An Expanded Evaluation of Protein Function Prediction Methods Shows an Improvement In Accuracy

Background: A major bottleneck in our understanding of the molecular underpinnings of life is the assignment of function to proteins. While molecular experiments provide the most reliable annotation of proteins, their relatively low throughput and restricted purview have led to an increasing role for computational function prediction. However, assessing methods for protein function prediction and tracking progress in the field remain challenging. Results: We conducted the second critical assessment of functional annotation (CAFA), a timed challenge to assess computational methods that automatically assign protein function. We evaluated 126 methods from 56 research groups for their ability to predict biological functions using Gene Ontology and gene-disease associations using Human Phenotype Ontology on a set of 3681 proteins from 18 species. CAFA2 featured expanded analysis compared with CAFA1, with regards to data set size, variety, and assessment metrics. To review progress in the field, the analysis compared the best methods from CAFA1 to those of CAFA2. Conclusions: The top-performing methods in CAFA2 outperformed those from CAFA1. This increased accuracy can be attributed to a combination of the growing number of experimental annotations and improved methods for function prediction. The assessment also revealed that the definition of top-performing algorithms is ontology specific, that different performance metrics can be used to probe the nature of accurate predictions, and the relative diversity of predictions in the biological process and human phenotype ontologies. While there was methodological improvement between CAFA1 and CAFA2, the interpretation of results and usefulness of individual methods remain context-dependent

An expanded evaluation of protein function prediction methods shows an improvement in accuracy

Background: A major bottleneck in our understanding of the molecular underpinnings of life is the assignment of function to proteins. While molecular experiments provide the most reliable annotation of proteins, their relatively low throughput and restricted purview have led to an increasing role for computational function prediction. However, assessing methods for protein function prediction and tracking progress in the field remain challenging. Results: We conducted the second critical assessment of functional annotation (CAFA), a timed challenge to assess computational methods that automatically assign protein function. We evaluated 126 methods from 56 research groups for their ability to predict biological functions using Gene Ontology and gene-disease associations using Human Phenotype Ontology on a set of 3681 proteins from 18 species. CAFA2 featured expanded analysis compared with CAFA1, with regards to data set size, variety, and assessment metrics. To review progress in the field, the analysis compared the best methods from CAFA1 to those of CAFA2. Conclusions: The top-performing methods in CAFA2 outperformed those from CAFA1. This increased accuracy can be attributed to a combination of the growing number of experimental annotations and improved methods for function prediction. The assessment also revealed that the definition of top-performing algorithms is ontology specific, that different performance metrics can be used to probe the nature of accurate predictions, and the relative diversity of predictions in the biological process and human phenotype ontologies. While there was methodological improvement between CAFA1 and CAFA2, the interpretation of results and usefulness of individual methods remain context-dependent. Keywords: Protein function prediction, Disease gene prioritizationpublishedVersio

Prolyl 4-hydroxylase:structural and functional characterization of the peptide-substrate-binding domain of the human enzyme, and cloning and characterization of a plant enzyme with unique properties

Abstract Collagen prolyl 4-hydroxylase is the key enzyme in the biosynthesis of collagens, a family of extracellular matrix proteins. Vertebrate collagen prolyl 4-hydroxylases are α2β2 tetramers, the β subunit being identical to the multifunctional protein disulphide isomerase (PDI). Several isoforms of the catalytic α subunit have been identified in various organisms. Prolyl 4-hydroxylases have also been isolated from plants, where they hydroxylate proline-rich structural glycoproteins of the cell walls. The structural and functional properties of the peptide-substrate-binding domain of human collagen prolyl 4-hydroxylase are characterized here. Data obtained from NMR studies indicate that the domain consists of five α helices and one short β strand, this structure being quite different from those of other proline-rich peptide-binding modules. Several residues involved in the binding of a short synthetic peptide were also identified by NMR. Kd values for the binding of several synthetic peptides to the α(I) and α(II) domains were determined by surface plasmon resonance and isothermal calorimetry, and the results indicated that the binding properties of the type I and type II collagen prolyl 4-hydroxylase tetramers can mainly be explained by the binding of peptides to this domain rather than to the catalytic domain. The peptide-substrate-binding domain of human type I collagen prolyl 4-hydroxylase was also crystallized. The crystals were well ordered and diffracted to at least 3 Å, the asymmetric unit most probably containing a domain dimer. The genome of Arabidopsis thaliana was found to encode at least six putative prolyl 4-hydroxylase polypeptides, one of which was cloned and characterized here as a recombinant protein. All the catalytically critical residues identified in animal prolyl 4-hydroxylases were also conserved in this plant prolyl 4-hydroxylase, and their mutagenesis led to inactivation of the enzyme. The recombinant plant enzyme was effective in hydroxylating poly(L-proline) and several synthetic proline-rich peptides. Surprisingly, contrary to previous reports on plant prolyl 4-hydroxylases, the collagen-like peptides were found to be good substrates, the enzyme preferentially hydroxylating prolines in the Y positions of the -X-Y-Gly- triplets, thus resembling the vertebrate collagen prolyl 4-hydroxylases even in this respect. The recombinant plant prolyl 4-hydroxylase also hydroxylated peptides representing the N and C-terminal hydroxylation sites present in the hypoxia-inducible transcription factor α. The fact that these peptides contain only one proline residue indicated that a poly(L-proline) type II conformation was not required for hydroxylation

The Structural Motifs for Substrate Binding and Dimerization of the α Subunit of Collagen Prolyl 4-Hydroxylase

SummaryCollagen prolyl 4-hydroxylase (C-P4H) catalyzes the proline hydroxylation of procollagen, an essential modification in the maturation of collagens. C-P4H consists of two catalytic α subunits and two protein disulfide isomerase β subunits. The assembly of these subunits is unknown. The α subunit contains an N domain (1–143), a peptide-substrate-binding-domain (PSB, 144–244) and a catalytic domain (245–517). Here, we report the dimeric structure of the N-terminal region (1–244) of the α subunit. It is shown that the N domain has an important role in the assembly of the C-P4H tetramer, by forming an extended four-helix bundle that includes an antiparallel coiled-coil dimerization motif between the two α subunits. Complexes of this construct with a C-P4H inhibitor and substrate show the mode of peptide-binding to the PSB domain. Both peptides adopt a poly-(L)-proline-type-II helix conformation and bind in a curved, asymmetric groove lined by conserved tyrosines and an Arg-Asp salt bridge

In silico tumor immune microenvironment (TiME) analysis of non-small cell lung cancer (NSCLC) to inform clinical development of CDR404: A first-of-its-kind MAGE-A4 targeted T-cell engager

e20024 Background: CDR404 is an antibody-based bivalent MAGE-A4 targeted T-cell engager (TCE). One key mechanism-of-action of TCEs is CD8 T-cell redirection which involves T-cell intravasation into tumors [Damato et al, 2019]. CDR404 mediated TiME remodeling will likely be dependent on the inflammatory (INFLAM) and vascular (VASC) phenotype especially since tumor vasculature constitutes functional and physical barriers to T-cell infiltration [Sahu et al, 2022] [Desbois et al 2020] [Duru et al, 2020]. To identify biomarkers for CDR404 anti-tumor responses in NSCLC, we evaluated the associations between MAGE-A4 mRNA expression, immune cell populations and frequency of 9p21 deletions mediating T-cell infiltration [Han et al, 2021]. Methods: Expression of MAGE-A4 mRNA was evaluated by the Tempus xR RNA-Seq assay (Tempus AI, Inc. Chicago IL). Consensus TME bulk RNA-Sequencing deconvolution [Jiménez-Sanchez et al, 2019] was used to analyze 16 TiME cell lineages in two NSCLC TCGA datasets – primary LUSC & LUAD [Giacomazzi et al, 2023]. Immune cell data was stratified by MAGE-A4 expression quartiles (Q): Null = not detected; MAGE-A4 LOW = Q1-Q3; MAGE-A4 HIGH = Q4. GISTIC2.0 was used to identify 9p21 gene deletions [Mermel et al, 2011]. Results: MAGE-A4 is enriched in LUSC. MAGE-A4 levels were similar across metastatic organ sites (e.g., liver vs. lymph nodes) in LUSC indicating that tumor location is not a confounding factor for TiME analysis. In LUSC, MAGE-A4 HIGH vs. MAGE-A4 NULL tumors had lower levels of 15/16 immune cell populations. Largest reductions were in endothelial cells (p=7.71-e05) and CD8 T-cells (p=0.00096). Deletions in 9p21 genes were more frequent in MAGE-A4 HIGH vs. MAGE-A4 NULL tumors, e.g., CDK2NA: 91% vs. 68% (p=0.0028), consistent with reduced CD8 T-cells in MAGE-A4 HIGH . In MAGE-A4 HIGH vs. MAGE-A4 LOW reductions in 4/16 immune cell populations were seen. In LUAD, immune cell levels were similar across MAGE-A4 subgroups except lower endothelial cells in MAGE-A4 HIGH vs. MAGE-A4 NULL (p=0.00055). Conclusions: LUSC MAGE-A4 HIGH tumors had a differentiated TiME profile. Our findings are consistent with an INFLAM LOW VASC LOW phenotype possibly indicative of an “immune desert” [Desbois et al 2020]. In contrast, LUAD MAGE-A4 HIGH tumors had an INFLAM HIGH VASC LOW phenotype indicating that MAGE-A4 associations with TiME may be histology dependent in NSCLC. Overall, in MAGE-A4 HIGH LUSC & LUAD, susceptibility to CDR404 mediated T-cell tumor intravasation may be better because of a lower angiogenic barrier. Translational baseline tumor biopsy sub-studies from the CDR404 Phase 1 trial are awaited to confirm if INFLAM/VASC phenotype is predictive of response in relapsed locally advanced/metastatic NSCLC patients. CDR-Life acknowledges Tempus AI, Inc. for the expression analysis using the Tempus xR RNA-Seq assay

1397 CDR404, an antibody-based bispecific & bivalent T-cell engager targeted against MAGE-A4, for Squamous Non-Small Cell Lung Cancer (SQ-NSCLC)

BackgroundSquamous non-small cell lung cancer (SQ-NSCLC) is the 2nd most common type of lung cancer. Given the paucity of actionable oncogene drivers, and lack of efficacy from multiple therapies in the Lung-MAP trial, there is a high unmet need in SQ-NSCLC to develop effective 2nd-line immunotherapies for patients with disease progression after immune checkpoint inhibitors (ICI).The melanoma antigen gene A4 (MAGE-A4) is exclusively expressed in cancer and absent in somatic tissues. MAGE-A4-derived peptides presented on HLA molecules at the cell surface recently emerged as a novel therapeutic opportunity. Thus, the two key objectives of this study were to: 1). Evaluate MAGE-A4 expression in human SQ-NSCLC; 2). Demonstrate the anti-cancer activity of CDR404, an antibody-based bispecific and bivalent T-cell engager targeted against MAGE-A4230–239 peptide in vitro and in vivo xenograft models of SQ-NSCLC.MethodsMAGE-A4 mRNA prevalence and expression in SQ-NSCLC was analyzed using the TCGA database (https://www.cancer.gov/tcga). Protein expression of MAGE-A4 was confirmed using immunohistochemistry (IHC) in fifty FFPE human SQ-NSCLC samples (clone E7O1U).CDR404 target cell killing in the presence of human PBMCs was assessed using the human SQ-NSCLC cell line NCI-H1703. HLA-A*02:01+MAGE-A4neg cancer cells were used as controls. To exclude reactivity of CDR404 in healthy tissues, HLA-A*02:01+ primary cells presenting peptides with high MAGE-A4 similarity were co-cultured with human PBMCs. In vivo activity of CDR404 in SQ-NSCLC was evaluated with an NCI-H1703 xenograft model in NSG mice.ResultsSQ-NSCLC had the highest MAGE-A4 mRNA expression levels among solid cancers in the TCGA database. IHC showed positive MAGE-A4 staining in 28/50 (56%) of SQ-NSCLC samples.In vitro, CDR404 showed efficient target cell lysis across all effector-to-target ratios tested. Similarly, simultaneous target engagement and resulting synapse formation induced T cell activation and secretion of cytolytic molecules in an effector-to-target ratio-dependent fashion. No reactivity was observed using co-cultured HLA-A*02:01+MAGE-A4neg cancer cells. Lack of T cell activation/cytolytic molecule release in the presence of HLA-A*02:01+ primary cells confirmed the specificity profile of CDR404. In vivo, treatment with four different doses of CDR404 induced complete tumor regression in the SQ-NSCLC NCI-H1703 xenograft model.ConclusionsThe high MAGE-A4 expression levels and the highly specific anti-cancer cell activity of CDR404 make it a highly attractive immunotherapy for development post-progression on ICI for patients with HLA-A*02:01+ SQ-NSCLC. A multi-tumor phase 1 trial of CDR404, including SQ-NSCLC, is expected to begin in 2024 with prospective patient selection for both HLA-A*02:01 and tumor MAGE-A4.Ethics ApprovalAnimal studies were performed in compliance with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals