Enhancement of antibody functions through Fc multiplications

Abstract

Antibodies carry out a plethora of functions through their crystallizable fragment (Fc) regions, which can be naturally tuned by the adoption of several isotypes and post-translational modifications. Protein engineering enables further Fc function modulations through modifications of the interactions between the Fc and its functional partners, including FcgR, FcRn, complement complex, and additions of auxiliary functional units. Due to the many functions embedded within the confinement of an Fc, a suitable balance must be maintained for a therapeutic antibody to be effective and safe. The outcome of any Fc engineering depends on the interplay among all the effector molecules involved. In this report, we assessed the effects of Fc multiplication (or tandem Fc) on antibody functions. Using IgG1 as a test case, we found that, depending on the specifically designed linker, Fc multiplication led to differentially folded, stable molecules with unique pharmacokinetic profiles. Interestingly, the variants with 3 copies of Fc improved in vitro opsonophagocytic killing activity and displayed significantly improved protective efficacies in a Klebsiella pneumoniae mouse therapeutic model despite faster clearance compared with its IgG1 counterpart. There was no adverse effect observed or pro-inflammatory cytokine release when the Fc variants were administered to animals. We further elucidated that enhanced binding to various effector molecules by IgG-3Fc created a "sink" leading to the rapid clearance of the 3Fc variants, and identified the increased FcRn binding as one strategy to facilitate "sink" escape. These findings reveal new opportunities for novel Fc engineering to further expand our abilities to manipulate and improve antibody therapeutics.

Considerations for the Design of Antibody-Based Therapeutics

“…Several studies have demonstrated the use of IgG1 molecules that contain 1 or 2 extra Fc domains tandemly linked to the IgG C-terminus. [204][205][206][207] Whether the additional Fc domains are linked by the IgG2 hinge or a flexible linker, they elicit significantly stronger ADCC and ADCP than the wild-type IgG. Intriguingly, the Fc domains can also be of distinct classes.…”

“…209 A potential drawback of these frameworks is faster clearance due to differences in FcRn binding avidity. 207 Regardless, Fc duplication is a creative engineering strategy for generating antibodies with stronger effector functions. In contrast to the standard approach of introducing framework mutations that alter FcR binding, this strategy uses the avidity effect to enhance binding.…”

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

“…Regarding ADCP and ADCC, incorporation of multiple Fc domains into each IgG molecule may amplify FcγR signaling by strengthening the avidity of the Fc‐FcγR interaction or by facilitating FcγR cross‐linking. In fact, studies with IgG variants containing two or three tandemly repeated Fc domains have shown enhanced FcγR‐mediated effector function compared to the wild‐type IgG . However, avidity effects are expected to be a function not only of the number of binding elements but also of their three‐dimensional arrangement.…”

“…In fact, studies with IgG variants containing two or three tandemly repeated Fc domains have shown enhanced FcγR-mediated effector function compared to the wild-type IgG. [19][20][21] However, avidity effects are expected to be a function not only of the number of binding elements but also of their three-dimensional arrangement. Avidity could vary between constructs with the same number of Fc regions, but with different structural topologies.…”

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

“…Additional efforts at engineering were made to create bispecific types of structures that can target two different antigens, [6] or even more recently, to produce antibodies that contain multiple crystallizable fragment (Fc)-domains, showing enhanced effector functions. [7] This additional variety of structures created in the course of the hunt for so-called biobetters, has shifted the focus away from the naturally occurring structural variety and heterogeneity found in unmodified Immunoglobulins (IgGs) ( Figure 1). However, the sheer size and complexity of these molecules by itself offers ample possibilities for modifications and changes that result in a rather large number of variants, which can differ in their biophysical properties and even in their biological activity.…”