Current status and future prospective of breast cancer immunotherapy.

The immune system is complicated, interconnected, and offers a powerful defense system that protects its host from foreign pathogens. Immunotherapy involves boosting the immune system to kill cancer cells, and nowadays, is a major emerging treatment for cancer. With the advances in our understanding of the immunology of cancer, there has been an explosion of studies to develop and evaluate therapies that engage the immune system in the fight against cancer. Nevertheless, conventional therapies have been effective in reducing tumor burden and prolonging patient life, but the overall efficacy of these treatment regimens has been somewhat mixed and often with severe side effects. A common reason for this is the activation of molecular mechanisms that lead to apoptosis of anti-tumor effector cells. The competency to block tumor escape entirely depends on our understanding of the cellular and molecular pathways which operate in the tumor microenvironment. Numerous strategies have been developed for activating the immune system to kill tumor cells. Breast cancer is one of the major causes of cancer death in women, and is characterized by complex molecular and cellular events that closely intertwine with the host immune system. In this regard, predictive biomarkers of immunotherapy, use of nanotechnology, personalized cancer vaccines, antibodies to checkpoint inhibitors, engineered chimeric antigen receptor-T cells, and the combination with other therapeutic modalities have transformed cancer therapy and optimized the therapeutic effect. In this chapter, we will offer a holistic view of the different therapeutic modalities and recent advances in immunotherapy. Additionally, we will summarize the recent advances and future prospective of breast cancer immunotherapies, as a case study.

An efficient computational protocol for template-based design of peptides that inhibit interactions involving SARS-CoV-2 proteins.

The RNA-dependent RNA polymerase (RdRp) complex of SARS-CoV-2 lies at the core of its replication and transcription processes. The interfaces between holo-RdRp subunits are highly conserved, facilitating the design of inhibitors with high affinity for the interaction interface hotspots. We, therefore, take this as a model protein complex for the application of a structural bioinformatics protocol to design peptides that inhibit RdRp complexation by preferential binding at the interface of its core subunit nonstructural protein, nsp12, with accessory factor nsp7. Here, the interaction hotspots of the nsp7-nsp12 subunit of RdRp, determined from a long molecular dynamics trajectory, are used as a template. A large library of peptide sequences constructed from multiple hotspot motifs of nsp12 is screened in-silico to determine sequences with high geometric complementarity and interaction specificity for the binding interface of nsp7 (target) in the complex. Two lead designed peptides are extensively characterized using orthogonal bioanalytical methods to determine their suitability for inhibition of RdRp complexation. Binding affinity of these peptides to accessory factor nsp7, determined using a surface plasmon resonance (SPR) assay, is slightly better than that of nsp12: dissociation constant of 133nM and 167nM, respectively, compared to 473nM for nsp12. A competitive ELISA is used to quantify inhibition of nsp7-nsp12 complexation, with one of the lead peptides giving an IC50 of 25µM . Cell penetrability and cytotoxicity are characterized using a cargo delivery assay and MTT cytotoxicity assay, respectively. Overall, this work presents a proof-of-concept of an approach for rational discovery of peptide inhibitors of SARS-CoV-2 protein-protein interactions.

Assessment of Functional Characterization and Comparability of Biotherapeutics: a Review.

The development of monoclonal antibody (mAb) biosimilars is a complex process. The key to their successful development and commercialization is an in-depth understanding of the key product attributes that impact safety and efficacy and the strategies to control them. Functional assessment of mAb is a crucial part of the comparability of biopharmaceutical drugs. The development of a relevant and robust functional assay requires an interdisciplinary approach and sufficient flexibility to balance regulatory concerns as well as dynamics and variability during the manufacturing process. Although many advanced tools are available to study and compare the potency and bioactivity of the protein, most of these techniques suffer from major shortcomings that limit their routine use. These include the complexity of the task, establishment of the relevance of the chosen method with the mechanism of action (MOA) of the biosimilar, cost and extended time of analysis, and often the ambiguity in interpretation of the resulting data. To overcome or to address these challenges, the use of multiple orthogonal state-of-the-art techniques is a necessary prerequisite.

A novel piperazine derivative that targets hepatitis B surface antigen effectively inhibits tenofovir resistant hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is a global problem. The loss of hepatitis B surface antigen (HBsAg) in serum is a therapeutic end point. Prolonged therapy with nucleoside/nucleotide analogues targeting the HBV-polymerase may lead to resistance and rarely results in the loss of HBsAg. Therefore, inhibitors targeting HBsAg may have potential therapeutic applications. Here, we used computational virtual screening, docking, and molecular dynamics simulations to identify potential small molecule inhibitors against HBsAg. After screening a million molecules from ZINC database, we identified small molecules with potential anti-HBV activity. Subsequently, cytotoxicity profiles and anti-HBV activities of these small molecules were tested using a widely used cell culture model for HBV. We identified a small molecule (ZINC20451377) which binds to HBsAg with high affinity, with a KD of 65.3 nM, as determined by Surface Plasmon Resonance spectroscopy. Notably, the small molecule inhibited HBsAg production and hepatitis B virion secretion (10 µM) at low micromolar concentrations and was also efficacious against a HBV quadruple mutant (CYEI mutant) resistant to tenofovir. We conclude that this small molecule exhibits strong anti-HBV properties and merits further testing.

Freeze thaw and lyophilization induced alteration in mAb therapeutics: Trastuzumab as a case study.

Long-term stability of therapeutic monoclonal antibody (mAb) products is necessary for their successful commercialization. Freeze-thaw (F/T) operations are often performed for a mAb product during processing, storage and distribution. Lyophilization (Lyo) is another unit operation that is commonly used for drug product manufacturing of mAbs. This paper aims to explore the impact of these operations on structure and function of a mAb therapeutic, as well as of biosimilars. Trastuzumab innovator and its five biosimilars were analysed for aggregation, charge heterogeneity, secondary structure, binding kinetics, and potency after each freeze-thaw and lyophilization cycle. It is observed that both F/T and Lyo induce protein aggregation, which in turn causes perturbations in the biological potency of the mAb therapeutic. The average value of the percentage of aggregation increased from 0.6 % (week 1) to 5.3 % (week 10) in F/T study and from 0.8 % (week 1) to 10.1 % (week 10) in Lyo study. The acidic pool increased from 26.5 % (week 1) to 44.4 % (week 10) and the basic variants from 13.9 % (week 1) to 24.0 % (week 10) in F/T study. Similarly, acidic pool increased from 27.1 % (week 1) to 42.0 % (week 10) and basic variants from 14.8 % (week 1) to 24.4 % (week 10) in Lyo study. The average percentage of beta-sheet increased from 58.4 % (week 1) to 60.9 % (week 10) in F/T study and from 59.7 % (week 1) to 72.6 % (week 10) in Lyo study. Lower binding affinity was found in week 7 as compared to week 1 in Lyo study whereas no change in binding affinity was observed in the F/T study. The average potency value gradually decreased from 0.97IU/ ml (week 1) to 0.75IU/ ml (week 10) in F/T study and from 1.0IU/ ml (week 1) to 0.66IU/ ml (week 10) in Lyo study. Results indicate that lyophilization has a bigger impact on binding affinity than freeze thaw and as expected, the impact was comparable across the innovator and biosimilar products.

Impact of mAb Aggregation on Its Biological Activity: Rituximab as a Case Study.

Monoclonal antibody (mAb) products are presently the dominant class of therapeutic proteins. When stressed, they are known to be prone to molecular instabilities like aggregation, fragmentation, oxidation and reduction, of which aggregation is typically the most significant concern. These stresses may be experienced during manufacturing, storage, filling, formulation development and shipping. This paper investigates how mAb aggregates generated by a variety of mechanical, thermal and chemical stresses impact the biological activity of a biotherapeutic. Increased aggregation resulted in a decrease in biological activity, as confirmed by cell based assays such as antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) and ligand binding assays such as surface plasmon resonance (SPR). It was observed that aggregates formed due to extreme pH (pH 3.5 and pH 11.0), stirring (1 d stir and 3 d stir), thermal stress, and oxidation via CuSO4 have the most impact on potency of the therapeutic. In contrast, aggregates formed due to stresses from pipetting, milder pH (4.3 and 8.5), oxidation via H2O2, freeze-thaw (Ft-slow and Ft-fast) have relatively less impact on the potency of the mAb biotherapeutic. The results affirm that understanding of the mechanism of aggregation is critical for achieving consistent product quality and the resulting efficacy.

Structure-Based Design of Small Peptide Ligands to Inhibit Early-Stage Protein Aggregation Nucleation.

We report a structure-based approach to design peptides that can bind to aggregation-prone, partially folded intermediates (PFI) of insulin, thereby inhibiting early stages of aggregation nucleation. We account for the important role of the modular architecture of protein-protein binding interfaces and tertiary structure heterogeneity of the PFIs in the design of peptide inhibitors. The determination of association hotspots revealed that two interface segments are required to capture majority contribution to insulin homodimer binding energy. The selection of peptides that will have a high probability to inhibit insulin self-association was done on the basis of similarity in binding interface coverage of PFI residues in the peptide-PFI complex and the native-PFI dimer. Data on aggregate growth rate and secondary structure for formulations incubated under amyloidogenic conditions show that designed peptides inhibit insulin aggregation in a concentration-dependent manner. The mechanism of aggregation inhibition was probed by determining the enthalpy of peptide-insulin binding and peptide micellization using isothermal titration calorimetry. Finally, the effect of designed peptides on insulin activity was quantified using a spectrophotometric assay for glucose uptake by HepG2 cells.

Assessment of structural and functional similarity of biosimilar products: Rituximab as a case study.

Biosimilars are products that are similar in terms of quality, safety, and efficacy to an already licensed reference/ innovator product and are expected to offer improved affordability. The most significant source of reduction in the cost of development of a biosimilar is the reduced clinical examination that it is expected to undergo as compared to the innovator product. However, this clinical relief is predicated on the assumption that there is analytical similarity between the biosimilar and the innovator product. As a result, establishing analytical similarity is arguably the most important step towards successful development of a biosimilar. Here, we present results from an analytical similarity exercise that was performed with five biosimilars of rituximab (Ristova®, Roche), a chimeric mouse/ human monoclonal antibody biotherapeutic, that are available on the Indian market. The results show that, while the biosimilars exhibited similarity with respect to protein structure and function, there were significant differences with respect to size heterogeneity, charge heterogeneity and glycosylation pattern.