WO2013087789A1

WO2013087789A1 - Antibody isoform arrays and methods thereof

Info

Publication number: WO2013087789A1
Application number: PCT/EP2012/075434
Authority: WO
Inventors: Tero Satomaa; Juhani Saarinen; Heidi Virtanen
Original assignee: Glykos Finland Ltd.
Priority date: 2011-12-13
Filing date: 2012-12-13
Publication date: 2013-06-20

Abstract

Aspects of the present invention describe methodology by which arrays of antibody isoforms can be created and employed for providing antibody isoforms with altered binding to effector molecules. The invention simplifies screening of various antibody isoforms by binding of effector molecules such as Fc receptors.

Description

Antibody isoform arrays and methods thereof

SUMMARY OF INVENTION

Microarrays have been extremely useful for investigating binding interactions among diverse types of molecular species, with the main advantage being the ability to examine many interactions using small amounts of samples and reagents. Microarrays are increasingly being used to advance research in the field of molecular biology. Several types of microarrays are being used in the study of antibodies, antigens, screening of biomarkers, etc.

The present invention provides methods of providing an antibody isoform with altered binding to an effector molecule compared with a reference antibody, the method comprising: (a) providing an array of a reference antibody and antibody isoforms of said reference antibody having a plurality of structures; (b) contacting said array with effector molecules; and (c) assessing binding of effector molecules to said array, whereby one or more antibody isoforms with altered effector molecule binding compared with the reference antibody are obtained.

The present invention describes a multiplexed approach to identify and characterize antibody- protein (effector molecule) interactions that are specific to or modulated by certain antibody isoforms. Previously such assays have been laborious, suffered from inter-sample and inter- assay variability causing inaccuracies and consumed lots of sample and materials. The present invention describes a microarray-based resolution to obtain the antibody-effector molecule (protein) interaction data, which is faster, more efficient, more accurate and reproducible, consuming many times less materials and allowing many times more experiments to be performed simultaneously in identical conditions.

An aspect of the invention provides a method of providing an antibody isoform with altered binding to an effector molecule compared with a reference antibody, the method comprising: (a) providing an array of a reference antibody and antibody isoforms of said reference antibody; (b) contacting said array with effector molecules; and (c) assessing binding of effector molecules to said array, whereby one or more antibody isoforms with altered effector molecule binding compared with the reference antibody are obtained. In an aspect of the invention said isoform is a glycoform.

In an aspect of the invention effector molecules are selected from the group consisting of Fc receptor, carbohydrate binding protein, antibody binding protein, and complement factor.

In an aspect of the invention Fc receptor is selected from the group consisting of FcyRI family, FcyRII family, FcyRIII family, FcaR, FcsR, FcμR, Fc5R, and FcRn.

In an aspect of the invention carbohydrate binding protein is selected from the group consisting of lectins, DC-SIGN, SIGN-Rl, macrophage mannose receptor, mannose binding protein, asialoglycoprotein receptor, and an antibody against carbohydrate structure of an antibody.

In an aspect of the invention antibody binding protein is selected from the group consisting of protein A, protein G, rheumatoid factor, an HAMA (Human Anti-Mouse Antibody) protein and an HAHA (Human Anti-Human Antibody) protein.

In an aspect of the invention complement factor is selected from the group consisting of complement factor Clq and complement factor C3b.

In an aspect of the invention the effector molecule is labeled.

In an aspect of the invention the label is a fluorochrome.

In an aspect of the invention binding is assessed by determining k_D value.

In an aspect of the invention array comprises between about 2 and 10 distinct antibody isoforms.

In an aspect of the invention the array is a microscope slide, plate, a chip, or a population of beads.

In an aspect of the invention antibodies are cross-linked to said array.

An aspect of the invention provides an antibody isoform obtained using the method of the invention.

An aspect of the invention provides an antibody isoform array. FIGURE LEGENDS

Figure 1. A schematic diagram indicating an array and assigning of codes for reference antibodies, antibody isoform(s) and control protein(s). "Rl" is assigned to first reference antibody, "R2" to second reference antibody, and finally "Rx" to the last reference antibody. "Rill" is assigned to the first antibody isoform (of reference antibody Rl), "R1I2" is assigned to the second antibody isoform (of reference antibody Rl), and "Rlln" is assigned to the last antibody isoform (of reference antibody Rl). "R2I1" is assigned to the first antibody isoform (of reference antibody R2), "R2I2" is assigned to the second antibody isoform (of reference antibody R2), and "R2In" is assigned to the last antibody isoform (of reference antibody R2). "Rxll" is assigned to the first antibody isoform (of reference antibody Rx), "RxI2" is assigned to the second antibody isoform (of reference antibody Rx), and "Rxln" is assigned to the last antibody isoform (of reference antibody Rx). The array may, optionally, comprise control protein(s) "CI", "C2", "Cy". On array there may be several parallel "spots" of Rl, and parallel "spots" of R2, etc. On array there may be several parallel "spots" of R1I1, parallel "spots" of R1I2, etc. On array there may be several parallel "spots" of Rxll, parallel "spots" of Rxln, etc. On array there may be several parallel "spots" of CI, parallel "spots" of C2, etc.

DETAILED DESCRIPTION OF THE INVENTION

The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as V_H or VH) and a heavy chain constant region. The heavy chain constant region typically is comprised of three domains, C_HI, C_H2, and C_H3- Each light chain typically is comprised of a light chain variable region (abbreviated herein as V_L or VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, C_L. The V_H and V_L regions may be further subdivided into regions of complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each V_H and V_L is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (phrases such as variable domain residue numbering as in Kabat or according to Kabat herein refer to this numbering system for heavy chain variable domains or light chain variable domains). Using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence. Immunoglobulin heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. An antibody Fc domain may be the Fc domain of an IgA, IgM, IgE, IgD or IgG antibody or a variant thereof. In certain embodiments, the domain is an IgG antibody Fc domain such as an IgGl, IgG2a, IgG2b, IgG3 or IgG4 antibody Fc domain.

For example, in the bevacizumab (Avastin ® ) light chain sequence:

DIOMTOSPSSLSASVGDRVTITCSASODISNYLNWYOOKPGKAPKVLIYFTSSLHSGVPS RFSGSGSGTDFTLTISSLOPEDFATYYCOOYSTVPWTFGOGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC the CDRs are underlined and correspond to amino acids 24-34 (CDR1), amino acids 50-56 (CDR2) and amino acids 89-98 (CDR3). Regions between CDRs are the framework regions.

In the heavy chain of bevacizumab the CDRs are the following (underlined):

EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTY AADFKRRFTFSLDTSKSTAYLOMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGOGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

The term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226 to the carboxyl-terminus thereof. The Fc region of an

immunoglobulin generally comprises two constant domains, CH2 and CH3. The "CH2 domain" of a human IgG Fc region usually extends from about amino acid 231 to about amino acid 340. The "CH3 domain" of a human IgG Fc region usually extends from about amino acid 341 to about amino acid residue 447 of a human IgG (i.e. comprises the residues C-terminal to a CH2 domain).

The term "antibody" (Ab) in the context of the present invention refers to an

immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq, the first component in the classical pathway of complement activation. Antibodies may also be bispecific antibodies, diabodies, or similar molecules. The term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that retain the ability to specifically bind to the antigen. It has been shown that the antigen- binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include (i) a Fab' or Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L and C_HI domains; (ii) F(ab')₂ fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the V_H and C_HI domains; (iv) a Fv fragment consisting essentially of the V_L and V_H domains of a single arm of an antibody, (v) camelid or nanobodies, or (vi) single chain antibodies or single chain Fv (scFv). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype. A "reference antibody" is an antibody comprising amino acid sequences which lacks one or more of the antibody modifications disclosed herein (amino acid insertions, substitution, and/or deletions and glycoforms) and which differs in effector molecule binding compared to the antibody isoforms of the reference antibody as herein disclosed. The reference antibody and (its) antibody isoforms bind with the same or similar affinity to the corresponding antigen.

For example, commercially obtained bevacizumab antibody and its most prevalent glycoform on an array according to the present invention may be a reference antibody (assigned as "Rl", see Figure 1) and bevacizumab isoforms, preferably bevacizumab glycoforms, may include non-limiting examples such as Man5, Man6, GO, etc. (assigned as "Rill", "R1I2", ... , "Rlln", see Figure 1). A second reference antibody may be assigned as "R2" (see Fig. 1) and its isoforms from "R2I1" to "R2In". The last reference antibody on the array is assigned as "Rx" and its isoforms are assigned as from "Rxll" to "Rxln". "n" and "x" may be any number 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1000, 10000, 100000 or more, or any meaningful number between.

In the present invention antibody isoforms also encompass IgG subclasses or isotypes, for example, on an array IgGl may be assigned as a reference antibody and IgG2, IgG3, and IgG4 may be considered to be antibody isoforms of the reference antibody IgGl. Antibody isoforms also encompass naturally occurring antibody and immunoglobulin variants.

An "antibody isoform" as used herein comprises amino acid sequences which differs from that of the reference antibody by virtue of one or more than one amino acid

alterations/modifications or is a different glycoform compared to the reference antibody. In some embodiments amino acid alterations/modifications are directed to non-CDR regions, preferably to Fc region.

In certain embodiments, the antibody isoform has more than one amino acid substitution compared to a reference antibody, e.g. from about two to about ten amino acid substitutions, and preferably from about two, three or four to about five amino acid substitutions, in an amino acid sequence of the reference antibody. Native antibodies contain carbohydrate at conserved positions in the constant region of the heavy chain. Each antibody has a distinct variety of N-linked carbohydrate structures. The Fc region of IgGl has typically a single N-linked biantennary carbohydrate at Asn297 of the CH2 domain. The term "glycoform" or "antibody glycoform" as used herein refers to an isoform of an antibody that differs with respect to the type of the attached glycan compared to the reference antibody. Glycoform purity as used herein refers to proportion of the structures sharing the common structural feature defining the glycoform of total glycan structures. A glycoform may comprise of a number of glycan structures, with substantial amount, majority, or nearly all of the structures being a single glycan structure. A glycoform may also comprise of a single glycan structure. Different glycoforms may result from differences in biosynthesis during the process of glycosylation, or due to the action of glycosidases or

glycosyltransferases. Glycoforms may be detected through structural analysis or through differential binding of glycoform-specific reagents.

The carbohydrate moieties of the present invention will be described with reference to commonly used nomenclature for the description of oligosaccharides. Carbohydrate nomenclature in this context is essentially according to recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (e.g. Carbohydrate Res. 1998, 312, 167;

Carbohydrate Res. 1997, 297, 1; Eur. J. Biochem. 1998, 257, 293). This nomenclature includes, for instance, Man, which represents D-mannose; GlcNAc, which represents N- acetyl-D-glucosamine (2-acetamido-2-deoxy-D-glucose); Gal which represents D-galactose; Fuc for L-fucose; and Glc, which represents D-glucose. Sialic acids are described by the shorthand notations NeuNAc and Neu5Ac, for 5-N-acetylneuraminic acid, and NeuNGc and Neu5Gc for 5-N-glycolylneuraminic acid. All monosaccharides are in pyranose form unless otherwise indicated.

Typical antibody glycoforms, i.e. antibody isoforms when compared to "a reference antibody", include but are not limited to high-mannose glycoform (characterized by majority of glycans being high-mannose type N-glycans), afucosylated glycoform (characterized by substantially reduced amounts of or nearly absent or totally absent N-glycan core al,6- fucosylation), bisecting glycoform (characterized by majority of glycans carrying "bisecting" pi,4-linked GlcNAc in the N-glycan core β 1,4- linked Man residue), GO glycoform

(characterized by majority of glycans being biantennary N-glycans having two non-reducing end pi,2-linked GlcNAc residues), Gl glycoform (characterized by majority of glycans being biantennary N-glycans having one non-reducing end pi,2-linked GlcNAc residue and one galactosylated pi,2-linked GlcNAc residue) and G2 glycoform (characterized by majority of glycans being biantennary N-glycans having two galactosylated pi,2-linked GlcNAc residues); and FGO, FGl and FG2 glycoforms that are N-glycan core al,6-fucosylated forms of the above glycoforms GO, Gl and G2, respectively.

As used herein, the term "effector molecule" refers to a protein capable of binding an antibody. In the present invention effector molecules comprise of Fc receptors, carbohydrate binding proteins, antibody binding proteins, and complement factors.

Some effector molecules may induce an immune response in vivo. Exemplary immune responses include an activation of cell of a myeloid or lymphoid origin, for instance lymphocytes (such as B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some of immune responses are induced by specific Fc receptors (FcR). In some embodiments, effector molecules, or FcRs, are capable of inducing antibody-dependent cellular cytotoxicity (ADCC) in vitro or in vivo, via natural killer cells. For example, monocytes, macrophages, which express FcR are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. Other effector molecule induce events include phagocytosis and down regulation of cell surface receptors (e.g. B cell receptor).

An FcR may have specificity for a particular type or subtype of Ig, such as IgA, IgM, IgE or IgG (e.g., IgGl, IgG2a, IgG2b, IgG3 or IgG4). The FcR polypeptide may comprise an eukaryotic, prokaryotic, or synthetic FcR domain. For example, an FcR may be Fc-gamma Rll-a (CD32; human protein encoded by FCGR2A), Fc-gamma Rll-b (human protein encoded by FCGR2B), Fc-gamma RII-c (human protein encoded by FCGR2C), Fc-gamma RHIa (human protein encoded by FCGR3A), Fc-gamma RHIb (human protein encoded by FCGR3B), Fc-gamma RI (human protein encoded by FCGR1A; CD64) and FcRn (human protein encoded by FCGRT), Fc-epsilon RI IgE receptor and Fc-alpha RI IgA (CD86).

FcyRs are part of the immunoglobulin gene superfamily. They are expressed constitutively on haemopoeitic cells such as lymphocytes, macrophages, eosinophils, neutrophils and natural killer cells and can be differentially up-regulated when the cells are exposed to activating agents such as cytokines. They have an IgG binding cc-chain with an extracellular portion composed of either two (FcyRII and FcyRIII) or three (FcyRI) Ig-like domains. FcyRI and FcyRIII also have accessory proteins (gamma and zeta respectively) associated with the alpha- chain that function in signal transduction. FcyRs have specificity for the Fc region of the gamma heavy chain of IgG.

The three types of FcyR: FcyRI(CD64), II(CD32) and III(CD16) can be further classified into different forms: FcyRIa, lb, Ic, Ila, Ilbl, IIb2, lie, Ilia and Illb. It is the C_H2 domains of the antibody, including the lower hinge region, that contain the sequences responsible for difference in binding to FcyRs. Stimulation of these receptors may result in the activation of effector functions such as ADCC, phagocytosis, superoxide burst and release of inflammatory mediators. Effector molecules also include polymorphs of FcRs, one relevant polymorphism with clinical significance being V158/F158 FcyRIIIa. Human IgGl binds with greater affinity to the V158 allotype than to the F158 allotype.

Human FcyRIIIa is expressed on leucocytes that include Natural Killer (NK) cells, where it can mediate ADCC in response to target cells that have been sensitised with IgG antibody. Whilst the affinity of the receptor for the Fc region of monomeric IgG is only in the μΜ range, complexed IgG is able to bind multiple receptors with high avidity, and subsequently to trigger ADCC. The importance of the receptor for therapy with mAbs is illustrated by the treatment of follicular non-Hodgkin's lymphoma using rituximab. In this case patient survival is correlated with the allotype of FcyRIIIa, such that possession of the V158 allotype is preferable to the F158 allotype. Rituximab mediates ADCC more effectively via the V158 allotype than the F158 allotype.

It is specifically contemplated that an FcR is a native sequence human FcR. In particular, an FcR is one which binds an IgG antibody (a gamma receptor; FcyR) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. In the present invention FcR lack intracellular and transmembrane domains, i.e. FcR are soluble. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus. FcR also encompass an FcRn that regulates the serum half-lives of IgGs. Enhancement or diminishment of the half-life of the Fc (or Fc-containing polypeptide) is reflected, respectively, in the increase or decrease of the Fc region affinity for FcRn (neonatal Fc receptor). The correlation of FcRn binding affinity and serum half-life is consistent with its proposed biological role in salvaging an antibody from lysosomal degradation. In addition, FcRn transfers IgGs from mother to fetus.

Other effector molecules of the present invention include molecules part of complement dependent cytotoxicity (CDC) pathway or "complement factor" or "complement". Clq and two serine proteases, Clr and Cls, form the complex CI, the first component of the CDC pathway. To activate the complement cascade, it is necessary for Clq to bind to at least two molecules of IgGl, IgG2 or IgG3 but only one molecule of IgM. Clq and two serine proteases, Clr and Cls, form the complex CI, the first component of the complement dependent cytotoxicity (CDC) pathway. Clq is a hexavalent molecule with a molecular weight of approximately 460,000 and a structure likened to a bouquet of tulips in which six collagenous "stalks" are connected to six globular head regions. To activate the complement cascade, it is necessary for Clq to bind to at least two molecules of IgGl, IgG2, or IgG3 (the consensus is that IgG4 does not activate complement), but only one molecule of IgM, attached to the antigenic target.

As used herein, the term "carbohydrate binding protein" refers to a protein which binds carbohydrate molecules or glycans. Typically, carbohydrate binding protein of the invention include but are not limited to lectins, DC-SIGN, SIGN-R1, macrophage mannose receptor, mannose binding protein, mannose binding lectin, asialoglycoprotein receptor, and an antibody against carbohydrate structure of an antibody.

In some embodiments carbohydrate binding protein is a dendritic cell specific ICAM-3 grabbing nonintegrin (DC-SIGN) protein that is expressed on the surface of dendritic cells (DC) in the periphery and participates in the primary contact between the antigen-presenting cells and resting T-cells in the lymphatic system via ICAM-3 on the T-cells. DC-Sign also interacts with ICAM-2 on epithelial cells during migration of DCs to lymphoid tissues. DC- Sign also binds strongly to the HIV envelope protein gpl20 and facilitates viral infection in trans of CD4+ T-cells. The DC-Sign protein consists of a short amino-terminal cytoplasmic tail, a transmembrane domain, a stalk of up to 71/2 repeats, followed by a C-terminal C-type carbohydrate recognition domain. The stalk promotes the formation of tetramers (coiled coil). In the present invention, binding affinity of DC-Sign to gpl20 protein may be used as a "control" to compare binding affinities of antibody isoforms to reference antibody on an array (a control protein may be assigned as "CI", see Figure 1). For example, gpl20 may be attached to an array and when the array is hybridized with DC-Sign proteins, the binding affinity of gpl20 to DC-Sign may be compared to the binding affinity of a reference antibody to an antibody isoform. In particular, this is preferable when an antibody isoform comprises high mannose structures. In general, "control" proteins on an array may be assigned as "CI", "C2", ... , "Cy". "y" may be any number 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, 200, 500, 1000, 10000, 100000 or more, or any meaningful number between. In a preferred

embodiment, number of control proteins is about the same as the number of reference antibodies. In an embodiment, number of control proteins is less than the number of reference antibodies. In an embodiment, number of control proteins is more than the number of reference antibodies. Any protein, which bind an effector molecule, can serve as "control" protein.

DC-Sign binds to high mannose structures (Man9) and may be used to determine binding of DC-Sign to antibody glycoforms comprising Man9 or closely related carbohydrate or glycan structures. Another carbohydrate or glycan structure to which DC-Sign binds comprises blood group-related Lewis tumor antigens (e.g. Lex and Ley). Lewis antigens are complex oligosaccharides and are both found as glycolipids, embedded in the cell membrane and linked to cell surface proteins (e.g. HER1, HER2 and CEA) with only limited expression on normal tissue. Lewis antigens have been shown to mediate dendritic cell adhesion and tumor cell infiltration. Lewis Y interacts with the DC-SIGN receptor on dendritic cells to escape immune surveillance by promoting immune suppression. Thus, Lewis antigens provide a non- limiting example of a carbohydrate structure to which a carbohydrate binding protein may bind or to which an antibody against carbohydrate structure of an antibody may bind.

Mannose-binding lectin (MBL), also called mannose binding protein (MBP), is a calcium- dependent serum protein that plays a role in the innate immune response by binding to carbohydrates on the surface of a wide range of pathogens (viruses, bacteria, fungi, protozoa) where it can activate the complement system. MBL serves also as a direct opsonin and mediates binding and uptake of pathogens by tagging the surface of a pathogen to facilitate recognition and ingestion by phagocytes. The mannose binding lectin (MBL) binds mannose containing oligosaccharides.

Mannose-binding lectin is a member of the collectin family of proteins, which are made in the liver. Collectins get their name because they have a collagen-like region and a lectin region. The MBL2 gene on human chromosome 10 produces MBL, an oligomer of 248-amino acid protein subunits composed of three identical polypeptide chains comprising a N-terminal cysteine rich region, a collagen-like region, a neck region, and a carbohydrate recognition domain. Three MBL polypeptide chains assemble into a biologically active trimer found in vivo.

When serum MBL interacts with carbohydrates on the surface of microorganisms, it forms the pathogen recognition component of the lectin pathway of complement activation. MBL binds to surface arrays containing repeated mannose or N-acetylglucosamine residues. It circulates as a complex with one or more MBP-associated serine proteases (MASPs) that autoactivate when the complex binds to an appropriate surface.

For example, effector molecule of the invention mannose binding protein (MBP) activates the complement system, which is a set of plasma proteins that work together to attack

extracellular pathogens. While the most important role of the complement system is opsonization (coating foreign organisms with a receptor recognized by phagocytes), it also recruits inflammatory cells and kills pathogens directly through membrane attack complexes. In mammals, activating or "fixing" complement generally means that MBP binds to the serum proteins CI, C2, C3, C4, C5, C6, C7, C8, and C9, collectively called "complement," and thereby stimulate the binding of macrophages to the protein and facilitate subsequent ingestion by those macrophages.

Other carbohydrate binding proteins of the present invention include the following non- limiting examples: tetranectin, lithostatin, mouse macrophage galactose lectin, Kupffer cell receptor, chicken neurocan, perlucin, asialoglycoprotein receptor, Endol80, cartilage proteoglycan core protein, IgE Fc receptor, pancreatitis-associated protein, mouse

macrophage receptor, macrophage mannose receptor (MMR; CD206), Natural Killer group, stem cell growth factor, factor ΓΧ/Χ binding protein, mannose binding protein, bovine conglutinin, bovine CL43, collectin liver 1, surfactant protein A, surfactant protein D, e- selectin, tunicate c-type lectin, CD94 NK receptor domain, LY49A NK receptor domain, chicken hepatic lectin, trout c-type lectin, HIV gp 120-binding c-type lectin, and dendritic cell immunoreceptor.

Both the MMR and DC-SIGN have the capacity to direct internalized antigens into endocytic pathways that result in MHC presentation and subsequent T cell activation. MMR

preferentially binds terminal mannoses, DC-SIGN has suggested selectivity towards high mannose N-glycans.

As used herein, the term "antibody against carbohydrate structure of an antibody" refers to an antibody which specifically binds to a carbohydrate moiety/structure or a glycan of an antibody. Typically, any antibody which can bind a antigen comprising a glycan or a carbohydrate moiety may be an "antibody against carbohydrate structure of an antibody" according to present invention. For example, a non-limiting antibody against carbohydrate structure of an antibody may be an anti-Neu5Gc antibody.

As used herein, the term "antibody binding protein" refers to a protein which binds antibody. Antibody binding proteins of the invention include but are not limited to human anti-mouse antibodies (HAMA), human anti-human antibodies (HAHA), protein A, protein G, and rheumatoid factor.

"Rheumatoid factor" or "RF" refers to an autoantibody that binds to the Fc region of IgG antibodies. Rheumatoid factor (RF) is a term used to describe a group of autoantibodies known as rheumatoid factors. RF is considered an early marker of rheumatoid arthritis since its presence is associated with an increased risk of developing RA in people with mild arthritic symptoms.

RF factors include three subclasses that react with the crystallizable fragment (Fc fragment) of immunoglobulin G (IgG). RF targets IgG proteins by combining with them to form deposits that lodge in the joints and tissues. The three subclasses of RF include IgM, IgA and IgG autoantibodies. In some embodiments, effector molecules such as RF are contacted with a reference antibody and antibody isoforms. The reference antibody and antibody isoforms are preferably at least one of human IgM, IgG or IgA that is immobilized onto an array.

The contacting step is performed under conditions to allow for binding of the effector molecule such as RF to the immobilized reference antibody and antibody isoform. The presence of the captured or bind RF is then detected and binding is compared between the reference antibody and antibody isoform.

For example, additional antibody binding proteins include the following non-limiting examples MRP (mrp4) Fibrinogen- and Ig-binding protein precursor of Streptococcus pyogenes (Stenberg et al., 1992); Protein B cAMP factor Streptococcus agalactiae (Ruhlmann et al., 1988); protein A (encoded by spa gene); protein G (encoded by spg gene); protein H; Protein sbi (sbi) (Zhang et al., 1998); Allergen Asp fl 1 (Aspergillus flavus) which binds to IgE and IgG; Allergen Asp fl 2 which binds to IgE and IgG; and Allergen Asp fl 3 which binds to IgE and IgG.

Additional antibody binding proteins include HAMA proteins (human anti-murine antibody). Generally, there is a risk associated with the addition of a mouse-based antibody to a human, resulting in the human patient launching an immune response against the mouse-based antibody. This immune response has also been termed a HAMA response. In the present invention an effector molecule comprises an antibody binding protein such as HAMA protein. In the present invention an antibody provoking a HAMA response is referred as HAMA protein.

A human therapeutic antibody can elicit a human anti-human antibody (HAHA) response. In the present invention an antibody provoking a HAHA response is referred as HAHA protein. This response can limit the antibodies' efficacy and can negatively affect their safety profile in the worst-case scenario. As an example, the fully human phage display-derived anti-TNF antibody HUMIRA® (Abbott Laboratories) unexpectedly provokes a HAHA response in approximately 12% of patients on monotherapy and about 5% in combination therapy with the methotrexate. In an embodiment, an effector molecule comprises an antibody binding protein such as a HAHA protein. In the present invention, the term "antibody binding protein" is considered not to encompass proteins or polypeptides which are referred to "carbohydrate binding proteins", Fc receptors or complement.

Effector molecules

Effector molecules are molecules or portions of molecules that demonstrate an affinity for an antibody, reference antibody, antibody isoform, control protein or a fragment thereof.

Antibodies and antibody isoforms are typically on a support as described herein. Effector molecules can bind with "low affinity," as used herein, is defined as an interaction with a dissociation constant (K_D) of more than 10^"5 M, "moderate affinity" as used herein is defined as a -5 -8

K_D between 10^" M and ^" M, and "high affinity" as used herein is defined as a K_D of less than 10 -^"8 M. Effector molecules may be based on a variety of molecules or substances. In various embodiments effector molecule(s) may include, but is not limited to, an Fc receptor, a carbohydrate binding protein, an antibody binding protein, a complement factor, or a fragment thereof. For each effector molecule, a preferred concentration may be empirically determined by arraying a number of antibody, antibody isoforms, and optionally control proteins

(subdivisions of an array), which include one or more antibodies, antibody isoforms, and optionally control proteins, at varying densities and identifying an optimal concentration for an effector molecule.

Modifications to Antibody Amino Acid Sequence and Antibody Isoforms

One embodiment of the invention includes antibodies that are modified to create variation in effector molecule binding. Amino acid sequence isoforms, or variants, of the antibody are prepared by introducing appropriate nucleotide changes into the polynucleotide encoding the antibody. Alternatively, the amino acid changes can be made during peptide synthesis of the antibodies. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the polypeptide sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct. The amino acid alterations may be introduced in the subject antibody amino acid sequence at the time that sequence is made. Amino acid substitution can be achieved by well-known molecular biology techniques, such as site directed mutagenesis of the nucleic acid sequence encoding the antibody that is to be modified. Suitable techniques include those described in Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 3.sup.rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2001) and Ausubel et al., 2006, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. Other techniques for generating antibody isoforms include synthesizing a polynucleotide having a sequence that encodes a modified antibody.

In another example, amino acids of an antibody can be deleted. Amino acid deletion can be achieved by standard molecular biology techniques, such as site directed mutagenesis of the nucleic acid sequence encoding the antibody. Suitable techniques include those described in Sambrook et al. and Ausubel et al., supra. Other techniques for generating antibody with modified amino acids include synthesizing an oligonucleotide comprising a sequence that encodes a region in which the codon that encodes the amino acid that is to be modified is deleted. This oligonucleotide can then be ligated into a vector backbone comprising other appropriate antibody sequences, such as variable regions and Fc sequences, as appropriate.

Amino acid sequence insertions include carboxyl-terminal fusions ranging in length from one residue to polypeptides containing e.g. dozens of or more residues within a light or heavy chain of an antibody. Other insertions may include intrasequence insertions of single or multiple amino acid residues within a light or heavy chain. In a typical embodiment, 1, 2, 3, 4, 5 or more amino acids are inserted into the antibody sequence. The inserted amino acid may be any amino acid.

Another type of an antibody isoform is an amino acid substitution modified antibody. These isoforms have at least one amino acid residue in the antibody molecule replaced by a different residue compared to the original antibody.

Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

(1) hydrophobic: met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

Nucleic acid molecules encoding amino acid sequence isoforms of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence isoforms such as IgGl, IgG2, IgG3, and IgG4 or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non- variant version of the antibody.

In a preferred embodiment, amino acid insertions, deletions or substitutions are directed to amino acids located in non-complementarity determining regions (non-CDRs). CDR regions include CDR1, CDR2, and CDR3. In the present invention, non-CDR regions include framework regions, hinge region, light chain constant region (CL), heavy chain constant regions CHI, CH2, and CH3 or Fc region. In a preferred embodiment, amino acid insertions, deletions or substitutions are directed to amino acids located in Fc region.

Mutagenesis may be performed in accordance with any of the techniques known in the art including, but not limited to, synthesizing an oligonucleotide having one or more

modifications within the sequence of the light or heavy chain an immunoglobulin to be modified. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to about 75 nucleotides or more in length is preferred, with about 10 to about 25 or more residues on both sides of the junction of the sequence being altered. A number of such primers introducing a variety of different mutations at one or more positions may be used to generate a library of mutants or isoforms.

An antibody isoform may have improved or reduced effector molecule binding compared to a reference antibody. The antibody isoform which displays improved binding to an effector molecule binds at least one effector molecule with better affinity than the reference antibody. The antibody isoform which displays reduced binding to an effector molecule binds at least one effector molecule with worse affinity than a reference antibody. Such antibody isoform which display reduced binding to an effector molecule may possess little or no appreciable binding to an effector molecule, e.g., 0-20% binding to the effector molecule compared to a reference antibody, e.g. as determined in the Examples. The antibody isoform which displays improved binding to an effector molecule, is one which binds any one or more of the above identified effector molecules with substantially better binding affinity than the reference antibody, when the amounts of antibody isoform and reference antibody in the binding assay are essentially the same. In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to Clq compared to a reference antibody. In certain embodiments, an antibody isoform with altered binding of an effector molecule may have altered binding to one or more FcRs and altered binding to Clq compared to a reference antibody. In other embodiments, an antibody isoform with altered recognition of an effector molecule may have improved binding to some effector molecules and reduced binding to other effector molecules. In other embodiments, an antibody isoform with altered recognition of an Fc receptor may have improved binding to some Fc receptor and reduced binding to other Fc receptor.

For example, an antibody isoform with improved effector molecule binding may display from about 1.10 fold to about 100 fold, e.g. from about 1.15 fold to about 50 fold improvement in effector molecule binding affinity compared to the reference antibody, where effector molecule binding affinity is determined, e.g. as disclosed in the Examples herein.

In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to an FcR compared to a reference antibody. In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to an FcyR compared to a reference antibody. In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to carbohydrate binding protein compared to a reference antibody. In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to DC-SIGN protein compared to a reference antibody. In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to mannose binding protein compared to a reference antibody. In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to an antibody against carbohydrate structure of an antibody compared to a reference antibody. In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to antibody binding protein compared to a reference antibody. In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to rheumatoid factor compared to a reference antibody. In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to protein A compared to a reference antibody. In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to protein G compared to a reference antibody. In an alternative embodiment, an antibody isoform with altered binding of an effector molecule may have improved or reduced binding to complement factor compared to a reference antibody.

Antibody isoforms and their capabilities to induce cytotoxic effects on cells may be determined by comparing ability of selected antibody isoforms with that of a known, or control, antibody to induce cell lysis etc. in an ADCC assay or in vivo models. An antibody isoform with higher binding compared with a reference antibody is one which in vitro or in vivo is substantially more effective at mediating ADCC or CDC, when the amounts of antibody isoform and reference antibody used in the assay are essentially the same. Generally, the antibody isoforms which mediate ADCC more effectively will be identified using the in vitro ADCC assay, but other assays or methods for determining ADCC activity, e.g. in an animal model etc., are contemplated. The preferred antibody isoform is from about 1.01 fold to about 100 fold, e.g. from about 1.10 fold to about 50 fold, more effective at mediating ADCC than the reference antibody, e.g. in the in vitro assay.

It has been demonstrated that absence of fucose correlates with improved ADCC activity. Similarly, presence of a bisecting N-acetylglucosamine (GlcNAc) has also been shown to increase ADCC activity. In addition, the presence of sialic acid correlates with improved anti- inflammatory activity of IgG. Accordingly, instant invention provides a novel method for screening and identifying antibody isoforms or antibody glycoforms which have altered binding to effector molecules.

It will be understood by one of skill in the art that any animal cell which is capable of producing a glycoprotein comprising may be used to produce reference antibodies and antibody isoforms and antibody glycoforms. Such animal host cells include, but are not limited to, CEK, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, NSO, and in particular, neuronal cell lines such as, for example, SK-N-AS, SK-N-FI, SK-N-DZ human neuroblastomas, SK-N-SH human neuroblastoma, Daoy human cerebellar medulloblastoma, DBTRG-05MG glioblastoma cells, IMR-32 human neuroblastoma, 1321N1 human astrocytoma, MOG-G-CCM human astrocytoma, U87MG human glioblastoma-astrocytoma, A 172 human glioblastoma, C6 rat glioma cells, Neuro-2a mouse neuroblastoma, NB41A3 mouse neuroblastoma, SCP sheep choroid plexus, G355-5, PG-4 Cat normal astrocyte, Mpf ferret brain, and normal cell lines such as, for example, CTX TNA2 rat normal cortex brain such as, for example, CRL7030 and Hs578Bst.

In other embodiments, an antibody isoform or glycoform of the present invention comprises one or more engineered glycoforms, i.e., a carbohydrate composition that is covalently attached to a molecule comprising an Fc region. Engineered antibody glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector molecule binding and inducing immune responses, ADCC, CDC, etc. functions. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example beta(l,4)-N-acetylglucosaminyltransferase III (GnTIII), by expressing a molecule, or an antibody, comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al, 1999, Nat. Biotechnol 17: 176- 180; Davies et al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) U.S. Pat. No.

6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland). See, e.g., WO 00061739;

EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.

A convenient way of producing an antibody isoform according to an embodiment of the present invention is to express it from the nucleic acid encoding it, by use of the nucleic acid in an expression system. Accordingly, an embodiment of the present invention also encompasses a method of making an antibody isoform, the method including expression from nucleic acid encoding the polypeptide (generally nucleic acid according to an embodiment of the invention). This may conveniently be achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow expression of the polypeptide. Antibody isoforms may also be expressed in in vitro systems, such as reticulocyte lysate.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, COS cells and many others. A common, preferred bacterial host is E. coli. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. "phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons, 2006.

In certain embodiments, an antibody isoform of the embodiments has improved binding affinity for an effector molecule such as FcR, as compared to the reference antibody. In some embodiments, the binding affinity of an antibody isoform to effector molecule is improved by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody, where FcR binding affinity is determined e.g. as disclosed in the Examples herein. In other embodiments, the binding affinity of an antibody isoform to effector molecule is improved by at least about 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about

1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, as compared to the reference antibody, where effector molecule binding affinity is determined as disclosed in the Examples herein. In one embodiment, the effector molecule that the antibody isoform has improved binding to is FcyRIIIa. In another embodiment, the FcR that antibody isoform has improved binding to is FcyRIIb (as compared to reference antibody). In still another embodiment, the FcR that the antibody isoform has improved binding to is FcyRIIa. In a specific embodiment, the FcR that the antibody isoform has improved binding to is FcyRIIIa F158. In another specific embodiment, the FcR that the antibody isoform has improved binding to is FcyRIIIa VI 58.

In other embodiments, an antibody isoform of the embodiments has reduced binding affinity for an effector molecule, as compared to the reference antibody. In some embodiments, the binding affinity of an antibody isoform to effector molecule is reduced by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody, where effector molecule binding affinity is determined as disclosed in the Examples herein. In other embodiments, the binding affinity of an antibody isoform to effector molecule is reduced by at least about 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about

1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, as compared to the reference antibody, where effector molecule binding affinity is determined as disclosed in the Examples herein. In one embodiment, the FcR that the antibody isoform has reduced binding to is FcyRIIIa. In another embodiment, the FcR that the antibody isoform has reduced binding to is FcyRIIb. In still another embodiment, the FcR that the antibody isoform has reduced binding to is FcyRIIa. In a specific embodiment, the FcR that the antibody isoform has reduced binding to is FcyRIIIa F158. In another specific embodiment, the FcR that the antibody isoform has reduced binding to is FcyRIIIa VI 58.

In certain embodiment, an antibody isoform of the embodiments has improved binding affinity for complement factor, or Clq, as compared to the reference antibody. In a specific embodiment, the binding affinity of an antibody isoform to Clq is improved by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody, where Clq binding affinity is determined as disclosed in the Examples herein. In other embodiments, the binding affinity of an antibody isoform to Clq is improved by at least about 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, as compared to the reference antibody, where Clq binding affinity is determined as disclosed in the Examples herein.

In other embodiments, an antibody isoform has reduced binding affinity for complement factor, or Clq, as compared to the reference antibody. In a specific embodiment, the binding affinity of an antibody isoform to Clq is reduced by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody, where Clq binding affinity is determined as disclosed in the Examples herein. In other embodiments, the binding affinity of an antibody isoform to Clq is reduced by at least about 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, as compared to the reference antibody, where Clq binding affinity is determined e.g. as disclosed in the

Examples herein. Antibody isoforms may also be assayed for their cellular activity, e.g., ability to mediate ADCC or CDC activity. To assess the ability of any particular antibody isoform to mediate lysis of the target cell by ADCC, an antibody isoform of interest is added to target cells in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cytolysis is generally detected by the release of label (e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells. Useful effector cells for such assays include peripheral blood

mononuclear cells (PBMC) and Natural Killer (NK) cells. Specific examples of in vitro ADCC assays are described in Wisecarver et al., 1985, 79:277; Bruggemann et al., 1987, J Exp Med 166: 1351; Wilkinson et al., 2001, J Immunol Methods 258: 183; and Patel et al., 1995 J Immunol Methods 184:29. Alternatively, or additionally, ADCC activity may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., 1998, PNAS USA 95:652. To assess complement activation, a CDC assay, e.g. as described in Gazzano- Santoro et al., 1996, J. Immunol. Methods, 202: 163, may be performed.

In certain embodiments, an antibody isoform of the embodiments has improved ADCC activity, as compared to the reference antibody. In some embodiments, ADCC activity improved by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the ADCC activity of an antibody isoform to effector molecule such as FcR is improved by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In other embodiments, an antibody isoform of the embodiments has reduced ADCC activity, as compared to the reference antibody. In some embodiments, ADCC activity is reduced by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the ADCC activity of an antibody isoform to effector molecule is reduced by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In certain embodiments, an antibody isoform of the embodiments has improved CDC activity, as compared to the reference antibody. In some embodiments, CDC activity is improved by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the CDC activity of an antibody isoform to effector molecule is improved by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In other embodiments, an antibody isoform of the embodiments has reduced CDC activity, as compared to the reference antibody. In some embodiments, CDC activity is reduced by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the CDC activity of an antibody isoform to effector molecule is reduced by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody. In certain embodiments, an antibody isoform of the embodiments has improved DC-SIGN binding activity, as compared to the reference antibody. In some embodiments, DC-SIGN binding activity is improved by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the DC-SIGN binding activity of an antibody isoform to effector molecule is improved by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In other embodiments, an antibody isoform of the embodiments has reduced DC-SIGN binding activity, as compared to the reference antibody. In some embodiments, DC-SIGN binding activity is reduced by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the DC-SIGN binding activity of an antibody isoform to effector molecule is reduced by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In certain embodiments, an antibody isoform of the embodiments has improved protein A binding activity, as compared to the reference antibody. In some embodiments, protein A binding activity is improved by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the protein A binding activity of an antibody isoform to effector molecule is improved by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In other embodiments, an antibody isoform of the embodiments has reduced protein A binding activity, as compared to the reference antibody. In some embodiments, protein A binding activity is reduced by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the protein A binding activity of an antibody isoform to effector molecule is reduced by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In certain embodiments, an antibody isoform of the embodiments has improved protein G binding activity, as compared to the reference antibody. In some embodiments, protein G binding activity is improved by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the protein G binding activity of an antibody isoform to effector molecule is improved by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In other embodiments, an antibody isoform of the embodiments has reduced protein G binding activity, as compared to the reference antibody. In some embodiments, protein G binding activity is reduced by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the protein G binding activity of an antibody isoform to effector molecule is reduced by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In certain embodiments, an antibody isoform of the embodiments has improved carbohydrate binding protein activity, as compared to the reference antibody. In some embodiments, carbohydrate binding protein activity is improved by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the carbohydrate binding protein activity of an antibody isoform to effector molecule is improved by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In other embodiments, an antibody isoform of the embodiments has reduced carbohydrate binding protein activity, as compared to the reference antibody. In some embodiments, carbohydrate binding protein activity is reduced by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the carbohydrate binding protein activity of an antibody isoform to effector molecule is reduced by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In certain embodiments, an antibody isoform of the embodiments has improved antibody binding protein activity, as compared to the reference antibody. In some embodiments, antibody binding protein activity is improved by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the antibody binding protein activity of an antibody isoform to effector molecule is improved by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

In other embodiments, an antibody isoform of the embodiments has reduced antibody binding protein activity, as compared to the reference antibody. In some embodiments, antibody binding protein activity is reduced by about 1.10 fold to about 100 fold, or about 1.15 fold to about 50 fold, or about 1.20 fold to about 25 fold, as compared to the reference antibody. In other embodiments, the antibody binding protein activity of an antibody isoform to effector molecule is reduced by at least about 1.10 fold, 1.10 fold, or at least about 1.20 fold, or at least about 1.30 fold, or at least about 1.4 fold, or at least about 1.5 fold, or at least about 1.6 fold, or at least about 1.70 fold, or at least about 1.8 fold, or at least about 1.9 fold, or at least about 2.0 fold, or at least about 2.5 fold, or at least about 3 fold, or at least about 3.5 fold, or at least about 4.0 fold, or at least about 4.5 fold, or at least about 5.0 fold, or at least about 5.5 fold, or at least about 6 fold, or at least about 7 fold, or at least about 8 fold, or at least about 10 fold, or at least about 25 fold, as compared to the reference antibody.

Antibody compositions

After an antibody isoform has been identified or obtained it may be provided in isolated and/or purified form, it may be used as desired, and it may be formulated into a composition comprising at least one additional component, such as a pharmaceutically acceptable excipient or carrier. Nucleic acid encoding the antibody isoform may be used to produce the antibody isoform for subsequent use. As noted, such nucleic acid may, for example, be isolated from a library or diverse population initially provided and from which the antibody isoform was produced and identified.

An antibody isoform in accordance with an embodiment of the present invention may be used in methods of diagnosis or treatment of the human or animal body of subjects, preferably human.

Accordingly, further aspects of the invention provide methods of treatment comprising administration of an antibody isoform as provided, pharmaceutical compositions comprising such an antibody isoform, and use of such an antibody isoform in the manufacture of a medicament for administration, for example in a method of making a medicament or pharmaceutical composition comprising formulating the antibody isoform with a

pharmaceutically acceptable excipient.

Such pharmaceutical compositions may comprise an antibody comprising an antibody isoform or a fusion protein comprising an antibody isoform, as provided herein.

The antibody, or the reference antibody, may be selected from one which binds to tumour associated antigens. A tumour associated antigen may be selected from the following list: 707-AP (707 alanine proline), AFP (alpha (alpha)-fetoprotein), AIM-2 (interferon-inducible protein absent in melanoma 2), ART-4 (adenocarcinoma antigen recognized by T cells 4), BAGE (B antigen), Bcr-abl (breakpoint cluster region- Abelson), CAMEL (CTL-recognized antigen on melanoma), CAP-1 (carcino-embryonic antigen peptide- 1), CASP-8 (caspase-8), CDC27 (cell-division-cycle 27), CDK4 (cyclin-dependent kinase 4), CEA (carcino-embryonic antigen), CLCA2 (calcium-activated chloride channel-2), CT (cancer/testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiation antigen melanoma), ELF2 (elongation factor 2), Ep- CAM (epithelial cell adhesion molecule), EphA2, 3 (Ephrin type-A receptor 2, 3), ETV6- AML1 (Ets variant gene 6/acute myeloid leukemia 1 gene ETS), FGF-5 (Fibroblast growth factor-5), FN (fibronectin), G250 (glycoprotein 250), GAGE (G antigen), GplOO

(glycoprotein 100 kD), HAGE (helicase antigen), HER-2/neu (human epidermal receptor- 2/neurological), HLA-A*0201-R170I (arginine (R) to isoleucine (I) exchange at residue 170 of the alpha-helix of the alpha2-domain in the HLA-A2 gene), HSP70-2M (heat shock protein 70-2 mutated), HST-2 (human signet ring tumor-2) hTERT (human telomerase reverse transcriptase), iCE (intestinal carboxyl esterase), IL-13Ralpha2 (interleukin 13 receptor alpha2 chain), KIAA0205, LAGE (L antigen), LDLR/FUT (low density lipid receptor/GDP- L-fucose: beta-D-galactosidase 2-alpha-L-fucosyltransferase), MAGE (melanoma antigen), MART-l/Melan-A (melanoma antigen recognized by T cells- 1/Melanoma antigen A), MART-2 (melanoma Ag recognized by T cells-2), MC1R (melanocortin 1 receptor), M-CSF (macrophage colony- stimulating factor gene), MUC1,2 (mucin 1,2), MUM-1, -2, -3

(melanoma ubiquitous mutated 1,2,3), NA88-A (NA cDNA clone of patient M88), Neo-PAP (Neo-poly(A) polymerase— NPM/ALK), nucleophosmin/anaplastic lymphoma kinase fusion protein), NY-ESO-1 (New York— esophageous 1), OA1 (ocular albinism type 1 protein), OS- 9, P15 (protein 15), pl90 minor bcr-abl (protein of 190 KD bcr-abl), Pml/RARalpha

(promyelocytic leukemia/retinoic acid receptor .alpha.), PRAME (preferentially expressed antigen of melanoma), PSA (prostate-specific antigen), PSMA (prostate-specific membrane antigen) PTPRK (receptor-type protein-tyrosine phosphatase kappa), RAGE (renal antigen), RU1,2 (renal ubiquitous 1,2), SAGE (sarcoma antigen), SART-1, -2, -3 (squamous antigen rejecting tumor 1, 2, 3), SSX-2 (synovial sarcoma, X breakpoint 2), Survivin-2B (intron 2- retaining surviving), SYT/SSX (synaptotagmin I/synovial sarcoma, X fusion protein), TEL/AML1 (translocation Ets-family leukemia/acute myeloid leukemia 1), TGFbetaRII (transforming growth factor beta receptor 2), TPI (triosephosphate isomerase), TRAG-3 (taxol resistant associated protein 3), TRG (testin-related gene), TRP-1 (tyrosinase related protein 1, or gp75), TRP-2 (tyrosinase related protein 2), TRP-2/INT2 (TRP-2/intron 2), TRP-2/6b (TRP-2/novel exon 6b), WT1 (Wilms' tumor gene). Further tumour associated antigens may be selected from those listed on the world wide web at

cancerimmunity.org/peptidedatabase/mutation.htm.

More specifically, exemplary target antibodies include, but are not limited to 2B8 and C2B8 (Zevalin ® and Rituxan ®, IDEC Pharmaceuticals Corp., San Diego), Lym 1 and Lym 2 (Techniclone), LL2 (Immunomedics Corp., New Jersey), Trastuzumab (Herceptin ®,

Genentech Inc., South San Francisco), Tositumomab (Bexxar ®, Coulter Pharm., San

Francisco), Alemtzumab (Campath ®, Millennium Pharmaceuticals, Cambridge),

Gemtuzumab ozogamicin (Mylotarg ®, Wyeth-Ayerst, Philadelphia), Cetuximab (Erbitux®., Imclone Systems, New York), Bevacizumab (Avastin®, Genentech Inc., South San

Francisco), BR96, BL22, LMB9, LMB2, MB1, BH3, B4, B72.3 (Cytogen Corp.), SSI (NeoPharm), CC49 (National Cancer Institute), Cantuzumab mertansine (ImmunoGen, Cambridge), MNL 2704 (Milleneum Pharmaceuticals, Cambridge), Bivatuzumab mertansine (Boehringer Ingelheim, Germany), Trastuzumab-DMl (Genentech, South San Francisco), My9-6-DMl (ImmunoGen, Cabridge), SGN-10, -15, -25, and -35 (Seattle Genetics, Seattle), and 5E10 (University of Iowa). In preferred embodiments, the starting antibodies or reference antibodies of the present invention will bind to the same tumor- associated antigens as the antibody isoforms enumerated immediately above.

In another embodiment, the antibody may be selected from one which binds to the target antigen CD20. Such antibodies are described in International Patent Application WO

06/130458. Details of the variable regions of a panel of anti-CD20 antibodies are given in Table 1 of WO 06/130458.

In accordance with an embodiment of the present invention, the antibody, or reference antibody, that binds to CD20 may comprise the heavy and light chain variable regions of the anti-CD20 antibody 1.5.3 as disclosed in Table 1 of International Patent Application WO 06/130458. The nucleic acid and protein sequences of the heavy and light chain variable regions of antibody 1.5.3 are given as SEQ ID NOS: 25 to 28 respectively. The anti-CD20 antibody may further comprise an antibody isoform as provided herein.

Clinical indications in which an antibody isoform may be used are those in which the polypeptide provides therapeutic benefit.

Such clinical conditions may include cancer, respiratory conditions, inflammation, cardiovascular diseases, gastrointestinal diseases and diseases of the central nervous system.

In accordance with an embodiment of the present invention an antibody isoform may be given to an individual, preferably by administration in a "prophylactic ally effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors. Antibodies, antibody isoform, and antibody glycoform arrays

One or more different types of antibodies, reference antibodies and antibody isoforms can be immobilized on a support surface. Antibodies, reference antibodies and antibody isoforms may be localized or segregated to particular regions on a support or on particular supports, e.g., latex beads. Each of these particular regions will be able to bind at least one effector molecule. These regions are referred to as sensing elements or regions. Typically, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 100,000 or more different sensing elements (including all values and ranges there between), can be immobilized on a support surface to form various arrays.

In various embodiments of the invention antibodies, reference antibodies and antibody isoforms may be coupled to a support. A "support" refers to a solid phase onto which an antibody and an antibody isoform can be provided, (e.g., by attachment, deposition, coupling and other known methods). One or more antibodies and antibody isoforms may be

immobilized on supports including, but not limited to glass (e.g., a chemically-modified glass slide), latex, plastic, membranes, microtiter wells, mass spectrometer plates, beads (e.g., cross-linked polymer beads) or the like. Antibody and antibody isoform array can include, but is not limited to a plate, a chip, and/or a population of beads. A variety of array formats are known in the art and can be adapted to the inventive methods based on the descriptions provided in this application.

In certain embodiments of the invention, a surface may comprise a plurality of addressable locations, each of which location has one or more reference antibodies and antibody isoforms. An antibody isoform can comprise an antibody glycoform. Antibody isoforms can comprise a purely random feature and a non-random feature. Antibody glycoforms can be non-random feature and single amino acid substitutions of an antibody can be a random feature.

Each antibody and antibody isoform can be arranged in regions on the support. The regions can be arranged in any pattern on the support, but are preferably in regular pattern, such as lines, orthogonal arrays, or regular curves (e.g., circles). Alternatively, antibodies and antibody isoforms can be placed on the support surface in continuous patterns, rather than in discontinuous patterns. Alternatively, the support can be a separate material. For example, a support can be a solid phase, such as a polymeric, paramagnetic, latex, or glass bead, upon which are immobilized antibodies and antibody isoforms. A solid phase material may be placed onto a probe or detectable media (e.g., fluorescently tagged bead) that is removably insertable into a gas phase ion spectrometer or passed by a detector such as a laser/spectrometer device. The solid phase with each type of antibody and antibody isoform(s) is typically placed at different addressable locations of the support surface.

The support can be made of any suitable material. For example, the support materials include, but are not limited to, insulating materials (e.g., glass such as silicon oxide, plastic, ceramic), semi-conducting materials (e.g. silicon wafers), or electrically conducting materials (e.g., metals, such as nickel, brass, steel, aluminum, gold, or electrically conductive polymers), organic polymers, biopolymers, or any combination thereof. The support material can also be solid or porous.

The support can be conditioned to bind antibodies and antibody isoforms. In some

embodiments, the surface of the support can be conditioned (e.g., chemically or mechanically (e.g., roughening)) to place antibodies and antibody isoforms on the surface. Typically, a support comprises reactive groups that can immobilize antibodies and antibody isoforms. For example, the support can comprise a carbonyldiimidazole group which covalently reacts with amine groups. In another example, the support can comprise an epoxy surface which covalently reacts with amine and thiol groups. In another example, the support could be a glass surface in which the surface is modified by first appending a poly-ethylene glycol chain followed by capping with a thiol-reactive moiety such as a maleimide, which reacts covalently with a thiol-containing ligand. Supports with these reactive surfaces are commercially available from Ciphergen Biosystems (Fremont, Calif.) or can be synthesized using protocols known to those knowledgeable in the art.

In an embodiment of the present invention, the support is according to the formula: SOL-[polymer]_n-[spacer]_m-L, wherein m and n are each independently either 0 or 1 and SOL, polymer, spacer and L are as described herein. In an embodiment the support comprises a solid phase (SOL) and/or a polymer to which the antibodies in the array are bound. In an embodiment the support further comprises a chemoselective ligation group (L). The ligation group is used for conjugating the antibody to the support. Preferred solid phases include glass, metal and plastic, and non-soluble polymers, in an embodiment the solid phase is glass. Preferred polymers include but are not limited to polyalkylene glycols such as polyethylene glycol, polypropylene glycol, polybutylene glycol, and branched derivatives thereof, polyamines, polyamides, and polypeptides. Preferred polymers or spacers further include silane molecules, in an embodiment for adhering to glass or metal surface e.g. similarly as described in

WO2004005477 and/or WO2005000760. Preferred silanes are carboxylic acid comprising silanes, e.g. aminosilanes modified by divalent activated carboxylic acids such as N- hydroxysuccinimide (NHS) activated divalent carboxylic acids or carboxylic acid anhydrides such as succinic anhydride. In an embodiment the support is chemically derivatized to comprise a chemical spacer structure comprising at least one carbon atom, in an embodiment an alkyl, such as an alkyl comprising 1-30 carbon atoms, 2-24 carbon atoms, 3-22 carbon atoms or 4-18 carbon atoms, and optionally one or more heteroatoms such as oxygen, nitrogen and/or sulfur, for example the spacer may be a polyethylene glycol comprising oxygen as every third atom in linear spacer. In an embodiment polyethylene glycol includes also derivatives of polyethylene glycols comprising side chain derivatives. Preferred polyethylene glycols comprise 1-30 carbon atoms, 2-24 carbon atoms, 3-22 carbon atoms or 4-18 carbon atoms. In some embodiments the spacer is a short range spacer comprising 2-10 carbon atoms and optionally 1-5 heteroatoms as a linear chain and/or a long range spacer comprising 8-24 carbon atoms and optionally 1-12 heteroatoms.

Various support material including coatings and functional groups on array surfaces have been described e.g. in WO/2006/115547 (especially dendritic support), WO2005000760 and WO2004005477 describing mirror metal surfaces such as aluminum and silane coatings such as aminosilane. In an embodiment the glass material is essentially as described in

WO2005000760. Although a variety of support materials are contemplated, preferred substrates are glass, preferably gold-coated or other coated glass or low self-fluorescent glass, for example borosilicate or soda lime silicate glass, and the like. In an embodiment, the ligation group is preferably a functionalized alkoxysilane, chlorosilane, hydrogel or functionalized alkanethiol. When the solid phase is gold-coated, the preferred ligation group is a functionalized alkanethiol. If the solid phase is glass, then the preferred ligation group is a hydrogel, chlorosilane or alkoxysilane and the most preferable functional compound is a multiaminoorganosilane such as N-(2-aminoethyl)-3- aminopropyltrimethoxysilane (EDA), trimethoxysilylpropyldiethylenetriamine (DETA) or (aminoethylaminomethyl)phenethyltrimethoxysilane (PEDA). In an embodiment the aminosilanes are modified to comprise carboxylic acid, activated carboxylic acid, or carboxylic acid ester such as NHS ester, aromatic alcohol ester or halogen aromatic alcohol ester.

WO2005000760 describes hydrogel and silane support glass surfaces suitable for

immobilizing proteins of the present invention. In an embodiment of the present invention, gold surface polymer support comprising thiol groups are attached to the surface by a condensation reaction or Au-S bonding. The preferred ligation groups include groups selected to impart functionality to a glass surface, including but not limited to primary, secondary or tertiary amine, aldehyde, carboxylate, cyanate, epoxide, ester, ether, chloro, bromo, iodo, ketone, vinyl (alkyl), acrylate, ethylene glycol, fluoro, hydroxy, isocyanate, isothiocyanate, NHS ester, thiol, and other well known groups. For example, amino and epoxy silane coated supports are commonly used for preparing DNA and protein microarrays. Preferred compounds further include alkanethiols and a wide variety of silanes, preferably epoxysilanes such as epoxycyclohexyl ethyltrimethoxysilane or glycidoxypropyl trimethoxysilane, and aminosilanes such as aminopropyl-trialkoxysilane, aminobutyldimethylmethoxysilane, and multiaminosilanes having more than one amine group. Suitable compounds for use in the homogenous coating mixture may be, for example, multiaminoalkyl monoalkoxysilane, multiamino-alkyl dialkoxy silane, and/or multiaminoalkyl trialkoxysilane. Also suitable are multiaminoorganosilanes such as trimethoxysilylpropyl-diethylenetriamine (DETA), N- (2- aminoethyl)-3-aminopropyitrimethoxysilane (EDA), and/or (aminoethyl aminomethyl) phenethyltrimethoxysilane (PEDA). Also suitable are hydrogels, which are polymer networks capable of swelling in water. Typical hydrogels are derived from carbohydrates (chitosan, alginates, hyaluronic acid, etc.), proteins (e.g. collagen), and synthetic polymers, the most predominant ones being polyethylene glycols, nitrocellulose, polyurethane, and the like.

There are numerous methods of producing hydrogels suitable for use in the present invention, for example as described in Hennink WE, Adv. Drug Deliv. Rev. 54: 13-36 (2000) and Gehrke SF, NY Acad. Sci. 831: 179-207 (1997). A suitable method of forming the fluorescent labeled compound is to react a portion of a chemically functional molecule with a fluorophore. For example, a reaction dependent fluorophore could be conjugated to the amine group of a DETA molecule (i.e., trimethoxysilylpropyl-diethylenetriamine). The fluorescent molecules thus formed are then mixed with non-reacted DETA molecules.

A variety of support materials, including polymeric membranes (such as nylon and nitrocellulose), magnetic particles, mica, glass, silica, gold, cellulose, and polystyrene, among others; and technologies to produce such are described in the following documents: U.S. Pat. Nos. 5,077,210; 5,242,974; 5,384,261 ; 5,405,783; 5,412,087; 5,424,186; 5,429,807;

5,436,327; 5,445,934; 5,472,672; 5,527,681; 5,529,756; 5,545,531 ; 5,554,501 ; 5,556,752; 5,561,071 ; 5,599,895; 5,624,711 ; 5,639,603; 5,658,734; 5,677,126; 5,688,642; 5,700,637; 5,744,305; 5,760,130; 5,837,832; 5,843,655; 5,861 ,242; 5,874,974; 5,885,837; 5,919,626; PCT/US98/26245; WO 93/17126; WO 95/11995; WO 95/35505; EP 742 287; and EP 799 897. There are numerous patents and patent applications describing methods of using arrays in various applications, including: U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661 ,028; 5,848,659; 5,874,219; WO 95/21265; WO 96/31622; WO 97/10365; WO 97/27317; EP 373203; and EP 785 280. The techniques and uses in these documents are all applicable herein.

Supports useful for the methods and products of the present invention include materials that may be modified to have surface reactive functional groups such as hydroxyls. These groups can further react with a homogeneous mixture of a fluorescent-tagged compound and a chemically functional compound to which other chemical moieties can be bound. Suitable supports include but are not limited to inorganic materials such as silicon, glass, silica, diamond, quartz, alumina, silicon nitride, platinum, gold, aluminum, tungsten, titanium and various other metals and various other ceramics. Alternatively, polymeric materials such as polyesters, polyamides, polyimides, acrylics, polyethers, polysulfones, fluoropolymers, and the like may be used as suitable organic supports. The support may be provided in any suitable form, such as slides, wafers, fibers, beads, particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, or the like. The support may have any convenient shape, such as that of a disc, square, sphere, circle, or the like. The support can further be fashioned as a bead, dipstick, test tube, pin, membrane, channel, capillary tube, column, or as an array of pins or glass fibers. In an embodiment of the present invention, the antibody isoform array is according to the formula:

SOL- ( [polymer] _n- [spacer] _m-L-S- Ab)_a, wherein SOL is solid phase support; n and m are independently either 0 or 1; L-S is covalent bond formed between the chemoselective ligation group L and amino acid side chain S, preferably lysine or cysteine, of Ab; each occurrence of Ab is independently an antibody isoform, with the proviso that the array comprises at least 2 different antibody isoforms; and a is an integer between 2 and about 100,000.

In a preferred embodiment of the present invention, the antibody isoform array comprises at least 2 different antibody glycoforms, preferably selected from the group of complex-type N- glycans GO, FG0, Gl, FG1, G2 and FG2, degraded complex-type N-glycan, hybrid-type N- glycan, high-mannose type N-glycan, oligomannose-type N-glycan with four Man residues and high-mannose type N-glycan with five Man residues.

In an embodiment of the present invention, the chemoselective ligation group L is a non- covalent ligation pair of, for example, biotin and streptavidin or an antibody and its ligand. In an embodiment, biotin is covalently bound to the amino groups of lysine side chains in the antibody isoform by use of e.g. NHS ester activated biotin or NHS ester activated

caproylbiotin by well-known methods; in another embodiment, biotin is covalently bound to the sulphydryl groups of cysteine side chains in the antibody isoform by use of e.g.

maleimidobiotin or maleimidocaproylbiotin by well-known methods; and e.g. streptavidin or avidin is covalently coupled to SOL from its amino acid side chains according to the present invention; and the biotin-(strept)avidin complex L is formed by contacting the thus produced solid support and biotinylated antibody isoform.

Due to the complex nature of the antibody molecules and the N-glycan structures comprised therein, the fact that the Asn297 N-glycans are buried inside the three-dimensional antibody structure, as well as the fact that the covalent conjugation of antibodies to the solid support chemically modifies their amino acid side chains, the performance of an antibody isoform array according to the invention was beforehand highly unpredictable to a person skilled in the art. However, the present inventors surprisingly found that the covalently conjugated antibody isoform array according to the invention can be used to successfully assay interaction between antibody isoforms and effector molecules.

Arrays utilized in this invention may include between about 2, 5, 10, 15, 20, 50, 100, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to 25,000, 50,000, 75,000, to about 100,000 distinct antibodies and antibody isoforms, including values and ranges there between.

Typically effector molecules are contacted with a support comprising an array of antibodies and antibody isoforms in any suitable manner, e.g., bathing, soaking, dipping, spraying, washing over, or pipetting. The effector molecules can contact the support comprising one or more antibodies and antibody isoforms for a period of time sufficient to allow the effector molecules to bind to the antibodies and antibody isoforms. Typically, the effector molecules and the support comprising the antibodies and antibody isoforms are contacted for a period of between about 30 seconds to about, 1, 5, 10, 20, 30, 40, 50 minutes to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, to 24 hours or so. In some embodiments, between about 30 seconds and about 15 minutes is sufficient for binding of the effector molecules. Typically, the effector molecules are contacted with the antibodies and antibody isoforms under ambient temperature and pressure conditions. For some samples, however, modified temperature (typically at about 4, 5, 10, 15, 20, 25°C to about 30, 32, 34, 36, to 37°C) and pressure (atmospheric pressure to 1, 5, 10, 15, 20, 25, 30 or more psi) conditions may be desirable. These conditions are determinable by those skilled in the art.

For some samples, the effector molecules can be contacted to the support in different concentrations in order to determine binding constant K_D.

After the support is contact with the effector molecules, it is preferred that unbound and weakly absorbed materials on the support surface are washed out or off so that only the more tightly bound materials remain on the support surface. Washing a support surface can be accomplished by, e.g., bathing, soaking, dipping, rinsing, spraying, or washing the support surface with an eluant. A microfluidics process may be used when an eluant is introduced to small spots of capture agents on the support. Typically, an eluant may be at a temperature of between less than 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 to 100°C or any value or range therebetween. In some embodiments, washing unbound materials from the probe surface may not be necessary if components bound by antibodies and antibody isoforms can be resolved by gas phase ion spectrometry without a wash or are detected using a high specificity sandwich reagent that will ignore molecules that might be present other than the target.

Any suitable eluants (e.g., organic or aqueous) that preserve the relevant interaction can be used to wash the support surface. Preferably, an aqueous solution is used. Exemplary aqueous solutions include, e.g., a HEPES buffer, a Tris buffer, or a phosphate buffered saline. To increase the wash stringency of the buffers, additives can be incorporated into the buffers. These include, but are limited to, ionic interaction modifier (both ionic strength and pH), hydrophobic interaction modifier, chao tropic reagents, affinity interaction displacers. The selection of a particular eluant or eluant additives is dependent on the conditions used (e.g., types of antibodies and antibody isoforms used, and/or types of effector molecules, etc.).

Prior to desorption and ionization of a molecule from a support surface, an energy absorbing molecule ("EAM") or a matrix material is typically applied to the support surface. The energy absorbing molecules can assist absorption of energy from an energy source from a gas phase ion spectrometer, and can assist desorption of molecules from the support surface. Exemplary energy absorbing molecules include cinnamic acid derivatives, sinapinic acid ("SPA"), cyano hydroxy cinnamic acid ("CHCA") and dihydroxybenzoic acid.

The energy absorbing molecule and the effector molecules, antibodies and antibody isoforms can be contacted in any suitable manner. For example, an energy absorbing molecule is mixed with the effector molecules, and the mixture is placed on the support surface. In another example, an energy absorbing molecule can be placed on the support surface prior to contacting the support surface with the effector molecules. In another example, the effector molecules can be placed on the support surface prior to contacting the support surface with an energy absorbing molecule. Then the molecules bound to the antibodies, antibody isoforms or capture reagents on the support surface are desorbed, ionized and detected as described in detail below.

Detection Methods

Methods detecting effector molecules captured or bound on a solid support comprising reference antibodies, antibody isoforms, and optionally control proteins, can generally be divided into photometric methods of detection and non-photometric methods of detection.

Photometric methods of detection include, without limitation, those methods that detect or measure absorbance, fluorescence, refractive index, polarization or light scattering. Methods involving absorbance include measuring light absorbance of an analyte directly (increased absorbance compared to background) or indirectly (measuring decreased absorbance compared to background). Measurement of ultraviolet, visible and infrared light all are known. Methods involving fluorescence also include direct and indirect fluorescent measurement. Methods involving fluorescence include, for example, fluorescent tagging in immunological methods such as ELISA or sandwich assay. Methods involving measuring refractive index include, for example, surface plasmon resonance ("SPR"), grating coupled methods (e.g., sensors uniform grating couplers, wavelength-interrogated optical sensors ("WIOS") and chirped grating couplers), resonant mirror and interferometric techniques. Methods involving measuring polarization include, for example, ellipsometry. Light scattering methods (nephelometry) may also be used.

Non-photometric methods of detection include, without limitation, magnetic resonance imaging, gas phase ion spectrometry, atomic force microscopy and multipolar coupled resonance spectroscopy. Magnetic resonance imaging (MRI) is based on the principles of nuclear magnetic resonance (NMR), a spectroscopic technique used by scientists to obtain microscopic chemical and physical information about molecules. Gas phase ion spectrometers include mass spectrometers, ion mobility spectrometers and total ion current measuring devices.

Mass spectrometers measure a parameter which can be translated into mass-to-charge ratios of ions. Generally ions of interest bear a single charge, and mass-to-charge ratios are often simply referred to as mass. Mass spectrometers include an inlet system, an ionization source, an ion optic assembly, a mass analyzer, and a detector. Several different ionization sources have been used for desorbing and ionizing analytes from the surface of a support or biochip in a mass spectrometer. Such methodologies include laser desorption/ionization (MALDI, SELDI), fast atom bombardment, plasma desorption, and secondary ion mass spectrometers. In such mass spectrometers the inlet system comprises a support interface capable of engaging the support and positioning it in interrogatable relationship with the ionization source and concurrently in communication with the mass spectrometer, e.g., the ion optic assembly, the mass analyzer and the detector.

Solid supports for use in bioassays that have a generally planar surface for the capture of targets and adapted for facile use as supports with detection instruments are generally referred to as biochips.

In certain embodiments, methods for detecting effector molecules of a biological cytotoxicity pathway, e.g., ADCC pathway, wherein the methods may comprise: providing a support comprising a plurality of antibodies and antibody isoforms immobilized on a surface of the support, contacting effector molecules with a support, and detecting the effector molecules of the biological pathway, such as ADCC, bound to the antibodies and antibody isoforms on the support by photometric methods and determining binding constant K_D. By comparing binding constants between the antibody and antibody isoforms desired antibody isoform(s) can be identified. In some applications, antibody isoforms having high affinity to effector molecules are desired as they may have enhanced capabilities of inducing ADCC.

Analysis of Data

Data generated by quantitation of the amount of an effector molecule of interest bound to each antibody and antibody isoform, and optionally control protein, on the array can be analyzed using any suitable means. In one embodiment, data is analyzed with the use of a

programmable digital computer. The computer program generally contains a readable medium that stores codes. Certain code can be devoted to memory that includes the location of each feature on a support, the identity of the antibody or antibody isoform at that feature and the elution conditions used to wash the support surface. The computer also may contain code that receives as input, data on the strength of the signal at various addressable locations on the support. This data can indicate the number of targets detected, including the strength of the signal generated by each target.

Data analysis can include the steps of determining signal strength (e.g., height of peaks) of a effector molecules detected and removing "outliers" (data deviating from a predetermined statistical distribution). The observed peaks can be normalized, a process whereby the height of each peak relative to some reference is calculated. For example, a reference can be background noise generated by instrument and chemicals (e.g., energy absorbing molecule) which is set as zero in the scale. Then the signal strength detected for each target can be displayed in the form of relative intensities in the scale desired. Alternatively, a standard may be admitted with the sample so that a peak from the standard can be used as a reference to calculate relative intensities of the signals observed for each target detected. Data analysis may also include comparison between binding of an antibody (reference antibody) and its isoform(s) to an effector molecule and determining whether the effector molecule binds with higher, similar or lower affinity to an antibody isoform than to the (reference) antibody.

Data generated by reference antibody can be compared to control protein to determine if the effector molecule(s) bind with similar (or different) affinity to the reference antibody and control protein. In some embodiments the reference antibody and the control protein can be the same. For example, if the reference antibody is an IgGl molecule, the control protein may also be IgGl molecule. Thus, antibody isoforms may comprise IgGl glycoforms and the effector molecule may be FcyRIIIA. Data obtained from reference antibody and control protein on binding between IgGl and FcyRIIIA can be compared to the binding affinity between an IgGl glycoform and FcyRIIIA.

In some embodiments the reference antibody and the control protein can be different molecules. For example, if the reference antibody is a glycoform of an IgGl molecule, the control protein may be gpl20 molecule. Thus, antibody isoforms may comprise IgGl glycoforms and the effector molecule may be DC-Sign molecule. Data obtained from reference antibody IgGl and control protein gpl20 on binding of DC-Sign can be compared to the binding affinity between an IgGl glycoform and DC-Sign.

In an embodiment, multiple concentrations or a dilution series of antibody isoforms and reference antibodies can be used on a single array. Data obtained from the different concentrations can be compared to more accurately measure binding affinities between antibody isoforms and effector molecule. In an embodiment, multiple concentrations or a dilution series of the effector molecules can be used. The concentrations are selected so that a dose-response curve is formed and the data obtained from the different concentrations can be used to determine kinetic constants such as Kd or EC50 for the binding between antibody isoform or reference antibody and effector molecule. EXAMPLES

EXAMPLE 1 Production of arrays

Preparation of antibody isoforms. CHO cell line DP- 12 was obtained from the American Tissue Type Collection (ATCC product number CRL- 12445, clone 1934). The cell line producing humanized IgGl against IL8 was cultured in serum- free medium according to ATCC's instructions (available also at www.atcc.org). 5 mg/1 kifunensine was added for the duration of antibody production to inhibit mannosidase I activity. After four days' culture, the supernatants were harvested and subjected to protein G affinity column (HiTrap Protein G HP, GE Healthcare) chromatography in HPLC system according to manufacturer's instructions. N-glycans were liberated from the purified IgG preparates by N-glycosidase F (Glyko) digestion, purified and subjected to MALDI-TOF mass spectrometric N-glycan profiling (Ultraflex III TOF/TOF, Bruker Daltonics) essentially as described (Satomaa et al. BMC Cell Biol. 10:42, 2009). The analysis showed that the Fc domain N-glycans of the CHO cell supernatant-derived IgG were normal biantennary complex-type glycoform N-glycans with the major glycan signals at m/z 1485.6, 1647.6 and 1809.9 corresponding to the

[M+Na]⁺ ions of Hex₃HexNAc4dHexi, Hex₄HexNAc₄dHex₁ and HexsHexNAo^dHexi oligosaccharides, respectively and the IgG preparate produced with kifunensine was essentially completely of the high-mannose type glycoform with the major glycan signals at m/z 1905 and 1743 corresponding to the [M+Na]⁺ ions of Hex₉HexNAc₂ and Hex₈HexNAc₂ oligosaccharides, respectively.

Humanized anti-CD20 IgGl monoclonal antibody produced in CHO cells, mouse-human chimeric anti-EGFR IgGl monoclonal antibody produced in Sp2/0 cells and human anti- EGFR IgG2 monoclonal antibody produced in CHO cells were diluted in printing solution (see below). Sialidase treatment of the antibody produced in Sp2/0 cells was performed with A. ureafaciens enzyme (Calbiochem) according to manufacturer' s instructions and the removal of sialic acids from the N-glycans was verified by mass spectrometry as above. The antibody was purified by protein G affinity chromatography as above. Printing of arrays. Arrays were printed onto Schott Nexterion H MPX -16 slides (Schott Technical Glass Solutions GmbH, Jena, Germany). Antibody isoform and control protein samples were diluted to concentrations of 0.1 mg/ml and 0.5 mg/ml in a buffer that had been made by bringing 100 mM sodium citrate buffer pH 2.6 to pH 7 by adding 1 M Na₂HP0₄. The samples were printed at a volume of -400 pL per spot using a Scienion

sciFLEXARRAYER S5 non-contact printer (Scienion AG, Berlin, Germany). For each sample concentration, 6 replicates were printed. 6 replicate spots of Cy3-labeled protein served as positive control and 0-6 replicate spots of printing buffer solution served as negative controls. In the arrays the distance between adjacent spots was approximately 380 μιη. Arrays of up to 24 different isoforms and control substances were printed resulting in up to 144 spots/array. The printed array slides were incubated in 75% humidity in room temperature overnight, allowed to dry in room temperature and stored until use in -20 °C in a desiccator.

EXAMPLE 2

Hybridization with effector molecules and reading of arrays

Preparation of binding proteins for assays. Recombinant human Fc gamma receptors (FcyRI and FcyRIIIa) was from R&D Systems Inc. (USA) and anti-Neu5Gc chicken antibody was from Sialix Inc. (USA). These binding proteins were labeled with NHS-activated Cy3 or Cy5 (GE Healthcare, UK) according to manufacturer's instructions and purified from excess reagent by changing the buffer to phosphate buffered saline (PBS) either in NAP-5 columns (GE Healthcare, UK) or Amicon Ultra 0.5 mL 10K centrifugal filters (Millipore, USA).

Assay procedure to evaluate Fc gamma receptor binding affinities. Printed slides were blocked with 25 mM ethanolamine in 100 mM borate buffer, pH 8.5 for at least one hour in room temperature. Slides were rinsed three times with PBS-Tween (0.05-0.1% Tween), once with PBS and once with water. A Schott Nexterion MPX superstructure (Schott Technical Glass Solutions GmbH, Jena, Germany) was attached to create wells. Arrays were incubated with various concentrations of labeled binding proteins in 50-60 μΐ volume of PBS buffer. Incubations were carried out for 2.5 h at room temperature, after which the slides were washed five times in PBS-Tween, once with PBS, rinsed with water and dried using nitrogen gas stream. Arrays were imaged using Tecan's LS Reloaded laser scanner (Tecan Group Ltd., Switzerland) at excitation wavelengths of 532 and 633 nm and detection wavelengths of 575 and 692 nm for Cy3 and Cy5, respectively. The images were quantified using Array Pro software.

Assay procedure to evaluate anti-Neu5Gc binding affinities. Printed slides were blocked with 25 mM ethanolamine in 100 mM borate buffer, pH 8.5 for at least one hour in room temperature. Slides were rinsed three times with PBS-Tween (0.1% Tween), once with PBS and once with MilliQ water. A Schott Nexterion MPX superstructure (Schott Technical Glass Solutions GmbH, Jena, Germany) was attached to create wells. Arrays were incubated with various concentrations of anti-Neu5Gc (Sialix Inc., USA) in 60 μΐ volume of PBS buffer. Incubations were carried out overnight in +4 °C after which the slides were washed five times with PBS-Tween. After this, arrays were incubated with various concentrations of Cy3- labeled anti-chicken IgY (Millipore, USA) for 1 h in room temperature. Arrays were washed five times with PBS-Tween, once with PBS, rinsed with MilliQ water and and dried using nitrogen gas stream. Arrays were imaged using Tecan's LS Reloaded laser scanner (Tecan Group Ltd., Switzerland) at excitation wavelengths of 532 and 633 nm and detection wavelengths of 575 and 692 nm for Cy3 and Cy5, respectively. The images were quantified using Array Pro software.

EXAMPLE 3

Protein binding results to antibody isoform arrays

Effect of antibody glycoform to binding to FcyRIIIa. Normal CHO-produced glycoform as well as high-mannose glycoforms of humanized anti-IL8 IgGl antibody were printed to an array as described in Example 1 and analyzed for binding to human recombinant FcyRIIIa soluble extracellular domain as described in Example 2. At antibody printing concentration 0.5 mg/ml and FcyRIIIa assay concentration 50 μg/ml, the relative signal intensities of high- mannose glycoforms were 1.4-fold greater compared to normal CHO-produced glycoform. This demonstrated that the binding affinities of the antibody glycoforms and the reference antibody were in the order: high-mannose > CHO-produced; and the Kd values were in the order CHO-produced > high-mannose glycoform; which is consistent with published results obtained by conventional assays (Kanda et al. Glycobiology 17: 104-118, 2006).

Effect of antibody isoform to binding to FcyRI. Mouse-human chimeric anti-EGFR IgGl monoclonal antibody produced in Sp2/0 cells and human anti-EGFR IgG2 monoclonal antibody produced in CHO cells were printed to an array as described in Example 1 and analyzed for binding to human recombinant FcyRI soluble extracellular domain as described in Example 2. At antibody printing concentration 0.5 mg/ml and FcyRIa assay concentration 50 μg/ml, the relative signal intensity of anti-EGFR IgG2 was less than 20% of anti-EGFR IgGl, which is consistent with published results obtained by conventional assays (Bruhns et al. Blood 113:3716-3725, 2009).

Effect of antibody glycoform to binding to anti-Neu5Gc antibody. Mouse-human chimeric anti-EGFR IgGl monoclonal antibody produced in Sp2/0 cells and the same antibody after sialidase treatment and purification were printed to an array as described in Example 1 and analyzed for binding to anti-Neu5Gc antibody as described in Example 2. At antibody printing concentration 0.5 mg/ml, the relative signal intensity of sialidase-treated antibody was less than 5% of anti-EGFR IgGl, demonstrating sensitive detection of different amounts of xenoantigenic Neu5Gc carbohydrate structure in antibody isoforms.

Claims

1. A method of providing an antibody isoform with altered binding to an effector molecule compared with a reference antibody, the method comprising: (a) providing an array of a reference antibody and antibody isoforms of said reference antibody; (b) contacting said array with effector molecules; and (c) assessing binding of effector molecules to said array, whereby one or more antibody isoforms with altered effector molecule binding compared with the reference antibody are obtained and wherein said effector molecules are selected from the group consisting of Fc receptor, carbohydrate binding protein, antibody binding protein, and complement factor.

2. The method of claim 1, wherein said isoform is a glycoform.

3. The method of claim 1 or 2, wherein the antibody class is selected from the group consisting of: a) IgGl, IgG2 and IgG3; b) IgGl and IgG2; c) IgGl and IgG3; d) IgGl; e) IgG2; and f) IgG3; optionally the antibody is a human antibody, or a mutant, point mutant, chimeric or an antibody class suffling of two human IgG antibody classes and optionally the antibody comprises at least Fc domain of the preferred antibody type.

4. The method of any one of claims 1-3, wherein the antibody is bound or covalently bound to a solid support or a polymer support.

5. The method of any one of claims 1-4, wherein the antibody is covalently bound to a support by an amino or thiol group of the antibody.

6. The method of any one of claims 1-5, wherein the antibody is covalently bound to a support by an amino group of the antibody, optionally by an activated carboxylic acid.

7. The method of any one of claims 1-6, wherein the effector molecule binds to three- dimensional structure of the antibody.

8. The method of any one of claims 1-7, wherein the effector molecule binds to the glycan of the antibody.

9. The method any one of claims 1-8, wherein the Fc domain N-glycan of the antibody is selected from the group consisting of complex-type N-glycans GO, FGO, Gl, FG1, G2 and FG2, degraded complex-type N-glycan, hybrid-type N-glycan, high-mannose type N-glycan, oligomannose-type N-glycan with four Man residues and high-mannose type N-glycan with five Man residues.

10. The method of claim 9, wherein the array comprises at least two or at least three antibody glycoforms with major N-glycan structures selected from at least two or at least three of complex-type N-glycans GO, FGO, Gl, FG1, G2 and FG2, degraded complex-type N-glycan, hybrid-type N-glycan, high-mannose type N-glycan, oligomannose-type N-glycan with four Man residues and high-mannose type N-glycan with five Man residues.

11. The method of any of claims 1-10, wherein the effector molecule binds to the glycan of the antibody and the native three-dimensional structure of the Fc domain of the antibody.

12. The method of any one of claims 1-11, wherein the effector molecule binds both to the glycan of the antibody and the native three-dimensional structure of the Fc domain of the antibody.

13. The method of claim 1, wherein Fc receptor is selected from the group consisting of FcyRI family, FcyRII family, FcyRIII family, FcaR, FcsR, FcμR, Fc5R, and FcRn.

14. The method of claim 1, wherein carbohydrate binding protein is selected from the group consisting of lectins, DC-SIGN, SIGN-Rl, macrophage mannose receptor, mannose binding protein, asialoglycoprotein receptor, and an antibody against carbohydrate structure of an antibody.

15. The method of claim 1, wherein antibody binding protein is selected from the group consisting of protein A, protein G, rheumatoid factor, an HAMA protein and an HAHA protein.

16. The method of claim 1, wherein complement factor is selected from the group consisting of complement factor Clq and complement factor C3b.

17. The method of claim 1, wherein the effector molecule is labeled.

18. The method of claim 17, wherein the label is a fluorochrome.

19. The method of claim 1, wherein binding is assessed by determining k_D value.

20. The method of claim 1, wherein said array comprises between about 2 and 10 distinct antibody isoforms.

21. The method of claim 1, wherein said array is a microscope slide, plate, a chip, or a population of beads.

22. The method of claim 1, further comprising cross-linking said antibody and antibody isoforms to said array.

23. An antibody isoform obtained using the method of claim 1.

24. An antibody isoform array of claim 1.

25. An antibody isoform array of claim 24 that is according to the formula: SOL- ( [polymer] _n- [spacer] _m-L-S- Ab)_a, wherein SOL is solid phase support; n and m are independently either 0 or 1; L-S is covalent bond formed between the chemoselective ligation group L and amino acid side chain S, preferably lysine or cysteine, of Ab; each occurrence of Ab is independently an antibody isoform, with the proviso that the array comprises at least 2 different antibody isoforms; and a is an integer between 2 and about 100,000.

26. An antibody isoform array of claim 25 comprising at least 2 different antibody glycoforms, preferably selected from the group of complex-type N-glycans GO, FG0, Gl, FG1, G2 and FG2, degraded complex-type N-glycan, hybrid-type N-glycan, high-mannose type N-glycan, oligomannose-type N-glycan with four Man residues and high-mannose type N-glycan with five Man residues.