.

[0611] 14. The method according to any one of items 8 to 13, wherein the antigen-binding protein in the library includes VL FWR4 of SEQ ID NO: 29 or SEQ ID NO: 154, except that it has one or two amino acid mutations.

[0612] 15. The method according to item 8, 10, or 12, wherein the antigen-binding protein in the library includes VL FWR4 of SEQ ID NO: 29, except that it has one or two amino acid mutations.

[0613] 16. The method according to any one of the preceding items, wherein the antigen-binding protein of the library comprises an antibody heavy chain variable region (VH), the VH comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, and at least one of the FWR1, FWR2, FWR3, and FWR4 contains one or more mutations compared to the parental FWR1, FWR2, FWR3, and FWR4.

[0614] 17. The method according to item 16, wherein the antigen-binding protein in the library includes VH FWR1 of SEQ ID NO: 30, SEQ ID NO: 155, SEQ ID NO: 160, or SEQ ID NO: 166, except that it has one or two amino acid mutations.

[0615] 18. The method according to any one of items 16 to 17, wherein the antigen-binding protein in the library includes VH FWR2 of SEQ ID NO: 31, SEQ ID NO: 156, SEQ ID NO: 161, or SEQ ID NO: 167, except that it has one or two amino acid mutations.

[0616] 19. The method according to item 16, wherein the antigen-binding protein in the library comprises VH FWR2 of SEQ ID NO: 31, except that it has one or two amino acid mutations.

[0617] 20. The method according to any one of items 16 to 19, wherein the antigen-binding protein in the library includes VH FWR3 of SEQ ID NO: 32, SEQ ID NO: 157, SEQ ID NO: 162, or SEQ ID NO: 168, except that it has one or two amino acid mutations.

[0618] 21. The method according to item 16 or 19, wherein the antigen-binding protein in the library includes VH FWR3 of SEQ ID NO: 32, except that it has one or two amino acid mutations.

[0619] 22. The method according to any one of items 16 to 21, wherein the antigen-binding protein in the library includes VH FWR4 of SEQ ID NO: 33, except that it has one to two amino acid mutations.

[0620] 23. The method according to any one of the preceding items, wherein the one or more mutations are not located at a residue position occupied by a histidine residue.

[0621] 24. The method described in any one of the preceding items, wherein one or more of the mutations are not mutations in histidine residues.

[0622] 25. The method according to any one of the preceding items, wherein the one or more mutations are located at a residue position at least one amino acid away from a histidine residue, for example, at a residue position at least two amino acids away from a histidine residue, for example, at least five amino acids, for example, at least eight amino acids, for example, at least ten amino acids, for example, at least fifteen amino acids, for example, at least 20 amino acids, for example, at least 25 amino acids, for example, at least 50 amino acids.

[0623] 26. The method according to any one of the preceding items, wherein one or more of the mutations are located at VH residues 39, 44, 45, 47, 89, 91, 103, or 105 according to Kabat numbering.

[0624] 27. The method according to any one of the preceding items, wherein one or more mutations are located in VH residues selected from the group consisting of 39, 44, 89, and 105 according to Kabat numbering.

[0625] 28. The method according to any one of the preceding items, wherein one or more mutations are located in a VH residue selected from the group consisting of Q39, G44, V89, and Q105 according to Kabat numbering.

[0626] 29. The method according to any one of the preceding items, wherein one or more of the mutations are located at VL residues 32, 36, 38, 43, 44, 46, 49, 50, 85, 87, 98, or 100 according to Kabat numbering.

[0627] 30. The method according to any one of the preceding items, wherein one or more mutations are located at a VL residue position selected from the group consisting of 38, 43, 85, and 100 according to Kabat numbering.

[0628] 31. The method according to any one of the preceding items, wherein one or more mutations are located in a VL residue selected from the group consisting of Q38, S43, D85, and G100 according to Kabat numbering.

[0629] 32. The antigen-binding protein in the library is a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) The method according to any one of the preceding items, comprising, wherein one or more mutations are introduced at the residue positions of VH39, VH44, VH89, VH105, VL38, VL43, VL85 and / or VL100 according to Kabat numbering.

[0630] 33. The antigen-binding protein in the library is a) VH FWR2 of sequence number 156 b) VH FWR3 of sequence number 157 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 152 e) VL FWR3 with sequence number 153 f) VL FWR4, sequence number 154 The method according to any one of the preceding items, comprising, wherein one or more mutations are introduced at the residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably, one or more mutations are introduced at the residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering.

[0631] 34. The antigen-binding protein in the library is a) VH FWR2 of sequence number 161 b) VH FWR3 of sequence number 162 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 158, e) VL FWR3, sequence number 159 f) VL FWR4, sequence number 154 The method according to any one of the preceding items, comprising, wherein one or more mutations are introduced at the residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably, one or more mutations are introduced at the residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering.

[0632] 35. The antigen-binding protein in the library is a) VH FWR2 of sequence number 164 b) VH FWR3 of sequence number 165 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 167, e) VL FWR3, sequence number 168 f) VL FWR4, sequence number 154 The method according to any one of the preceding items, comprising, wherein one or more mutations are introduced at the residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably, one or more mutations are introduced at the residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering.

[0633] 36. The method according to any one of the preceding items, wherein the step of selecting an antigen-binding protein from the library that exhibits a lower KD at an acidic pH than neutral pH, or a higher KD at an acidic pH than neutral pH, includes using an in vitro display technique.

[0634] 37. The method according to item 36, wherein the in vitro display technology is selected from the group consisting of phage displays, ribosome displays, yeast displays, bacterial displays, mammalian displays, and CIS displays.

[0635] 38. A pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acid of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parent FWR1, FWR2, FWR2', FWR3, and FWR4, the pH-dependent antigen-binding protein having a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, and the pH-dependent antigen-binding protein not comprising heavy chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, respectively, and light chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 4, 5, and 6, respectively.

[0636] 39. A pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acid of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parent FWR1, FWR2, FWR2', FWR3, and FWR4, and the pH-dependent antigen-binding protein is more acidic than neutral at pH A pH-dependent antigen-binding protein having high binding affinity or lower binding affinity at acidic pH than neutral pH, wherein the pH-dependent antigen does not contain heavy chain complementarity-determining regions 1, 2, and 3 of any of SEQ ID NOs: 1, 2, and 3, SEQ ID NOs: 178, 179, and 180, SEQ ID NOs: 181, 182, and 183, and SEQ ID NOs: 184, 185, and 186, respectively, nor light chain complementarity-determining regions 1, 2, and 3 of any of SEQ ID NOs: 4, 5, and 6, SEQ ID NOs: 169, 170, and 171, SEQ ID NOs: 172, 173, and 174, and SEQ ID NOs: 175, 176, and 177, respectively.

[0637] 40. A pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, the FWR2 and the adjacent amino acid of CDR2 are called FWR2', at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parent FWR1, FWR2, FWR2', FWR3, and FWR4, and the pH-dependent antigen-binding protein is more effective at a pH lower than neutral pH. The pH-dependent antigen-binding protein also has high binding affinity at acidic pH, and the pH-dependent antigen-binding protein does not contain heavy chain complementarity-determining regions 1, 2, and 3 of any of SEQ ID NOs: 1, 2, and 3, SEQ ID NOs: 178, 179, and 180, SEQ ID NOs: 181, 182, and 183, and SEQ ID NOs: 184, 185, and 186, respectively, nor light chain complementarity-determining regions 1, 2, and 3 of any of SEQ ID NOs: 4, 5, and 6, SEQ ID NOs: 169, 170, and 171, SEQ ID NOs: 172, 173, and 174, and SEQ ID NOs: 175, 176, and 177, respectively.

[0638] 41. The pH-dependent antigen-binding protein described in any one of items 38 to 40, wherein the pH-dependent antigen-binding protein is defined in any one of items 1 to 37.

[0639] 42. The pH-dependent antigen-binding protein according to any one of items 38 to 41, wherein the one or more mutations are located at a residue position at the interface between the VL and VH of the pH-dependent antigen-binding protein.

[0640] 43. A pH-dependent antigen-binding protein according to any one of items 38 to 42, wherein the mutation is not located at a residue position occupied by a histidine residue.

[0641] 44. A pH-dependent antigen-binding protein as described in any one of items 38 to 43, wherein one or more of the mutations are not mutations in histidine residues.

[0642] 45. A pH-dependent antigen-binding protein according to any one of items 38 to 44, wherein one or more mutations are located at a residue position at least one amino acid away from a histidine residue, for example, at a residue position at least two amino acids away from a histidine residue, for example, at least five amino acids, for example, at least eight amino acids, for example, at least ten amino acids, for example, at least fifteen amino acids, for example, at least 20 amino acids, for example, at least 25 amino acids, for example, at least 50 amino acids away from a histidine residue.

[0643] 46. ​​The pH-dependent antigen-binding protein according to any one of items 38 to 45, wherein the one or more mutations are located at VH residues selected from the group consisting of 39, 44, 89, and 105 according to Kabat numbering, preferably the mutations are located at VH residues selected from the group consisting of 44, 89, and 105.

[0644] 47. A pH-dependent antigen-binding protein according to any one of items 38 to 46, wherein one or more of the mutations are located in VH residues selected from the group consisting of 45, 47, 91, and 103 according to Kabat numbering.

[0645] 48. A pH-dependent antigen-binding protein according to any one of items 38 to 47, wherein one or more of the mutations are located in a VH residue selected from the group consisting of L45, W47, Y91, and W103.

[0646] 49. The pH-dependent antigen-binding protein according to any one of items 38 to 48, wherein the one or more mutations are located at VH residues selected from the group consisting of 38, 43, 85, and 100 according to Kabat numbering, preferably the mutation is at the VH residue position of 38.

[0647] 50. A pH-dependent antigen-binding protein according to any one of items 38 to 49, wherein one or more of the mutations are located at a VL residue position selected from the group consisting of 32, 46, and 49 according to Kabat numbering.

[0648] 51. A pH-dependent antigen-binding protein according to any one of items 38 to 50, wherein one or more of the mutations are located at a VL residue position selected from the group consisting of 46 and 49 according to Kabat numbering.

[0649] 52. A pH-dependent antigen-binding protein according to any one of items 38 to 51, wherein one or more of the mutations are located at a VL residue position selected from the group consisting of 36, 44, 87, and 98, according to Kabat numbering.

[0650] 53. A pH-dependent antigen-binding protein according to any one of items 38 to 52, wherein one or more of the mutations are located in a VL residue selected from the group consisting of Y32, T46, and Y49 according to Kabat numbering.

[0651] 54. A pH-dependent antigen-binding protein according to any one of items 38 to 53, wherein one or more of the mutations are located in a VL residue selected from the group consisting of T46 and Y49 according to Kabat numbering.

[0652] 55. A pH-dependent antigen-binding protein according to any one of items 38 to 54, wherein one or more of the mutations are located in a VL residue selected from the group consisting of Y36, P44, Y87, and F98 according to Kabat numbering.

[0653] 56. A pH-dependent antigen-binding protein according to any one of items 38 to 55, wherein one or more of the mutations are located in VH residues selected from the group consisting of 39, 44, 45, 47, 89, 91, 103, and 105 according to Kabat numbering.

[0654] 57. The VH comprises one or more of the following amino acid residues, for example, two, three, or four: 39th place: S, E, R, T, or A 44th place: P, R, N, S, K, Q, A, Y, or T 89th place T, N, I, Q, A, L, Y, D, F, S, or K and / or A pH-dependent antigen-binding protein as described in any one of items 38 to 56, where all positions in the formula are indicated according to Kabat numbering, with T, R, K, P, D, I, S, or T at position 105.

[0655] 58. The VH comprises one or more of the following amino acid residues, for example, two, three, or four: 39th place S, E, or A 44th place: P, R, N, S, K, Q, A, or T 89th place T, N, I, Q, A, L, Y, D, F, S, or K and / or A pH-dependent antigen-binding protein as described in any one of items 38 to 57, where all positions in the formula are indicated according to Kabat numbering, with T, R, K, P, D, I, S, or T at position 105.

[0656] 59. A pH-dependent antigen-binding protein according to any one of items 38 to 58, wherein one or more of the mutations are located in a VL residue selected from the group consisting of 32, 36, 38, 43, 44, 46, 49, 85, 87, 98, and 100 according to Kabat numbering.

[0657] 60. A pH-dependent antigen-binding protein according to any one of items 38 to 59, wherein one or more of the mutations are located in a VL residue selected from the group consisting of 36, 38, 43, 44, 46, 49, 85, 87, 98, and 100 according to Kabat numbering.

[0658] 61. The VL contains one or more of the following amino acid residues, for example, two, three, four, five, six, or seven: N, ranked 36th. 38th place: L, Y, S, I, T, A, R, F, or V 43rd place: P, T, A, R, Q, K, or V 46th place A or S, 49th place A, H, or S, 85th place S, N, T, F, V, L, S, A, H, or R and / or A pH-dependent antigen-binding protein as described in any one of items 38 to 60, where all positions in the formula are indicated according to Kabat numbering, with S, Y, W, or L at position 100.

[0659] 62. The VL contains one or more of the following amino acid residues, for example, two, three, four, five, six, or seven: N, ranked 36th. 38th place: L, Y, S, I, T, A, R, F, or V 43rd place: P, T, A, or V 46th place A or S, 49th place A, H, or S, 85th place S, N, T, F, V, L, S, A, or R and / or A pH-dependent antigen-binding protein as described in any one of items 38 to 61, where S, Y, or L is at position 100, and all positions in the formula are indicated according to Kabat numbering.

[0660] 63. A pH-dependent antigen-binding protein as described in any one of items 38 to 62, wherein one or more of the mutations are located in a VH residue selected from the group consisting of Q39, G44, L45, W47, V89, Y91, W103, and Q105 according to Kabat numbering.

[0661] 64. A pH-dependent antigen-binding protein according to any one of items 38 to 63, wherein one or more of the mutations are located in a VL residue selected from the group consisting of Y32, Y36, Q38, D43, P44, T46, Y49, D85, Y87, F98, and G100, according to Kabat numbering.

[0662] 65. A pH-dependent antigen-binding protein according to any one of items 38 to 64, wherein one or more of the mutations are located in a VL residue selected from the group consisting of Y36, Q38, D43, P44, T46, Y49, D85, Y87, F98, and G100, according to Kabat numbering.

[0663] 66. The antigen-binding protein is a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) A pH-dependent antigen-binding protein as described in any one of items 38 to 65, comprising one or more mutations introduced at a residue position selected from the group consisting of VH39, VH44, VH89, VH105, VL38, VL43, VL85, and VL100, according to Kabat numbering.

[0664] 67. The antigen-binding protein is a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) A pH-dependent antigen-binding protein as described in any one of items 38 to 66, comprising one or more mutations introduced at a residue position selected from the group consisting of VH45, VH47, VH91, VH103, VL36, VL44, VL87, and VL98 according to Kabat numbering.

[0665] 68. The antigen-binding protein is a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) A pH-dependent antigen-binding protein as described in any one of items 38 to 67, comprising one or more mutations introduced at a residue position selected from the group consisting of VL32, VL46, and VL49 according to Kabat numbering.

[0666] 69. The antigen-binding protein is a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) A pH-dependent antigen-binding protein as described in any one of items 38 to 68, comprising one or more mutations introduced at a residue position selected from the group consisting of VL46 and VL49 according to Kabat numbering.

[0667] 70. The antigen-binding protein, a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) A pH-dependent antigen-binding protein as described in any one of items 38 to 69, comprising, and having one or more mutations introduced at a residue position selected from the group consisting of VH39, VH44, VH45, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL46, VL49, VL85, VL87, VL98, and VL100, according to Kabat numbering.

[0668] 71. The antigen-binding protein is a) VH FWR2 of sequence number 31 b) VH FWR3 of sequence number 32 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 27 e) VL FWR3, sequence number 28 f) VL FWR4 (Sequence No. 29) A pH-dependent antigen-binding protein as described in any one of items 38 to 70, comprising one or more mutations introduced at a residue position selected from the group consisting of VH39, VH44, VH45, VH89, VH91, VH103, VH105, VL36, VL38, VL43, VL46, VL49, VL85, VL87, VL98, and VL100, according to Kabat numbering.

[0669] 72. The antigen-binding protein is a) VH FWR2 of sequence number 156 b) VH FWR3 of sequence number 157 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 152 e) VL FWR3 with sequence number 153 f) VL FWR4, sequence number 154 A pH-dependent antigen-binding protein according to any one of items 38 to 71, comprising, wherein one or more mutations are introduced at the residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably, one or more mutations are introduced at the residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering.

[0670] 73. The antigen-binding protein is a) VH FWR2 of sequence number 161 b) VH FWR3 of sequence number 162 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 158 e) VL FWR3, sequence number 159 f) VL FWR4, sequence number 154 A pH-dependent antigen-binding protein according to any one of items 38 to 72, comprising, wherein one or more mutations are introduced at the residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably, one or more mutations are introduced at the residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering.

[0671] 74. The antigen-binding protein is a) VH FWR2 of sequence number 164 b) VH FWR3 of sequence number 165 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 167 e) VL FWR3, sequence number 168 f) VL FWR4 of SEQ ID NO: 154 comprising, wherein one or more mutations are introduced at residue positions of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and / or VL100 according to Kabat numbering, preferably, one or more mutations are introduced at residue positions of VH44, VH89, VH105, and / or VL38 according to Kabat numbering, the pH-dependent antigen-binding protein according to any one of items 38 to 73.

[0672] 75. The pH-dependent antigen-binding protein according to any one of items 38 to 74, wherein the antigen-binding protein comprises: a) VH FWR2 of WVRX1APGQX2X3EX4MG (SEQ ID NO: 98) b) VH FWR3 of RVTITADX5STSTAYMX6LX7SLRSDDTAX8YX9CAR (SEQ ID NO: 99) c) X 10 GX 11 VH FWR4 of GTLVTVSS (SEQ ID NO: 100), wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 each may be any amino acid, provided that at least one of X1, X2, X8, X 11 is selected from the following: X1 is any amino acid other than Q, preferably, X1 is S, E, or A, and / or X2 is any amino acid other than G, preferably, X2 is P, R, N, S, K, Q, A, or T, and / or X8 is any amino acid other than V, preferably, X8 is T, N, I, Q, A, L, or K, and / or X 11 is any amino acid other than Q, more preferably, X 11 is T, R, K, P, D, I, or T, and d) The light chain CDR1 of TRSX1GSIGSDX2VH (SEQ ID NO: 104) e) The VL FWR2 of WX3QX4RPGSX5X6TX7VIX8 (SEQ ID NO: 101) f) GVPDRFSGSIDSSSNSASLTISGLKTEDEAX9YX 10 C (SEQ ID NO: 102) of VL FWR3 g) X 11 GX 12 GTKLTVX 13 (SEQ ID NO: 103) of VL FWR4, wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 , X 13 each of which may be any amino acid, provided that at least one of X2, X4, X5, X7, X8, X9, X 12 is selected from the following: X2 is any amino acid other than Y, preferably X2 is A, S, T, or H, and / or X4 is any amino acid other than Q, preferably X4 is L, Y, S, I, T, A, R, or V, and / or X5 is any amino acid other than S, preferably X5 is P, T, A, or V, and / or X7 is any amino acid other than T, preferably X7 is A, or S, and / or X8 is any amino acid other than Y, preferably X8 is A, H, or S, and / or X9 is any amino acid other than D, preferably X9 is S, N, T, F, V, L, S, A, or R, and / or X 12 is any amino acid other than G, preferably X 12 is S, Y, or L.

[0673] 76. The antigen-binding protein is a pH-dependent antigen-binding protein according to any one of items 38 to 75, comprising: a) VH FWR2 of WVRX1APGQX2X3EX4MG (Sequence ID 98) b) VH FWR3 of RVTITADX5STSTAYMX6LX7SLRSDDTAX8YX9CAR (Sequence ID 99) c)X 10 GX 11 GTLVTVSS (Sequence ID 100) VH FWR4, In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X8, X 11 At least one of the following is selected: X1 is any amino acid other than Q, preferably X1 is S, E, or A, and / or X2 is any amino acid other than G, preferably X2 is P, R, N, S, K, Q, A, or T, and / or X8 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, or K, and / or X 11 is any amino acid other than Q, and more preferably X 11 is T, R, K, P, D, I, or T. and d) Light chain CDR1 of TRSX1GSIGSDX2VH (Sequence ID 104) e) VL FWR2 of WX3QX4RPGSX5X6TX7VIX8 (Sequence ID 101) f)GVPDRFSGSIDSSSNSASLTISGLKTEDEAX9YX 10 VL FWR3 of C (Sequence ID 102) g)X 11 GX 12 GTKLTVX 13 VL FWR4 (Sequence ID 103), In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10, X 11 , X 12 , X 13 Each of these can be any amino acid, however X2, X3, X4, X5, X7, X8, X9, X 12 At least one of the following is selected: X2 is any amino acid other than Y, preferably X2 is A, S, T, or H, and / or X3 is any amino acid other than Y, preferably X3 is N, and / or X4 is any amino acid other than Q, preferably X4 is L, Y, S, I, T, A, R, or V, and / or X5 is any amino acid other than S, preferably X5 is P, T, A, or V, and / or X7 is any amino acid other than T, preferably X7 is A or S, and / or X8 is any amino acid other than Y, preferably X8 is A, H, or S, and / or X9 is any amino acid other than D, preferably X9 is S, N, T, F, V, L, S, A, or R, and / or X 12 is any amino acid other than G, preferably X 12 It is S, Y, or L.

[0674] 77. The antigen-binding protein is a pH-dependent antigen-binding protein according to any one of items 38 to 76, comprising: a) VH FWR2 of WVRX1APGQX2X3EX4MG (Sequence ID 98) b) VH FWR3 of RVTITADX5STSTAYMX6LX7SLRSDDTAX8YX9CAR (Sequence ID 99) c)X 10 GX 11 GTLVTVSS (Sequence ID 100) VH FWR4, In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11Each of these can be any amino acid, however X1, X2, X8, X 11 At least one of the following is selected: X1 is any amino acid other than Q, preferably X1 is S, E, or A, and / or X2 is any amino acid other than G, preferably X2 is P, R, N, S, K, Q, A, or T, and / or X8 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, or K, and / or X 11 is any amino acid other than Q, preferably X 11 is T, R, K, P, D, I, or T. and d) VL FWR2 of WX1QX2RPGSX3X4TX5VIX6 (Sequence ID 101) e) VL FWR3 of GVPDRFSGSIDSSSNSASLTISGLKTEDEAX7YX8C (Sequence ID 102) f)X9GX 10 GTKLTVX 11 VL FWR4 (Sequence ID 103), In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X2, X3, X5, X6, X7, X 10 At least one of the following is selected: X2 is any amino acid other than Q, preferably X2 is L, Y, S, I, T, A, R, or V, and / or X3 is any amino acid other than S, preferably X3 is P, T, A, or V, and / or X5 is any amino acid other than T, preferably X5 is A or S, and / or X6 is any amino acid other than Y, preferably X6 is A, H, or S, and / or X7 is any amino acid other than D, preferably X7 is S, N, T, F, V, L, S, A, or R, and / or X 10 is any amino acid other than G, preferably X 10 It is S, Y, or L.

[0675] 78. The antigen-binding protein is a pH-dependent antigen-binding protein according to any one of items 38 to 77, comprising: a) VH FWR2 of WVRX1APGQX2X3EX4MG (Sequence ID 98) b) VH FWR3 of RVTITADX5STSTAYMX6LX7SLRSDDTAX8YX9CAR (Sequence ID 99) c)X 10 GX 11 GTLVTVSS (Sequence ID 100) VH FWR4, In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X8, X 11 At least one of the following is selected: X1 is any amino acid other than Q, preferably X1 is S, E, or A, and / or X2 is any amino acid other than G, preferably X2 is P, R, N, S, K, Q, A, or T, and / or X8 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, or K, and / or X 11 is any amino acid other than Q, preferably X 11 is T, R, K, P, D, I, or T. and d) VL FWR2 of WX1QX2RPGSX3X4TX5VIX6 (Sequence ID 101) e) VL FWR3 of GVPDRFSGSIDSSSNSASLTISGLKTEDEAX7YX8C (Sequence ID 102) f)X9GX10 GTKLTVX 11 VL FWR4 (Sequence ID 103), In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X3, X5, X6, X7, X 10 At least one of the following is selected: X1 is any amino acid other than Y, preferably X1 is N, and / or X2 is any amino acid other than Q, preferably X2 is L, Y, S, I, T, A, R, or V, and / or X3 is any amino acid other than S, preferably X3 is P, T, A, or V, and / or X5 is any amino acid other than T, preferably X5 is A or S, and / or X6 is any amino acid other than Y, preferably X6 is A, H, or S, and / or X7 is any amino acid other than D, preferably X7 is S, N, T, F, V, L, S, A, or R, and / or X 10 is any amino acid other than G, preferably X 10 It is S, Y, or L.

[0676] 79. The antigen-binding protein is a pH-dependent antigen-binding protein according to any one of items 38 to 78, comprising: a) VH FWR2 of WVRX1APGKX2X3EX4VS (Sequence ID 187) b) VH FWR3 of RFTISRDX5AKNSLYLX6MX7SLRAEDTAX8YX9CAK (Sequence ID 188) c)X 10 GX 11 GTLVTVSS (Sequence ID 100) VH FWR4, In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X11 Each of these can be any amino acid, however X1, X2, X8, X 11 At least one of the following is selected: X1 is any amino acid other than Q, preferably X1 is S, E, or A, and / or X2 is any amino acid other than G, preferably X2 is P, R, N, S, K, Q, A, Y, or T, more preferably X2 is Y, R, or T, and / or X8 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, Y, D, F, S, or K, more preferably X8 is Y, D, F, or S, and / or X 11 is any amino acid other than Q, preferably X 11 is T, R, K, P, D, I, S, or T, and more preferably X 11 is T, S, or P, and d) VL FWR2 of WX1QX2KPGKX3X4KX5LIX6 (Sequence ID 190) e) VL FWR3 of GVPSRFSGSGSGTDFTLTISSLQPEDVAX7YX8C (Sequence ID 191) f)X9GX 10 GTKVEIX 11 VL FWR4 (Sequence ID 192), In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X3, X5, X6, X7, X 10 At least one of the following is selected: X1 is any amino acid other than Y, preferably X1 is N, and / or X2 is any amino acid other than Q, preferably X2 is L, Y, S, I, T, A, R, F, or V, more preferably X2 is S, R, or L, and / or X3 is any amino acid other than A, preferably X3 is P, T, or V, and / or X5 is any amino acid other than L, preferably X5 is A or S, and / or X6 is any amino acid other than Y, preferably X6 is A, H, or S, and / or X7 is any amino acid other than T, preferably X7 is N, F, V, L, S, A, or R, and / or X 10 is any amino acid other than Q, preferably X 10 It is S, Y, or L.

[0677] 80. The antigen-binding protein is a pH-dependent antigen-binding protein as described in any one of items 38 to 79, comprising: a) VH FWR2 of WVRQAPGKX1LEWVS (Sequence ID 193) b) VH FWR3 of RFTISRDNAKNSLYLQMNSLRAEDTAX2YYCAK (SEQ ID NO: 194) c) VH FWR4 of WGX3GTLVTVSS (Sequence ID 195) and d) VL FWR2 of WYQX4KPGKAPKLLIY (Sequence ID 196), In the formula, each of X1, X2, X3, and X4 can be any amino acid, provided that at least one of X1, X2, X3, and X4 is selected from the following: X1 is any amino acid other than G, preferably X1 is P, R, N, S, K, Q, A, Y, or T, more preferably X1 is Y, R, or T, and / or X2 is any amino acid other than V, preferably X2 is T, N, I, Q, A, L, Y, D, F, S, or K, more preferably X2 is Y, D, F, or S, and / or X3 is any amino acid other than Q, preferably X3 is T, R, K, P, D, I, S, or T, more preferably X3 is S, T, or P, and / or X4 is any amino acid other than Q, preferably X4 is L, Y, S, I, T, A, R, F, or V, and more preferably X4 is R, S, or L.

[0678] 81. The antigen-binding protein is a pH-dependent antigen-binding protein according to any one of items 38 to 80, comprising: a) VH FWR2 of WVRX1APGKX2X3EX4VG (Sequence ID 197) b)RFTFSLDX5SKSTAYLX6MX7SLRX8EDTAX9YX 10 CAK (Sequence ID 198) VH FWR3 c)X 11 GX 12 GTLVTVSS (Sequence ID 189) VH FWR4, In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X8, X 11 At least one of the following is selected: X1 is any amino acid other than Q, preferably X1 is S, E, or A, and / or X2 is any amino acid other than G, preferably X2 is P, R, N, S, K, Q, A, or T, more preferably X2 is A, P, S, or T, and / or X8 is any amino acid other than A, preferably X8 is T, and / or X9 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, or K, more preferably X8 is A, T, I, or L, and / or X 12 is any amino acid other than Q, preferably X 11is T, R, K, P, D, I, S, or T, and more preferably X 11 is T, D, S, or P, and d) VL FWR2 of WX1QX2KPGKX3X4KX5LIX6 (Sequence ID 190) e) VL FWR3 of GVPSRFSGSGSGTDFTLTISSLQPEDFAX7YX8C (Sequence ID 199) f)X9GX 10 GTKVEIX 11 VL FWR4 (Sequence ID 192), In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X3, X5, X6, X7, X 10 At least one of the following is selected: X1 is any amino acid other than Y, preferably X1 is N, and / or X2 is any amino acid other than Q, preferably X2 is L, Y, S, I, T, A, R, F, or V, more preferably X2 is F, L, S, T, or I, and / or X3 is any amino acid other than A, preferably X3 is P, T, or V, and / or X5 is any amino acid other than V, preferably X5 is A or S, and / or X6 is any amino acid other than Y, preferably X6 is A, H, or S, and / or X7 is any amino acid other than T, preferably X7 is S, N, F, V, L, A, or R, and / or X 10 is any amino acid other than Q, preferably X 10 It is S, Y, or L.

[0679] 82. The antigen-binding protein is a pH-dependent antigen-binding protein according to any one of items 38 to 81, comprising: a) VH FWR2 of WVRQAPGKX1LEWVG (Sequence ID 200) b) VH FWR3 of RFTFSLDTSKSTAYLQMNSLRX2EDTAX3YYCAK (Sequence ID 201) c) VH FWR4 of WGX4GTLVTVSS (Sequence ID 195) and d) VL FWR2 of WYQX5KPGKAPKVLIY (Sequence ID 202), In the formula, each of X1, X2, X3, and X4 can be any amino acid, provided that at least one of X1, X2, X3, and X4 is selected from the following: X1 is any amino acid other than G, preferably X1 is P, R, N, S, K, Q, A, Y, or T, more preferably X1 is A, P, S, or T, and / or X2 is any amino acid other than A, preferably X8 is T, and / or X3 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, Y, D, F, S, or K, preferably X2 is A, T, I, or L, and / or X4 is any amino acid other than Q, preferably X3 is T, R, K, P, D, I, S, or T, preferably X3 is T, D, S, or P, and / or X5 is any amino acid other than Q, preferably X4 is L, Y, S, I, T, A, R, F, or V, and more preferably X4 is F, L, S, T, or I.

[0680] 83. The antigen-binding protein is a pH-dependent antigen-binding protein as described in any one of items 38 to 82, comprising: a) VH FWR2 of WVRX1APGKX2X3EX4MG (Sequence ID 203) b) VH FWR3 of RVTMTEDX5STDTAYMX6LX7SLRSEDTAX8YX9CST (Sequence ID 204) c)X 10 GX11 GTLVTVSS (Sequence ID 189) VH FWR4, In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X8, X 11 At least one of the following is selected: X1 is any amino acid other than Q, preferably X 1は , S, E, or A, and / or X2 is any amino acid other than G, preferably X2 is P, R, N, S, K, Q, A, or T, and / or X8 is any amino acid other than V, preferably X8 is T, N, I, Q, A, L, or K, and / or X 11は , any amino acid other than Q, preferably X 11 is T, R, K, P, D, I, S, or T. and d) VL FWR2 of WX1QX2KPGKX3X4KX5LIX6 (Sequence ID 190) e) VL FWR3 of GVPSRFSGSGSGTEFTLTISSLQPEDLAX7YX8C (Sequence ID 205) f)X9GX 10 GTKVEIX 11 VL FWR4 (Sequence ID 192), In the formula, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 Each of these can be any amino acid, however X1, X2, X3, X5, X6, X7, X 10 At least one of the following is selected: X1 is any amino acid other than Y, preferably X1 is N, and / or X2 is any amino acid other than Q, preferably X2 is L, Y, S, I, T, A, R, F, or V, and / or X3 is any amino acid other than A, preferably X3 is P, T, or V, and / or X5 is any amino acid other than R, preferably X5 is A or S, and / or X6 is any amino acid other than Y, preferably X6 is A, H, or S, and / or X7 is any amino acid other than S, preferably X7 is N, F, V, L, A, or R, and / or X 10 is any amino acid other than Q, preferably X 10 It is S, Y, or L.

[0681] 84. The antigen-binding protein is a pH-dependent antigen-binding protein according to any one of items 38 to 83, comprising: a) VH FWR2 of sequence number 39 b) VH FWR3 of sequence number 40 c) VH FWR4 of sequence number 33 and d) VL FWR2 of sequence number 43 e) FWR2' is S50 according to Kabat numbering. f) VL FWR3, sequence number 45 g) VL FWR4 of sequence number 47.

[0682] 85. An antigen-binding protein comprising an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein one or more mutations are introduced at residue positions selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98 and VL100 according to Kabat numbering.

[0683] 86. An antigen-binding protein comprising an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein one or more mutations are introduced at residue positions selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98 and VL100 according to Kabat numbering. The aforementioned VH includes one or more of the following amino acid residues, for example, two, three, or four: - S, E, or A in 39th place, - P, R, N, S, K, Q, A, or T, ranked 44th - T, N, I, Q, A, L, Y, D, F, S, or K, and / or - 105th place T, R, K, P, D, I, S, or T, and / or The VL includes one or more of the following amino acid residues, for example, two, three, four, five, six, or seven: - N, ranked 36th. - 38th place: L, Y, S, I, T, A, R, F, or V - P, T, A, or V in 43rd place, - 46th place A or S, - 49th place A, H, or S, - 85th place S, N, T, F, V, L, S, A, or R and / or - S, Y, or L in 100th place, The antigen-binding protein, wherein all positions in the formula are indicated according to Kabat numbering.

[0684] 87. One or more of the above mutations are: i. FWR1 of sequence number 26 ii. FWR2 of sequence number 27 iii. FWR3 of sequence number 28, and iv. FWR4 of sequence number 29 or i. FWR1 of sequence number 151 ii. FWR2 of sequence number 152 iii. FWR3 of sequence number 153, and iv. FWR4 of sequence number 154 or i. FWR1 of sequence number 151 ii. FWR2 of sequence number 158 iii. FWR3 of sequence number 159, and iv. FWR4 of sequence number 154 or i. FWR1 of sequence number 163 ii. FWR2 of sequence number 164 iii. FWR3 of sequence number 165, and iv. FWR4 of sequence number 154 Within the VL region of the antigen-binding protein, including the framework region, And the following: i. FWR1 of sequence number 30 ii. FWR2 of Sequence ID 31 iii. FWR3 of sequence number 32, and iv. FWR4 of sequence number 33 or i. FWR1 of sequence number 155 ii. FWR2 of sequence number 156 iii. FWR3 of sequence number 157, and iv. FWR4 of sequence number 33 or i. FWR1 of sequence number 160 ii. FWR2 of sequence number 161 iii. FWR3 of sequence number 162, and iv. FWR4 of sequence number 33 or i. FWR1 of sequence number 166 ii. FWR2 of sequence number 167 iii. FWR3 of sequence number 168, and iv. The antigen-binding protein according to either item 85 or 86, which is introduced into the VH region of the antigen-binding protein including the framework region of FWR4 of SEQ ID NO: 33.

[0685] 88. An antigen-binding protein according to any one of items 85 to 87, wherein one or more of the above mutations are introduced at a residue position selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and VL100, according to Kabat numbering.

[0686] 89. An antigen-binding protein according to any one of items 85 to 88, wherein one or more of the mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH L45, VH W47, VH V89, VH Y91, VH W103, VH Q105, VL Y32, VL Y36, VL Q38, VL S43, VL P44, VL T46, VL Y49, VL E50, VL D85, VL Y87, VL F98, VL G100, according to the Kabat numbering of another amino acid.

[0687] 90. An antigen-binding protein according to any one of items 85 to 89, wherein one or more of the mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH L45, VH W47, VH V89, VH Y91, VH W103, VH Q105, VL Y36, VL Q38, VL S43, VL P44, VL T46, VL Y49, VL E50, VL D85, VL Y87, VL F98, VL G100, according to the Kabat numbering of another amino acid.

[0688] 91. An antigen-binding protein according to any one of items 85 to 90, wherein one or more of the mutations are amino acid substitutions selected from the group consisting of VH39, VH44, VH89, VH105, VL38, VL43, VL50, VL85, and VL100, according to the Kabat numbering of another amino acid.

[0689] 92. An antigen-binding protein according to any one of items 85 to 89, wherein one or more of the mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH V89, VH Q105, VL Q38, VL S43, VL E50, VL D85, and VL G100, according to the Kabat numbering of another amino acid.

[0690] 93. An antigen-binding protein according to any one of items 85 to 92, wherein one or more of the above mutations are introduced at a residue position selected from the group consisting of VH39, VH44, VH89, VH105, VL38, VL43, VL85, and VL105 according to Kabat numbering.

[0691] 94. An antigen-binding protein according to any one of items 85 to 93, wherein one or more of the mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH V89, VH Q105, VL Q38, VL S43, VL D85, and VL G100, according to the Kabat numbering of another amino acid.

[0692] 95. An antigen-binding protein according to any one of items 85 to 94, wherein one or more of the above mutations are introduced at a residue position selected from the group consisting of VH45, VH47, VH91, and VH103 according to Kabat numbering.

[0693] 96. An antigen-binding protein according to any one of items 85 to 95, wherein one or more of the above mutations are introduced at a residue position selected from the group consisting of VH L45, VH W47, VH Y91, and VH W103 according to Kabat numbering.

[0694] 97. An antigen-binding protein according to any one of items 85 to 96, wherein one or more of the above mutations are introduced at a residue position selected from the group consisting of VL32, VL46, and VL49 according to Kabat numbering.

[0695] 98. An antigen-binding protein according to any one of items 85 to 97, wherein one or more of the above mutations are introduced at residue positions selected from the group consisting of VL46 and VL49 according to Kabat numbering.

[0696] 99. An antigen-binding protein according to any one of items 85 to 98, wherein one or more of the above mutations are introduced at a residue position selected from the group consisting of VL Y32, VL T46, and VL Y49 according to Kabat numbering.

[0697] 100. An antigen-binding protein according to any one of items 85 to 99, wherein one or more of the above mutations are introduced at a residue position selected from the group consisting of VL T46 and VL Y49, according to Kabat numbering.

[0698] 101. An antigen-binding protein according to any one of items 85 to 100, wherein one or more of the above mutations are introduced at a residue position selected from the group consisting of VL36, VL44, VL87, and VL98 according to Kabat numbering.

[0699] 102. An antigen-binding protein according to any one of items 85 to 101, wherein one or more of the above mutations are introduced at a residue position selected from the group consisting of VL Y36, VL P44, VL Y87, and VL F98 according to Kabat numbering.

[0700] 103. The antigen-binding protein according to any one of items 85 to 102, wherein the antigen-binding protein is a pH-dependent antigen-binding protein having a higher binding affinity at an acidic pH than at a neutral pH, or a lower binding affinity at an acidic pH than at a neutral pH.

[0701] 104. The antigen-binding protein is as follows: Sequence numbers 1, 2, and 3, respectively Sequence numbers 178, 179, and 180, respectively. The numbers 181, 182, 183, and Each of the heavy chain complementarity determination regions 1, 2, and 3 of sequence numbers 184, 185, and 186, respectively, and below: Sequence numbers 4, 5, and 6, respectively These are sequence numbers 169, 170, and 171 respectively. Sequence numbers 172, 173, 174, and An antigen-binding protein as described in any one of items 85 to 103, which does not contain light chain complementarity-determining regions 1, 2, and 3 of any of sequence numbers 175, 176, or 177, respectively.

[0702] 105. The antigen-binding protein according to any one of items 85 to 104, wherein the antigen-binding protein does not contain the heavy chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, respectively, and the light chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 4, 5, and 6, respectively.

[0703] A pH-dependent antigen-binding protein as described in any one of items 38 to 84, or an antigen-binding protein as described in any one of items 85 to 105, including VH FWR1 of SEQ ID NO: 30, SEQ ID NO: 155, SEQ ID NO: 160, or SEQ ID NO: 166, except that it has mutations in 1 to 2 amino acids.

[0704] A pH-dependent antigen-binding protein as described in any one of items 38 to 84, or an antigen-binding protein as described in any one of items 85 to 106, comprising VH FWR1 of SEQ ID NO: 30, SEQ ID NO: 155, SEQ ID NO: 160, or SEQ ID NO: 166, except that 1 to 2 amino acids are mutated, and comprising VH FWR2 of SEQ ID NO: 31, SEQ ID NO: 156, SEQ ID NO: 161, or SEQ ID NO: 167, except that 1 to 2 amino acids are mutated.

[0705] 108. A pH-dependent antigen-binding protein as described in any one of items 38 to 84, or an antigen-binding protein as described in any one of items 85 to 107, comprising VH FWR1 of SEQ ID NO: 30, SEQ ID NO: 155, SEQ ID NO: 160, or SEQ ID NO: 166, except that 1 to 2 amino acids are mutated, and comprising VH FWR2 of SEQ ID NO: 31, except that 1 to 2 amino acids are mutated.

[0706] A pH-dependent antigen-binding protein as described in any one of items 38 to 84, or an antigen-binding protein as described in any one of items 85 to 108, comprising VH FWR1 of SEQ ID NO: 30, SEQ ID NO: 155, SEQ ID NO: 160, or SEQ ID NO: 166, except that 1 to 2 amino acids are mutated, and comprising VH FWR3 of SEQ ID NO: 32, SEQ ID NO: 157, SEQ ID NO: 162, or SEQ ID NO: 168, except that 1 to 2 amino acids are mutated.

[0707] A pH-dependent antigen-binding protein as described in any one of items 38 to 84, or an antigen-binding protein as described in any one of items 85 to 109, comprising VH FWR1 of SEQ ID NO: 30, SEQ ID NO: 155, SEQ ID NO: 160, or SEQ ID NO: 166, except that 1 to 2 amino acids are mutated, and comprising VH FWR3 of SEQ ID NO: 32, except that 1 to 2 amino acids are mutated.

[0708] A pH-dependent antigen-binding protein as described in any one of items 38 to 84, or an antigen-binding protein as described in any one of items 85 to 110, comprising VH FWR1 of SEQ ID NO: 30, SEQ ID NO: 155, SEQ ID NO: 160, or SEQ ID NO: 166, except that 1 to 2 amino acids are mutated, and comprising VH FWR4 of SEQ ID NO: 33, except that 1 to 2 amino acids are mutated.

[0709] 112. The antigen-binding protein according to any one of items 85 to 111, wherein the antigen-binding protein is a pH-dependent antigen-binding protein as defined in any one of items 38 to 84.

[0710] 113. The method according to any one of items 1 to 37, wherein the antigen-binding protein in the library comprises or consists of a pH-dependent antigen-binding protein as described in any one of items 38 to 84, or an antigen-binding protein as described in any one of items 85 to 112.

[0711] 114. A method for generating a pH-dependent antigen-binding protein directed toward an antigen of interest, wherein: - The step of providing an antigen-binding protein that binds to the antigen of interest; - The step of introducing one or more mutations in the antigen-binding protein at residue positions selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL32, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and VL100, according to Kabat numbering, The method for generating a pH-dependent antigen-binding protein targeted to the antigen of interest.

[0712] 115. The method according to item 114, wherein one or more of the mutations are introduced at residue positions selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL36, VL38, VL43, VL44, VL46, VL49, VL50, VL85, VL87, VL98, and VL100, according to Kabat numbering.

[0713] 116. The method according to any one of items 114 and 115, wherein the one or more mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH L45, VH W47, VH V89, VH Y91, VH W103, VH Q105, VL Y32, VL Y36, VL Q38, VL S43, VL P44, VL T46, VL Y49, VL E50, VL D85, VL Y87, VL F98, VL G100, according to the Kabat numbering of another amino acid.

[0714] 117. The method according to any one of items 114 to 116, wherein the one or more mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH L45, VH W47, VH V89, VH Y91, VH W103, VH Q105, VL Y36, VL Q38, VL S43, VL P44, VL T46, VL Y49, VL E50, VL D85, VL Y87, VL F98, VL G100, according to the Kabat numbering of another amino acid.

[0715] 118. The method according to any one of items 114 to 117, wherein the one or more mutations are introduced at residue positions selected from the group consisting of VH39, VH44, VH89, VH105, VL38, VL43, VL50, VL85 and VL100, according to the Kabat numbering of another amino acid.

[0716] 119. The method according to any one of items 114 to 118, wherein one or more mutations are introduced at residue positions selected from the group consisting of VH39, VH44, VH89, VH105, VL38, VL43, VL85 and VL100 according to Kabat numbering.

[0717] 120. The method according to any one of items 114 to 119, wherein the one or more mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH V89, VH Q105, VL Q38, VL S43, VL E50, VL D85, and VL G100, according to the Kabat numbering of another amino acid.

[0718] 121. The method according to any one of items 114 to 120, wherein the one or more mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH V89, VH Q105, VL Q38, VL S43, VL D85, and VL G100, according to the Kabat numbering of another amino acid.

[0719] 122. The method according to any one of items 114 to 121, wherein one or more of the mutations are introduced at a residue position selected from the group consisting of VH45, VH47, VH91, and VH103 according to Kabat numbering.

[0720] 123. The method according to any one of items 114 to 122, wherein one or more mutations are introduced at residue positions selected from the group consisting of VH L45, VH W47, VH Y91, and VH W103 according to Kabat numbering.

[0721] 124. The method according to any one of items 114 to 123, wherein one or more mutations are introduced at residue positions selected from the group consisting of VL32, VL46, and VL49 according to Kabat numbering.

[0722] 125. The method according to any one of items 114 to 124, wherein one or more mutations are introduced at residue positions selected from the group consisting of VL46 and VL49 according to Kabat numbering.

[0723] 126. The method according to any one of items 114 to 125, wherein one or more mutations are introduced at residue positions selected from the group consisting of VL Y32, VL T46, and VL Y49 according to Kabat numbering.

[0724] 127. The method according to any one of items 114 to 126, wherein one or more mutations are introduced at residue positions selected from the group consisting of VL T46 and VL Y49 according to Kabat numbering.

[0725] 128. The method according to any one of items 114 to 127, wherein one or more mutations are introduced at residue positions selected from the group consisting of VL36, VL44, VL87, and VL98 according to Kabat numbering.

[0726] 129. The method according to any one of items 114 to 128, wherein one or more mutations are introduced at residue positions selected from the group consisting of VL Y36, VL P44, VL Y87, and VL F98 according to Kabat numbering.

[0727] 130. The method according to any one of items 114 to 129, further comprising the step of testing whether the antigen-binding protein containing the one or more mutations has a higher binding affinity at an acidic pH than a neutral pH, or a lower binding affinity at an acidic pH than a neutral pH.

[0728] 131. The method according to any one of items 114 to 130, further comprising the step of selecting an antigen-binding protein that has a higher binding affinity at an acidic pH than a neutral pH, or a lower binding affinity at an acidic pH than a neutral pH.

[0729] 132. The method according to any one of items 114 to 131, further comprising the step of selecting an antibody that exhibits the best thermal stability, for example, the best Fab fragment melting temperature.

[0730] 133. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the one or more mutations are located at a residue position occupied by a charged residue, and the mutations are, for example, substitutions for an uncharged amino acid or an amino acid that cannot participate in hydrogen bonding.

[0731] 134. A pH-dependent antigen-binding protein or a method described in any one of the preceding items, wherein one or more of the mutations are mutations in charged residues.

[0732] 135. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the one or more mutations are located at a residue position occupied by a residue capable of participating in hydrogen bonding, and the mutation is, for example, a substitution to an amino acid that cannot participate in hydrogen bonding.

[0733] 136. The method described in any one of the preceding items or a pH-dependent antigen-binding protein, wherein one or more of the mutations are mutations in residues that can participate in hydrogen bonding.

[0734] 137. The method described in any one of the preceding items or a pH-dependent antigen-binding protein, wherein one or more of the mutations are mutations to residues that cannot participate in hydrogen bonding.

[0735] 138. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the one or more mutations are located at a residue position occupied by a residue selected from the group consisting of arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, threonine, tryptophan, and tyrosine, and the mutation is, for example, a substitution for an uncharged amino acid or an amino acid that cannot participate in hydrogen bonding.

[0736] 139. The method described in any one of the preceding items or a pH-dependent antigen-binding protein, wherein one or more of the mutations are mutations in residues selected from the group consisting of arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, threonine, tryptophan, and tyrosine.

[0737] 140. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the one or more mutations are located at a residue position occupied by a residue selected from the group consisting of arginine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, serine, threonine, tryptophan, and tyrosine, and the mutation is, for example, a substitution for an uncharged amino acid or an amino acid that cannot participate in hydrogen bonding.

[0738] 141. The method described in any one of the preceding items or a pH-dependent antigen-binding protein, wherein one or more of the mutations are mutations in residues selected from the group consisting of arginine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, serine, threonine, tryptophan, and tyrosine.

[0739] 142. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the one or more mutations are located at a residue position occupied by a residue selected from the group consisting of arginine, aspartic acid, glutamic acid, histidine, and lysine, and the mutation is, for example, a substitution for an uncharged amino acid or an amino acid that cannot participate in hydrogen bonding.

[0740] 143. The method described in any one of the preceding items or a pH-dependent antigen-binding protein, wherein one or more of the mutations are mutations in residues selected from the group consisting of arginine, aspartic acid, glutamic acid, histidine, and lysine.

[0741] 144. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the one or more mutations are located at a residue position occupied by a residue selected from the group consisting of arginine, aspartic acid, glutamic acid, histidine, and lysine, and the mutation is, for example, a substitution for an uncharged amino acid or an amino acid that cannot participate in hydrogen bonding.

[0742] 145. The method described in any one of the preceding items or a pH-dependent antigen-binding protein, wherein one or more of the mutations are mutations in residues selected from the group consisting of arginine, aspartic acid, glutamic acid, histidine, and lysine.

[0743] 146. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the pH-dependent antigen-binding protein has a lower Kd value for the antigen at acidic pH compared to its Kd value at neutral pH.

[0744] 147. The method according to item 146 or a pH-dependent antigen-binding protein, wherein the Kd value at acidic pH is reduced by 2 times, for example, 5 times, for example, 10 times, for example, 25 times, for example, 50 times, for example, 75 times, for example, 100 times, for example, 125 times, for example, 250 times, for example, 500 times, for example, 750 times, for example, 1000 times, compared to neutral pH.

[0745] 148. The method described in any one of the preceding items or a pH-dependent antigen-binding protein, wherein the pH-dependent antigen-binding protein has a higher Kd value for the antigen at acidic pH compared to its Kd value at neutral pH.

[0746] 149. The method according to item 148 or a pH-dependent antigen-binding protein, wherein the Kd value at acidic pH increases by 2 times, for example, 5 times, for example, 10 times, for example, 25 times, for example, 50 times, for example, 75 times, for example, 100 times, for example, 125 times, for example, 250 times, for example, 500 times, for example, 750 times, for example, 1000 times, compared to neutral pH.

[0747] 150. The method described in any one of the preceding items or the pH-dependent antigen-binding protein, wherein the pH-dependent antigen-binding protein is selected from the group consisting of a full-length antibody, a Fab fragment, an F(ab') fragment, an F(ab')2 fragment, scFv, a diabody, and a triabody.

[0748] 151. The method described in any one of the preceding items or a pH-dependent antigen-binding protein, wherein the pH-dependent antigen-binding protein includes an immunoglobulin constant region.

[0749] 152. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the constant region of the pH-dependent antigen-binding protein immunoglobulin is selected from the group consisting of IgG, IgM, IgA, IgD, and IgE.

[0750] 153. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the constant region of the pH-dependent antigen-binding protein immunoglobulin is selected from the group consisting of IgG and IgA.

[0751] 154. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the constant region of the pH-dependent antigen-binding protein immunoglobulin is selected from the group consisting of IgG1, IgA1, and IgA2.

[0752] 155. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the constant region of the pH-dependent antigen-binding protein immunoglobulin is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0753] 156. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the pH-dependent antigen-binding protein is a monoclonal antibody.

[0754] 157. The method according to any one of the preceding items or a pH-dependent antigen-binding protein, wherein the pH-dependent antigen-binding protein is a human antibody or a chimeric antibody.

[0755] 158. The method described in any one of the preceding items or a pH-dependent antigen-binding protein having one or more mutations selected from the group consisting of PEGylation, polysialylation, Fc region mutation, and N-glycosylation.

[0756] 159. The pH-dependent antigen-binding protein is a method according to any one of the preceding items, comprising a detection label or a pH-dependent antigen-binding protein.

[0757] 160. The method according to item 159 or a pH-dependent antigen-binding protein, wherein the detection label is selected from the group consisting of colorimetric, fluorescent, luminescent, magnetic, and paramagnetic labels.

[0758] 161. The method described in item 159 or a pH-dependent antigen-binding protein, wherein the detection label is biotin.

[0759] 162. The method described in item 159 or a pH-dependent antigen-binding protein, wherein the detection label is gold nanoparticles.

[0760] 163. A composition comprising a pH-dependent antigen-binding protein as described in any one of items 38 to 113, and a pharmaceutically acceptable excipient.

[0761] 164. A pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL) for use in a method of treatment for a patient requiring treatment for cancer, autoimmune disease, metabolic disease, or hematological disorder, wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, or the composition described in item 163.

[0762] 165. The aforementioned cancer is a solid tumor carcinoma, and the pH-dependent antigen-binding protein is as described in item 164.

[0763] 166. The pH-dependent antigen-binding protein according to any one of items 164 to 165, wherein the pH-dependent antigen-binding protein is a protein according to any one of items 38 to 113.

[0764] 167. A method for treating cancer, autoimmune disease, metabolic disease, or hematological disease, comprising administering to a patient in need of treatment for cancer, autoimmune disease, metabolic disease, or hematological disease a pH-dependent antigen-binding protein comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, or the composition described in item 163.

[0765] 168. The method according to item 167, wherein the cancer is a solid tumor carcinoma.

[0766] 169. The method according to any one of items 167 to 168, wherein the pH-dependent antigen-binding protein is a protein described in any one of items 38 to 113.

[0767] 170. Use of the pH-dependent antigen-binding protein, comprising an antibody light chain variable region (VL), in the manufacture of a pharmaceutical product for the treatment of cancer, autoimmune disease, metabolic disease, or hematological disorder, wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acid of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH, or use of the composition described in item 163.

[0768] 171. Use as described in item 170, wherein the cancer is a solid tumor carcinoma.

[0769] 172. The use according to any one of items 170 to 171, wherein the pH-dependent antigen-binding protein is a protein described in any one of items 38 to 113.

[0770] 173. Use in in vitro methods for the detection and / or diagnosis of cancer of the pH-dependent antigen-binding protein, or the composition described in item 163, comprising an antibody light chain variable region (VL), wherein the VL comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parental FWR1, FWR2, FWR2', FWR3, and FWR4, wherein the pH-dependent antigen-binding protein has a higher binding affinity at acidic pH than neutral pH, or a lower binding affinity at acidic pH than neutral pH.

[0771] 174. The method according to any one of items 167 to 169, wherein the plasma recycling of the pH-dependent antigen-binding protein is improved compared to antigen-binding proteins including parent FWR1, FWR2, FWR2', FWR3, and FWR4.

[0772] 175. The method according to any one of items 167 to 169, 174, wherein the pH-dependent antigen binding improves the clearance of the antigen from plasma compared to antigen-binding proteins including parental FWR1, FWR2, FWR2', FWR3, and FWR4.

[0773] 176. The method according to any one of items 167 to 169, 174 to 175, wherein the pH-dependent antigen binding improves the release of the antigen from the endosome compared to antigen-binding proteins including parental FWR1, FWR2, FWR2', FWR3, and FWR4.

[0774] 177. The method according to any one of items 167 to 169, 174 to 175, wherein the intracellular uptake of the pH-dependent antigen-binding protein is improved in an acidic microenvironment compared to antigen-binding proteins including parental FWR1, FWR2, FWR2', FWR3, and FWR4.

[0775] 178. - To provide a pH-dependent antigen-binding protein as described in any one of items 38 to 113, - The pH-dependent antigen-binding protein is brought into contact with the antigen to which it binds, -Includes detecting contact between the pH-dependent antigen-binding protein and the antigen, This provides an in vitro antigen detection method for detecting the aforementioned antigen.

[0776] 179. - To provide a pH-dependent antigen-binding protein as described in any one of items 38 to 113, - The pH-dependent antigen-binding protein is brought into contact with the antigen to which it binds in a complex mixture. - Includes separating the pH-dependent antigen-binding protein / antigen complex from the complex mixture, This provides an in vitro antigen purification method for purifying the aforementioned antigen.

[0777] 180. Each of the VL and VH comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acids of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parent FWR1, FWR2, FWR2', FWR3, and FWR4, and the pH-dependent antigen-binding protein is An antigen-binding protein as described in any one of items 85 to 113, which does not include heavy chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs. 1, 2, and 3, respectively, SEQ ID NOs. 178, 179, and 180, respectively, SEQ ID NOs. 181, 182, and 183, respectively, and SEQ ID NOs. 184, 185, and 186, respectively, nor does it include light chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs. 4, 5, and 6, respectively, SEQ ID NOs. 169, 170, and 171, respectively, SEQ ID NOs. 172, 173, and 174, respectively, and SEQ ID NOs. 175, 176, and 177, respectively.

[0778] 181. The antigen-binding protein according to any one of items 85 to 113, wherein each of the VL and VH comprises three complementarity-determining regions CDR1, CDR2, and CDR3 surrounded by four framework regions FWR1, FWR2, FWR3, and FWR4, wherein the FWR2 and the adjacent amino acid of CDR2 are called FWR2', and at least one of the FWR1, FWR2, FWR2', FWR3, and FWR4 contains at least one mutation compared to the parent FWR1, FWR2, FWR2', FWR3, and FWR4, and the pH-dependent antigen-binding protein does not contain heavy chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, respectively, and light chain complementarity-determining regions 1, 2, and 3 of SEQ ID NOs: 4, 5, and 6, respectively.

[0779] 182. The antigen-binding protein according to any one of items 180 and 181, wherein the three complementarity-determining regions CDR1, CDR2, and CDR3, the four framework regions FWR1, FWR2, FWR3, and FWR4, and FWR2' are as described in any one of items 38 to 84.

[0780] 183. The method according to any one of items 114 to 162, wherein one or more of the mutations are introduced at a residue position selected from the group consisting of VH39, VH44, VH45, VH47, VH89, VH91, VH103, VH105, VL36, VL38, VL43, VL44, VL46, VL49, VL85, VL87, VL98, and VL100, according to Kabat numbering.

[0781] 184. The method according to any one of items 114 to 162 and 183, wherein the one or more mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH L45, VH W47, VH V89, VH Y91, VH W103, VH Q105, VL Y32, VL Y36, VL Q38, VL S43, VL P44, VL T46, VL Y49, VL D85, VL Y87, VL F98, VL G100, according to the Kabat numbering of another amino acid.

[0782] 185. The method according to any one of items 114 to 162 and 183 to 184, wherein the one or more mutations are amino acid substitutions selected from the group consisting of VH Q39, VH G44, VH L45, VH W47, VH V89, VH Y91, VH W103, VH Q105, VL Y36, VL Q38, VL S43, VL P44, VL T46, VL Y49, VL D85, VL Y87, VL F98, VL G100, according to the Kabat numbering of another amino acid.

[0783] 186. The method according to any one of items 114 to 162 and 183 to 185, wherein the one or more mutations are introduced at residue positions selected from the group consisting of VH39, VH44, VH89, VH105, VL38, VL43, VL85 and VL100, according to the Kabat numbering of another amino acid.

[0784] 187. The method, pH-dependent antigen-binding protein, antigen-binding protein, composition, or use according to any one of the preceding items, wherein the binding affinity is higher at an acidic pH than neutral pH, or lower at an acidic pH than neutral pH, respectively, and the KD is lower or higher.

[0785] 188. The method, pH-dependent antigen-binding protein, antigen-binding protein, composition, or use according to any one of the preceding items, wherein the binding affinity is higher at an acidic pH than at a neutral pH, or lower at an acidic pH than at a neutral pH, is measured as the ratio of DELFIA signals at an acidic pH and a neutral pH.

[0786] 189. The method, pH-dependent antigen-binding protein, antigen-binding protein, composition, or use according to any one of the preceding items, wherein the binding affinity, which is higher at an acidic pH than a neutral pH or lower at an acidic pH than a neutral pH, is at least 1.25 times, for example, at least 1.3 times, for example, at least 1.5 times, for example, at least 1.6 times, for example, at least 1.7 times, for example, at least 1.8 times, at least 1.9 times, for example, at least 2 times, for example, at least 2.2 times, for example, at least 2.5 times, for example, at least 5 times, for example, at least 6 times, for example, at least 10 times, for example, at least 10.5 times.

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