Bernard Scallon

An engineered Fc variant of an IgG eliminates all immune effector functions via structural perturbations.

The Fc variant of IgG2, designated as IgG2σ, was engineered with V234A/G237A /P238S/H268A/V309L/A330S/P331S substitutions to eliminate affinity for Fcγ receptors and C1q complement protein and consequently, immune effector functions. IgG2σ was compared to other previously well-characterized Fc muted variants, including aglycosylated IgG1, IgG2m4 (H268Q/V309L/A330S/P331S, changes to IgG4), and IgG4 ProAlaAla (S228P/L234A/L235A) in its capacity to bind FcγRs and activate various immune-stimulatory responses. In contrast to the previously characterized muted Fc variants, which retain selective FcγR binding and effector functions, IgG2σ shows no detectable binding to the Fcγ receptors in affinity and avidity measurements, nor any detectable antibody-dependent cytotoxicity, phagocytosis, complement activity, or Fc-mediated cytokine release. Moreover, IgG2σ shows minimal immunogenic potential by T-cell epitope analysis. The circulating half-life of IgG2σ in monkeys is extended relative to IgG1 and IgG2, in spite of similar in vitro binding to recombinant FcRn. The three-dimensional structure of the Fc, needed for assessing the basis for the absence of effector function, was compared with that of IgG2 revealing a number of conformational differences near the hinge region of the CH2 domain that result from the amino acid substitutions. Modeling reveals that at least one of the key interactions with FcγRs is disrupted by a conformational change that reorients P329 to a position that prevents it from interacting with conserved W90 and W113 residues of the FcγRs. Inspection of the structure also indicated significant changes to the conformations of D270 and P329 in the CH2 domain that could negatively impact C1q binding. Thus, structural perturbations of the Fc provide a rationale for the loss of function. In toto, these properties of IgG2σ suggest that it is a superior alternative to previously described IgG variants of minimal effector function, for future therapeutic applications of non-immunostimulatory mAb and Fc-fusion platforms.

Fc glycans terminated with N-acetylglucosamine residues increase antibody resistance to papain

Glycosylation in the CH2 domain of Fc is required for immunoglobulins G (IgGs) to exhibit immune effector functions including complement‐dependent cytotoxicity (CDC) and antibody‐dependent (Ab‐dependent) cellular cytotoxicity (ADCC). We recently established that glycosylated Abs are more resistant to papain digestion than non‐glycosylated IgGs (Biochem. Biophys. Res.Commun. 2006, 341, 797–803). To test whether specific Fc glycan structures affect Ab resistance to papain, we used in vitro glycoengineering methods to prepare homogeneous Ab glycoforms terminated with either sialic acid (G2S2), β‐galactose (G2), or N‐acetylglucosamine (G0) and subjected them to papain digestions. Analyses of aliquots taken at different times during the digestions by matrix‐assisted laser desorption‐time‐of‐flight‐mass spectroscopy (MALDI‐TOF‐MS) and high‐performance liquid chromatography (HPLC) methods showed that the G0 glycoform was at least two times more resistant to papain digestion than the G2 and G2S2 glycoforms. The increased resistance of the G0 glycoform over the G2 and G2S2 glycoforms was independent of the specific Ab analyzed. A mouse/human chimeric version of Ab1, a fully human version of Ab2, and a humanized version of Ab3 exhibited a similar pattern of glycoform‐dependent resistance. These data suggest that terminal sugars of Fc glycans may play important roles in Ab stability and affect resistance to proteases in addition to impacting Ab effector functions.

Correlations between pharmacokinetics of IgG antibodies in primates vs. FcRn-transgenic mice reveal a rodent model with predictive capabilities

Transgenic mice expressing human neonatal Fc receptor (FcRn) instead of mouse FcRn are available for IgG antibody pharmacokinetic (PK) studies. Given the interest in a rodent model that offers reliable predictions of antibody PK in monkeys and humans, we set out to test whether the PK of IgG antibodies in such mice correlated with the PK of the same antibodies in primates. We began by using a single research antibody to study the influence of: (1) different transgenic mouse lines that differ in FcRn transgene expression; (2) homozygous vs. hemizygous FcRn transgenic mice; (3) the presence vs. absence of coinjected high-dose human intravenous immunoglobulin (IVIG), and (4) the presence vs. absence of coinjected high-dose human serum albumin (HSA). Results of those studies suggested that use of hemizygous Tg32 mice (Tg32 hemi) not treated with IVIG or HSA offered potential as a predictive model for PK in humans. Mouse PK studies were then done under those conditions with a panel of test antibodies whose PK in mice and primates is not significantly affected by target binding, and for which monkey or human PK data were readily available. Results from the studies revealed significant correlations between terminal half-life or clearance values observed in the mice and the corresponding values reported in humans. A significant relationship in clearance values between mice and monkeys was also observed. These correlations suggest that the Tg32 hemi mouse model, which is both convenient and cost-effective, can offer value in predicting antibody half-life and clearance in primates.

A review of antibody therapeutics and antibody-related technologies for oncology.

INTRODUCTION The antibodies currently approved for the treatment of diseases, including cancer, have been developed predominantly based on the understanding and identification of key targets involved in disease pathology. Thus, for oncology, currently marketed antibodies to epidermal growth factor receptors (EGFR/HER1 and HER2) and vascular endothelial growth factor (VEGF) treat cancer by blocking the function of these targets that are crucial for tumor progression. Other targets for launched products are those that are highly up-regulated on neoplastic cells, including CD20, CD52, and CD33. Antibodies are generally highly specific for their molecular targets and can be used to affect disease-specific targets, thereby sparing normal cells and causing less toxicity than traditional cytotoxic chemotherapies. Effective antibodies act through one or more of a variety of mechanisms, including (a) blocking essential cellular growth factors or receptors, (b) directly inducing apoptosis, (c) binding to target cells and recruiting ‘‘effector functions’’ such as antibodydependent cellular cytotoxicity (ADCC), or complementdependent cytotoxicity (CDC), and (d) delivering cytotoxic payloads such as chemotherapies, radioisotopes and toxins. The use of informatics will be essential as we develop new waves of products that provide additional efficacy, specificity, or safety over currently marketed products. Informatics approaches can be used to (a) identify novel targets either upstream or downstream of already validated targets to enhance or complement efficacy, (b) identify targets that are more specific for tumors, thereby enhancing safety and providing a means of directing toxic agents to the tumor, (c) identify novel pathways essential for disease progression, and (d) identify Fc mutants that have enhanced immune effector function. ANTIBODY TECHNOLOGY The first monoclonal antibodies from mice were generated in 1975. In humans, mouse-derived antibodies are highly immunogenic, and therefore ‘‘chimeric’’ antibodies were created by replacing mouse constant domains (non-antigen binding domains) with human constant domains. This improvement considerably reduced the immune response to therapeutic antibodies. Additional modifications of framework regions within the antigenbinding variable regions further reduce immunogenicity and result in what are termed ‘‘humanized’’ antibodies. Fully human antibodies can be derived from human cells or from genetically engineered mice transgenic for human antibody genes. Human antibodies can also be generated from antibody-expressing phage libraries as single chain Fv or Fab fragments that can subsequently be converted to full-length antibodies.

Chimeric antibodies with extended half-life in ferrets

Ferrets have long been used as a disease model for the study of influenza vaccines, but a more recent use has been for the study of human monoclonal antibodies directed against influenza viruses. Published data suggest that human antibodies are cleared unusually quickly from the ferret and that immune responses may be partially responsible. This immunogenicity increases variability within groups and may present an obstacle to long‐term studies.