Effect of Trastuzumab–HER2 Complex Formation on Stress-Induced Modifications in the CDRs of Trastuzumab

Asparagine deamidation and aspartic acid isomerization in the complementarity determining regions (CDRs) of monoclonal antibodies may alter their affinity to the target antigen. Trastuzumab has two hot spots for deamidation and one position for isomerization in the CDRs. Little is known how complex formation with its target antigen HER2 affects these modifications. Modifications in the CDRs of trastuzumab were thus compared between the free antibody and the trastuzumab–HER2 complex when stressed under physiological conditions at 37°C. Complex formation and stability of the complex upon stressing were assessed by size-exclusion chromatography. Deamidation of light-chain Asn-30 (Lc-Asn-30) was extensive when trastuzumab was stressed free but reduced about 10-fold when the antibody was stressed in complex with HER2. Almost no deamidation of heavy-chain (Hc-Asn-55) was detected in the trastuzumab–HER2 complex, while deamidation was observed when the antibody was stressed alone. Hc-Asp-102 isomerization, a modification that critically affects biological activity, was observed to a moderate degree when the free antibody was stressed but was not detected at all in the trastuzumab–HER2 complex. This shows that complex formation has a major influence on critical modifications in the CDRs of trastuzumab.

Creation of stable water-free antibody based protein liquids

Antibodies represent highly specific and high binding affinity biomolecular recognition elements for diagnostic assays, biosensors, and therapeutics, but are sensitive to denaturation and degradation. Consequently, the combination of existing in a hydrated state with a large and complex biomolecular structure results in loss of antibody-antigen binding, limited shelf-life, and decreased sensor response over time and under non-optimal conditions. The development and use of water-free protein liquids has led to stabilization of labile biomolecules, solvents for biotransformation reactions, and formation of new bio-composites with incompatible materials. Here, we exploit the polycationic nature of modified antibodies and their ability to form ion pairs for the conversion of primary Immunoglobulin G antibodies into stable protein liquids that retained more than 60% binding activity after repeated heating up to 125 °C, and demonstrate compatibility with thermoplastics.

Correlation of N-glycan dynamics and interaction network with allosteric antigen binding and Fc receptor recognition

Aim: Fragment crystallizable (Fc) glycans modulate Fc conformations and functions, and glycan may also regulate antigen recognition. In the antibody drug development, glycosylation patterns affect antibody drug characteristics and quality control. In order to provide a global feature of N-glycan interactions in response to antigen and Fc receptor bindings, the interactions among Fc N-glycans and N-glycans’ interaction with Fc CH2 and CH3 domains have been studied. Methods: Molecular dynamics simulations were used to generate conformation ensembles of free antibody, antibody-antigen complex, antibody-human Fc-gamma-receptor-I (hFcγRI) and antibody-antigen-hFcγRI, the hydrogen bonds and radial distance distribution involving N-glycans carbohydrate chains have been analyzed. Results: Two important interaction patterns have been observed. The first is the strong but non-specific interactions between two carbohydrate chains in free antibody. Secondly, it has been found that N-glycans carbohydrate chains can directly interact with CH3 domain in free antibody, and that the distance distribution between carbohydrate chains and CH3 domain clearly differentiate the free antibody, antibody-antigen complex, antibody-hFcγRI complex, and final antibody-antigen-hFcγRI complex. Conclusions: N-glycans partially acts as allosteric sensor and respond to antigen and hFcγRI binding.

A cell-free antibody engineering platform rapidly generates SARS-CoV-2 neutralizing antibodies

Antibody engineering technologies face increasing demands for speed, reliability and scale. We developed CeVICA, a cell-free antibody engineering platform that integrates a novel generation method and design for camelid heavy-chain antibody VHH domain-based synthetic libraries, optimized in vitro selection based on ribosome display and a computational pipeline for binder prediction based on CDR-directed clustering. We applied CeVICA to engineer antibodies against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike proteins and identified >800 predicted binder families. Among 14 experimentally-tested binders, 6 showed inhibition of pseudotyped virus infection. Antibody affinity maturation further increased binding affinity and potency of inhibition. Additionally, the unique capability of CeVICA for efficient and comprehensive binder prediction allowed retrospective validation of the fitness of our synthetic VHH library design and revealed direction for future refinement. CeVICA offers an integrated solution to rapid generation of divergent synthetic antibodies with tunable affinities in vitro and may serve as the basis for automated and highly parallel antibody generation.

Linker-free antibody conjugation for sensitive hydrogel microparticle-based multiplex immunoassay

Graphically encoded hydrogel microparticles were directly conjugated with reduced antibodies without linkers for highly sensitive multiplex immunoassay.

Convergent synthesis of hydrophilic monomethyl dolastatin 10 based drug linkers for antibody–drug conjugation

A cytotoxic reagent-free fragment coupling methodology was developed to produce hydrophilic drug linkers to prepare aggregation free antibody–drug conjugates.

Use of Immunohistochemistry to Establish Ethiology in the Case of Primary Spontaneous Pneumotorax Patients

Primary spontaneous pneumothorax has a complex morphopathological substrate, in which active smoking plays an essential etiopathogenic role. Inflammation of the distal airways, bronchial anomalies, perivascular eosinophilic infiltrate associated with hereditary factors and physiognomy (longilli patients) lead to obstruction of distal airwayswhich is the essential element in the emergence of emphysematous changes. Immunohistochemistry (IHC) is a technique used to identify cellular or tissue (antigens) constituents by Ag-Ac, the Ac link site being identified either by direct labeling of the antibody or by a secondary labeling method. IHC reactions are based on tissue-antibody antigen binding, the latter being evidenced by direct conjugation to tracer molecules (direct reaction) or by another chain of other labeled free antibody linkages. We can consider the immunohistochemical method as having a potential utility, especially in selected patients, where there are sufficient clinical and epidemiological reasons to suspect a pneumothorax-causing disease but where the classical investigations did not provide diagnostic performance.

Immunochemical engineering of cell surfaces to generate virus resistance

Modern immunochemical engineering allows the creation of cells that either secrete antibodies or incorporate them into various cellular compartments, including the plasma membrane. Because the receptors for most viruses are known, if one can achieve the proper stoichiometry and geometry, plasma membrane-associated antibodies to these receptors should block viral infection. In this report, we test this concept for two different viruses, human rhinovirus and HIV. Plasma membrane-tethered antibodies efficiently rendered cells permanently nonpermissive for infection by both these viruses. Membrane-bound antibodies were much more efficient than free antibody in preventing infection, likely because of the effective molarity of membrane bound antibodies. Such resistant cells may restore immune-competence to otherwise compromised HIV patients.