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The classical complement system represents a central effector mechanism of Abs initiated by the binding of C1q to target bound IgG. Human C1q contains six heterotrimeric globular head groups that mediate IgG interaction, resulting in an avidity-driven binding event involving multiple IgG molecules binding a single C1q. Accordingly, surface bound IgG molecules are thought to assemble into noncovalent hexameric rings for optimal binding to the six-headed C1q. To study the C1q–Fc interaction of various Abs and screen for altered C1q binding mutants, we developed, to our knowledge, a novel HPLC-based method. Employing a single-chain form of C1q representing one C1q head group, our HPLC methodology was able to detect the interaction between the single-chain monomeric form of C1q and various ligands. We show that, despite a narrow window of specific binding owing to the low affinity of the monomeric C1q–IgG interaction, this approach clearly distinguished between IgG subclasses with established C1q binding properties. IgG3 displayed the strongest binding, followed by IgG1, with IgG2 and IgG4 showing the weakest binding. Fc mutants known to have increased C1q binding through oligomerization or enhanced C1q interaction showed greatly increased column retention, and IgG glycovariants displayed a consistent trend of increasing retention upon increasing galactosylation and sialylation. Furthermore, the column retention of IgG isotypes and glycovariants matches both the cell surface recruitment of C1q and complement-mediated cytotoxicity induced by each variant on an anti-CD20 Ab backbone. This methodology therefore provides a valuable tool for testing IgG Ab (glyco)variants for C1q binding, with clear relevance for therapeutic Ab development.

Bispecific antibodies (bsAbs) with avidity-enhanced specificity can be used to address target cells with increased specificity, ideally binding efficiently to cells that express two cognate antigens, yet not to cells that express only one of those. Building blocks required to generate such bsAbs are binders that recognize the two antigens with high specificity yet with various (including very low monovalent) affinities. The herein described &lsquo, back-to-germline&rsquo, (B2G) procedure defines such derivatives. It converts parent antibodies with high specificity to derivatives that retain specificity but modulate affinity. The approach defines mutations to be introduced into antibody complementarity-determining regions (CDRs) regions without requiring structures of antibody-antigen complexes. Instead, it reverses the B-cell maturation process that increases affinities, with preference on CDR residues with high antigen contact probability. Placing germline residues at those positions generates VH and VL domains and Fv-combinations thereof that retain specificities but are &lsquo, de-matured&lsquo, to different degrees. De-maturation influences on-rates and off-rates, and can produce entities with extremely low affinity for which binding can only be detected in bivalent formats. A comparison with alanine replacement in CDRs (so far, the most frequently applied technology) indicates that B2G may be more reliable/predictable without introduction of stickiness or poly-reactivity. The applicability for generating sets of affinity-modulated monospecific variants is exemplarily shown for antibodies that bind CD138, Her2/neu, and EGFR.

Purpose The Göttingen minipig is a relevant non-rodent species for regulatory toxicological studies. Yet, its use with therapeutic antibodies has been limited by the unknown binding properties of human immunoglobulins (huIgG) to porcine Fc gamma receptors (poFcγR) influencing safety and efficacy readouts. Therefore, knowing IgG-FcγR interactions in the animal model is a prerequisite for the use of minipigs in preclinical safety and efficacy studies with therapeutic antibodies. Methods Here, we describe the cloning and expression of poFcγRs and their interactions with free and complexed human therapeutic IgG1 by surface plasmon resonance and flow cytometry. Results We show here that poFcγRIa, poFcγRIIa, and poFcγRIIb bind huIgG1 antibodies with comparable affinities as corresponding huFcγRs. Importantly, poFcγRs bind huIgG immune complexes with high avidity, thus probably allowing human-like effector functions. However, poFcγRIIIa binds poIgG1a but not to huIgG1. Conclusions The lack of binding of poFcγRIIIa to huIgG1 might cause underestimation of FcγRIIIa-mediated efficacy or toxicity as mediated by porcine natural killer cells. Therefore, the suitability of minipigs in preclinical studies with human therapeutic antibodies has to be assessed case by case. Our results facilitate the use of Göttingen minipigs for assessment of human therapeutic antibodies in preclinical studies. Electronic supplementary material The online version of this article (10.1007/s11095-019-2574-y) contains supplementary material, which is available to authorized users.

A novel bispecific antibody format was applied to generate T cell-engaging antibodies. The TriFab format is a trivalent IgG-shaped entity composed of two Fab arms that bind to antigens on the surface of tumor cells, which are linked via flexible peptides to a CD3 binding moiety that replaces the CH2 domains of conventional IgGs. The distinctive feature of these T cell recruiting bispecifics is that their CD3 variable regions are incorporated between domains, rather than N- or C-terminally fused to an Fc or antibody fragments. T cell recruiting TriFabs resemble in size and shape, are expressed and show biophysical properties similar to regular IgGs. Transmission electron microscopy (TEM) demonstrates high flexibility of the cell surface binding arms as well as target antigen accessibility of the interspersed CD3 binding domain. Functional co-culturing assays of peripheral blood mononuclear cells (PBMCs) and different tumor cell lines (MCF7 and A431) revealed a dose-dependent T cell-mediated cytotoxicity that was induced by the TriFabs targeting either LeY or EGFR cell surface antigens.

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.

Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.

Recombinant human IgG antibodies (hIgGs) completely devoid of binding to Fcγ receptors (FcγRs) and complement protein C1q, and thus with abolished immune effector functions, are of use for various therapeutic applications in order to reduce FcγR activation and Fc-mediated toxicity. Fc engineering approaches described to date only partially achieve this goal or employ a large number of mutations, which may increase the risk of anti-drug antibody generation. We describe here two new, engineered hIgG Fc domains, hIgG1-P329G LALA and hIgG4-P329G SPLE, with completely abolished FcγR and C1q interactions, containing a limited number of mutations and with unaffected FcRn interactions and Fc stability. Both 'effector-silent' Fc variants are based on a novel Fc mutation, P329G that disrupts the formation of a proline sandwich motif with the FcγRs. As this motif is present in the interface of all IgG Fc/FcγR complexes, its disruption can be applied to all human and most of the other mammalian IgG subclasses in order to create effector silent IgG molecules.