High-Throughput and Format-Agnostic Mispairing Assay for Multispecific Antibodies Using Intact Mass Spectrometry.

Multispecific antibodies have gained significant importance in a broad indication space due to their ability to engage multiple epitopes simultaneously and to thereby overcome therapeutic barriers. With growing therapeutic potential, however, the molecular complexity increases, thus intensifying the demand for innovative protein engineering and analytical strategies. A major challenge for multispecific antibodies is the correct assembly of light and heavy chains. Engineering strategies exist to stabilize the correct pairing, but typically individual engineering campaigns are required to arrive at the anticipated format. Mass spectrometry has proven to be a versatile tool to identify mispaired species. However, due to manual data analysis procedures, mass spectrometry is limited to lower throughputs. To keep pace with increasing sample numbers, we developed a high-throughput-capable mispairing workflow based on intact mass spectrometry with automated data analysis, peak detection, and relative quantification using Genedata Expressionist. This workflow is capable of detecting mispaired species of ∼1000 multispecific antibodies in three weeks and thus is applicable to complex screening campaigns. As a proof of concept, the assay was applied to engineering a trispecific antibody. Strikingly, the new setup has not only proved successful in mispairing analysis but has also revealed its potential to automatically annotate other product-related impurities. Furthermore, we could confirm the assay to be format-agnostic, as shown by analyzing several different multispecific formats in one run. With these comprehensive capabilities, the new automated intact mass workflow can be applied as a universal tool to detect and annotate peaks in a format-agnostic approach and in high-throughput, thus enabling complex discovery campaigns.

Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections.

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.

An end-to-end automated platform process for high-throughput engineering of next-generation multi-specific antibody therapeutics.

Next-generation multi-specific antibody therapeutics (MSATs) are engineered to combine several functional activities into one molecule to provide higher efficacy compared to conventional, mono-specific antibody therapeutics. However, highly engineered MSATs frequently display poor yields and less favorable drug-like properties (DLPs), which can adversely affect their development. Systematic screening of a large panel of MSAT variants in very high throughput (HT) is thus critical to identify potent molecule candidates with good yield and DLPs early in the discovery process. Here we report on the establishment of a novel, format-agnostic platform process for the fast generation and multiparametric screening of tens of thousands of MSAT variants. To this end, we have introduced full automation across the entire value chain for MSAT engineering. Specifically, we have automated the in-silico design of very large MSAT panels such that it reflects precisely the wet-lab processes for MSAT DNA library generation. This includes mass saturation mutagenesis or bulk modular cloning technologies while, concomitantly, enabling library deconvolution approaches using HT Sanger DNA sequencing. These DNA workflows are tightly linked to fully automated downstream processes for compartmentalized mammalian cell transfection expression, and screening of multiple parameters. All sub-processes are seamlessly integrated with tailored workflow supporting bioinformatics. As described here, we used this platform to perform multifactor optimization of a next-generation bispecific, cross-over dual variable domain-Ig (CODV-Ig). Screening of more than 25,000 individual protein variants in mono- and bispecific format led to the identification of CODV-Ig variants with over 1,000-fold increased potency and significantly optimized production titers, demonstrating the power and versatility of the platform.