The effector functions mediated by the antibody Fc region can be divided into (1) effector functions that operate after binding of antiobdy to an antigen (these funcitons involved the participation of the complement cascade or Fc receptor (FcR) bearing cells) and (2) effector functions that operate independently of antigen binding (thse functions ocnfer persistence in the circualtion and the ability to transfer across cellular barriers by transcytosis. Presta (US 6,737,056). 

Modifying effector functions can be acheived by engineering the Fc region to either improve or reduce binding of FcyRs or the complement factors. When designing an IgG for a particular function, one must consider not only the human IgG isotype but also which of the FcyR would be the preferential target, the immune cell types epxressing the target receptor(s) and the differential binding of the various polymorphs (Presta, 2008, Curr. Opin. Immunol. 20, 460-470).

Numerous studies have shed light on the effector functions of antibodies as important mechanisms of action of therpaeutic antibodies in addition to theri binding affintiy and specificity for targets, in particular antibody-dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and a long half-life/clearance rate. Each of these efector functions is primarily triggered through direct interaciton of the Fc domain of the antibody with its corresponding ligands: ACC through interaction with the Fc gamma receptor IIIa (FcyRIIIa), CDC through interaction with the series of soluble blood rpteoins that constitute the antibody-dependent complement activaiton pathway (e.g., C1q, C3 and C4) and serum persistance through itneraction with the neonatal Fc receptor (FcRn). Kubota “Engineered therapeutic antibodies with improved effector functions” Cancer Sci, September 2009, 100 (9) 2009).

A number of preclinical studies have suggested that ADCC is a major mechanism of action of antitumor antibodies, such as rituximab. The importance of ADCC has also been recognized in clinical settings, as evidenced by significant correlation between FcyRIIIa funcitonal polymorphisms and clinical outcomes of multiple therapeutic antibodies. (Kubota “Engineered therapeutic antibodies with improved effector functions” Cancer Sci, September 2009, 100 (9) 2009). 

Antibody Engineering for Increased Immunogenicity:

While binding of an antibody to the requisite antigen has a neutralizing effect that might prevent the bidning of foreign antigen to its endogenous target (e.g., reeceptor or ligand), binding alone may not remove the foreign antigen. To be efficient in removing and/or destruction foreign antigen, an antibody should be endowed with both high affinity binding to its antigen, and efficient effector functions. The interaction of antibodies and antibody-antigen complexes with cells of the immune system effects a variety of responses, including antibody-dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC)).

Several antibody effector functions are mediated by Fc receptors (FcRs) which bind the Fc region of an antibody. FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FcepisolonR, for IgA as FcalphaR and so on. Three subclasses of FcyR have been identified: FcyR1 (CD64), FcyRII (CD32) and FcyRIII (CD16). Because each FcyR subclass is encoded by two or three genes, and alternative RNA splicing leads to multiple transcirpts, a broad diversity in FcyR isoforms exists. The three genes encoding the FcyRI subclass (FcyRIA, RcyRIB and FcyRIC) are clusted in region 1q21.1 of the long arm of chromosome 1; the genes encoded FcyRII isoforms (FcyRIIA, FcyRIIB FcyRIIIB) are all clustered in region 1q22. Tehse different FcR subtypes are expressed on different cell types. For example, in humans, FcyRIIIB is ofund only on neutrophiles, whereas FcyRIIA is found on macrophages, monocytes and NK cells and a subpopulation of T cells. Notably, FcyRIIIA is the only FcR present on NK cells, one of the cell types impolicated in ADCC. (Presta (US 6,737,056)), 

Enhancing ADCC activity by modifying the amino acid sequence of the Fc domain has been extensively studies, mainly through the random mutational analysis of human IgG1 Fc. For example, Fc domain variants with up to three mutations (S298A, E333A and K334A) which improved binding to FcyRIIIa and enhanced capacity for ADCC. (Kubota “Engineered therapeutic antibodies with improved effector functions” Cancer Sci, September 2009, 100 (9) 2009).

Antibodies such as Xencar: CmAb and MacroGenics by engineering of amino acid in the Fc region as been done to develop more potent ADCC (“Proprietary Innovative antibody engineering technologies in Chugai Pharmaceutical”, 12/18/2012).

Presta (US 6,737,056) discloses a varaint of a parent polypetpide that includes an Fc region which meidates ADCC in the presence of human effector cells more effectively or binds an Fc gamma recetpor (FcyR) with better afifnity that includes an amino acid modificiation (e.g., a substitution) at any one of amino acid positions256, 290, 298, 312, 326, 330, 333, 334, 360, 378 or 430 of teh Fc region.

Exchanging Fc isotypes:  

One study showed that exchanging the human IgG1 of an antibody with a murine IgG2a significantly improved ADCC using mouse effector cells and significantly better anti-tumor activity in a mouse model (Lutterbuese, Cancer Immunol Immunother 2007, 56, 459-468).

Another approach for enhancing CDC actvity is engineering of the H chain by shuffling IgG1 and IgG3 sequences within the H constant region. Several variant H constant regions, screened form a set of IgG1/IgG3 mixed sequences showed unexpectedly strong C1q binding and CDC activity that exceeded the levels observed for either parental IgG1 or IgG3. (Kubota “Engineered therapeutic antibodies with improved effector functions” Cancer Sci, September 2009, 100 (9) 2009).

Antibody engineering for Reduced Immunogenicity:

Select appropriate isotypes: There are several strategies that can be used in the design of antiboides that avoid Fcgamma receptor interactions. For monoclonal antibodies, one approach is to select the human y4IgG isotype during construction of a humanized antibody. The y4IgG isotype does not bind Fcy receptors. Alternatively, a monoclonal antibody agent can be genetically enineered that lacks the Fc region, including for example single chain antibodies and antigen-binding domains. Yet another approach is to chemically remove the Fc region of a monoclonal antibody using proteolytic enzymes thereby generating antigen-binding antibody fragments such as Fab or F(ab)2 fragments. (Gupta-Bansal (US 2003/0198636).

Remove sugars: Another approach to reduce effector function is to remove sugars that are linked to particular residues in the Fc region, for rexample, by deleting or altering the residue the sugar is attached to, removing the sugars enzymatically, by producing the antibody in cells cultured in the presence of a glycosylation inhbitor, or by expressing the antibody in cells unable to glycosylate proteins. However, the forgoing approaches have residual effector function both in the form of complement-dependent cytolytic activity and Fc receptor binding. (Taylor, US 2007/0048300)

Taylor, US 2007/0048300) discloses a method for prodcing aglycosylated antibodies suitable as therapeutics because of their reduced effector function, by introducing an amino alteration at a first amino acid residue position which results in the reduced glycosylation of the antibody at a different or second amino acid residue position. In one embodiment, the preferred amino acid residue is of sufficient steric bulk and charge such that the residue inhibits glycosylation at a second amino acid position. Such amino acids include lysine, arginine and throsine. In another embodiment, the polypeptide has a first amino acid residue and second amino acid residue that are near or within a glycosylation motif, for example, an N-linked glycosylation motif tht contains the amino acid sequence NXT or NXS. In a particular embodiment, the polyeptide has a first amiho acid 299 and the second amino acid is 297, according to the Kabat numbeirng. In a particular embodiemnt, the amino acid substitution is T299C or T299A.

Exchanging Fc isotypes: Under certain cirumstances, abrogating or diminishing effector functions may be required. Abrogation of effector function has to some extent already been supplied by nature in the form of human IgG2 and IgG4 that exhibit decreased susceptibility to ADCC and CDC.

Alter the Fc region: There are several known ways to reduce the effector function of an antibody while retaining the other valuable attributres of the Fc region (US 2007/0048300A1) . One approach is to replace amino acid residues in the Fc portion (US 5,648,260 and 5,624,821). Another approach to reduce effector function is to remove sugars that are linked to partciular residues in the Rc region.

Moore (US 2006/0275282) describes antibodies and Fc fusion proteins with reduced immunogicity such as having reduced ability to bind one or more human class II MHC molecules. 

Presta (US 6,737,056) discloses variants with reduced binding to an FcyRII having amino acid modification at any one of positions 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 33, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 of the Fc region. The varaint may display reduced binding to an FcyrIII with modificaiton at positions 238, 239, 248, 249, 252, 254… 

Modification in Fc for Altered binding to FcRn:

FcRn is structurally similar to major histocompatbility complex (MHC) and consists of an alpha chain nocovalently bound to beta2-microglobulin. The binding site of human and murine antibodies for FcyR has been mapped to the “lower hinge region” consisteng of residues 233-239 (EU index).

Modification for increased binding to FcRn: Another direction in the improvement of the therapeutic eficiency of antibodies might be the further prolongation of their long in vivo half-lives. With this aim, there have been extensive studies attempting to introduce mutations in the Fc domain that render antibodies capable of more strongly binding the neonatal Fc receptor (FcRn). Kubota “Engineered therapeutic antibodies with improved effector functions” Cancer Sci, September 2009, 100 (9) 2009).

Presta (US 6,737,056) discloses a polypeptides varaint with increased binding to FcRn that incldues modification at any of the positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434  of the Fc region. 

Modificaiton for decreased binding to FcRn: 

Presta (US 6,737,056) discloses a polypeptides variant with reduced binding to an FcRn that includes an amino acid modificaiton at any of 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435, 436, 439 or 447 of the Fc region. 

Modification for C1Q binding:

C1q and two serine proteases, C1r and C1s, form the complex C1, the first component of the complement dependent cyytoxicity (CDC) pathway. F1q is a hexavalent molecule with a MW of about 460k and a structure liikened to a bouqet of tulips in which six collagenous stalks are connected to six globular head regions. To activate the complement cascade, it is necessary for C1q to bind to at least two molecules of IgG1, IgG2 or IgG3 (the consensus is that IgG4 does not activate complement), but only one molecule of IgM, attached to the antigenic target. Presta (US 6,737,056)

Glu318, Lys320 and Lys322 has been reported as forming the binding site to c1q. The residue Pro331 has also been implicated in 1q binding. Presta (US 6,737,056)

It has also been proposed that the ability of IgG to bind C1q and activate the complement cascade also depends on the presence, absence or modificaiton of the carbohydrate moeity positioned between the two CH2 domains Presta (US 6,737,056). 

Engineering of glycosylation in the Fc region; De-fucosylated antibodies : 

One IgG molecule contains two N-linked oligosacharide sites in the Fc region. The general structure of N-linked oligosacharide on IgG is complex-type, characterized by a mannosyl-chitobiose core (Man3GlcNAc2-Asn) with or without bisecting GlcNAc/l-fucose (Fuc) and other cahin variants including the presence or absence of Ga1 and sialic acid. In addition, oligosaccharides may contain zero (G(0) one (G1) or two (G2) Gal. Recent studies have shown that engineering the oligosaccharides of IgGs may hield optimized ADCC. (Shinkawa, J. Biological Chemistry, 278(5), (2003)

Removing fucose in the Fc region has been used to increase binding affinity to FcyRIIIA. This has been done with Roche/Glycart: Glycomab  and Kyowa Hakko Kirin/BioWa: Potelligent (“Proprietary Innovative antibody engineering technologies in Chugai Pharmaceutical”, 12/18/2012).