Determining the affinities of high-affinity antibodies using KinExA and surface plasmon resonance.

Accurate and efficient affinity measurement techniques are essential for the biophysical characterization of therapeutic monoclonal antibodies, one of the fastest growing drug classes. Surface plasmon resonance (SPR) is widely used for determining antibody affinity, but does not perform well with extremely high affinity (low picomolar to femtomolar range) molecules. In this study, we compare the SPR-based Carterra LSA and the kinetic exclusion assay (KinExA) for measuring the affinities of 48 antibodies generated against the SARS-CoV-2 receptor-binding domain. These data reveal that high-affinity antibodies can be generated straight from selections using high-quality in vitro library platforms with 54% correspondence between affinities measured using LSA and KinExA. Generally, where there was a 2-fold or greater difference between LSA and KinExA, KinExA reported that affinities were tighter. We highlight the differences between LSA and KinExA, identifying the benefits and pitfalls of each in terms of dynamic range and throughput. Furthermore, we demonstrate for the first time that single-point screening with KinExA can significantly improve throughput while maintaining a strong correlation with full binding curve equilibrium measurements, enabling the accurate rank-ordering of clones with exceptionally tight binding properties.

Insights into next generation sequencing guided antibody selection strategies.

Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random colony picking and Sanger sequencing, which has limitations. Next-generation sequencing (NGS) offers a cost-effective solution with increased read depth, allowing a comprehensive understanding of diversity. Our study establishes NGS guidelines for antibody drug discovery, demonstrating its advantages in expanding the number of unique HCDR3 clusters, broadening the number of high affinity antibodies, expanding the total number of antibodies recognizing different epitopes, and improving lead prioritization. Surprisingly, our investigation into the correlation between NGS-derived frequencies of CDRs and affinity revealed a lack of association, although this limitation could be moderately mitigated by leveraging NGS clustering, enrichment and/or relative abundance across different regions to enhance lead prioritization. This study highlights NGS benefits, offering insights, recommendations, and the most effective approach to leverage NGS in therapeutic antibody discovery.

High throughput purification of monoclonal recombinant antibodies using a Protein-A coated membrane plate system.

The therapeutic use of monoclonal antibodies (mAbs) ranges from cancer treatment to immune-mediated conditions, covering infectious and cardiovascular disorders, among others. The development of improved methods for therapeutic antibody discovery has accelerated the identification of numerous mAbs: a discovery campaign can be deeply mined, resulting in hundreds, even thousands, of potential antibody leads for a given target of interest. High throughput mAb expression and purification methods are required for the rapid validation of those leads. In this work, we describe the implementation of a Protein-A coated membrane plate system, the Purexa™ AHT membrane plate, for robust preparative purification of hundreds of recombinant mAbs, without the need for automation. The high efficiency (>80%) recovery generated sufficient mAb for downstream screening analyses such as ELISA and surface plasmon resonance (SPR). This new system allows the functional validation of hundreds of lead antibodies from discovery campaigns in a timely manner regardless of operational size.

Pandemic's silver lining.

The intense international focus on the COVID-19 pandemic has provided a unique opportunity to use a wide array of novel tools to carry out scientific studies on the SARS-CoV-2 virus. The value of these comparative studies extends far beyond their consequences for SARS-CoV-2, providing broad implications for health-related science. Here we specifically discuss the impacts of these comparisons on advances in vaccines, the analysis of host humoral immunity, and antibody discovery. As an extension, we also discuss potential synergies between these areas.Abbreviations: CoVIC: The Coronavirus Immunotherapeutic Consortium; EUA: Emergency Use Authorization.

Simultaneous affinity maturation and developability enhancement using natural liability-free CDRs.

ABBREVIATIONS: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; ka: association rate; kd: dissociation rate; KD: dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.

A pandemic-enabled comparison of discovery platforms demonstrates a naïve antibody library can match the best immune-sourced antibodies.

As a result of the SARS-CoV-2 pandemic numerous scientific groups have generated antibodies against a single target: the CoV-2 spike antigen. This has provided an unprecedented opportunity to compare the efficacy of different methods and the specificities and qualities of the antibodies generated by those methods. Generally, the most potent neutralizing antibodies have been generated from convalescent patients and immunized animals, with non-immune phage libraries usually yielding significantly less potent antibodies. Here, we show that it is possible to generate ultra-potent (IC50 < 2 ng/ml) human neutralizing antibodies directly from a unique semisynthetic naïve antibody library format with affinities, developability properties and neutralization activities comparable to the best from hyperimmune sources. This demonstrates that appropriately designed and constructed naïve antibody libraries can effectively compete with immunization to directly provide therapeutic antibodies against a viral pathogen, without the need for immune sources or downstream optimization.

Drug-like antibodies with high affinity, diversity and developability directly from next-generation antibody libraries.

Therapeutic antibodies must have "drug-like" properties. These include high affinity and specificity for the intended target, biological activity, and additional characteristics now known as "developability properties": long-term stability and resistance to aggregation when in solution, thermodynamic stability to prevent unfolding, high expression yields to facilitate manufacturing, low self-interaction, among others. Sequence-based liabilities may affect one or more of these characteristics. Improving the stability and developability of a lead antibody is typically achieved by modifying its sequence, a time-consuming process that often results in reduced affinity. Here we present a new antibody library format that yields high-affinity binders with drug-like developability properties directly from initial selections, reducing the need for further engineering or affinity maturation. The innovative semi-synthetic design involves grafting natural complementarity-determining regions (CDRs) from human antibodies into scaffolds based on well-behaved clinical antibodies. HCDR3s were amplified directly from B cells, while the remaining CDRs, from which all sequence liabilities had been purged, were replicated from a large next-generation sequencing dataset. By combining two in vitro display techniques, phage and yeast display, we were able to routinely recover a large number of unique, highly developable antibodies against clinically relevant targets with affinities in the subnanomolar to low nanomolar range. We anticipate that the designs and approaches presented here will accelerate the drug development process by reducing the failure rate of leads due to poor antibody affinities and developability.Abbreviations: AC-SINS: affinity-capture self-interaction nanoparticle spectroscopy; CDR: complementarity-determining region; CQA: critical quality attribute; ELISA: enzyme-linked immunoassay; FACS: fluorescence-activated cell sorting; Fv: fragment variable; GM-CSF: granulocyte-macrophage colony-stimulating factor; HCDR3: heavy chain CDR3; IFN2a: interferon α-2; IL6: interleukin-6; MACS: magnetic-activated cell sorting; NGS: next generation sequencing; PCR: polymerase chain reaction; SEC: size-exclusion chromatography; SPR: surface plasmon resonance; TGFß-R2: transforming growth factor ß-R2; VH: variable heavy; VK: variable kappa; VL: variable light; Vl: variable lambda.

Polymerized laminin incorporation into alginate-based microcapsules reduces pericapsular overgrowth and inflammation.

Cell encapsulation coats cells with an artificial membrane to preserve their physical and functional integrity. Different approaches try to develop more functional and biocompatible materials to avoid cell loss after transplantation due to inflammatory reaction, one of the main causes for graft failure. In this study, the LN-Biodritin biomaterial, based on alginate, chondroitin sulfate, and laminin, previously developed by our group, was further improved by replacing laminin by polylaminin, an artificial laminin polymer with anti-inflammatory properties, generating the new biomaterial polyLN-Biodritin. Capsules containing polylaminin are stable, do not induce macrophage activation in vitro, and are also able to prevent macrophage activation by encapsulated human pancreatic islets in vitro, preserving their glucose-stimulated insulin secretion potential. In addition, when empty capsules containing polylaminin were implanted into immunocompetent mice, the inflammatory response towards the implant was attenuated, when compared with capsules without polylaminin. The results indicate that polylaminin incorporation leads to lower levels of pericapsular growth on the capsules surface, lower infiltration of cells into the peritoneal cavity, and lower production of proinflammatory cytokines, both at the implant site (interleukin-12p70 (IL-12p70), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interferon-γ (IFN-γ)) and systemically (IL-12p70 and TNF-α). Therefore, polylaminin incorporation into the microcapsules polymer attenuates the host posttransplantation immune response against implanted microcapsules, being likely to favor maintenance of engrafted encapsulated cells.

Roles of Commensal Microbiota in Pancreas Homeostasis and Pancreatic Pathologies.

The pancreas plays a central role in metabolism, allowing ingested food to be converted and used as fuel by the cells throughout the body. On the other hand, the pancreas may be affected by devastating diseases, such as pancreatitis, pancreatic adenocarcinoma (PAC), and diabetes mellitus (DM), which generally results in a wide metabolic imbalance. The causes for the development and progression of these diseases are still controversial; therefore it is essential to better understand the underlying mechanisms which compromise the pancreatic homeostasis. The interest in the study of the commensal microbiome increased extensively in recent years, when many discoveries have illustrated its central role in both human physiology and maintenance of homeostasis. Further understanding of the involvement of the microbiome during the development of pathological conditions is critical for the improvement of new diagnostic and therapeutic approaches. In the present review, we discuss recent findings on the behavior and functions played by the microbiota in major pancreatic diseases and provide further insights into its potential roles in the maintenance of pancreatic steady-state activities.