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Living species vary significantly in phenotype and genomic content. Sophisticated statistical methods linking genes with phenotypes within a species have led to breakthroughs in complex genetic diseases and genetic breeding. Despite the abundance of genomic and phenotypic data available for thousands of species, finding genotype-phenotype associations across species is challenging due to the non-independence of species data resulting from common ancestry. To address this, we present CALANGO (comparative analysis with annotation-based genomic components), a phylogeny-aware comparative genomics tool to find homologous regions and biological roles associated with quantitative phenotypes across species. In two case studies, CALANGO identified both known and previously unidentified genotype-phenotype associations. The first study revealed unknown aspects of the ecological interaction between Escherichia coli , its integrated bacteriophages, and the pathogenicity phenotype. The second identified an association between maximum height in angiosperms and the expansion of a reproductive mechanism that prevents inbreeding and increases genetic diversity, with implications for conservation biology and agriculture., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line-Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody-drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti-GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line-derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker-payload based ADCs. Overall, these data suggest that anti-GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638-49. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Bispecific antibodies offer a clinically validated platform for drug discovery. In generating functionally active bispecific antibodies, it is necessary to identify a unique parental antibody pair to merge into a single molecule. However, technologies that allow high-throughput production of bispecific immunoglobulin Gs (BsIgGs) for screening purposes are limited. Here, we describe a novel bispecific antibody format termed tethered-variable CLBsIgG (tcBsIgG) that allows robust production of intact BsIgG in a single cell line, concurrently ensuring cognate light chain pairing and preserving key antibody structural and functional properties. This technology is broadly applicable in the generation of BsIgG from a variety of antibody isotypes, including human BsIgG1, BsIgG2 and BsIgG4. The practicality of the tcBsIgG platform is demonstrated by screening BsIgGs generated from FGF21-mimetic anti-Klotho-β agonistic antibodies in a combinatorial manner. This screen identified multiple biepitopic combinations with enhanced agonistic activity relative to the parental monoclonal antibodies, thereby demonstrating that biepitopic antibodies can acquire enhanced functionality compared to monospecific parental antibodies. By design, the tcBsIgG format is amenable to high-throughput production of large panels of bispecific antibodies and thus can facilitate the identification of rare BsIgG combinations to enable the discovery of molecules with improved biological function., (© The Author 2017. Published by Oxford University Press.)

The ability to obtain bacterial genomes from the same host has allowed for comparative studies that help in the understanding of the molecular evolution of specific pathotypes. Avian pathogenic Escherichia coli (APEC) is a group of extraintestinal strains responsible for causing colibacillosis in birds. APEC is also suggested to possess a role as a zoonotic agent. Despite its importance, APEC pathogenesis still has several cryptic pathogenic processes that need to be better understood. In this work, a genome-wide survey of eight APEC strains for genes with evidence of recombination revealed that ∼14% of the homologous groups evaluated present signs of recombination. Enrichment analyses revealed that nine Gene Ontology (GO) terms were significantly more represented in recombinant genes. Among these GO terms, several were noted to be ATP-related categories. The search for positive selection in these APEC genomes revealed 32 groups of homologous genes with evidence of positive selection. Among these groups, we found several related to cell metabolism, as well as several uncharacterized genes, beyond the well-known virulence factors ompC, lamB, waaW, waaL, and fliC. A GO term enrichment test showed a prevalence of terms related to bacterial cell contact with the external environment (e.g., viral entry into host cell, detection of virus, pore complex, bacterial-type flagellum filament C, and porin activity). Finally, the genes with evidence of positive selection were retrieved from genomes of non-APEC strains and tested as were done for APEC strains. The result revealed that none of the groups of genes presented evidence of positive selection, confirming that the analysis was effective in inferring positive selection for APEC and not for E. coli in general, which means that the study of the genes with evidence of positive selection identified in this study can contribute for the better understanding of APEC pathogenesis processes.

Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2 + basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and Nippostrongylus brasiliensis infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2 + basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2-specific antibodies may be a novel and more efficacious treatment for asthma.

Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

Cancer stem cells (CSCs) are hypothesized to actively maintain tumors similarly to how their normal counterparts replenish differentiated cell types within tissues, making them an attractive therapeutic target for the treatment of cancer. Because most CSC markers also label normal tissue stem cells, it is unclear how to selectively target them without compromising normal tissue homeostasis. We evaluated a strategy that targets the cell surface leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a well-characterized tissue stem cell and CSC marker, with an antibody conjugated to distinct cytotoxic drugs. One antibody-drug conjugate (ADC) demonstrated potent tumor efficacy and safety in vivo. Furthermore, the ADC decreased tumor size and proliferation, translating to improved survival in a genetically engineered model of intestinal tumorigenesis. These data demonstrate that ADCs can be leveraged to exploit differences between normal and cancer stem cells to successfully target gastrointestinal cancers., (Copyright © 2015, American Association for the Advancement of Science.)

Background: Detection of genes evolving under positive Darwinian evolution in genome-scale data is nowadays a prevailing strategy in comparative genomics studies to identify genes potentially involved in adaptation processes. Despite the large number of studies aiming to detect and contextualize such gene sets, there is virtually no software available to perform this task in a general, automatic, large-scale and reliable manner. This certainly occurs due to the computational challenges involved in this task, such as the appropriate modeling of data under analysis, the computation time to perform several of the required steps when dealing with genome-scale data and the highly error-prone nature of the sequence and alignment data structures needed for genome-wide positive selection detection., Results: We present POTION, an open source, modular and end-to-end software for genome-scale detection of positive Darwinian selection in groups of homologous coding sequences. Our software represents a key step towards genome-scale, automated detection of positive selection, from predicted coding sequences and their homology relationships to high-quality groups of positively selected genes. POTION reduces false positives through several sophisticated sequence and group filters based on numeric, phylogenetic, quality and conservation criteria to remove spurious data and through multiple hypothesis corrections, and considerably reduces computation time thanks to a parallelized design. Our software achieved a high classification performance when used to evaluate a curated dataset of Trypanosoma brucei paralogs previously surveyed for positive selection. When used to analyze predicted groups of homologous genes of 19 strains of Mycobacterium tuberculosis as a case study we demonstrated the filters implemented in POTION to remove sources of errors that commonly inflate errors in positive selection detection. A thorough literature review found no other software similar to POTION in terms of customization, scale and automation., Conclusion: To the best of our knowledge, POTION is the first tool to allow users to construct and check hypotheses regarding the occurrence of site-based evidence of positive selection in non-curated, genome-scale data within a feasible time frame and with no human intervention after initial configuration. POTION is available at http://www.lmb.cnptia.embrapa.br/share/POTION/.