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ABSTRACTRabbits produce robust antibody responses and have unique features in their antibody repertoire that make them an attractive alternative to rodents for in vivo discovery. However, the frequent occurrence of a non-canonical disulfide bond between complementarity-determining region (CDR) H1 (C35a) and CDRH2 (C50) is often seen as a liability for therapeutic antibody development, despite limited reports of its effect on antibody binding, function, and stability. Here, we describe the discovery and humanization of a human-mouse cross-reactive anti-programmed cell death (PD-1) monoclonal rabbit antibody, termed h1340.CC, which possesses this non-canonical disulfide bond. Initial removal of the non-canonical disulfide resulted in a loss of PD-1 affinity and cross-reactivity, which led us to explore protein engineering approaches to recover these. First, guided by the sequence of a related clone and the crystal structure of h1340.CC in complex with PD-1, we generated variant h1340.SA.LV with a potency and cross-reactivity similar to h1340.CC, but only partially recovered affinity. Side-by-side developability assessment of both h1340.CC and h1340.SA.LV indicate that they possess similar, favorable properties. Next, and prompted by recent developments in machine learning (ML)-guided protein engineering, we used an unbiased ML- and structure-guided approach to rapidly and efficiently generate a different variant with recovered affinity. Our case study thus indicates that, while the non-canonical inter-CDR disulfide bond found in rabbit antibodies does not necessarily constitute an obstacle to therapeutic antibody development, combining structure- and ML-guided approaches can provide a fast and efficient way to improve antibody properties and remove potential liabilities.

Antibodies are fundamental effectors of humoral immunity, and have become a highly successful class of therapeutics. There is increasing evidence that antibodies utilize transient homotypic interactions to enhance function, and elucidation of such interactions can provide insights into their biology and new opportunities for their optimization as drugs. Yet the transitory nature of weak interactions makes them difficult to investigate. Capitalizing on their rich structural data and high conservation, we have characterized all the ways that antibody fragment antigen-binding (Fab) regions interact crystallographically. This approach led to the discovery of previously unrealized interfaces between antibodies. While diverse interactions exist, β-sheet dimers and variable-constant elbow dimers are recurrent motifs. Disulfide engineering enabled interactions to be trapped and investigated structurally and functionally, providing experimental validation of the interfaces and illustrating their potential for optimization. This work provides first insight into previously undiscovered oligomeric interactions between antibodies, and enables new opportunities for their biotherapeutic optimization.

Abstract Background One of the mechanisms by which tumors evade immune surveillance is through shedding of the major histocompatibility complex (MHC) class I chain-related protein A and B (MICA/B) from their cell surface. MICA/B are ligands for the activating receptor NKG2D on NK and CD8 T cells. This shedding reduces cell surface levels of MICA/B and impairs NKG2D recognition. Shed MICA/B can also mask NKG2D receptor and is thought to induce NKG2D internalization, further compromising immune surveillance by NK cells. Methods We isolated human primary NK cells from normal donors and tested the suppressive activity of soluble recombinant MICA in vitro. Utilizing a panel of novel anti-MICA antibodies, we further examined the stimulatory activities of anti-MICA antibodies that reversed the suppressive effects of soluble MICA. Results We show that suppressive effects of soluble MICA (sMICA) on NK cell cytolytic activity was not due to the down-regulation of cell surface NKG2D. In the presence of an α3 domain-specific MICA antibody, which did not obstruct NKG2D binding, sMICA-mediated NK cell suppression was completely reversed. Reversal of NK cell inhibition by sMICA was mediated by immune complex formation that agonized NKG2D signaling. Furthermore, this restorative activity was dependent on antibody Fc effector function as the introduction of Fc mutations to abrogate Fc receptor binding failed to reverse sMICA-mediated NK cell suppression. Furthermore, MICA immune complexes preformed with an α3 domain-specific antibody (containing a wild-type Fc) induced IFN-γ and TNF-α secretion by NK cells in the absence of cancer cells, whereas MICA immune complexes preformed with the Fc effectorless antibody failed to induce IFN-γ and TNF-α secretion. Finally, we demonstrated that MICA immune complexes formed with the α3 domain-specific antibody activates NKG2D on NK cells leading to the release of IFN-γ. Conclusions Our results demonstrate that an α3 domain-specific MICA antibody can circumvent sMICA-mediated suppression of NK cell cytolytic activity. Moreover, our data suggest that MICA immune complexes formed with α3-specific antibodies can activate NKG2D receptor and restore NK cell function in a Fc-dependent manner. The clinical utility of α3 domain-specific MICA/B antibodies may hold great promise as a new strategy for cancer immunotherapy.