Inflamed immune phenotype predicts favorable clinical outcomes of immune checkpoint inhibitor therapy across multiple cancer types.

BACKGROUND: The inflamed immune phenotype (IIP), defined by enrichment of tumor-infiltrating lymphocytes (TILs) within intratumoral areas, is a promising tumor-agnostic biomarker of response to immune checkpoint inhibitor (ICI) therapy. However, it is challenging to define the IIP in an objective and reproducible manner during manual histopathologic examination. Here, we investigate artificial intelligence (AI)-based immune phenotypes capable of predicting ICI clinical outcomes in multiple solid tumor types. METHODS: Lunit SCOPE IO is a deep learning model which determines the immune phenotype of the tumor microenvironment based on TIL analysis. We evaluated the correlation between the IIP and ICI treatment outcomes in terms of objective response rates (ORR), progression-free survival (PFS), and overall survival (OS) in a cohort of 1,806 ICI-treated patients representing over 27 solid tumor types retrospectively collected from multiple institutions. RESULTS: We observed an overall IIP prevalence of 35.2% and significantly more favorable ORRs (26.3% vs 15.8%), PFS (median 5.3 vs 3.1 months, HR 0.68, 95% CI 0.61 to 0.76), and OS (median 25.3 vs 13.6 months, HR 0.66, 95% CI 0.57 to 0.75) after ICI therapy in IIP compared with non-IIP patients, respectively (p<0.001 for all comparisons). On subgroup analysis, the IIP was generally prognostic of favorable PFS across major patient subgroups, with the exception of the microsatellite unstable/mismatch repair deficient subgroup. CONCLUSION: The AI-based IIP may represent a practical, affordable, clinically actionable, and tumor-agnostic biomarker prognostic of ICI therapy response across diverse tumor types.

Impact of Trisulfide on the Structure and Function of Different Antibody Constructs.

Trisulfide is a post-translational modification (PTM) commonly found in recombinant antibodies. It has been demonstrated that trisulfide had no impact on the bioactivity of mono-specific antibodies (MsAbs). However, the impact of trisulfide on multi-specific antibodies has not been evaluated. In this study, two mass spectrometric methods were developed for comprehensive trisulfide characterization. The non-reduced peptide mapping method combined with the unique electron activated dissociation (EAD) provided signature fragments for confident trisulfide identification as well as trisulfide quantitation at individual sites. A higher throughput method using Fab mass analysis was also developed and qualified to support routine monitoring of trisulfide during process development. Fab mass analysis features simpler sample preparation and shorter analysis time but provides comparable results to the non-reduced peptide mapping method. In this study, a bi-specific (BsAb) and a tri-specific antibody (TsAb) were compared side-by-side with a MsAb to evaluate the impact of trisulfide on the structure and function of multi-specific antibodies. Results indicated that trisulfide dominantly formed at similar locations across different antibody constructs and had no impact on the size heterogeneity, charge heterogeneity, or bioactivities of any assessed antibodies. Together with the in vitro stability under heat stress (25 °C and 40 °C for up to four weeks) and rapid conversion from trisulfide to disulfide during in vivo circulation, trisulfide could be categorized as a non-critical quality attribute (non-CQA) for antibody products.

Structure Based Prediction of Asparagine Deamidation Propensity in Monoclonal Antibodies.

Identification of asparagine (Asn) sites that are prone to deamidation is critical for the development of therapeutic monoclonal antibodies (mAbs). Despite a common chemical degradation pathway, the rates of Asn deamidation can vary dramatically among different sites, and prediction of the sensitive deamidation sites is still challenging. In this study, characterization of Asn deamidation for five IgG1 and five IgG4 mAbs under both normal and stressed conditions revealed dramatic differences in the Asn deamidation rates. A comprehensive analysis of the deamidation sites indicated that the deamidation rate differences could be explained by differences in the local structure conformation, structure flexibility and solvent accessibility. A decision tree was developed to predict the deamidation propensity for all Asn sites in IgG mAbs based on the analysis of these three structural parameters. This decision tree will allow potential Asn deamidation hot spots to be identified early in development.

Myocardial Ablation of G Protein-Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway.

Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.

Structure-based engineering of a monoclonal antibody for improved solubility.

Protein aggregation is of great concern to pharmaceutical formulations and has been implicated in several diseases. We engineered an anti-IL-13 monoclonal antibody CNTO607 for improved solubility. Three structure-based engineering approaches were employed in this study: (i) modifying the isoelectric point (pI), (ii) decreasing the overall surface hydrophobicity and (iii) re-introducing an N-linked carbohydrate moiety within a complementarity-determining region (CDR) sequence. A mutant was identified with a modified pI that had a 2-fold improvement in solubility while retaining the binding affinity to IL-13. Several mutants with decreased overall surface hydrophobicity also showed moderately improved solubility while maintaining a similar antigen affinity. Structural studies combined with mutagenesis data identified an aggregation 'hot spot' in heavy-chain CDR3 (H-CDR3) that contains three residues ((99)FHW(100a)). The same residues, however, were found to be essential for high affinity binding to IL-13. On the basis of the spatial proximity and germline sequence, we reintroduced the consensus N-glycosylation site in H-CDR2 which was found in the original antibody, anticipating that the carbohydrate moiety would shield the aggregation 'hot spot' in H-CDR3 while not interfering with antigen binding. Peptide mapping and mass spectrometric analysis revealed that the N-glycosylation site was generally occupied. This variant showed greatly improved solubility and bound to IL-13 with affinity similar to CNTO607 without the N-linked carbohydrate. All three engineering approaches led to improved solubility and adding an N-linked carbohydrate to the CDR was the most effective route for enhancing the solubility of CNTO607.