Site-specific antibody-drug conjugation through glycoengineering

Abstract

To provide binding polypeptides (e.g., antibodies), and effector moiety conjugates thereof.SOLUTION: The present disclosure provides a binding polypeptide comprising at least one modified glycan comprising at least one moiety of Formula (IV): -Gal-Sia-C(H)=N-Q-CON-X (where, A) Q is NH or O; B) CON is a connector moiety; C) X is an effector moiety; D) Gal is a component derived from galactose; and E) Sia is a component derived from sialic acid, and Sia is present or absent). The present disclosure also provides nucleic acids encoding an antigen-binding polypeptide, recombinant expression vectors, and host cells for producing such an antigen-binding polypeptide. The present disclosure further provides methods for using the disclosed antigen-binding polypeptides to treat diseases.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 61 / 776,724, filed on March 11, 2013, entitled "Site-Specific Antibody Drug Conjugation by Carbohydrate Engineering"; U.S. Provisional Patent Application No. 61 / 776,710, filed on March 11, 2013, entitled "Highly Glycosylated Binding Polypeptide"; and U.S. Provisional Patent Application No. 61 / 776,715, filed on March 11, 2013, entitled "Fc-Containing Polypeptide with Altered Glycosylation and Reduced Effector Function". The contents of the foregoing applications are hereby incorporated by reference in their entirety.

Background Art

[0002] The treatment of cancer remains a significant challenge for humanity. Current standard treatments, including surgery, radiation, and chemotherapy, have saved the lives of many patients, but there is a great need for more effective treatments, particularly target-specific treatments that are more effective and have a large therapeutic window. One of these target-specific treatments uses antibody-drug conjugates (ADCs), in which an antigen-specific antibody targets a chemotherapy drug that is non-specific to the tumor site. These molecules have been shown to have efficacy and a favorable safety profile in clinical settings. However, the development of such treatments can be difficult due to many factors, including the stability of the antibody itself and the linkage, which can have a significant impact on tumor specificity and thus reduce efficacy. ADCs have high non-specific binding and low stability in circulation, so they are removed by normal tissues before reaching the tumor. In addition, ADCs with a significant subpopulation having high drug loading can produce aggregates that are eliminated by macrophages, thus shortening the half-life. Therefore, there is an increasing need for control and improvement of critical processes and prevention of complications such as aggregation of the product and non-specific toxicity from IgG.

[0003] The ADCs produced by current methods are effective, but the development of such therapies can be difficult because the results of the conjugation chemical reactions used often have heterogeneous mixtures. For example, the conjugation of drugs to the lysine residues of antibodies is complicated by the fact that there are many lysine residues (approximately 30) in the antibodies available for conjugation. Since the optimal number of drug-to-antibody ratios (DAR) is very low (e.g., approximately 4:1), lysine conjugation often produces a very non-uniform profile. Furthermore, many lysines are located in the critical antigen-binding sites of the CDR regions, and drug conjugation can lead to a decrease in the affinity of the antibody. On the other hand, thiol-mediated conjugation mainly targets the 8 cysteines involved in the hinge disulfide bonds, but it is still difficult to predict and identify which 4 of the 8 cysteines will consistently conjugate in various preparations. More recently, site-specific conjugation in thiol-based chemical reactions has been made possible by genetically engineering free cysteine residues, but such linkages often exhibit extremely variable stability, and the drug-linker undergoes exchange reactions with albumin and other thiol-containing serum molecules. Therefore, a site-specific conjugation strategy that produces ADCs with defined conjugation sites and stable linkages is extremely useful in ensuring drug conjugation and minimizing adverse effects on the structure or function of the antibody. Summary of the Invention Means for Solving the Problems

[0004] The present disclosure provides a binding polypeptide (e.g., an antibody), and a conjugate thereof with an effector portion (e.g., a drug conjugate). In certain embodiments, the co The conjugate comprises a site-specifically modified drug-glycan linkage within the native or modified glycan of a binding polypeptide. The present disclosure also provides a nucleic acid encoding an antigen-binding polypeptide, a recombinant expression vector, and a host cell for making such antigen-binding polypeptides. Also provided are methods of treating a disease using the antigen-binding polypeptides disclosed herein.

[0005] In certain embodiments, the binding polypeptide of the invention can be obtained by coupling an effector moiety (e.g., a drug moiety) by a stable (e.g., oxime) linkage. This strategy provides highly defined products with enhanced in vivo stability and reduced aggregation. In other embodiments, to provide additional site selectivity and uniformity, a conjugate of an effector moiety (e.g., a drug conjugate) can be formed by coupling to the terminal sugar residue (e.g., terminal sialic acid or galactose residue) of an IgG glycan. The terminal sugar residue can be readily converted to a reactive aldehyde form by mild oxidation (e.g., with sodium periodate). The oxidized sugar residue can then be conjugated to an aldehyde-reactive aminooxy drug-linker to provide a stable and homogeneous population of protein-drug conjugates (e.g., ADCs).

[0006] Accordingly, in one aspect, the invention provides at least one formula (IV): -Gal-Sia-C(H)=N-Q-CON-X Formula (IV) [wherein, A) Q is NH or O, B) CON is a connecting moiety, C) X is an effector moiety (e.g., a drug moiety or a targeting moiety), D) Gal is a component derived from galactose, E) Sia is a component derived from sialic acid, Sia is present or absent] Provided is a binding polypeptide comprising at least one modified glycan comprising a moiety.

[0007] In one embodiment, the modified glycan is a bi-branched glycan. In another embodiment, the bi-branched glycan is fucosylated or non-fucosylated. In another embodiment, the modified glycan comprises at least two moieties of formula (IV), and Sia is present in only one of the two moieties. In another embodiment, the modified glycan comprises at least two moieties of formula (IV), and Sia is present in both of the two moieties. In another embodiment, the modified glycan is N-linked to the binding polypeptide.

[0008] In another embodiment, the binding polypeptide comprises an Fc domain. In another embodiment, the modified glycan is N-linked to the binding polypeptide via the asparagine residue at amino acid position 297 of the Fc domain according to EU numbering. In another embodiment, the modified glycan is N-linked to the binding polypeptide via the asparagine residue at amino acid position 298 of the Fc domain according to EU numbering. In another embodiment, the Fc domain is human.

[0009] In another embodiment, the binding polypeptide comprises a CH1 domain. In one embodiment, the modified glycan is N-linked to the binding polypeptide via the asparagine residue at amino acid position 114 of the CH1 domain according to Kabat numbering. In one embodiment, the binding polypeptide is an antibody or an immunoadhesin.

[0010] In one embodiment, the effector moiety is a cytotoxin. In another embodiment, the cytotoxin is selected from the group consisting of the cytotoxins listed in Table 1. In another embodiment, the effector moiety is a detection agent. In a given embodiment, the effector moiety is a targeting moiety. In one embodiment, the targeting moiety is a carbohydrate or a glycopeptide. In another embodiment, the targeting moiety is a glycan. In another embodiment, the effector moiety is a detection agent. In a given embodiment, the effector moiety is a targeting moiety. In one embodiment, the targeting moiety is a carbohydrate or a glycopeptide. In another embodiment, the targeting moiety is a glycan.

[0011] In another embodiment, the linking moiety comprises a pH-sensitive linker, a disulfide linker, an enzyme-sensitive linker, or other cleavable linker moiety. In another embodiment, the linking moiety comprises a linker moiety selected from the group of linker moieties shown in Table 2 or 14.

[0012] In other aspects, the invention provides a composition comprising the binding polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. In one embodiment, the ratio of the effector moiety, for therapeutic or diagnostic use, to the binding polypeptide is less than 4. In another embodiment, the ratio of the effector moiety, for therapeutic or diagnostic use, to the binding polypeptide is about 2.

[0013] In another aspect, the invention provides a method of treating a patient in need thereof, comprising administering an effective amount of the composition of the invention.

[0014] In another aspect, the invention provides an isolated polynucleotide encoding the binding polypeptide of the invention. In another aspect, the invention provides a vector comprising the polynucleotide. In another aspect, the invention provides a host cell comprising the polynucleotide or the vector.

[0015] In yet another aspect, the invention provides a method of making the binding polypeptide of the invention, comprising reacting an effector moiety of formula (I): NH2-Q-CON-X Formula (I) [wherein, A) Q is NH or O, B) CON is a linking moiety, C) X is an effector moiety] with an altered binding polypeptide comprising oxidized glycan.

[0016] In one embodiment, the modified binding polypeptide comprises an oxidized glycan produced by reacting a binding polypeptide comprising a glycan with a mild oxidizing agent. In certain embodiments, the mild oxidizing agent is sodium periodate. In certain embodiments, less than 1 mM sodium periodate is used. In one embodiment, the oxidizing agent is galactose oxidase. In another embodiment, the binding polypeptide comprising a glycan comprises one or two terminal sialic acid residues. In another embodiment, the terminal sialic acid residue is introduced by treating the binding polypeptide with a sialyltransferase, or a combination of a sialyltransferase and a galactosyltransferase.

Brief Description of the Drawings

[0017]

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Mode for Carrying Out the Invention

[0018] The present disclosure provides a binding polypeptide (e.g., an antibody) and a conjugate thereof with an effector moiety (e.g., a drug conjugate). In certain embodiments, the conjugate comprises a site-specifically modified drug-glycan linkage within the native or modified glycan of an antigen-binding polypeptide such as an IgG molecule. The present disclosure also provides a nucleic acid encoding an antigen-binding polypeptide, a recombinant expression vector, and a host cell for producing such an antigen-binding polypeptide. The present disclosure also provides a method of treating a disease using the antigen-binding polypeptide disclosed herein.

[0019] I. Definitions As used herein, the term "binding polypeptide" or "binding polypeptide" means a polypeptide (e.g., an antibody) comprising at least one binding site responsible for selective binding to a target antigen of interest (e.g., a human antigen). Exemplary binding sites include antibody variable domains, ligand-binding sites of receptors, or receptor-binding sites of ligands. In certain embodiments, the binding polypeptide of the present invention comprises a plurality (e.g., two, three, four, or more) of binding sites.

[0020] As used herein, the term "native residue" means an amino acid residue that occurs naturally at a particular amino acid position in a binding polypeptide (e.g., an antibody or a fragment thereof) and has not been modified, introduced, or altered by human hand. As used herein, the term "modified binding polypeptide" or "altered binding polypeptide" includes a binding polypeptide (e.g., an antibody or a fragment thereof) that contains at least one non-naturally mutated amino acid residue.

[0021] As used herein, the term "specifically binds" means the ability of an antibody or an antigen-binding fragment thereof to bind to an antigen with a dissociation constant (Kd) of at most about 1×10 -6 M, 1×10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, 1×10 -12 M, or less, and / or the ability to bind to an antigen with an affinity that is at least 2-fold higher than the affinity for a non-specific antigen.

[0022] As used herein, the term "antibody" means an assembly (e.g., an intact antibody molecule, an antibody fragment, or a variant thereof) having significant known specific immunoreactive activity against a target antigen. Antibodies and immunoglobulins include light and heavy chains that may or may not have interchain covalent linkages. The basic immunoglobulin structure of the vertebrate system is relatively well understood.

[0023] As discussed in more detail below, the general term "antibody" includes five distinct classes of antibodies that can be biochemically distinguished. Although all five classes of antibodies are clearly within the scope of the present disclosure, the following discussion is generally directed to immunoglobulin molecules of the IgG class. With respect to IgG, an immunoglobulin comprises two identical light chains with a molecular weight of approximately 23,000 daltons and two identical heavy chains with a molecular weight of 53,000 - 70,000. The four chains are linked in a "Y" shape by disulfide bonds, and the light chains are connected to the heavy chains at a portion starting from the opening of the "Y" and continuing into the variable region.

[0024] The light chains of immunoglobulins are classified into either kappa or lambda (κ, λ). The class of each heavy chain can bind to either a kappa or lambda light chain. Generally, the light chains and heavy chains are covalently bonded to each other, and the positions of the "tails" of the two heavy chains are joined to each other by either covalent disulfide linkage or non-covalent linkage when the immunoglobulin is produced by either a hybridoma, B cell, or genetically engineered host cell. In the heavy chains, the amino acid sequence is arranged from the N-terminus of the fork-like end of the "Y" shape to the C-terminus at the bottom of each chain. Those skilled in the art will understand that the heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and there are several subclasses among them (e.g., γ1 - γ4). It is this property of the chains that determines the "class" of the antibody to be IgG, IgM, IgA, IgG, or IgE, respectively. The subclasses of immunoglobulin isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known to result in functional specialization. Those skilled in the art will recognize that modified forms of each of these classes and isotypes are also within the scope of the present disclosure since they can be readily recognized in view of the present disclosure.

[0025] Both the light chains and heavy chains are divided into structurally homologous regions and functionally homologous regions. The term "region" means an element or a portion of a chain of an immunoglobulin or antibody includes a constant region or a variable region, and yet another distinct element or portion of said region. For example, a light chain variable region includes "complementary determining regions" or "CDRs" that are interspersed within the "framework regions" or "FRs" as defined herein.

[0026] Regions of an immunoglobulin heavy or light chain may be defined as "constant" (C) regions or "variable" (V) regions, where in the case of "constant regions", it is so defined based on relatively little variation in the sequences within the regions of various class members, or in the case of "variable regions", it is so defined based on significant variation within the regions of various class members. The terms "constant region" and "variable region" may also be used functionally. In this regard, it will be understood that the variable region of an immunoglobulin or antibody determines antigen recognition and specificity. Conversely, the constant region of an immunoglobulin or antibody confers important effector functions such as, for example, secretion, transplacental transfer, Fc receptor binding, and complement binding. The subunit structure and three-dimensional arrangement of the constant regions of the various classes of immunoglobulins are well known.

[0027] The constant and variable regions of immunoglobulin heavy and light chains fold into domains. The term "domain" refers to a globular region of a heavy or light chain that includes peptide loops stabilized by, for example, β-pleated sheets and / or interchain disulfide bonds (e.g., including three to four peptide loops). The constant region domains on an immunoglobulin light chain are each referred to in the same sense as "light chain constant region domain", "CL region", or "CL domain". The constant domains on a heavy chain (e.g., hinge, CH1, CH2, or CH3 domains) are each referred to in the same sense as "heavy chain constant region domain", "CH" region domain, or "CH domain". The variable domains on a light chain are each referred to in the same sense as "light chain variable region domain", "VL region domain", or "VL domain". The variable domains on a heavy chain are each referred to in the same sense as "heavy chain variable region domain", "VH region domain", or "VH domain".

[0028] By convention, the numbering of the variable constant region domains increases as the variable constant region domain becomes more distal from the antigen-binding site or amino terminus of the immunoglobulin or antibody. The N-terminus of each of the heavy and light chains of an immunoglobulin is the variable region, and the C-terminus is the constant region, although in reality the carboxy termini of the heavy and light chains are included in the CH3 and CL domains, respectively. Thus, the light chain domains of an immunoglobulin are arranged in the VL-CL configuration, and the heavy chain domains are arranged in the VH-CH1-hinge-CH2-CH3 configuration.

[0029] Amino acid positions in the heavy chain constant region, such as amino acid positions in the CH1, hinge, CH2, CH3, and CL domains, may be numbered according to the Kabat index numbering system (see Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Department of Health and Human Services, 5th ed., 1991). Alternatively, the amino acid positions of an antibody may be numbered according to the EU index numbering system (see Kabat et al., ibid).

[0030] As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" includes the amino-terminal variable domain of an immunoglobulin light chain.

[0031] As used herein, the term "CH1 domain" includes, for example, the first (amino-terminal most) constant region domain of an immunoglobulin heavy chain spanning positions approximately 114 to 223 (EU positions 118 to 215) of the Kabat numbering system. The CH1 domain is adjacent to the VH domain of the immunoglobulin heavy chain and the amino terminus of the hinge region and does not form part of the Fc region.

[0032] As used herein, the term "hinge region" includes a portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three individual domains, the upper, middle, and lower hinge domains (Roux et al., J. Immunol., 1998, 161, 4083).

[0033] As used herein, the term "CH2 domain" includes a portion of the heavy chain immunoglobulin molecule that spans positions approximately 244 to 360 (EU positions 231 to 340) in, for example, the Kabat numbering system. The CH2 domain is unique in that it does not form tight pairs with other domains. In contrast, two N-linked branched carbohydrate chains intervene between the two CH2 domains of an intact native IgG molecule. In one embodiment, the binding polypeptide of the present disclosure includes a CH2 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0034] As used herein, the term "CH3 domain" includes a portion of the heavy chain immunoglobulin molecule that spans approximately 110 residues from the N-terminus of the CH2 domain, e.g., positions approximately 361 to 476 (EU positions 341 to 445) in the Kabat numbering system. The CH3 domain typically forms the C-terminal portion of the antibody. However, in some immunoglobulins, additional domains may extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ chain of IgM and the e chain of IgE). In one embodiment, the binding polypeptide of the present disclosure includes a CH3 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0035] As used herein, the term "CL domain" includes, for example, the constant region domain of an immunoglobulin light chain spanning positions approximately 107A to 216 of Kabat. The CL domain is adjacent to the VL domain. In one embodiment, the binding polypeptide of the present disclosure includes a CL domain derived from a kappa light chain (e.g., a human kappa light chain).

[0036] As used herein, the term "Fc region" is defined as the portion of the heavy chain constant region that begins at the hinge region immediately upstream of the papain cleavage site (i.e., residue 216 of IgG with the first residue of the heavy chain constant region being 114) and ends at the C-terminus of the antibody. Thus, a complete Fc region includes at least the hinge domain, the CH2 domain, and the CH3 domain.

[0037] As used herein, the term "native Fc" means a molecule that includes the sequence of a non-antigen-binding fragment obtained by digestion of an antibody or generated by other means, whether in monomeric or multimeric form, and may include the hinge region. The origin of the original immunoglobulin of native Fc is preferably of human origin and can be any immunoglobulin, but IgG1 and IgG2 are preferred. Native Fc molecules are composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from 1 to 4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of native Fc is a disulfide-linked dimer obtained by papain digestion of IgG. As used herein, the term "native Fc" is a general term for monomeric, dimeric, and multimeric forms.

[0038] As used herein, the term "Fc variant" is modified from native Fc Rather, it means a molecule or sequence that still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with these salvage receptors are known in the art. Thus, the term "Fc variant" can include humanized molecules or sequences from non-human native Fc. Further, native Fc contains regions that can be removed to provide structural features or biological activities not required for the antibody-like binding polypeptides of the present invention. Thus, the term "Fc variant" refers to a molecule or sequence in which one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cell-mediated cytotoxicity (ADCC) are deleted or one or more Fc sites or residues are modified.

[0039] As used herein, the term "Fc domain" encompasses native Fc and Fc variants, as well as the sequences defined above. Together with Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether obtained by digestion from whole antibodies or produced by other means.

[0040] As pointed out above, an antibody can selectively recognize and specifically bind to an epitope on an antigen through its variable region. That is, the VL domain and VH domain of the antibody combine to form a variable region (Fv) that defines a three-dimensional antigen-binding site. This quaternary structure of the antibody forms the antigen-binding sites present at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by three complementarity-determining regions (CDRs) on each of the variable regions of the heavy and light chains. As used herein, the term "antigen-binding site" includes a site that specifically binds to (and immunoreacts with) an antigen (e.g., a cell surface or soluble antigen). The antigen-binding site includes the variable regions of the heavy and light chains of the immunoglobulin, and the binding site formed by these variable regions determines the specificity of the antibody. The antigen-binding site is formed by variable regions that vary for each antibody. The modified antibodies of the present disclosure include at least one antigen-binding site.

[0041] In certain embodiments, the binding polypeptide of the present disclosure includes at least two antigen-binding domains that result in the association of the binding polypeptide with a selected antigen. The antigen-binding domains need not necessarily be from the same immunoglobulin molecule. In this regard, the variable regions may be from any type of animal that can initiate a humoral response and be induced to produce immunoglobulins against a desired antigen. Thus, the variable regions of the binding polypeptide may be of mammalian origin, for example, human, mouse, rat, goat, sheep, non-human primates (e.g., cynomolgus monkey, macaque, etc.), wolf, or camelid (e.g., camel, llama, and related species).

[0042] In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short non - contiguous sequences of amino acids that are specifically arranged such that the antigen - binding site is formed when the antibody assumes its three - dimensional conformation in an aqueous environment. The remainder of the heavy - and light - chain variable regions show little intermolecular variability in amino acid sequence and are called framework regions. The framework regions generally adopt a β - sheet conformation, and the CDRs form loops that connect to the β - sheet structure or, in some cases, form part of the β - sheet structure. Thus, these framework regions act to form a scaffold that positions the six CDRs in the correct positions by non - covalent interactions between the chains. The antigen - binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates non - covalent binding of the antibody to the immunoreactive antigen epitope.

[0043] Exemplary binding polypeptides of the present invention include antibody variants. As used herein, the term "antibody variant" refers to an antibody in synthetic, modified forms that are altered such that they do not occur naturally, e.g., an antibody that contains a portion of at least two heavy chains but does not contain two complete heavy chains (e.g., a domain - deleted antibody or a minibody), an antibody in a multispecific form that is altered to bind to two or more different antigens or different epitopes on a single antigen (e.g., bispecific, trispecific, etc.), a heavy - chain molecule linked to an scFv molecule, etc. Further, the term "antibody variant" includes antibodies in multivalent forms (e.g., trivalent, tetravalent, etc.), antibodies that bind to three, four, or more copies of the same antigen.

[0044] As used herein, the term "valence" means the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to one target molecule or a specific site on a target molecule. If a polypeptide contains more than one target binding site, each target binding site can specifically bind to the same or different molecules (e.g., can bind to different ligands or different antigens, or different epitopes on the same antigen). It is preferred that the binding polypeptide of interest has at least one binding site specific for a human antigen molecule.

[0045] The term "specificity" means the ability to specifically bind (e.g., immunoreact) to a given target antigen (e.g., a human target antigen). A binding polypeptide can be monospecific and contain one or more binding sites that specifically bind to a target, or the polypeptide can be multispecific and contain two or more binding sites that specifically bind to the same or different targets. In certain embodiments, the binding polypeptides of the invention are specific for two different (e.g., non-overlapping) portions of the same target. In certain embodiments, the binding polypeptides of the invention are specific for more than one target. Exemplary binding polypeptides (e.g., antibodies) containing antigen-binding sites that bind to antigens expressed on tumor cells are known in the art, and one or more CDRs from such antibodies may be included in the antibodies of the invention.

[0046] The term "linker moiety" includes a moiety to which an effector moiety can be linked to the binding polypeptides disclosed herein. The linker moiety can be selected such that it is cleavable (e.g., cleavable by an enzyme or pH-sensitive) or non-cleavable. Exemplary linker moieties are described in Table 2 herein.

[0047] As used herein, the term "effector portion" includes agents having biological or other functional activity (e.g., proteins, nucleic acids, lipids, carbohydrates, glycopeptides, drug moieties, and fragments thereof). For example, a modified binding polypeptide comprising an effector portion conjugated to a binding polypeptide has at least one additional function or property compared to an unconjugated antibody. For example, conjugation of a cytotoxic agent (e.g., an effector portion) to a binding polypeptide results in the formation of a binding polypeptide having drug cytotoxicity as a second function (i.e., in addition to antigen binding). In another example, conjugation of a second binding polypeptide to a binding polypeptide can confer additional binding properties. In certain embodiments, when the effector portion is a therapeutically or diagnostically useful protein or nucleic acid that is genetically encoded, the effector portion can be synthesized or expressed by any of the peptide synthesis methods or recombinant DNA methods well known in the art. In another aspect, when the effector portion is a non-genetically encoded peptide or drug moiety, the effector portion may be synthetically produced or purified from a natural source. As used herein, the term "drug moiety" includes anti-inflammatory agents, anti-cancer agents, anti-infective agents (e.g., antifungal agents, antibacterial agents, anti-parasitic agents, anti-viral agents, etc.), and anesthetic therapeutic agents. In a further embodiment, the drug moiety is an anti-cancer agent or a cytotoxic agent. Applicable drug moieties can also include prodrugs. Exemplary effector portions are described in Table 1 of this specification.

[0048] In certain embodiments, "effector portion" includes "targeting portion". As used herein, the term "targeting portion" means an effector portion that binds to a target molecule. Targeting portions can include, without limitation, proteins, nucleic acids, lipids, carbohydrates (e.g., glycans), and combinations thereof (e.g., glycoproteins, glycopeptides, and glycolipids).

[0049] As used herein, the term "prodrug" means a precursor or derivative form of a pharmaceutically active agent that is less active, less reactive, or less likely to cause side effects compared to the parent drug, and can be enzymatically activated or otherwise converted in vivo to a more active form. Prodrugs applicable to the compositions of the present disclosure include, but are not limited to, phosphate-containing prodrugs, amino acid-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, and other 5-fluorouridine prodrugs, which can be converted to a more active free cytotoxic drug. One of ordinary skill in the art can perform chemical modifications on the desired drug moiety or its prodrug to make the reaction of this compound more convenient for the purpose of preparing the modified binding polypeptide of the present disclosure. The drug moiety also includes derivatives, pharmaceutically acceptable salts, esters, amides, and ethers of the drug moieties described herein. Derivatives include modifications to the drugs identified herein that can improve or not significantly reduce the desired therapeutic activity of a particular drug.

[0050] As used herein, the term "anticancer agent" includes agents that are detrimental to the growth and / or proliferation of neoplastic or tumor cells and that can act to reduce, inhibit, or destroy a malignant disease. Examples of such agents include, but are not limited to, cell division inhibitors, alkylating agents, antibiotics, cytotoxic nucleosides, tubulin binders, hormones, hormone antagonists, cytotoxic agents, etc. Cytotoxic agents include, but are not limited to, tomamycin derivatives, maytansine derivatives, cryptophycine derivatives, anthracycline derivatives, bisphosphonate derivatives, leptomycin derivatives, streptonigrin derivatives, auristatin derivatives, and duocarmycin derivatives. Any agent that acts to delay or slow the growth of immunoreactive cells or malignant cells is within the scope of the present disclosure.

[0051] As used herein, the term "antigen" or "target antigen" means a molecule or a part of a molecule that can be bound by the binding site of a binding polypeptide. A target antigen can have one or more epitopes.

[0052] II. Binding Polypeptide In one aspect, the present disclosure provides a binding polypeptide (e.g., an antibody, an antibody fragment, an antibody variant, and a fusion protein) that includes a glycosylated domain, such as a glycosylated constant domain. The binding polypeptides disclosed herein include any binding polypeptide that includes a domain having an N-linked glycosylation site. In certain embodiments, the binding polypeptide is an antibody, or a fragment or derivative thereof. Any antibody from any source or species can be used in the binding polypeptides disclosed herein. Suitable antibodies include, without limitation, human antibodies, humanized antibodies, or chimeric antibodies.

[0053] In certain embodiments, the glycosylated domain is the Fc domain. In certain embodiments, the glycosylated domain is the native glycosylation domain of N297.

[0054] In other embodiments, the glycosylation domain is a modified glycosylation domain. Exemplary modified glycosylation domains in the Fc domain include an asparagine residue at amino acid position 298 according to EU numbering; and a serine or threonine residue at amino acid position 300 according to EU numbering.

[0055] In the binding polypeptides disclosed herein, Fc domains from any class of immunoglobulin (e.g., IgM, IgG, IgD, IgA, and IgE) as well as species can be used. Chimeric Fc domains that include portions of Fc domains from different species or Ig classes can also be used. In certain embodiments, the Fc domain is the Fc domain of human IgG1. In the case of the Fc domain of human IgG1, mutations to asparagine at Kabat position 298 of the wild-type amino acid, and to serine or threonine at Kabat position 300 result in the formation of an N-linked glycosylation consensus site (i.e., an N-X-T / S sequence where X is any amino acid other than proline). However, for Fc domains of other species and / or Ig classes or isotypes, one of ordinary skill in the art will understand that it may be necessary to mutate Kabat position 299 of the Fc domain if a proline residue is present in order to recreate the N-X-T / S sequence.

[0056] In other embodiments, the present disclosure provides a binding polypeptide (e.g., an antibody, an antibody fragment, an antibody variant, and a fusion protein) that includes at least one CH1 domain having an N-linked glycosylation site. Such exemplary binding polypeptides can include, for example, a modified glycosylation site at position 114 according to Kabat numbering.

[0057] All classes of immunoglobulins (e.g., IgM, IgG, IgD, IgA, and IgE) as well as CH1 domains from species can be used in the binding polypeptides disclosed herein. Chimeric CH1 domains containing portions of CH1 domains from different species or Ig classes can also be used. In certain embodiments, the CH1 domain is the CH1 domain of human IgG1. In the case of the human IgG1 domain, mutation of the wild-type amino acid asparagine at position 114 results in the formation of an N-linked glycosylation consensus site (i.e., the N-X-T / S sequence where X is any amino acid other than proline). However, for other species and / or other CH1 domains of Ig classes or isotypes, one of ordinary skill in the art will understand that it may be necessary to mutate positions 115 and / or 116 of the CH1 domain to create an N-X-T / S sequence.

[0058] In certain embodiments, the binding polypeptides of the present disclosure can include antigen-binding fragments of antibodies. The term "antigen-binding fragment" means a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with an intact antibody with respect to antigen binding (i.e., specific binding) (i.e., the intact antibody from which the antigen-binding fragment is derived). Antigen-binding fragments can be generated by recombinant or biochemical methods well known in the art. Exemplary antigen-binding fragments include Fv, Fab, Fab’, and (Fab’)2. In preferred embodiments, the antigen-binding fragments of the present disclosure are modified antigen-binding fragments that include at least one modified glycosylation site. In one exemplary embodiment, the modified antigen-binding fragment of the present disclosure includes the modified VH domain described above. In another exemplary embodiment, the modified antigen-binding fragment of the present disclosure includes the modified CH1 domain described above.

[0059] In an exemplary embodiment, the binding polypeptide comprises a single-chain variable region sequence (ScFv). The sequence of the single-chain variable region comprises a single polypeptide having one or more antigen-binding sites, such as a VL domain linked to a VH domain by a flexible linker. The ScFv molecule can be constructed in a VH-linker-VL arrangement or a VL-linker-VH arrangement. The flexible hinge connecting the VL domain and the VH domain that constitutes the antigen-binding site preferably contains from about 10 to about 50 amino acid residues. Linker peptides are known in the art. The binding polypeptide of the present invention may comprise at least one scFv and / or at least one constant region. In one embodiment, the binding polypeptide of the present disclosure comprises at least one scFv linked or fused to an antibody or fragment comprising a CH1 domain (e.g., a CH1 domain containing an asparagine residue at position 114 of Kabat), and / or a CH2 domain (e.g., a CH2 domain containing an asparagine residue at position EU298 and a serine or threonine residue at position EU300).

[0060] In certain exemplary embodiments, the binding polypeptides of the present disclosure are multivalent (e.g., tetravalent) antibodies produced by fusing a DNA sequence encoding an antibody having an ScFv molecule (e.g., a modified ScFv molecule). For example, in one embodiment, these sequences are combined such that the ScFv molecule (e.g., a modified ScFv molecule) is linked to the Fc fragment of the antibody by a flexible linker (e.g., a gly / ser linker) at its N-terminus or C-terminus. In another embodiment, the tetravalent antibodies of the present disclosure can be produced by fusing an ScFv molecule to a linker peptide fused to a CH1 domain (e.g., a CH1 domain containing an asparagine residue at position 114 of Kabat) to construct a tetravalent ScFv-Fab molecule.

[0061] In another embodiment, the binding polypeptide of the present disclosure is a modified minibody. The modified minibody of the present disclosure is a dimeric molecule composed of two polypeptide chains, each containing a ScFv molecule (e.g., a modified ScFv molecule containing the modified VH domain described above), fused to the CH3 domain or a part thereof by a connecting peptide. The minibody can be produced by constructing the ScFv component and connecting the peptide-CH3 component using methods described in the art (see, for example, U.S. Patent No. 5,837,821 or WO94 / 09817Al). In another embodiment, a tetravalent minibody can be constructed. The tetravalent minibody can be constructed in the same manner as the minibody, except that two ScFv molecules are linked using a flexible linker. The linked scFv-scFv construct is then linked to the CH3 domain.

[0062] In another embodiment, the binding polypeptide of the present disclosure includes a bispecific antibody. The bispecific antibody is a dimeric, tetravalent molecule that usually has a short (less than 10, preferably 1 to 5) amino acid residue linker connecting both variable domains such that each has a polypeptide similar to the ScFv molecule, but the VL and VH domains on the same polypeptide chain cannot interact. Instead, the VL and VH domains of one polypeptide chain interact (respectively) with the VH and VL domains on the second polypeptide chain (see, for example, WO02 / 02781). The bispecific antibody of the present disclosure includes a ScFv molecule fused to the CH3 domain.

[0063] In other embodiments, the binding polypeptide of the present invention includes a multispecific or multivalent antibody that contains one or more variable domains consecutively on the same polypeptide chain, e.g., a It includes tandem variable domain (TVD) polypeptides. Exemplary TVD polypeptides include the "double head" or "dual Fv" configurations described in U.S. Patent No. 5,989,830. In the dual Fv configuration, the variable domains of two different antibodies are expressed in a tandem arrangement on two separate chains (one heavy chain and one light chain), where in one polypeptide chain, there are two consecutive VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains continuously connected by a peptide linker (VL1-linker-VL2). In the crossed double head configuration, the variable domains of two different antibodies are expressed in a tandem arrangement on two separate polypeptide chains (one heavy chain and one light chain), where in one polypeptide chain, there are two consecutive VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of complementary VL domains continuously connected by a peptide linker in the reverse arrangement (VL2-linker-VL1). Further antibody variants based on the "dual-Fv" format include dual-variable domain IgG (DVD-IgG) bispecific antibodies (see U.S. Patent No. 7,612,181) and the TBT1 format (see US2010 / 0226923A1). By adding constant domains to each chain of the dual-Fv (CH1-Fc to the heavy chain and kappa or lambda constant domains to the light chain), functional bispecific antibodies are provided without the need for any further modification (i.e., constant domains are clearly added to enhance stability).

[0064] In another exemplary embodiment, the binding polypeptide comprises a bispecific antibody having a crossed double variable domain IgG (CODV-IgG) based on a "double head" conformation (see US20120251541A1, the entire text of which is incorporated herein by reference). The CODV-IgG antibody variant has a single polypeptide chain having a VL domain (VL1-L1-VL2-L2-CL) continuously connected to the CL domain, and a complementary VH domain (VH2-L3-VH1-L4-CH1) continuously connected to the CH1 domain in the opposite orientation. The polypeptide chains form a crossed light chain-heavy chain pair. In a given embodiment, the second polypeptide may be further connected to the Fc domain (VH2-L3-VH1-L4-CH1-Fc). In a given embodiment, linker L3 is at least twice as long as linker L1 and / or linker L4 is at least twice as long as linker L2. For example, L1 and L2 may be 1 to 3 amino acid residues in length, L3 may be 2 to 6 amino acid residues in length, and L4 may be 4 to 7 amino acid residues in length. Examples of suitable linkers include a single glycine (Gly) residue, a diglycine peptide (Gly-Gly), a tripeptide (Gly-Gly-Gly), a peptide of 4 glycine residues (Gly-Gly-Gly-Gly), a peptide of 5 glycine residues (Gly-Gly-Gly-Gly-Gly), a peptide of 6 glycine residues (Gly-Gly-Gly-Gly-Gly-Gly), a peptide of 7 glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly), a peptide of 8 glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly). Other combinations of amino acid residues such as the peptide Gly-Gly-Gly-Gly-Ser and the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser may also be used.

[0065] In certain embodiments, the binding polypeptide comprises an immunoadhesin molecule comprising a non-antibody binding region (e.g., a receptor, ligand, or cell adhesion molecule) fused to an antibody constant region (see, e.g., Ashkenazi et al., Methods, Vol. 8(2), pp. 104-115, 1995, the entire disclosure of which is incorporated herein by reference).

[0066] In certain embodiments, the binding polypeptide comprises an immunoglobulin-like domain. Suit Examples of agonistic immunoglobulin-like domains include, but are not limited to, fibronectin domains (see, e.g., Koide et al. (2007), Methods Mol. Biol., Vol. 352, pp. 95-109, which is incorporated herein by reference in its entirety), DARPins (see, e.g., Stumpp et al. (2008), Drug Discov. Today, Vol. 13(15-16), pp. 695-701, which is incorporated herein by reference in its entirety), the Z domain of protein A (see, e.g., Nygren et al. (2008), FEBS J., Vol. 275(11), pp. 2668-76, which is incorporated herein by reference in its entirety), lipocalins (see, e.g., Skerra et al. (2008), FEBS J., Vol. 275(11), pp. 2677-83, which is incorporated herein by reference in its entirety), Affilins (see, e.g., Ebersbach et al. (2007), J. Mol. Biol., Vol. 372(1), pp. 172-85, which is incorporated herein by reference in its entirety), Affitins (see, e.g., Krehenbrink et al. (2008), J. Mol. Biol., Vol. 383(5), pp. 1058-68, which is incorporated herein by reference in its entirety), Avimers (see, e.g., Silverman et al. (2005), Nat. Biotechnol., Vol. 23(12), pp. 1556-61, which is incorporated herein by reference in its entirety), Fynomers (see, e.g., Grabulovski et al. (2007), J Biol Chem, Vol. 282(5), pp. 3196-3204, which is incorporated herein by reference in its entirety), and Kunitz domain peptides (see, e.g., Nixon et al. (2006), Curr Opin Drug Discov Devel, Vol. 9(2), pp. 261-8, which is incorporated herein by reference in its entirety).

[0067] III. N-linked glycans In certain embodiments, the binding polypeptide of the present invention uses an N-linked glycan that is "N-linked" via an asparagine residue to a glycosylation site in the polypeptide backbone of the binding polypeptide. The glycosylation site may be a natural or a modified glycosylation site. Additionally, or alternatively, the glycan may be a natural glycan or a modified glycan that includes non-natural linkages.

[0068] In certain exemplary embodiments, the binding polypeptide of the present invention includes a natural glycosylation site of an antibody Fc domain. This natural glycosylation site includes a wild-type asparagine residue at position 297 of the Fc domain according to EU numbering (N297). The natural N-linked glycan at this position is generally linked to the nitrogen group of the N297 side chain by a β-glycosylamide linkage. However, other suitable linkages recognized in the art can also be used. In other exemplary embodiments, the binding polypeptide of the present invention includes one or more modified glycosylation sites. Such modified glycosylation sites include substitution of one or more wild-type amino acids in the polypeptide backbone of the binding polypeptide with asparagine residues that can be N-glycosylated by cellular glycosylation enzymes. Exemplary modified glycosylation sites of the present invention include introduction of an asparagine mutation at position 298 (298N) of the Fc domain or at position 114 (114N) of the CH1 domain.

[0069] Any type of naturally occurring or synthetic (i.e., non-natural) N-linked glycan can be linked to the glycosylation site of the binding polypeptide of the present invention. In certain embodiments, the glycan includes a sugar (e.g., a sugar residue located at the end of an oligosaccharide), and the sugar can be oxidized (e.g., by periodate treatment or galactose oxidase) to generate a group suitable for conjugation to an effector moiety (e.g., a reactive aldehyde group). Suitable oxidizable sugars include, but are not limited to Examples include galactose and sialic acid (e.g., N-acetylneuraminic acid). In certain embodiments, the glycan is a bi-branched glycan. In certain embodiments, the glycan is a naturally occurring mammalian glycoform.

[0070] Glycosylation can be achieved by any means known in the art. In certain embodiments, glycosylation is achieved by expressing a binding polypeptide in a cell capable of N-linked glycosylation. Any natural or modified cell (e.g., prokaryotic or eukaryotic) can be used. Generally, mammalian cells are used to achieve glycosylation. N-glycans produced in mammalian cells are typically referred to as complex-type, high-mannose-type, and hybrid-type N-glycans (see, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology, 2nd ed., which is hereby incorporated by reference in its entirety). These complex-type N-glycans typically have a structure with two to six outer branches having a sialyllactosamine sequence linked to the internal core structure Man3GlcNAc2. Complex-type N-glycans end with an oligosaccharide and have at least one, preferably at least two branches with alternating GlcNAc and galactose (Gal) residues, such as, for example, NeuNAc-; NeuAcα2,6 GalNAcα1-; NeuAcα2,3 Galβ1,3 GalNAcα1-; and NeuAcα2,3 / 6 Galβ1,4 GlcNAcβ1. Furthermore, sulfate esters may be present on galactose, GalNAc, and GlcNAc residues. NeuAc may be O-acetylated or replaced with NeuGl (N-glycolylneuraminic acid). Complex-type N-glycans may have bisecting GlcNAc and interchain substitution of core fucose (Fuc).

[0071] Furthermore, or alternatively, glycosylation can be achieved or modified by enzymatic means in vitro. For example, one or more glycosyltransferases can be used to add specific sugar residues to the native or modified N-glycans of the binding polypeptide, or one or more glycosidases can be used to remove unwanted sugars from N-linked glycans. Such enzymatic means are well known in the art (see, e.g., WO2007 / 005786, the entire disclosure of which is incorporated herein by reference).

[0072] IV. Immunological effector functions and Fc modifications In certain embodiments, the binding polypeptide of the invention can include an antibody constant region that mediates one or more effector functions (e.g., an IgG constant region, e.g., a human IgG constant region, e.g., a human IgG1 or IgG4 constant region). For example, when the C1 complex binds to the antibody constant region, it can activate the complement system. Activation of the complement system is important in opsonization and lysis of cellular pathogens. Activation of the complement system can also stimulate an inflammatory response and be involved in autoimmune hypersensitivity. Further, antibodies bind via the Fc region to receptors on a variety of cells (the Fc receptor binding site on the antibody Fc region binds to the Fc receptor (FcR) on the cell). There are numerous Fc receptors specific for various classes of antibodies, such as IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). When an antibody binds to an Fc receptor on the cell surface, it induces numerous important and diverse biological responses such as phagocytosis and destruction of the particle coated by the antibody, clearance of immune complexes, lysis of the target cell coated by the antibody by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production. In a preferred embodiment, the binding polypeptide of the invention (e.g., an antibody or an antigen-binding fragment thereof) binds to the Fc-gamma receptor. In an alternative embodiment, the binding polypeptide of the invention can include a constant region lacking one or more effector functions (e.g., ADCC activity) and / or unable to bind to the Fcγ receptor. and / or may not be able to bind to the Fcγ receptor.

[0073] Certain embodiments of the invention include antibodies in which at least one amino acid in one or more constant region domains is deleted or otherwise altered to provide desired biochemical characteristics such as reduced or enhanced effector function, the ability to non-covalently dimerize, increased ability to localize to tumor sites, reduced serum half-life, or increased serum half-life, compared to an unmodified full antibody having approximately the same immunogenicity. For example, certain antibodies for use in the diagnostic and treatment methods described herein include polypeptide chains similar to immunoglobulin heavy chains but are domain-deleted antibodies in which at least a portion of one or more heavy chain domains is deleted. For example, in certain antibodies, an entire domain of the constant region of the modified antibody is deleted, e.g., the entire or a portion of the CH2 domain is deleted.

[0074] In other certain embodiments, the binding polypeptide includes constant regions derived from various antibody isotypes (e.g., constant regions from two or more of human IgG1, IgG2, IgG3, or IgG4). In other embodiments, the binding polypeptide includes a chimeric hinge (i.e., a hinge including a hinge portion derived from hinge domains of various antibody isotypes, e.g., the upper hinge domain from an IgG4 molecule and the IgG1 central hinge domain). In one embodiment, the binding polypeptide includes an Fc region or a portion thereof from a human IgG4 molecule and a Ser228Pro mutation (EU numbering) in the core hinge region of the molecule.

[0075] In certain embodiments, the Fc portion may be mutated to increase or reduce effector function using techniques known in the art. For example, deletion or inactivation of the constant region domain (by point mutation or other means) can reduce Fc receptor binding of the circulating modified antibody, thereby increasing tumor localization. In other cases, modifications of the constant region consistent with the present invention relax complement binding, thus reducing serum half-life and non-specific association of conjugated cytotoxic agents. Still other modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties, and these modifications enhance localization by increasing antigen specificity or flexibility. The resulting physiological profile, bioavailability, and other biochemical effects of the modification, such as tumor localization, biodistribution, and serum half-life, can be readily measured and quantified using well-known immunological techniques without undue experimentation.

[0076] In certain embodiments, the Fc domain used in the antibodies of the present invention is an Fc variant. As used herein, the term "Fc variant" means an Fc domain having at least one amino acid substitution compared to the wild-type Fc domain from which the Fc domain is derived. For example, when the Fc domain is derived from a human IgG1 antibody, an Fc variant of the Fc domain of the human IgG1 contains at least one amino acid substitution compared to the Fc domain.

[0077] The amino acid substitution(s) of the Fc variant may be located at any position within the Fc domain (i.e., any amino acid position of the EU numbering). In one embodiment, the Fc variant contains a substitution at an amino acid position located in the hinge domain or a portion thereof. In another embodiment, the Fc variant contains a substitution at an amino acid position located in the CH2 domain or a portion thereof. In another embodiment, the Fc variant contains a substitution at an amino acid position located in the CH3 domain or a portion thereof. In another embodiment, the Fc variant contains a substitution at an amino acid position located in the CH4 domain or a portion thereof.

[0078] The binding polypeptides of the present invention can use any Fc variant known to result in an improvement (e.g., reduction or enhancement) in effector function and / or FcR binding in any technical field. The Fc variants are, for example, International PCT Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO03 / 074569A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351A2, WO04 / 074455A2, WO04 / 099249A2, WO05 / 040217A2, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350A2, and WO06 / 085967A2, or U.S. Patent Nos. 5,648,260, 5,739,277, 5,834,250, 5,869,046, 6,096,871, 6,121,022, 6,194,551, 6,242,195, 6,277,375, 6,528,624, 6,538,124, 6,737,056, 6,821,505, 6,998,253, and 7,083,784, each of which is incorporated herein by reference, and may contain any one of the amino acid substitutions disclosed therein. In an exemplary embodiment, the binding polypeptide of the present invention may contain an Fc variant (e.g., H268D or H268E) with an amino acid substitution at position EU268. In another exemplary embodiment, the binding polypeptide of the present invention may contain amino acid substitutions at positions EU239 (e.g., S239D or S239E) and / or EU332 (e.g., I332D or I332Q).

[0079] ​In certain embodiments, the binding polypeptide of the invention can comprise an Fc variant that includes amino acid substitutions that alter the antigen-independent effector functions of the antibody, particularly the circulating half-life of the binding polypeptide. Such binding polypeptides exhibit either increased or decreased binding to FcRn as compared to binding polypeptides without these substitutions, and thus have either increased or decreased serum half-lives, respectively. The serum half-life of an Fc variant with improved affinity for FcRn is expected to be longer, and such molecules are advantageously applicable in methods of treating mammals, such as in the treatment of chronic diseases or disorders, where a long half-life of the administered antibody is desirable. In contrast, the half-life of an Fc variant with reduced FcRn binding affinity is expected to be shorter, and such molecules can also be useful, for example, in in vivo diagnostic imaging methods, or in the administration to mammals in situations where a shortened circulation time can be advantageous, such as when the primary antibody has toxic side effects when present in the circulation for an extended period of time. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta and are thus useful for treating diseases or disorders in pregnant women. Further, other applications where reduced FcRn binding affinity may be desirable include applications localized to the brain, kidney, and / or liver. In an exemplary embodiment, the modified binding polypeptide of the invention (e.g., an antibody or an antigen-binding fragment thereof) exhibits a reduced transport across the epithelium of the renal glomerulus from the vasculature. In another exemplary embodiment, the modified binding polypeptide of the invention (e.g., an antibody and an antigen-binding fragment thereof) exhibits a reduced transport across the blood-brain barrier (BBB) from the brain into the interstitial space of the vasculature. In one embodiment, an antibody having modified FcRn binding comprises an Fc domain having one or more amino acid substitutions within the "FcRn-binding loop" of the Fc domain. The FcRn-binding loop consists of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions that alter FcRn binding activity are disclosed in International PCT Publication No. WO05 / 047327, which is hereby incorporated by reference in its entirety.In an exemplary, predetermined embodiment, the binding polypeptide of the present invention (e.g., an antibody or an antigen-binding fragment thereof) comprises an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering). In yet other exemplary embodiments, the binding molecule of the present invention is a double mutant H4. comprises a human Fc domain having 33K / N434F (see, e.g., U.S. Patent No. 8,163,881).

[0080] In other embodiments, the binding polypeptide for use in the diagnostic and treatment methods described herein has a constant region, such as an IgG1 or IgG4 heavy chain constant region, that has been modified such that glycosylation is reduced or eliminated. For example, the binding polypeptide of the present invention (e.g., an antibody or an antigen-binding fragment thereof) may also include an Fc variant that includes an amino acid substitution that modifies the glycosylation of the antibody Fc. For example, the glycosylation of the Fc variant may be reduced (e.g., N- or O-linked glycosylation). In an exemplary embodiment, the Fc variant includes a reduction in the glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif, such as an N-linked glycosylation motif that includes the amino acid sequence NXT or NXS. In a particular embodiment, the antibody includes an Fc variant having an amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more particular embodiment, the antibody includes an IgG1 or IgG4 constant region that includes the S228P and T299A mutations (EU numbering).

[0081] Exemplary amino acid substitutions that confer reduced or altered glycosylation are disclosed in International PCT Publication No. WO05 / 018572, which is incorporated herein by reference in its entirety. In preferred embodiments, the binding polypeptides of the invention are modified to eliminate glycosylation. Such binding polypeptides can be referred to as "agly" binding polypeptides (e.g., "agly" antibodies). Without being bound by theory, "agly" binding polypeptides are thought to have an improved safety and stability profile in vivo. Agly binding polypeptides can be of any isotype or subclass, e.g., of IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the agly binding polypeptide comprises the non-glycosylated Fc region of an IgG4 antibody lacking Fc-effector function, thus eliminating the potential for Fc-mediated toxicity to normal vital organs that express IL-6. In still other embodiments, the binding polypeptides of the invention comprise an altered glycan. For example, the antibody can have a reduced number of fucose residues on the N-glycan of Asn297 in the Fc region, i.e., be afucosylated. Afucosylation increases FcγRII binding on NK cells and potentially increases ADCC. Diabodies comprising an anti-IL-6 scFv and an anti-CD3 scFv have been shown to induce killing of IL-6-expressing cells by ADCC. Thus, in one embodiment, an afucosylated anti-IL-6 antibody is used to target and kill IL-6-expressing cells. In another embodiment, the binding polypeptide can have an altered number of sialic acid residues on the N-glycan of Asn297 in the Fc region. A number of methods recognized in the art can be utilized to generate "agly" antibodies or antibodies with altered glycans. For example, such antibodies can be produced using genetically modified host cells (e.g., modified yeast, e.g., Pichia or CHO cells) having a modified glycosylation pathway (e.g., glycosyltransferase deletion).

[0082] V. Effector portion In certain embodiments, the binding polypeptide of the present disclosure includes an effector portion (e.g., a drug portion and a targeting portion). Generally, these effector portions conjugate to the N-linked glycans on the binding polypeptide (either directly or via a linker portion) (e.g., the N-linked glycans are linked to N298 of the CH2 domain (EU numbering) and / or N114 of the CH1 domain (Kabat numbering)). In certain embodiments, the binding polypeptide has a glycan at Kabat position 114 is a full-length antibody containing two CH1 domains having glycans, in which case both glycans are conjugated to one or more effector portions.

[0083] Any effector portion can be added to the binding polypeptides disclosed herein. The effector portion preferably adds a non-native function to the modified antibody or its fragment without significantly altering the intrinsic activity of the binding polypeptide. The effector portion can be, for example, but not limited to, a therapeutic or diagnostic agent. The modified binding polypeptides (e.g., antibodies) of the present disclosure may contain one or more effector portions, which may be the same or different.

[0084] In one embodiment, the effector portion has the formula (I): H2N-Q-CON-X Formula (I) wherein wherein A) Q is NH or O, B) CON is a connecting portion, C) X is an effector portion (e.g., a therapeutic or diagnostic agent as defined herein).

[0085] The connecting moiety connects the therapeutic agent to H2N-Q-. The connecting moiety may include at least one suitable moiety known to those skilled in the art, such as, for example, an alkenylenyl moiety, a polyethylene glycol moiety, a poly(glycine) moiety, a poly(oxazoline) moiety, a carbonyl moiety, a moiety derived from cysteine amide, a moiety derived from valine coupled to citrulline, and a moiety derived from 4-aminobenzyl carbamate, or any combination thereof.

[0086] In another embodiment, the effector moiety of formula (I) is of formula (Ia): H2N-Q-CH2-C(O)-Z-X Formula (Ia) may be wherein A) Q is NH or O, B) Z is -Cys-(MC) a -(VC) b -(PABC) c -(C 16 H 32 O8C2H4) f and wherein i. Cys is a moiety derived from cysteine amide, ii. MC is a moiety derived from maleimide, iii. VC is a moiety derived from valine coupled to citrulline, iv. PABC is a moiety derived from 4-aminobenzyl carbamate, v. X is an effector moiety (e.g., a therapeutic or diagnostic agent as defined herein), vi. a is 0 or 1, vii. b is 0 or 1, viii. c is 0 or 1, ix. f is 0 or 1.

[0087] The "constituent derived from cysteine amide" is the attachment point to H2N-Q-CH2-C(O)-. In one embodiment, the "constituent derived from cysteine amide" has the structure:

Chemical formula

[0088] In one embodiment, the "Cys" constituent of the effector moiety may contain one such part. For example, the following structure shows an effector moiety having one such part (wherein the "Cys" constituent is indicated by a dotted box):

Chemical formula

[0089] In another embodiment, the "Cys" constituent of the effector moiety may contain two or more such parts. For example, the following moiety contains two such parts.

Chemical formula

[0090] As can be seen from the structure, the "Cys" constituent has a -(MC) a -(VC) b -(PABC) c -(C 16 H 32 O8C2H4) f -X group.

[0091] In one embodiment, the expression "constituent derived from maleimide" refers to any part of the effector moiety having the structure:

Chemical formula

[0092] In one embodiment, the "Cys" component may be connected to the "MC" component via the sulfur atom in the "Cys" component, as shown by the dotted box in the following structure:

Chemical formula

[0093] In one embodiment, the expression "component derived from valine that couples with citrulline" may refer to any part of the effector moiety having the following structure:

Chemical formula

[0094] Any Cys-(MC) in the effector moiety a -(VC) b -(PABC) c -(C 16 H 32 O8C2H4) f The number of VC components included in the -X group is indicated by the subscript letter "b", and can be 0 or 1. In one embodiment, b is 1. In another embodiment, b is 0.

[0095] In one embodiment, the expression "component derived from 4-aminobenzyl carbamate" may refer to any part of the effector moiety having the following structure:

Chemical formula

[0096] Any Cys-(MC) in the effector moiety a -(VC) b -(PABC) c -(C 16 H 32 O8C2H4) f The number of PABC components included in the -X group is indicated by the subscript "c" and may be 0 or 1. In one embodiment, c is 1. In another embodiment, c is 0.

[0097] In one embodiment, "C 16 H 32 O8C2H4" refers to the following structure:

Chemical formula

[0098] Any Cys-(MC) in the effector moiety a -(VC) b -(PABC) c -(C 16 H 32 O8C2H4) f The number of units of C 16 H 32 O8 included in the -X group is indicated by the subscript "f". In one embodiment, f is 1. In another embodiment, f is 0.

[0099] In one embodiment, a is 1, b is 1, c is 1, and f is 0.

[0100] a) Therapeutic effector moiety In certain embodiments, the conjugating polypeptide of the present disclosure is conjugated to an effector moiety that includes a therapeutic agent, such as a drug moiety (or a prodrug thereof), or a radiolabeled compound. In one embodiment, the therapeutic agent is a cytotoxic agent. Exemplary cytotoxic therapeutic agents are described in Table 1 herein.

[0101]

Table 1

[0102]

Table 2

[0103] Additional exemplary drug moieties include anti-inflammatory agents, anti-cancer agents, anti-infective agents (e.g., antifungal agents, antibacterial agents, anti-parasitic agents, anti-viral agents, etc.), and anesthetic therapeutic agents. In a further embodiment, the drug moiety is an anti-cancer agent. Exemplary anti-cancer agents include, but are not limited to, cell division inhibitors, enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin-binding drugs or tubulin inhibitors, proteasome inhibitors, hormones and hormone antagonists, angiogenesis inhibitors, and the like. Exemplary cell division inhibitory anti-cancer agents include alkylating agents, such as drugs of the anthracycline family (e.g., adriamycin, carminomycin, cyclosporin A, chloroquine, methopterin, mitomycin, porfiromycin, streptozocin, porfiromycin, anthracenedione, and aziridine). Other cell division inhibitory anti-cancer agents include DNA synthesis inhibitors (e.g., methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin D, and mitomycin C), DNA intercalators or cross-linking agents (e.g., bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin), and DNA-RNA transcription regulators (e.g., actinomycin D, daunorubicin, doxorubicin, homohalichondrin B, and idarubicin). Other exemplary cell division inhibitors applicable in the present disclosure include ansamycin benzoquinone, quinonoid derivatives (e.g., quinolone, genistein, bactacyclin), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazilquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0104] Exemplary cytotoxic nucleoside anti-cancer agents include, but are not limited to, for example, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine. Exemplary anti-cancer tubulin-binding agents include, but are not limited to, taxoids (e.g., paclitaxel, docetaxel, taxane), nocodazole, rizoxin, laulastatin (e.g., laulastatin-10, -11, or -15), colchicine and colchicinoids (e.g., ZD6126), combretastatin (e.g., combretastatin A-4, AVE-6032, and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)). Exemplary anti-cancer hormones and hormone antagonists include, but are not limited to, corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroxyprogesterone), estrogens (e.g., diethylstilbestrol), anti-estrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminoglutethimide), 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprorelin (leuprolide), luteinizing hormone releasing hormone, pifithrin α, rapamycin, sex hormone binding globulin, and thapsigargin. Exemplary anti-cancer anti-angiogenic compounds include, but are not limited to, angiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

[0105] Exemplary anti-cancer enzyme inhibitors include, but are not limited to, S(±)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside, etoposide, formestane, fostriecin, histipidine, 2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG34, and tyrphostin AG879.

[0106] Exemplary anti-cancer gene regulators include, but are not limited to, 5-aza-2'-deoxycytidine, 5-azacytidine, colecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin A aldehyde), retinoic acid, tretinoin, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0107] Other preferred classes of anti-cancer agents include, but are not limited to, for example, drugs of the pteridine family, diynes, and podophyllotoxins. Particularly useful species of these classes include, for example, methopterin, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, leurosidine, vindesine, leurosine, and the like.

[0108] Still other anti-cancer agents applicable in the teachings herein include auristatins (e.g., auristatin E and monomethyl auristatin E), geldanamycin, calicheamicin, gramicidin D, maytansinoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, teniposide, colchicine, dihydroxyanthracenedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0109] Still other anti-cancer agents applicable in the teachings of this specification include tomamycin derivatives, maytansine derivatives, cryptophycin derivatives, anthracycline derivatives, bisphosphonate derivatives, leptomycin derivatives, streptozocin derivatives, auristatin derivatives, and duocarmycin derivatives.

[0110] Another class of applicable anti-cancer agents that can be used as a drug moiety are radiosensitizing drugs that can effectively target tumor or immunoreactive cells. Such drug moieties increase the efficacy of radiotherapy by enhancing sensitivity to ionizing radiation. Without wishing to be limited by theory, antibodies modified with a radiosensitizing drug moiety and internalized into tumor cells are expected to deliver the radiosensitizer closer to the nucleus, where radiosensitization is maximized. Antibodies that have lost the radiosensitizing drug moiety are rapidly removed from the blood, and the remaining radiosensitizer localizes to the target tumor, minimizing uptake into normal tissues and. After removal from the blood, adjuvant radiotherapy can be administered by external irradiation locally applied to the tumor, direct implantation of radioactivity into the tumor, or radioimmunotherapy with the same modified antibody.

[0111] In one embodiment, the therapeutic agent comprises a radionuclide or a radiolabel with high-energy ionizing radiation, and the high-energy ionizing radiation can cause multiple strand breaks in the nuclear DNA, leading to cell death. Exemplary high-energy radionuclides include 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, and 188Re. These isotopes typically produce high-energy α or β particles with short track lengths. Such radionuclides kill cells that are very proximal, such as neoplastic cells to which the conjugate is attached or has invaded. They have little or no effect on non-localized cells and are essentially non-immunogenic. Alternatively, high-energy isotopes can be produced by thermal irradiation of otherwise stable isotopes, as in the case of boron neutron capture therapy (Guan et al., PNAS, Vol. 95, pp. 13206-10, 1998).

[0112] In one embodiment, the therapeutic agent is selected from MMAE, MMAF, and PEG8-Do110.

[0113] Exemplary therapeutic effector moieties include the following structures:

Chemical formula

[0114] In one embodiment, the effector moiety is selected from the following:

Chemical formula

[0115] In certain embodiments, the effector moiety comprises more than one therapeutic agent. These multiple therapeutic agents may be the same or different.

[0116] b) Diagnostic effector moiety In certain embodiments, the binding polypeptide of the present disclosure is conjugated to an effector moiety that includes a diagnostic agent. In one embodiment, the diagnostic agent is a detectable small molecule label such as biotin, fluorophore, chromophore, spin resonance probe, or radiolabel. Exemplary fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, etc.), and other emissive molecules (e.g., luminol). The fluorophore may be environmentally sensitive such that its fluorescence changes when it is in proximity to one or more residues in a modified binding polypeptide that undergoes a structural change upon binding to a substrate (e.g., dansyl probe). Exemplary radiolabels include small molecules that include atoms having one or more low-sensitivity nuclei (13C, 15N, 2H, 125I, 124I, 123I, 99Tc, 43K, 52Fe, 64Cu, 68Ga, 111In, etc.). The radionuclide is preferably a gamma, photon, or positron-emitting radionuclide having a half-life suitable to enable activity or detection after the elapsed time between administration and localization at the imaging site.

[0117] In one embodiment, the diagnostic agent is a polypeptide. Exemplary diagnostic polypeptides include enzymes having fluorescence-generating or dye-generating activity, such as the ability to cleave a substrate that forms a fluorophore or chromophore as a product (i.e., a reporter protein such as luciferase). Other diagnostic proteins may have intrinsic fluorescence-generating or dye-generating activity (e.g., green, red, and yellow bioluminescent aequorin proteins from bioluminescent marine organisms), or may include proteins that include one or more low-energy radionuclides (13C, 15N, 2H, 125I, 124I, 123I, 99Tc, 43K, 52Fe, 64Cu, 68Ga, 111In, etc.).

[0118] Regarding the use of radiolabeled conjugates in combination with the present disclosure, the binding polypeptides of the present disclosure may be directly labeled (e.g., by iodination) or indirectly labeled by the use of a chelating agent. As used herein, both the expressions "indirect labeling" and "indirect labeling approach" mean that the chelating agent is covalently attached to the binding polypeptide and at least one radionuclide is associated with the chelating agent. Such chelating agents, since they bind to both the polypeptide and the radioisotope, are typically referred to as bifunctional chelating agents. Exemplary chelating agents include 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid ("MX-DTPA") and cyclohexyldiethylenetriaminepentaacetic acid ("CHX-DTPA") derivatives. Other chelating agents include P-DOTA and EDTA derivatives. Radionuclides particularly preferred for indirect labeling include 111In and 90Y. In most imaging studies, 5 mCi of 111In-labeled antibody is utilized, because this dose is safe, the imaging efficiency is increased compared to low doses, and optimal imaging occurs 3 to 6 days after antibody administration. See, for example, Murray, (1985), J. Nuc. Med., Vol. 26, p. 3328, and Carraguillo et al. (1985), J. Nuc. Med., Vol. 26, p. 67. A radionuclide particularly preferred for direct labeling is 131I. One of ordinary skill in the art will understand that non-radiolabeled conjugates can be assembled depending on the agent selected for conjugation.

[0119] In certain embodiments, the diagnostic effector moiety is a FRET (fluorescence resonance energy transfer) probe. FRET is used in a variety of diagnostic applications such as cancer diagnosis. The FRET probe may include a cleavable linker (enzyme-sensitive or pH linker) that connects the donor and acceptor moieties of the FRET probe, and cleavage results in enhanced fluorescence (including near-infrared light) (see, for example, A. Cobos-Correa et al., Membrane-bound FRET probe visualizes MMP12 activity in pulmonary inflammation, Nature Chemical Biology (2009), 5(9), 628-634; S. Gehrig et al., Spatially Resolved Monitoring of Neutrophil Elastase Activity with Ratiometric Fluorescent Reporters (2012) Angew. Chem. Int. Ed., 51, 6258-6261).

[0120] In one embodiment, the effector moiety is selected from:

Chemical Structure

[0121] c) a functionalized effector moiety In certain embodiments, the effector moiety of the present invention may be functionalized such that additional groups are included in addition to the effector moiety itself. For example, the effector moiety may include a cleavable linker that releases the effector moiety from the binding polypeptide under certain conditions. In an exemplary embodiment, the effector moiety may include a linker that is cleaved by a cellular enzyme and / or is pH-sensitive. Additionally or alternatively, the effector moiety may include a disulfide bond that is cleaved by intracellular glutathione when taken up into the cell. Exemplary disulfide and pH-sensitive linkers are shown below:

Chem.

[0122] In yet other embodiments, the effector moiety may include hydrophilic and biocompatible moieties such as poly(glycine), poly(oxazoline), or PEG moieties. Exemplary structures (“Y”) are provided below:

Chem.

[0123] In certain embodiments, the effector moiety includes an aminooxy group that facilitates conjugation to the binding polypeptide by a stable oxime bond. Exemplary effector moieties including the aminooxy group are described in Table 2 herein.

[0124]

Table 3

[0125]

Table 4

[0126] In other embodiments, the effector moiety includes a hydrazide and / or N-alkylated hydrazine group to facilitate conjugation to the binding polypeptide by a stable hydrazone bond. Exemplary effector moieties including the aminooxy group are described in Table 14 herein.

[0127]

Table 5

[0128] d) targeting moiety In certain embodiments, the effector portion comprises a targeting moiety that specifically binds to one or more target molecules. Without limitation, any type of targeting moiety can be used, including proteins, nucleic acids, lipids, carbohydrates (e.g., glycans), and combinations thereof (e.g., glycoproteins, glycopeptides, and glycolipids). In certain embodiments, the targeting moiety is a carbohydrate or a glycopeptide. In certain embodiments, the targeting moiety is a glycan. The targeting moiety can be a naturally or non-naturally occurring molecule.

[0129] VI. Conjugation of the Effector Portion to a Binding Polypeptide In certain embodiments, the effector portion conjugates (either directly or via a linker moiety) to an oxidized glycan (e.g., an oxidized N-linked glycan) of a modified binding polypeptide (e.g., a glycan in which N114 of the CH1 domain of an antibody is modified, or a natural glycan at N297 of the F domain of an antibody). The term "oxidized glycan" means that an alcohol substituent on the glycan has been oxidized to a carbonyl substituent. The carbonyl substituent can react with a suitable nitrogen nucleophile to form a carbon-nitrogen double bond. For example, when a carbonyl group reacts with an aminooxy group or a hydrazine group, an oxime or a hydrazone is formed, respectively. In one embodiment, the carbonyl substituent is an aldehyde. Suitable oxidized glycans include oxidized galactose and oxidized sialic acid.

[0130] In one embodiment, the modified polypeptide of formula (II) is of formula (II): Ab(Gal-C(O)H) x (Gal-Sia-C(O)H) y Formula (II) can be represented by wherein A) Ab is an antibody or another binding polypeptide as defined herein, B) Gal is a component derived from galactose, C) Sia is a component derived from sialic acid, D) x is from 0 to 5, E) y is from 0 to 5, At least one of x and y is not 0.

[0131] Using any chemical reaction recognized in the art, an effector moiety (e.g., an effector moiety containing a linker moiety) can be conjugated to a glycan (see, for example, Hermanson, G.T., Bioconjugate Techniques., Academic Press (1996), the entire text of which is incorporated herein by reference). In certain embodiments, the sugar residues of the glycan (e.g., sialic acid or galactose residues) are first oxidized (e.g., using sodium periodate treatment of sialic acid or galactose oxidase treatment of galactose) to produce reactive aldehyde groups. These aldehyde groups are reacted with an effector moiety of an aminooxy group or a hydrazine group to form an oxime or hydrazone linker, respectively. Exemplary methods using this general reaction scheme are described in Examples 10 to 15.

[0132] In certain embodiments, the native or modified glycan of the binding polypeptide is first pretreated in vitro with a glycosyltransferase enzyme to yield a terminal sugar residue with appropriate reactivity. For example, sialylation may first be achieved using a combination of galactosyltransferase (GalT) and sialyltransferase (SialT). In certain embodiments, a biantennary glycan lacking galactose (G0F or G0) or containing only one galactose (G1F or G1) may be converted to a higher-order galactosylated or sialylated structure (G1F, G1, G2F, G2, G1S1F, G1S1, G2S1F, G2S1, G2S2F, or G2S2) suitable for conjugation.

[0133] An exemplary conjugation scheme for generating sialylated glycoconjugates is shown in Figure 25C. Sialic acid residues are introduced enzymatically and site-specifically onto the glycans of an antibody (e.g., where Asn-297 is the native glycan) using a combination of galactosyltransferase (GalT) and sialyltransferase (SialT). The introduced sialic acid residues are subsequently oxidized with low concentrations of sodium periodate to obtain reactive sialic acids with appropriate reactivity for a drug-linker (e.g., an aminooxy drug linker) to produce an antibody-drug conjugate (ADC) (e.g., an oxime-linked ADC). By controlling the number of glycans and the number of sialic residues in in vitro reconstruction, one of ordinary skill in the art can achieve precise control over the drug-antibody ratio (DAR) of the ADC. For example, when adding approximately one sialic acid onto a single biantennary glycan (A1F) in each heavy chain, an antibody or binding polypeptide with a DAR of 2 can be obtained uniformly.

[0134] VII. Modified Binding Polypeptide In certain embodiments, the present invention provides a modified polypeptide that is a product of conjugating an effector moiety (either directly or via a linker moiety) to an oxidized glycan (e.g., an oxidized N-linked glycan) of an altered binding polypeptide (e.g., a glycan with modified N114 in the antibody CH1 domain or a native glycan at N297 in the antibody F domain).

[0135] In one embodiment, the binding polypeptide has the formula (III): Ab(Gal-C(H)=N-Q-CON-X) x (Gal-Sia-C(H)=N-Q -CON-X) y Formula (III) can be represented as, wherein, A) Ab is an antibody as defined herein, B) Q is NH or O, C) CON is the connecting moiety as defined herein, D) X is a therapeutic or diagnostic agent as defined herein, E) Gal is a component derived from galactose, F) Sia is a component derived from sialic acid, G) x is from 0 to 5, H) y is from 0 to 5, At least one of x and y is not 0.

[0136] In one embodiment, the binding polypeptide has the formula (III): Ab(Gal-C(H)=N-Q-CH2-C(O)-Z-X) x (Gal-Sia-C(H)=N-Q-CH2-C(O)-Z-X) y Formula (IIIa) and can be represented by wherein A) Ab is an antibody, B) Q is NH or O, C) Z is Cys-(MC) a -(VC) b -(PABC) c -(C 16 H 32 O8C2H4) f - and wherein i. Cys is a component derived from cysteine amide, ii. MC is a component derived from maleimide, iii. VC is a component derived from valine coupled with citrulline, iv. PABC is a component derived from 4-aminobenzyl carbamate, v. X is an effector moiety (e.g., a therapeutic or diagnostic agent as defined herein), vi. a is 0 or 1, vii. b is 0 or 1, viii. c is 0 or 1, ix. f is 0 or 1, D) X is a therapeutic agent as defined herein, E) Gal is a component derived from galactose, F) Sia is a component derived from sialic acid, G) x is from 0 to 5, H) y is from 0 to 5, At least one of x and y is not 0.

[0137] It should be understood that formula (III) is not intended to show that the antibody, Gal substituent, and Gal - Sia substituent are connected in a chain. Instead, when such substituents are present, the antibody is directly connected to each substituent. For example, a binding polypeptide of formula (III) where x is 1 and y is 2 can have the following arrangement:

Chemical formula

[0138] The CON substituent in formula (III) and the components therein are as described for formula (I) with respect to the effector part.

[0139] In one embodiment, Q is NH. In another embodiment, Q is O.

[0140] In one embodiment, x is 0.

[0141] The antibody Ab of formula (III) can be any suitable antibody described herein.

[0142] In one embodiment, a method for preparing a binding polypeptide of formula (III) is provided, the method being formula (I): NH2 - Q - CON - X Formula (I) [Wherein, A) Q is NH or O, B) CON is a connecting part, C) X is an effector moiety (e.g., a therapeutic or diagnostic agent as defined herein) and reacting the effector moiety of Ab(OXG) r Formula (II) [wherein, A) OXG is an oxidized glycan, B) r is selected from 0 to 4 with a modified antibody of

[0143] In one embodiment, a method for preparing a binding polypeptide of formula (III) is provided, the method comprising reacting the effector moiety of formula (I): NH2-Q-CON-X Formula (I) [wherein, A) Q is NH or O, B) CON is a linking moiety, C) X is an effector moiety (e.g., a therapeutic or diagnostic agent as defined herein) with the effector moiety of formula (IIa): Ab(Gal-C(O)H) x (Gal-Sia-C(O)H) y Formula (IIa), [wherein, A) Ab is an antibody as described herein, B) Gal is a component derived from galactose, C) Sia is a component derived from sialic acid, D) x is from 0 to 5, E) y is from 0 to 5, and at least one of x and y is not 0 with a modified antibody of

[0144] VII. Method of treating with a modified antibody In one aspect, the present invention provides a method of treating or diagnosing a patient in need thereof, comprising administering an effective amount of a binding polypeptide disclosed herein. Preferred embodiments of the present disclosure provide kits and methods for diagnosing and / or treating disorders such as neoplastic disorders in mammalian subjects in need of such treatment. The subject is preferably a human.

[0145] The binding polypeptides of the present disclosure are useful in a number of different applications. For example, in one embodiment, the binding polypeptide of a subject is useful for reducing or eliminating cells bearing an epitope recognized by binding to the binding domain of the binding polypeptide. In another embodiment, the binding polypeptide of a subject is effective for reducing or eliminating the concentration of soluble antigen in circulation. In one embodiment, the binding polypeptide may reduce tumor size, inhibit tumor growth, and / or extend the survival time of tumor-bearing animals. Accordingly, the present disclosure also relates to a method of treating tumors in such humans or other animals by administering to the human or animal an effective, non-toxic amount of a modified antibody. One of ordinary skill in the art can determine, by routine experimentation, what an effective non-toxic amount of a modified binding polypeptide would be for the purpose of treating a malignant tumor. For example, the therapeutically effective amount of a modified antibody or fragment thereof may vary according to factors such as disease stage (e.g., stage I vs. stage IV), age, gender, medical comorbidities (e.g., immunosuppressive conditions or diseases), and the weight of the subject, as well as the ability of the modified antibody to elicit a desired response in the subject. The dosage regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dosage may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0146] Generally, the compositions provided by the present disclosure can be used to prophylactically or therapeutically treat any neoplasm, including an antigen marker that enables targeting of cancerous cells by a modified antibody.

[0147] VIII. Method for Administering a Modified Antibody or Fragment Thereof Methods for preparing and administering the binding polypeptides of the present disclosure are well known to those skilled in the art or can be readily determined by those skilled in the art. The route of administration of the binding polypeptides of the present disclosure may be oral, parenteral, by inhalation, or topical. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Intravenous, intraarterial, subcutaneous, and intramuscular forms of parenteral administration are generally preferred. All of these administration forms are clearly contemplated to be within the scope of the present disclosure, and the form for administration is an injectable, particularly an intravenous or intraarterial injection or infusion solution. Usually, a pharmaceutical composition suitable for injection may contain a buffer (e.g., acetate buffer, phosphate buffer, or citrate buffer), a surfactant (e.g., polysorbate), and optionally a stabilizer (e.g., human albumin), etc. However, in other methods applicable with the teachings herein, the modified antibody can be delivered directly to the site of a population of harmful cells, thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0148] In one embodiment, the binding polypeptide to be administered has the formula (III): Ab(Gal-C(H)=N-Q-CON-X) x (Gal-Sia-C(H)=N-Q-CON-X) y The binding polypeptide of formula (III) wherein A) Ab is an antibody as defined herein, B) Q is NH or O, C) CON is a connecting moiety as defined herein, D) X is an effector moiety (e.g., a therapeutic or diagnostic agent as defined herein), E) Gal is a constituent component derived from galactose, F) Sia is a constituent component derived from sialic acid, G) x is from 0 to 5, ​H) y ranges from 0 to 5, at least one of x and y is not 0.

[0149] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, for example, saline and buffered media. In the compositions and methods of the present disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01 - 0.1 M, preferably 0.05 M phosphate buffer, or 0.8% saline. Other commonly used parenteral vehicles include sodium phosphate solution, dextrose plus Ringer's solution, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include liquids and nutrient replenishers, electrolyte replenishers, for example, those based on dextrose plus Ringer's solution. Preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases may also be present. More specifically, a pharmaceutical composition suitable for injectable use includes a sterile aqueous solution (if water-soluble), or a dispersing agent for immediately preparing a sterile injectable solution or dispersion and sterile powder. In such cases, the composition must be sterile and must have a fluidity that facilitates syringability. The composition must be stable under the conditions of manufacture and storage and is preferably protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium including, for example, water, ethanol, polyols (for example, glycerin, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. For example, the use of coatings such as lecithin can maintain the required particle size in the case of dispersions and the use of surfactants can maintain appropriate fluidity.

[0150] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it is preferred to include in the composition an isotonic agent such as, for example, sugar, polyalcohol such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of an injectable composition can be achieved by including in the composition an agent that delays absorption such as aluminum monostearate and gelatin.

[0151] In any case, a sterile injectable solution can be prepared by placing the active compound (for example, a modified binding polypeptide alone or in combination with other active substances) in a suitable solvent in the required amount, which includes one or a combination of the components listed herein as appropriate, and then filter sterilizing. Generally, a dispersion is prepared by placing the active compound in a sterile vehicle which includes a basic dispersion medium and other components required from those listed above. In the case of a sterile powder for preparing a sterile injectable solution, the preferred methods of preparation are vacuum drying and lyophilization, in which case the powders are obtained from a pre-sterile filtered solution containing all additional desired components in addition to the active ingredient. Injectable preparations are processed according to methods known in the art, filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under aseptic conditions. Further, the preparations may be packaged and sold in the form of kits such as those described in co-pending U.S.S.N. 09 / 259,337 and U.S.S.N. 09 / 259,338, each of which is incorporated herein by reference. Such manufactured products preferably have a label or package insert indicating that the relevant composition is useful for treating subjects suffering from or predisposed to autoimmune or neoplastic disorders.

[0152] The effective dosage of the compositions of the present disclosure for treating the above-described conditions will vary depending on many different factors including the means of administration, the target site, the physiological state of the patient, whether the patient is human or an animal, other drug therapies being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages can be determined using conventional methods known to those of ordinary skill in the art to optimize safety and efficacy.

[0153] In passive immunization with a binding polypeptide, the dosage can be, for example, in the range of about 0.0001 to 100 mg / kg of the host body weight, more commonly 0.01 to 5 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, etc.). For example, the dosage can be 1 mg / kg body weight, or 10 mg / kg body weight, or within the range of 1 - 10 mg / kg, preferably at least 1 mg / kg. Dosages at intermediate values within the above ranges are also considered to be within the scope of the present disclosure. Such dosages can be administered to the subject daily, every other day, weekly, or according to any other schedule determined by empirical analysis. An exemplary treatment involves administration in multiple doses over a long period such as at least 6 months. Further exemplary treatment regimens involve administration once every two weeks, or once a month, or once every three to six months. Exemplary dosage schedules include 1 - 10 mg / kg or 15 mg / kg daily, 30 mg / kg every other day, or 60 mg / kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered is within the indicated range.

[0154] The binding polypeptides of the present disclosure can be administered on multiple occasions. The interval between administrations can be weekly, monthly, or yearly. The interval may be irregular, as indicated by measuring the blood levels of the modified binding polypeptide or antigen in the patient. In some methods, by adjusting the dosage, a plasma concentration of the modified binding polypeptide of 1 to 1000 μg / ml is achieved, and in some methods, 25 to 300 μg / ml is achieved. Alternatively, the binding polypeptide may be administered as a sustained-release formulation, in which case it may not need to be administered as frequently. For antibodies, the dosage and frequency are varied according to the half-life of the antibody in the patient. Generally, humanized antibodies exhibit the longest half-life, followed by chimeric antibodies and non-human antibodies.

[0155] The dosage and frequency of administration may be varied according to whether the treatment is prophylactic or therapeutic. In prophylactic applications, a composition containing the antibody or a cocktail thereof of the present invention may be administered to a patient who is not yet in a disease state to enhance the patient's resistance. Such an amount is defined as a "prophylactically effective dosage." In this use, the exact amount also depends on the patient's health status and overall immunity, but generally ranges from 0.1 to 25 mg per dose, particularly in the range of 0.5 to 2.5 mg per dose. Relatively low dosages are administered at relatively infrequent intervals over a long period. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages at relatively short intervals (e.g., about 1 to 400 mg / kg of antibody per dose, a dosage of 5 to 25 mg is more commonly used for radioimmunoconjugates, and more dosages are used for cytotoxic-drug-modified antibodies) may be required until the progression of the disease slows or ends, preferably until the patient shows a partial or complete remission of the symptoms of the disease. Thereafter, a prophylactic regimen may be administered to the patient.

[0156] The binding polypeptide of the present disclosure can optionally be administered in combination with other agents effective to treat a disorder or condition in need of treatment (e.g., prophylactic or therapeutic). The effective single treatment dosage (i.e., therapeutically effective amount) of the 90Y-labeled modified antibody of the present disclosure ranges from about 5 to about 75 mCi, more preferably from about 10 to about 40 mCi. The dosage for treatment other than myeloablative in the case of the 131I modified antibody ranges from about 5 to about 70 mCi, more preferably from about 5 to about 40 mCi. The dosage for effective treatment for myeloablative in the case of the 131I-labeled antibody (i.e., may require autologous bone marrow transplantation) ranges from about 30 to about 600 mCi, more preferably from about 50 to less than about 500 mCi. When combined with a chimeric antibody, the dosage for treatment other than myeloablative in the case of the iodine-131I modified chimeric antibody ranges from about 5 to about 40 mCi, more preferably less than about 30 mCi. For example, the imaging criteria for 111In labeling are typically less than about 5 mCi.

[0157] The conjugating polypeptide can be administered as described immediately above, but in other embodiments, it must be emphasized that the conjugating polypeptide may be administered as a first-line treatment to otherwise healthy patients in other respects. In such embodiments, the conjugating polypeptide may be administered to patients with normal or average red bone marrow accumulation and / or to patients who have not experienced and are not currently experiencing it. As used herein, the administration of a modified antibody or fragment thereof that is made in cooperation with or in combination with adjuvant therapy means the sequential, simultaneous, over a predetermined period, concomitant, or contemporaneous administration or application of the therapy and the disclosed antibody. One of ordinary skill in the art will understand that the administration or application of the various components of the combined treatment regimen can be timed to enhance the overall effectiveness of the treatment. For example, a chemotherapeutic agent may be administered within a few weeks after being administered in a standard well-known treatment course, followed by the radioimmunoconjugate of the present disclosure. Conversely, after the cytotoxic agent associated with the conjugating polypeptide is administered intravenously, external irradiation applied locally to the tumor may be performed. In yet other embodiments, the modified conjugating polypeptide may be administered simultaneously with one or more selected chemotherapeutic agents in a single visit. One of ordinary skill in the art (e.g., an experienced oncology specialist) can readily identify an effective combined treatment regimen based on the selected adjuvant therapy and the teachings herein without undue experimentation.

[0158] In this regard, it will be understood that the combination of the conjugating polypeptide and the chemotherapeutic agent can be administered to the patient in any order and within any time frame that provides a therapeutic benefit to the patient. That is, the chemotherapeutic agent and the conjugating polypeptide can be administered in any order or simultaneously. In a selected embodiment, the conjugating polypeptide of the present disclosure is administered to a patient who has previously received chemotherapy. In yet other embodiments, the conjugating polypeptide and the chemotherapy treatment are administered substantially simultaneously or in parallel. For example, while administering the conjugating polypeptide to the patient, the patient may experience the course of chemotherapy. In a preferred embodiment, the modified antibody is administered within one year of all chemotherapeutic agents or treatments. In other preferred embodiments, the conjugating polypeptide is administered within 10, 8, 6, 4, or 2 months of all chemotherapeutic agents or treatments. In still other preferred embodiments, the conjugating polypeptide is administered within 4, 3, 2, or 1 week of all chemotherapeutic agents or treatments. In yet other embodiments, the conjugating polypeptide is administered within 5, 4, 3, 2, or 1 day of a selected chemotherapeutic agent or treatment. It will be further understood that the two agents or treatments may be administered to the patient within hours or minutes (i.e., substantially simultaneously).

[0159] The binding polypeptides of the present disclosure can further be understood to be used in combination with, or in conjunction with, any chemotherapeutic agent or agents that eliminate, reduce, inhibit, or control the growth of neoplastic cells in vivo (e.g., to provide a combined treatment regimen). Exemplary chemotherapeutic drugs applicable in the present disclosure include alkylating agents, vinca alkaloids (e.g., vincristine and vinblastine), procarbazine, methotrexate, and prednisone. The combination of four drugs, MOPP (mechlorethamine (nitrogen mustard), vincristine (Oncovin), procarbazine, and prednisone), is very effective in treating various types of lymphoma and is included in the preferred embodiments of the present invention. In patients resistant to MOPP, combinations of ABVD (e.g., doxorubicin, bleomycin, vinblastine, and dacarbazine), ChIVPP (chlorambucil, vinblastine, procarbazine, and prednisone), CABS (lomustine, doxorubicin, bleomycin, and streptozotocin), MOPP plus ABVD, MOPP plus ABV (doxorubicin, bleomycin, and vinblastine), or BCVPP (carmustine, cyclophosphamide, vinblastine, procarbazine, and prednisone) can be used. Arnold S. Freedman and Lee M. Nadler, Malignant Lymphomas, HARRISON’S PRINCIPLES OF INTERNAL MEDICINE, pages 1774 - 1788 (edited by Kurt J. Isselbacher et al., 13th edition, 1994), as well as V. T. DeVita et al. (1997), and the references cited herein for standard dosing and regimens. These therapies can be used as such or modified as appropriate for a particular patient and used in combination with one or more of the binding polypeptides of the present disclosure described herein.

[0160] Additional regimens useful in the context of the present disclosure include the use of a single alkylating agent such as cyclophosphamide or chlorambucil, or combinations such as CVP (cyclophosphamide, vincristine, and prednisone), CHOP (CVP and doxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone, and procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD (CHOP plus methotrexate, bleomycin, and leucovorin), ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, and leucovorin, plus standard MOPP), ProMACE-CytaBOM (prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate, and leucovorin), and MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, fixed-dose prednisone, bleomycin, and leucovorin). Those skilled in the art can readily determine the standard dosages and schedules for each of these regimens. CHOP has also been used in combination with bleomycin, methotrexate, procarbazine, nitrogen mustard, cytosine arabinoside, and etoposide. Other applicable chemotherapeutic agents include, but are not limited to, 2-chlorodeoxyadenosine (2-CDA), 2'-deoxycoformycin, and fludarabine.

[0161] Salvage therapy is used in patients with intermediate- and high-grade NHL who are unable to achieve remission or relapse. Salvage therapy includes cytarabine, carboplatin, cisplatin, etoposide, and ifosfamide, administered alone or in combination. Drugs such as doxorubicin are used. In certain neoplastic disorders with a recurrent or rapidly progressive form, the following protocols are often used: IMVP-16 (ifosfamide, methotrexate, and etoposide), MIME (methyl-gag, ifosfamide, methotrexate, and etoposide), DHAP (dexamethasone, high-dose cytarabine, and cisplatin), ESHAP (etoposide, methylprednisolone, HD cytarabine, cisplatin), CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone, and bleomycin), and CAMP (lomustine, mitoxantrone, cytarabine, and prednisone) at well-known dosing rates and schedules for each.

[0162] The amount of chemotherapeutic agent used in combination with the modified antibody of the present disclosure may be varied depending on the subject and can be administered according to those known in the art. See, for example, Bruce A, Chabner et al., Antineoplastic Agents, GOODMAN&GILMAN’S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, pages 1233-1287 (edited by Joel G. Hardman et al., 9th edition, 1996).

[0163] As discussed above, the binding polypeptide, immunoreactive fragment, or recombinant thereof of the present disclosure may be administered in a pharmaceutically effective amount for treating a mammalian disorder in vivo. In this regard, the binding polypeptide of the present disclosure is formulated to facilitate the administration of the active substance and to promote its stability.

[0164] The pharmaceutical composition according to the present disclosure preferably contains a pharmaceutically acceptable, non-toxic, sterile carrier, such as physiological saline, a non-toxic buffer, a preservative, etc. For the purposes of the present application, a pharmaceutically effective amount of a modified binding polypeptide, immunoreactive fragment, or recombinant thereof, conjugated or not conjugated to a therapeutic agent, is an amount sufficient to achieve effective binding to an antigen and, to achieve advantages, for example, to reduce the symptoms of a disease or disorder or to detect a substance or cell. In the case of tumor cells, it is preferred that the modified binding polypeptide can interact with selected immunoreactive antigens on the neoplasm or immunoreactive cells and result in an increase in the death of these cells. Of course, the pharmaceutical composition of the present disclosure may be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

[0165] In maintaining the scope of the present invention, the binding polypeptide of the present disclosure may be administered to humans or other animals in an amount sufficient to provide a therapeutic or prophylactic effect according to the aforementioned treatment methods. The binding polypeptide of the present disclosure can be administered to such humans or other animals in a conventional dosage form prepared by combining the antibody of the present disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. Those skilled in the art will recognize that the form and characteristics of the pharmaceutically acceptable carrier or diluent will depend on the amount of the active ingredient to be combined, the route of administration, and other well-known variables. Those skilled in the art will further understand that a cocktail containing one or more species of the binding polypeptide described in the present disclosure may prove to be particularly effective.

[0166] IX. Expression of the Binding Polypeptide In one aspect, the present invention provides a polynucleotide encoding the binding polypeptide disclosed herein. A method for producing a binding polypeptide is also provided, which includes expressing these polynucleotides.

[0167] The polynucleotides encoding the binding polypeptides disclosed herein can be inserted into an expression vector for introduction into a host cell that can be used to generate the desired amount of the claimed antibody, or a fragment thereof. Thus, in one aspect, the invention provides expression vectors comprising the polynucleotides disclosed herein, as well as vectors and host cells containing these polynucleotides.

[0168] As used herein, the term "vector" or "expression vector" is used for the purposes of this specification and the claims to mean a vector used according to the invention as a vehicle for introducing and expressing a desired gene in a cell. As is known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors applicable in the present invention include a selectable marker, restriction sites suitable for facilitating the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0169] A number of expression vector systems can be used for the purposes of the present invention. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV, or MoMLV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Further, cells that incorporate DNA into their chromosomes can be selected by introducing one or more markers that allow the transfected host cells to be selected. The markers may confer prototrophy on auxotrophic hosts, biocide resistance (e.g., antibiotic resistance), or resistance to heavy metals such as copper. The gene for the selectable marker may be ligated directly to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may be required for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In a particularly preferred embodiment, as discussed above, the cloned variable region genes are synthetically inserted into an expression vector together with the constant region genes (preferably human) of the heavy and light chains.

[0170] In other preferred embodiments, the binding polypeptides of the invention may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as the heavy and light chains of an antibody, may be produced from a single polycistronic construct. These systems are advantageous for providing relatively high levels of the polypeptides of the invention in the eukaryotic cells of the host using an internal ribosome entry site (IRES). Applicable IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference. Those skilled in the art will appreciate that such expression systems can be used to effectively produce the full range of polypeptides disclosed in this application.

[0171] More generally, after preparing a vector or DNA sequence encoding an antibody, or a fragment thereof, an expression vector may be introduced into a suitable host cell. That is, the host cell may be transformed. Introduction of a plasmid into a host cell can be accomplished by a variety of techniques well known to those skilled in the art. Such techniques include, but are not limited to, transfection (including electroporation and electropermeabilization), protoplast fusion, calcium phosphate precipitation, cell fusion with coated DNA, microinjection, and infection with intact virus. See Ridgway, A.A.G., "Mammalian Expression Vectors", Chapter 24.2, pages 470-472, Vectors, edited by Rodriguez and Denhardt (Butterworths, Boston, Mass., 1988). It is most preferred to introduce the plasmid into the host by electroporation. The transformed cells are grown under conditions suitable for the production of the light and heavy chains and assayed for the synthesis of the heavy and / or light chain proteins. Exemplary assay techniques include enzyme-linked immunosorbent assay (E LISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry, and the like.

[0172] As used herein, the term "transformation" is used in a broad sense and means the introduction of DNA into a recipient host cell that changes the genotype and consequently results in a change in the recipient cell.

[0173] Following the same principle, "host cell" means a cell transformed with a vector encoding at least one heterologous gene constructed using recombinant DNA technology. In the description of the process for isolating polypeptides from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably and, unless otherwise specifically indicated, mean the source of the antibody. In other words, the recovery of polypeptides from "cells" can mean either the recovery from whole centrifuged cells or the recovery from cell cultures containing both the medium and the suspended cells.

[0174] In one embodiment, the host cell line used for antibody expression is of mammalian origin, and one of ordinary skill in the art can determine the specific host cell line that is optimal for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary line, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), 293 (human kidney). In one embodiment, the cell line results in altered glycosylation of the antibody expressed therefrom, for example, afucosylation (e.g., PER.C6.RTM. (Crucell) or FUT8-knockout CHO cell line (Potelligent.RTM. cells) (Biowa, Princeton, N.J.)). In one embodiment, NS0 cells can be used. CHO cells are particularly preferred. Host cell lines are typically available from commercial services, the American Type Culture Collection, or published literature.

[0175] It is also possible to scale up in vitro production to obtain large quantities of the desired peptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension cultures such as in an airlift reactor or in a continuously stirred reactor, or cell cultures immobilized or trapped in hollow fibers, microcapsules, on agarose microbeads, or on ceramic cartridges. If necessary and / or desired, the polypeptide solution can be purified by conventional chromatography methods such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose, and / or (immuno)affinity chromatography.

[0176] The gene encoding the binding polypeptide of the present invention can also be expressed in non-mammalian cells such as bacteria, yeast, or plant cells. In this regard, it will be understood that various unicellular non-mammalian microorganisms such as bacteria, i.e., those that can be grown in culture or in fermentation, can also be transformed. Bacteria are readily transformable and include species of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella, the Bacillaceae family, such as Bacillus subtilis, Pneumococcus, Streptococcus, and species of Haem ophilus influenzae. It will be further understood that when expressed in bacteria, the polypeptide may be part of inclusion bodies. The polypeptide must be isolated, purified, and then assembled into a functional molecule.

[0177] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, i.e., common baker's yeast, is the most commonly used eukaryotic microorganism, but numerous other strains are also generally available. For expression in Saccharomyces cerevisiae, plasmid YRp7, for example (Stinchcomb et al., Nature, Vol. 282, p. 39 (1979); Kingsman et al., Gene, Vol. 7, p. 141 (1979); Tschemper et al., Gene, Vol. 10, p. 157 (1980)) is commonly used. This plasmid already contains the TRP1 gene, and the TRP1 gene provides a selectable marker for mutant yeast strains that are unable to grow in tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, Genetics, Vol. 85, p. 12 (1977)). The presence of a disruption of trp1 as a characteristic of the genome of the yeast host cell provides an environment effective for detecting transformation by growth in the absence of tryptophan.

Example

[0178] The present invention will be further illustrated by the following examples, which should not be construed as a further limitation. The sequence listings, figures, and the content of all references, patents, and published patent applications cited throughout this application are hereby specifically incorporated herein by reference.

Example

[0179] Design, Preparation, and Characterization of Variants of the 2C3 Anti-CD-52 Hyperglycosylated Antibody For the purpose of adding bulky groups to the interaction interface (e.g., the FcRn binding site for modulating the pharmacokinetics of an antibody) to regulate the effector function of an antibody by altering its interaction with FcγR, or for introducing chemical modifications to the sequence of a novel cross-linking site for conjugation of effector moieties (including but not limited to drugs, toxins, cytotoxic agents, and radionuclides), a plurality of highly glycosylated mutations were designed in the heavy chain of 2C3, an anti-CD-52 antibody. The highly glycosylated 2C3 variants are described in Table 3.

[0180]

Table 6

[0181] 1A. Preparation of highly glycosylated variants of the 2C3 anti-CD-52 antibody The A114N mutation designed based on the Kabat numbering system was introduced into the CH1 domain of 2C3 by mutagenic PCR. To generate the full-length antibody, the VH domain plus the mutated A114N residue was inserted into the pENTR-LIC-IgG1 vector encoding antibody CH domains 1-3 by ligation-independent cloning (LIC). All other mutations were introduced onto pENTR-LIC-IgG1 by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA). The VH of WT2C3 was cloned into the mutated vector by LIC. The full-length variants were cloned into the pCEP4(-E+I)Dest expression vector by Gateway cloning. The Fc mutations were designed based on the EU numbering system. The mutations were confirmed by DNA sequencing. The amino acid sequences of the heavy and light chains of WT2C3, and the amino acid sequence of the mutated 2C3 heavy chain are described in Table 4. The mutated amino acids are highlighted in gray and the consensus glycosylation target sites created by the mutations are underlined.

[0182]

Table 7

[0183]

Table 8

[0184] Variants and WT controls were transfected into HEK293-EBNA cells in a 6-well plate format. As shown in Figure 9, when analyzed by SDS-PAGE and Western blot, the expression levels were found to be approximately 0.1 μg / ml. Furthermore, the expression of the variants in the conditioned medium was also measured by capture of Protein A on Biacore. Concentrations were determined using the dissociation response 6 minutes after injection into immobilized Protein A. WT2C3 produced by CHO, serially diluted from 90 μg / mL to 1.5 ng / mL in the medium, was used as a standard curve. Concentrations were calculated to approximately 0.2 μg / mL by a calibration curve using 4-parameter fitting. As shown in Figure 9, the relative expression levels were low and generally consistent with the Western blot results.

[0185] 1B. Verification of Hyperglycosylation To determine whether additional glycosylation sites were introduced by the mutation, the 2C3 variants and wild-type proteins were treated with PNGaseF, a universal deglycosylation enzyme, and the protein samples were analyzed by SDS-PAGE and Western blot. As shown in Figure 10, an apparent increase in molecular weight was observed only for the A114N variant, indicating the presence of additional N-linked carbohydrates.

[0186] Small-scale antibody preparations were generated and the 2C3 variants were purified to further verify the introduction of glycosylation sites. As shown in Figure 11, it was confirmed that only the A114N variant had the introduction of additional glycosylation sites.

[0187] 1C. Binding Characteristics of 2C3 Anti-CD-52 Variants Using Biacore, the binding properties of the purified proteins were compared. Mouse and human FcRn-HPC4 purified by SEC were immobilized on a CM5 chip by amine coupling. Each antibody was diluted to 200, 50, and 10 nM and injected onto the immobilized Fc receptor. Campath, WT 2C3 produced by CHO, and DEPC-treated Campath were included as positive and negative controls. As shown in Figure 13, the Y436 mutant showed approximately a 2-fold reduction in binding to human FcRn. Interestingly, the binding of this mutant to mouse FcRn was not affected. None of the other 2C3 mutants had any significant effect on human or mouse FcRn binding.

[0188] Using Biacore, the antigen-binding properties of the purified proteins were compared using a Biacore binding assay of 741, a CD-52 peptide. 741, a CD-52 peptide, and 777, the peptide of interest, were immobilized on a CM5 chip. The antibody was serially diluted 2-fold from 60 to 0.2 nM in HBS-EP, injected twice for 3 minutes each, and then dissociated for 5 minutes in buffer at a flow rate of 50 μL / min. GLD52 lot 17200-084 was included as a control. The surface was regenerated with a single pulse of 40 mM HCl. A 1:1 binding model was used to fit the curve from 7.5 to 0.2 nM. As shown in Figure 16, the CD-52 binding affinity of the A114N mutant was slightly lower, and the affinity of the NGT mutant was slightly higher than that of the remaining mutants in this assay. The Biacore binding assay of 741, a CD-52 peptide, was repeated with proteins purified from a larger-scale preparation (prep). As shown in Figure 17, the A114N mutant showed CD-52 peptide binding comparable to that of WT 2C3.

[0189] 1D. Characterization of the charge of the A114N mutant Isoelectric focusing (IEF) was performed to characterize the charge of the 2C3 variant. The purified protein was run on an immobilized pH gradient (pH 3 - 10) acrylamide (IPG) gel. As shown in Figure 18A, A114N was found to have a more negative charge, presumably due to sialic acid residues. Intact MS data confirmed a complex structure with sialic acid on the A114N variant. In contrast, WT 2C3 was shown to have G0F and G1F as the dominant glycosylated species (Figures 18C and 18D, respectively).

Example

[0190] Preparation of highly glycosylated variants in the backbone of several antibodies In addition to the 2C3 anti-CD-52 antibody, the A114N mutation was introduced into several other antibody backbones to confirm that unique highly glycosylated sites can be introduced into unrelated heavy chain variable domain sequences. Highly glycosylated anti-TEM1, anti-FAP, and anti-Her2 variants are described in Table 5.

[0191]

Table 9

[0192] 2A. Preparation of highly glycosylated variants of anti-TEM1 and anti-FAP antibodies The A114N mutation named based on the Kabat numbering system was introduced by mutagenic PCR into the CH1 domains of anti-TEM1 and anti-FAP. To generate the full-length antibody, the mutated VH plus residue 114 was inserted by ligation-independent cloning (LIC) into the pENTR-LIC-IgG1 vector encoding antibody CH domains 1-3. The full-length mutant was then cloned by Gateway cloning into the pCEP4(-E+I)Dest expression vector. The mutation was confirmed by DNA sequencing. The amino acid sequences of the anti-TEM1 wild type, as well as the mutated heavy and light chains, are shown in Table 6. The mutated amino acids are highlighted in gray and the consensus glycosylation target sites created by the mutation are underlined.

[0193]

Table 10

[0194] Mutants and wild-type controls were transfected into HEK298-EBNA cells in a triple-flask format and purified on a HiTrap Protein A column (GE Healthcare Biosciences, Pittsburgh, PA, USA). Analysis at A280 on a NanoDrop spectrophotometer showed that the expression of anti-FAP A114N and anti-FAP A114C was approximately 3 μg / ml and approximately 1 μg / ml, respectively. The expression of anti-TEM1 A114N was approximately 0.04 μg / ml.

[0195] 2B. Verification of High Glycosylation To confirm that additional glycosylation sites were introduced into the A114N variant, the protein purified from the A114N variant was analyzed on a reducing SDS-PAGE together with the wild-type control protein. An additional glycosylation site adds 2000 - 3000 daltons to the molecular weight of the heavy chain. As shown in Figure 20, SDS-PAGE indicated that the apparent molecular weight of the heavy chain bands of the A114N variants of anti-FAP and anti-TEM1 increased, suggesting that additional glycosylation sites were successfully introduced into both antibodies.

[0196] 2C. Preparation of highly glycosylated variants of anti-Her2 antibody The A114N of Her-2, A114N / NNAS of Her-2, and WT Her- 2 antibodies were prepared by ligation-independent cloning. The VH domain of Herceptin was synthesized and PCR amplified with either of two sets of LIC-compatible primers with WT or the A114N mutation. To obtain the full-length antibody, the amplified VH insert (WT or A114N) was cloned into two pENTR vectors encoding the CH1-3 domains, WT of pENTR-LIC-IgG1, and NNAS of pENTR-LIC-IgG1, resulting in three full-length variants (A114N, NNAS, A114N / NNAS) and a WT control as entry clones on pENTR. These variants were cloned into the pCEP4(-E+I)Dest expression vector by Gateway cloning. Mutations were confirmed by DNA sequencing. The wild-type amino acid sequence of anti-Her-2, as well as the mutated heavy and light chain amino acid sequences, are listed in Table 7. The mutated amino acids are highlighted in gray, and the consensus glycosylation target sites created by the mutations are underlined.

[0197]

Table 11

[0198]

Table 12

[0199] Expression of highly glycosylated variants of the anti-Her2 antibody of 2D.A114N The anti-Her2 and wild-type constructs of A114N were transfected into HEK293-EBNA cells in 12 triple flasks with Lipofectamine-2000 (reagent to DNA ratio 2.5:1) and XtremeGene HP (reagent to DNA ratio 3:1). When aliquots were measured by Octet from the conditioned medium (CM) on day 3, protein expression was shown to be constant across 6 flasks for both Lipofectamine 2000 and XtremeGene HP. As shown in Table 8, the overall transfection efficiency was 30% higher with XtremeGene HP. The conditioned medium collected on day 3 was pooled together for each of the two transfection conditions and purified by a Protein A column. By measuring with Octet, the antibody in the serum-containing mock medium was shown to be 1.8 μg / ml, whereas it was 0 μg / ml in the serum-free mock medium.

[0200]

Table 13

[0201] The conditioned medium from day 6 was collected and purified separately for each transfection condition. Both eluates were separately buffer-exchanged into PBS, pH 7.2 and concentrated approximately 15-fold using an Amicon-4 (cut-off 50 kD) column. The CM on day 6 showed higher expression levels compared to the CM on day 3. As shown in Table 8, a total of 3 mg of Herceptin A114N at 15.59 mg / ml (from Lipofectamine transfection) and 6 mg of Herceptin A114N at 16.86 mg / ml (from XtremeGene HP transfection) were generated from the conditioned medium on day 6 for further downstream applications such as antibody-drug conjugation.

[0202] SDS-PAGE and HIC Analysis of 2E.A114N Anti-Her2 Variant Prior to conjugation, purified A114N Herceptin was characterized by SDS-PAGE and HIC (hydrophobic interaction chromatography). As shown in Figure 21, the quality of the purified A114N Herceptin was determined to be suitable for further downstream applications.

[0203] 2F. Conjugation to Modified Glycosylation The following was demonstrated: a) the glycosylation site was introduced at the Kabat 114 position on anti-TEM1, b) the A114N variant had high glycosylation on the heavy chain by reducing SDS-PAGE, and c) the A114 high-glycosylated variant had a complex carbohydrate structure containing terminal sialic acid and galactose ideal for SAM and GAM conjugation by intact LC / MS. To confirm that the modified glycosylation site was suitable for conjugation, anti-TEM1 A114N was conjugated to 5 kDa PEG by an aminooxy chemical reaction. As shown in Figure 22, PEG was successfully conjugated to anti-TEM1 A114N by aminooxy ligation. This variant was also successfully prepared on the 2C3 backbone of anti-FAP and anti-CD-52 (not shown). These data demonstrate that the glycosylation site at N114 is useful for conjugation of effector moieties.

Example

[0204] Production of Fc Variants of S298N / Y300S A modified Fc variant was designed and produced by introducing a new glycosylation site at Ser298 of EU, adjacent to the naturally occurring Asn297 site. The glycosylation of Asn297 was either maintained or removed by mutation. The results of the mutations and desired glycosylation are described in Table 9.

[0205]

Table 14

[0206] Generation of Altered Glycosylation Variants of 3A.H66αβ-TCR Antibody Using the pENTR_LIC_IgG1 template, mutations were introduced onto the heavy chain of the αβ T cell receptor antibody clone #66 by QuikChange. After amplifying the VH domain of HEBE1 Δab IgG1 #66 with LIC primers, it was cloned by LIC into mutant or wild-type pENTR_LIC_IgG1 to generate full-length mutant or wild-type antibodies. Subcloning was verified with DraIII / XhoI double digest, generating an insert of approximately 1250 bp size in successful clones. These full-length mutants were then cloned into the expression vector pCEP4(-E+I)Dest by Gateway cloning. Mutations were confirmed by DNA sequencing. The amino acid sequences of the WT H66 anti-αβTCR heavy and light chains, and the amino acid sequence of the mutated H66 heavy chain are shown in Table 10. The mutated amino acids are highlighted in grey and the consensus glycosylation target sites created by the mutations are underlined.

[0207]

Table 15

[0208]

Table 16

[0209] Constructs of the variant, wild type, and two non-glycosylated controls (HEBE1 Agly IgG4 and HEBE1 Δab IgG1 in pCEP4) were transfected into HEK293-EBNA cells in triple flasks for expression. Proteins were purified from 160 ml of conditioned medium (CM) using a 1 ml HiTrap Protein A column (GE) with a multi-channel peristaltic pump. 5 micrograms of each resulting supernatant were analyzed on 4-20% Tris-Glycine reducing and non-reducing SDS-PAGE gels (see Figure 2). The heavy chains of the non-glycosylated variants (N297Q, T299A, and Agly controls) migrated further (arrows), consistent with the loss of glycans in these antibodies. However, the heavy chains of the modified glycosylated antibodies (NSY, STY, SY, Δab, and wt controls, arrows) migrated similarly to the wild type control. This result was consistent with the presence of the modified glycosylation site at EU position 298. SEC-HPLC analysis indicated that all variants were expressed as monomers.

[0210] 3B. Glycosylation analysis by LC-MS The Fc variant of the modified H66 IgG1 was partially reduced with 20 mM DTT at 37 °C for 30 minutes. The sample was then analyzed by capillary LC / MS on an Agilent 1100 capillary HPLC system coupled to a QSTAR qq TOF hybrid system (Applied Biosystems). Protein reconstruction by Bayesian theory with baseline correction and computer modeling in Analyst QS 1.1 (Applied Bisoystem) were used for data analysis. In the H66 antibody variant of S298N / T299A / Y300S, one glycosylation site was observed at amino acid 298, and bi- and tri-branched complex glycans were detected as the main species aligned in G0F, G1F, and G2F (see Figure 34). This altered glycosylation profile was consistent with the shifted N298 glycosylation instead of the wild type N297 glycosylation site.

[0211] Binding characteristics of αβ TCR antibody variants to human FcγRIIIa and FcγRI using 3C.Biacore Binding to recombinant human FcγRIIIa (V158 and F158) and FcγRI was evaluated using Biacore. All four flow cells of the CM5 chip were immobilized with anti-HPC4 antibody by the standard amine coupling procedure provided by Biacore. The anti-HPC4 antibody was diluted to 50 μg / mL in 10 mM sodium acetate pH 5.0 for the coupling reaction and injected at 5 μL / min for 25 min. Approximately 12000 RU of the antibody was immobilized on the chip surface. Recombinant human FcγRIIIa-V158 and Fc γRIIIa-F158 were diluted to 0.6 μg / mL in binding buffer (HBS-P containing 1 mM CaCl2) and injected into flow cells 2 and 4 at 5 μL / min for 3 min, respectively, to capture 300 - 400 RU of receptor on the anti-HPC4 chip. To distinguish between low binders, three-fold more rhFcγRIIIa than normally used in this assay was captured on the anti-HPC4 surface. Flow cells 1 and 3 were used as reference controls. Each antibody was diluted to 200 nM in binding buffer and injected over all four flow cells for 4 min, followed by dissociation in buffer for 5 min. The surface was regenerated with 10 mM EDTA in HBS-EP buffer at 20 μL / min for 3 min. The results of these experiments are shown in Figure 3.

[0212] Biacore was also used here to compare FcγRI binding. Anti-TetraHis antibody was buffer-exchanged to 10 mM sodium acetate pH 4.0 using a Zeba Desalting column and diluted to 25 μg / mL in acetate buffer for amino coupling. Two flow cells of a CM5 chip were injected at 5 μL / min for 20 min and then immobilized with approximately 9000 RU of anti-TetraHis antibody. To compare samples with weak binding, 10-fold more FcγRI was captured on the anti-TetraHis surface as in the previous experiment. Recombinant human FcγRI was diluted to 10 μg / mL in HBS-EP binding buffer and injected into flow cell 2 at 5 μL / min for 1 min to capture approximately 1000 RU of receptor on the anti-TetraHis chip. A single concentration of 100 nM antibody was injected onto the captured receptor and control surfaces at 30 μL / min for 3 min. Subsequently, dissociation was monitored for 3 min. The surface was then regenerated by injecting 10 mM glycine pH 2.5 twice at 20 μL / min for 30 s. The results of these experiments are shown in Figure 4.

[0213] These results demonstrate a significant reduction in the binding of the sugar-modified mutants to FcγRIIIa or FcγRI. In particular, the binding of the S298N / T299A / Y300S of H66 to both receptors was almost completely disrupted. This mutant was selected for further detailed analysis.

[0214] 3D. Characterization of stability using circular dichroism The stability of the S298N / T299A / Y300S mutant of the antibody was monitored by far-UV CD thermal melting experiments that monitored the CD signals at 216 nm and 222 nm as a function of the temperature increase that brought about the unfolding (denaturation) of the antibody.

[0215] The temperature was controlled by a thermoelectric Peltier (Jasco model AWC100) and raised from 25 to 89 °C at a rate of 1 °C / min. CD spectra were collected on a Jasco815 spectrophotometer at a protein concentration of approximately 0.5 mg / mL in PBS buffer in a quartz cuvette (Hellma, Inc) with an optical path length of 10 mm. The scan speed was 50 nm / min and the data pitch was 0.5 nm. The sensitivity setting was medium and a bandwidth of 2.5 nm was used. CD signals and HT potentials were collected from 210 to 260 nm at a data interval of 0.5 nm and a temperature interval of 1 °C, and four duplicate scans were performed for each sample. The results demonstrate that both the H66 of delta AB and the H66 variant of S298N / T299A / Y300S show similar thermal behavior and have approximately the same onset temperature (around 63 °C) for degradation (Figure 35), further suggesting that they have comparable stability.

Example

[0216] Functional analysis of Fc-modified mutants Fc-modified mutants were evaluated by PBMC proliferation assays and cytokine release assays. In the PBMC proliferation assay, human PBMC were cultured with increasing concentrations of the therapeutic antibody for 72 hours, 3 H-thymidine was added, and the cells were collected after 18 hours. In the T cell depletion / cytokine release assay, human PBMC were cultured with increasing concentrations of the therapeutic antibody, and cell number and viability were analyzed daily until day 7 (Vi-Cell, Beckman Coulter). The cell supernatants were also collected, stored at -20 °C, and analyzed on a cytokine panel (8-plex) with 8 components (Bio-Rad).

[0217] PBMCs from normal donors were thawed and processed under the following conditions (in media containing all complement): untreated; BMA031, moIgG2b 10 μg / ml; OKT3, moIgG2a 10 μg / ml; H66, huIgG1 delta AB 10 μg / ml, 1 μg / ml, and 0.1 μg / ml; H66, huIgG1 S298N / T299A / Y300S 10 μg / ml, 1 μg / ml, and 0.1 μg / ml.

[0218] Cytokines were collected on Day 2 (D2) and Day 4 (D4) for BioPlex analysis (IL2, IL4, IL6, IL8, IL10, GM-CSF, IFNg, TNFa). On D4, cells were stained for CD4, CD8, CD25, and abTCR expression.

[0219] The results shown in Figures 5 - 8 demonstrate that S298N / T299A / Y300S of H66 behaves similarly to delta AB of H66 in all cell-based assays performed, showing minimal activation of T cells by CD25 expression, binding to abTCR (with slightly different kinetics to delta AB), and minimal cytokine release at both D2 and D4 time points. The S298N / T299A / Y300S variant thus eliminated effector function as effectively as the delta AB mutation.

Example

[0220] Preparation and Characterization of Modified Fc Variants in the Backbone of Anti-CD52 Antibodies In addition to the H66 anti-αβ TCR antibody, the S298N / Y300S mutation was also introduced into the backbone of an anti-CD52 antibody (clone 2C3). This variant was then tested to determine whether the observed modulation of effector function seen in the H66 anti-α TCR antibody with S298N / Y300S was consistent with another antibody backbone.

[0221] Generation of Glycosylation Variants Altered by the 5A.2C3 Anti-CD52 Antibody First, the 2C3 variant DNA of S298N / Y300S was prepared by quick-change mutagenesis using pENTR_LIC_IgG1, and the WT 2C3 VH was cloned by LIC into the mutated vector. The full-length variant was cloned into the pCEP4(-E+I)Dest expression vector using Gateway technology. Subsequently, the mutations were confirmed by DNA sequencing, and the sequences were those described in Table 11. The variant was then transfected into HEK293-EBNA cells in a 6-well plate format, and the protein was purified from the conditioned medium. The anti-CD52 2C3 wild-type antibody was generated in parallel as a control. The expression level was found to be 0.1 μg / mL using SD-PAGE and Western blot analysis (Figure 9A). The expression of the variant in neat conditioned medium was also measured by capture on Protein A on a Biacore. The concentration was determined using the dissociation response after injecting immobilized Protein A for 6 minutes. The WT2C3 produced by CHO was serially diluted from 90 μg / mL to 1.5 ng / mL in the medium and used as a standard curve. The concentration was calculated to be within approximately 0.2 μg / mL by a calibration curve using a four-parameter fit. The relative expression level was low and generally consistent with the Western blot data (Figure 9B).

[0222]

Table 17

[0223] 5B. Glycosylation analysis using PNGaseF To evaluate the additional glycosylation sites introduced by the mutation, the concentrated S298N / Y300S variant was deglycosylated with PNGaseF. According to that, it was pointed out that no apparent change in molecular weight was demonstrated, indicating the absence of additional carbohydrates (Figure 10). For further purification of these variants for additional characterization, small-scale preparations were performed, and the results reconfirmed the absence of additional carbohydrates on the S298N / Y300S variant (Figure 11).

[0224] Binding characteristics of the 2C3 anti-CD-52 antibody variant to human FcγRIIIa using 5C.Biacore Biacore was also used to characterize the antigen-binding, FcγRIII, and binding properties of the purified antibodies (see Figures 12, 13, and 14). The 2C3 variant of S298N / Y300S bound strongly to the CD52 peptide, and the binding sensorgram was indistinguishable from the wild-type control, demonstrating that this mutation did not affect antigen binding (Figure 12A).

[0225] To assay for effector functions of Fc, the FcγRIII receptor (Val158) was used in the binding assay. Mutant and wild-type control antibodies were diluted to 200 nM, It was injected into FcγRIIIa captured by the HPC4-tag. FcγRIII binding was hardly detectable for the S298N / Y300S variant, indicating loss of effector function by this variant (Figures 12B and 14A). To further assay for Fc effector function, the FcγRIII receptor (Phe158) was also used in the binding assay. Variant and wild-type control antibodies were diluted to 200 nM and injected into FcγRIIIa captured by the HPC4-tag. FcγRIII binding was hardly detectable for the S298N / Y300S variant, indicating loss of effector function with the Phe158 variant (Figure 14B). Finally, Biacore was used to compare the FcRn binding characteristics of the purified proteins. Mouse and SEC-purified human FcRn-HPC4 were immobilized on a CM5 chip by amine coupling. Each antibody was diluted to 200, 50, and 10 nM and injected over the receptor. Campath, WT2C3 produced by CHO, and DEPC-treated Campath were included as positive and negative controls. These data indicate that the variant binds to both human and mouse FcRn receptors with the same affinity as the wild-type antibody control and is unlikely to have alterations in circulating half-life or other pharmacokinetic properties (see Figures 12C, 13A, and B). Thus, the S298N / Y300S mutation can generally be applied to antibodies to reduce or eliminate unwanted Fc effector function, such as through involvement of human Fc receptors.

Example

[0226] Detection of circulating immune complexes in the S298N / Y300S variant The detection of circulating immune complexes was also investigated using a C1q binding assay against the S298N / Y300S variant and WT controls. High-binding Costar 96-well plates were coated overnight at 4 °C with 100 μl of 2C3Ab serially diluted two-fold in coating buffer (0.1 M NaCHO3, pH 9.2) at concentrations ranging from 10 to 0.001 μg / ml. ELISA analysis showed that C1q binding was reduced in the S298N / Y300S variant compared to WT (Figure 15A). Equal coating of the wells was confirmed by binding of anti-FabAb to the coated 2C3Ab (Figure 15B).

Example

[0227] Separation and analysis of the S298N / Y300S variant using isoelectric focusing The S298N / Y300S variant was characterized by running an isoelectric focusing (IEF) gel with a pH range of 3 - 10. S298N / Y300S was found to have a more negative charge and thus is likely a sialic acid molecule (Figure 18A). Both the S298N / Y300S variant and WT 2C3 were shown by intact MS to have G0F and G1F as the dominant glycosylated species (Figures 18B and D, respectively).

Example

[0228] Antigen-binding affinity of S298N / Y300S Using Biacore, the antigen-binding affinities of the 2C3Ab of WT anti-CD-52 and the S298N / Y300S variant, prepared and purified from both small-scale (Figure 16) and large-scale (Figure 17) expression, were compared. CM5 chips immobilized with the CD52 peptide 741 and the control peptide 777 were obtained. The antibodies were serially diluted 2-fold from 60 to 0.2 nM in HBS-EP and then injected over the chip surface for 3 minutes, followed by dissociation in buffer at a flow rate of 50 μl / min for 5 minutes. The surface was then regenerated with a pulse of 40 mM HCl. These analyses were performed twice each, and it was demonstrated that the S298N / Y300S variant and the WT 2C3 antibody exhibit comparable CD52 peptide-binding properties.

[0229] To screen the antibodies produced during small-scale transfection, a culture medium screening platform was designed to test the functional binding properties before purification. These tests were performed using Octet (Figure 19A) to determine the concentration, with a protein A biosensor and a GLD52 standard curve. The samples were diluted to 7.5 and 2 nM in HBS-Ep for comparison of CD52 binding using Biacore (Figure 19B). The results of the peptide-binding assay showed that both the S298N / Y300S variant and the WT 2C3 antibody have comparable CD52 peptide-binding properties. Furthermore, these analyses demonstrated that Octet and Biacore are sufficient to predict antigen-binding by antibodies from small-scale transfection.

Example

[0230] Preparation of glycosylation variants in which S298N / Y300S, S298N / T299A / Y300S, and S297Q / S298N / Y300S in the backbone of additional antibodies are altered In addition to the anti-αβ-TCR antibody and the 2C3 anti-Cd-52 antibody, it was confirmed that the S298 / Y300S, S298N / T299A / Y300S, and N297Q / S298N / Y300S mutations can be introduced into the backbone of other antibodies to introduce additional tandem glycosylation sites into unrelated heavy chain variable domain sequences. The alternately glycosylated anti-CD-52 12G6 and anti-Her2 variants are described in Tables 12 and 13.

[0231]

Table 18

[0232]

Table 19

[0233]

Table 20

Examples

[0234] Production of Altered Antibodies Containing Reactive Glycan Moieties To produce an antibody comprising a glycan moiety capable of reacting with a derivatized effector moiety, an anti-HER antibody was first glycosylated in vitro using a glycosyltransferase and a related sugar nucleotide donor. For example, to introduce sialic acid residues, the donor antibody was first galactosylated with β-galactosyltransferase and subsequently sialylated with α2,6-sialyltransferase according to the method of Kaneko et al. (Kaneko, Y., Nimmerjahn, F., and Ravetch, J. V. (2006), Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation., Science, 313, pp. 670-3). The reaction was carried out in a one-pot synthesis step using β-galactosyltransferase (50 mU / mg, Sigma) and α2,6-sialyltransferase (5 μg / mg, R&D system) with UDP-galactose (10 mM), the sugar nucleotide substrate of the donor, and CMP-sialic acid in 50 mM MES buffer (pH 6.5) containing 5 mM MnCl2. The reaction mixture containing 5 mg / ml anti-HER2 antibody was incubated at 37°C for 48 hours. Sialylation was verified using MALDI-TOF MS analysis of the overmethylated glycan released from the antibody with PNGaseF, sialic acid content analysis using Dionex HPLC, and lectin blotting with SNA, a lectin specific for α2,6-sialic acid.

[0235] MALDI-TOF analysis of the glycan released by PNGaseF treatment of the sialylated anti-HER2 antibody indicated that the native glycan was completely reconstructed with A1F, a predominantly monosialylated and bifurcated structure, along with a small amount of disialylated species (Figure 27A). Treatment of the antibody with a large amount of α2,6-sialyltransferase generated a more homogeneous population of the A1F glycoform, suggesting that complete sialylation can be hindered either by enzyme activity or glycan localization. The sialic acid content was determined to be approximately 2 mol per mole of antibody, which is consistent with the A1F glycan as the major glycoform species (Figure 27B). Lectin blotting with the Sambucus nigra agglutinin (SAN lectin), specific for α2,6-linked sialic acid, confirmed the presence of sialic acid in the α2,6-linkage configuration (Figure 27C).

[0236] In conclusion, the native protein glycan is somewhat heterogeneous, but reconstruction with galactosyl and sialyltransferases yields an almost homogeneous antibody with monosialylated but fully galactosylated bifurcated glycans (A1F). The fact that there was only approximately one sialic acid introduced to the two galactose acceptors on each branched glycan could be due to the glycan being mostly buried within the antibody and thus having access to only one galactose, or due to non-covalent interactions of the glycan with the protein surface.

Example

[0237] Oxidation of the Altered Antibody Containing the Reactive Glycan Moiety After verifying sialylation, the oxidation of sialylated anti-HER2 antibody during the manufacturing process with various concentrations of periodate (0.25 to 2 mM) was investigated. The sialylated antibody was first buffer-exchanged into 25 mM Tris-HCl (pH 7.5) containing 5 mM EDTA and subsequently buffer-exchanged with PBS buffer. Then, the buffered antibody mixture was applied to a Protein A Sepharose column pre-equilibrated with PBS buffer. The column was washed with 15 column volumes of PBS, 15 column volumes of PBS containing 5 mM EDTA, and 30 column volumes of PBS, and then eluted with 25 mM citrate phosphate buffer (pH 2.9). The eluate was immediately neutralized with dibasic phosphate buffer, and the antibody was concentrated using Amicon ultra from Millipore. After purification, The sialylated anti-HER2 antibody was then oxidized with sodium periodate (Sigma) in 100 mM sodium acetate buffer (pH 5.6) for 30 minutes in the dark on ice, and the reaction was quenched with 3% glycerol on ice for 15 minutes. The product was desalted and exchanged into 100 mM sodium acetate (pH 5.6) by 5 rounds of ultrafiltration on a 50 kDa Amicon. Figure 28A shows the analysis of sialic acid content of the sialylated antibody determined with various amounts of periodate. Complete oxidation of sialic acid residues was achieved at periodate concentrations higher than 0.5 mM. In fact, a periodate concentration as low as about 0.5 mM is sufficient to completely oxidize the introduced sialic acid. Therefore, a periodate concentration of 1 mM was chosen for the oxidation of sialylated antibody for drug conjugate.

[0238] Oxidation can have a detrimental effect on the integrity of the antibody. For example, oxidation of methionine residues including Met-252 and Met-428 located in the CH3 region of Fc near the FcRn binding site is known to affect FcRn binding, which is crucial for the extension of the serum half-life of the antibody (Wang, W. et al. (2011) Impact of methionine Oxidation in human IgG1 Fc on the serum half-life of monoclonal antibodies. (Mol Immunol, Vol. 48, pp. 860 - 866). Therefore, to investigate the potential side effects of periodate oxidation on methionine residues (e.g., Met-252) that are crucial for FcRn interaction, the oxidation state of sialylated antibodies was determined by LC / MS analysis of tryptic peptide digests. The analysis revealed approximately 30% oxidation of Met-252 and <10% oxidation of Met-428 after treating trastuzumab sialylated with 1 mM periodate. To determine the impact of this level of methionine oxidation on FcRn binding, the kinetics of FcRn binding to each antibody were evaluated using surface plasmon resonance (BIACORE). This analysis revealed that the oxidation state correlates with a minor loss of FcRn binding (12% and 26% decreases for mouse and human FcRn, respectively; see Figures 28B and 28C). Notably, it has been reported that a ~25% decrease in Ka for human FcRn has no effect on plasma half-life in human FcRn transgenic mice because a single intact FcRn site on each antibody is sufficient to confer functional and PK advantages (Wang et al., ibid).

[0239] In summary, these data indicate that introducing sialic acid residues sensitive to periodate by sialyltransferase treatment allows for the use of very low concentrations of periodate, resulting in minimal side effects on the antibody-FcRn interaction and antibody integrity (≦1%) as evaluated by aggregation. Thus, using sialylated antibodies by the method of the present invention provides a wider window of oxidation conditions to be used and enables the reproducible production of active glycoconjugates without affecting serum half-life.

[0240] Galactose in the highly glycosylated antibody variants can also be specifically oxidized using galactose oxidase to generate aldehyde groups for conjugation. To confirm this approach, the A114N anti-TEM1 antibody was concentrated to 13 - 20 mg / ml and then treated with 20 mU / mg sialidase in PBS at 37 °C for 6 hours. The desialylated product was then oxidized with galactose oxidase (「GAO」), first adding 5 μg of GAO per mg of protein at 37 °C overnight, followed by adding 2 μg of GAO per mg of protein and incubating for an additional 5 hours. Sodium acetate was added to adjust the pH to 5.6 (0.1 v / v, pH 5.6), and DMSO was added to achieve a final reaction concentration of 16% and was added prior to conjugation. The highly glycosylated variant A114N anti-HER antibody (15 mg / ml) was similarly desialylated with sialidase (20 mU / mg) and oxidized with 5 μg of G AO in a single reaction overnight at 37 °C.

Example

[0241] Synthesis of Reactive Effector Moieties To facilitate the conjugation of the aldehyde-derivatized antibodies of the invention with the glycoforms, candidate drug effector moieties (e.g., monomethyl auristatin E (MMAE) and dolastatin 10 (Dol10)) were derivatized with aminooxysystamide to contain a functional group (e.g., aminooxy-cys) that is specifically reactive with aldehydes.

[0242] Briefly stated, to produce aminooxy-cystamide as the starting material, S-trityl-L-cysteinamide (362 mg, 1 mmol) was added to 3 mL of a DMF solution of t-BOC-aminooxyacetic acid N-hydroxysuccinamide ester (289 mg, 1 mmol). The reaction was completed after 3 hours as revealed by HPLC analysis. Subsequently, the reaction mixture was diluted with 30 ml of dichloromethane and washed with 0.1 M sodium bicarbonate solution (2 × 20 mL), water (2 × 20 mL), and brine (2 × 20 mL). The solution was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. 3 mL of TFA was added to this dried residue, followed by 150 μL of triethylsilane. The resulting solution was precipitated from t-butyl methyl ether, and this process was repeated 3 times. After filtration, the residue was dried under reduced pressure to obtain 205 mg of an off-white solid (yield 67%). The compound was used in the next step without further purification.

[0243] To produce aminooxy-derivatized MMAE (aminooxy-Cys-MC-VC-PABC-MMAE), 30.1 mg (0.098 mmol, 2 equivalents) of aminooxyscystamide in 3 mL of DMF was combined with 64.6 mg of MC-VC-PABC-MMAE (0.049 mmol) and 100 μL of triethylamine. The resulting reaction mixture was stirred at room temperature for 15 minutes, but according to HPLC analysis, the reaction was completed by that time. The compound was purified by preparative HPLC to obtain 45 mg (62%) of the desired product as an off-white solid. Reverse-phase HPLC analysis suggested that the purity of the compound was >96%. ESI: C73H116N14O18S (MH) + Calculated value of 1509.8501; found value, m / z 1509.8469.

[0244] To produce aminooxy-derivatized Dol10 (aminooxy-Cys-MC-VC-PABC-PEG8-Dol10), aminooxysuccinamide 7.4 mg (0.024 mmol, 3 equiv), MC-VC-PABC-PEG8-Dol10 12 mg (0.008 mmol), and triethylamine 30 μL were combined in 3 mL of DMF. The reaction was complete within 15 minutes as determined by HPLC analysis. Purification by preparative HPLC afforded 6.2 mg (46%) of the desired product as an off-white solid. Reverse-phase HPLC analysis suggested that the purity of the compound was >96%. ESI: C80H12 4N16O19S2 (MH) + Calculated value of 1678.0664; found, m / z 1678.0613.

Example

[0245] Sialic acid-mediated (SAM) conjugation of the reactive effector moiety After desalting, the drug-linker of Example 11 was combined with the oxidized, sialylated antibody of Example 10 at a concentration of 25 mM in 75% DMSO (0.167 v / v) to achieve a molar ratio of drug-linker to antibody of 24:1 and a final antibody concentration of 5 mg / ml. The mixture was incubated overnight at room temperature. Unincorporated drug-linker and any free drug were captured using BioBeads. The product was buffer-exchanged into histidine-Tween buffer using a PD-10 column and sterile filtered. The level of endotoxin was determined and an ADC of <0.1 EU / mg was achieved for in vivo testing.

[0246] Figures 29A - C show the hydrophobic interaction chromatography (HIC) of variously sialylated antibodies conjugated with AO - MMAE (anti - FAP B11 and G11 of Example 11, and anti - HER2 antibody). The sialylated HER2 antibody was also conjugated with the drug - linker AO - Cys - MC - VC - PABC - PEG8 - Dol10 (Figure 29D). This analysis revealed that there were mainly 1 to 2 drug conjugates per antibody, and the drug - to - antibody ratio (DAR) was in the range of 1.3 - 1.9. The extended retention time of the Do110 glycoconjugate (Figure 29D) compared to the MMAE glycoconjugate (Figure 29C) is likely due to the greater hydrophobicity of Do110.

[0247] LC - MS analysis was also performed on anti - HER antibodies conjugated with two different drug - linkers (AO - MMAE or AO - PEG8 - Dol10) at a 30 mg scale. This analysis showed that the DAR values after conjugation were similar at 1.7 and 1.5, which is comparable to the HIC analysis. Size - exclusion chromatography (SEC) showed very low levels (1%) of aggregates in these conjugates.

Example

[0248] Galactose - mediated (GAM) conjugate of the reactive effector moiety On the A114N anti-TEM1 highly glycosylated mutant antibody described in Example 11, galactose aldehyde produced by galactose oxidase was incubated overnight at 25 °C to conjugate with 24 molar excess of aminooxy-MC-VC-PABC-MMAE drug-linker over the antibody, and an ADC conjugate with DAR 1.72 was obtained. For the galactose oxidase-treated anti-HER antibody prepared as described in Example 11, 1M sodium acetate, pH 5.6 of 1 / 10 of the reaction volume was added to adjust the pH to 5.6, DMSO was added to a final concentration of 14%, and then 24 equivalents of aminooxy MC-VC-PABC-MMAE drug linker were added. The reaction was incubated overnight at room temperature. Free drug and drug-linker were collected with Biobeads and the product buffer was exchanged by SEC (yield 65%). The product conjugate was analyzed by HIC. As shown in Figure 30, AO-MMAE was conjugated to approximately 60% of the molecules.

Example

[0249] In vitro ADC cell proliferation assay The in vitro activity of the anti-HER and anti-FAP glycoconjugate molecules of the present invention was also compared with the corresponding thiol conjugate containing the same drug moiety linked to the hinge region cysteine of the same donor antibody by thiol ligation. The thiol conjugate contained approximately twice the amount of drug (DAR) per antibody than the glycoconjugate. Thiol-based conjugation was performed as described by Stefano et al. (publication, Methods in Molecular Biology, 2013). Then, the relative efficacy of each ADC was evaluated using the Her2+ SK-BR-3 and Her2-MDA-MB-231 cell lines. The results of this analysis are presented in Table 15 below.

[0250]

Table 21

[0251] Figure 31 shows a comparison of the in vitro efficacy of the anti-HER glycoconjugate and its corresponding thiol conjugate. Cell viability was determined after exposing the conjugate to Her2 antigen-expressing (SK-BR-3) cells (Figures 31A and C), or non-expressing (MDA-MB-231) cells (Figures 31 and D) for 72 hours. The ADCs either had MMAE or PEG8-Dol119 linked to the glycan (the "glyco"), or contained a cysteine in the hinge region (the "thiol") by a conventional chemical reaction. As shown in Figures 31A and C, an approximately 2-fold lower EC50 was observed for the thiol conjugate compared to the glycoconjugate, which is consistent with the DAR being 2-fold higher in the former than in the latter. No toxicity was observed in the Her2 cell line with any antibody up to 100 μg / ml.

[0252] A similar trend was also observed for cell proliferation of ADCs prepared with antibodies against the tumor antigen (FAP), which is highly expressed by reactive stromal fibroblasts in epithelial cancers, including colon cancer, pancreatic cancer, and breast cancer (Teicher, B.A. (2009), Antibody-drug conjugate targets., Curr Cancer Drug Targets, 9, 982-1004). These conjugates were also prepared by conjugating either an aminooxy MMAE drug-linker or a maleimide MMAE drug-linker to the glycan or the thiol group. The cell proliferation assays of these conjugates showed that the EC 50has approximately 100-fold higher potency against CHO cells transfected with human FAP than the same cells lacking FAP expression, as shown in Figure 32, which shows a comparison of the in vitro potencies of the anti-FAP B11 glycoconjugate versus the thiol conjugate. Cell viability was determined after exposure of the conjugate to CHO cells transfected with or without FAP antigen. The ADCs contained MMAE conjugated to the glycan ("glyco") or MMAE conjugated to the cysteine in the hinge region by a conventional chemical component ("thiol"). The approximately 2-fold lower EC50 for the thiol compared to the glycoconjugate is consistent with the relative amount of drug delivered per antibody, assuming similar efficiencies for target binding and internalization in antigen-expressing CHO cells. In parallel, as described previously, the glycoconjugate of the anti-FAP (B11) ADC with a DAR of 1.5 was assayed and an EC 50 was shown that was approximately 2-fold higher (DAR 3.3).

[0253] As shown in Figure 36, a similar trend was observed in cell proliferation assays for ADCs prepared with the A114N hyperglycosylated variant and AO-MMAE anti-HER antibody described in Example 14 against SK-BR-3 expressing cells or MDA-MB-231 cells. The A114N glycoconjugate clearly shows enhanced cytotoxicity against Her2-expressing cell lines across non-expressing lineages. The relative toxicity compared to the SialT glycoconjugate prepared with the same antibody is consistent with the lower drug loading of this preparation. Cell proliferation assays were also performed on ADCs prepared with the A114N hyperglycosylated variant and AO-MMAE anti-TEM1 antibody prepared as described in Example 14. Higher toxicity was observed in the TEM1-expressing cell lines SJSA-1 and A673 compared to the non-expressing MDA-MB-231 line. The level of toxicity compared to the conventional thiol conjugate with the same antibody was consistent with the drug loading (DAR) of this preparation.

[0254] Cell proliferation assays were also performed on ADCs prepared with the A114N hyperglycosylated variant and AO-MMAE anti-TEM1 antibody prepared as described in Example 14. Higher toxicity was observed in the TEM1-expressing cell lines SJSA-1 and A673 compared to the non-expressing MDA-MB-231 line. The level of toxicity compared to the conventional thiol conjugate with the same antibody was consistent with the drug loading (DAR) of this preparation.

[0255]

Table 22

[0256] In summary, site-specific conjugation of drugs with glycans having cleavable linkers generates in vitro efficacy comparable to that of toxic ADCs and conventional thiol-based conjugates, as demonstrated using various antibodies and various drug-linkers. Furthermore, at periodate concentrations below 2 mM, the level of drug conjugate correlates with the decrease in sialic acid. As expected from the complete conversion of sialic acid to its oxidized form, increasing the periodate concentration above 2 mM provides little advantage. However, under all conditions, the number of drugs per antibody is slightly less than the sialic acid content, and it is pointed out that some of the oxidized sialic acid may not be available for coupling either by being buried or by steric hindrance caused by the bulk of the drug-linker at other points.

Example

[0257] In Vivo Characterization of Antibody-Drug Conjugates The efficacy of the anti-HER glycoconjugate was also evaluated in a Her2+ tumor cell xenograft model and compared to a comparator of a thiol conjugate having an approximately two-fold higher DAR. SK-OV-3 Her2+ tumor cells were implanted into Beige / SCID mice, and after establishing tumors of approximately 150 mm 3 , treatment was initiated. ADCs at 3 or 10 mg / kg doses were injected via the tail vein on days 38, 45, 52, and 59. There were approximately 10 mice per group. Tumor volumes of mice in different groups were measured and their survival was recorded. Survival curves were plotted based on the Kaplan-Meier method.

[0258] Figure 33 shows the comparison of the in vivo efficacy of anti-HER glycoconjugates and thiol conjugates in a Her2+ tumor cell xenograft model. Beige / SCID mice implanted with SK-OV-3 Her2+ tumor cells were administered MMAE (Figures 33A and B) and PEG8-Dol10 (Figures 33C and D), including a comparison control of a glycoconjugate or thiol conjugate containing approximately twice the DAR. The kinetics of tumor growth of the MMAE conjugate are shown in Figure 33A. In this case, the glycoconjugate showed significantly higher efficacy than the naked antibody alone (black), but lower efficacy than the comparison control of the thiol conjugate with approximately twice the DAR (green). The MMAE glycoconjugate showed significant tumor regression and a delay of approximately 20 days in tumor growth (Figure 33A), and showed an approximately two-fold increase in survival time from the first dose and showed an approximately two-fold increase during the survival time from the first dose (Figure 33B). The thiol MMAE conjugate showed almost complete tumor suppression at the same dose of ADC (10 mg / kg).

[0259] The in vivo efficacy of the PEG8-Dol10 glycoconjugate ("glycoDol10") and a comparison control of a thiol conjugate with approximately twice the DAR ("thiolDol10") was also determined in the same Her2+ tumor cell xenograft model. Both conjugates showed lower efficacy than the MMAE conjugate described above. However, the 10 mg / kg aminooxy-PEG8-Dol10 glycoconjugate ("glycoDol10") showed a 15-day delay in tumor growth (Figure 33C) and showed an increase of approximately 20 days (1.7-fold) in survival time after the first administration (Figure 33D). The thiol conjugate was more effective at the same dose and showed an approximately two-fold increase in survival. At a low dose (3 mg / kg), the thiol conjugate showed lower efficacy than the 10 mg / kg glycoconjugate. This dose corresponds to a dose of 80 μmol of PEG8-Dol10 drug per kg, compared to a dose of 110 μmol of PEG8-Dol10 drug per kg for the glycoconjugate.

[0260] These data demonstrate that site-specific conjugation of drugs to the sialic acid of the glycans of antibodies produces molecules with potency comparable to that of ADCs produced by thiol-based chemical reactions. The somewhat lower in vivo efficacy may be due to the internalization of the antigen bound to each antibody, resulting in fewer drugs being delivered into tumor cells by each antibody. Although the inventors have not compared these glycoconjugates to thiol conjugates with the same DAR, the efficacy observed at various doses of two ADCs, where the administered drugs are at comparable levels, indicates that the glycoconjugates have an intrinsic efficacy comparable to that of their thiol counterparts, pointing to no deleterious effects of this site of conjugation. Furthermore, a 10 mg / kg dose of the Dol10 glycoconjugate, which introduces only slightly more (28%) drug, results in a two-fold increase in survival over the thiol conjugate (3 mg / kg), suggesting that these conjugates can result in excellent efficacy even with the same DAR. Higher drug contents can be achieved by numerous different strategies, including the use of branched drug linkers or the introduction of additional glycosylation sites, and by the same method, when there are obvious limitations in the incorporation of natural glycan sialic acid.

Claims

1. At least one compound of formula (IV): -Gal-Sia-C(H)=N-Q-CON-X Formula (IV) [In the formula, A) Q is NH or O; B) CON is a connecting portion; C) X is an effector moiety; D) Gal is a galactose-derived component; E) Sia is a component derived from sialic acid; Sia is present or absent. A binding polypeptide comprising at least one modified glycan, comprising a moiety.

2. The binding polypeptide of claim 1 , wherein the modified glycan is a biantennary glycan.

3. 3. The binding polypeptide of claim 1 or 2, wherein the biantennary glycan is fucosylated or non-fucosylated.

4. The binding polypeptide of any one of claims 1 to 3, wherein the modified glycan comprises at least two moieties of formula (IV) and Sia is present in only one of the two moieties.

5. The binding polypeptide of any one of claims 1 to 3, wherein the modified glycan comprises at least two moieties of formula (IV), and Sia is present in both of the two moieties.

6. The binding polypeptide of any one of claims 1 to 5, wherein the modified glycan is N-linked to the binding polypeptide.

7. The binding polypeptide of any one of claims 1 to 6, wherein the binding polypeptide comprises an Fc domain.

8. 8. The binding polypeptide of claim 7, wherein the modified glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 297 of the Fc domain according to EU numbering.

9. 9. The binding polypeptide of claim 8, wherein the modified glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 298 of the Fc domain according to EU numbering.

10. The binding polypeptide of any one of claims 1 to 9, wherein the Fc domain is human.

11. A binding polypeptide according to any one of claims 1 to 10, wherein the binding polypeptide comprises a CH1 domain.

12. 12. The binding polypeptide of claim 11, wherein the modified glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 114 of the CH1 domain according to the Kabat numbering system.

13. The binding polypeptide of any one of claims 1 to 12, wherein the effector moiety is a cytotoxin. peptide.

14. 14. The binding polypeptide of claim 13, wherein the cytotoxin is selected from the group consisting of the cytotoxins listed in Table 1.

15. The binding polypeptide of any one of claims 1 to 13, wherein the effector moiety is a detection agent.

16. The binding polypeptide of any one of claims 1 to 13, wherein the effector moiety is a targeting moiety.

17. The binding polypeptide of claim 16 , wherein the targeting moiety is a carbohydrate or a glycopeptide.

18. 17. The binding polypeptide of claim 16, wherein the targeting moiety is a glycan.

19. The binding polypeptide of any one of claims 1 to 18, wherein the connecting moiety comprises a pH-sensitive linker, a disulfide linker, an enzyme-sensitive linker, or other cleavable linker moiety.

20. 20. The binding polypeptide of any one of claims 1 to 19, wherein the connecting moiety comprises a linker moiety selected from the group of linker moieties shown in Tables 2 or 14.

21. 21. The binding polypeptide of any one of claims 1 to 20 which is an antibody or an immunoadhesin.

22. A composition comprising a binding polypeptide according to any one of claims 1 to 21 and a pharma- ceutically acceptable carrier or excipient.

23. 23. The composition of claim 22, wherein the ratio of therapeutic or diagnostic effector moiety to binding polypeptide is less than 4.

24. 24. The composition of claim 23, wherein the ratio of therapeutic or diagnostic effector moiety to binding polypeptide is about 2.

25. 25. A method of treating a patient in need thereof comprising administering an effective amount of the composition of claim 24.

26. An isolated polynucleotide encoding a binding polypeptide according to any one of claims 1 to 21.

27. A vector comprising the polynucleotide of claim 26.

28. 28. A host cell comprising a polynucleotide or vector according to claim 26 or 27.

29. A method for producing a binding polypeptide according to any one of claims 1 to 28, comprising the step of: NH ２ -Q-CON-X Formula (I) [In the formula, A) Q is NH or O; B) CON is a connecting portion; C) X is an effector moiety. reacting the effector moiety of with an altered binding polypeptide comprising an oxidized glycan.

30. 30. The method of claim 29, wherein the modified binding polypeptide comprising an oxidized glycan is produced by reacting a binding polypeptide comprising the glycan with a mild oxidizing agent.

31. 31. The method of claim 30, wherein the mild oxidizing agent is sodium periodate.

32. 32. The method of claim 31 , wherein less than 1 mM sodium periodate is used.

33. 31. The method of claim 30, wherein the oxidizing agent is galactose oxidase.

34. The method of any one of claims 29 to 33, wherein the glycan-containing binding polypeptide comprises one or two terminal sialic acid residues.

35. 35. The method of claim 34, wherein the terminal sialic acid residues are introduced by treating the binding polypeptide with a sialyltransferase or a combination of a sialyltransferase and a galactosyltransferase.

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