Methods and compositions for reducing antibody viscosity

Abstract

This specification provides a method for identifying antigen-binding polypeptide variants (e.g., antibody variants, antigen-binding fragments, or variants of antibody-based constructs) that have reduced viscosity compared to a parent antigen-binding polypeptide (e.g., an antibody, its antigen-binding fragment, or an antibody construct). Antigen-binding polypeptide variants produced by this method are also provided. Related libraries and methods for screening such libraries are further provided. Methods for identifying one or more amino acids in an antibody, an antigen-binding fragment of an antibody, or an antibody construct that affect the viscosity of the antibody, antigen-binding fragment, or antibody construct are also provided. Exemplary antibody variable domains with reduced viscosity are also provided.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 466,219, filed on May 12, 2023, and U.S. Provisional Patent Application No. 63 / 587,649, filed on October 3, 2023, the contents of each of which are hereby incorporated by reference in their entirety.

[0002] This application relates to a method for reducing the viscosity of an antibody composition, and to an antibody produced by that method.

[0003] Sequence Listing The content of the electronic sequence listing (146392066640seqlist.xml; size: 22,642 bytes; creation date: May 8, 2024) is hereby incorporated by reference in its entirety.

Background Art

[0004] Over the past 30 years, the U.S. Food and Drug Administration (US FDA) has approved approximately 125 biological drugs, including antibody-based therapies such as monoclonal antibodies (mAbs), antibody-drug conjugates, and Fc fusion proteins. The majority of antibody-based therapies are administered intravenously (IV). However, IV administration typically requires visits to a medical facility and is usually associated with longer administration times, higher medical costs, and lower patient compliance. As frequent administration of biological drugs may be required throughout a patient's life, alternative routes, namely subcutaneous administration, are increasingly being used for patients with chronic diseases. Currently, approximately 30% of approved antibody therapies are administered by subcutaneous injection. See, for example, Carter and Rajpal (2022) Cell, 185(15):P2789-2805. Subcutaneous injection can sometimes be self-administered by patients using ready-to-use delivery devices, which is advantageous for comfort and privacy. Concentrated solutions of antibody-based therapeutics contained in such devices may offer the further advantage of longer intervals between injections, which can reduce healthcare costs by minimizing hospital or clinic visits and improving patient compliance and adherence to treatment regimens.

[0005] The development of antibody-based therapeutic formulations for subcutaneous administration is a critical consideration. While the volume of drugs that can be subcutaneously injected is typically 1–2 mL, volumes up to 3 mL are possible. Such low volumes necessitate the formulation of antibody-based therapeutics at concentrations typically ≥150 mg / mL to deliver the required dose. However, such highly concentrated solutions can sometimes exhibit high viscosity, adding complexity to the development of antibody-based therapeutics as drug products suitable for subcutaneous administration. High-viscosity formulations can increase injection time, cause pain at the injection site, and negatively impact patient compliance. Furthermore, high-viscosity formulations also present difficulties during the manufacturing of antibody-based therapeutics.

[0006] While factors contributing to the viscosity of high-concentration antibody solutions have been widely studied, predicting the viscosity of antibodies, their antigen-binding fragments, or antibody-based constructs remains a challenge. What is needed in the art is an improved method for predicting and reducing the viscosity of compositions containing high-concentration antibody-based therapeutics, including antibodies, their antigen-binding fragments, and antibody-based constructs. [Overview of the project]

[0007] In some embodiments, a method is provided for identifying antigen-binding polypeptide variants having reduced viscosity compared to a parent antigen-binding polypeptide, comprising: (a) identifying one or more wild-type amino acids in a parent antigen-binding polypeptide having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) substituting one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to the wild-type amino acids; (c) measuring the viscosity of the antigen-binding polypeptide variant; and (d) identifying antigen-binding polypeptide variants having reduced viscosity compared to the parent polypeptide. In some embodiments, one or more wild-type amino acids in the parent antigen-binding polypeptide are surface-exposed. In some embodiments, one or more wild-type amino acids are aromatic amino acids, and one or more aromatic amino acids are substituted with one or more charged amino acids or amino acids having smaller side chains. In some embodiments, one or more aromatic amino acids are selected from the group consisting of: W, Y, and F. In some embodiments, one or more charged amino acids or amino acids having smaller side chains are selected from the group consisting of A, I, L, V, D, E, H, K, R, N, Q, S, and T. In some embodiments, one or more wild-type amino acids have smaller side chains, and one or more amino acids having smaller chains are substituted with one or more charged amino acids. In some embodiments, one or more wild-type amino acids have smaller side chains selected from the group consisting of: A, I, L, V, N, Q, S, G, P, C, M, or T. In some embodiments, one or more charged amino acids are selected from the group consisting of: R, K, H, D, and E.

[0008] In some embodiments, a high SAP value is 2 or greater, for example, 2.3 or greater. In some embodiments, a high SASA ratio is 0.5 or greater. In some embodiments, a high SASA ratio is 0.25 or greater. In some embodiments, the viscosity of the antigen-binding polypeptide variant is evaluated by dynamic light scattering (DLS). In some embodiments, the viscosity of the antigen-binding polypeptide variant is measured by cone-plate rheometry. In some embodiments, the viscosity of the antigen-binding polypeptide variant is measured at high concentrations. In some embodiments, high concentrations are approximately 50 mg / ml to 300 mg / ml.

[0009] In some embodiments, the parent antigen-binding polypeptide is an antibody, an antigen-binding fragment of an antibody, or an antibody construct, the antibody or antibody construct comprising one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and one or more wild-type amino acids in one or more CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are identified as having a high SAP value and / or a high SASA ratio. In some embodiments, the parent antigen-binding polypeptide is an antibody. In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody contains a human IgG Fc region. In some embodiments, the human IgG constant region is an IgG1, IgG2, or IgG4 Fc region. In some embodiments, the parent polypeptide is an antigen-binding fragment of an antibody. In some embodiments, the antigen-binding fragment is Fab, F(ab')2, triplicate specificity Fab3, scFv, monovalent IgG, diabody, triabody, scFv-Vc, minibody, V HThe parent polypeptide is H, V-NAR, hcIgG, or IgNAR. In some embodiments, the parent polypeptide is an antibody construct. In some embodiments, the antibody construct is CrossMab, dual-acting Fab (DAF), DVD-IgG, or a knob-in-hole bispecific antibody.

[0010] In some embodiments, the methods provided herein further include subjecting an antigen-binding polypeptide variant to at least one affinity maturation step. In some embodiments, one or more substituted amino acids in the antigen-binding polypeptide variant introduced to replace one or more wild-type amino acids having high SAP values ​​and / or high SASA ratios are not randomized during affinity maturation. In some embodiments, polypeptide variants produced by the methods described herein are provided.

[0011] In some embodiments, a library is provided comprising multiple antigen-binding polypeptide variants, where at least one variant comprises one or more amino acid substitutions compared to a parent antigen-binding polypeptide, and the one or more amino acid substitutions compared to the parent antigen-binding polypeptide replace one or more wild-type amino acids identified as having a high SAP value and a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to wild-type amino acids. In some embodiments, the multiple comprises at least 1,000 unique variants. In some embodiments, the multiple antigen-binding variants are multiple antibodies, antigen-binding fragments of antibodies, or antibody constructs.

[0012] A method for predicting one or more amino acids in an antigen-binding polypeptide that affect the viscosity of the antigen-binding polypeptide is also provided herein, comprising identifying one or more amino acids in the antigen-binding polypeptide having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio, wherein one or more amino acids having a high SAP value and / or a high SASA ratio are predicted to affect the viscosity of the antigen-binding polypeptide. A method for predicting one or more amino acids in an antibody, an antigen-binding fragment of an antibody, or an antibody construct that affects the viscosity of an antibody, an antigen-binding fragment, or an antibody construct is also provided herein, wherein the antibody, antigen-binding fragment, or antibody construct comprises one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, the method comprising identifying one or more amino acid positions in one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having high spatial aggregation tendency (SAP) values ​​and high solvent-accessible surface area (SASA) ratios, the one or more amino acid positions having SAP values ​​and high SASA ratios are predicted to affect the viscosity of the antibody, antigen-binding fragment, or antibody construct.

[0013] In some embodiments, the antibody heavy chain variable domain (V) described in any one of SEQ ID NOs: 3-17 or 19-22 H ) is provided. In some embodiments, a heavy chain variable domain (V) described in any one of sequence numbers 3-17 or 19-22 is provided. H ) and the light chain variable domain (V) described in Sequence ID No. 2 L Antibodies containing ) are provided.

[0014] It should be understood that other embodiments of the invention can be formed by combining one, some, or all of the characteristics of the various embodiments described herein. These and other aspects of the invention will be apparent to those skilled in the art. These and other embodiments of the invention will be further described by the following detailed description. [Brief explanation of the drawing]

[0015] [Figure 1] We provide the results of experiments conducted to measure the viscosity of alanine-substituted variants of anti-GCGR IgG1 antibodies, including VH as described in SEQ ID NO: 1 and VL as described in SEQ ID NO: 2. See Tilegenova et al. (2020) mAbs, 12(1):1692764.

[0016] [Figure 2] This image provides a surface electrostatic potential map (left) and spatial aggregation tendency (SAP) (right) of anti-GCGR Fab fragments containing VH (SEQ ID NO: 1) and VL (SEQ ID NO: 2). On the left, surface charge patches were analyzed based on electrostatic potential maps calculated by an adaptive Poisson-Boltzmann solver (APBS) against the averaged structure obtained from molecular dynamics (MD) trajectories. On the right, circles indicate large hydrophobic patches overlapping with four CDRs. The representations are the average SAP values ​​calculated for each atom across all representational MD trajectories.

[0017] [Figure 3] The SAP shows six CDRs, including four CDRs (L3, H1, H2, and H3) that have multiple (but not all) residues with high SAP values ​​(e.g., ≥2, e.g., SAP values ​​of 2.3 or higher) for anti-GCGR Fab, as shown on the right side of Figure 2.

[0018] [Figure 4] The aromatic amino acid residues in the CDR of the anti-GCGR Fab substituted in Example 1A are shown.

[0019] [Figure 5]This paper presents viscosity analysis of anti-GCGR IgG1 variants targeting aromatic residues with high SAP scores. Specifically, individual alanine mutations were introduced for each of the 10 parental aromatic residues with high SAP scores. The viscosity of these single mutants, along with a negative control mutant (VH Y79A), was measured by rheometry at a concentration of 180 mg / mL in 20 mM histidine acetate, pH 5.5, at 25.0°C (bars, left axis). The SAP scores of selected sites are also shown (right axis). Furthermore, three double mutants combining two viscosity-reducing mutations were tested to evaluate arbitrary additive effects. The viscosity of the parental anti-GCGR antibody is shown as a reference dotted line.

[0020] [Figure 6] Figure 5 shows a comparison of the viscosity of the single-substituted variant IgG1 antibody against the parent IgG1 antibody, versus the SAP values ​​of each amino acid in the original wild-type antibody.

[0021] [Figure 7] This paper presents the selection of anti-GCGR viscosity hotspot sites combining high SAP scores and high SASA ratios. SAP scores and SASA ratios were calculated and plotted against each other for all sites in the anti-GCGR Fab fragment. The dotted lines indicate the cutoff values ​​used for high SASA ratios (0.25) and high SAP scores (2.3). Sites exhibiting both high SASA ratios and high SAP scores (upper right quadrant) were selected for mutation analysis to assess their contribution to high viscosity. Furthermore, to provide a more comprehensive analysis, representative sites within high SASA ratio and low SAP regions (labeled residues in the upper left quadrant) as well as low SASA ratio and high SAP regions (labeled residues in the lower right quadrant) were also included.

[0022] [Figure 8]Plots of DLS interaction parameters (kD, ml / g) and viscosity (η, cP) for an exemplary anti-GCGR IgG1 antibody variant are shown. The Pearson correlation efficiency between the two sets of measurements was calculated to be -0.73. The thick black solid line represents the Pearson linear regression fit. DLS interaction parameters were measured between 2 and 10 mg / mL. Viscosity was measured at 180 mg / mL.

[0023] [Figure 9] This table reports the screening of DLS interaction parameters for approximately 200 anti-GCGR IgG1 variants. DLS interaction parameters are shown, measured at 25.0°C in 20 mM histidine acetate, pH 5.5. Variants with exceptionally low diffusion coefficients are highlighted (gray) to indicate their polydisperse behavior in solution. Parent residues are highlighted with (P). Mutants with expression or purification problems, or those showing significant aggregation, are also highlighted (black).

[0024] [Figure 10] Figure 9 shows plots of DLS interaction parameters (kD, ml / g) and dissociation constants (KD, M) for each anti-GCGR IgG1 antibody variant tested. The KD of anti-GCGR mutants to the extracellular domain of GCGR was determined by SPR. Variants with interaction parameters >12 mL / g and binding affinity within 5 times that of the parent antibody are shown in the upper left quadrant. The parent antibody is also highlighted using labeling. [Modes for carrying out the invention]

[0025] overview This specification provides a method for identifying antigen-binding polypeptide variants (e.g., antibodies, their antigen-binding fragments, and antibody-based constructs) having reduced viscosity compared to the parent antigen-binding polypeptide. Antigen-binding polypeptide variants (e.g., antibodies, their antigen-binding fragments, and antibody-based constructs) produced by this method are also provided. A library containing multiple antigen-binding polypeptide variants having reduced viscosity is also described herein. Furthermore, this application provides a method for predicting one or more amino acids in an antibody, an antigen-binding fragment of an antibody, or an antibody construct that affect the viscosity of the antibody, antigen-binding fragment, or antibody construct.

[0026] This application is based on the applicant's unexpected finding that key amino acids in antigen-binding polypeptides (e.g., antibodies, their antigen-binding fragments, or antibody-based constructs) that influence viscosity are characterized by having high spatial aggregation tendency (SAP) values ​​(e.g., ≥2, e.g., SAP values ​​of 2.3 or higher) and / or high solvent-accessible surface area (SASA) ratios (e.g., SASA ratio ≥0.25, e.g., ≥0.5). The applicant also unexpectedly found that antigen-binding polypeptide variants with reduced viscosity can be produced by substituting one or more amino acids in a parent antigen-binding polypeptide identified as having high SAP values ​​and / or high SASA ratios with amino acids having lower SAP values ​​and / or lower SASA ratios. The inventors also unexpectedly found a strong negative correlation between dynamic light scattering (DLS) interaction parameters and viscosity, highlighting the potential to use DLS independently as a predictive assay or to complement other activity assays in early-stage drug discovery. DLS screening methods require lower protein content than certain other viscosity assays, e.g., cone-and-plate rheometry. Furthermore, the DLS screening method can be used alone or in combination with binding assays to identify binding proteins with desired viscosity and / or target binding affinity, further enabling the therapeutic development of binding proteins such as antibodies.

[0027] Accordingly, in some embodiments, a method is provided for identifying antigen-binding polypeptide variants having reduced viscosity compared to the parent antigen-binding polypeptide, the method comprising: (a) identifying one or more wild-type amino acids in the parent antigen-binding polypeptide having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) substituting one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to the wild-type amino acids; and (c) identifying antigen-binding polypeptide variants having reduced viscosity compared to the parent polypeptide. In some embodiments, the method further comprises determining the viscosity of the antigen-binding polypeptide variant by measurement or the like.

[0028] In certain embodiments, a method is provided for identifying antigen-binding polypeptide variants having reduced viscosity compared to a parent antigen-binding polypeptide, the method comprising: (a) identifying one or more wild-type amino acids in a parent antigen-binding polypeptide having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) substituting one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to the wild-type amino acids; (c) determining the viscosity of the antigen-binding polypeptide variant by measurement or other means; and (d) identifying the antigen-binding polypeptide variant having reduced viscosity compared to the parent polypeptide.

[0029] As described herein, in some embodiments, the methods may be carried out using one or more processors, for example, using a computer. Thus, in some embodiments, polypeptides and polypeptide sequences, for example, antigen-binding polypeptide variants, may refer to various forms based on context, such as data representing molecular structure or sequence.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to the extent of this invention. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 3D Ed., John Wiley and Sons, New York (2006) provides those skilled in the art with a general dictionary of many of the terms used in this invention. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the invention, but preferred methods and materials are described herein. Numerical ranges include the number defining the range. Unless otherwise indicated, nucleic acids are written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxy direction. Experts refer to Sambrook et al., 1989 and Ausubel FM et al., 1993 in particular for definitions and terms in the art. It should be understood that this invention is not limited to the specific methods, protocols, and reagents described, and these may vary.

[0031] A numerical range includes the number that defines the range.

[0032] Unless otherwise indicated, nucleic acids are written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction.

[0033] The headings provided herein are not limitations on the various aspects or embodiments that can be obtained by referring to this specification as a whole. Therefore, the terms defined below are further defined by referring to the entire specification.

[0034] definition Before describing embodiments in detail, it should be understood that this disclosure is not limited to any particular composition or biological system and is naturally subject to change. It should also be understood that the terminology used herein is intended solely to describe a particular embodiment and is not intended to limit it.

[0035] When used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple subjects unless otherwise explicitly indicated by the content. Thus, for example, a reference to “a molecule” may optionally include a combination of two or more such molecules.

[0036] As used herein, the term “approximately” refers to the normal range of error for each value, as readily understood by those skilled in the art. References to values ​​or parameters referred to “approximately” herein include (and describe) embodiments relating to the value or parameter itself.

[0037] It should be understood that the aspects and embodiments of this disclosure include the terms "includes," "consist of," and "essentially consist of."

[0038] As used herein, the term "antibody" is used in the broadest sense and specifically includes intact antibodies (e.g., full-length antibodies), antibody fragments (including but not limited to Fab, F(ab’)2, Fab’-SH, Fv, diabody, scFv, scFv-Fc, single domain antibodies, single heavy chain antibodies, and single light chain antibodies), monoclonal antibodies, and polyclonal antibodies, as long as they exhibit the desired biological activity (e.g., epitope binding). "Antibody" (or "Ab") and "immunoglobulin" (or "Ig") are glycoproteins having the same structural features. Antibodies exhibit binding specificity for a particular antigen, while immunoglobulins include both antibodies and other antibody-like molecules lacking antigen specificity. Polypeptides of the latter type are produced, for example, at low levels by the lymphatic system and at high levels by myelomas.

[0039] As a reference frame, as used herein, immunoglobulin refers to the structure of immunoglobulin G (IgG). However, those skilled in the art will understand / recognize that antibodies of any immunoglobulin class can be utilized in the methods of the present invention described herein. For clarity, an IgG molecule contains a pair of heavy chains (HC) and a pair of light chains (LC). Each LC has one variable domain (V L ) and one constant domain (C L ), and each HC has one variable domain (V H ) and three constant domains (C H 1, C H 2, C H 3). The C H 1 and C H 2 domains are linked by a hinge region. This structure is well known in the art.

[0040] In short, a basic quadruple antibody unit is a heterotetrameric glycoprotein composed of two light (L) chains and two heavy (H) chains (IgM antibodies consist of five basic heterotetrameric units along with an additional polypeptide called a J chain, thus containing 10 antigen-binding sites, and secreted IgA antibodies can polymerize to form a multivalent aggregate containing 2-5 basic quadruple units along with the J chain). In the case of IgG, a quadruple unit is typically about 150,000 daltons. Each L chain is linked to the H chain by one disulfide covalent bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Both the H and L chains also have regularly spaced intrachain disulfide crosslinks. Each H chain has a variable domain (V) at its N-terminus. H ) has, and subsequently each of the α and γ chains has three constant domains (C H ), as well as for μ and ε isotypes, four C H It has a domain. Each L chain has a variable domain (V) at its N-terminus. L ) has a constant domain (C) at the opposite end. L ) has. V L V H They are aligned as C L This is the first constant domain of the heavy chain (C H 1) is aligned with the following. Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain. V H and V L When these two molecules pair together, they form a single antigen-binding site. For the structures and properties of various classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0041] The light chains (L chains) derived from any vertebrate species can be assigned to one of two distinct types called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each having a heavy chain denoted as α, δ, γ, ε, and μ, respectively. The γ and α classes are C H Based on relatively minor differences in sequence and function, they are further classified into subclasses. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0042] As used herein, the term “isolated” antibody may refer to an antibody substantially free of other cellular material. In one embodiment, the isolated antibody is substantially free of other proteins from the same species. In another embodiment, the isolated antibody is expressed by cells from different species and is substantially free of other proteins from different species. In some embodiments, the “isolated” antibody is identified, separated, and / or recovered from components of its natural environment. These contaminating components from its natural environment are substances that may interfere with the diagnostic and therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Antibodies can be made substantially free of naturally associated components (or components associated with the cell expression system used to produce the antibody) by isolating them using protein purification techniques well known in the art. In some embodiments, the antibody is purified (1) to more than 75% by weight, most preferably more than 80%, 90%, 95%, or 99% by weight, if obtained by the Lowry method, or (2) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. Isolated antibodies include in-situ antibodies in recombinant cells, because at least one component of the antibody's natural environment is absent. However, typically, isolated antibodies are prepared by at least one purification step.

[0043] As used herein, the term “epitope” means any antigenic determinant on an antigen to which an antibody paratope binds. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural and charge properties.

[0044] As used herein, the terms “native antibodies and immunoglobulins” typically refer to heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is attached to a heavy chain by one covalent disulfide bond (also called a “VH / VL pair”), although the number of disulfide bonds differs between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly separated intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other, with the constant domain of the light chain aligned with the first constant domain of the heavy chain, and the light chain variable domain aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain. For example, see Chothia et al., J.Mol.Biol., 186:651 (1985); Novotny and Haber, Proc.Natl.Acad.Sci.USA, 82:4592 (1985).

[0045] As used herein, the term “variable” refers to the fact that the sequences of specific portions of the variable domain vary widely among antibodies and are used in the binding and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portion of the variable domain is called the framework (FR). The native heavy-chain and light-chain variable domains each contain four FR regions, primarily employing a β-sheet arrangement, connected by three CDRs, which form loops that connect (and sometimes form part of) the β-sheet structure. The CDRs of each chain are closely spaced and held together by the FR regions, and together with the CDRs from the other chain, contribute to the formation of the antibody’s antigen-binding site. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991). The constant domain does not directly participate in antibody-antigen binding, but exhibits various effector functions, such as antibody participation in antibody-dependent cytotoxicity. The target variable region sequence includes the humanized variable region sequence of the CD47 antibody, which is described in detail elsewhere in this specification.

[0046] The terms "hypervariable regions (HVRs)" or "complementarity-determining regions (CDRs)" may refer to subregions of the VH and VL domains characterized by enhanced sequence variability and / or the formation of defined loops. These include three CDRs in the VH domain (H1, H2, and H3) and three CDRs in the VL domain (L1, L2, and L3). H3 is thought to be important for conferring fine binding specificity, while L3 and H3 exhibit the highest levels of variability. See Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0047] Several CDR / HVR descriptions are known. The Kabat complementarity-determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, on the other hand, refers to the location of the structural loop (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVR presents a compromise between Kabat HVR and the Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software. The “contact” HVR is based on the analysis of available complex crystal structures. The residues derived from each of these HVR / CDRs are shown below. “Framework” or “FR” residues are variable domain residues other than the HVR / CDR residues. TIFF2026517933000002.tif51170

[0048] "Extended" HVRs are also known: in VL, 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3); and in VH, 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) (Kabat numbering).

[0049] "Kabat numbering" may refer to the numbering system used for the heavy-chain variable domain or light-chain variable domain of antibody aggregates as described by Kabat et al. The actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of FR or HVR in the variable domain. Kabat numbering of residues can be determined for a given antibody by the alignment of the antibody sequence and the "standard" Kabat-numbered sequence in homologous regions. Typically, Kabat numbering is used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), while the EU numbering system or index is generally used when referring to residues in the heavy-chain constant region (e.g., EU index, EU IgG1 numbering as in Kabat).

[0050] As used herein, “monoclonal” antibody refers to a substantially homogeneous antibody, for example, an antibody obtained from a population of antibodies that are substantially identical but allow for trace amounts of background mutations and / or modifications. “Monoclonal” exhibits substantially homogeneous characteristics of the antibody and does not require antibody production by a specific method. In some embodiments, monoclonal antibodies are selected by their HVR, VH and / or VL sequences and / or binding properties, for example, from a pool of clones (e.g., recombinant, hybridoma, or phage-derived). Monoclonal antibodies can be manipulated to include one or more mutations, for example, to affect the antibody's binding affinity or other properties, to produce humanized or chimeric antibodies, to improve antibody production and / or homogeneity, and to manipulate multispecific antibodies, the resulting antibodies still considered essentially monoclonal. A population of monoclonal antibodies can be distinguished from polyclonal antibodies because the individual monoclonal antibodies in that population recognize the same antigenic site. Various techniques are known for the production of monoclonal antibodies; for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA method (e.g., U.S. Patent No. 4,816,567), phage display technology (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al.) al.,J.Mol.Biol.222:581-597(1992);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al.,J.Mol.Biol.340(5):1073-1093(2004);Fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004);and Lee et al., J.Immunol.Methods 284(1-2):119-132(2004), and techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin locus or genes encoding human immunoglobulin sequences (see, for example, International Publication No. 1998 / 24893; International Publication No. 1996 / 34096; International Publication No. 1996 / 33735; International Publication No. 1991 / 10741; Jakobovits et al., Proc.Natl.Acad.Sci.USA 90:2551(1993);Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature See Biotechnol. 14:826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0051] As used herein, the “Fc region” generally refers to the dimer complex containing the C-terminal polypeptide sequence of an immunoglobulin heavy chain, which can be obtained by papain digestion of an intact antibody. The Fc region may contain native or variant Fc sequences. While the boundaries of the Fc sequence of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc sequence includes the carboxyl terminus of the Fc sequence from approximately Cys226 or approximately Pro230. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The Fc sequence of an immunoglobulin generally consists of two constant domains, C H 2 domains and C H Includes 3 domains, optionally C H It contains four domains. In this specification, “Fc polypeptide” means one of the polypeptides that constitute the Fc region, e.g., monomer Fc. Fc polypeptides can be obtained from any suitable immunoglobulin, such as human IgG1, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD, or IgM. Fc polypeptides can also be obtained from mice, e.g., mouse IgG2a. The Fc region contains the carboxyl terminus of both H chains held together by a disulfide. The effector function of the antibody is determined by the sequence in the Fc region. This region is also the part recognized by the Fc receptor (FcR) found in specific cell types. In some embodiments, the Fc polypeptide contains some or all of the wild-type hinge sequence (generally at its N-terminus). In some embodiments, the Fc polypeptide does not contain a functional or wild-type hinge sequence.

[0052] As used herein, "Fc component" refers to the hinge region of the Fc region, CH 2 domains or C H This refers to 3 domains.

[0053] In certain embodiments, the Fc region preferably comprises an IgG Fc region derived from a wild-type human IgG Fc region. In certain embodiments, the Fc region is derived from “wild-type” mouse IgG, such as mouse IgG2a. “Wild-type” human IgG Fc or “wild-type” mouse IgG Fc refers to an amino acid sequence that is naturally present in the human population or mouse population, respectively. Of course, one or more modifications may be made to the wild-type sequence, just as the Fc sequence may differ slightly between individuals, and these may still be within the scope of the present invention. For example, the Fc region may contain modifications such as mutations in glycosylation sites or the inclusion of non-natural amino acids.

[0054] A “chimeric” antibody may refer to an antibody having a heavy and / or light chain portion from a particular isotype, class, or organism, and another portion from a different isotype, class, or organism. In some embodiments, the variable region originates from one origin or organism, and the constant region originates from another origin or organism.

[0055] A “humanized antibody” may refer to an antibody that primarily contains human sequences and minimal amounts of non-human (e.g., mouse or chicken) sequences. In some embodiments, a humanized antibody has one or more HVR sequences (having the desired binding specificity) derived from antibodies of a non-human (e.g., mouse or chicken) organism grafted onto a human recipient antibody framework (FR). In some embodiments, non-human residues are further grafted onto the human framework (which are not present in either the source antibody or the recipient antibody), for example, to improve antibody properties. Generally, a humanized antibody contains at least one, typically substantially all of two variable domains, all or substantially all of the hypervariable loops, corresponding to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the FRs are FRs of a human immunoglobulin sequence. Additionally, a humanized antibody optionally contains at least a portion of the constant region (Fc) of an immunoglobulin, typically at least a portion of the constant region of a human immunoglobulin. See Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0056] "Human" antibodies may refer to antibodies having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or antibodies produced using any of the techniques for producing human antibodies disclosed herein. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries; preparation of human monoclonal antibodies as described in Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991); Marks et al., J.Mol.Biol., 222:581 (1991); Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991); and transgenic animals modified to produce such antibodies in response to antigen challenge, but whose endogenous gene locus has been deactivated, e.g., immunized xenos (e.g., XENOMOUSE). (商標) By administering the antigen to chickens having a human immunoglobulin sequence (see U.S. Patents 6,075,181 and 6,150,584 relating to the technology) or to chickens having a human immunoglobulin sequence (see, for example, International Publication No. 2012162422, International Publication No. 2011019844 and International Publication No. 2013059159).

[0057] There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0058] As used herein, the term “antibody fragment” and all its grammatical variants are defined, in certain cases, as part of an intact antibody that includes the antigen-binding site or variable region of the intact antibody, excluding the constant heavy chain domain of the Fc region of the intact antibody (i.e., CH2, CH3, and / or CH4, depending on the antibody isotype). Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment (but not limited to (1) a single-chain Fv(scFv) molecule) having a primary structure consisting of a single uninterrupted sequence of consecutive amino acid residues (referred herein to as a “single-chain antibody fragment” or “single-chain polypeptide”); (2) a single-chain polypeptide containing only one light chain variable domain, or a fragment thereof containing three CDRs of a light chain variable domain and not the associated heavy chain portion; and (3) a single-chain polypeptide containing only one heavy chain variable region, or a fragment thereof containing three CDRs of a heavy chain variable region and not the associated light chain portion; and multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chains may contain any constant domain sequence found in the non-Fc region of an intact antibody (e.g., CH1 in an IgG isotype), and / or any hinge region sequence found in an intact antibody, and / or a leucine zipper sequence fused to or located within the hinge region sequence or constant domain sequence of the heavy chain.

[0059] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each possessing a single antigen-binding site, while the other is named the "Fc" fragment, reflecting its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites that can still crosslink antigens. "Fv" is the smallest antibody fragment containing complete antigen recognition and antigen-binding sites. In double-chain Fv species, this region consists of a dimer in which one heavy-chain variable domain and one light-chain variable domain are tightly and non-covalently associated. In single-chain Fv species (scFv), one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a mobile peptide linker so that the light and heavy chains can associate in a "dimer" structure similar to that in double-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site. See, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0060] The Fab fragment also includes the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines derived from the antibody hinge region. Fab'-SH is the herein designation for Fab' having a free thiol group in the cysteine ​​residue of the constant domain. The F(ab')2 antibody fragment was originally produced as a pair with the Fab' fragment, which has a hinge cysteine ​​in between. Other chemical couplings of antibody fragments are also known.

[0061] As used herein, the term “monoclonal antibody” (mAb) refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Each mAb is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma cultures that are not contaminated with other immunoglobulins. The modifier “monoclonal” indicates the characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced in immortalized B cells or their hybridomas, or by recombinant DNA methods.

[0062] Monoclonal antibodies include hybrid and recombinant antibodies produced by splicing the variable (including hypervariable) domain of a CD47 antibody with a constant domain (e.g., "humanized" antibodies), or by splicing the light chain with the heavy chain, or by splicing a chain from one species with a chain from another species, or by fusing with a heterologous protein, as long as they exhibit the desired biological activity, regardless of the designation of the species of origin or immunoglobulin class or subclass, as well as antibody fragments (e.g., Fab, F(ab')2, and Fv).

[0063] The monoclonal antibodies described herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is derived from a particular species or is identical or identical to a corresponding sequence in an antibody belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or identical to a corresponding sequence in an antibody belonging to a different species or another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity.

[0064] Antibodies that "bind to a target antigen" (such as monospecific or multispecific antibodies) are useful as diagnostic and / or therapeutic agents when targeting proteins or cells or tissues that express proteins, and are antibodies that bind to antigens, e.g., proteins, with sufficient affinity so as not to cross-react significantly with other proteins. In such embodiments, the degree of antibody binding to "non-target" proteins is less than about 10% of the antibody binding to that particular target protein, when determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA) or ELISA. With respect to antibody binding to target molecules, the terms "specific binding," "specifically binding," or "specific" to a particular polypeptide or epitope on a particular polypeptide target mean binding that is measurably different from nonspecific interactions (e.g., nonspecific interactions may be binding to bovine serum albumin or casein). Specific binding can be measured, for example, by determining the binding of the molecule compared to the binding of a control molecule. For example, specific binding can be determined against the target by competition with a similar control molecule, for example, against excessive unlabeled binding. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled targets. As used herein, the terms “specific binding,” “specifically binding,” or “specific” to an epitope on a particular polypeptide or a particular polypeptide target are, for example, K to a target. DThis can be indicated by molecules having affinity of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM, or more. In one embodiment, the term “specific binding” refers to binding in which a multispecific antibody binds to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0065] The commercially available reagents mentioned in the examples were used according to the manufacturer's instructions unless otherwise specified. Throughout the following examples and this specification, the cell source identified by the ATCC accession number is the American Type Culture Collection (Manassas, VA). Unless otherwise specified, the present invention uses standard procedures for recombinant DNA technology, such as those described in the textbooks above and below in this specification: Sambrook et al., supra; Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, NY, 1989); Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc., NY, 1990); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press, Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al., Current Protocols in Immunology, 1991.

[0066] All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0067] Method for identifying antigen-binding polypeptide variants with reduced viscosity compared to the parent antigen-binding polypeptide. In some embodiments, a method is provided for identifying antigen-binding polypeptide variants (e.g., antibody variants, antigen-binding fragments of antibody variants, or variants of antibody constructs) having reduced viscosity compared to a parent antigen-binding polypeptide (e.g., an antibody, its antigen-binding fragment, or antibody construct), comprising: (a) identifying one or more wild-type amino acids in the parent antigen-binding polypeptide having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) substituting one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to wild-type amino acids; (c) measuring the viscosity of the antigen-binding polypeptide variant; and (d) identifying the antigen-binding polypeptide variant having reduced viscosity compared to the parent antigen-binding polypeptide. In some embodiments, the method comprises (a) identifying one or more wild-type amino acids in the parent antigen-binding polypeptide having a high SAP value and a high SASA ratio. In some embodiments, the method includes (b) substituting one or more wild-type amino acids identified as having a high SAP value and a high SASA ratio with amino acids having a lower SAP value and a lower SASA ratio compared to the wild-type amino acids. In some embodiments, the method includes determining the SAP value and / or SASA ratio of one or more substituted amino acids in the antigen-binding polypeptide variant after step (b) and before step (c).

[0068] In some embodiments, a method is provided for reducing the viscosity of an antigen-binding polypeptide (e.g., an antibody, an antigen-binding fragment thereof, or an antibody construct), comprising: (a) identifying one or more wild-type amino acids in the antigen-binding polypeptide having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) substituting one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to the wild-type amino acids to produce an antigen-binding polypeptide variant (e.g., an antibody variant, an antigen-binding fragment of an antibody variant, or a variant of an antibody construct); (c) measuring the viscosity of the antigen-binding polypeptide variant; and (d) identifying an antigen-binding polypeptide variant having a reduced viscosity compared to the wild-type antigen-binding polypeptide, thereby reducing the viscosity of the antigen-binding polypeptide. In some embodiments, the method comprises (a) identifying one or more wild-type amino acids in an antigen-binding polypeptide having a high SAP value and a high SASA ratio. In some embodiments, the method includes (b) substituting one or more wild-type amino acids identified as having a high SAP value and a high SASA ratio with amino acids having a lower SAP value and a lower SASA ratio compared to wild-type amino acids to produce an antigen-binding polypeptide variant (e.g., an antibody variant, an antigen-binding fragment of an antibody variant, or a variant of an antibody construct).

[0069] In some embodiments of any of the methods described herein, the terms “parent antigen-binding polypeptide” and “antigen-binding polypeptide” are used interchangeably.

[0070] In some embodiments, one or more wild-type amino acids in an antigen-binding polypeptide (such as a parent antigen-binding polypeptide) identified as having a high SAP value and / or a high SASA ratio are surface-exposed amino acids. In some embodiments, surface-exposed amino acids are identified by structural modeling of the antigen-binding polypeptide (such as computational modeling) and / or by studying the elucidated structure of the antigen-binding polypeptide (such as the elucidated crystal structure). In some embodiments, one or more wild-type amino acids in a parent antigen-binding polypeptide identified as having a high SAP value and / or a high SASA ratio have large side chains. In some embodiments, “large side chain” refers to an amino acid side chain having a volume greater than about 189 Å, e.g., about 189 Å to about 228 Å. In some embodiments, one or more wild-type amino acids in a parent antigen-binding polypeptide identified as having a high SAP value and / or a high SASA ratio have large hydrophobic side chains. In some embodiments, one or more wild-type amino acids in a parent antigen-binding polypeptide identified as having a high SAP value and / or a high SASA ratio are aromatic amino acids. In some embodiments, aromatic amino acids are selected from the group consisting of: W, Y, and F. In some embodiments, one or more wild-type amino acids having large side chains or large hydrophobic side chains, identified as having high SAP values ​​and / or high SASA ratios, are substituted with one or more charged amino acids or amino acids having smaller side chains. In some embodiments, "smaller side chain" refers to an amino acid side chain having a volume of less than approximately 174 Å. In some embodiments, one or more charged amino acids or amino acids having smaller side chains are selected from the group consisting of A, I, L, V, D, E, H, K, R, N, Q, S, and T. In some embodiments, one or more aromatic amino acids identified as having high SAP values ​​and / or high SASA ratios are substituted with one or more charged amino acids or amino acids having smaller side chains. In some embodiments, one or more charged amino acids or amino acids having smaller side chains are selected from the group consisting of A, I, L, V, D, E, H, K, R, N, Q, S, and T.In some embodiments, one or more wild-type amino acids in a parent antigen-binding polypeptide identified as having a high SAP value and / or a high SASA ratio have smaller side chains. In some embodiments, one or more amino acids having smaller side chains are selected from the group consisting of: A, I, L, V, N, Q, S, G, P, C, M, or T. In some embodiments, one or more amino acids having smaller side chains are substituted with one or more charged amino acids. In some embodiments, one or more charged amino acids are selected from the group consisting of: R, K, H, D, and E. In some embodiments, one or more charged amino acids are selected from the group consisting of: R, K, and H. In some embodiments, one or more wild-type amino acids in a parent antigen-binding polypeptide identified as having a high SAP value and / or a high SASA ratio are substituted with R, K, or H. In some embodiments, one of the wild-type amino acids in a parent antigen-binding polypeptide identified as having a high SAP value and / or a high SASA ratio is substituted with, for example, H to produce an antigen-binding polypeptide variant containing a single histidine substitution. In some embodiments, histidine substitution is the only amino acid substitution in the antigen-binding polypeptide variant. In some embodiments, histidine substitution is the only amino acid substitution introduced into the antigen-binding polypeptide variant to reduce the viscosity of the antigen-binding polypeptide variant compared to the parent antigen-binding polypeptide.

[0071] In some embodiments, a method is provided for identifying antigen-binding polypeptide variants (e.g., antibody variants, antigen-binding fragments of antibody variants, or variants of antibody constructs) having reduced viscosity compared to a parent antigen-binding polypeptide (e.g., an antibody, its antigen-binding fragment, or antibody construct), comprising: (a) identifying one or more wild-type amino acids selected from the group consisting of W, Y, and F in a parent antigen-binding polypeptide having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) substituting one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to wild-type amino acids; (c) measuring the viscosity of the antigen-binding polypeptide variant; and (d) identifying the antigen-binding polypeptide variant having reduced viscosity compared to a parent antigen-binding polypeptide. In some embodiments, the method comprises (a) identifying one or more wild-type amino acids in a parent antigen-binding polypeptide having a high SAP value and a high SASA ratio. In some embodiments, the method includes (b) substituting one or more wild-type amino acids identified as having a high SAP value and a high SASA ratio with amino acids having a lower SAP value and a lower SASA ratio compared to the wild-type amino acids. In some embodiments, the method includes determining the SAP value and / or SASA ratio of one or more substituted amino acids in the antigen-binding polypeptide variant after step (b) and before step (c).In some embodiments, a method is provided for reducing the viscosity of an antigen-binding polypeptide (e.g., an antibody, an antigen-binding fragment thereof, or an antibody construct), comprising: (a) identifying one or more wild-type amino acids in the antigen-binding polypeptide selected from the group consisting of W, Y, and F having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) substituting one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to the wild-type amino acids to produce an antigen-binding polypeptide variant (e.g., an antibody variant, an antigen-binding fragment of an antibody variant, or a variant of an antibody construct); (c) measuring the viscosity of the antigen-binding polypeptide variant; and (d) identifying an antigen-binding polypeptide variant having a reduced viscosity compared to the wild-type antigen-binding polypeptide, thereby reducing the viscosity of the antigen-binding polypeptide. In some embodiments, one or more wild-type amino acids selected from the group consisting of W, Y, and F, which are identified as having a high SAP value and / or a high SASA ratio, are substituted with one or more charged amino acids or amino acids having a smaller side chain. In some embodiments, one or more charged amino acids or amino acids having a smaller side chain are selected from the group consisting of A, I, L, V, D, E, H, K, R, N, Q, S, and T. In some embodiments, one or more wild-type amino acids in the parent antigen-binding polypeptide, which are identified as having a high SAP value and / or a high SASA ratio, are substituted with R, K, or H. In some embodiments, one of the wild-type amino acids in the parent antigen-binding polypeptide, which are identified as having a high SAP value and / or a high SASA ratio, is substituted with, for example, H to produce an antigen-binding polypeptide variant containing a single histidine substitution. In some embodiments, the histidine substitution is the only amino acid substitution in the antigen-binding polypeptide variant. In some embodiments, the histidine substitution is the only amino acid substitution introduced into the antigen-binding polypeptide variant to reduce the viscosity of the antigen-binding polypeptide variant compared to the parent antigen-binding polypeptide.

[0072] In some embodiments, a method is provided for identifying antigen-binding polypeptide variants (e.g., antibody variants, antigen-binding fragments of antibody variants, or variants of antibody constructs) having reduced viscosity compared to a parent antigen-binding polypeptide (e.g., an antibody, its antigen-binding fragment, or antibody construct), comprising: (a) identifying one or more wild-type amino acids selected from the group consisting of W, Y, and F in a parent antigen-binding polypeptide having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) substituting one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with (i) Y or F (the wild-type amino acid is W), (ii) Y or W (the wild-type amino acid is F), and / or (iii) F or W (the wild-type amino acid is Y); (c) measuring the viscosity of the antigen-binding polypeptide variant; and (d) identifying the antigen-binding polypeptide variant having reduced viscosity compared to a parent antigen-binding polypeptide. In some embodiments, the method comprises (a) identifying one or more wild-type amino acids in a parent antigen-binding polypeptide having a high SAP value and a high SASA ratio. In some embodiments, the method comprises (b) substituting one or more wild-type amino acids identified as having a high SAP value and a high SASA ratio with amino acids having a lower SAP value and a lower SASA ratio compared to the wild-type amino acids. In some embodiments, the method comprises determining the SAP value and / or SASA ratio of one or more substituted amino acids in the antigen-binding polypeptide variant after step (b) and before step (c).In some embodiments, a method is provided for reducing the viscosity of an antigen-binding polypeptide (e.g., an antibody, an antigen-binding fragment thereof, or an antibody construct), comprising: (a) identifying one or more wild-type amino acids in the antigen-binding polypeptide selected from the group consisting of W, Y, and F having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) substituting one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with (i) Y or F (the wild-type amino acid is W), (ii) Y or W (the wild-type amino acid is F), and / or (iii) F or W (the wild-type amino acid is Y) to produce an antigen-binding polypeptide variant (e.g., an antibody variant, an antigen-binding fragment of an antibody variant, or a variant of an antibody construct); (c) measuring the viscosity of the antigen-binding polypeptide variant; and (d) identifying an antigen-binding polypeptide variant having a reduced viscosity compared to the wild-type antigen-binding polypeptide, thereby reducing the viscosity of the antigen-binding polypeptide.

[0073] SAP provides the effective, dynamically exposed hydrophobicity of a specific patch on the surface of an antigen-binding polypeptide. SAP is calculated for a spherical region centered on all atoms in the antigen-binding polypeptide. This gives a unique SAP value for each atom. The SAP of an amino acid residue is obtained by averaging the SAP of all its constituent atoms. Further details regarding SAP and the calculation of SAP values ​​are provided, for example, in Chennamsetty et al. (2009) PNAS USA, 106(29):11937-11942; Lauer et al. (2012) J Pharm Sci, 101(1):102-115; and elsewhere. In some embodiments, molecular dynamics simulations are used to prepare an average model of SAP that takes into account the flexibility of antibody molecules in solution. For example, see R. Salomon-Ferrer, DACase, RC Walker (2013) "An overview of the Amber biomolecular simulation package." WIREs Comput. Mol. Sci. 3, 198-210; and DACase, TECheatham, III, T. Darden, H. Gohlke, R. Luo, KMMerz, Jr., A. Onufriev, C. Simmerling, B. Wang and R. Woods (2005) "The Amber biomolecular simulation programs." J. Computat. Chem. 26, 1668-1688. Exemplary molecular dynamics tools include, for example, DACase, HMAktulga, K. Belfon, IYBen-Shalom, JTBerryman, SRBrozell, DSCerutti, TECheatham, III, GACisneros, VWDCruzeiro, TADarden, N. Forouzesh, G. Giambasu, T. Giese, MK Gilson, H. Gohlke, AWGoetz, J. Harris, S. Izadi, SAIzmailov, K. Kasavajhala, MC Kaymak, E. King, A. Kovalenko, T. Kurtzman, TSLee, P. Li, C. Lin, J. Liu, T. Luchko, R. Luo, M. Machado, V. Man, M. Manathunga, KMMerz, Y. Miao, O. Mikha ilovskii,G.Monard,H.Nguyen,KAO'Hearn,A.Onufriev,F.Pan,S.Pantano,R.Qi,A.Rahnamoun,DRRoe ,A.Roitberg,C.Sagui,S.Schott-Verdugo,A.Shajan,J.Shen,CLSimmerling,NRSkrynnikov,J.Smith ,J.Swails,RCWalker,J.Wang,J.Wang,H.Wei,X.Wu,Y.Wu,Y.Xiong,Y.Xue,DMYork,S.Zhao,Q.Zhu,and PAKollman(2023),Amber 2023,University of California,San Francisco;C.Tian,K.Kasavajhala,KAABelfon,L.Raguette,H.Huang,ANMigues J.Bickel,Y.Wang,J.Pincay,Q.Wu and C.Simmerling.(2019)「ff19SB:Amino-Acid-Specific Protein Backbone Parameters Trained against Quantum Mechanics Energy Surfaces in Solution.」J.Chem.Theory Comput.16,528-552; GPUs.2.Explicit Solvent Particle Mesh Ewald.」J.Chem.Theory Comput.9,3878-3888 is available for download from the SAP program Thanks for watching the 1st of the 1000s SAP applications ≧2 and 2.0, 2.1, 2.2,The SAP values ​​are any one of 3, 2.4, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0, or greater than 8.0, and include any value in between. In some embodiments, the SAP values ​​of one or more wild-type amino acids in the parent antigen-binding polypeptide were identified as having high SAP values ​​based on having an SAP value of 2.3 or greater.

[0074] The SASA ratio is typically calculated by a method involving in silicic rolling of a spherical probe approximating a water molecule around a complete atomic protein model. Various methods for calculating the SASA ratio of soluble proteins in folded and unfolded states are known in the art. See, for example, Ali et al. (2014) Current Protein and Peptide Science, 15(5):456-76. In some embodiments, the SASA ratio of wild-type amino acids in the parent antigen-binding protein is calculated according to the method described in ssbio(dot)readthedocs(dot)io / en / latest / instructions / msms(dot)html and Lee and Richards (1971) JMB 55(3):379-400, IN3-IN4. In some embodiments, the SASA ratio of one or more wild-type amino acids in the parent antigen-binding polypeptide identified as having a high SASA ratio is ≥0.25, e.g., 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, or greater than 1.0, and includes any value in between. In some embodiments, a high SASA ratio is 0.25 or greater. In some embodiments, a high SASA ratio is 0.5 or greater.

[0075] As discussed elsewhere in this specification, in some embodiments, the viscosity of antigen-binding polypeptide variants produced by substituting one or more wild-type amino acids identified as having high SAP values ​​and / or SASA ratios with amino acids having lower SAP values ​​and / or lower SASA ratios compared to wild-type amino acids is measured, for example, to determine whether the viscosity of the antigen-binding polypeptide variant is lower than that of the parent antigen-binding polypeptide. In some embodiments, the viscosity of the antigen-binding polypeptide variant is measured by dynamic light scattering (DLS) (e.g., indirectly). As discussed in the examples, the DLS interaction parameter (k D The viscosity (η, cP) may correlate with the viscosity (ml / gram). In some embodiments, the viscosity of the antigen-binding polypeptide variant is measured by DLS at concentrations of approximately 1 mg / ml to approximately 10 mg / ml (e.g., any one of approximately 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 mg / ml, including any value in between). In some embodiments, the viscosity of the antigen-binding polypeptide variant is measured by cone-plate rheometry. See, for example, Lang et al. (2020) Appl Sci, 10(1), 172 and Zhang et al. (2017) Curr Opin in Chem Eng, 16:48-55. In some embodiments, the viscosity of the antigen-binding polypeptide variant is measured by cone-plate rheometry, for example, at high concentrations. In some embodiments, high concentrations are approximately 50 mg / ml to approximately 300 mg / ml, for example, any one of approximately 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mg / ml, including any value in between.

[0076] In some embodiments, the parent antigen-binding polypeptide is an antibody, an antigen-binding fragment of an antibody, or an antibody construct, and the antibody, its fragment, or antibody construct comprises one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and one or more wild-type amino acids in one or more CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are identified as having a high SAP value and / or a high SASA ratio. In some embodiments, one or more wild-type amino acids in CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 identified as having high SAP values ​​and / or high SASA ratios are substituted with amino acids having lower SAP values ​​and / or lower SASA ratios, for example, as described above, to produce antibody variants, antigen-binding fragments of antibody variants, or antibody construct variants. In some embodiments, one or more wild-type amino acids in the parent antibody, its antigen-binding fragment, or antibody construct identified as having high SAP values ​​and / or high SASA ratios have large side chains. In some embodiments, one or more wild-type amino acids identified as having high SAP values ​​and / or high SASA ratios have large hydrophobic side chains. In some embodiments, one or more wild-type amino acids identified as having high SAP values ​​and / or high SASA ratios are aromatic amino acids. In some embodiments, the aromatic amino acids are selected from the group consisting of: W, Y, and F. In some embodiments, one or more wild-type amino acids having large side chains or large hydrophobic side chains, which are identified as having high SAP values ​​and / or high SASA ratios, are substituted with one or more charged amino acids or amino acids having smaller side chains. In some embodiments, one or more charged amino acids or amino acids having smaller side chains are selected from the group consisting of A, I, L, V, D, E, H, K, R, N, Q, S, and T.In some embodiments, one or more aromatic amino acids identified as having a high SAP value and / or a high SASA ratio are substituted with one or more charged amino acids or amino acids having a smaller side chain. In some embodiments, one or more charged amino acids or amino acids having a smaller side chain are selected from the group consisting of A, I, L, V, D, E, H, K, R, N, Q, S, and T. In some embodiments, one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio have a smaller side chain. In some embodiments, one or more amino acids having a smaller side chain are selected from the group consisting of: A, I, L, V, N, Q, S, G, P, C, M, or T. In some embodiments, one or more amino acids having a smaller side chain are substituted with one or more charged amino acids. In some embodiments, one or more charged amino acids are selected from the group consisting of: R, K, H, D, and E. In some embodiments, one or more charged amino acids are selected from the group consisting of: R, K, and H. In some embodiments, one or more wild-type amino acids in a parent antibody, its antigen-binding fragment, or antibody construct identified as having a high SAP value and / or a high SASA ratio are substituted with R, K, or H. In some embodiments, one of the wild-type amino acids in a parent antibody, its antigen-binding fragment, or antibody construct identified as having a high SAP value and / or a high SASA ratio is substituted with, for example, H to produce an antibody variant, its antigen-binding fragment, or antibody construct variant containing a single histidine substitution. In some embodiments, the histidine substitution is the only amino acid substitution in the antibody variant, its fragment, or antibody construct variant. In some embodiments, the histidine substitution is the only amino acid substitution introduced into the antibody variant, its fragment, or antibody construct variant to reduce the viscosity of the antibody variant, its fragment, or antibody construct variant compared to the parent antibody, its fragment, or antibody construct.

[0077] In some embodiments, the method includes (a) identifying one or more wild-type amino acids selected from the group consisting of W, Y, and F in one or more of the parent antibody, its antigen-binding fragment, or antibody construct CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) producing an antibody variant, its antigen-binding fragment, or antibody construct variant by substituting one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to the wild-type amino acids; (c) measuring the viscosity of the variant; and (d) identifying an antibody variant, its antigen-binding fragment, or antibody construct variant with reduced viscosity compared to the parent antibody, the antibody antigen-binding fragment, or the antibody construct. In some embodiments, the method includes (a) identifying one or more wild-type amino acids selected from the group consisting of W, Y, and F in one or more parent antibodies, antigen-binding fragments, or antibody constructs CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having a high spatial aggregation tendency (SAP) value and a high solvent-accessible surface area (SASA) ratio. In some embodiments, the method includes (b) substituting one or more wild-type amino acids identified as having a high SAP value and a high SASA ratio with amino acids having a lower SAP value and a lower SASA ratio compared to the wild-type amino acids to produce an antibody variant, its antigen-binding fragment, or an antibody construct variant. In some embodiments, the method includes determining the SAP value and / or SASA ratio of one or more substituted amino acids in the antibody variant, its antigen-binding fragment, or antibody construct variant after step (b) and before step (c).

[0078] In some embodiments, a method is provided for reducing the viscosity of an antibody, its antigen-binding fragment, or an antibody construct (or for identifying an antibody variant, its antigen-binding fragment, or an antibody construct variant with reduced viscosity), comprising: (a) identifying one or more wild-type amino acids in an antigen-binding polypeptide selected from the group consisting of W, Y, and F having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) substituting one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to the wild-type amino acids to produce an antibody variant, an antigen-binding fragment of an antibody variant, or a variant of an antibody construct; (c) measuring the viscosity of the antibody variant or the variant of an antibody construct; and (d) identifying a variant with reduced viscosity compared to a wild-type antibody, its antigen-binding fragment, or an antibody construct, thereby reducing the viscosity of the antibody, its antigen-binding fragment, or an antibody construct. In some embodiments, one or more wild-type amino acids selected from the group consisting of W, Y, and F, which are identified as having a high SAP value and / or a high SASA ratio, are substituted with one or more charged amino acids or amino acids having a smaller side chain. In some embodiments, one or more charged amino acids or amino acids having a smaller side chain are selected from the group consisting of A, I, L, V, D, E, H, K, R, N, Q, S, and T. In some embodiments, one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio are substituted with R, K, or H. In some embodiments, one of the wild-type amino acids identified as having a high SAP value and / or a high SASA ratio is substituted with, for example, H to produce an antibody variant, its antigen-binding fragment, or antibody construct variant containing a single histidine substitution. In some embodiments, the histidine substitution is the only amino acid substitution in the variant.In some embodiments, histidine substitution is the only amino acid substitution introduced into an antibody variant, its antigen-binding fragment, or antibody construct variant to reduce the viscosity of the variant compared to the parent antibody, its antigen-binding fragment, or antibody construct.

[0079] In some embodiments, a method for reducing the viscosity of an antibody, its antigen-binding fragment, or antibody construct (or a method for identifying an antibody variant, its antigen-binding fragment, or antibody construct variant with reduced viscosity), comprising: (a) identifying one or more wild-type amino acids in an antigen-binding polypeptide selected from the group consisting of W, Y, and F having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; and (b) one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio, (i) Y or F( A method is provided comprising: (ii) producing an antibody variant, an antigen-binding fragment of an antibody variant, or a variant of an antibody construct by substituting (i) the wild-type amino acid is W, (ii) Y or W (the wild-type amino acid is F), and / or (iii) F or W (the wild-type amino acid is Y); (c) measuring the viscosity of the antibody variant or the variant of the antibody construct; and (d) identifying a variant with reduced viscosity compared to a wild-type antibody, its antigen-binding fragment, or antibody construct, thereby reducing the viscosity of the antibody, its antigen-binding fragment, or antibody construct.

[0080] In some embodiments, the parent antigen-binding polypeptide is an antibody. In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the parent antigen-binding polypeptide is an antibody, its antigen-binding fragment, or an antibody construct containing the human IgG Fc region or a portion thereof, e.g., the CH2 and / or CH3 domain. In some embodiments, the human IgG constant region is the IgG1, IgG2, or IgG4 constant region.

[0081] In some embodiments, the parent antigen-binding polypeptide is an antibody construct (such as an antibody-based construct), for example, a multispecific antibody construct. Exemplary antibody constructs that can be used with the methods described herein include, but are not limited to, CrossMab, dual-action Fab ("DAF"), e.g., two-in-one DAF and four-in-one DAF, DutaMab, DT-IgG, knob-in-hole ("KIH") bispecific antibodies, e.g., common light chain KIH bispecific antibody and KIH bispecific antibody containing two different light chains, SEEDbodies, TrioMab, Dock and Lock bispecific constructs, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-Vs, V(H)-IgG, IgG(L)-Vs, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, scGv4-Ig, Zybodies, and DVI-IgG, e.g., four-in-one DVI-IgG. These and other multispecific antibody constructs are described, for example, in Spiess et al. (2015) Mol Immunol, 67(2 Pt A):95-106; Labrijn et al. (2019) Nat Revs Drug Discovery, 18:585-608; and elsewhere.

[0082] In some embodiments, the parent antigen-binding polypeptide is an antigen-binding fragment of an antibody, or a construct comprising an antigen-binding fragment of an antibody. Exemplary antigen-binding fragments and constructs comprising such fragments include, but are not limited to, Fab, Fab2, e.g., single-specific and bispecific Fab2, F(ab')2, e.g., single-specific and bispecific F(ab')2, trispecific Fab3, scFv, monovalent IgG, one-arm antibody, diabody, triabody, scFv-Vcs, minibody, V HExamples include H, V-NAR, hcIgG, IgNAR, nanobody, nanobody-HSA, bispecific T cell engager (BiTE), biaffinity retargeting molecule (DART), tandem diabody (TandAbs), scDiabodies, scDiabody-CH3, diabody-CH3, triplebody, mini antibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-VL-scFv, F(ab')2, -scFv2s, scFv-KIH, Fab-scFv-Fc, and intrabody. These and other antigen-binding fragments, as well as constructs containing such fragments, are described in more detail elsewhere, for example, Spiess et al. (2015) Mol Immunol, 67(2 Pt A):95-106; Labrijn et al. (2019) Nat Revs Drug Discovery, 18:585-608; and elsewhere.

[0083] In some embodiments, an antigen-binding polypeptide (e.g., an antibody, an antigen-binding fragment of an antibody, or an antibody construct) is subjected to at least one affinity maturation step (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 affinity maturation steps) before being used in the methods described herein (e.g., before substituting one or more wild-type amino acids having a high SAP and / or high SASA ratio with amino acids having a lower SAP and / or lower SASA ratio). In some embodiments, an antigen-binding polypeptide variant (e.g., an antibody variant, an antigen-binding fragment of an antibody variant, or an antibody construct variant) produced by the methods described herein is subjected to at least one affinity maturation step (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 affinity maturation steps) after substituting one or more wild-type amino acids having a high SAP and / or high SASA ratio with amino acids having a lower SAP and / or lower SASA ratio. Further details regarding affinity maturation are provided elsewhere in this specification. In some embodiments, the substituted amino acids in the antigen-binding polypeptide variant (i.e., amino acids with lower SAP values ​​and / or lower SASA ratios introduced into the variant to replace wild-type amino acids with high SAP values ​​and / or high SASA ratios) are immobilized during the affinity maturation process (e.g., not randomized or further substituted). In some embodiments, one or more amino acids in the antigen-binding polypeptide variant (e.g., amino acids with lower SAP values ​​and / or lower SASA ratios other than the wild-type amino acids with high SAP values ​​and / or high SASA ratios) are randomized to produce a library of randomized variants. In some embodiments, one or more amino acids in the randomized variants substituted to improve the affinity of the antigen-binding polypeptide variant to its target are located at or near the site of the antigen-binding polypeptide variant that contacts the target (e.g., target ligand or target antigen). The library of randomized variants is then screened to identify those variants that have the desired affinity to the target.

[0084] Accordingly, in certain embodiments, affinity maturation includes the steps of: mutagenerating or randomizing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 of an antibody variant, antigen-binding fragment of an antibody variant, or antibody construct variant obtained by the methods herein, at one or more positions to produce a library of randomized variants; contacting the library of randomized variants with a target (e.g., a target ligand or target antigen); detecting the binding of a target to one or more randomized variants; and obtaining one or more randomized variants that specifically bind to a target. In some embodiments, the randomized variants thus identified are referred to as “affinity-matured variants.” In some embodiments, one or more amino acids in CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 in antibody variants, antigen-binding fragments of antibody variants, or antibody construct variants having lower SAP values ​​and / or lower SASA ratios, introduced to replace wild-type amino acids having high SAP values ​​and / or high SASA ratios, are immobilized and not targeted for further randomization (e.g., without further mutagenesis). Methods for mutagenerating CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 in antibodies (or their antigen-binding fragments) are known in the art and are discussed elsewhere in this specification. Details regarding libraries and library screening are provided elsewhere in this specification.

[0085] In certain embodiments, the methods described herein further include the step of determining the nucleic acid sequence of an antigen-binding polypeptide variant obtained after at least one affinity maturation. In certain embodiments, the methods described herein further include the step of producing a randomized variant (e.g., a randomized antibody variant, an antigen-binding fragment of a randomized antibody variant, or a randomized antibody construct variant). In some embodiments, the method for producing a randomized variant (e.g., a randomized antibody variant, an antigen-binding fragment of a randomized antibody variant, or a randomized antibody construct variant) is a production-scale production process (e.g., a fermentation process).

[0086] In some embodiments, antigen-binding polypeptide variants (e.g., antibody variants, antigen-binding fragments of antibody variants, or antibody construct variants) produced according to the methods described herein are provided herein. In some embodiments, compositions are provided comprising antigen-binding polypeptide variants (e.g., antibody variants, antigen-binding fragments of antibody variants, or antibody construct variants) produced according to the methods described herein at concentrations of about 20 mg / ml to 500 mg / ml, for example, about 30 mg / ml to about 400 mg / ml, about 40 mg / ml to about 350 mg / ml, or about 50 mg / ml to about 300 mg / ml.

[0087] The amino acid sequences of exemplary VH domain variants of anti-GCGR antibodies produced according to the method described herein are provided below.

[0088] In some embodiments, affinity-mature antigen-binding polypeptide variants (e.g., affinity-mature antibody variants, antigen-binding fragments of affinity-mature antibody variants, or affinity-mature antibody construct variants) obtained after affinity maturation of antigen-binding polypeptide variants produced according to the method described herein are provided. In some embodiments, compositions are provided comprising affinity-mature antigen-binding polypeptide variants (e.g., affinity-mature antibody variants, antigen-binding fragments of antibody variants, or affinity-mature antibody construct variants) at concentrations of about 20 mg / ml to 500 mg / ml, for example, about 30 mg / ml to about 400 mg / ml, about 40 mg / ml to about 350 mg / ml, or about 50 mg / ml to about 300 mg / ml.

[0089] Method using dynamic light scattering (DLS) interaction parameters In certain embodiments, methods are provided herein that include the use of dynamic light scattering (DLS) interaction parameters for evaluating the viscosity of antigen-binding polypeptides. As described herein, the inventors have found a strong linear correlation between measured DLS interaction parameters and viscosity for antigen-binding polypeptides, such as antigen-binding polypeptide variants described herein. In some embodiments, methods for evaluating viscosity using DLS interaction parameters are configured to enable medium-throughput screening of multiple antigen-binding polypeptides, e.g., at least about 100 antigen-binding polypeptides, e.g., at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,000 antigen-binding polypeptides.

[0090] In some embodiments, the method includes measuring the DLS interaction parameters of a composition containing an antigen-binding polypeptide, for example, an antigen-binding polypeptide variant. In some embodiments, measuring the DLS interaction parameters of a composition containing an antigen-binding polypeptide includes subjecting the composition to DLS technology, wherein the composition contains an antigen-binding polypeptide in a concentration of about 0.1 mg / ml to about 100 mg / ml, for example, about 2 mg / ml to about 10 mg / ml, and each interaction parameter is measured using the following formula: D=D0(1+k D *c) In the formula, D = translational diffusion coefficient; D0 is the self-diffusion coefficient (value of D at concentration 0); c = protein concentration; and k D = Interaction parameter. Variant k D The more positive the value, the weaker the self-interaction of the variant. Conversely, the k of the variant... D The more negative the value, the stronger the self-interaction of the variant. See, for example, Sorret et al. (2016) Biophysical Journal, 111:1831-1842. Techniques for measuring DLS are publicly known in the art, including, for example, techniques involving fixed-angle multi-angle instruments. Furthermore, there are several techniques for analyzing DLS data, including: e.g., CUMULANT, CONTIN, and CORENN. For example, accessed September 1, 2023.<https: / / lsinstruments.ch / en / theory / dynamic-light-scattering-dls / introduction> The entire text is incorporated herein by reference.

[0091] In some embodiments, the method includes determining the viscosity of an antigen-binding polypeptide based on its DLS interaction parameters by comparing the measured DLS interaction parameters of the antigen-binding polypeptide with a standard curve and / or reference. In some embodiments, the standard curve and / or reference includes known viscosities and DLS interaction parameters of other polypeptides, e.g., variants of the antigen-binding polypeptide.

[0092] In certain embodiments provided herein, a method for evaluating the viscosity of an antigen-binding polypeptide is further paired with an assay (e.g., a binding assay, an activity assay, or an efficacy assay) for evaluating the affinity between the antigen-binding polypeptide and a target antigen, for example, in order to evaluate the antigen-binding polypeptide. Thus, such methods taught herein enable the evaluation of the viscosity and desired attributes of the antigen-binding polypeptide (e.g., binding affinity, activity, or potency to a target antigen), which can aid in the development of therapeutic agents for identifying useful antigen-binding polypeptides with desired properties, such as low viscosity and high binding affinity. Techniques for measuring the binding affinity of antigen-binding polypeptides are known in the art and include techniques involving surface plasmon resonance (SPR) and biolayer interference (BLI). For example, Regenmortel & Azimzadeh, J Immunoassay, 21, 2000, which is incorporated herein by reference in its entirety. Techniques for performing activity and efficacy assays are known in the art and include animal model and cell-based assays. For example, Jiang & Mire-Sluis, Biosimilars, 2018, which is incorporated herein by reference in its entirety.

[0093] In certain embodiments, methods are provided herein that include: (a) mutagenerating an antigen-binding polypeptide based on the SAP value and / or SASA ratio described herein (e.g., based on a variant having a high SAP value and / or a high SASA ratio); and (b) identifying one or more mutants having a desirable viscosity described herein (e.g., low viscosity or viscosity lower than that of the parent antigen-binding polypeptide on which the mutagenesis was based) using DLS. In some embodiments, the method further includes performing one or more additional assays, e.g., binding assays, activity assays or efficacy assays, to evaluate the characteristics of the mutant antigen-binding polypeptide. For example, in some embodiments, such methods are useful for identifying antigen-binding polypeptide variants having reduced viscosity and a desired (e.g., higher) binding affinity or activity compared to the parent antigen-binding polypeptide.

[0094] A method for predicting amino acids that affect viscosity in antigen-binding polypeptides. In some embodiments, a method is provided for predicting one or more amino acids in an antigen-binding polypeptide that will affect the viscosity of the antigen-binding polypeptide. In some embodiments, the method comprises identifying one or more amino acids in an antigen-binding polypeptide having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio, wherein one or more amino acids having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio are predicted to affect the viscosity of the antigen-binding polypeptide. In some embodiments, the method comprises identifying one or more amino acids in an antigen-binding polypeptide having a high spatial aggregation tendency (SAP) value and a high solvent-accessible surface area (SASA) ratio, wherein one or more amino acids having a high spatial aggregation tendency (SAP) value and a high solvent-accessible surface area (SASA) ratio are predicted to affect the viscosity of the antigen-binding polypeptide.

[0095] In some embodiments, a method is provided herein for predicting one or more amino acids in an antibody, an antigen-binding fragment of an antibody, or an antibody construct that affect the viscosity of the antibody, antigen-binding fragment, or antibody construct, wherein the antibody, antigen-binding fragment, or antibody construct comprises one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, the method comprising identifying one or more amino acid positions in one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that have a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio, the one or more amino acid positions having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio are predicted to affect the viscosity of the antibody, antigen-binding fragment, or antibody construct. In some embodiments, the method involves identifying one or more amino acid positions in one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that have high spatial aggregation tendency (SAP) values ​​and high solvent-accessible surface area (SASA) ratios, where one or more amino acid positions with high spatial aggregation tendency (SAP) values ​​and high solvent-accessible surface area (SASA) ratios are predicted to affect the viscosity of the antibody, antigen-binding fragment, or antibody construct.

[0096] In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody contains the human IgG Fc region or a portion thereof, e.g., the CH2 and / or CH3 domain. In some embodiments, the human IgG constant region is the IgG1, IgG2, or IgG4 constant region. In some embodiments, the antibody fragment is Fab, F(ab')2, trispecific Fab3, scFv, monovalent IgG, diabody, tribody, scFv-Vc, minibody, V HThese are H, V-NAR, hcIgG, or IgNAR. Other exemplary antibody fragments used with this method are, but are not limited to, those described in detail elsewhere in this specification. In some embodiments, the antibody construct is, for example, CrossMab, dual-acting Fab (DAF), DVD-IgG, or a knob-in-hole bispecific antibody. Other exemplary antibody constructs used with this method are, but are not limited to those described in detail elsewhere in this specification.

[0097] Libraries and library screening In some embodiments, a library comprising multiple antigen-binding polypeptide variants is provided herein, wherein at least one variant in the library comprises one or more amino acid substitutions compared to a parent antigen-binding polypeptide, wherein one or more amino acid substitutions compared to the parent antigen-binding polypeptide replace one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to wild-type amino acids. In some embodiments, each variant in the library comprises one or more amino acid substitutions compared to a parent antigen-binding polypeptide, wherein one or more amino acid substitutions compared to the parent antigen-binding polypeptide replace one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to wild-type amino acids. In some embodiments, the library is screened to identify antigen-binding polypeptide variants with reduced viscosity compared to the parent antigen-binding polypeptide. In some embodiments, the library is screened to identify antigen-binding polypeptide variants that have reduced viscosity compared to the parent antigen-binding polypeptide, and affinities to targets (e.g., target ligands or target antigens) that are equivalent to, at least as high as, or improved compared to, the affinity of the parent antigen-binding polypeptide to the target. In some embodiments, an affinity to a target that is "equivalent" to the affinity of the parent antigen-binding polypeptide to the target is an affinity within five times the affinity of the parent antigen-binding polypeptide to the target (e.g., any one of approximately 4.5 times, 4.0 times, 3.5 times, 3.0 times, 2.5 times, 2.0 times, 1.5 times, or less than 1.5 times (including any value in between)).

[0098] In some embodiments, the library is a polypeptide library (e.g., multiple antigen-binding polypeptide variants, e.g., antibody variants, antigen-binding fragments of antibody variants, or antibody construct variants). In some embodiments, the polypeptide library is a polypeptide display library. Such polypeptide display libraries can be screened to select and / or evolve antigen-binding polypeptide variants with desired properties for a wide variety of uses, including but not limited to therapeutic, prophylactic, veterinary, diagnostic, reagent, or material applications. In certain embodiments, the library is a nucleic acid library where each nucleic acid (or group of nucleic acids) encodes a different antigen-binding polypeptide variant. In some embodiments, the library is a group of host cells (e.g., prokaryotic or eukaryotic host cells) each containing (and, for example, expressing) a different nucleic acid (or group of nucleic acids), where each different nucleic acid (or group of nucleic acids) encodes a different antigen-binding polypeptide variant.

[0099] In certain embodiments, the plurality of antigen-binding polypeptide variants in the library described herein include at least 2, 3, 4, 5, 10, 30, 100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000, 25000, 50000, 75000, 100000, 250000, 500000, 750000, 1000000, 2500000, 5000000, 7500000, 10000000, or more than 10000000 different antigen-binding polypeptide variants (e.g., unique antigen-binding polypeptide variants), and any value in between. In certain embodiments, the library of antigen-binding polypeptide variants includes about 2, about 5, about 10, about 50, about 100, about 250, about 500, about 750, about 10 3 , about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 1012 , about 10 13 , about 10 14 , or about 10 14 Over (for example, about 10 15 or about 10 16 It has array diversity (including any value in between).

[0100] In certain embodiments, libraries are generated via genetic engineering. Various methods for mutagenesis and subsequent library construction have been previously described (along with appropriate methods for screening or selection). Such mutagenesis methods include, but are not limited to, error-prone PCR, loop shuffling, oligonucleotide-directed mutagenesis, random nucleotide insertion, or other pre-recombination methods. Further details on these methods are described, for example, in Abou-Nadler et al. (2010) Bioengineered Bugs 1,337-340; Firth et al. (2005) Bioinformatics 21,3314-3315; Cirino et al. (2003) Methods Mol Biol 231,3-9; Pirakitikulr (2010) Protein Sci 19,2336-2346; Steffens et al. (2007) J. Biomol Tech 18,147-149; and others. Therefore, in certain embodiments, a library of multispecific antigen-binding proteins generated via genetic engineering techniques is provided.

[0101] In certain embodiments, the library is produced by in vitro translation. Briefly, in vitro translation involves cloning a polypeptide-encoding sequence(s) into a promoter-containing vector, producing mRNA by transcribing the cloned sequence(s) with RNA polymerase, and synthesizing the polypeptide by translating this mRNA in vitro, for example, using a cell-free extract. The desired antigen-binding polypeptide variant can be produced simply by modifying the sequence encoding the cloned polypeptide. Many mRNAs can be efficiently translated in wheat germ extract or rabbit reticulocyte lysate. Further details regarding in vitro translation can be found, for example, in Hope et al. (1985) Cell 43, 177-188; Hope et al. (1986) Cell 46, 885-894; Hope et al. (1987) EMBO J. 6, 2781-2784; Hope et al. (1988) Nature 333, 635-640; and Melton et al. (1984) Nucl. Acids Res. 12, 7057-7070.

[0102] Accordingly, in some embodiments, a plurality of nucleic acid molecules encoding the polypeptide display library described herein are provided. Expression vectors operably linked to the plurality of nucleic acid molecules are also provided herein. A plurality of nucleic acids encoding the plurality of antigen-binding domains described herein are also provided, as well as a method for constructing the library provided herein by expressing the nucleic acids.

[0103] In certain embodiments, the libraries provided herein are produced by chemical synthesis. Methods for solid-phase and liquid-phase peptide synthesis are well known in the art, for example: Fmoc Solid Phase Peptide Synthesis, A Practical Approach, (WCChan, PD White Eds), Oxford University Press, 2000; Solid Phase Synthesis, A Practical Guide, (SFKates, F Albericio Eds), Marcel Dekker, 2000; P. Seneci, Solid-Phase Synthesis and Combinatorial Technologies, John Wiley & Sons, 2000; Synthesis of Peptides and Peptidomimetics (M. Goodman, Editor-in-chief, A. Felix, L. Moroder, C. Tmiolo Eds), Thieme, 2002; NLBenoiton, Chemistry of Peptide Synthesis, CRC Press, 2005; Methods in Molecular Biology, 298, Peptide Synthesis and Applications, (J. Howl Ed) Humana This is described in detail in Press, 2005; and Amino Acids, Peptides and Proteins in Organic Chemistry, Volume 3, Building Blocks, Catalysts and Coupling Chemistry, (AB Hughs, Ed.) Wiley-VCH, 2011. Thus, in certain embodiments, a multispecific antigen-binding protein library produced by chemical synthesis techniques is provided.

[0104] In certain embodiments, the libraries provided herein are display libraries. In certain embodiments, the display libraries are phage display libraries, phagemide display libraries, virus display libraries, bacterial display libraries, yeast display libraries, λgt11 libraries, CIS display libraries, and in vitro compartmentalized libraries, or ribosome display libraries. Methods for preparing and screening such display libraries are well known to those skilled in the art, for example, Molek et al. (2011) Molecules 16,857-887; Boder et al. (1997) Nat Biotechnol 15,553-557; Scott et al. (1990) Science 249,386-390; Brisette et al. (2007) Methods Mol Biol 383,203-213; Kenrick et al. (2010) Protein Eng Des Sel 23,9-17; Freudl et al. (1986) J Mol Biol 188,491-494; Getz et al. (2012) Methods Enzymol 503,75-97; Smith et al. (2014) Curr Drug Discov Technol This information is described in 11,48-55; Hanes, et al. (1997) Proc Natl Acad Sci USA 94,4937-4942; Lipovsek et al. (2004) J Imm Methods 290,51-67; Ullman et al. (2011) Brief. Funct. Genomics,10,125-134; Odegrip et al. (2004) Proc Natl Acad Sci USA 101,2806-2810; and Miller et al. (2006) Nat Methods 3,561-570.

[0105] In certain embodiments, the libraries provided herein are RNA-protein fusion libraries produced by techniques described, for example, Szostak et al., U.S. Patent No. 6,258,558, U.S. Patent No. 6,261,804, U.S. Patent No. 5,643,768, and U.S. Patent No. 5,658,754. In certain embodiments, the libraries provided herein are DNA-protein libraries, such as those described, for example, U.S. Patent No. 6,416,950.

[0106] Screening method In some embodiments, the libraries described herein are screened to identify antigen-binding polypeptide variants (e.g., antibody variants, antigen-binding fragments of antibody variants, or antibody construct variants) with reduced viscosity compared to the parent antigen-binding polypeptide (e.g., parent antibody, antigen-binding fragment of parent antibody, or parent antibody construct). In some embodiments, the libraries provided herein can be screened to identify antigen-binding polypeptide variants (e.g., antibody variants, antigen-binding fragments of antibody variants, or antibody construct variants) that have low viscosity (e.g., reduced viscosity compared to the parent antigen-binding protein) and / or affinity for the target antigen that is equivalent to, at least as high as, or improved compared to, the affinity of the parent antigen-binding polypeptide (e.g., parent antibody, antigen-binding fragment of parent antibody, or parent antibody construct) for the target antigen.

[0107] In certain embodiments, screening a library includes the steps of: contacting the library described herein with a target of interest and an antigen-binding polypeptide variant in the library that specifically binds to that target; detecting the binding of the target to an antigen-binding polypeptide variant that specifically binds to the target (e.g., detecting a complex comprising the target and an antigen-binding polypeptide variant that specifically binds to the target); and obtaining an antigen-binding polypeptide variant that specifically binds to the target. In some embodiments, the method further includes measuring the viscosity of the antigen-binding polypeptide variant obtained in this manner (e.g., according to the method described herein) to identify antigen-binding polypeptide variants having a lower viscosity (e.g., less than about 50 cP, less than about 20 cP, or less than about 10 cP when measured at a high concentration such as 180 mg / mL, 20 mM histidine acetate, pH 5.5, 25°C) or reduced viscosity compared to the parent antigen-binding polypeptide.

[0108] In some embodiments, library screening further includes subjecting the antigen-binding polypeptide variants identified by library screening or obtained by using the methods herein to at least one affinity maturation step (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 affinity maturation steps). In some embodiments, the antigen-binding polypeptide is subjected to at least one affinity maturation step (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 affinity maturation steps) to obtain affinity-mature antigen-binding polypeptide variants, and the affinity-mature antigen-binding polypeptide variants are used in the methods herein or in libraries or library screening described herein. Affinity maturation is the process of subjecting antigen-binding polypeptide variants (e.g., antibody variants, antigen-binding fragments of antibody variants, or antibody construct variants) produced by methods provided herein or by screening libraries provided herein to a scheme for selection due to increased affinity for a target (e.g., a target ligand or target antigen) (see Wu et al. (1998) Proc Natl Acad Sci USA. 95, 6037-42).Further details regarding antibody affinity maturation can also be found in, for example, Merchant et al. (2013) Proc Natl Acad Sci US A.110(32):E2987-96; Julian et al. (2017) Scientific Reports.7:45259; Tiller et al. (2017) Front.Immunol.8:986; Koenig et al. (2017) Proc Natl Acad Sci US A.114(4):E486-E495; Yamashita et al. (2019) Structure.27,519-527; Payandeh et al. (2019) J Cell Biochem.120:940-950; Richter et al. (2019) mAbs.11(1):166-177; and Cisneros et al. This is also described in detail in al. (2019) Mol.Syst.Des.Eng.4:737-746.

[0109] In some embodiments, the substituted amino acids in the antigen-binding polypeptide variant (i.e., amino acids with lower SAP values ​​and / or lower SASA ratios introduced into the variant to replace wild-type amino acids with high SAP values ​​and / or high SASA ratios) are immobilized during the affinity maturation process (e.g., not randomized or further substituted). In some embodiments, one or more amino acids in the antigen-binding polypeptide variant (e.g., amino acids with lower SAP values ​​and / or lower SASA ratios other than wild-type amino acids with high SAP values ​​and / or high SASA ratios) are randomized to produce a library of randomized variants. In some embodiments, one or more amino acids in the randomized variants are substituted to improve the affinity of the antigen-binding polypeptide variant to its target. In some embodiments, the one or more amino acids in the randomized variants that are substituted to improve the affinity of the antigen-binding polypeptide variant to its target are located at or near the site of the antigen-binding polypeptide variant that contacts the target (e.g., target ligand or target antigen). The library of randomized variants is then screened to identify those variants that have the desired affinity to the target. In some embodiments, variants obtained after affinity maturation are called "affinity-matured variants."

[0110] In some embodiments, where the antigen-binding polypeptide is an antibody, its antigen-binding fragment, or an antibody construct, or any of the aforementioned variants obtained by using the methods herein, one or more amino acids that are randomized during affinity maturation are V H and / or V LIt is located within. In some embodiments, one or more amino acids that are randomized during affinity maturation are located within CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3. Therefore, in certain embodiments, the method described herein is used to obtain an antibody variant, an antigen-binding fragment of an antibody variant, or a V of an antibody construct variant obtained by the method herein. H and / or V LThe process includes (e.g., further including) the steps of: mutagenerating or randomizing (e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3) at one or more positions to produce a library of randomized variants; contacting the library of randomized variants with a target (e.g., a target ligand or target antigen); detecting the binding of the target to one or more randomized variants; and obtaining one or more randomized variants that specifically bind to the target. In some embodiments, one or more amino acids in the antibody variants, antigen-binding fragments of the antibody variants, or CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 of the antibody are immobilized (e.g., not targeted for further randomization or mutagenesis). In some embodiments, antibody variants having lower SAP values ​​and / or lower SASA ratios are introduced into the variant to replace wild-type amino acids having high SAP values ​​and / or high SASA ratios, antigen-binding fragments of the antibody variant, or one or more amino acids in CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 in the antibody are not immobilized (e.g., targeting further randomization or mutagenesis). In certain embodiments, affinity-mature antibody variants, their antigen-binding fragments, or affinity-mature antibody construct variants include at least one or at least two randomized CDRs that have not been previously randomized. In some embodiments, affinity-matured antibody variants, their antigen-binding fragments, or affinity-matured antibody construct variants obtained after two or more rounds of affinity maturation have low viscosity (e.g., lower viscosity compared to the parent antibody, fragment, or construct) and / or bind to the target of interest with an affinity at least as high as that of affinity-matured antibody variants, their antigen-binding fragments, or affinity-matured antibody construct variants obtained after one round of affinity maturation.In some embodiments, affinity-matured antibody variants, their antigen-binding fragments, or affinity-matured antibody construct variants that have undergone two or more rounds of affinity maturation have a lower viscosity than the parent antibody, its antigen-binding fragment, or the parent antibody construct, and bind to the target of interest with an affinity that is equivalent to, at least as high as, or better than, that of the parent antibody, its antigen-binding fragment, or the parent antibody construct for binding to the target of interest.

[0111] In some embodiments, the method further includes measuring the viscosity of affinity-matured antigen-binding polypeptide variants (i.e., variants obtained after at least one affinity-mature step) according to, for example, the method described herein, to identify affinity-matured antigen-binding polypeptide variants having high affinity for a target of interest (e.g., affinity for a target of interest that is equivalent to, at least as high as, or improved compared to, the affinity of the parent antigen-binding polypeptide to the target) and low viscosity (e.g., reduced viscosity compared to the parent antigen-binding polypeptide). In some embodiments, low viscosity refers to a viscosity of less than about 50 cP, less than about 20 cP, or less than about 10 cP when measured at high concentrations (e.g., 180 mg / mL, 20 mM histidine acetate, pH 5.5, 25°C).

[0112] In some embodiments, affinity matured variants are subjected to further characterization. For example, in some embodiments, further characterization includes (a) identifying one or more amino acids in the affinity matured variant having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) substituting the amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to the affinity matured variant to produce a substituted affinity matured variant; (c) measuring the viscosity of the substituted affinity matured variant; and (d) identifying the substituted affinity matured variant having a reduced viscosity compared to the affinity matured variant. In some embodiments, the affinity of the substituted affinity matured variant for its target is measured, for example, according to methods described elsewhere in this specification. In some embodiments, the substituted affinity-matured variant has a low viscosity (e.g., reduced viscosity compared to the parent antigen-binding polypeptide) and a high affinity for the target of interest (e.g., an affinity for the target of interest that is equivalent to, at least as high as, or improved compared to, the affinity of the parent affinity-matured variant for the target). In some embodiments, the substituted affinity-matured variant undergoes one or more further affinity-mature steps (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 further affinity-mature steps).

[0113] Antigen-binding polypeptides (e.g., antibody variants, antigen-binding fragments, or antibody construct variants obtained by screening the libraries herein or by using the methods herein) H and / or V LMethods for mutagenerating one or more of the following (or one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and / or CDR-L3) are known in the art and may include, for example, random mutagenesis, CDR walking mutagenesis or sequential and parallel optimization, mutagenesis by structure-based rational design, site-directed mutagenesis, enzyme-based mutagenesis, chemical-based mutagenesis, and gene synthesis methods for synthetic antibody gene production. For example, see Yang et al., 1995, CDR Walking Mutagenesis for the Affinity Mutation of a Potent Human Anti-HIV-1 Antibody into the Picomolar Range, J.Mol.Biol.254:392-40, and Lim et al., 2019, Review: Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation, Int.J.Mol.Sci, 20:1861 (the contents of both are incorporated herein by reference in their entirety).

[0114] The libraries of antigen-binding polypeptide variants described herein may be screened by any technique known in the art to evolve novel or improved binding proteins that specifically bind to target ligands. In certain embodiments, the target ligand is immobilized on a solid support (such as a column resin or microtiter plate well) and the target ligand is brought into contact with a library of candidate multispecific antigen-binding proteins (such as any library described herein). The selected techniques may include, for example, phage display (Smith (1985) Science 228, 1315-1317), mRNA display (Wilson et al. (2001) Proc Natl Acad Sci USA 98:3750-3755), bacterial display (Georgiou, et al. (1997) Nat Biotechnol 15:29-34), yeast display (Boder and Wittrup (1997) Nat. Biotechnol. 15:553-5577), or ribosome display (Hanes and Pluckthun (1997) Proc Natl Acad Sci USA 94:4937-4942 and International Publication No. 2008 / 068637).

[0115] In certain embodiments, the library of antigen-binding domain polypeptide variants is a phage display library. In certain embodiments, phage particles presenting the antigen-binding domain variants described herein are provided. In certain embodiments, phage particles presenting the antigen-binding domain variants described herein that can bind to a target ligand are provided.

[0116] Phage display is a technique that presents multiple multispecific antigen-binding protein variants as fusion proteins with the coating protein on the surface of bacteriophage particles (Smith, GP (1985) Science, 228:1315-7; Scott, J.K. and Smith, GP (1990) Science 249:386; Sergeeva, A., et al. (2006) Adv. Drug Deliv. Rev. 58:1622-54). The usefulness of phage display lies in the fact that it allows for the rapid and efficient sorting of large libraries of selectively randomized protein variants (or randomly cloned cDNAs) for sequences that bind to target molecules with high affinity.

[0117] Displaying libraries of peptides on phages (Cwirla, SE et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378) or proteins (Lowman, H B et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352:624; Marks, J Det al. (1991), J. Mol. Biol., 222:581; Kang, A Set al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) has been used to screen millions of polypeptides or oligopeptides for those with specific binding properties (Smith, GP (1991) Current Opin. Biotechnol., 2:668; Wu et al. (1998) Proc Natl Acad Sci USA. May 95,6037-42). Multivalent phage display methods have been used to display random small peptides and small proteins by filamentous phage fusion to either gene III or gene VIII. (Wells and Lowman, Curr. Opin. Struct. Biol., 3:355-362 (1992), and references cited therein.) In monovalent phage display, a protein or peptide library is fused to gene III or a portion thereof and expressed at low levels in the presence of wild-type gene III protein, so that the phage particle displays one copy of the fusion protein or none at all. The binding force effect is reduced for multivalent phages so that selection is based on endogenous ligand affinity, and phagemid vectors are used, which simplifies DNA manipulation. (Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:205-0216 (1991).)

[0118] Selecting phage libraries of antigen-binding polypeptide variants requires procedures for constructing and amplifying numerous variants, affinity purification using target ligands, and means for evaluating the results of binding enrichment (see, for example, U.S. Patents 5,223409, 5,403484, 5,571689, and 5,663143).

[0119] Most phage display methods use filamentous phages (such as M13 phage). Examples include the Lambdoid phage display system (see International Publication No. 1995 / 34683, U.S. Patent No. 5627024), the T4 phage display system (Ren et al. (1998) Gene 215:439; Zhu et al. (1998) Cancer research, 58:3209-3214; Jiang et al., (1997) Infection & Immunity, 65:4770-4777; Ren et al. (1997) Gene, 195:303-311; Ren (1996) Protein Sci., 5:1833; Efimov et al. (1995) Virus Genes, 10:173), and the T7 phage display system (Smith and Scott (1993) Methods in Enzymology, 217:228-257; U.S. Patent No. 5766905 is also known.

[0120] Many other improvements and modifications of the basic phage display concept are currently under development. These improvements enhance the display system's ability to screen peptide libraries for binding to selected target molecules and present functional proteins that have the potential to screen these proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (International Publication 1998 / 14277), and phage display libraries have been used to analyze and control bimolecule interactions (International Publication 1998 / 20169; International Publication 1998 / 20159) and the characterization of constrained helical peptides (International Publication 1998 / 20036). International Publication 1997 / 35196 describes a method for isolating affinity ligands by contacting a phage display library with one solution in which the ligand binds to the target molecule and a second solution in which the affinity ligand does not bind to the target molecule, thereby selectively isolating the binding ligand. International Publication No. 1997 / 46251 describes a method for biopanning a random phage display library with affinity-purified antibodies, then isolating the bound phages, and subsequently isolating high-affinity bound phages by performing a micropanning process using microplate wells. Such a method may be applied to libraries of antigen-binding polypeptide variants disclosed herein. The use of Staphylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mol Biotech. 9:187). International Publication No. 1997 / 47314 describes the use of substrate subtraction libraries to distinguish enzyme specificity using combinatorial libraries that may be phage display libraries. Further methods for selecting specific binding proteins are described in U.S. Patent Nos. 5498538, 5432018 and International Publication No. 1998 / 15833.Methods for generating peptide libraries and screening these libraries are also disclosed in U.S. Patents 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and 5,723,323.

[0121] Variants, their antigen-binding fragments, or antibody construct variants, or any affinity-mature variants described herein, produced by screening the libraries described herein or by using the methods described herein, can be characterized for their physical / chemical properties and biological functions by a variety of assays known in the art. Such assays include, but are not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size-exclusion high-pressure liquid chromatography (HPLC), mass spectrometry, ion-exchange chromatography, and papain digestion. In some embodiments, the viscosity of any of the above antibody variants, their antigen-binding fragments, antibody construct variants, or affinity-mature variants is measured, for example, by methods known in the art and / or described elsewhere in this specification.

[0122] In certain embodiments, antibody variants, their antigen-binding fragments, or antibody construct variants, or any of the aforementioned affinity-mature variants, obtained by screening the libraries described herein or by using the methods described herein, are analyzed for their biological activity. In some embodiments, antibody variants, their antigen-binding fragments, or antibody construct variants are tested for their antigen-binding activity. Antigen-binding assays known in the Art and used herein include, but are not limited to, any direct or competitive binding assays using techniques such as Western blotting, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, fluorescence immunoassays, and protein A immunoassays.

[0123] Production and purification of antigen-binding polypeptide variants In some embodiments, the method further includes producing affinity-matured antigen-binding polypeptide variants (e.g., antibody variants, antigen-binding fragments of antibody variants, or antibody construct variants). In some embodiments, the method for producing affinity-matured variants (e.g., affinity-matured antibody variants, antigen-binding fragments of affinity-matured antibody variants, or affinity-matured antibody construct variants) is a manufacturing process on a production scale (e.g., a fermentation process). Antigen-binding polypeptide variants obtained by screening the library herein, by using the method herein, or by affinity maturation of variants obtained by screening the library or using the method herein may be produced by any means known in the art. Exemplary techniques for the production of antigen-binding polypeptide variants are described below; however, these exemplary techniques are provided for illustrative purposes only and are not intended to limit the scope of the method.

[0124] Antigen-binding polypeptide variants obtained by screening the libraries herein, by using the methods herein, or by affinity maturation of variants obtained by screening the libraries or using the methods herein can be produced using recombinant methods. For recombinant production of antigen-binding polypeptide variants, the nucleic acid encoding the antigen-binding polypeptide variant is isolated and inserted into a replicable vector (or vector) for further cloning (DNA amplification) or expression. The DNA encoding the antigen-binding polypeptide variant can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of an antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selectable marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0125] Antigen-binding polypeptide variants obtained by screening the libraries herein, by using the methods herein, by affinity maturation of variants obtained by screening the libraries, or by using the methods herein can be recombinantly produced as fusion polypeptides with heterologous polypeptides, such as signal sequences or mature proteins, or other polypeptides having a specific cleavage site at the N-terminus of the polypeptide. The selected heterologous signal sequence may be recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize or process the native antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, alkaline phosphatases, penicillinases, lpp, or a thermostable enterotoxin II reader. For yeast secretion, the native signal sequence may be replaced by, for example, a yeast invertase reader, a factor reader (including the α-factor reader of Saccharomyces and Kluyveromyces), or an acid phosphatase reader, a C. albicans glucoamylase reader, etc. In mammalian cell expression, mammalian signal sequences and viral secretion leaders, such as the herpes simplex gD signal, are available.

[0126] Both expression vectors and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, for example, enabling the vector to replicate independently of host chromosomal DNA. These sequences may include origins of replication or autonomous replication sequences. Such sequences are well known for various bacteria, yeasts, and viruses. Generally, origin of replication components are not required for mammalian expression vectors (the SV40 origin may be used because it contains an initial promoter).

[0127] Expression vectors and cloning vectors may contain select genes or select markers. Typical select genes encode (a) proteins that confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline; (b) proteins that supplement nutritional requirement deficiencies; or (c) proteins that supply essential nutrients unavailable from complex media. Examples of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin. Another example of select markers suitable for mammalian cells is those that allow identification of cells capable of taking up nucleic acids encoding antigen-binding polypeptide variants, e.g., DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably the primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc. For example, Chinese hamster ovary (CHO) cell lines lacking endogenous DHFR activity, transformed with the DHFR gene, can be identified by culturing the transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR.

[0128] Alternatively, host cells transformed or co-transformed with a DNA sequence encoding the antibody of interest, a wild-type DHFR gene, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH) (specifically, a wild-type host containing endogenous DHFR), may be selected by cells grown in a medium containing the selectable marker, such as an aminoglycoside antibiotic, such as kanamycin, neomycin, or a selector for G418.

[0129] Expression vectors and cloning vectors generally contain a promoter that is recognized by the host organism and operably ligated to a nucleic acid encoding an antigen-binding polypeptide variant. Suitable promoters for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoters, tryptophan (trp) promoter systems, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Eukaryotic promoter sequences are known. Yeast promoters are well known in the art and may include inducible promoters / enhancers that are regulated by growth conditions. Substantially all eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the transcription start site. Examples include, but are not limited to, promoters of 3-phosphoglycerate kinases or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, glucose phosphate isomerase, and glucokinase. Transcription of antigen-binding polypeptide variants from vectors in mammalian host cells can be controlled, for example, by promoters obtained from viral genomes. Early and late promoters of the SV40 virus are conveniently obtained as SV40 restriction fragments that also contain the SV40 virus origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as the HindIII E restriction fragment. Alternatively, the Roussarcoma virus long-terminal repeat can be used as a promoter.

[0130] The transcription of DNA encoding antigen-binding polypeptide variants obtained by screening the libraries herein, by using the methods herein, or by subjecting variants obtained by screening the libraries or using the methods herein to affinity maturation by higher eukaryotes is often increased by inserting enhancer sequences into vectors. Many enhancer sequences derived from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) are currently known. However, generally, enhancers derived from eukaryotic virus will be used.

[0131] Expression vectors used in eukaryotic host cells (nucleated cells derived from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) also include sequences necessary for transcription termination and mRNA stabilization.

[0132] Suitable host cells for cloning or expressing DNA in vectors as described herein are the prokaryotes, yeasts, or higher-order eukaryotic cells mentioned above. Suitable prokaryotes for this purpose include eubacteria, e.g., Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae, e.g., Escherichia, e.g., Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans and Shigella. In addition to prokaryotes, eukaryotic microorganisms, e.g., filamentous fungi or yeasts are suitable cloning or expression hosts for vectors encoding antigen-binding polypeptide variants. Among lower-order eukaryotic host microorganisms, Saccharomyces cerevisiae or common baker's yeast are the most commonly used. Certain fungal and yeast strains may be selected in which the glycosylation pathway is "humanized," resulting in the production of antigen-binding polypeptide variants with a partially or completely human glycosylation pattern. For example, see Li et al., Nat. Biotech. 24:210-215 (2006).

[0133] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also be used as hosts.

[0134] Furthermore, suitable host cells for expressing glycosylated antigen-binding polypeptide variants are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants, as well as corresponding tolerant insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori, have been identified.

[0135] Vertebrate cells can be used as hosts, and the propagation of vertebrate cells in culture (tissue culture) is a routine procedure. Examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed with SV40 (COS-7, ATCCCRL1651); and the human embryonic kidney cell line (293 or 293 cells subcloned for proliferation in suspension culture, Graham et al., J. Gen). Virol.36:59(1977)); Baby hamster kidney cells (BHK, ATCCCCL10); Mouse Sertoli cells (TM4, Mather, Biol. Reprod.23:243-251(1980)); Monkey kidney cells (CV1ATCCCCL70); African green monkey kidney cells (VERO-76, ATCCCCL-1587); Human cervical tumor cells (HELA, ATCCCCL2); Canine kidney cells (MDCK, ATCCCCL34); Buffalo rat hepatocytes (BRL3A, ATCCCCL1442); Human lung cells (W138, ATCCCCL75); Human hepatocytes (HepG2, HB8065); Mouse mammary gland tumors (MMT060562, ATCCCCL51); TRI cells (Mather et al., Annals NYAcad. Sci.383:44-68(1982)); MRC These include 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2). Other useful mammalian host cell lines include DHFR -Examples include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), as well as myeloma cell lines such as NS0 and Sp2 / 0. For an overview of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268. In some embodiments, the host cell is CHO-K1 cells. The CHO-K1 cell line is a subclone of the CHO cell line (see, for example, www(dot)phe-culturecollections(dot)org(dot)uk / media / 128263 / chok1-cell-line-profile(dot)pdf and web(dot)expasy(dot)org / cellosaurus / CVCL_0214).

[0136] Host cells can be cultured in various media. Commercial media such as Ham F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. Furthermore, Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. Any of the media described in al., Analyst Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; International Publication No. 90 / 03430; International Publication No. 87 / 00195; or U.S. Reissue Patent No. 30,985 may be used as a culture medium for host cells. Any of these media may optionally contain hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), or antibiotics (such as GENTAMYCIN). (商標) Drugs, trace elements (usually defined as inorganic compounds present at final densities in the micromolar range), and glucose or equivalent energy sources may be supplemented. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, are those already used in the host cells selected for expression and will be apparent to those skilled in the art.

[0137] When recombinant technology is used, antigen-binding polypeptide variants can be secreted intracellularly, within the periplasmic space, or directly into the culture medium. If antigen-binding polypeptide variants are produced intracellularly, the first step is to remove any particulate residue, host cells, or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E. coli.

[0138] Antigen-binding polypeptide variant compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a typically preferred purification step. In some embodiments, the antigen-binding polypeptide variants obtained according to the methods herein include an epitope tag (e.g., a tag bound to the antigen-binding polypeptide variant via a cleavable linker) to facilitate purification. Exemplary epitope tags include, but are not limited to, 6×His (also known as the His tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, e.g., enhanced green fluorescent protein or EGFP), GST (glutathione-S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose-binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0139] Antibody collection or recovery and purification In relevant embodiments, producing an antigen-binding polypeptide variant obtained according to the method herein includes culturing the host cells under conditions that enable the expression of the modified antigen-binding polypeptide variant, and recovering (e.g., harvesting) the antigen-binding polypeptide variant. In certain embodiments, producing an antigen-binding polypeptide variant prepared according to the method herein further includes purifying the recovered antigen-binding polypeptide variant to obtain a substantially homogeneous preparation, for example, for further assays and use.

[0140] Antigen-binding polypeptide variants prepared according to the methods described herein may be produced intracellularly or secreted directly into the culture medium. If such antigen-binding polypeptide variants are produced intracellularly, the first step is to remove any particulate residue, host cells, or lysed fragments, for example, by centrifugation or ultrafiltration. If antigen-binding polypeptide variants prepared according to the methods described herein are secreted into the culture medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. To inhibit proteolysis, a protease inhibitor such as PMSF may be included in one of the aforementioned steps, and antibiotics may be included to prevent the growth of foreign contaminants.

[0141] Using standard protein purification methods known in the art, substantially homogeneous preparations of antigen-binding polypeptide variants prepared according to the method described herein can be obtained from cells. The following procedure is an example of a suitable purification procedure: fractionation on an immunoaffinity or ion-exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or cation-exchange resin (such as DEAE), chromatographic fractionation, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using Sephadex G-75, for example.

[0142] Additionally or alternatively, modified antigen-binding polypeptide variants prepared using the methods described herein can be purified, for example, by hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0143] In certain embodiments where the antigen-binding polypeptide variant includes a human IgG region or a portion thereof, the preparation derived from the cell culture medium described above is applied to a protein A-immobilized solid phase to enable specific binding of the antigen-binding polypeptide variant to protein A. The solid phase is then washed to remove any nonspecifically bound contaminants. The antigen-binding polypeptide variant is recovered from the solid phase by elution into a solution containing a chaotropic agent or a neutral detergent. Exemplary chaotropic agents and neutral detergents include, but are not limited to, guanidine-HCl, urea, lithium perchlorate, arginine, histidine, SDS (sodium dodecyl sulfate), Tween, Triton, and NP-40, all of which are commercially available.

[0144] The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antigen-binding polypeptide variant. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J.Immunol.Meth.62:1-13(1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J.5:15671575(1986)). The matrix to which the affinity ligand binds is most often agarose, but other matrices are also available. Mechanically stable matrices such as controlled-pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Modified antigen-binding polypeptide variants are C HFor 3-domain variants, Bakerbond ABX® resin (JTBaker, Phillipsburg, NJ) is useful for purification. Other techniques for purifying proteins, such as fractionation on ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin Sepharose®, chromatography on anion or cation exchange resins (such as polyaspartate columns), chromatographic fractionation, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antigen-binding polypeptide variant to be recovered.

[0145] After any optional pre-purification step(s), the mixture containing the antigen-binding polypeptide variant and impurities can be subjected to low-pH hydrophobic interaction chromatography, preferably performed with an elution buffer at a pH of about 2.5–4.5 and a low salt concentration (e.g., about 0–0.25 M salt). The production of the modified antigen-binding polypeptide variant may be performed (alternatively or additionally to any of the specific methods described above) by dialyzing the solution containing the polypeptide mixture.

[0146] Example of an antibody variable domain with reduced viscosity In some embodiments, antibody variable domain variants produced using the methods described herein are provided. In some embodiments, the antibody variable domain variant is an antibody heavy chain variable domain (V) shown in SEQ ID NO: 1. H It is a variant of ). In some embodiments, V H The variant contains the amino acid sequence described in either SEQ ID NOs: 3-17 or 19-22. The amino acid sequences of SEQ ID NOs: 1, 3-17, and 19-22 are provided below. The variable domains are numbered according to Kabat's numbering scheme. EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWYDYFKGFDYWGQGTLVTVSS(Sequence ID 1) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIKDWYDYFKGFDYWGQGTLVTVSS V99K (Sequence ID 3) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDFYDYFKGFDYWGQGTLVTVSS W100aF (Sequence ID 4) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDVYDYFKGFDYWGQGTLVTVSS W100aV (Sequence ID 5) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDLYDYFKGFDYWGQGTLVTVSS W100aL (Sequence ID 6) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDAYDYFKGFDYWGQGTLVTVSS W100aA (Sequence ID 7) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWVDYFKGFDYWGQGTLVTVSS Y100bV (Sequence ID 8) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWADYFKGFDYWGQGTLVTVSS Y100bA (Sequence ID 9) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWHDYFKGFDYWGQGTLVTVSS Y100bH (Sequence ID 10) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWKDYFKGFDYWGQGTLVTVSS Y100bK (Sequence ID 11) EVQLVESGGGLVQPGGSLRLSCAASGFNIHYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWYDYFKGFDYWGQGTLVTVSS Y30H (Sequence ID 12) EVQLVESGGGLVQPGGSLRLSCAASGFNIYHNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWYDYFKGFDYWGQGTLVTVSS Y31H (Sequence ID 13) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYRGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWYDYFKGFDYWGQGTLVTVSS S54R (Sequence ID 14) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGRTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWYDYFKGFDYWGQGTLVTVSS S56R (Sequence ID 15) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWQDYFKGFDYWGQGTLVTVSS Y100bQ (Sequence ID 16) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWRDYFKGFDYWGQGTLVTVSS Y100bR (Sequence ID 17) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDHYDYFKGFDYWGQGTLVTVSS W100aH (Sequence ID 19) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDKYDYFKGFDYWGQGTLVTVSS W100aK (Sequence ID 20) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDRYDYFKGFDYWGQGTLVTVSS W100aR (Sequence ID 21) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWHDYFKGFDYWGQGTLVTVSS Y100bH (Sequence ID 22)

[0147] In some embodiments, V described in any one of sequence numbers 3-17 or 19-22 H and the antibody light chain variable domain (V) described in Sequence ID No. 2 L An antibody containing ) is provided. In some embodiments, V described in any one of SEQ ID NOs. 3-17 or 19-22 is provided. H and the antibody light chain variable domain (V) described in Sequence ID No. 18. L Antibodies containing ) are provided.

[0148] As shown in the examples, V described in any one of sequence numbers 3-17 or 19-22 H and V as described in Sequence ID No. 2 or 18 L Antibodies containing V are described in SEQ ID NO: 1. H and V as described in Sequence ID No. 2 L It exhibits lower viscosity compared to antibodies containing [specific component]. The variable domain is numbered according to Kabat's numbering scheme. The amino acid sequence of SEQ ID NO: 2 is provided below: DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGSYLYTFGQGTKVEIK (Sequence ID 2).

[0149] In some embodiments, the antibody variable domain variant is the antibody light chain variable domain (V) shown in SEQ ID NO: 2. L It is a variant of ). In some embodiments, V L The variant contains the amino acid sequence described in any one of SEQ ID NOs: 18. The amino acid sequence of SEQ ID NOs: 18 is provided below: DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYYCQQYGSKLYTFGQGTKVEIK Y94K (Sequence ID 18)

[0150] As shown in the examples, V shown in Sequence ID No. 18 L Antibodies containing V are shown in SEQ ID NO: 2. L It exhibits lower viscosity compared to antibodies containing the antibody light chain variable domain (V) shown in SEQ ID NO: 18. In some embodiments, it exhibits lower viscosity compared to antibodies containing the antibody light chain variable domain (V) shown in SEQ ID NO: 18. L ) and V shown in any one of sequence numbers 1, 3-17 or 19-22 H Antibodies containing the following are provided.

[0151] All publications and patent applications cited herein are incorporated herein by reference in such a way that each individual publication or patent application is specifically and individually indicated to be incorporated by reference. [Examples]

[0152] The following examples are provided to those skilled in the art to provide a complete disclosure and explanation of how to prepare and use the present invention, and are not intended to limit the scope of what the inventors consider to be the present invention, nor are they intended to represent all or only experiments that have been performed. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental error and deviation should be taken into consideration. Unless otherwise specified, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0153] Example 1A: Deep mutagenesis analysis of the variable domain of an anti-glucagon receptor (GCGR) antibody The following experiment was conducted using the V described in Sequence ID No. 1, which is set as an exemplary antibody showing increased viscosity at high concentrations. H Domain and V as described in Sequence ID No. 2 L The procedure was performed using an anti-GCGR (G protein-coupled glucagon receptor) IgG1 antibody containing the domain. Sequence IDs 1 and 2 are provided below. Further details regarding this anti-GCGR antibody are provided in International Publication No. 2013 / 059531, the contents of which are incorporated herein by reference in their entirety. EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWYDYFKGFDYWGQGTLVTVSS(Sequence ID 1) DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASSLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQYGSYLYTFGQGTKVEIK (Sequence ID 2)

[0154] The CDR of antibodies is generally rich in aromatic residues that contribute to antigen-binding affinity (Traxlmayr et al. (2016) J Biol Chem, 291:22496-508). In a previous study (see Tilegenova et al. (2020) mAbs, 12(1):1692764), 16 aromatic residues in the variable region of anti-GCGR IgG1 were mutated to alanine to investigate the possible role of aromatic residues in the high viscosity of this antibody. Twelve of the 16 mutants were expressed in sufficient quantities for further study. See Figure 1. Viscosity was measured at a final concentration of 180 mg / mL in 20 mM histidine-acetate buffer at pH 5.5. Seven different alanine variants were identified that substantially reduced viscosity (approximately 2- to 8-fold reduction). See Figure 1. These results suggest the general involvement of aromatic amino acid residues in the intermolecular interactions underlying the high viscosity exhibited by concentrated antibody solutions. Two of these seven variants, namely V L Y55A and Y91A also retained antigen-binding affinity to GCGR, a desirable attribute for potential antibody clinical candidates. See Table 1. Antigen-binding affinity (K D The value was evaluated by surface plasmon resonance. [Table 1]

[0155] As shown in Figure 1, not all alanine-substituted variants showed a reduction in viscosity, suggesting that other types of intermolecular interactions may also contribute to high viscosity. To investigate such molecular interactions, the crystal structure of anti-GCGR Fab was elucidated (2.7 Å resolution), and surface analysis was performed to identify the locations of charged and / or hydrophobic patches on the Fab. Briefly, the electrostatic potential map of the Fab was calculated using the Adaptive Poisson-Boltzmann Solver (APBS, see, e.g., www(dot)cgl(dot)ucsf(dot)edu / chimera / docs / ContributedSoftware / apbs / apbs(dot)html and Baker et al. (2001) PNAS USA, 98(18):10037-10041). See the left side of Figure 2. For example, the spatial aggregation tendency (SAP) of Fab was also determined, as described in Chennamsetty et al. (2009) PNAS USA, 106(29):11937-11942 and Lauer et al. (2012) J Pharm Sci, 101(1):102-115. See the right side of Figure 2. Molecular dynamics (MD) simulations were incorporated to prepare an average model of SAP that takes into account the flexibility of antibody molecules in solution. For example, see R. Salomon-Ferrer, DACase, RC Walker. (2013) "An overview of the Amber biomolecular simulation package." WIREs Comput. Mol. Sci. 3, 198-210; and DACase, TECheatham, III, T. Darden, H. Gohlke, R. Luo, KMMerz, Jr., A. Onufriev, C. Simmerling, B. Wang and R. Woods. (2005) "The Amber biomolecular simulation programs." J. Computat. Chem. 26, 1668-1688. Hydrophobic patches on the anti-GCGR Fab were identified (circled on the right side of Figure 2).Furthermore, four high-SAP regions (e.g., those with SAP values ​​≥2, such as 2.3) were identified within the CDRs of anti-GCGR Fab. See Figure 3. Of the six CDRs shown, four (L3, H1, H2, and H3) have multiple (but not all) residues with SAP values ​​>2.

[0156] The applicant hypothesized that disruption of hydrophobic patches within the CDR would reduce viscosity. To test this hypothesis, substituted variants were generated in which one or more aromatic residues in the hydrophobic patches of CDR-H1, CDR-H2, CDR-H3, and CDR-L3 were replaced with alanine. Figure 4 shows the aromatic residues in the substituted anti-GCGR Fab CDRs.

[0157] Figure 5 shows the relationship between V and viscosity at 180 mg / ml IgG1, 20 mM histidine acetate, pH 5.5, and 25°C. H and V L The effects of single substitutions and double substitutions in VH are shown. Figure 6 compares the viscosity of the single-substituted variant IgG1 antibody shown in Figure 5 against the parent IgG1 antibody with the SAP values ​​of the original wild-type aromatic residues at each position. Consistent with the results reported by Tilegenova et al. (2020) mAbs, 12(1):1692764, not all alanine substitutions reduced viscosity. For example, the Y31A substitution reduced the viscosity of the variant compared to the parent antibody, but the Y30A substitution adjacent to the Y31A substitution did not reduce the viscosity of the variant compared to the parent antibody. Similarly, the Y100bA substitution reduced the viscosity of the variant compared to the parent antibody, but the Y100dA substitution adjacent to the Y100bA substitution did not. Such results suggest that SAP values ​​alone are not sufficient to predict the important residues that affect viscosity.

[0158] Next, we calculated the solvent-accessible surface area (SASA) ratio for each amino acid identified as having a high SAP value (e.g., ≥2, e.g., an SAP value of 2.3 or higher), as described, for example, in ssbio(www)readthedocs(dot)io / en / latest / instructions / msms(dot)html. Simply put, the SASA ratio is calculated by dividing the surface-accessible area of ​​the amino acid side chain by the total surface area of ​​the side chain. Figure 7 shows the SASA ratios for specific amino acid positions in the CDR of the anti-GCGR IgG1 antibody, including residues with SAP values ​​≥2. The dotted line in Figure 7 indicates the cutoff values ​​used for high SASA ratios (0.25) and high SAP scores (2.3). Amino acids with high SAP values ​​and high SASA, such as Y31, Y100b, Y53, and Y100a in the VH domain of the anti-GCGR antibody, are predicted to play a significant role in the viscosity of the antibody and to play a role in reducing viscosity when substituted. Furthermore, to provide a more comprehensive analysis, we included representative sites within the high SASA ratio and low SAP region (labeled residues in the upper left quadrant) as well as the low SASA ratio and high SAP region (labeled residues in the lower right quadrant). Other amino acid positions, shown as unlabeled circles in Figure 7, are not expected to affect viscosity if, for example, they are mutated to alanine.

[0159] Next, the applicant constructed approximately 200 antibody single-mutation variants in which amino acids in the CDR were substituted, determined to have high SAP values ​​(e.g., ≥2, e.g., ≥2.3) and high SASA ratios (e.g., ≥0.25 or ≥0.5). (Gly, Pro, Cys, and Met substitution variants were not constructed.) The antibody mutants were initially screened by dynamic light scattering (DLS) at concentrations of approximately 2 mg / ml to approximately 10 mg / ml, and the interaction parameters of each variant were measured using the following formula: D=D0(1+k D *c) In the formula, D = translational diffusion coefficient; D0 is the self-diffusion coefficient (value of D at concentration 0); c = protein concentration; and k D = Interaction parameter. Variant kD The more positive the value, the weaker the self-interaction of the variant. Conversely, the k of the variant... D The more negative the value, the stronger the self-interaction of the variant. For example, see Sorret et al. (2016) Biophysical Journal, 111:1831-1842.

[0160] Figure 9 provides a table showing the effect of specific substitution amino acids of anti-GCGR IgG1 antibodies on DLS interaction parameters (ml / g). DLS interaction parameters are shown, measured at 25.0°C in 20 mM histidine acetate, pH 5.5. Variants with exceptionally low diffusion coefficients are highlighted (gray) to indicate their polydisperse behavior in solution. Parent residues are highlighted with (P). Mutants with expression or purification problems, or those showing significant aggregation, are also highlighted (black). A subset of anti-GCGR IgG1 variants from this DLS study (Figure 9) were evaluated for viscosity at high concentrations. Specifically, approximately 30 variants were selected across a wide range of interaction parameter values ​​(0–26 mL / g) equivalent to, above, or below the parent antibody (8 mL / g). These variants represent diverse substitution amino acid types, including aliphatic, aromatic, positively charged polar, and uncharged polar. Further variants with negative DLS interaction parameters were planned for this study but were excluded from viscosity measurements because they were lost due to precipitation during the spin enrichment process. Additionally, approximately 10 single-alanine-substituted variants were included from Figure 5. Figure 8 shows the DLS interaction parameters (k) for these exemplary anti-GCGR IgG1 antibody variants. D The plots for φ(ml / g) and viscosity (η, cP) are shown. The plots suggest a correlation between DLS interaction parameters and the viscosity of the anti-GCGR antibody variant. DLS interaction parameter values ​​lower than wild-type values ​​correlate with higher viscosity, while DLS interaction parameter values ​​higher than wild-type values ​​correlate with lower viscosity.

[0161] Figure 10 shows the DLS interaction parameters (k D , ml / g) and dissociation constants (K D , M) plots for each anti-GCGR IgG1 antibody variant tested in Figure 9. Some variants, namely VH domains containing W100aF, Y30H, W100aV, W100aL, Y100bK, Y100bH, Y100bR or Y100bQ substitutions and VL domains shown in SEQ ID NO: 2 or variants containing Y94K VL domain substitutions showed an increase in DLS interaction parameters with little effect on antigen binding affinity (e.g., less than a 5-fold decrease in binding affinity compared to the parent). See the upper left quadrant of Figure 10.

[0162] Table 2 provides additional specific information on the variants in the upper left quadrant of Figure 10. [Table 2]

[0163] Several additional parameters were tested for both the parent and variant. Specifically, computational developmental analysis was performed using the Therapeutic Antibody Profiler (TAP). The amber TAP flag identified for the VH W100aL variant was for surface hydrophobicity. Also shown are the calculated isoelectric point for the Fv fragment, multispecificity from baculovirus ELISA, and predicted immunogenic T cell epitopes using NetMHCIIpan-4.0 EL. Humanity scores were determined from OASis percentiles. The results are shown in Tables 3A and 3B. Specifically, Tables 3A-3B provide characterization data for the tested anti-GCGR IgG1 antibody variants described herein. The experimental data presented include dynamic light scattering interaction parameters (kD), antigen binding kinetics (kon, koff, and KD) measured by surface plasmon resonance, percentage of IgG1 monomer determined by analytical size exclusion chromatography, and viscosity measured by cone-plate rheometry with 180 mg / mL of IgG1 at 25.0° in 20 mM histidine acetate, pH 5.5. The calculated data presented include TAP scores and flags, isoelectric point, and SAP values. nd indicates no binding detected; nm indicates not measured. [Table 3A] TIFF2026517933000006.tif250170TIFF2026517933000007.tif250170TIFF20265179330 00008.tif250170TIFF2026517933000009.tif250170TIFF2026517933000010.tif250170 [Table 3B] TIFF2026517933000012.tif250170TIFF2026517933000013.tif250170TIFF20265179330 00014.tif250170TIFF2026517933000015.tif250170TIFF2026517933000016.tif250170

[0164] The amino acid sequences of the VH domains of Y100bV, W100aF, S56R, Y100bA, Y100bH, Y30H, W100aV, W100aL, and Y100bK are shown below. W100aA, S54R, V99K, and Y31H are shown below: EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIKDWYDYFKGFDYWGQGTLVTVSS V99K (Sequence ID 3) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDFYDYFKGFDYWGQGTLVTVSS W100aF (Sequence ID 4) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDVYDYFKGFDYWGQGTLVTVSS W100aV (Sequence ID 5) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDLYDYFKGFDYWGQGTLVTVSS W100aL (Sequence ID 6) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDAYDYFKGFDYWGQGTLVTVSS W100aA (Sequence ID 7) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWVDYFKGFDYWGQGTLVTVSS Y100bV (Sequence ID 8) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWADYFKGFDYWGQGTLVTVSS Y100bA (Sequence ID 9) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWHDYFKGFDYWGQGTLVTVSS Y100bH (Sequence ID 10) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWKDYFKGFDYWGQGTLVTVSS Y100bK (Sequence ID 11) EVQLVESGGGLVQPGGSLRLSCAASGFNIHYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWYDYFKGFDYWGQGTLVTVSS Y30H (Sequence ID 12) EVQLVESGGGLVQPGGSLRLSCAASGFNIYHNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWYDYFKGFDYWGQGTLVTVSS Y31H (Sequence ID 13) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYRGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWYDYFKGFDYWGQGTLVTVSS S54R (Sequence ID 14) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGRTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWYDYFKGFDYWGQGTLVTVSS S56R (Sequence ID 15)

[0165] Based on the information provided herein, for example, Figure 9, additional test amino acid sequences including the following are also included in this application: EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDHYDYFKGFDYWGQGTLVTVSS W100aH (Sequence ID 19) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDKYDYFKGFDYWGQGTLVTVSS W100aK (Sequence ID 20) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDRYDYFKGFDYWGQGTLVTVSS W100aR (Sequence ID 21) EVQLVESGGGLVQPGGSLRLSCAASGFNIYYNYIHWVRQAPGKGLEWVAEFSPYSGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSAAIVDWHDYFKGFDYWGQGTLVTVSS Y100bH (Sequence ID 22)

[0166] Summary of Example 1A The structure of the anti-GCGR Fab, including the VH domain shown in SEQ ID NO: 1 and the VL domain shown in SEQ ID NO: 2, was elucidated to a resolution of 2.7 Å.

[0167] To investigate the contribution of hydrophobic patches to self-interaction, extensive mutagenesis of the VH and VL domains was performed. The applicants found that residues influencing significant viscosity were characterized by SAP values ​​≥ 2 and SASA ratios ≥ 0.5.

[0168] The applicant found that substituting residues with SAP values ​​≥ 2 and SASA ratios ≥ 0.5 with charged or polar residues increased the DLS interaction parameters of the resulting variants. Such variants may exhibit reduced viscosity.

[0169] The applicant also identified a mutation in Sequence ID No. 1 that reduces viscosity while maintaining antigen-binding affinity.

[0170] Example 1B: Interaction parameter (k) compared with parental anti-GCGR antibody D Determination of the viscosity of anti-glucagon receptor (GCGR) antibody variants that were found to show an increase in viscosity. Increased interaction parameters compared to parental anti-GCGR IgG1 antibody ( k The viscosity of the anti-GCGR IgG1 antibody variant discussed in Example 1A, which was found to exhibit D), was analyzed by rheometry at a concentration of ≤180 mg / ml, for example, as described in Lang et al. (2020) Appl Sci, 10(1), 172 and Zhang et al. (2017) Curr Opin in Chem Eng, 16:48-55; and Sharma et al. (2011) Soft Matter 10.1039 / c0sm01312a.

[0171] The present invention is described with respect to specific embodiments found or proposed by the inventors that include preferred modes for carrying out the invention. In light of this disclosure, it will be understood by those skilled in the art that numerous modifications and changes can be made in the specific embodiments exemplified without departing from the intended scope of the invention. For example, changes can be made to the underlying DNA sequence without affecting the protein sequence due to codon redundancy. Furthermore, by considering biological and functional equivalence, the protein structure can be altered without affecting the biological action in any way or amount. All such modifications are intended to be included within the scope of the appended claims.

Claims

1. A method for identifying antigen-binding polypeptide variants having reduced viscosity compared to the parent antigen-binding polypeptide, (a) Identifying one or more wild-type amino acids in a parent antigen-binding polypeptide having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio; (b) Replacing one or more wild-type amino acids identified as having a high SAP value and / or a high SASA ratio with amino acids having a lower SAP value and / or a lower SASA ratio compared to the wild-type amino acids; (c) Measuring the viscosity of the antigen-binding polypeptide variant; and (d) Identify the antigen-binding polypeptide variant having a reduced viscosity compared to the parent polypeptide. Methods that include...

2. The method according to claim 1, wherein one or more wild-type amino acids in the parent antigen-binding polypeptide are exposed on the surface.

3. The method according to claim 1 or 2, wherein the one or more wild-type amino acids are aromatic amino acids, and the one or more aromatic amino acids are substituted with one or more charged amino acids or amino acids having smaller side chains.

4. The method according to claim 3, wherein one or more aromatic amino acids are selected from the group consisting of: W, Y, and F.

5. The method according to claim 3 or 4, wherein the one or more charged amino acids or amino acids having smaller side chains are selected from the group consisting of A, I, L, V, D, E, H, K, R, N, Q, S, and T.

6. The method according to any one of claims 1 to 5, wherein one or more wild-type amino acids have a smaller side chain, and the one or more amino acids having the smaller chain are substituted with one or more charged amino acids.

7. The method according to claim 6, wherein one or more wild-type amino acids have a smaller side chain selected from the group consisting of: A, I, L, V, N, Q, S, G, P, C, M, or T.

8. The method according to claim 6 or 7, wherein one or more charged amino acids are selected from the group consisting of: R, K, H, D, and E.

9. The method according to any one of claims 1 to 8, wherein the high SAP value is 2 or more.

10. The method according to claim 9, wherein the aforementioned high SAP value is 2.3 or higher.

11. The method according to any one of claims 1 to 10, wherein the high SASA ratio is 0.25 or more.

12. The method according to any one of claims 1 to 11, wherein the high SASA ratio is 0.5 or more.

13. The method according to any one of claims 1 to 12, wherein the viscosity of the antigen-binding polypeptide variant is evaluated by dynamic light scattering (DLS).

14. The method according to any one of claims 1 to 13, wherein the viscosity of the antigen-binding polypeptide variant is measured by cone-plate rheometry.

15. The method according to claim 14, wherein the viscosity of the antigen-binding polypeptide variant is measured at a high concentration.

16. The method according to claim 15, wherein the high concentration is approximately 50 mg / ml to 300 mg / ml.

17. A method according to any one of claims 1 to 16, The aforementioned parent antigen-binding polypeptide is an antibody, an antigen-binding fragment of an antibody, or an antibody construct. The antibody or antibody construct comprises one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. One or more wild-type amino acids in the aforementioned CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are identified as having a high SAP value and / or a high SASA ratio. method.

18. The method according to claim 17, wherein the parent antigen-binding polypeptide is an antibody.

19. The method according to claim 17 or 18, wherein the antibody is a therapeutic antibody.

20. The method according to any one of claims 17 to 19, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

21. The method according to any one of claims 17 to 20, wherein the antibody is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

22. The method according to any one of claims 17 to 21, wherein the antibody comprises a human IgG Fc region.

23. The method according to claim 22, wherein the human IgG constant region is the IgG1, IgG2, or IgG4Fc region.

24. The method according to claim 17, wherein the parent polypeptide is an antigen-binding fragment of an antibody.

25. The antigen-binding fragment is Fab, F(ab')2, triple-specific Fab ３ scFv, monovalent IgG, diamond body, triabody, scFv-Vc, minibody, V Ｈ The method according to claim 24, wherein the material is H, V-NAR, hcIgG, or IgNAR.

26. The method according to claim 17, wherein the parent polypeptide is an antibody construct.

27. The method according to claim 26, wherein the antibody construct is a CrossMab, a dual-acting Fab (DAF), DVD-IgG, or a knob-in-hole bispecific antibody.

28. The method according to any one of claims 1 to 27, further comprising the step of subjecting the antigen-binding polypeptide variant to at least one affinity maturation step.

29. The method according to claim 28, wherein the substituted amino acids in the antigen-binding polypeptide variant are not randomized during affinity maturation.

30. A polypeptide variant produced by the method described in any one of claims 1 to 29.

31. A library containing multiple antigen-binding polypeptide variants, At least one variant contains one or more amino acid substitutions compared to the parent antigen-binding polypeptide, The one or more amino acid substitutions to the parent antigen-binding polypeptide involve replacing one or more wild-type amino acids, identified as having a high SAP value and / or a high SASA ratio, with amino acids having a lower SAP value and / or a lower SASA ratio compared to the wild-type amino acids. Library.

32. The library according to claim 31, wherein the plurality includes at least 1,000 unique variants.

33. The library according to claim 31 or 32, wherein the plurality of antigen-binding variants are plurality of antibodies, antigen-binding fragments of antibodies, or antibody constructs.

34. A method for predicting one or more amino acids in an antigen-binding polypeptide that affect the viscosity of the antigen-binding polypeptide, Identifying one or more amino acids in antigen-binding polypeptides having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio, Includes, One or more amino acids having a high spatial aggregation tendency (SAP) value and / or a high solvent-accessible surface area (SASA) ratio are predicted to affect the viscosity of the antigen-binding polypeptide. method.

35. A method for predicting one or more amino acids in an antibody, an antigen-binding fragment of an antibody, or an antibody construct that affect the viscosity of the antibody, the antigen-binding fragment, or the antibody construct, wherein the antibody, the antigen-binding fragment, or the antibody construct comprises one or more of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and the method is: Identifying one or more amino acid positions in one or more of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having a high spatial aggregation tendency (SAP) value and a high solvent-accessible surface area (SASA) ratio, Includes, The one or more amino acid positions having the aforementioned high spatial aggregation tendency (SAP) value and the aforementioned high solvent-accessible surface area (SASA) ratio are predicted to affect the viscosity of the antibody, the antigen-binding fragment, or the antibody construct. method.

36. The antibody heavy chain variable domain (V) described in any one of SEQ ID NOs: 3-17 or 19-22 Ｈ ).

37. The heavy chain variable domain (V) described in any one of sequence numbers 3-17 or 19-22 Ｈ ) and the light chain variable domain (V) described in Sequence ID No. 2 or 18. Ｌ Antibodies containing )

38. Light chain variable domain (V) described in Sequence ID No. 18 Ｌ Antibodies containing )

39. Light chain variable domain (V) described in Sequence ID No. 18 Ｌ ) and the heavy chain variable domain (V) described in any one of Sequence IDs 1, 3-17, or 19-22. Ｈ Antibodies containing )

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