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.

Structural basis for the recognition of human cytomegalovirus glycoprotein B by a neutralizing human antibody.

Human cytomegalovirus (HCMV) infections are life-threating to people with a compromised or immature immune system. Upon adhesion, fusion of the virus envelope with the host cell is initiated. In this step, the viral glycoprotein gB is considered to represent the major fusogen. Here, we present for the first time structural data on the binding of an anti-herpes virus antibody and describe the atomic interactions between the antigenic domain Dom-II of HCMV gB and the Fab fragment of the human antibody SM5-1. The crystal structure shows that SM5-1 binds Dom-II almost exclusively via only two CDRs, namely light chain CDR L1 and a 22-residue-long heavy chain CDR H3. Two contiguous segments of Dom-II are targeted by SM5-1, and the combining site includes a hydrophobic pocket on the Dom-II surface that is only partially filled by CDR H3 residues. SM5-1 belongs to a series of sequence-homologous anti-HCMV gB monoclonal antibodies that were isolated from the same donor at a single time point and that represent different maturation states. Analysis of amino acid substitutions in these antibodies in combination with molecular dynamics simulations show that key contributors to the picomolar affinity of SM5-1 do not directly interact with the antigen but significantly reduce the flexibility of CDR H3 in the bound and unbound state of SM5-1 through intramolecular side chain interactions. Thus, these residues most likely alleviate unfavorable binding entropies associated with extra-long CDR H3s, and this might represent a common strategy during antibody maturation. Models of entire HCMV gB in different conformational states hint that SM5-1 neutralizes HCMV either by blocking the pre- to postfusion transition of gB or by precluding the interaction with additional effectors such as the gH/gL complex.

Characterization of a discontinuous neutralizing epitope on glycoprotein B of human cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and newborn infants infected in utero. The viral envelope glycoprotein B (gB) is an attractive molecule for active vaccination and passive immunoprophylaxis and therapy. Using human monoclonal antibodies (MAbs), we have recently identified antigenic region 4 (AD-4) on gB as an important target for neutralizing antibodies. AD-4 is formed by a discontinuous sequence comprising amino acids 121 to 132 and 344 to 438 of gB of HCMV strain AD169. To map epitopes for human antibodies on this protein domain, we used a three-dimensional (3D) model of HCMV gB to identify surface-exposed amino acids on AD-4 and selected juxtaposed residues for alanine scans. A tyrosine (Y) at position 364 and a lysine (K) at position 379 (the YK epitope), which are immediate neighbors on the AD-4 surface, were found to be essential for binding of the human MAbs. Recognition of AD-4 by sera from HCMV-infected individuals also was largely dependent on these two residues, indicating a general importance for the antibody response against AD-4. A panel of AD-4 recombinant viruses harboring mutations at the crucial antibody binding sites was generated. The viruses showed significantly reduced susceptibility to neutralization by AD-4-specific MAbs or polyclonal AD-4-specific antibodies, indicating that the YK epitope is dominant for the AD-4-specific neutralizing antibody response during infection. To our knowledge, this is the first molecular identification of a functional discontinuous epitope on HCMV gB. Induction of antibodies specific for this epitope may be a desirable goal following vaccination with gB.

B cell repertoire analysis identifies new antigenic domains on glycoprotein B of human cytomegalovirus which are target of neutralizing antibodies.

Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133-343 of gB and domain II, a discontinuous domain, built from residues 121-132 and 344-438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization.

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.