Facile repurposing of peptide-MHC-restricted antibodies for cancer immunotherapy.

Monoclonal antibodies (Abs) that recognize major histocompatability complex (MHC)-presented tumor antigens in a manner similar to T cell receptors (TCRs) have great potential as cancer immunotherapeutics. However, isolation of 'TCR-mimic' (TCRm) Abs is laborious because Abs have not evolved the structurally nuanced peptide-MHC restriction of αß-TCRs. Here, we present a strategy for rapid isolation of highly peptide-specific and 'MHC-restricted' Abs by re-engineering preselected Abs that engage peptide-MHC in a manner structurally similar to that of conventional αß-TCRs. We created structure-based libraries focused on the peptide-interacting residues of TCRm Ab complementarity-determining region (CDR) loops, and rapidly generated MHC-restricted Abs to both mouse and human tumor antigens that specifically killed target cells when formatted as IgG, bispecific T cell engager (BiTE) and chimeric antigen receptor-T (CAR-T). Crystallographic analysis of one selected pMHC-restricted Ab revealed highly peptide-specific recognition, validating the engineering strategy. This approach can yield tumor antigen-specific antibodies in several weeks, potentially enabling rapid clinical translation.

Global analysis of shared T cell specificities in human non-small cell lung cancer enables HLA inference and antigen discovery.

To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRß chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A∗02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer.

A protein scaffold, engineered SPINK2, for generation of inhibitors with high affinity and specificity against target proteases.

Proteases are one of attractive therapeutic targets to play key roles in pharmacological action. There are many protease inhibitors in nature, and most of them structurally have cystine knot motifs. Their structures are favorable for recognition of active pockets of proteases, leading to the potent inhibition. However, they also have drawbacks, such as broad cross-reactivity, on the therapeutic application. To create therapeutic proteins derived from a disulfide-rich scaffold, we selected human serine protease inhibitor Kazal type 2 (SPINK2) through a scaffold screening, as a protein scaffold with requirements for therapeutic proteins. We then constructed a diverse library of the engineered SPINK2 by introducing random mutations into its flexible loop region with the designed method. By phage panning against four serine proteases, we isolated potent inhibitors against each target with picomolar KD and sub-nanomolar Ki values. Also, they exhibited the desired specificities against target proteases without inhibiting non-target proteases. The crystal structure of kallikrein related peptidase 4 (KLK4)-engineered SPINK2 complex revealed the interface with extensive conformational complementarity. Our study demonstrates that engineered SPINK2 can serve as a scaffold to generate therapeutic molecules against target proteins with groove structures.

Proteins improving recombinant antibody production in mammalian cells.

Mammalian cells have been successfully used for the industrial manufacture of antibodies due to their ability to synthesize antibodies correctly. Nascent polypeptides must be subjected to protein folding and assembly in the ER and the Golgi to be secreted as mature proteins. If these reactions do not proceed appropriately, unfolded or misfolded proteins are degraded by the ER-associated degradation (ERAD) pathway. The accumulation of unfolded proteins or intracellular antibody crystals accompanied by this failure triggers the unfolded protein response (UPR), which can considerably attenuate the levels of translation, folding, assembly, and secretion, resulting in reduction of antibody productivity. Accumulating studies by omics-based analysis of recombinant mammalian cells suggest that not only protein secretion processes including protein folding and assembly but also translation are likely to be the rate-limiting factors for increasing antibody production. Here, this review describes the mechanism of antibody folding and assembly and recent advantages which could improve recombinant antibody production in mammalian cells by utilizing proteins such as ER chaperones or UPR-related proteins.

Chicken ovalbumin promoter is demethylated upon expression in the regions specifically involved in estrogen-responsiveness.

Here we report the methylation status of the chicken ovalbumin promoter. Genomic DNA of oviduct from immature chickens and laying hens was analyzed through bisulfite genomic sequencing. In the ovalbumin control locus up to the 6 kb upstream region, CpG sites were methylated in immature chickens, except for several sites, and almost all CpGs residing in DNase I hypersensitive sites I, II, and III, but not IV, were selectively unmethylated in ovalbumin expressing chickens. Chromatin immunoprecipitation assays showed that the ovalbumin control region was associated with acetylated histone H3 but not with dimethylated histone H3 at Lys 27. These results demonstrate that DNA demethylation was restricted to short DNA regions of DNase I hypersensitive sites, especially to those which participated in estrogen-responsiveness, even when cells expressed extremely high levels of ovalbumin and these sites were associated with acetylated histones.