High-throughput Fab antibody fragment generation and grading

Abstract

A method and assay for high-throughput antigen-binding grading by generating and testing antibody fragments from full-length antibodies is provided. In one embodiment, the method for high-throughput antibody grading by antigen binding may include (a) processing a pool of antibodies to generate a pool of antibody fragments; (b) purifying the pool of antibody fragments; and (c) grading the antibody fragments in the purified pool of antibody fragments according to their binding affinity using a high-throughput antigen-binding assay.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority and benefit of U.S. Provisional Application No. 63 / 463,995, filed on May 4, 2023, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Antibody production involves the preparation of an antigen sample and their injection into an animal to cause a high expression level of antigen - specific antibodies in the serum, and the antibodies can then be recovered from the animal. Polyclonal antibodies are recovered directly from the serum. Monoclonal antibodies are produced by fusing antibody - secreting spleen cells from an immunized mouse with immortalized myeloma cells to generate a monoclonal hybridoma cell line that expresses the specific antibody in the cell culture supernatant. For antibody production, a screening step is required to identify antibodies with favorable binding properties. This is a costly and time - consuming step in antibody production. There remains a need in the art for a rapid and efficient screening and identification of antibodies with desired binding characteristics. The need for a rapid and efficient screening for antibodies with desired binding characteristics mainly stems from the fact that the antibody binding properties define how a particular antibody functions as a therapeutic agent. An effective blocking antibody therapeutic agent, for example, typically requires the highest possible binding affinity of a given antibody to its target.

Summary of the Invention

Means for Solving the Problems

[0003] In one embodiment, a method for high - throughput ranking of antibodies by antigen binding may include: (a) processing a pool of antibodies to generate a pool of antibody fragments; (b) purifying the pool of antibody fragments; and (c) using a high - throughput antigen - binding assay to rank the antibody fragments in the purified pool of antibody fragments according to their binding affinities.

[0004] In one embodiment, a pool of antibody fragments can be generated by treating the pool of antibody fragments with a protease or chemical process, thereby enabling the antibody fragments in the pool to bind to the antigen with or without a reduced avidity effect on antigen binding. The pool of antibody fragments may be a pool of Fab fragments.

[0005] A pool of Fab fragments can be generated by digesting a pool of antibody fragments with papain.

[0006] In one embodiment, the method may further include determining the degree of antibody treatment by SDS-PAGE.

[0007] In one embodiment, the method may further include determining the degree of antibody treatment by size exclusion chromatography (SEC).

[0008] In one embodiment, the method may further include determining the purity of the antibody fragment pool by SDS-PAGE.

[0009] In one embodiment, the method may further include determining the purity of the antibody fragment pool by size exclusion chromatography (SEC).

[0010] In one embodiment, the antibody pool may include 2 to 24 different antibodies, 5 to 24 different antibodies, 10 to 24 different antibodies, 15 to 24 different antibodies, 20 to 24 different antibodies, 24 different antibodies, or more than 24 different antibodies.

[0011] In one embodiment, a high-throughput antigen-binding assay includes label-free biosensing. Label-free biosensing may be selected from the group consisting of polarization analysis; surface plasmon resonance (SPR); localized surface plasmon resonance using noble metal nanoparticles in solution or on a transparent surface; surface acoustic wave (SAW) devices; attenuated quartz crystal microbalance (QCM-D); atomic force microscopy; plasmon-photon coupling; transmission sensing via subwavelength nanoholes (enhanced light transmission); photonic crystal sensing; refractive index sensing; waveguide mode resonance sensing; ring resonator sensing; isothermal titration calorimetry (ITC); microscale thermophoresis; and biolayer interferometry (BLI).

[0012] In one embodiment, the high-throughput antigen-binding assay includes biolayer interference (BLI).

[0013] In one embodiment, steps (a) and (b) are performed on a plate.

[0014] In one embodiment, steps (a) to (c) are automated.

[0015] For a further understanding of the characteristics, purposes, and merits of this disclosure, the following detailed description, to be read in conjunction with the following drawings, should be referenced. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1 shows 24 Fab fragments produced by the method described herein and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All 24 Fab fragments were confirmed to have been successfully purified. [Figure 2-1] Figure 2 shows the results of size exclusion chromatography (SEC) for 24 Fab fragments produced by the method described herein. All 24 Fab fragments were confirmed to have been successfully purified. [Figure 2-2]Figure 2 shows the results of size exclusion chromatography (SEC) for 24 Fab fragments produced by the method described herein. All 24 Fab fragments were confirmed to have been successfully purified. [Figure 2-3] Figure 2 shows the results of size exclusion chromatography (SEC) for 24 Fab fragments produced by the method described herein. All 24 Fab fragments were confirmed to have been successfully purified. [Figure 2-4] Figure 2 shows the results of size exclusion chromatography (SEC) for 24 Fab fragments produced by the method described herein. All 24 Fab fragments were confirmed to have been successfully purified. [Figure 3-1] Figure 3 shows 24 Fab fragments generated by the method described herein and analyzed by biolayer interferometry (BLI). As shown, variants #1, #5, #6, and #24 did not bind to the antigen as Fab and were excluded from consideration. The remaining variants were graded by Koff. [Figure 3-2] Figure 3 shows 24 Fab fragments generated by the method described herein and analyzed by biolayer interferometry (BLI). As shown, variants #1, #5, #6, and #24 did not bind to the antigen as Fab and were excluded from consideration. The remaining variants were graded by Koff. [Figure 3-3] Figure 3 shows 24 Fab fragments generated by the method described herein and analyzed by biolayer interferometry (BLI). As shown, variants #1, #5, #6, and #24 did not bind to the antigen as Fab and were excluded from consideration. The remaining variants were graded by Koff. [Figure 4]Figure 4 shows the BLI ranking results of 24 Fab fragments. The method described herein successfully ranked 24 antibodies as Fab fragments by Koff. By using Fab fragments instead of full-length antibodies, rapid and efficient exclusion of the weakest binders became possible. The use of Fab fragments changed the ranking of the entire variant by Koff compared to full-length antibody ranking. Variants #1, #5, #6, and #24 were excluded because they did not show binding to the antigen. The Koff affinity ranking generated as a result of this experiment was used directly for the final candidate molecule selection.

Mode for Carrying Out the Invention

[0017] It should be understood that the present disclosure is not limited to specific embodiments since variations of the specific embodiments described below can be created and still fall within the scope of the appended claims. It should also be understood that the technical terms used are for the purpose of describing specific embodiments and are not intended to be limiting. Instead, the scope of the present disclosure is established by the appended claims.

[0018] The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0019] "Antibody" (Ab) and "immunoglobulin" (Ig) are glycoproteins having the same structural features. Ab exhibits binding specificity for a specific antigen, whereas immunoglobulins include both Ab and other Ab-like molecules lacking antigen specificity. The term "antibody" specifically includes monoclonal antibodies, including antibody fragment clones.

[0020] Native antibodies and immunoglobulins are typically heterotetrameric glycoproteins with approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among heavy chains of different Ig isotypes. Each heavy and light chain also has regularly spaced 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; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Certain amino acid residues are thought to form interfaces between the light and heavy chain variable domains. Examples of native antibodies and immunoglobulins that are not heterotetramers exist, such as camelid antibodies containing only the heavy chain, e.g., llama or alpaca antibodies (these are single-chain immunoglobulins, sometimes referred to as "VHH" antibodies).

[0021] The term "variable," in relation to antibody variable domains, broadly refers to the fact that certain portions of a variable domain have widely different sequences among antibodies, and that these differences are used in the binding and specificity of each particular antibody to its specific antigen. Variability is not uniformly distributed throughout the antibody's variable domain. It is concentrated in three segments called complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) or hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called frameworks (FRs). The native heavy and light chain variable domains each contain four FR regions, primarily β-sheet structures, connected by three CDRs, where the three CDRs connect the β-sheet structures, forming loops that in some cases form parts of them. The CDRs in each chain are held together in close proximity by the FR regions, and together with CDRs from other chains, contribute to the formation of the antibody's antigen-binding site. The constant domain does not directly contribute to the binding of antibodies to antigens, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity.

[0022] "Antibody fragment" refers to any antibody fragment, e.g., Fab, Fab', F(ab')2, Fv, and Fd fragments; diabodies; single-chain antibody molecules containing single-domain antibody molecules (dAb) and single-chain Fv molecules (scFv); multispecific antibodies formed from antibody fragments; or other compositions that retain antigen-binding function. "Antibody fragment" refers to a portion of an intact antibody, usually containing the antigen-binding or variable region of an intact antibody. Antibody fragments may originate from sources including, but not limited to, bacterial cell lines, insect cell lines, plant cell lines, yeast cell lines, or cell lines of other origins, in their natural or genetically modified forms, e.g., humanized, human, chimeric, synthetic, recombinant, hybrid, mutant, grafted, and in vitro-produced forms, including human or other mammalian cell lines. Antibody fragments may also be induced by controlled lysis of purified antibodies using enzymes, e.g., ficin, papain, or pepsin, but not limited to these. Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments (each with a single antigen-binding site) and the remaining "Fc" fragment (its name reflects its ability to easily crystallize). Pepsin digestion of antibodies produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of crosslinking antigens. Ficin digestion of antibodies produces either an F(ab')2 or a Fab fragment, depending on the concentration of cysteine ​​present when digestion is performed.

[0023] "Fv" is the minimal antibody fragment containing a complete antigen recognition and binding site. In the double-chain Fv species, this region consists of a dimer of one heavy chain and one light chain variable domain that are tightly and non-covalently associated. In the single-chain Fv species, one heavy chain and one light chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimer" structure similar to that in the double-chain Fv species. In this configuration, the three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. In total, six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site. VHH is not an antibody fragment, but it exhibits monovalent antigen binding with only three CDRs. Antibody fragments derived from VHH molecules can be fused with or without a suitable linker peptide to other VHH fragments, for example, to increase avidity-mediated binding or to generate bispecific or multispecific fusion proteins containing VHH fragments that bind to an antigen or to more antigens.

[0024] The Fab fragment also contains a constant domain of the light chain and a 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 from the antibody hinge region. Fab'-SH is the name used herein for Fab' fragments in which the cysteine ​​residues of the constant domain possess a free thiol group. The F(ab')2 antibody fragment was initially produced as a pair of Fab' fragments (with a hinge cysteine ​​between them). Other chemical couplings of antibody fragments are also known.

[0025] The "light chains" of Ab from any vertebrate species can be assigned to one of two distinct types called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0026] Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0027] As used herein, "monoclonal antibody" broadly refers to an antibody obtained from a substantially homogeneous population of antibodies, where, for example, the individual antibodies constituting the population are identical except for naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized by hybridoma culture methods that are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the characteristic of an antibody as being 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, the monoclonal antibodies described herein may be produced by hybridoma or recombinant DNA methods. The term "monoclonal antibody" also includes clones (Fv clones) of antigen-recognition and binding site-containing antibody fragments isolated from phage antibody libraries.

[0028] Monoclonal antibodies as used herein include, in particular, “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain originates from a particular species or is identical or homologous to a corresponding sequence in an antibody belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody belonging to a different species or another antibody class or subclass, and fragments of such antibodies insofar as they exhibit the desired biological activity.

[0029] A "human antibody" (also called a "fully human antibody") is an antibody that contains a human framework region and a CDR from human immunoglobulin. In one example, the framework and CDR are from the same human heavy and / or light chain amino acid sequence. The framework region from one human antibody may be manipulated to contain a CDR from a different human antibody.

[0030] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing minimal sequences derived from non-human immunoglobulins. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's CDR are replaced with residues from the CDR of a non-human species (e.g., mouse, camelid, rat, or rabbit) or synthetic sequence (donor antibody) that has the desired specificity, affinity, and capability. In some examples, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Humanized antibodies may also contain residues not found in the recipient antibody or the introduced CDR or framework sequence. These modifications are made to further improve and optimize antibody performance. In one embodiment, all CDRs in the humanized immunoglobulin may be from the donor immunoglobulin. Constant regions are not required to exist, but if present, they should be substantially identical to the constant regions of human immunoglobulins, e.g., at least about 85-90%, e.g., about 95% or more identical. Therefore, all parts of humanized immunoglobulins, except in some cases the CDR, are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. A "humanized antibody" is an antibody containing humanized light chain and humanized heavy chain immunoglobulins. Humanized antibodies bind to the same antigen as the donor antibody that provides the CDR. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids obtained from the donor framework. Humanized or other monoclonal antibodies may have additional conserved amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed using genetic engineering.

[0031] A "single-stranded Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, and these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form a desired structure for antigen binding.

[0032] As used herein, "diabody" broadly refers to a small antibody fragment having two antigen-binding sites. Such a fragment may, for example, include a heavy chain variable domain (VH) (VH-VL) attached to a light chain variable domain (VL) on the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with a complementary domain on another chain, thereby generating two antigen-binding sites.

[0033] "Isolated" antibodies are those identified, separated, and / or recovered from components of their natural environment. Contaminating components from that natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Antibodies can be purified, for example, (1) to more than 95% by weight (or optimally more than 99% by weight) as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibodies contain antibodies present in situ within recombinant cells because at least one component of the antibody's natural environment is absent. Isolated antibodies are prepared by at least one purification step.

[0034] As used herein, "variant" broadly refers to a polypeptide that has the same or identical function as an antibody or its antibody fragment, but does not necessarily contain the same or identical amino acid sequence as the antibody or its antibody fragment, nor does it necessarily possess the same or identical structure as the antibody or its antibody fragment.Variants having similar amino acid identity refer to polypeptides that satisfy at least one of the following: (a) a polypeptide containing, or alternatively consisting thereof, an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of an antibody containing VHH, or an antibody fragment thereof (containing a VH domain, VHCDR, VL domain, or VLCDR having any one of the amino acid sequences described herein); (b) a polypeptide containing at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, and at least 99% identical; (b) a polypeptide containing at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, and at least 99% identical amino acid sequence; (c) a polypeptide encoded by a nucleotide sequence having 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues, wherein the complementary sequence of the nucleotide sequence hybridizes under stringent conditions with a nucleotide sequence encoding an antibody or an antibody fragment (including a VH domain, VHCDR, VL domain, or VLCDR having any one of the amino acid sequences described herein); and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a nucleotide sequence encoding an antibody or an antibody fragment (including a VH domain, VHCDR, VL domain, or VLCDR having any one of the amino acid sequences described herein).Polypeptides having a similar structure to the antibodies or antibody fragments described herein refer to polypeptides having a similar secondary, tertiary, or quaternary structure to the antibodies or antibody fragments described herein. The structure of a polypeptide may be determined by methods known to those skilled in the art, including but not limited to X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy. To determine the degree of identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps may be introduced in the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). Then, amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. The degree of identity between two sequences depends on the number of identical positions shared by the sequences (for example, identity % is equal to [number of identical overlapping positions] / [total number of positions] × 100%). In one embodiment, the two sequences are of the same length.

[0035] The determination of the degree of identity between two sequences can be achieved using mathematical algorithms known to those skilled in the art. The BLASTn and BLASTx programs (Altschul, S. et al., J. Mol. Biol., 215:403-10, 1990) incorporate such algorithms. A BLAST nucleotide search (available at blast.ncbi.nlm.nih.gov) can be performed, for example, using the BLASTn program, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search can be performed, for example, using the BLASTx program, score=50, word length=3, to obtain amino acid sequences homologous to the protein molecules described herein. Gapped BLAST can be used to obtain gapped alignments for comparison purposes (Altschul, S. et al., Nucleic Acids Res., 25:3389-402, 1997). Alternatively, PSI-BLAST can be used to perform iterative searches to detect distance relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of each program (e.g., BLASTx and BLASTn) may be used.

[0036] "Conservative" amino acid substitutions are those that do not substantially affect or reduce the affinity of an antibody or other protein to a target epitope. For example, a monoclonal antibody that immunospecifically binds to human light chain amyloid fibrils may contain up to approximately 1, 2, 5, 10, or 15 conservative substitutions and be able to immunospecifically bind to human light chain amyloid fibril polypeptides. The term "conservative variant" also includes the use of substituted amino acids instead of unsubstituted parent amino acids, as long as the antibody immunospecifically binds to the target epitope. Non-conservative substitutions reduce binding to the target epitope.

[0037] Tables of conserved amino acid substitutions that provide functionally similar amino acids are well known to those skilled in the art. The following six groups are examples of amino acids that can be considered conserved substitutions when the amino acids within the group are substituted for each other: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0038] As used herein, “derivative” refers to a variant polypeptide described herein that includes, or alternatively consists of, the amino acid sequence of an antibody modified by the introduction of substitution, deletion, or addition of amino acid residues. The term “derivative” also refers, as used herein, to an antibody modified, for example, by covalent bonding of any type of molecule to the polypeptide. For example, but not limited to, antibodies may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cell ligands or other proteins, etc. Derivatives of antibodies or their antigen-binding fragments may be modified by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, and metabolic synthesis of tunicamycin. Furthermore, derivatives of antibodies or their antigen-binding fragments may contain one or more non-classical amino acids. Polypeptide derivatives possess similar or identical functions to those of their antigen-binding fragments described herein.

[0039] As used herein, "epitope" refers to a portion of a peptide having antigenic or immunogenic activity in an animal, such as a mammal, such as a human. Antigenic epitopes do not necessarily have to be immunogenic.

[0040] As used herein, “fragment” broadly refers to a polypeptide having an amino acid sequence of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 35 amino acid residues, at least 40 amino acid residues, at least 45 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues of an antibody (including an antibody fragment or variant thereof, or alternatively, a molecule such as a Fab or scFv consisting thereof).

[0041] When used herein, "fusion protein" broadly refers to a polypeptide comprising, or alternatively comprising, the amino acid sequence of an antibody described herein and the amino acid sequence of a heterologous polypeptide (e.g., an antibody or polypeptide not related to an antibody domain).

[0042] High-throughput Fab antibody fragment generation and grading The methods described herein enable the generation of antibodies or antibody fragments, such as Fab fragments (typically produced by targeted degradation of antibodies from full-length antibodies via the use of proteases, such as papain, pepsin, or ficin), and their grading by binding affinity using label-free biosensing techniques in a high-throughput (HT) manner.

[0043] Classifying antibody variants by their ability to bind to their respective antigens is part of the monoclonal antibody drug development process. Antibody classification procedures are used to differentiate hits from various antibody discovery efforts, antibody humanization programs, or other programs in order to improve lead molecule selection. However, this procedure has at least two limitations: (1) the need to compare a large number of Ig molecules simultaneously, and (2) the avidity effect observed with full-length Ig, which can significantly alter the desired affinity classification. This specification describes a screening method that utilizes antibody fragments, e.g., Fab, to identify and classify binders in a rapid, high-throughput process that avoids the avidity effect.

[0044] This specification describes (1) performing all operations simultaneously with a pool of up to 24 (or more, depending on the plate capacity) antibodies using 96-well flat-bottom plates and 96-well filter plates, and (2) a method for solving both problems at once by generating an antibody fragment pool containing fragments (e.g., Fab fragments) from the antibody pool by digesting them with papain (or another suitable protease or chemical reaction to generate binding fragments corresponding to the antibody pool) and removing the Fc domain via protein A purification.

[0045] The degree of sufficient digestion and Fc removal was confirmed by SDS-PAGE and size exclusion chromatography (SEC) before proceeding to label-free biosensing-based rating experiments. Antibody rating by using the corresponding fragment (e.g., Fab) instead of full-length Ig eliminated avidity effects and enabled true affinity-based rating. off It becomes possible to obtain the value. Furthermore, K off By using this method, it becomes possible to grade antibody fragments without having to determine the antibody concentration, thereby allowing for, for example, K on or K D This grants a significant advantage to ratings based on [the specified criteria].

[0046] The method described herein eliminates the avidity effect from label-free biosensing-based antibody grading procedures by using, for example, Fab antibody fragments instead of full-length Ig molecules. A high-throughput format that allows for rapid grading of approximately 24 antibodies in, for example, less than one week, depending on the plate capacity used in the method. Using the exemplary system described herein, up to 24 antibodies can be graded in a single experiment by their antigen-binding affinity (using Fab antibody fragments) rather than avidity (when full-length Ig is used). Those skilled in the art will understand that the system capacity can be adjusted to grade smaller or larger antibody pools. All 24 Fab fragments were generated simultaneously under the same conditions, which provided the advantage of direct comparison and ensured reliable affinity grading.

[0047] Label-free biosensing Label-free biosensing can be used to determine antibody fragment binding ratings. Label-free biosensing can be carried out from the group consisting of, for example, (a) polarization analysis; (b) surface plasmon resonance (SPR); (c) localized surface plasmon resonance using noble metal nanoparticles in solution or on a transparent surface; (d) surface acoustic wave (SAW) devices; (e) attenuated quartz crystal microbalance (QCM-D); (e) atomic force microscopy; (f) plasmon-photon coupling; (g) transmission sensing via subwavelength nanoholes (enhanced light transmission); (h) photonic crystal sensing; (i) refractive index sensing; (j) waveguide mode resonance sensing; (k) ring resonator sensing; (l) isothermal titration calorimetry (ITC); (m) microscale thermophoresis; and (n) biolayer interferometry (BLI).

[0048] High-throughput screening of antibody fragments The method described herein facilitates the development of antibody drug candidates by rapidly screening Fab fragments through a combination of preparation of Fab fragments via enzymatic digestion of full-length antibodies (e.g., IgG), isolation of Fab fragments via Fc fragment removal using protein A, and label-free high-throughput biosensing. The method described herein is superior to label-free biosensing-based antibody grading of full-length Ig because it eliminates the avidity element by using Fab antibody fragments instead. Simultaneous plate-based Fab generation using up to, for example, 24 antibodies provides a new level of reliability through actual direct comparisons essential for reliable affinity grading. Furthermore, from digestion to K off The entire rating process requires only 2-3 days for up to 24 types of antibodies, offering unprecedented economic efficiency in terms of time and human resources, thus presenting a significant economic advantage over previous methods.

[0049] Method for generating antibodies The antibodies described herein (including scFv and antibody fragments or variants described herein, or alternatively, other molecules comprising them) can be produced by any method known in the art for the synthesis of antibodies, in particular, for example, by chemical synthesis or by recombinant expression techniques.

[0050] Single-chain Fv (scFv) can be generated (Brinkmann, U. et al., J. Immunol. Methods, 182:41-50, 1995; Ames, R. et al., J. Immunol. Methods, 184:177-86, 1995; Kettleborough, C. et al. al.,Eur.J.Immunol.,24:952-8,1994;Persic,L.et al.,Gene,187:9-18,1997;Burton,D.and Barbas, C., Adv. Immunol., 57:191-280, 1994; International Publication No. 92 / 001047; International Publication No. 90 / 02809; International Publication No. 91 / 10737; International Publication No. 92 / 01047; International Publication No. 92 / 18619; International Publication No. 93 / 11236; International Publication No. 95 / 15982; International Publication No. 95 / 20401; Country International publication pamphlet No. 97 / 13844; and U.S. pamphlets No. 5,698,426; No. 5,223,409; No. 5,403,484; No. 5,580,717; No. 5,427,908; No. 5,750,753; No. 5,821,047; No. 5,571,698; No. 5,427,908; No. 5,516,637; No. 5,780,225; No. 5,658,727; No. 5,733,743 and No. 5,969,108). Antibody-coding regions from phages can be isolated and used to generate complete antibodies, including human or humanized antibodies, or any other desired antigen-binding fragments, and can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria (as described below, for example). Techniques for recombinantly generating Fab, Fab', and F(ab')2 fragments can also be used (International Publication No. 92 / 22324; Mullinax, R. et al., BioTechniques, 12:864-9, 1992; Sawai, H. et al., Am.J. Reprod.Immunol., 34:26-34, 1995; Better, M. et al., Science, 240:1041-3, 1988).

[0051] To generate a complete antibody, PCR primers containing a VH or VL nucleotide sequence, a restriction site, and an adjacent sequence to protect the restriction site may be used to amplify the VH or VL sequence in the scFv clone. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain may be cloned into a vector expressing the VH constant region, e.g., the human gamma-4 constant region, and the PCR-amplified VL domain may be cloned into a vector expressing the VL constant region, e.g., the human kappa or lambda constant region. The vector for expressing the VH or VL domain may contain a suitable promoter for inducing heavy and light chain expression in a selected expression system, a secretory signal, a cloning site for the Ig variable domain, the Ig constant domain, and a selection marker such as neomycin. The VH and VL domains may also be cloned into a single vector expressing the required constant region. The heavy chain conversion vector and light chain conversion vector are then co-transfected into a cell line to generate a stable or transient cell line expressing a full-length antibody, e.g., IgG, using techniques known to those skilled in the art.

[0052] Once the antibodies described herein (including antibody fragments, or variants thereof, or alternatively molecules comprising them) are isolated, chemically synthesized, or recombinantly expressed, they may be purified by any method known in the art for the purification of immunoglobulin molecules, or more commonly, protein molecules, for example, by chromatography (e.g., by ion exchange, affinity, particularly affinity to specific antigens after protein A, and sizing column chromatography), centrifugation, solubility difference, or any other standard technique for protein purification. Furthermore, the humanized antibodies described herein may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification. The identified antibody fragments may be re-engineered into full-length antibodies or other manipulated conjugated molecules based on the identified CDR.

[0053] The host cells used to express antibodies may be any of the following: bacterial cells, e.g., Escherichia coli (E. coli), yeast (e.g., Saccharomyces cerevisiae), or eukaryotic cells (e.g., mammalian cell lines). For example, clearly defined types of mammalian cells for this purpose, such as myeloma cells, 3T3, HeLa, C6A2780, Vero, MOCK II, Chinese hamster ovary (CHO), Sf9, Sf21, COS, NS0, or HEK293 cell lines, may be used.

[0054] Conventional techniques are included in general methods for constructing vectors, transfection methods required to generate host cells, and culture methods required to generate antibodies and their fragments from the host cells. The cell line used to generate antibodies is preferably a mammalian cell line, but any other suitable cell line may be used, such as a bacterial cell line, for example, a bacterial strain derived from Escherichia coli (E. coli), or a yeast cell line.

[0055] antibody specificity The binding specificity of an antibody can be determined by any suitable means. Examples of suitable assays for measuring binding specificity include, but are not limited to, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Other means, such as SPR or any techniques described above or known, may also be used.

[0056] Antibody binding affinity can be determined, for example, by scatchard analysis (Frankel, M. & Gerhard, W., Mol. Immunol., 16:101-6, 1979). In another embodiment, binding affinity is measured by the antigen / antibody dissociation rate. High binding affinity can be measured by competitive radioimmunoassay. Binding affinity can be measured by ELISA. Antibody affinity can be measured by flow cytometry.

[0057] Antibodies that "specifically bind" to an antigen or "immunologically bind" are antibodies that bind to the antigen with high affinity and do not significantly bind to other unrelated antigens. Antibodies can bind to epitopes with binding affinities ranging from picomolar concentrations (very high affinity) to low micromolar concentrations (weak binder). Most antibody drugs have a dissociation constant (K) ranging from low nanomolar to picomolar concentrations. d For example, it has concentrations of approximately 1 pM, approximately 900 nM, approximately 80 pM, approximately 700 nM, approximately 200 pM, approximately 500 nM, approximately 500 pM, approximately 400 nM, approximately 750 pM, approximately 900 nM, approximately 500 pM, approximately 900 nM, approximately 100 pM, approximately 900 nM, approximately 50 pM, or approximately 500 nM.

[0058] Antibody variant The antibodies described herein may be "chimeric," meaning an antibody in which a portion of the heavy or light chain originates from a particular source or species, while at least the remaining portion of the heavy or light chain originates from a different source or species. In some embodiments, a chimeric antibody refers to an antibody comprising at least one variable region from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (e.g., human, dog, cat, horse, etc.). A "humanized antibody" means an antibody in which at least one amino acid in a portion of the non-human variable region is replaced with a corresponding amino acid from the human variable region. In some embodiments, a humanized antibody comprises at least one human constant region (e.g., γ constant region, α constant region, δ constant region, ε constant region, μ constant region, etc.) or a fragment thereof. In some embodiments, a humanized antibody is an antibody fragment, e.g., Fab, scFv, (Fab')2, etc. The term "humanization" also refers to a form of non-human (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of the antibody) containing the minimal sequence of a non-human immunoglobulin. Humanized antibodies may include human immunoglobulin (recipient antibody) in which residues from the recipient's CDR are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capability. In some examples, Fv framework region (FR) residues of human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may include residues not found in the recipient antibody or the introduced CDR or framework sequence, but which are included to further improve and optimize antibody performance.

[0059] Monoclonal antibodies The monoclonal antibodies disclosed herein may be of any isotype. A monoclonal antibody may be, for example, an IgM or IgG antibody, e.g., IgG1 or IgG2. The class of an antibody may be switched to another according to known procedures (e.g., IgG may be switched to IgM). Class switching may also be used to convert one IgG subclass to another, for example, from IgG1 to IgG2.

[0060] The humanized antibodies described herein may be monovalent, divalent, trivalent, or polyvalent. For example, a monovalent scFv may be polyvalent either chemically or by association with another protein or substance. scFv fused to a hexahistidine tag or Flag tag may be polyvalent using Ni-NTA agarose (Qiagen) or anti-Flag antibody (Stratagene, Inc.).

[0061] Humanized antibodies can be monospecific, bispecific, tripspecific, or more multispecific. Multispecific antibodies can be specific to different epitopes or heterologous epitopes, such as heterologous polypeptides or solid support materials. [Examples]

[0062] A full-length antibody pool was obtained and digested with papain according to the following protocol to generate Fc and Fab fragments: (1) A new digestion buffer solution was prepared: 20 mM sodium phosphate, 10 mM EDTA, 20 mM cysteine-HCl (adjusted to pH 7.0); (2) Washed papain resin was prepared: 500 μL of papain resin (Fisher Scientific, cat#20341) was washed twice with 4 mL of digestion buffer and resuspended in 500 μL of digestion buffer; (3) Antibody samples were prepared by adding 50 μL of 1 mg / mL antibody to 50 μL of digestion buffer and transferred to a 96-well plate; (4) 50 μL of washed papain resin was added to 100 μL of antibody sample on the plate. PBS was added to all the other wells, covered with aluminum seal, the edges of the plate were covered with tape, and incubated overnight at 37°C at 900 rpm in a shaker (Incu-Mixer MP, Benchmark, H6004); (5) After 24 hours, the sample was transferred to a MultiScreen-GV filter plate (Millipore / Sigma, cat#MAGVS2210) and centrifuged at 1,000 g for 2 minutes; the flow-through was collected. 150 μL of 10 mM Tris-HCl, pH 7.0 was added to the plate containing the resin, the resin was resuspended, centrifuged again, and the flow-through was collected in the same collection plate; (6) The degree of IgG digestion was tested by flow-through SDS-PAGE and size exclusion chromatography (SEC).

[0063] The results from experiments generating Fab fragments and quality control using SDS-PAGE are shown in Figure 1.

[0064] The results from experiments generating Fab fragments and quality control using size exclusion chromatography (SEC) are shown in Figure 2.

[0065] After generating Fc and Fab fragments by papain digestion, the Fab fragments were purified using protein A as described below: (1) Binding buffer was prepared: 20 mM sodium phosphate, pH 7.4; (2) 500 μL of Protein A resin (Fisher Scientific, cat#15918014) was washed twice with 4 mL of binding buffer and resuspended in 500 μL of binding buffer; (3) As described above, 100 μL of Fc and Fab fragments produced by papain digestion were added to a 96-well plate; (4) 50 μL of Protein A resin (Fisher Scientific, cat#15918014) was added to each 100 μL of IgG fragment. The samples were covered with aluminum seals and incubated at 900 rpm with shaking for 1 hour at room temperature (approximately 25°C). (5) The sample was transferred to a MultiScreen-GV filter plate (Millipore / Sigma, cat#MAGVS2210), the plate was centrifuged at 1,000 g for 2 minutes, and the flow-through was collected; (6) The degree of Fab fragment purification was tested by flow-through SDS-PAGE and SEC.

[0066] As described above, after generating and purifying the Fab fragments, they were graded by antigen binding using a high-throughput biolayer interference (BLI) system including the following: (1) Using biotinylated antigen and streptavidin biosensor Octet (Fortebio cat#18-5019); (2) BLI scouting experiments were conducted to determine the optimal concentrations of biotinylated antigen and Fab fragment to be used; (3) BLI dynamics experiments were performed using a high-throughput BLI system (Octet HTX system); (4) Analyze the data and K for each Fab fragment off A value was determined, and the binder was rated accordingly.

[0067] The results of the BLI trial are shown in Figure 3.

[0068] K off The results of the value test are shown in Figure 4.

[0069] All references cited herein are incorporated herein by reference as if each reference were specifically and individually indicated to be incorporated by reference. No reference to any reference should be construed as an acknowledgment that this disclosure does not have prior rights to such reference by prior invention with respect to its disclosure prior to the filing date. It will also be understood that each or more of the elements described above may find useful applications in other types of ways different from those described above. Without further analysis, the foregoing sufficiently illustrates the essence of this disclosure so that others can easily adapt it for various applications by applying current knowledge without omitting features that properly constitute the essential features of the comprehensive or specific aspects of this disclosure as shown in the appended claims, from the perspective of the prior art. The embodiments described above are presented as examples only; the scope of this disclosure should be limited only by the following claims.

Claims

1. A method for high-throughput rating of antibodies by antigen binding, (a) Processing a pool of antibodies to produce a pool of antibody fragments, wherein the antibody fragments, when bound to an antigen, do not exhibit a binding avidity effect; (b) Purifying the pool of antibody fragments; and (c) Using a high-throughput antigen-binding assay, classify the antibody fragments in the pool of purified antibody fragments according to their binding affinity. Methods that include...

2. The method according to claim 1, wherein the pool of antibody fragments is produced by treating the pool of antibody fragments with a protease or a chemical process, so that the antibody fragments in the pool of antibody fragments bind to the antigen with or without an avidity effect on antigen binding.

3. The method according to claim 2, wherein the pool of antibody fragments is a pool of Fab fragments.

4. The method according to claim 3, wherein the pool of Fab fragments is produced by digesting the pool of antibody fragments with papain.

5. The method according to any one of claims 1 to 4, further comprising determining the degree of antibody treatment by SDS-PAGE.

6. The method according to any one of claims 1 to 5, further comprising determining the degree of antibody treatment by size exclusion chromatography (SEC).

7. The method according to any one of claims 1 to 6, further comprising determining the purity of the antibody fragment pool by SDS-PAGE.

8. The method according to any one of claims 1 to 6, further comprising determining the purity of the antibody fragment pool by size exclusion chromatography (SEC).

9. The method according to any one of claims 1 to 8, wherein the antibody pool comprises 2 to 24 different antibodies, 5 to 24 different antibodies, 10 to 24 different antibodies, 15 to 24 different antibodies, 20 to 24 different antibodies, 24 different antibodies, or more than 24 different antibodies.

10. The method according to any one of claims 1 to 9, wherein the high-throughput antigen-binding assay includes label-free biosensing.

11. The method according to claim 10, wherein the biosensing without labeling is selected from the group consisting of polarization analysis; surface plasmon resonance (SPR); localized surface plasmon resonance using noble metal nanoparticles in solution or on a transparent surface; surface acoustic wave (SAW) devices; attenuated quartz crystal microbalance (QCM-D); atomic force microscopy; plasmon-photon coupling; transmission sensing via subwavelength nanoholes (enhanced light transmission); photonic crystal sensing; refractive index sensing; waveguide mode resonance sensing; ring resonator sensing; isothermal titration calorimetry (ITC); microscale thermophoresis; and biolayer interferometry (BLI).

12. The high-throughput antigen-binding assay according to any one of claims 1 to 11, comprising biolayer interference (BLI).

13. (a) and (b) the method according to any one of claims 1 to 12, which is carried out on a plate.

14. (a) to (c) the method according to any one of claims 1 to 13, which is automated.

15. The antibody fragment is related to the antigen K ｏｆｆ The method according to any one of claims 1 to 14, which is graded according to speed.

16. An isolated antibody identified by the method described in any one of claims 1 to 15.

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