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.

EVALUATION OF THE EFFECT OF HYDRATED LIME ON THE SCAVENGING OF FERAL SWINE (SUS SCROFA) CARCASSES AND IMPLICATIONS FOR MANAGING CARCASS-BASED TRANSMISSION OF AFRICAN SWINE FEVER VIRUS.

African swine fever (ASF) is a devastating hemorrhagic disease marked by extensive morbidity and mortality in infected swine. The recent global movement of African swine fever virus (ASFV) in domestic and wild swine (Sus scrofa) populations has initiated preparedness and response planning activities within many ASF-free countries. Within the US, feral swine are of utmost concern because they are susceptible to infection, are wide-spread, and are known to interact with domestic swine populations.

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.

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.

Development of novel ultra-sensitive IL-11 target engagement assays to support mechanistic PK/PD modeling for an anti-IL-11 antibody therapeutic.

An in-house antibody generation campaign identified a diverse, high affinity set of anti-interleukin-11 (IL-11) monoclonal antibodies (mAbs) to enable successful development of novel, custom ultra-sensitive target engagement assays for detection of "free" (unbound to the dosed anti-IL-11 therapeutic mAb) and "total" (free and mAb-IL-11 complexed form) IL-11 in preclinical species and human. Antibody hits from distinct epitope communities were screened on various platforms, including enzyme-linked immunosorbent assay, Meso Scale Discovery, Simoa HD-1 and Simoa Planar Array (SP-X), and used for assay development and sensitivity optimization. The ultra-sensitive SP-X format achieved a lower limit of quantitation of 0.

Effect of the ADCC-Modulating Mutations and the Selection of Human IgG Isotypes on Physicochemical Properties of Fc.

Monoclonal antibodies, particularly IgGs and Ig-based molecules, are a well-established and growing class of biotherapeutic drugs. In order to improve efficacy, potency and pharmacokinetics of these therapeutic drugs, pharmaceutical industries have investigated significantly in engineering fragment crystallizable (Fc) domain of these drugs to optimize the interactions of these drugs and Fc gamma receptors (FcγRs) in recent ten years. The biological function of the therapeutics with the antibody-dependent cellular cytotoxicity (ADCC) enhanced double mutation (S239D/I332E) of isotype IgG1, the ADCC reduced double mutation (L234A/L235A) of isotype IgG1, and ADCC reduced isotype IgG4 has been well understood.

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.

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 (K) value reported previously; however, the analytical approach and data analysis suffered from various technical limitations at the time and therefore warrants re-examination.

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.

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 characterization, is vital to achieving a more complete picture of antibody function in a biological context.

Current status and future recommendations for feral swine disease surveillance in the United States.

USDA APHIS Wildlife Services (WS) responded to the threat of feral swine as a pathogen reservoir as early as 2004. To increase awareness and knowledge on that risk, WS began opportunistic sampling of animals harvested by its operational component to curtail swine damage to agriculture and property. Initially, pseudorabies and swine brucellosis were of most concern, as both serve as a potential threat to the domestic swine industry and the latter also possesses zoonotic implications.

Controversy and debate: Memory-Based Methods Paper 1: the fatal flaws of food frequency questionnaires and other memory-based dietary assessment methods.

There is an escalating debate over the value and validity of self-reported dietary intake as estimated by Food Frequency Questionnaires and other forms of memory-based dietary assessment methods. Proponents argue that despite limitations, memory-based methods provide valid and valuable information about consumed foods and beverages and therefore can be used to assess diet-disease relations. In fact, over the past 60 years, thousands of memory-based dietary research reports were used to inform public policy and establish the Dietary Guidelines for Americans.

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.

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.

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.

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.

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.