Matrixed CDR grafting: A neoclassical framework for antibody humanization and developability.

Discovery and optimization of a biotherapeutic monoclonal antibody requires a careful balance of target engagement and physicochemical developability properties. To take full advantage of the sequence diversity provided by different antibody discovery platforms, a rapid and reliable process for humanization of antibodies from nonhuman sources is required. Canonically, maximizing homology of the human variable region (V-region) to the original germline was believed to result in preservation of binding, often without much consideration for inherent molecular properties. We expand on this approach by grafting the complementary determining regions (CDRs) of a mouse anti-LAG3 antibody into an extensive matrix of human variable heavy chain (VH) and variable light chain (VL) framework regions with substantially broader sequence homology to assess the impact on complementary determining region-framework compatibility through progressive evaluation of expression, affinity, biophysical developability, and function. Specific VH and VL framework sequences were associated with major expression and purification phenotypes. Greater VL sequence conservation was correlated with retained or improved affinity. Analysis of grafts that bound the target demonstrated that initial developability criteria were significantly impacted by VH, but not VL. In contrast, cell binding and functional characteristics were significantly impacted by VL, but not VH. Principal component analysis of all factors identified multiple grafts that exhibited more favorable antibody properties, notably with nonoptimal sequence conservation. Overall, this study demonstrates that modern throughput systems enable a more thorough, customizable, and systematic analysis of graft-framework combinations, resulting in humanized antibodies with improved global properties that may progress through development more quickly and with a greater probability of success.

Embedding Dynamics in Intrinsic Physicochemical Profiles of Market-Stage Antibody-Based Biotherapeutics.

Adequate stability, manufacturability, and safety are crucial to bringing an antibody-based biotherapeutic to the market. Following the concept of holistic in silico developability, we introduce a physicochemical description of 91 market-stage antibody-based biotherapeutics based on orthogonal molecular properties of variable regions (Fvs) embedded in different simulation environments, mimicking conditions experienced by antibodies during manufacturing, formulation, and in vivo. In this work, the evaluation of molecular properties includes conformational flexibility of the Fvs using molecular dynamics (MD) simulations. The comparison between static homology models and simulations shows that MD significantly affects certain molecular descriptors like surface molecular patches. Moreover, the structural stability of a subset of Fv regions is linked to changes in their specific molecular interactions with ions in different experimental conditions. This is supported by the observation of differences in protein melting temperatures upon addition of NaCl. A DEvelopability Navigator In Silico (DENIS) is proposed to compare mAb candidates for their similarity with market-stage biotherapeutics in terms of physicochemical properties and conformational stability. Expanding on our previous developability guidelines (Ahmed et al. Proc. Natl. Acad. Sci. 2021, 118 (37), e2020577118), the hydrodynamic radius and the protein strand ratio are introduced as two additional descriptors that enable a more comprehensive in silico characterization of biotherapeutic drug candidates. Test cases show how this approach can facilitate identification and optimization of intrinsically developable lead candidates. DENIS represents an advanced computational tool to progress biotherapeutic drug candidates from discovery into early development by predicting drug properties in different aqueous environments.

Determination of the SLAMF1 self-association affinity constant with sedimentation velocity ultracentrifugation.

Signaling lymphocytic activating molecule family member 1 (SLAMF1 or CD150) is a cell surface glycoprotein expressed on various immune populations, regulating cell-cell interactions, activation, differentiation, and inflammatory responses and has been suggested as a potential target for inflammatory diseases. Signaling is believed to be mediated by high-affinity homophilic interactions; the recombinant soluble form of SLAMF1 has optimal activity in the range of 20 µg/mL. This contradicts with a rather weak homo-dimerization binding constant (KD) value reported previously; however, the analytical approach and data analysis suffered from various technical limitations at the time and therefore warrants re-examination. To address this apparent discrepancy, we determined the KD of soluble SLAMF1 using sedimentation velocity analytical ultracentrifuge (SV-AUC). A globally fitted monomer-dimer model properly explains the data from a wide concentration range obtained with both UV and fluorescence detection systems. The analysis suggests the dimerization KD value for human SLAMF1 is 0.48 µM. Additionally, our data show that SLAMF1 self-association is not driven by non-specific binding to glycans supporting the view of specific protein-protein interaction. We anticipate antibody biotherapeutics capable of modulating the biological consequences of SLAMF1 interactions will be readily identified.

Conformational entropy in molecular recognition by proteins.

Molecular recognition by proteins is fundamental to almost every biological process, particularly the protein associations underlying cellular signal transduction. Understanding the basis for protein-protein interactions requires the full characterization of the thermodynamics of their association. Historically it has been virtually impossible to experimentally estimate changes in protein conformational entropy, a potentially important component of the free energy of protein association. However, nuclear magnetic resonance spectroscopy has emerged as a powerful tool for characterizing the dynamics of proteins. Here we employ changes in conformational dynamics as a proxy for corresponding changes in conformational entropy. We find that the change in internal dynamics of the protein calmodulin varies significantly on binding a variety of target domains. Surprisingly, the apparent change in the corresponding conformational entropy is linearly related to the change in the overall binding entropy. This indicates that changes in protein conformational entropy can contribute significantly to the free energy of protein-ligand association.

The role of conformational entropy in molecular recognition by calmodulin.

The physical basis for high-affinity interactions involving proteins is complex and potentially involves a range of energetic contributions. Among these are changes in protein conformational entropy, which cannot yet be reliably computed from molecular structures. We have recently used changes in conformational dynamics as a proxy for changes in conformational entropy of calmodulin upon association with domains from regulated proteins. The apparent change in conformational entropy was linearly related to the overall binding entropy. This view warrants a more quantitative foundation. Here we calibrate an 'entropy meter' using an experimental dynamical proxy based on NMR relaxation and show that changes in the conformational entropy of calmodulin are a significant component of the energetics of binding. Furthermore, the distribution of motion at the interface between the target domain and calmodulin is surprisingly noncomplementary. These observations promote modification of our understanding of the energetics of protein-ligand interactions.

Human Serum Albumin Mitigates Formation of Fatty Acid Particles in Polysorbate-Containing Solutions.

Over seventy percent of marketed monoclonal antibody therapeutics contain between 0.001% and 0.1% (w/v) polysorbate, as it has a generally beneficial stabilizing effect that increases drug product shelf life. However, polysorbate has also been shown to contribute to particle formation due to auto-oxidation and hydrolysis,1 which results in free fatty acids and subsequent fatty acid particle formation. Although the impact of fatty acid particles on the safety and efficacy of drug products has not been fully evaluated, it is advantageous to mitigate particle formation due to degradation of polysorbate, improving the consistency of a product's quality attributes (in this case particulate levels) throughout its lifecycle. In this report, we describe a simple experimental assay to rapidly generate fatty acid particles. Further, we show that the presence of human serum albumin (HSA) is sufficient to prevent the formation of fatty acid particles. Separately, we demonstrate that HSA can also rapidly and completely solubilize pre-formed particles. These results point to a highly plausible mechanistic explanation of previous observations and diminishes concern regarding low levels of particles in the final drug product.

An adapted consensus protein design strategy for identifying globally optimal biotherapeutics.

Biotherapeutic optimization, whether to improve general properties or to engineer specific attributes, is a time-consuming process with uncertain outcomes. Conversely, Consensus Protein Design has been shown to be a viable approach to enhance protein stability while retaining function. In adapting this method for a more limited number of protein sequences, we studied 21 consensus single-point variants from eight publicly available CD3 binding sequences with high similarity but diverse biophysical and pharmacological properties. All single-point consensus variants retained CD3 binding and performed similarly in cell-based functional assays. Using Ridge regression analysis, we identified the variants and sequence positions with overall beneficial effects on developability attributes of the CD3 binders. A second round of sequence generation that combined these substitutions into a single molecule yielded a unique CD3 binder with globally optimized developability attributes. In this first application to therapeutic antibodies, adapted Consensus Protein Design was found to be highly beneficial within lead optimization, conserving resources and minimizing iterations. Future implementations of this general strategy may help accelerate drug discovery and improve success rates in bringing novel biotherapeutics to market.

Intranasal Administration of a Monoclonal Neutralizing Antibody Protects Mice against SARS-CoV-2 Infection.

Despite the recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of the SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2, retains full activity against the variant of concern (VOC) B.1.1.7 and still neutralizes the VOC B.1.351, although with reduced potency. Importantly, not only systemic but also intranasal application of DZIF-10c abolished the presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology when administered prophylactically. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies.

Measuring the effects of macromolecular crowding on antibody function with biolayer interferometry.

Biotherapeutic proteins are commonly dosed at high concentrations into the blood, which is an inherently complex, crowded solution with substantial protein content. The effects of macromolecular crowding may lead to an appreciable level of non-specific hetero-association in this physiological environment. Therefore, developing a method to characterize the diverse consequences of non-specific interactions between proteins under such non-ideal, crowded conditions, which deviate substantially from those commonly employed for in vitro characterization, is vital to achieving a more complete picture of antibody function in a biological context. In this study, we investigated non-specific interactions between human serum albumin (HSA) and two monoclonal antibodies (mAbs) by static light scattering and determined these interactions are both ionic strength-dependent and mAb-dependent. Using biolayer interferometry (BLI), we assessed the effect of HSA on antigen binding by mAbs, demonstrating that these non-specific interactions have a functional impact on mAb:antigen interactions, particularly at low ionic strength. While this effect is mitigated at physiological ionic strength, our in vitro data support the notion that HSA in the blood may lead to non-specific interactions with mAbs in vivo, with a potential impact on their interactions with antigen. Furthermore, the BLI method offers a high-throughput advantage compared to orthogonal techniques such as analytical ultracentrifugation and is amenable to a greater variety of solution conditions compared to nuclear magnetic resonance spectroscopy. Our study demonstrates that BLI is a viable technology for examining the impact of non-specific interactions on specific biologically relevant interactions, providing a direct method to assess binding events in crowded conditions.

Solution structure of the chick TGFbeta type II receptor ligand-binding domain.

The transforming growth factor beta (TGFbeta) signaling pathway influences cell proliferation, immune responses, and extracellular matrix reorganization throughout the vertebrate life cycle. The signaling cascade is initiated by ligand-binding to its cognate type II receptor. Here, we present the structure of the chick type II TGFbeta receptor determined by solution NMR methods. Distance and angular constraints were derived from 15N and 13C edited NMR experiments. Torsion angle dynamics was used throughout the structure calculations and refinement. The 20 final structures were energy minimized using the generalized Born solvent model. For these 20 structures, the average backbone root-mean-square distance from the average structure is below 0.6A. The overall fold of this 109-residue domain is conserved within the superfamily of these receptors. Chick receptors fully recognize and respond to human TGFbeta ligands despite only 60% identity at the sequence level. Comparison with the human TGFbeta receptor determined by X-ray crystallography reveals different conformations in several regions. Sequence divergence and crystal packing interactions under low pH conditions are likely causes. This solution structure identifies regions were structural changes, however subtle, may occur upon ligand-binding. We also identified two very well conserved molecular surfaces. One was found to bind ligand in the crystallized human TGFbeta3:TGFbeta type II receptor complex. The other, newly identified area can be the interaction site with type I and/or type III receptors of the TGFbeta signaling complex.

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity.

An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.

Conformational dynamics of calmodulin in complex with the calmodulin-dependent kinase kinase alpha calmodulin-binding domain.

As the primary intracellular calcium sensor, calcium-saturated calmodulin (CaM) regulates numerous and diverse proteins. Several mechanisms, including tissue-specific expression, localization, and sequestration, work in concert to limit the total number of available targets of calmodulin within a cell. While the free energies of binding of calmodulin-binding domains of regulated proteins by CaM have been shown to be highly similar, they result from vastly different enthalpic and entropic contributions. Here, we report the backbone and side-chain methyl dynamics of calcium-activated calmodulin in complex with a peptide corresponding to the CaM-binding domain of calmodulin kinase kinase, along with the thermodynamic underpinnings of complex formation. The results show a considerable reduction in side-chain mobility throughout CaM upon binding the CaMKKalpha peptide, which is consistent with the enthalpically driven nature of the binding. Site-specific comparison to another kinase-derived peptide complex with similar thermodynamic values reveals significant differences in dynamics largely localized to the hydrophobic binding sites.