WO2014184545A2

WO2014184545A2 - Antibodies

Info

Publication number: WO2014184545A2
Application number: PCT/GB2014/051469
Authority: WO
Inventors: Matthew David Max Crispin; Xiaojie Yu; Christopher Neil Scanlan
Original assignee: Isis Innovation Limited
Priority date: 2013-05-14
Filing date: 2014-05-14
Publication date: 2014-11-20
Also published as: WO2014184545A3; GB201308658D0

Abstract

The present invention relates to polypeptides, proteins and antibodies comprising a FcγRIIB-binding domain. The invention further relates to methods of suppressing immune cell activation or immuno-suppression, and particularly to methods of treating inflammatory and autoimmune disorders using such polypeptides, proteins and antibodies.

Description

ANTIBODIES

The present invention relates to polypeptides, proteins and antibodies comprising a FcyRI IB-binding domain. The invention further relates to methods of suppressing immune cell activation or immuno-suppression, and particularly to methods of treating inflammatory and autoimmune disorders using such polypeptides, proteins and antibodies. Fc receptors are proteins which are found on the surface of certain cells (including B lymphocytes, natural killer cells, macrophages, neutrophils, and mast cells) and which contribute to the protective functions of the immune system. The name is derived from its binding specificity for the Fc region of an antibody. Fc receptors bind to antibodies that are attached to infected cells or invading pathogens. Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity.

There are several different types of Fc receptors (abbreviated FcR), which are classified based on the type of antibody that they recognize. For example, those that bind the most common class of antibody, IgG, are called Fc-gamma receptors (FcyR); those that bind IgA are called Fc-alpha receptors (FcaR); and those that bind IgE are called Fc- epsilon receptors (FcsR).

All of the Fey receptors (FcyR) belong to the immunoglobulin superfamily and are the most important Fc receptors for inducing phagocytosis of opsonized (coated) microbes. This family includes several members, FcyRI (CD64), FcvRI I A (CD32), FcyRIIB (CD32a and CD32b), FcyRIIIA (CD16a), FcyRIIIB (CD16b), which differ in their antibody affinities due to their different molecular structures. For instance, FcyRI binds to IgG more strongly than FcyRII or FcyRIII does. FcyRI also has an extracellular portion composed of three immunoglobulin (lg)-Hke domains, i.e. one more domain than FcyRII or FcyRIII. This property allows activation of FcyRI by a sole IgG molecule (or monomer), while the latter two Fey receptors must bind multiple IgG molecules within an immune complex to be activated.

FcvRIIB (CD32) is a surface receptor protein and is part of a large population of B cell co-receptors which act to modulate signalling. It has a low-affinity for IgG antibodies and down-regulates antibody production in the presence of IgG. FcyRIIB is known to mediate general immunosuppressive effects in various immune cell populations, including B cells, monocytes and dendritic cells. The binding of IgG Fc to cell surface and soluble serum ligands triggers a variety of immunological processes including phagocytosis, cytotoxicity, inflammation and immunosuppression^1,2. The structure of the Fc domain is stabilized by the /V-linked glycan, attached to Asn297 of each IgG heavy chain This glycan is critical for Fc function: genetic or enzymatic removal leads to an almost complete loss of antibody effector functions⁷*⁸. Moreover, composition of the glycan is a key parameter in determining the balance between pro-inflammatory or anti-inflammatory effects. For example, removal of the core a1 ,6-linked fucose residue of the lgG1 Fc glycan enhances binding for FcYRIIIa^9,10 whilst elevation of the levels of terminal a2,6- sialylation leads to decreased natural killer cell activation¹¹ and other potent cell- mediated immunosuppressive effects^12"14. In addition to natural variations in Fc glycosylation, a growing number of enriched and engineered Fc glycoforms are finding application in therapeutic monoclonal antibodies when a particular balance of effector functions is desirable^15"17. The three-dimensional structure of the Fc glycoforms have been investigated^3,18"23 as have the independent effects of glycan and protein engineering on receptor binding^9,10,24"27. By comparison, however, relatively little is known about the interdependence of glycan composition and protein structure on receptor binding^28,29.

The AZ-linked glycans in lgG1 Fc are complex, mostly core-fucosylated, biantennary- type structures with varying amounts of bisecting GlcNAc, terminal galactose and sialic acid residues³⁰. Levels of sialylation are low with less than 10% of total Fc glycans from serum IgG being sialylated³⁰. Tri- or tetra-antennary glycans are generally not found in serum IgG Fc. The absence of larger, branched and/or sialylated structures is notable, especially when compared to the glycosylation of other serum or cell-surface glycoproteins^30,31. X-ray crystallographic^3,4,20,21 and NMR studies^32-34 of the IgG Fc domain have defined the conformation of the /V-linked glycans at Asn297. In the complex-type IgG Fc glycoforms, the conformation of the oligosaccharide is well- conserved and contacts over 500 A² of the surface of each C 2 domains^3,4. The 6-arm of the glycan chain makes several stable interactions with hydrophobic amino acid residues of the Cy2 domain. The terminal Gal6' (see legend to Fig. 1 for terminology) on the 6 arm has been shown to restrict glycan flexibility through inter-action with the Fc protein backbone³². Key amino acid residues that interact with the 6-arm glycans through hydrogen bonds and hydrophobic interactions include: Phe241 , Phe243, Val262, Val264, Asp265, Lys246 and Arg301. Aromatic rings of Phe241 and Phe243 form CH-TT interactions with the Glc-NAc2 and Glc Ac5' residues of the Fc glycan and contribute to the stability of the Fc domain ^²⁹·³⁵·³⁶. The 3-arm on the other hand, makes fewer contacts with the protein backbone with hydrophobic interactions between Man4 and Lys334 being the only observable protein-glycan interaction ³'⁴. Whilst the presence of extensive protein-glycan interactions suggests a relatively immobile carbohydrate conformation and reduced enzymatic processing, recent NMR

spectroscopic studies indicate a more dynamic and mobile role for Fc glycan ^33,34. Nonetheless, the relatively limited processing of the Fc glycan indicates a reduced accessibility to glycan reactive enzymes in the Golgi apparatus.

The influence of hydrophobic residues in the protein-glycan interface of the Fc on glycan processing was first observed in mutational studies on mouse-human chimeric lgG3 antibodies, where replacement of Phe241 , Phe243 or Val264 with Ala resulted in elevated levels of mono- and di-sialylated glycans and decreased binding to C1q and FcyR activity²⁸. In addition, ribosomal display has been used to discover site-specific Fc mutants with enhanced FcvRllla binding²⁹. This approach identified the F243L mutant which, in addition to enhanced FcyRllla binding, also exhibited altered glycan processing including decreased fucosylation²⁹. These studies left open the question, however, of whether the alteration of the protein-carbohydrate interaction directly affected the Fc protein conformation or the increased glycan processing affected FCYR binding.

The inventors have therefore isolated and characterised a series of Fc mutants with chemically-defined glycosyiation. These glycoform-controlled mutants showed similar reduced FcyR binding indicating that the conformation of the Cv2 domain is modulated by glycan-protein interaction independently of glycan type.

It is one object of the invention therefore to provide polypeptides, proteins and antibodies which have increased or decreased binding to particular FcyRs. In particular, it has been found that replacement of certain amino acids in IgG antibodies results in antibodies with enhanced affinity for FcvRIIB and greater selectivity for FcyRIIB.

Some Fc-FcyR interface mutations are known to selectively enhance FcyRllB binding, with a glycan-protein interface mutation that decreases binding to all FcvRs, to provide an IgG which more broadly eliminates activatory (but not inhibitory) receptor binding. In particular, US 2007/023129 (Xencor, Inc.) discloses IgG antibodies with the double mutant S267E/L328F which was shown to significantly increase affinity for the inhibitory FcvRIIB and decrease affinity for the activatory FcyRIIIA, while the binding for the other FcyRs remain similar to the wild type²⁶.

The inventors have now discovered that certain triple mutants exhibit enhanced affinity for the FcvRIIB. Furthermore, by comparison to the double mutant S267E/L328F, these triple mutants have significantly decreased affinity for FcyRIA, FcyRIIA, FCYRIIIA and FcvRIIIB. These new triple mutants therefore have enhanced specificity for FcyRllB. Despite identifying over 900 Fc variants with altered binding affinity for various FcyRs, these new triple mutants have not previously been identified by Xencor²⁵. Polypeptides, protein and antibodies of the invention comprising such triple mutations may therefore be used to treat diseases and disorders which would benefit from suppressing immune cell activation or immuno-suppression, such as autoimmune diseases and inflammatory diseases. ln one embodiment, therefore, the invention provides a polypeptide comprising a FcyRI IB-binding domain, wherein:

(a) the amino acids in the FcyRIIB-binding domain which correspond to the amino acids at positions 264, 267 and 328 of SEQ ID NO: 1 are each different from the amino acids which are present at the corresponding positions in SEQ ID NO: 1 ; or

(b) the amino acids in the FcyRIIB-binding domain which correspond to the amino acids at positions 262, 267 and 328 of SEQ ID NO: 1 are each different from the amino acids which are present at the corresponding positions in SEQ ID NO: 1.

In another embodiment, the invention provides a polypeptide comprising a FcyRIIB- binding domain, wherein: (a) the amino acids in the FcyRIIB-binding domain which correspond to the amino acids at positions 264, 267 and 328 of SEQ ID NO: 2 are each different from the amino acids which are present at the corresponding positions in SEQ ID NO: 2;

or

(b) the amino acids in the FcyRIIB-binding domain which correspond to the amino acids at positions 262, 267 and 328 of SEQ ID NO: 2 are each different from the amino acids which are present at the corresponding positions in SEQ ID NO: 2. Preferably, the FcyRIIB-binding domain comprises an amino acid sequence having at least 70% sequence identity to amino acids 224-446 of SEQ ID NO: 1 or 2.

One of more of the following abbreviations are used herein: 2-AA, 2-aminobenzoic acid; a.s.u., asymmetric unit; ADCC, antigen-dependent cellular cytotoxicity; B4GALTI, β-1 ,4-galactosyltransferase I; Bis-tris, 2,2-Bis(hydroxymethyl)- 2,2',2"-nitrilotriethanol; BSA, bovine serum albumin; CID, collision-induced dissociation; DMEM, Dulbecco's Modified Eagle Medium; ESI, electrospray ionization; FcDR, FcD recep-tor; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GnT, GlcNAc transferase; HEK, human embryonic kidney; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1- yl]ethanesulfonic acid; HPLC, high performance liquid chro-matography; HRP, horse radish peroxidase; Ig, immunoglobu-lin; Man, mannose; Man, mannose; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; PDB, Protein Data Bank; PEI, Polyethylenimine; PNGase F, Peptide-N-glycanase F; r.m.s.d., root-mean-square deviation; ST6GALI, a 2,6-sialyltransferase I; TMB, 3,3' ,5,5' -Tetramethylbenzidine. The polypeptide of the invention comprises or consists of a FcyRIIB-binding domain.

Preferably, the polypeptide of the invention has a higher selectivity for the Fc RIIB receptor than a control polypeptide comprising or consisting of the sequence of SEQ ID NO: 1 with the double substitution S267E/L328F or than a control polypeptide

comprising or consisting of the sequence of SEQ ID NO: 2 with the double substitution S267E/L328F. In this context, the term "higher selectivity" refers to the ability of the polypeptide of the invention to bind with lower affinity to one or more of FcvRIA, FcyRIIA, Fc RIIIA and FcyRIIIB. Most preferably, the polypeptide of the invention has an affinity for the FcyRIIB receptor greater than a control polypeptide comprising or consisting of the sequence of SEQ ID NO: 1 or than a control polypeptide comprising or consisting of the sequence of SEQ ID NO: 2. Preferably, the polypeptide of the invention has a lower affinity for one or more receptors selected from the group consisting of FcyRIA, FcvRIIA, FcyRIIIA and FcyRIIIB than a control polypeptide comprising or consisting of the sequence of SEQ ID NO: 1 with the double substitution S267E/L328F or than a control polypeptide comprising or consisting of the sequence of SEQ ID NO: 2 with the double substitution S267E/L328F. Most preferably, the polypeptide of the invention has an affinity for FcyRIA lower than a control polypeptide comprising or consisting of the sequence of SEQ ID NO: 1 or 2 with the double substitution S267E/L328F. Most preferably, the polypeptide of the invention has an affinity for FcvRIIA lower than a control polypeptide comprising or consisting of the sequence of SEQ ID NO: 1 or 2 with the double substitution S267E/L328F.

Most preferably, the polypeptide of the invention has an affinity for FCYRMIA lower than a control polypeptide comprising or consisting of the sequence of SEQ ID NO: 1 or 2 with the double substitution S267E/L328F.

Most preferably, the polypeptide of the invention has an affinity for FCYRIIIB lower than a control polypeptide comprising or consisting of the sequence of SEQ ID NO: 1 or 2.

In the above comparisons, the polypeptides of the invention may be in the form of a full length human lgG1 or an isolated Fc dimer, and in either biantennary complex glycoform or uniform Man5GlcNAc₂ glycoform. The FcvRHB-binding domain preferably comprises an amino acid sequence having at least 70% sequence identity to amino acids 224-446 of SEQ ID NO: 1 or 2, more preferably at least 80%, 85%, 90%, 95% or 99% sequence identity to amino acids 224- 446 of SEQ ID NO: 1 or 2. SEQ ID NO: 1 is the full amino acid sequence of the human lgG1 immunoglobulin (as given in Figure 3 of Edelman, G. M. et al. (1969) The covalent structure of an entire gamma G immunoglobulin molecule. Proc Natl. Acad. Sci. U S A 63, 78-85). SEQ ID NO: 2 is the corresponding sequence from Uniprot P01857. Embodiments of the invention which refer to SEQ ID NO: 1 apply equally to SEQ ID NO: 2, mutatis mutandis, and vice versa. Amino acids 224-446 of SEQ ID NO: 1 or 2 correspond to the Fc domain of the human lgG1 immunoglobulin. Amino acids 224-340 comprise the lower hinge and CH2 domain; amino acids 340-347 comprise the linker; and amino acids 348-446 comprise the CH3 domain.

In other embodiments, the polypeptide preferably comprises an amino acid sequence having at least 70% sequence identity to amino acids 200-446 of SEQ ID NO: 1 or 2, more preferably at least 80%, 85%, 90%, 95% or 99% sequence identity to amino acids 200-446 of SEQ ID NO: 1 or 2.

In other embodiments, the polypeptide preferably comprises an amino acid sequence having at least 70% sequence identity to amino acids 100-446 of SEQ ID NO: 1 or 2, more preferably at least 80%, 85%, 90%, 95% or 99% sequence identity to amino acids 100-446 of SEQ ID NO: 1 or 2.

In other embodiments, the polypeptide preferably comprises an amino acid sequence having at least 70% sequence identity to amino acids 1-446 of SEQ ID NO: 1 or 2, more preferably at least 80%, 85%, 90%, 95% or 99% sequence identity to amino acids 1-446 of SEQ ID NO: 1 or 2.

Percentage amino acid sequence identities and nucleotide sequence identities may be obtained using the BLAST methods of alignment (Altschul ef a/. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; and http://www.ncbi.nlm.nih.gov/BI.AST). Preferably the standard or default alignment parameters are used.

Standard protein-protein BLAST (blastp) may be used for finding similar sequences in protein databases. Like other BLAST programs, blastp is designed to find local regions of similarity. When sequence similarity spans the whole sequence, blastp will also report a global alignment, which is the preferred result for protein identification purposes.

Preferably the standard or default alignment parameters are used. In some instances, the "low complexity filter" may be taken off. BLAST protein searches may also be performed with the BLASTX program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. (See Altschul et al. (1997) supra). When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs may be used. With regard to nucleotide sequence comparisons, MEGABLAST, discontiguous- megablast, and blastn may be used to accomplish this goal. Preferably the standard or default alignment parameters are used.

MEGABLAST is specifically designed to efficiently find long alignments between very similar sequences.

Discontiguous MEGABLAST may be used to find nucleotide sequences which are similar, but not identical, to the nucleic acids of the invention. The BLAST nucleotide algorithm finds similar sequences by breaking the query into short sub-sequences called words. The program identifies the exact matches to the query words first (word hits). The BLAST program then extends these word hits in multiple steps to generate the final gapped alignments. In some embodiments, the BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12.

One of the important parameters governing the sensitivity of BLAST searches is the word size. The most important reason that blastn is more sensitive than MEGABLAST is that it uses a shorter default word size (11 ). Because of this, blastn is better than MEGABLAST at finding alignments to related nucleotide sequences from other organisms. The word size is adjustable in blastn and can be reduced from the default value to a minimum of 7 to increase search sensitivity. A more sensitive search can be achieved by using the newly-introduced discontiguous megablast page (vvvw.ncbi.nlm.nih.gov Web/Newsltr/FallWinter02/blastlab.html). This page uses an algorithm which is similar to that reported by Ma et al. (Bioinformatics. 2002 Mar; 18(3): 440-5). Rather than requiring exact word matches as seeds for alignment extension, discontiguous megablast uses non-contiguous word within a longer window of template. In coding mode, the third base wobbling is taken into consideration by focusing on finding matches at the first and second codon positions while ignoring the mismatches in the third position. Searching in discontiguous

MEGABLAST using the same word size is more sensitive and efficient than standard blastn using the same word size. Parameters unique for discontiguous megablast are: word size: 11 or 12; template: 16, 18, or 21 ; template type: coding (0), non-coding (1), or both (2). Preferably, the amino acid sequence identities are obtained using BLASTp.

The amino acids in the FcyRIIB-binding domain which correspond to the amino acids at

(a) positions 264, 267 and 328 of SEQ ID NO: 1 , or

(b) positions 262, 267 and 328 of SEQ ID NO: 1

are each different from the amino acids which are present at the corresponding positions in SEQ ID NO: 1.

The amino acids in the FcyRIIB-binding domain which correspond to the amino acids at (a) positions 264, 267 and 328 of SEQ ID NO: 2, or

(b) positions 262, 267 and 328 of SEQ ID NO: 2

are each different from the amino acids which are present at the corresponding positions in SEQ ID NO: 2.

It will be appreciated by the person skilled in the art that the invention relates primarily to polypeptides with substitutions which correspond (a) to positions 264, 267 and 328, or (b) to positions 262, 267 and 328 in the coding sequence of the IgG polypeptide as given in SEQ ID NO: 1 or 2. Consequently, the precise amino acid sequence of the polypeptide at positions other than these positions is not of primary importance in the context of the present invention, i.e. the amino acid sequence of the claimed polypeptide might differ from that of SEQ ID NO: 1 or 2. However, these differences should not prevent the skilled person from identifying in the polypeptide the positions S which correspond to the above-mentioned positions as given in SEQ ID NO: 1 or 2. It is not a requirement of the invention, therefore, that the claimed polypeptide has the precise coding sequence given in SEQ ID NO: 1 or 2 or any fragments thereof. In other words, the sequences given in SEQ ID NO: 1 and 2 are given merely for reference purposes.

0

The alignment of polypeptide sequences in order to determine "corresponding" amino acids may be performed using programs such as BLAST, as discussed above.

The following amino acids are given in the sequence given in SEQ ID NO: 1 or 2:5

The polypeptide of the invention has an amino acid other than valine at the position in the amino acid sequence of the FcyRIIB-binding domain which corresponds to position 262 in SEQ ID NO: 1 or 2 or an amino acid other than valine at the position which0 corresponds to position 264 in SEQ ID NO: 1 or 2.

The polypeptide of the invention has an amino acid other than serine at the position in the amino acid sequence of the FcyRIIB-binding domain which corresponds to position 267 in SEQ ID NO: 1 or 2. The polypeptide of the invention has an amino acid other than leucine at the position in the amino acid sequence of the FcYRIIB-binding domain which corresponds to position 328 in SEQ ID NO: 1 or 2. The substituted amino acids may independently be selected from alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, glycine, serine, threonine, tyrosine, cysteine, lysine, arginine and histidine. Other (e.g. non-natural) amino acids may also be used. Preferably, the amino acid at position 262 is glutamic acid, aspartic acid, lysine or arginine, most preferably glutamic acid.

Preferably, the amino acid at position 264 is glutamic acid or aspartic acid, lysine or arginine, most preferably glutamic acid.

Preferably, the amino acid at position 267 is glutamic acid or aspartic acid, most preferably glutamic acid.

Preferably, the amino acid at position 328 is phenylalanine or tryptophan, most preferably phenylalanine.

In yet a further embodiment, the invention provides a protein comprising a dimer of the polypeptide of the invention. In some embodiments, the protein is a homo-dimer; in other embodiments it is a hetero-dimer. The polypeptide chains of ttie hetero-dimer may or may not both be polypeptides of the invention.

In some embodiments of the invention, the polypeptide consists of or comprises an Fc monomer or dimer from an immunoglobulin G (IgG). As used herein, the term "Fc" monomer includes the lower-hinge region (between the cysteines of the interchain disulphide bonds), the CH2 domain and the CH3 domain. Preferably, the protein of the invention comprises two Fc region monomers (one or both of which are polypeptides of the invention) which are covalently linked at the lower hinge via a disulfide bond. In some embodiments, the invention provides an antibody heavy chain comprising a polypeptide of the invention.

In other embodiments, the invention provides a protein comprising a dimer of two antibody heavy chains, at least one of which is a polypeptide of the invention.

In some embodiments, the invention provides an antibody comprising at least one polypeptide of the invention. Preferably, the antibody comprises two polypeptides of the invention. Preferably, the antibody comprises two heavy chains, one or both of which are polypeptides of the invention.

Antibodies of the invention include, but are not limited to monoclonal, bispecific, human, humanized and chimeric antibodies. The term antibodies also includes single chain antibodies and anti-idiotypic (anti-Id) antibodies. Preferably, the antibody is an IgG, e.g. IgGI, lgG2, lgG3 or lgG4.

The polypeptides, proteins or antibodies of the invention may be N-glycosylated by linkage to an N-glycan. As used herein, the term "N-glycan" is used to refer to an N-linked oligosaccharide, e.g. one that is or was attached by an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in a protein (e.g. Asn297). Preferably, the

polypeptides, proteins or antibodies of the invention comprise one or more types of glycan structures on the Fc domain. The predominant sugars found on such glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine

(GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (e.g., N-acetyl-neuraminic acid (NANA)). The processing of the sugar groups occurs co-translationally in the lumen of the ER and continues in the Golgi apparatus for N-linked glycoproteins.

Preferably, the polypeptides, antibodies or proteins of the invention are unglycosylated or in the native biantennary complex or homogenous Man₅GlcNAc₂ glycoform. Both latter forms maintain binding to the inhibitory receptor, FcvRIIB (Fig. S1) while showing decreased binding to all activatory receptors. These observations confirm that mutations disrupting the hydrophobic protein-glycan interface could modulate Fc binding to FcyRs independently of Fc glycoform.

In other embodiments, the polypeptides or proteins of the invention are in other homogenous naturally-occurring glycoforms, such as homogenous Man₉GlcNAc2, or homogenous hybrid glycoforms. In some embodiments, the invention provides an antibody comprising at least one (preferably one or two) antibody heavy chain, wherein at least one (preferably one or two) of the antibody heavy chains comprises a polypeptide of the invention, and wherein the antigen-binding domain of the antibody binds to a desired target. Targets of interest for therapeutic antibodies include CD2, CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD40, CD52, CD56, CD64, CD70, CD74, CD79, CD80, CD86, CD105, CD138, CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphA2, EphB2, FAP, av integrin, Mesothelin, EGFR, TAG-72, GD2, CA1X, 5T4, α4β7 integrin, Her2.

Other targets are cytokines, such as interleukins IL-I through IL- 13, tumour necrosis factors a & β, interferons a, β and γ, tumour growth factor Beta (TGF-β), colony stimulating factor (CSF) and granulocyte monocyte colony stimulating factor (GMCSF). See Human Cytokines: Handbook for Basic & Clinical Research (Aggrawal ef a/, eds., Blackwell Scientific, Boston, MA 1991). Other targets are hormones, enzymes, and intracellular and intercellular messengers, such as, adenyl cyclase, guanyi cyclase, and phospholipase C. Other targets of interest are leukocyte antigens, such as CD20, and CD33. Drugs may also be targets of interest. Target molecules can be human, mammalian or bacterial. Other targets are antigens, such as proteins, glycoproteins and carbohydrates from microbial pathogens, both viral and bacterial, and tumours. Still other targets are described in U.S. 4,366,241 (the contents of which are incorporated herein by reference).

In some embodiments, the target is a component of the B-cell receptor (BCR) complex. Preferably, the target is B-cell surface antigen, e.g. CD19, CD21, CD22, CD72, CD79a, CD79b or CD81 , preferably CD19.

In other embodiments, the polypeptide, protein or antibody of the invention is a fusion with an immunogenic therapeutic protein. Alternatively, the therapeutic protein may be linked or conjugated to the polypeptide, protein or antibody of the invention. Such a fusion conjugate may be capable of suppressing differentiation, survival or proliferation only of B cell populations possessing BCRs specific for epitopes of the therapeutic protein.

The invention therefore further provides a method of reducing the immunogenicity of a therapeutic protein, the method comprising administering an effective amount of a polypeptide, protein or antibody of the invention which is fused, linked or conjugated to the immunogenic therapeutic protein, to a patient to whom the therapeutic protein has, is being or will (independently) be administered.

The invention also provides a combined composition comprising:

(i) a polypeptide, protein or antibody of the invention which is fused, linked or conjugated to an immunogenic therapeutic protein, and

(ii) the immunogenic therapeutic protein,

for simultaneous, sequential or separate use in reducing the immunogenicity of the therapeutic protein.

For the avoidance of any doubt, it will be understood that the immunogenic therapeutic protein in (i) and (ii) are separate entities (i.e. the immunogenic therapeutic protein of (i) is fused/linked or conjugated to a polypeptide, protein or antibody of the invention whereas the immunogenic therapeutic protein of (ii) is not). The identity of the immunogenic therapeutic protein in both cases, however, is preferably the same. Polypeptides, proteins and antibodies of the invention can be incorporated into pharmaceutical compositions comprising the polypeptide, protein or antibody as an active therapeutic agent and a variety of pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pennsylvania, 1980). The preferred form depends on the intended mode of

administration and therapeutic application. The compositions can include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carrier or diluents, which are defined as vehicles commonly used to formulate pharmaceutical

compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition the pharmaceutical composition or formulation can also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. Hence the invention also provides a pharmaceutical composition comprising a polypeptide, protein or antibody of the invention, optionally together with one or more carriers, diluents or excipients.

The pharmaceutical carrier may be a liquid and the pharmaceutical composition would be in the form of a solution. Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurised compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhdric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the carrier can also be an oiiy ester such as ethyl oleate and S isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration.

Pharmaceutical compositions for parenteral administration are sterile, substantially isotonic, pyrogen-free and prepared in accordance with GMP of the FDA or similar body.0 Antibodies can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oils, saline, glycerol or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering

substances and the like can be present in compositions. Other components of

5 pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Antibodies can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained0 release of the active ingredient. Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polyactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed5 above (see Langer, Science 249, 1527(1990) and Hanes, Advanced Drug Delivery

Reviews 28, 97-119 (1997).

The polypeptide, protein, antibody or composition of the invention may be administered by any suitable route. Preferably, they are administered intra-venously (i.v.).

0

The invention may also be used in the treatment of diseases such as rheumatoid arthritis where direct or localised administration of the polypeptide, protein, antibody or composition of the invention is more desired. For example, the polypeptide, protein, antibody or composition of the invention may be injected directly into a joint.

In other embodiments, it is desirabe to administer the polypeptide, protein, antibody or composition of the invention to the lungs, e.g. via an inhaler or a nebuliser.

In yet other embodiments, it is desirable to administer the polypeptide, protein, antibody or composition of the invention to an eye, e.g. via an intra-ocular or intra-vitreol device. Methods for making the polypeptides, proteins and antibodies of the invention are well known in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See e.g., Sambrook et al.

Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press (1976); .;

Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999); Immunobiology, Janeway ef a/., 6th Edition, 2004, Garland Publishing, New York).

The invention further provides a polypeptide, protein or antibody of the invention for use as a medicament or for use in therapy.

The invention further provides a polypeptide, protein or antibody of the invention for use in the prophylaxis, prevention or treatment of an autoimmune disorder or an

inflammatory disorder. The invention further provides a method of prophylaxis, prevention or treatment of an autoimmune disorder or an inflammatory disorder, the method comprising administering an effective amount of a polypeptide, protein or antibody of the invention to a patient in need thereof.

In yet a further embodiment, the invention provides a polypeptide, protein or antibody of the invention for use in a method of suppressing immune cell activation or of immunosuppression.

The invention also provides a method of suppressing immune cell activation or of immuno-suppression comprising administering an effective amount of a polypeptide, protein or antibody of the invention to a patient in need thereof. The invention also provides a method of immuno-suppression or B-cell suppression, the method comprising administering an effective amount of an antibody of the invention, wherein the antibody comprises an antigen-binding domain having affinity for a B-cell surface antigen, to a patient in need thereof. The invention further provides an antibody of the invention, wherein the antibody comprises an antigen-binding domain having affinity for a B-cell surface antigen, for use in a method of immuno-suppression or B-cell suppression.

Preferably, the B-cell surface antigen is CD19, CD20, CD21, CD22, CD72, CD79a, CD79b or CD81.

In some embodiments, the autoimmune disease is a B-cell mediated autoimmune disease. The invention further provides a method of treating B cell lymphoma, the method comprising administering an effective amount of an antibody of the invention, wherein the antibody comprises an antigen-binding domain having affinity for CD20 on the B cell surface, wherein the Fc region of the antibody engages the FcyRllb on the same B cell, leading to B cell suppression or apoptosis, to a patient in need thereof.

The invention also provides a method of treating non-Hodgkin's lymphoma, the method comprising administering an effective amount of an antibody of the invention to a patient in need thereof.

In some embodiments, the autoimmune disorder is Type 1 diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE) or autoimmune thyroid disease (AITD).

Preferably the autoimmune disorder is rheumatoid arthritis. In this case, the antibody of the invention comprises an antigen-binding region which binds to an antigen which is associated with cartilage, e.g. Type II collagen.

In some embodiments, the inflammatory disorder is a diseases related to the

inflammation of the joints. Specific examples include Bechterew's disease, psoriatic arthritis, rheumatoid arthritis, arthritis in colitis ulcerosa, arthritis in morbus Crohn, affection of joints in systemic lupus erythematosus (SLE), systemic sclerosis, mixed connective tissue disease, reactive arthritis, Reiter^*s syndrome. Moreover, included in this embodiment of the invention is treatment of arthritis of any joint, in particular arthritis of a finger joint, the knee or the hip.

Preferably the inflammatory disorder is rheumatoid arthritis. In this case, the antibody of the invention comprises an antigen-binding region which binds to an antigen which is associated with rheumatoid arthritis, e.g. collagen, cartilage.

The invention may also be used to increase the immuno-modulatory function of therapeutic mAbs during vaccination and cancer treatment by enhancing the affinity of mAb Fc for FcyRllb. For example, the addition of anti-CD40 mAb (CD40 is a co- stimulatory molecule on surface of antigen presenting cells) during OVA vaccination of mouse, the use of murine lgG1 isotype, which has higher affinity for FcgRllb than lgG2a isotype, stimulates stronger anti-OVA immune response than lgG2a isotype, with stronger OVA-specific CD4 and CD8 T cell stimulation. This effect is abolished when FcgRllb is knocked out (see White, A. L, Chan, H. T., French, R. R., Beers, S. A., Cragg, . S., Johnson, P. W. & Glennie, M. J. (2013). FcyRllb controls the potency of agonistic anti-TNFR mAbs. Cancer Immunol Immunother 62, 941-8.; Li, F. & Ravetch, J. V. (2011 ). Inhibitory Fcgamma receptor engagement drives adjuvant and anti-tumor activities of agonistic CD40 antibodies. Science 333, 1030-4.)

In yet a further embodiment, therefore, the invention provides a method of vaccination, the method comprising administering an effective amount of an antibody of the invention, wherein the antibody comprises an antigen-binding domain having affinity for CD40, to a patient in need thereof.

The invention further provides a method of prevention, prophylaxis or treatment of cancer, the method comprising administering an effective amount of an antibody of the invention, wherein the antibody comprises an antigen-binding domain having affinity for CD40, to a patient in need thereof.

Preferably, the subject or patient to be treated is a mammal, most preferably a human.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Mass spectrometry analysis of N-glycans released from IgG Fc-F241A. (A) Negative ion ESI spectrum. (B) The data from panel A processed with the Maximum Entropy 3 function of MassLynx to convert multiply charged ions to singly charged ions. The position of the fucose residue in the triantennary glycans was not determined. The ion at m/z 3169 gave a composition corresponding to the tetra-sialylated triantennary glycan but this was not confirmed by fragmentation. (C) An example of negative ion collision-induced dissociation spectrum of the monosialylated, fucosylated biantennary glycan. Fragment ions are labelled according to the scheme devised by Domon and Costello³⁹. (D) Spectra showing tri-galactosylated structures with three (triply charged) and four (quadruply charged) sialic acids attached , respectively. Key: Integrated oligosaccharide nomenclature follows that of Bowden et al. . Residue labelling follows that of Vliegenhart et a/.⁴⁰ with the additional modifications of 7 for sialic acid, 1' for a1→6~linked core fucose⁴¹. These residue labels are in bold-face throughout the manuscript. The symbolic representation of glycans follows that of Harvey et al ⁴² with residues in both the schematic diagrams and molecular graphics following the colour scheme of the Consortium for Functional Glycomics.

Figure 2. Packing of N-link glycans in native (A-C) and F241A mutant (D-F) IgG Fc. Glycans are displayed as blue (GlcNAc), red (Fuc), and green (Man) sticks. Protein is displayed as a gray cartoon with four hydrophobic residues at the protein-glycan interface highlighted in pink (sticks). Overall structure of (A) native (PDB ID 3AVE) and (B) F241 A mutant lgG1 Fc. (B and E) The Cy2 domain is shown in together with a close-up of the hydrophobic interface (C and F). Four hydrophobic residues located on the protein-glycan interface are highlighted in pink (sticks). Electron density

corresponding to carbohydrate is depicted as a blue mesh (2F_Q-F_C map contoured at 1σ) around the carbohydrate moiety of the mutant Fc reported herein. Integrated

oligosaccharide nomenclature follows that of Bowden et al.²⁰, see legend to Fig. 1 for further details. Secondary structure was defined by Ksdssp⁴³. Figure 3. HPLC analysis of 2AA-labelled N-linked glycans from monoclonal lgG1 b12 mutants expressed in HEK 293T and HEK 293S cells. Normal-phase HPLC analysis of 2-AA-labelled N-linked glycans, released from target antibody glycoforms by in-gel protein PNGase F digestion. Glycan profile of lgG1 b12 expressed in HEK 293T (black) and HEK 293S (blue) for the following variants: (A) Wild type. (B) F241A. (C) F243A. (D) V262E. (E) V264E. The y-axis displays relative fluorescence (RF).

Figure 4. Generation of differentially glycosylated lgG1 Fc. Normal-phase HPLC analysis of 2-AA-labelled N-linked glycans, released from target antibody glycoforms by in-gel PNGase F digestion. (A) Glycan profile of monoclonal lgG1 b12. (B) Glycan profile of lgG1 incubated with 50 U/mL Clostridium perfringens neuraminidase for 48 hours at 37'C. (C) Glycan profile of lgG1 incubated with 25 pg/mL β1 ,4- galactosyltransferase (B4GALTI) and 80 μΜ uridine 5'-diphosphogalactose in 50 mM HEPES, 10 m nCI₂, pH 7.5 for 48 hours at 37'C. (D) Glycan profile of lgG1 sequentially treated with B4GALTI and a2,6-sialyltransferase I (ST6GALI) as described above. Figure 5. ELISA of monoclonal IgG variants binding human FcyRIA, FcyRIIA, FcyRIIB, FcyRIIIA and FcyRIIIB. The FcyRs were plated at 5 pg/mL overnight at 4°C, IgG variants F241A, F243A, V262E and V264E were incubated for 1.5 hrs and binding was detected by HRP-conjugated goat anti-human Fab antibody. Symbolic representation of IgG mutation and glycovariants:■ = wild type native,■ = wild type an₅GlcNAc2, Δ= mutant native, Δ = mutant Man₅GlcNAc₂,■ = wild type hypergalactosylated and hypersialylated, ELISA binding curves of the four IgG hydrophobic mutants for (A) FcyRIA, IgG variant starting concentration at 10 g/mL (B) FcyRIIA, IgG variant starting concentration at 100 pg mL. (C) FcyRIIB, IgG variant starting concentration at 300 pg/mL. (D) FcyRIIIA, IgG variant starting concentration at 100 pg mL. (E) FCYRIIIB, IgG variant starting concentration at 300 pg/mL (F) FcyRIIIA, IgG variant starting

concentration at 100 pg/mL All data points represent the calculated mean of two independent measurements from a total of at least two experiments.

Figure 6. ELISA of monoclonal IgG variants binding human FcyRIA, FcyRIIA, FcyRIIB, FcyRIIIA and FcyRIIIB. The FcyRs were plated at 5 pg mL overnight at 4°C, IgG variants S267E/L328F, V262E/S267E/L328F and V264E/S267E/L328F were incubated for 1.5 hrs and binding was detected by HRP-conjugated goat anti-human Fab antibody. Symbolic representation of IgG mutation and glycovariants:■ = wild type native,■ = wild type Man₅Glc Ac₂, Δ = mutant native, Δ = mutant ansGlcNAc2, ELISA binding curves of the four IgG hydrophobic mutants for (A) FcyRIA, IgG variant starting concentration at 10 pg/mL. (B) FcyRIIA, IgG variant starting concentration at 100 pg/mL. (C) FcyRIIB, IgG variant starting concentration at 300 pg mL. (D) FcyRIIIA, IgG variant starting concentration at 100 pg/mL. (E) FcyRIIIB, IgG variant starting concentration at 300 pg/mL. All data points represent the calculated mean of two independent

measurements from a total of at least two experiments. Flgure 7. SPR analysis of monoclonal IgG variants binding to human FcvRIIIA. The human FCYRIIIA was immobilized on the CM5 sensorchip by amine coupling. The IgG variants were injected at 5 different concentrations at a flow rate of 30 μΙ/min: IgG and IgG Hypersialylated (0.67, 0.33, 0.17, 0.083, and 0.042 μΜ); IgG Man₅GlcNAc₂ (0.33, 0.17, 0.083, 0.042 and 0.021 μΜ). The association time was 2 minutes and dissociation time was 3 minutes. The chip was regenerated with 10 mM glycine-HCI, pH1.7.

Sensorgrams were fitted with a global 1 :1 interaction, and the g, _d, and Ko were calculated, all using BIAevaluation software 2.0.3. KD values are reported as Mean+SD, and sensorgrams are representative a total of 3 independent experiments.

Figure S1. Electrospray ionization mass spectrometry analysis of N-glycans released from lgG1 b12 mutants. Spectra shown represent singly charged ions and were extracted using ion mobility from their respective negative ion ESI spectra. Spectra showing doubly charged ions from (A) Native, (B) F241A, (C) F243A, (D) V262E.

Masses, compositions and structures of the N-glycans are shown in Table S1.

Figure S2. Electrospray ionization mass spectrometry analysis of N-glycans released from lgG1 b12 mutants. Spectra shown represent doubly charged ions and were extracted using ion mobility from their respective negative ion ESI spectra. Spectra showing doubly charged ions from (A) F241A, (B) F243A, (C) V262E. Key: The symbolic representation of glycans follows that of Harvey et al (S5) with residues in both the schematic diagrams and molecular graphics following the color scheme of the Consortium for Functional Glycomics. Masses, compositions and structures of the N- glycans are shown in Table S1.

Figure S3. MALDI-TOF mass spectrometry analysis of N-linked glycans released from IgG b12 variants and their desialylated counterparts. N-linked glycans were released from IgG by PNGase F digestion. For desiaiylation, released sugars were treated with neuraminidase. N-linked glycans were analyzed by MALDI-TOF mass spectrometry, DHB was used as the matrix. (A) Wild type. (B) F241A. (C) F243A. (D) V262E. (E) V264E. Figure S4. HPLC analysis of 2AA-labelled, desialylated N-linked glycans from monoclonal lgG1 b12 mutants expressed in HEK 293T. Normal-phase HPLC analysis of 2AA-labelled N-linked glycans, released from target antibody glycoforms by in-gel PNGase F digestion, followed by neuraminidase digestion. Glycan profile of

neuraminidase-treated lgG1 b12 variants expressed in HEK 293T: (A) Wild type. (B) F241A. (C) F243A. (D) V262E. (E) V264E. (F) V264E treated with only a2,3-specific neuraminidase.

Figure S5. HPLC analysis of sialylated structures of mutant V264E. Individual HPLC sialylated peaks were collected, neuraminidase-treated, and re-analyzed by HPLC.

(A) Complete HPLC profile of V264E expressed in HEK 293T.

(B) HPLC profile of neuraminidase-digested V264E.

(C) HPLC spectrum of desialylated peak 1.

(D) HPLC spectrum of desialylated peak 2.

(E) HPLC spectrum of desialylated peak 3.

(F) HPLC spectrum of desialylated peak 4.

(G) HPLC spectrum of desialylated peak 5.

(H) HPLC spectrum of desialylated peak 6.

(I) HPLC spectrum of desialylated peak 7.

Figure S6. Mass spectra of trypsin-digested, native and deglycosylated IgG. IgG variants were PNGase-treated, then reduced, alkylated and digested with trypsin for 6 hours at 37°C, before analysis by MALDI-TOF MS. The tryptic peptide mass of the native and deglycosylated IgG were calculated online using the PeptideMass program at the ExPASy SIB Bioinformatics Resource Portal. (A) Mass spectrum of trypsin- digested native IgG. (B) Mass spectrum of trypsin-digested, deglycosylated IgG. The deglycosylated peptide containing the conserved N-glycosylation site, Asn297, is highlighted in red. Figure S7. Stacked plot of mass spectra of trypsin digested, deglycosylated native IgG and deglycosylated hydrophobic mutants. IgG deglycosylation was achieved by

PNGase F-digestion overnight. For trypsin digestion, IgG variants were reduced, alkylated and digested with trypsin for 6 hours before analysis by MALDI-TOF MS. The tryptic peptide mass of the native and degiycosylated IgG were calculated online using the PeptideMass program at the ExPASy SIB Bioinformatics Resource Portal. (A) Native glycosylated IgG. (B) Degiycosylated IgG. (C) Degiycosylated F241A IgG variant. (D) Degiycosylated F243A IgG variant. (E) Degiycosylated V262E IgG variant. (F) Degiycosylated V264E IgG variant. The position of peptide peaks corresponding to the native degiycosylated peptide containing the conserved N-glycosylation site, Asn297, is indicated by a red vertical line. The position of the peptide peaks corresponding to non- glycosylated peptide containing the conserved N-glycosylation site, Asn297, is indicated by a blue vertical line.

Figure S8. HPLC analysis of 2AA-labelled N-linked glycans from IgG b12 mutants expressed in HEK 293T and HEK 293S cells. Normal-phase HPLC analysis of 2-AA- labelled N-linked glycans, released from target antibody glycoforms by in-gel PNGase F digestion. Glycan profile of IgG expressed in HEK 293T (black) and HEK 293S (blue) for the following variants: (A) Wild type. (B) V262E/S267E/L328F. (C) V264E/S267E/L328F. Symbolic representation of glycan structures follows that of Harvey et a/.(S5): = NeuNAc, = Gal,■ = GlcNAc, · = Man,♦ - Fuc. The linkage position is shown by the angle of the lines linking the sugar residues (vertical line = 2-link, forward slash = 3-link, horizontal line = 4-link, back slash = 6-link). Anomericity is indicated by full lines for β- bonds and broken lines for a-bonds.

Table S1. List of masses, compositions and structures of the N-giycans derived from electrospray mass spectrometry and then converted into singly and doubly charged ions using ion mobility extraction.

Table S2. Summary of single IgG b12 mutants' affinity for FcyRs relative to the native (black) or MansGlcNAc2 (blue) wild type glycoforms. Each value represents the apparent affinity of a particular mutant glycoform over the same wild type glycoform (i.e. value <1 indicates weaker binding than the same wild type glycoform and vice versa). All data points represent the calculated mean of two Independent measurements from a total of at least two experiments. Table S3. Summary of double and triple IgG b12 mutants' affinity for FcyRs relative to the native (black) or Man₅GlcNAc2 (blue) wild type glyooforms. Each value represents the apparent affinity of a particular mutant glycoform over the same wild type glycoform (i.e. value <1 indicates weaker binding than the same wild type glycoform and vice versa). All data points represent the calculated mean of two independent measurements from a total of at least two experiments.

EXAMPLES

The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

The disclosure of each reference set forth herein is incorporated herein by reference in its entirety. Example 1 : Experimental procedures

Molecular cloning and mutagenesis. The pFUSE vector with the human lgG1 Fc insert was obtained from Invivogen, UK. The vectors encoding lgG1 b12 light and heavy chains were kindly provided by Prof. Ian A. Wilson (The Scripps Research institute, CA, USA). Protein mutagenesis was performed using the QuikChange kit (Agilent

Technology, UK) to generate the lgG1 Fc mutant F241A and the full-length lgG1 b12 mutants F241A, F243A, V262E, and V264E. The mutated Fc (residues 225-447, SWISS-PROT accession number P01857.1) encompassing hinge, Cy2 and Cy3 domains was cloned into the mammalian expression vector, pHLSec. The vectors containing fulMength Fc RIA, FcyRIIA (His131 variant), FcyRIIB, FCYRIIIA (Val158 variant), FCYRIIIB, and mouse β-1 ,4-galactosyltransferase I (B4GALTI), were ail purchased from Open Biosystems, UK. The vector containing full-length rat a2,6- sialyltransferase I (ST6GALI) was a gift from Prof. Karen Colley (University of Illinois, IL, USA). The soluble extracellular regions of each FCYR, B4GALTI and ST6GALI were cloned into the pHLSec vector as described for the Fc: FcyRIA (residues 16-288;

SWISS-PROT accession number BC152383); FcyRIIA (residues 34-217; SWISS- PROT accession number BC020823); FcyRIIB (residues 42-225; SWISS-PROT accession number N _001190828) FcyRIIIA (residues 16-288; SWISS-PROT accession number BC033678); FCYRIIIB (residues 17-200; SWISS-PROT accession number BC128562); ST6GAL1 (residues 89-403; SWISS-PROT accession number NP_001106815); and B4GALTI (residues 127-399; SWISS-PROT accession number BC053006). For B4GALTI, the residue Cys339 was mutated to Thr to minimise the potential for aggregation. The pHLSec vector encodes a hexahistidine tag at the C- terminus.

Protein expression. The Fc, FcyRs, B4GALTI and ST6GALI were expressed in HEK 293T cells. Briefly, HEK 293T cells (ATCC number CRL-1573) were grown to 90% confluence and transiently transfected with polyethyleneimine (PEI)55, using a transfection mix with DNA and PEI in ratio of 1 :1.5. Following transfection, cells were grown in DM EM/1% fetal bovine serum at 37°C, 5% CO2 for 5 days. Protein was purified from cell supernatant by immobilized metal affinity chromatography using Chelating Sepharose Fast Flow Ni²⁺ -agarose beads (GE Healthcare, UK) followed by size exclusion chromatography using a Superdex S-200 column equilibrated in phosphate buffered saline (PBS) (for FCYRS) or 10 mM HEPES pH 7.4, 150 mM NaCI (for Fc). Full-length lgG1 b12 was transiently expressed in HEK 293T or GnT l-deficient HEK 293S cells. Prior to transfection, light and heavy chain plasmids were mixed in a mass ratio of 4:1 ; and the total DNA was mixed with PEI in a mass ratio of 1 :1.5. After incubation for 4 days at 37°C, cell culture supernatant was harvested and lgG1 b12 was purified using Protein A Sepharose (GE Healthcare, UK) according to the

manufacturer's directions. Enzymatic release of N-linked qlvcans. Oligosaccharides were released from

Coomassie blue-stained reducing SDS-PAGE gel bands containing approximately 40 pg of IgG Fc. Gel bands were excised, washed with acetonitrile and water, and dried. Gel bands were rehydrated with 30 pl_ of 30 mM NaHCC>3 pH 7.0 containing 100 Units/mL PNGase F (New England Biolabs, UK) and incubated for 12 h at 37^*C. The enzymatically released N-linked glycans were eluted with water. Desialylation was carried out using linkage non-specific neuraminidase from Clostridium perfringens (New England Biolabs, UK) for 48 hours at 37'C.

Structural determination of N-alvcans. Glycans were labelled with anthranilic acid (2-AA) and separated using TSK amide-column (Sigma-Aldrich, UK). The released N-glycans, dissolved in water and 2-AA labelling buffer (3/8, v/v) were mixed with 2-AA and sodium cyano-borohydride and incubated for 1 h at 80^*C; excess 2-AA dye was removed using a Speed Amide-2 column (Systematic Systems, UK). High pressure liquid

chromatography (HPLC) was carried out in a linear gradient of solvents at room temperature. Solvent A was acetonitrile, solvent B was MilliQ water, and solvent C was 800 mM ammonium hydroxide adjusted to pH 3.85 using acetic acid. Solvent C was in a constant gradient of 2.5% throughout the run. The gradient was a constant 71.6% A for 6 mins at a flow rate of 0.8 mlJmin, followed by a linear gradient of 71.6-35% A over 80 mins at 0.8 mL min. Afterwards, the gradient was a linear 35-71.6% A for 1 min at a flow rate of 0.8 mL/min; then at the same gradient, the flow rate increases from 0.8 mL/min to 1.2 mlJmin over 1 minute and followed by the same gradient and flow rate for 13 minutes. The run finished by returning the flow rate to 0.8 mL/min over 1 min.

Fluorescence was detected at 425 nm and the excitation wavelength was 360 nm. Chromatography data were processed by the Empower software (all instruments and software from Interlink Scientific Services Limited, UK). Assignments were consistent with previously reported serum IgG N-linked glycan profiles ³⁰ and were confirmed by MALDI-TOF MS and exoglycosidase analysis. ln vitro modulation of laG alvcosylation. Hyper-a2,6-sialylated IgG was generated by incubating with B4GALTI in the presence of 80 μΜ Uridine 5'-diphosphogalactose (Sigma-Aldrich, UK) in 50 mM HEPES, 10 mM MnCi₂, pH 7.5 for 48 hours at 37'C. The hyper-βΐ ,4-galactosylated IgG was treated with ST6GALI in the presence of 70 μΜ cytidine-5'-monophospho-N-acetylneuraminic acid (Sigma-Aldrich, UK) in 50 mM HEPES, 10 mM MnCI₂, pH 6.5 for 48 hours at 37'C. The composition of the glycoform was verified by HPLC analysis after each enzymatic treatment. IgG deglycosylation was confirmed by a protein band shift in SDS-PAGE.

Crystallization and Structure Determination.

Recombinantly expressed mutant lgG1 Fc (F241 A) was concentrated to 7.0 mg/mL and crystallised after 10 days using the sitting drop vapour diffusion method using 100 nL protein plus 100 nL precipitant equilibrated against 95 \il reservoirs. Crystals of F241A lgG1 Fc grew at room temperature in a precipitant containing 28% polyethylene glycol monomethyl ether 2,000 in 0.1 M BIS-TRIS buffer at pH 6.5. Crystals were flash frozen by immersion in a cryoprotectant containing the mother liquor diluted in 30%

polyethylene glycol and then rapidly transferred to a gaseous nitrogen stream.

Crystallographic data were collected to 1.9 A resolution at beamline 104-1 at the Diamond Light Source (Oxfordshire, UK). Images were indexed, integrated, and scaled using HKL2000. The structure was solved using molecular replacement with the program PHASER59 using native Fc (PDB accession no. 3AVE) as a search model. Model building was performed with COOT and iteratively refined using restrained refinement in the CCP4 supported program, Refmac5, with the incorporation of translation-libration-screw (TLS) parameterization and automatically generated local non-crystallographic symmetry restraints. Model quality was validated with Molprobity. Data processing and refinement statistics are presented in Table 1.

FcvR binding assays. Recombinant FcyRs at 5 pg/mL in PBS were coated on high- binding microtitre plates (3690, Corning, NY, U.S.A.) overnight at 4^*C. Coated plates were washed with PBS containing 0.05% Tween 20 (Sigma-Aldrich-aldrich, U.S.A.) and blocked for 1 hour at room temperature with 5% bovine serum albumin (BSA) in PBS. ecombinant lgG1 b12 expressed from HEK 293T cells or from GnT l-deficient HEK 293S cells were then added and allowed to bind for 1.5 hours at room temperature. Plates were washed five times with PBS containing 0.05% Tween and binding was detected using a horse radish peroxidase (HRP)-conjugated Fab fragment specific for Human IgG Fab (Abeam, UK). The 3,3' ,5,5' -tetramethylbenzidine substrate (TMB; Thermo Scientific, U.S.A.) was used for development according to the manufacturer's directions and was stopped by the addition of 2M H₂S0₄. Absorbance was measured at 450 nm on a Spectramax 5 (Molecular Devices, California, U.S.A.) multiwell plate reader. Apparent affinity was calculated as the concentration of lgG1 b12 corresponding to half-maximal binding on the ELISA binding curve.

Accession Code.

The atomic coordinates and crystallographic structure factors of the F241A lgG1 Fc have been deposited in the Protein Data Bank (PDB) under the accession code 4BM7.

Surface Plasmon Resonance.

SPR experiments were carried out using the BIAcore T100 instrument (GE Healthcare, UK). Briefly, the FcyRIIIA (Val158 variant) was immobilized onto the surface of CM5 sensor chip (GE Healthcare, UK) to about 1000 RU during each independent experiment. All experiments were carried out in the HBS-EP running buffer (10 mM HEPES, 150 mM NaCI, 3mM EDTA and 0.005% Surfactant P20), at a flow rate of 30 μΙ/min. The monoclonal lgG1 b12 protein and its hypersialylated and high-mannose glycol-variants were injected at 5 different concentrations, allowed 2 minutes for association, and 3 minutes for dissociation. After each run, the sensor chip was regenerated using 10 mM glycine-HCI, pH1.7. The sensorgrams were fitted to a global 1:1 interaction, and the ka, kef, and KD were calculated, all using BIAevaluation software 2.0.3 (GE Healthcare, UK).

Electrosprav ionization (ESI) mass spectrometry

Electrospray mass spectrometry was performed with a Waters Synapt G2 instrument (Waters MS Technologies, Manchester, UK) in negative ion mode. Samples in 1 :1 (v:v) methanohwater were infused through Waters thin-wall borosilicate nanospray capillaries. The ion source conditions were: ion source temperature, 120^*C; infusion capillary potential, 1.2 kV; cone voltage 180 V; RF-1 voltage 150 V. Spectra (2 sec scans) were acquired with a digitization rate of 4 GHz and accumulated until a satisfactory signal.noise ratio had been obtained. The ions from neutral glycans were observed as [Μ+Η₂Ρ0₄Γ adducts, the phosphate arising from residual phosphate in the solution whereas acidic (sialylated) glycans gave [Μ-ΗΓ ions. For MS/MS data acquisition, the parent ion was selected at low resolution (about 5 m/z mass window) to allow transmission of isotope peaks and fragmented with argon at a pressure (recorded on the instrument's pressure gauge) of 0.5 mBar. The voltage on the collision cell was adjusted with mass and charge to give an even distribution of fragment ions across the mass scale (typically 80D120 V). Other voltages were applied as recommended by the manufacturer. Instrument control, data acquisition, and processing were performed with MassLynx software Version 4.1 (Waters). The negative ion collision-induced

dissociation (CID) spectra provide highly diagnostic fragmentation spectra that enable structural assignment of the glycans. For a more extensive discussion of these techniques, see Harvey er al. (Si, S2).

Fine structural and compositional analysis of sialylated glycans from the generated Fc mutants was also performed using negative ion ESI CID mass spectrometry (S2) (Figure S1). This method is illustrated in the spectrum of the [ -H]^~ ion of the monosialylated biantennary glycan (Figure S1 C). The presence of NeuSAc is indicated by the Bi ion at m/z 290 and the absence of an ion at m/z 306 indicates that this ion is a2-3-linked (S3). The C| ion at m/z 179 shows hexose (galactose) at the non- reducing terminus of the other antenna. The ^1,3A$ and ^1,3A /Yi ion at m/z 424 contains the Gal-GlcNAc sequence from the antennae and the ion at m/z 655 (not labelled) is the B3 ion Neu5Ac-Gal-GlcNAc. D and D-18 ions arising from the 6-antenna appear at m/z 688 and 670 respectively. Ions at m/z 1478, 1418 and 1275 are the diagnostic ^2,4A₇, B₆ and ^Ae ions from the /V-acetylchitobiose core, with additional loss of the sialic acid and locate the fucose residue to the reducing-terminal GlcNAc. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALD1- TOF MS) analysis

Oligosaccharides were released from target glycoproteins with PNGase F (New

England BioLabs) from Coomassie blue-stained NuPAf2455561GE gels (S4). Excised bands were washed five times alternately with acetonitiile and deionized water and rehydrated with 3000 U/ml aqueous PNGase F solution. After 12 h of incubation at 37°C, the enzymatically released N-linked giycans were eluted with water. Samples were analyzed by MALDi-TOF mass spectrometry with a Shimadzu AXIMA MALDI TOF/TOF mass spectrometer (Kratos Analytical, Manchester, U.K.) equipped with delayed extraction and a nitrogen laser (337 nm). Samples were cleaned on a Nafion 117 membrane (Sigma-Aldrich) and then prepared for MALDI-mass spectrometry by adding 0.5 μΙ aqueous solution of the giycans to the matrix solution (0.3 μΙ solution of 2,5- dihydroxybenzoic acid in acetonitrile/water; 1:1, v/v) on the stainless steel target plate and allowing it to dry at room temperature. The sample/matrix mixture was then recrystallized from ethanol.

In-Gel Trvptlc digestion of native and dealvcosylated laG variants

Deglycosylated IgG were prepared by PNGase F digestion (New England Biolabs, UK).

In-gel digestion of native and hydrophobic IgG mutants was carried out using the In-Gel Tryptic Digestion Kit (Thermo Scientific, UK) according to manufacturer's protocol.

Briefly, gel bands containing IgG were excised and destained extensively, followed by reduction and alkylation. The gel slices were then digested with trypsin for 6 hours at 37'C and the peptides were extracted with 1% trifluoracetic acid (Sigma-Aldrich, UK). The peptides were analyzed on a MALDI-TOF MS instrument (Kratos Analytical, Manchester, U.K.), using a-Cyano-4-hydroxycinnamic acid as the matrix. The expected tryptic peptide mass of the native and deglycosylated IgG were calculated online using the PeptideMass program at the ExPASy SIB Bioinformatics Resource Portal. The lack of intrinsic N-glycosylation at Fc Asn297 was assessed by the detection of the tryptic peptide EEQYNSTYR, which contains the N-glycosylation site Asn297 and results from non-glycosylated lgG1 Fc. The equivalent tryptic peptide resulting from the normal glycosylated, PNGase F-treated lgG1 Fc, is EEQYDSTYR. Example 2: Crystal lographic analysis of lgG1 Fc F241A mutant

To investigate the structural impact of mutations at the lgG1 Fc protein-glycan interface, we determined the crystal structure of recombinant lgG1 Fc F241A. This mutant has been previously reported by Lund et al. to reduce FcyR binding and increase glycan processing²⁸. Prior to crystallization, we performed electrospray ionization (ESI) mass spectrometric analysis of the N-linked glycans which confirmed extensive branching and terminal sialylation (Fig.1 ).

The lgG1 Fc F241A crystallized in the primitive ortho-rhombic spacegroup, P2i2i2i, with one molecule of the Fc homodimer in the asymmetric unit. Data were collected to a resolution of 1.9 A (Table 1). As observed in many crystal structures of the Fc, there was notable asymmetry between the two chains of the Fc. The protein and glycan moieties of the Cy2 domain from one chain (chain B) exhibited a high degree of disorder (Fig. 2D).

Table 1. Crystallographic data and refinement statistics F241 A Fc.

"Numbers in parentheses refer to the relevant outer resolution shell.

Emerge =∑hki Z\\l( kl;i) - </ M/ >|/∑_hki∑il(hki;i), where l(hkl;i) is the intensity of an individual measurement and <l(hkl)> is the average intensity from multiple observations.

"Rwoik ~ ShklHFobsl - /fl calcH Shkl |Fo s|

d/¾ree is calculated as for Rwork, but using only 5% of the data which were sequestered prior to refinement.

"r.m.s.d.: root mean square deviation from ideal geometry.

fa.s.u.: asymmetric unit.

⁹Ramachandran plots were calculated with olprobity³⁷. Interpretable electron density was observable for seven monosaccharide residues on chain A (Fig. 2E and 2F). In the structure of the wild type Fc, Phe241 participates in well-characterized CH-TT interaction with the GlcNAc2 residue ^4,28 (Fig. 2B and 2C). In contrast, electron density in our structure of the F241 A mutant, revealed the F241 A site- directed substitution and a destabilization of the protein-carbohydrate inter-face (Fig. 2E and 2F). The N-linked glycan rests in the established position along the Cv2 domain with defined electron density corresponding to the 6-arm whilst only diffuse density corresponding to the 3-arm is observed in the 2Fc—Fc map.

This weak density indicates that the 3-arm is either conformationally disordered or glycoforms differing in 3-arm composition are adopting different conformations where the X-ray scattering does not sum to yield consensus electron density. Previous reports had suggested that the intrinsic flexibility of the 3-arm causes the lack of electron density of the galactose in native galactosylated Fc ³·³⁸. Consistent with a loss of electron density of the 3-arm, detailed negative-ion electros-pray ionization mass spectrometry with fragmentation analysis revealed extensive branching on the 3-arm but not the 6-arm (Fig. 1).

The localized induction of disorder at the 3-arm does not fully account for the apparent increase in accessibility of the glycans to Golgi-resident glycosyltransferases (e.g. increase in 6-arm galactosylation) or the reduction in FcyR bind-ing affinity ²⁸ (Fig. 2). The mutation may influence the dynamics of the glycan-protein interface, not sufficiently captured by low-temperature X-ray crystallographic methods ³³. For example, the hydrophobic interface mutations may affect the position of equilibrium between the protein 'bound' and 'free' conformations proposed by NMR studies to yield more accessible glycans and potentially more widely spaced Cv2 domains ³³. Interestingly, NMR studies have indicated that a2,6-sialylation has minimal impact on glycan dynamics in the Fc domain ^M. This finding is entirely consistent with our observation of an elevation in 6-arm sialylation by electrospray mass spectrometry and the conserved conformation of the 6-arm observed by X-ray crystallography (Fig. 2C and 2F).

Having established that mutations at the glycan-protein interface can induce at least localized glycan disorder, we next sought to determine to what extent the reduction in FCYRIIIA binding can be attributed to disruption of the glycan-protein interface or to changes in glycan processing. To this end we used a combination of site- directed mutagenesis and glycan engineering. Example 3: HPLC analysis of lgG1 glycoforms.

A series of human lgG1 Fc mutants was generated containing mutations that modulated the protein-carbohydrate interactions within the Fc: F241A, F243A, V262E, and V264E. The panel of lgG1 b12 mutants were expressed in either human embryonic kidney (HEK) 293T or GlcNAc transferase (GnT) l-deficient HEK 293S cells ^M. The glycans were released from the purified antibody via protein N-glycosidase F (PNGase F). The free sugars were fluorescently labelled and resolved via normal-phase high- performance liquid chromatography (HPLC) using a TSK-amide column. The HPLC spectra from lgG1 Fc mutants expressed in HEK 293T (black spectra) or GnT l-deficient HEK 293S cells (blue spectra) are shown in Fig. 3.

The glycans from IgG b12 expressed in HEK 293T cells, show a series of fucosylated, biantennary, complex-type carbohydrates, typical of the protein-directed glycosylation observed for IgG (Fig. 3A; black spectrum). The most abundant species observed were agalactosylated structures with smaller amounts of mono and di-galactosylated structures. A small population of sialylated material was also present, showing the typical glycan profile for recombinant lgG1 Fc as reported previously ^20,3S. Consistent with previous analyses of lgG328, mutations interrupting the hydrophobic protein-glycan interface led to dramatic increases in terminal β1 ,4 linked galactose levels, with digalactosylated species representing the most abundant glycan populations for all mutants generated from HEK 293T cells (Fig. 3B-E; black spectra). Tri-antennary and tetra-antennary species, not normally observed on Fc, were detected, most notably on V262E and V264E. Additionally, increased bisecting GlcNAc and terminal sialylation were also evident for these mutants. An unusual digalactosylated, trisialylated species was also detected in the HPLC spectra of all of the mutants. Similar unusually siaylated structures have been detected in mouse serum glycoproteins ^'⁴⁶. This structural assignment was confirmed by electrospray mass spectrometry of recombinant lgG1 Fc (Fig. 1) and b12 mutants (Fig. S1 and Fig. S2). Further matrix-assisted laser

desorption/ionization time-of-flight mass spectrometry ( ALDI-TOF-MS) (Fig. S3), and HPLC analysis of desialylated mutants (Fig. S4; S5) were carried out to validate the HPLC assignments. ln contrast to the heterogeneous complex-type glycan spectra observed when the IgG panel was expressed in HEK 293T cells, expression in GnT (-deficient HEK 293S cells led to a purely oligomannose-type glycan profile composed of an₅GlcNAc₂ together with a small population of fucosylated Man₅GlcNAc₂ (Fig. 3; blue spectra). The latter structure has previously been shown to arise via the inefficient GnT (-independent fucosylation pathway ⁴⁷. Thus mutations that disrupt the glycan-protein interface affected neither oligomannose processing to an₅GlcNAc2 nor the proportion of GnT I- independent fucosylation. In addition to glycan analysis, the N-glycosylation site occupancy at the Asn297 was examined, as previous report showed decreased glycan site occupancy for the hydrophobic mutants ²⁸. A combination of PNGase F treatment and trypsin digestion, followed by MALDI-TOF- S, showed that the N-glycosylation site on Fc Asn297 is fully occupied for all the mutants (Fig. S6; S7). The discrepancy might be due to the different methods used to examine site occupancy.

To investigate the impact of sialylation on FcyR binding, in vitro

glycosyltransferase reactions were used to generate hyper-a2,6-sialylated IgG (Fig. 4). As there is a potential for a2,3-iinked sialic acid to be present in glycoproteins derived from HEK 293T cells, we first performed a sialidase digestion. Sialylation was not detected by HPLC analysis (Fig. 4A and 4B). The sialidase-treated material was then subjected to sequential pi,4-galactosylation and a2,6-sialylation (Figs. 4C and 4D, respectively). HPLC analysis revealed that a trace population (~5%) of

monogalctosylated glycans remained after the galactosyltransferase reaction (Fig. 4C) which corresponded to processing of the 3-arm but not 6-arm. This observation supports the model of steric inaccessibility of the 6-arm GlcNAc5' residue ³² (Fig. 1 ). Example 4: Affinity of lgG1 Fc mutants for Fc receptors.

We determined the role of the disrupted protein-glycan interactions in IgG Fc binding to Fc-FcvRs by determining the binding of our panel of IgG mutants to recombinant FcvRIA, FcyRIIA, FcyRIIB, FcvRIIIA, and FcyRIIIB (Fig. 5).

Consistent with studies with FcvRIIIA, the data show that abolition of the Fc glycan-protein hydrophobic interaction resulted in a decreased Fc affinity for the FcyRIIIA (Fig. 5D)²⁸. A similar pattern, albeit to different extents, was observed in the binding analysis with FcyRIA, FCYRIIA, FcyRIIB, and FcvRIIIB (Fig. 5). The high affinity, activatory FcyRIA and low-affinity, inhibitory FcyRIIB were least prone and FcyRIIIA. Interestingly, the V264E mutant contains the highest level of sialylated structures among mutants tested (Fig. 3E). V264 packs directly against the core residue, Glc-NAc2 (Fig. 2C). The V264E mutation would be predicted to perturb the overall trajectory of the glycan away from the surface of the Cv2 domain and may account for the extensive glycan terminal processing due to increased steric accessibility.

To investigate the effect of these hydrophobic mutations on FcyR binding independent of the differential Fc glycoforms, we compared FcyR binding to the panel of uniformly glycosylated lgG1 Fc mutants expressed in the GnT l-deficient HEK 293S cells (Fig. 3; blue spectra).

Consistent with previous reports, wild type lgG1 Fc with ansGlcNAc2 glycosylation exhibits increased affinity for FcyRIIIA (Fig. 5D), and decreased affinity for the FcyRIIB (Fig. 5E) compared with Fc with native biantennary complex glycans ^ ⁴⁸. We also demonstrate that the Man5GlcNAc₂ glycoform exhibits decreased Fc affinity for FcvRIIA which is highly homologous to FcyRIIB (Fig. 5B and 5C).

Mutations that disrupt the hydrophobic interface significantly decrease the Fc affinity for FcvRIIA, FcyRIIIA and FcyRIIIB, largely independently of the glycoform. This effect is evident by comparison of the wild-type and mutants expressed in GnTI-deficient HEK 293S cells (Fig. 3; blue spectra). Mutations affecting the hydrophobic interface resulted in significantly decreased Fc affinity for FcyRIIA, FcyRIIIA and FcyRIIIB (Fig. 5B, D, E), whilst interestingly, no significant changes were observed for FcyRIIB binding (Fig. 5C).

For the high affinity FcyRIA, only V264E caused a significant decrease in binding

(Fig. 5A). Together, the results show that the hydrophobic mutations disrupt the Fc binding to the activatory FcyRs independently of the Fc glycan, indicating that the productive engagement of Fc-FcyR requires the protein-glycan interaction at the Fc Cv2 domain.

Unlike F241A, V262E, and V264E, the F243A mutant expressed as the

Man₅Glc Ac₂ glycoform has a minimal effect on FcyRs binding (Fig. 5). This minimal effect can be explained by the different protein-glycan interfaces of oligo-mannose-, hy rid- and complex-type antibody glycoforms as revealed by X-ray crystallography . In contrast to the other residues, F243 exhibits minimal van der Waals contacts with the 6-arm mannose residues in the predicted structure of the ansGlcNAc2 glycoform²⁰. Therefore, in contrast to the significant effect of F243A mutation on the mobility of complex-type glycans, F243A would be predicted to have a minimal impact on the mobility of an₅GlcNAc2 structures. The relative affinity of the single mutants for FcyRs is summarized in Table S2.

Remarkably, the interaction between FcyRIIB and lgG1 Fc is relatively

unperturbed by the disruption of Fc protein-glycan interactions (Fig. 5C). We

hypothesized that further enhancements to the FcyRIIB selectivity could be achieved by combining our glycan-protein interface mutations with previously reported hinge- proximal Cv2 mutations (S267E/L328F) that exhibit selective FcyRIIB-binding ^24,25. We generated two novel lgG1 Fc mutants: V262E/S267E/L328F and V264E/S267E/L328F which exhibited enhanced affinity for the FcyRIIB and, by comparison to the double mutant alone ^24,25, significantly decreased affinity for FcyRIA, FcyRIIA and FcyRIIIB (Fig. 6; Table S3). The double mutant S267E/L328F shows significantly increased affinity for the inhibitory FcyRIIB and decreased affinity for the activatory FCYRIIIA, while the binding for the other FcyRs remain similar to the wild type (Fig. 6), consistent with published data ²⁵. The increased levels of glycan terminal processing and bisection of these triple mutants are comparable to those of the single V262E and V264E mutants. (Fig. S8).

Example 5: Affinity of hypersialylated lgG1 Fc mutants for FCYRIIIA. The results indicate that destabilisation of glycan-protein in-teractions modify the Fc structure relevant for FcyR binding (Fig. 5). However, it has previously been shown that hypersialylation of lgG1 Fc, regardless of the linkage type, decreases Fc binding to the FcyRIA, FcyRIIB and FcyRIIIA, which also reduces antibody-mediated cytotoxicity both in vivo and in vitro ^{11 ,49,5}°. Therefore, we generated hypergalactosylated and

hypersialylated human lgG1 Fc (Fig. 4) and examined their affinity for FcyRIIIA, a critical determinant of natural killer cell-mediated ADCC. Usually, the Fc-FcyR affinity strongly correlates with effector functions measured by cellular assays ¹⁷·²⁴·²⁵·^{51 5} . However, our data indicate that the hypersialylated Fc binds to the FcvRNIA with very similar affinity to the wild type, supported by both ELISA (Fig. 5F) and surface plasmon resonance (SPR) data (Fig. 7). One possible explanation for the reduced cytotoxicity is that terminal sialic acid might interact with inhibitory sialic acid binding Ig-like lectins (Siglec) present on the surface of immune cells including macrophages and natural killer cells ⁵⁵. This minimal effect of Fc sialylation on FCYRIIIA binding is also supported by the structural analysis presented here, which reveals that the protein-glycan interface, rather than glycan terminal processing, modulates Cy2 domain conformation (Fig. 5). REFERENCES

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(67) Davis, I. W.; Leaver-Fay, A.; Chen, V. B.; Block, J. N.; Kapral, G. J.; Wang, X.; Murray, L. W.; Arendall, W. B., 3rd; Snoeyink, J.; Richardson, J. S.; Richardson, D. C. Nucleic Acids Res 2007, 35, W375. Additional references

51. Harvey, D. J., Royle, L, Radcliffe, C. M., Rudd, P. M., and Dwek, R. A. (2008) Structural and quantitative analysis of /V-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry, Anal.

Biochem. 376, 44-60.

52. Harvey, D. J. (2005) Fragmentation of negative ions from carbohydrates: Part 1.

Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from /V-linked carbohydrates, J. Am. Soc. Mass Spectrom. 16, 622-630.

S3. Wheeler, S. F., and Harvey, D. J. (2000) Negative ion mass spectrometry of

sialylated carbohydrates: discrimination of N-acetylneuraminic acid linkages by ALDI-TOF and ESI-TOF mass spectrometry, Analytical chemistry 72, 5027- 5039.

54. Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A., and Harvey, D. J. (1997) Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography, Anal Biochem 250, 82-101.

55. Harvey, D. J., Merry, A. H., Royle, L, Campbell, M. P., Dwek, R. A., and Rudd, P.

M. (2009) Proposal for a standard system for drawing structural diagrams of N- and O-linked carbohydrates and related compounds, Proteomics 9, 3796-3801.

List of Sequences SEQ ID NO: 1

SEQ ID NO: 1 (as given in Figure 20/20) is the full amino acid sequence of the human lgG1 immunoglobulin heavy chain (from Edelman, G. M., et al. (1969). The covalent structure of an entire gammaG immunoglobulin molecule. Proc Natl Acad Sci USA 63, 78-85). SEQ ID NO: 2

SEQ ID NO: 2 is the EU heavy chain sequence which is corrected for consensus with modem Fc sequences (from Uniprot P01857):

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSRSAIIWVRQAPGQGLEWMGGIVPMFGP PNYAQKFQGRVTITADESTNTAYMELSSLRSEDTAFYFCAGGYGIYSPEEYNGGLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREQQYNSTYRWSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Claims

1. An IgG antibody comprising a FcyRIIB-binding domain, wherein: (a) a heavy chain of the antibody comprises an amino acid sequence having at least 90% sequence identity to amino acids 1-446 of SEQ ID NO: 1 or 2; and (b) the amino acids in the heavy chain which correspond to the amino acids at positions 264, 267 and 328 of SEQ ID NO: 1 or 2 are glutamic acid, glutamic acid and phenylalanine, respectively.

2. A polypeptide comprising a FcyRIIB-binding domain, wherein:

(a) the amino acids in the FcyRIIB-binding domain which correspond to the amino acids at positions 264, 267 and 328 of SEQ ID NO: 1 or 2 are each different from the amino acids which are present at the corresponding positions in SEQ ID NO: 1 or 2;

or

(b) the amino acids in the FcyRIIB-binding domain which correspond to the amino acids at positions 262, 267 and 328 of SEQ ID NO: 1 or 2 are each different from the amino acids which are present at the corresponding positions in SEQ ID NO: 1 or 2.

3. A polypeptide as claimed in claim 2, wherein the FcyRIIB-binding domain comprises an amino acid sequence having at least 70% sequence identity to amino acids 224-446 of SEQ ID NO: 1 or 2, preferably at least 80%, 85%, 90%, 95% or 99% sequence identity to amino acids 224-446 of SEQ ID NO: 1 or 2.

4. A polypeptide as claimed in claim 2, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to amino acids 200-446 of SEQ ID NO: 1 or 2, more preferably at least 80%, 85%, 90%, 95% or 99% sequence identity to amino acids 200-446 of SEQ ID NO: 1 or 2.

5. A polypeptide as claimed in claim 2, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to amino acids 100-446 of SEQ ID NO: 1 or 2, more preferably at least 80%, 85%, 90%, 95% or 99% sequence identity to amino acids 100-446 of SEQ ID NO: 1 or 2.

6. A polypeptide as claimed in claim 2, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to amino acids 1-446 of SEQ ID NO: 1 or 2, more preferably at least 80%, 85%, 90%, 95% or 99% sequence identity to amino acids 1-446 of SEQ ID NO: 1 or 2.

7. A polypeptide as claimed in any one of claims 1 to 6, wherein the polypeptide has a higher affinity for the FcvRIIB receptor than a control polypeptide comprising or consisting of the sequence of SEQ ID NO: 1 or 2 with the double substitution

S267E/L328F.

8. A polypeptide as claimed in any one of claims 1 to 7, wherein the polypeptide has a lower affinity for one or more receptors selected from the group consisting of FcyRIA, FcyRIIA, FcyRIIIA and FcyRIIIB than a control polypeptide comprising or consisting of the sequence of SEQ ID NO: 1 or 2 with the double substitution

S267E/L328F.

9. A polypeptide as claimed in any of claims 2 to 8, wherein the amino acid which corresponds to the amino acid at position 262 in SEQ ID NO: 1 or 2 is glutamic acid, aspartic acid, lysine or arginine, most preferably glutamic acid.

10. A polypeptide as claimed in any of claims 2 to 9, wherein the amino acid which corresponds to the amino acid at position 264 in SEQ ID NO: 1 or 2 is glutamic acid, aspartic acid, lysine or arginine, most preferably glutamic acid.

11. A polypeptide as claimed in any of claims 2 to 10, wherein the amino acid which corresponds to the amino acid at position 267 in SEQ ID NO: 1 or 2 is glutamic acid or aspartic acid, most preferably glutamic acid.

12. A polypeptide as claimed in any of claims 2 to 11 , wherein the amino acid which corresponds to the amino acid at position 328 in SEQ ID NO: 1 or 2 is phenylalanine or tryptophan, most preferably phenylalanine.

13. A polypeptide as claimed in any one of claims 2 to 12, wherein the polypeptide consists of or comprises an Fc monomer of an immunoglobulin G (IgG).

14. A protein comprising a dimer of the polypeptide as claimed in any one of claims 1 to 13.

15. An antibody heavy chain comprising a polypeptide as claimed in any one of claims 2 to 12.

16. An antibody comprising at least one polypeptide as claimed in any one of claims 2 to 12.

17. An antibody as claimed in claim 16, wherein the antibody is a monoclonal, bispecific, human, humanized, chimeric, single chain or anti-idiotypic antibody.

18. An antibody as claimed in claim 16 or claim 17, wherein the antibody is an IgG.

19. An antibody as claimed in any one of claims 16 to 18, wherein the antibody is N-glycosylated.

20. An antibody as claimed in any one of claims 16 to 19, wherein the antibody comprises an antigen-binding domain.

21. An antibody as claimed in claim 20, wherein the antigen-binding domain binds to a target selected from the group consisting of CD2, CD3, CD19, CD20, CD22, CD25,

CD30, CD33, CD40, CD62, CD56, CD64, CD70, CD74, CD79, CD80, CD86, CD105, CD138, CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED- B, T EFF2, EphA2, EphB2, FAP, av integrin, Mesothelin, EGFR, TAG-72, GD2, CA1X, 5T4, α4β7 integrin, Her2; a cytokine, such as an interleukin IL-! - IL- 13; tumour necrosis factor a or β, interferons©:, β or γ, tumour growth factor Beta (TGF-β), colony stimulating factor (CSF) or granulocyte monocyte colony stimulating factor (GMCSF); a hormone, an enzyme, an intracellular or intercellular messenger, such as adenyl cyclase, guanyl cyclase, or phospholipase C; a leukocyte antigen, such as CD20, or C033; or a drug.

22 An antibody as claimed in claim 20, wherein the antigen-binding domain binds to a target wherein the target is a component of the B-cell receptor (BCR) complex.

23. A polypeptide as claimed in any one of claims 2 to 13, a protein as claimed in claim 14 or an antibody as claimed in any one of claims 15 to 22, wherein the polypeptide, protein or antibody is fused, linked or conjugated to an immunogenic therapeutic protein.

24. A pharmaceutical composition comprising a polypeptide as claimed in any one of claims 2 to 13, a protein as claimed in claim 14 or an antibody as claimed in any one of claims 15 to 22, optionally together with one or more carriers, diluents or excipients.

25. A polypeptide as claimed in any one of claims 2 to 13, a protein as claimed in claim 14 or an antibody as claimed in any one of claims 15 to 22 for use as a medicament or for use in therapy.

26. A polypeptide as claimed in any one of claims 2 to 13, a protein as claimed in claim 14 or an antibody as claimed in any one of claims 15 to 22, for use in the prophylaxis, prevention or treatment of an autoimmune disorder or an inflammatory disorder.

27. A method of prophylaxis, prevention or treatment of an autoimmune disorder or an inflammatory disorder, the method comprising administering an effective amount of a polypeptide as claimed in any one of claims 2 to 13, a protein as claimed in claim 14 or an antibody as claimed in any one of claims 15 to 22 to a patient in need thereof.

28. A polypeptide, protein or antibody as claimed in claim 26 or a method as claimed in claim 27, wherein the autoimmune disorder is selected from the group consisting of a B-cell mediated autoimmune disease, type 1 diabetes, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE) and autoimmune thyroid disease (AITD).

29. A polypeptide, protein or antibody as claimed in claim 26 or a method as claimed in claim 27, wherein the inflammatory disorder is selected from the group consisting of Bechterew's disease, psoriatic arthritis, rheumatoid arthritis, arthritis in colitis ulcerosa, arthritis in morbus Crohn, affection of joints in systemic lupus erythematosus (SLE), systemic sclerosis, mixed connective tissue disease, reactive arthritis, and Reiter"s syndrome.

30. An antibody as claimed in any one of claims 15 to 20, for use in the prophylaxis, prevention or treatment of an autoimmune disorder, wherein the antigen binding domain of the antibody binds to an antigen associated with the autoimmune disorder.

31. An antibody as claimed in any one of claims 15 to 20, for use in the prophylaxis, prevention or treatment of an inflammatory disorder, wherein the antigen-binding domain of the antibody binds to an antigen associated with the inflammatory disorder.

32. An antibody as claimed in claim 31 , wherein the inflammatory disorder is rheumatoid arthritis and the antigen-binding domain binds to collagen or cartilage.

33. A polypeptide as claimed in any one of claims 1 to 13, a protein as claimed in claim 14 or an antibody as claimed in any one of claims 15 to 22 for use in a method of suppressing immune cell activation or of immuno-suppression.

34. A method of suppressing immune cell activation or of immuno-suppression comprising administering an effective amount of a polypeptide as claimed in any one of claims 2 to 13, a protein as claimed in claim 14 or an antibody as claimed in any one of claims 15 to 22 to a patient in need thereof.

35. A method of immuno-suppression or B-cell suppression, the method comprising administering an effective amount of an antibody as claimed in any one of claims 15 to 20, wherein the antibody comprises an antigen-binding domain having affinity for a B- cell surface antigen, to a patient in need thereof.

36. An antibody as claimed in any one of claims 15 to 20, wherein the antibody comprises an antigen-binding domain having affinity for a B-cell surface antigen, for use in a method of immuno-suppression or B-cell suppression.

37. A method as claimed in claim 35 or an antibody as claimed in claim 36, wherein the B-cell surface antigen is selected from the group consisting of CD19, CD21, CD22, CD72, CD79a, CD79b and CD81.

38. A method of reducing the immunogenicity of a therapeutic protein, the method comprising administering an effective amount of a polypeptide as claimed in any one of claims 2 to 13, a protein as claimed in claim 14 or an antibody as claimed in any one of claims 15 to 22, which is fused, linked or conjugated to the immunogenic therapeutic protein, to a patient to whom the therapeutic protein has, is being or will be administered.

39. A combined composition comprising:

(i) polypeptide as claimed in any one of claims 2 to 13, a protein as claimed in claim 14 or an antibody as claimed in any one of claims 15 to 22, which is fused, linked or conjugated to an immunogenic therapeutic protein, and

(ii) the immunogenic therapeutic protein,

40. A nucleic acid molecule coding for a polypeptide as claimed in any one of claims 2 to 13 or an antibody heavy chain as claimed in claim 15.

41. A vector comprising the nucleic acid molecule of claim 40, operably linked to one or more control sequences.

42. A host cell comprising the vector of claim 41.

5

43. A hybridoma producing an antibody as claimed in any one of claims 15 to 22.