High throughput fab antibody fragment generation and ranking

Abstract

Methods and assays are provided for high-throughput ranking of antigen binding by generating and testing of antibody fragments from full‑length antibodies.

Description

Alexion Reference: 0693WO HIGH THROUGHPUT FAB ANTIBODY FRAGMENT GENERATION AND RANKING CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0002] Antibody production involves preparation of antigen samples and their injection into an animal to evoke high expression levels of antigen-specific antibodies in the serum, which can then be recovered from the animal. Polyclonal antibodies are recovered directly from serum. Monoclonal antibodies are produced by fusing antibody-secreting spleen cells from immunized mice with immortalized myeloma cell to create monoclonal hybridoma cell lines that express the specific antibody in cell culture supernatant. For antibody production, a screening step to identify antibodies that have preferred binding properties is required. This is an expensive and time- consuming step in antibody production. There remains a need in the art for rapid and efficient screening and identification of antibodies with desirable binding characteristics. The need for a rapid and efficient screen for antibodies with desirable binding characteristics comes primarily from the fact that antibody binding properties define how a particular antibody functions as a therapeutic agent. Effective blocking antibody therapeutics, for example, usually require the highest possible binding affinity of a given antibody to its target. SUMMARY

[0003] In an embodiment, a method for high-throughput ranking of antibodies by antigen binding can comprise: (a) treating 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 pool of purified antibody fragment according to their binding affinities.

[0004] In an embodiment, the pool of antibody fragments can be generated by treating the pool of antibody fragments with a protease or chemical process, whereby the antibody fragments in the pool of antibody fragments are able to bind antigen with reduced or no avidity effects on antigen binding. The pool of antibody fragments can be a pool of Fab fragments.

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

[0006] In an embodiment, the method can further comprise determining the extent of antibody treatment by SDS-PAGE.Alexion Reference: 0693WO

[0007] In an embodiment, the method can further comprise determining the extent of antibody treatment by size exclusion chromatography (SEC).

[0008] In an embodiment, the method can further comprise determining the purity of the pool of antibody fragments by SDS-PAGE.

[0009] In an embodiment, the method can further comprise determining the purity of the pool of antibody fragments by size exclusion chromatography (SEC).

[0010] In an embodiment, the pool of antibodies can comprise 2 to 24 different antibodies, 5 to 24 different antibodies, 10-24 different antibodies, 15 to 24 different antibodies, 20 to 24 different antibodies, 24 different antibodies or more than 24 different antibodies.

[0011] In an embodiment, the high-throughput antigen binding assay comprises label-free biosensing. The label-free biosensing can be selected from the group consisting of: ellipsometry; surface plasmon resonance (SPR); localized surface plasmon resonance using noble metal nanoparticles in solution or on a transparent surface; surface acoustic wave (SAW) devices; quartz-crystal microbalance with dissipation (QCM-D); atomic force microscopy; plasmon- photon coupling; transmission sensing through sub-wavelength nanoholes (enhanced optical transmission); photonic crystal sensing; refraction sensing; guided mode resonance sensing; ring resonator sensing; isothermal titration calorimetry (ITC); microscale thermophoresis; and biolayer interferometry (BLI).

[0012] In an embodiment, the high-throughput antigen binding assay comprises biolayer interferometry (BLI).

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

[0014] In an embodiment, steps (a) through (c) are automated. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings.

[0016] FIG.1 depicts 24 Fab fragments generated by the methods described herein and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All 24 Fab fragments were confirmed to be successfully purified.

[0017] FIG.2 depicts size exclusion chromatography (SEC) results for 24 Fab fragments generated by the methods described herein. All 24 Fab fragment were confirmed to be successfully purified.Alexion Reference: 0693WO

[0018] FIG.3 depicts 24 Fab fragments generated by the methods described herein and analysed by Biolayer Interferometry (BLI). As shown, variants #1, #5, #6 and #24 did not bind antigen as Fabs, and were eliminated from consideration. The remaining variants were ranked by Koff.

[0019] FIG.4 depicts the BLI ranking results of the 24 Fab fragments. The methods described herein successfully ranked 24 antibodies as Fab fragments by Koff. The use of Fab fragments instead of full antibodies allowed for the rapid and efficient elimination of the weakest binders. The use of Fab fragments changed the overall variants ranking by Koff as compared to the full antibody ranking. Variants #1, #5, #6 and #24 were excluded as showing no binding to the antigen. The Koffaffinity ranking produced as a result of this experiment was directly used for final candidate molecule selection. DETAILED DESCRIPTION

[0020] It is to be understood that the disclosure is not limited to the particular embodiments described below, as variations of the particular embodiments can be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims.

[0021] The singular forms “a,” “an” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

[0022] “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While Abs exhibit binding specificity to a specific antigen, immunoglobulins include both Abs and other Ab-like molecules that lack antigen specificity. The term “antibody” specifically includes monoclonal antibodies, including antibody fragment clones.

[0023] “Native antibodies and immunoglobulins” are usually heterotetrameric glycoproteins of about 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 linkages varies between the heavy chains of different Ig isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and theAlexion Reference: 0693WO light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains. There are examples of native antibodies and immunoglobulins that are not heterotetrameric, e.g., camelid, e.g., llama or alpaca, antibodies, which comprise only a heavy chain (these are single chain immunoglobulins and are sometimes referred to as “VHH” antibodies).

[0024] The term “variable,” in the context of antibody variable domains, refers broadly to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. Variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs; i.e., CDR1, CDR2 and CDR3) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0025] “Antibody fragment” refers to any antibody fragment such as Fab, Fab′, F(ab′)2, Fv, and Fd fragments; diabodies; single-chain antibody molecules, including 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 fragments” comprise a portion of an intact antibody, generally the antigen binding or variable region of the intact antibody. Antibody fragments may be derived from human or other mammalian cell lines, including natural or genetically modified forms such as humanized, human, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated, from sources including but not limited to bacterial cell lines, insect cell lines, plant cell lines, yeast cell lines, or cell lines of other origin. Antibody fragments may also be derived by controlled lysis of purified antibody with enzymes such as, but not limited to ficin, papain or pepsin. Papain digestion of antibodies produces two identical antigen-binding fragments called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin digestion of antibodies yields an F(ab′)2 fragment that hasAlexion Reference: 0693WO two antigen-combining sites and is still capable of cross-linking antigen. Ficin digestion of antibodies yields either F(ab′)2 or Fab fragments, depending on the concentration of cysteine included when performing the digestion.

[0026] “Fv” is the minimum antibody fragment that contains a complete antigen-recognition and binding site. In a two-chain Fv species, this region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In a single-chain Fv species, one heavy and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two- chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an 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 an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. Although not an antibody fragment, a VHH exhibits monovalent antigen-binding with only three CDRs. Antibody fragments that are derived from VHH molecules can be fused, with or without a suitable linker peptide, to other VHH fragments to, for example, increase binding through avidity, or to create bi-specific or multi-specific fusion proteins comprising VHH fragments that bind to or more antigens.

[0027] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0028] The “light chains” of Abs from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0029] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.Alexion Reference: 0693WO

[0030] “Monoclonal antibody,” as used herein, refers broadly to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture methods, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies described herein can be made by a hybridoma method or by recombinant DNA methods. The term “monoclonal antibodies” also includes clones of antigen-recognition and binding-site containing antibody fragments (Fv clones) isolated from phage antibody libraries.

[0031] The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0032] A “human antibody” (also called a “fully human antibody”) is an antibody that includes human framework regions and the CDRs from a human immunoglobulin. In one example, the framework and the CDRs are from the same originating human heavy and / or light chain amino acid sequence. Framework regions from one human antibody can be engineered to include CDRs from a different human antibody.

[0033] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies), that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (such as mouse, camelid, rat or rabbit) or a synthetic sequence (donor antibody), having the desired specificity, affinity and capacity. InAlexion Reference: 0693WO some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In an embodiment, all CDRs can be from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they should be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, such as about 95% or more identical. All parts of a humanized immunoglobulin, except possibly the CDRs, are therefore substantially identical to corresponding parts of natural human immunoglobulin sequences. A “humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering.

[0034] “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0035] “Diabodies,” as used herein, refers broadly to small antibody fragments with two antigen-binding sites. Such fragments can comprise, for example, a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). 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 the complementary domains of another chain and create two antigen-binding sites.

[0036] An “isolated” antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The antibody can be purified, for example, (1) to greater than 95% by weight of antibody as determined by the Lowry method (or optimally, to more than 99% by weight), (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducingAlexion Reference: 0693WO conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. An isolated antibody is prepared by at least one purification step.

[0037] “Variant” as used herein refers broadly to a polypeptide that possesses a similar or identical function as an antibody or antibody fragment thereof, but does not necessarily comprise a similar or identical amino acid sequence of the antibody or antibody fragment thereof, or possess a similar or identical structure of an antibody or antibody fragment thereof. A variant having a similar amino acid identity refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide comprising, or alternatively consisting of, 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, including a VHH, or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those described herein); (b) a polypeptide encoded by a nucleotide sequence, the complementary sequence of which hybridizes under stringent conditions to a nucleotide sequence encoding an a antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of those described herein), 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 40 amino acid residues, at least 50 amino acid residues, at least 60 amino 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, or at least 150 amino acid residues; 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 the nucleotide sequence encoding an antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence described herein). A polypeptide with similar structure to an antibody or antibody fragment thereof, described herein, refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of an antibody, or antibody fragment thereof, described herein. The structure of a polypeptide can 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 percent identity of two amino acid sequences or of two nucleicAlexion Reference: 0693WO acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When 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 percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., % identity equals [number of identical overlapping positions] / [total number of positions] x 100%). In one embodiment, the two sequences are the same length.

[0038] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. The BLASTn and BLASTx programs (Altschul, S. et al., J. Mol. Biol., 215:403-10, 1990) have incorporated such an algorithm. BLAST nucleotide searches (available at blast.ncbi.nlm.nih.gov) can be performed, for example, with the BLASTn program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules described herein. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized (Altschul, S. et al., Nucleic Acids Res., 25:3389-402, 1997). Alternatively, PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST and PSI-BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used.

[0039] “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease the affinity of a protein, such as an antibody to a target epitope. For example, a monoclonal antibody that immunospecifically binds human light chain amyloid fibril can include at most about 1, at most about 2, at most about 5, at most about 10, or at most about 15 conservative substitutions and immunospecifically bind a human light chain amyloid fibril polypeptide. The term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibody immunospecifically binds the target epitope. Non-conservative substitutions are those that reduce binding to the target epitope.

[0040] Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples ofAlexion Reference: 0693WO amino acids that, when amino acids within a group are substituted one for another, can be considered conservative substitutions: 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).

[0041] “Derivative” as used herein, refers to a variant polypeptide described herein that comprises, or alternatively consists of, an amino acid sequence of an antibody, which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term “derivative” as used herein also refers to an antibody that has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, an antibody, can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of an antibody or antigen-binding fragment thereof, can be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of an antibody or antigen-binding fragment thereof, may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as an antigen-binding fragment thereof, described herein.

[0042] “Epitopes” as used herein refers to portions of a peptide having antigenic or immunogenic activity in an animal, e.g., a mammal, e.g., a human. Antigenic epitopes need not necessarily be immunogenic.

[0043] “Fragment” as used herein refers broadly to a polypeptide comprising 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 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 the amino acid sequence of an antibody (including molecules such as Fabs or scFvs, that comprise, or alternatively consist of, antibody fragments or variants thereof).Alexion Reference: 0693WO

[0044] “Fusion protein” as used herein refers broadly to a polypeptide that comprises, or alternatively consists of, an amino acid sequence of an antibody described herein and an amino acid sequence of a heterologous polypeptide (e.g., a polypeptide unrelated to an antibody or antibody domain). High Throughput Fab Antibody Fragment Generation and Ranking

[0045] The methods described herein allow for generation of antibodies or antibody fragments, e.g., Fab fragments, typically generated by targeted degradation of antibodies- typically through the use of a protease, e.g., papain, pepsin or ficin, from full antibodies and ranking them by binding affinity using label-free biosensing techniques in a high throughput (HT) manner.

[0046] Ranking antibody variants by their ability to bind to their antigen is a part of the monoclonal antibody drug development process. The antibody ranking procedures are used to differentiate between hits coming out from various antibody discovery efforts, antibody humanization projects, or other projects, to improve lead molecule selection. There are at least two limitations for this procedure, however: (1) the need to compare high numbers of Ig molecules at the same time, and (2) the avidity effect observed with full Igs that may significantly alter the desired affinity ranking. Described herein are screening methods that utilize antibody fragments, e.g., Fabs, to identify and rank binders in a rapid, high-throughput process that avoids the avidity effect.

[0047] Described herein are methods that solve both issues at once by: (1) performing all manipulations with a pool of up to 24 antibodies simultaneously using 96-well flat bottom plates and 96-well filter plates (or more, depending on the capacity of the plates), and (2) generating an antibody fragment pool comprising fragments (e.g., Fab fragments) from the antibody pool by, for example, 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 through Protein A purification.

[0048] The full extent of the digestion and the full extent of Fc removal was confirmed by SDS-PAGE and size exclusion chromatography (SEC) before proceeding to the label-free biosensing-based ranking experiment. Ranking of antibodies by using corresponding fragments (e.g., Fabs) rather than full Igs allows one to eliminate the avidity effect and obtain Koffvalues for a true affinity-based ranking. Using Koff further allows for ranking antibody fragments without having to determine the concentration of the antibody, thereby conferring a significant advantage to rankings based on, for example, Konor KD.Alexion Reference: 0693WO

[0049] The methods described herein eliminate the avidity effect from a label-free biosensing- based antibody ranking procedure by using, for example, Fab antibody fragments instead of full- length Ig molecules. The high-throughput format, allowing one to rapidly rank, depending on the capacity of the plates used in the methods, about 24 antibodies, e.g., in less than one week. Up to 24 antibodies can be ranked in a single experiment by their antigen binding affinity (using Fab antibody fragments) and not avidity (when full Igs are used), using the exemplary system described herein. One of skill in the art would understand that the capacity of the system can be adjusted to rank smaller or larger antibody pools. All 24 Fab fragments were produced under the same conditions, at the same time, which provided an advantage of a direct comparison and ensured a reliable affinity ranking. Label-Free Biosensing

[0050] Label-free biosensing can be used to determine the antibody fragment binding ranking. Label-free biosensing can be carried out, for example, by: (a) ellipsometry; (b) surface plasmon resonance (SPR); (c) localized SPR using noble metal nanoparticles in solution or on a transparent surface; (d) surface acoustic wave (SAW) devices; (e) quartz-crystal microbalance with dissipation (QCM-D) (e) atomic force microscopy; (f) plasmon-photon coupling; (g) transmission sensing through sub-wavelength nanoholes (enhanced optical transmission); (h) photonic crystal sensing; (i) refraction sensing; (j) guided mode resonance sensing; (k) ring resonator sensing; (l) isothermal titration calorimetry (ITC); (m) microscale thermophoresis; or (n) biolayer interferometry (BLI). High-throughput Screening of Antibody Fragments

[0051] The methods described herein combine preparation of Fab fragments via enzymatic digestion of full-length antibodies (e.g., IgG), isolation of the Fab fragments via Fc fragment removal using Protein A, and high-throughput label-free biosensing, to facilitate the development of antibody drug candidates by quickly screening the Fab fragments. The methods described herein are superior to label-free biosensing-based antibody ranking of full Igs because the instant methods eliminate the avidity component by using Fab antibody fragments instead. Performing plate-based Fab generation with up to, for example, 24 antibodies simultaneously provides a new level of confidence because of actual direct comparison that is indispensable for a reliable affinity ranking. Moreover, the whole process from digestion to Koff ranking results requires only 2–3 days for up to 24 antibodies, which provides an unprecedented economy of time and human resources, thus representing a significant economic advantage over prior methods.Alexion Reference: 0693WO Methods for Producing Antibodies

[0052] The antibodies described herein (including scFvs and other molecules comprising, or alternatively consisting of antibody fragments or variants described herein) 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.

[0053] Single chain Fvs (scFvs) 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., 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; WO 92 / 001047; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; W097 / 13844; and U.S. Patent Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108). Antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human or humanized antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below. Techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed (WO 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).

[0054] To generate whole antibodies, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains can be cloned into vectors expressing a VH constant region, e.g., the human gamma 4 constant region, and the PCR amplified VL domains can be cloned into vectors expressing a VL constant region, e.g., human kappa or lambda constant regions. Vectors for expressing the VH or VL domains can comprise a promoter suitable to direct expression of the heavy and light chains in the chosen expression system, a secretion signal, a cloning site for the Ig variable domain, Ig constant domains, and a selection marker such as neomycin. The VH and VL domains may also be cloned into one vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., lgG, using techniques known to those of skill in the art.

[0055] Once an antibody described herein (including molecules comprising, or alternatively consisting of, antibody fragments, or variants thereof) has been isolated, chemically synthesized or recombinantly expressed, it can be purified by any method known in the art for purification ofAlexion Reference: 0693WO an immunoglobulin molecule, or more generally, a protein molecule, such as, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the humanized antibodies described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification. Identified antibody fragments can be engineered back into fill-length antibodies or other engineered binding molecules based on identified CDRs.

[0056] Host cells used to express the antibodies can be either a bacterial cell such as E.coli, yeast (e.g., S. cerevisiae), or a eukaryotic cell (e.g., a mammalian cell line). For example, a mammalian cell of a well-defined type for this purpose, such as a myeloma cell, 3T3, HeLa, C6A2780, Vero, MOCK II, a Chinese hamster ovary (CHO), Sf9, Sf21, COS, NS0, or HEK293 cell line can be used.

[0057] The general methods for constructing vectors, the transfection methods required to produce the host cell, and the culturing methods required to produce the antibodies and fragments thereof from said host cells include conventional techniques. Although the cell line used to produce antibodies is preferably a mammalian cell line, any other suitable cell line, such as a bacterial cell line such as an E. coli-derived bacterial strain, or a yeast cell line, can be used. Antibody Specificity

[0058] The binding specificity of antibodies can be determined by any suitable means. Examples of suitable assays to measure binding specificity include, but are not limited to, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme- linked immunoadsorbent assay (ELISA). Other means, such as, for example, SPR or any techniques described above or that become known may also be used.

[0059] The binding affinity of antibodies can, for example, 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 an antigen / antibody dissociation rate. High binding affinity can be measured by a competition radioimmunoassay. Binding affinity can be measured by ELISA. Antibody affinity can be measured by flow cytometry.

[0060] An antibody that “specifically binds” or “immunospecifically binds” an antigen is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens. The antibodies can bind an epitope with a binding affinity from picomolar range (very high affinity) to low micromolar range (weak binders). Most antibody drugs have a dissociationAlexion Reference: 0693WO constant (Kd) in low nanomolar to picomolar range, e.g., about 1 pM, about 900 nM, about 80 pM, about 700 nM, about 200 pM, about 500 nM, about 500 pM, about 400 nM, about 750 pM, about 900 nM, about 500 pM, about 900 nM, about 100 pM, about 900 nM, about 50 pM or about 500 nM. Antibody Variants

[0061] The antibodies described herein may be “chimeric,” which refers to an antibody in which a portion of the heavy chain or light chain is derived from a particular source or species, while at least a part of the remainder of the heavy chain or light chain is derived 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 (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as human, dog, cat, equine, etc.). A “humanized antibody” means an antibody in which at least one amino acid in a portion of a non-human variable region has been replaced with the corresponding amino acid from a human variable region. In some embodiments, a humanized antibody comprises at least one human constant region (e.g., a γ constant region, an α constant region, a δ constant region, an ε constant region, a μ constant region, or etc.) or fragment thereof. In some embodiments, a humanized antibody is an antibody fragment, such as Fab, scFv, (Fab’)2, etc. The term “humanized” also denotes forms of non-human (for example, murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen- binding sequences of antibodies) that contain minimal sequence of non-human immunoglobulin. Humanized antibodies can include human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are substituted by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. Monoclonal antibodies

[0062] The monoclonal antibodies disclosed herein can be of any isotype. The monoclonal antibody can be, for example, an IgM or an IgG antibody, such as IgG1 or an IgG2. The class of an antibody can be switched with another (for example, IgG can be switched to IgM), accordingAlexion Reference: 0693WO to known procedures. Class switching can also be used to convert one IgG subclass to another, such as from IgG1 to IgG2.

[0063] The humanized antibodies described herein can be monovalent, bivalent, trivalent or multivalent. For example, monovalent scFvs can be multimerized either chemically or by association with another protein or substance. An scFv that is fused to a hexahistidine tag or a Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or using anti-Flag antibodies (Stratagene, Inc.).

[0064] Humanized antibodies can be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies can be specific for different epitopes or a heterologous epitope, such as a heterologous polypeptide or solid support material. EXAMPLE

[0065] A full-length antibody pool was obtained and digested by papain to produce Fc and Fab fragments according to the following protocol: (1) Prepared fresh digestion buffer: 20 mM sodium phosphate, 10 mM EDTA, 20 mM Cysteine-HCl, adjust to pH 7.0; (2) Prepared washed papain resin: wash 500 µL of papain resin (Fisher Scientific, cat #20341) in 4 mL of digestion buffer twice, resuspend in 500 µL digestion buffer; (3) Prepared antibody samples by adding 50 µL of 1 mg / mL antibody to 50 µL of digestion buffer, and transferred to a 96-well plate; (4) Added 50 µL of washed papain resin to 100 µL antibody samples on the plate. Added PBS to all other wells, covered with aluminum seal, covered plate edges with tape, and incubated overnight at 37oC on a shaker (Incu-Mixer MP, Benchmark, H6004) at 900 rpm; (5) After 24 hours, samples were moved to MultiScreen-GV filter plates (Millipore / Sigma, cat#MAGVS2210) and centrifuged for 2 minutes at 1,000 g; the flow through was collected. Added 150 µL of 10 mM Tris-HCl, pH 7.0 to the plate with resin, resuspended the resin, centrifuged again and collected the flow through to the same collector plate; and (6) tested the degree of IgG digestion by SDS-PAGE and size exclusion chromatography (SEC) of the flow-through.

[0066] Results from an experiment creating Fab fragments and quality control by SDS-PAGE are depicted in FIG.1.

[0067] Results from an experiment creating Fab fragments and quality control by size exclusion chromatography (SEC) are depicted in FIG.2.Alexion Reference: 0693WO

[0068] After production of the Fc and Fab fragments by papain digestion, the Fab fragments were purified using Protein A, as described below: (1) Prepared binding buffer: 20 mM sodium phosphate, pH 7.4; (2) Washed 500 µL of Protein A resin (Fisher Scientific, cat#15918014) in 4 mL of binding buffer twice, and resuspend in 500 µL binding buffer; (3) Added 100 µL of the Fc and Fab fragments generated by papain digestion, as described above, to a 96-well plate; (4) Added 50 µL of Protein A resin (Fisher Scientific, cat#15918014) to each 100 µL of IgG fragments. Covered with aluminum seal, incubated for 1 hour at RT (about 25oC) with shaking at 900 rpm; (5) Moved samples to the MultiScreen-GV filter plates (Millipore / Sigma, cat#MAGVS2210), centrifuge the plate 2 minutes at 1,000 g and collected the flow through; and (6) Tested the degree of Fab fragment purification by SDS-PAGE and SEC of the flow- through.

[0069] After the Fab fragments were produced and purified, as described above, they were ranked by antigen binding using a high-throughput biolayer interference (BLI) system, comprising: (1) Used biotinylated antigen and Streptavidin biosensors Octet (Fortebio cat#18-5019); (2) Performed BLI scouting experiments to determine optimal biotinylated antigen and Fab fragments concentration to use; (3) Ran BLI kinetics experiment on a high throughput BLI instrument (Octet HTX system); and (4) Analyzed data to determine Koff values for each Fab fragment and rank binders accordingly.

[0070] The results of the BLI testing are shown in FIG.3.

[0071] The results of the Koffvalue testing are shown in FIG.4.

[0072] All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention. It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of theAlexion Reference: 0693WO present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.

Claims

Alexion Reference: 0693WO CLAIMS What is claimed is:

1. A method for high-throughput ranking of antibodies by antigen binding comprising: (a) treating a pool of antibodies to generate a pool of antibody fragments, wherein the antibody fragments do not exhibit binding avidity effects when bound to antigen; (b) purifying the pool of antibody fragments; and (c) using a high-throughput antigen binding assay to rank the antibody fragments in the pool of purified antibody fragment according to their binding affinities.

2. The method of Claim 1, wherein the pool of antibody fragments is generated by treating the pool of antibody fragments with a protease or chemical process, whereby the antibody fragments in the pool of antibody fragments bind antigen with reduced or no avidity effects on antigen binding.

3. The method of Claim 2, wherein the pool of antibody fragments is a pool of Fab fragments.

4. The method of Claim 3, wherein the pool of Fab fragments is generated by digesting the pool of antibody fragments with papain.

5. The method of any one of Claims 1-4, further comprising determining the extent of antibody treatment by SDS-PAGE.

6. The method of any one of Claims 1-5, further comprising determining the extent of antibody treatment by size exclusion chromatography (SEC).

7. The method of any one of Claims 1-6, further comprising determining the purity of the pool of antibody fragments by SDS-PAGE.

8. The method of any one of Claims 1-6, further comprising determining the purity of the pool of antibody fragments by size exclusion chromatography (SEC).

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

10. The method of any one of Claims 1-9, wherein the high-throughput antigen binding assay comprises label-free biosensing.Alexion Reference: 0693WO 11. The method of Claim 10, wherein the label-free biosensing is selected from the group consisting of: ellipsometry; surface plasmon resonance (SPR); localized surface plasmon resonance using noble metal nanoparticles in solution or on a transparent surface; surface acoustic wave (SAW) devices; quartz-crystal microbalance with dissipation (QCM-D); atomic force microscopy; plasmon-photon coupling; transmission sensing through sub- wavelength nanoholes (enhanced optical transmission); photonic crystal sensing; refraction sensing; guided mode resonance sensing; ring resonator sensing; isothermal titration calorimetry (ITC); microscale thermophoresis; and biolayer interferometry (BLI).

12. The method of any one of Claims 1-11, wherein the high-throughput antigen binding assay comprises biolayer interferometry (BLI).

13. The method of any one of Claims 1-12, wherein (a) and (b) are performed on a plate.

14. The method of any one of Claims 1-13, wherein (a) through (c) are automated.

15. The method of any one of Claims 1-14, wherein the antibody fragments are ranked according to Koffrate with respect to the antigen.

16. An isolated antibody identified through the method of any of Claims 1-15.

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