A large body of data exists demonstrating the key role of FcRn in extending the half-life of therapeutic antibodies by rescuing them from lysosomal degradation. This led to the widely accepted hypothesis that FcRn binding of an IgG via the CH2-CH3 interface of Fc correlates with IgG half-life. Several studies have demonstrated that in vivo half-life can be modified by changing the binding affinity of IgG to FcRn. These modifications were generated by mutating the coding sequence for the Fc region that resulted in enhanced or reduced FcRn binding at endosomal pH without enhancing binding at neutral pH. In contrast to this, we have observed that the half-lifes of IgG molecules that had showed no target-mediated disposition or off-target binding varies widely, even when they share identical Fc domains. This led us to hypothesize that domains of IgG molecules other than Fc could contribute to the modulation of FcRn binding and affect in vivo half-life. This hypothesis was strengthened by recent publications by other groups showing a correlation between antibody charge and the FcRn affinity and/or in vivo half-life. In this study we explored the role of IgG domains other than the FcRn binding domain in altering the affinity between IgG and FcRn and its relation to the in vivo half-life. Here we describe a surface plasmon resonance (SPR) based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn. We systematically dissected the contributions of IgG variable domain regions in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 100 unique IgG molecules against more than 25 different therapeutic targets we have demonstrated that variable domains and in particular CDRs significantly alter binding affinity to FcRn, by 10 to 80-fold, whereas heavy and light chain isotypes do not. Because CDRs modulate the affinity between IgG and FcRn in our in vitro studies, it is important to understand the role they play in modulation of IgG half-life in vivo as this would have far-reaching implications in the half-life optimization efforts of IgG therapeutics.

In the age of highly active antiretroviral therapy (HAART), patients infected with Human Immunodeficiency Virus Type 1 (HIV-1) are now living significantly healthier and longer lives. However, HIV prevention and cure still remain significant challenges. Globally, women face specific barriers to using and accessing both female and male condoms, the primary method recommended by the World Health Organization (WHO) to prevent sexual transmission of HIV-1. Although HAART treatment as prevention (TasP) of HIV has shown promising preliminary results, poor economic feasibility of the method in resource poor settings has yet to be resolved. Since women carry over 50% of the disease burden, there is a significant need for the development of a female-controlled method of prevention. One such approach is the reformulation of topical vaginal microbicides. Our laboratory is developing HIV-targeted microbicide formulations that utilize highly specific, broadly neutralizing anti-HIV antibodies (bNAbs). The purpose of my research project was to characterize Fc receptor expression in epithelial cell models of the lower female genital tract with a particular focus on the neonatal Fc receptor (FcRn). Fc receptors are a large family of proteins that bind to the Fc region of immunoglobulins (Igs) and function in Ig transport and effector functions. These receptors could function in enhancing the delivery of bNAbs in microbicide formulations or potentially serve as a mechanism of delivering HIV to target cells in tissues via transport of HIV-antibody complexes. Thus, this thesis assesses Fc receptor expression in human vaginal and endocervical organotypic cultures via microarray, quantitative RT-PCR, immunohistology and preliminary functional assays. Microarray results revealed significant expression of Fc receptor family genes in the epithelial cells of the lower female reproductive tract (FRT). The polymeric immunoglobulin receptor (pIgR), a well-characterized receptor that transports secretory IgA across mucosal epithelia, was abundantly expressed and hormonally regulated in epithelial cells of the vagina and endocervix. FcRn, a receptor originally characterized in the placenta and gut where it confers passive immunity from mother-to-child via bidirectional IgG transcytosis, was expressed in both tissue models. Moreover, several members of the novel FC receptor-like (FCRL) family were detected by microarray in both models. Immunohistological staining revealed pIgR protein in the endocervical mucosal epithelium, confirming current literature describing its expression in the FRT and role in local production of cervicovaginal secretions. FcRn protein expression was detected in the basal cell layer of the stratified squamous vaginal epithelium and in the columnar cells of the endocervix. Preliminary functional assays did not observe FcRn-specific transcytosis of human IgG across vaginal or endocervical epithelia by ELISA or immunohistology. VRCO1, a monoclonal antibody in development for application in microbicide formulation, crossed the epithelium, but was likely not transcytosed via FcRn because immunohistology revealed the presence of antibody between epithelial cells rather than the expected intracellular localization of IgG utilized in the FcRn mechanism. These preliminary findings indicate that Fc receptors, pIgR and Fc receptor-like proteins may play an important role in antibody-mediated immune responses in the FRT. Further research is require to determine whether FcRn functions in HIV-antibody complex-mediated HIV transmission or monoclonal antibody transcytosis.

La pharmacocinétique (PK) des anticorps monoclonaux (mAbs) est sujette à d’importantes variations interindividuelles. Le récepteur néonatal au Fc des IgG (FcRn) et le statut immun à l’encontre de ces mAbs sont des déterminants de cette PK. La bioconjugaison des mAbs à des cytotoxiques entraîne une altération de leur PK. Nous montrons que le taux de couplage modifie l’affinité de ces espèces pour le FcRn à pH6. La proportion d’agrégats au sein des solutions d’anticorps armés augmente avec le taux de couplage et pourrait entraîner une altération de leur PK. Par ailleurs, cette agrégation est impliquée dans l’immunogénicité des mAbs, et nous avons donc cherché à identifier des acides aminés impliqués dans l’agrégation de mAbs indiqués en clinique. Il apparait que la nature biochimique de résidus des paratopes pourrait augmenter cette agrégation. Les anti-TNF- présentent très peu d'agrégats et figurent pourtant parmi les plus immunogènes chez l’Homme. Nous avons donc exploré le rôle des complexes immuns dans leur immunogénicité chez la souris. Il apparait que la présence du FcRn n’est pas à l’origine de l’immunisation contre ces mAbs, contrairement à celle des complexes immuns. Ces résultats donnent des pistes pour la production de mAbs plus efficients et mieux tolérés. / The pharmacokinetic (PK) profile of monoclonal antibodies (mAbs) shows interindividudal variability. The neonatal Fc receptor (FcRn) and the immounogenicity of these mAbs are determinative factors of mAb PK. Generation of antibody-drug-conjugates alters their PK profile. We show that the the affinity for FcRn at pH6 increases with the drug-to-mAb ratio, as does the amount of aggregates inside the mAb-drug-conjugate. The amount of aggregates could be responsible for an avidity effect towards FcRn. These aggregates are known to cause immunogenicity, so we studied biochemical determinants inside the aminoacid sequence of marketed mAbs. We show that the biochemical nature of some aminoacids inside the paratope has an impact on the amount of aggregation. Anti-TNF- mAbs show very little aggregation but are very immunogenic in humans. We studied the role of the formation of immune complexes in the immunization against anti-TNF- mAbs in mice, and showed that immune complexes, but not FcRn are essential in the immunization process against anti- TNF- mAbs. These results give leads towards the generation of more efficient, better tolerated mAbs.