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Abstract Background Transcripts can exhibit significant variation in tissue samples from inbred laboratory mice. We have designed and carried out a microarray experiment to examine transcript variation across samples from adipose, heart, kidney, and liver tissues of C57BL/6J mice and to partition variation into within-mouse and between-mouse components. Within-mouse variance captures variation due to heterogeneity of gene expression within tissues, RNA-extraction, and array processing. Between-mouse variance reflects differences in transcript abundance between genetically identical mice. Results The nature and extent of transcript variation differs across tissues. Adipose has the largest total variance and the largest within-mouse variance. Liver has the smallest total variance, but it has the most between-mouse variance. Genes with high variability can be classified into groups with correlated patterns of expression that are enriched for specific biological functions. Variation between mice is associated with circadian rhythm, growth hormone signaling, immune response, androgen regulation, lipid metabolism, and the extracellular matrix. Genes showing correlated patterns of within-mouse variation are also associated with biological functions that largely reflect heterogeneity of cell types within tissues. Conclusions Genetically identical mice can experience different individual outcomes for medically important traits. Variation in gene expression observed between genetically identical mice can identify functional classes of genes that are likely to vary in the absence of experimental perturbations, can inform experimental design decisions, and provides a baseline for the interpretation of gene expression data in interventional studies. The extent of transcript variation among genetically identical mice underscores the importance of stochastic and micro-environmental factors and their phenotypic consequences.

The intestinal epithelial receptor Guanylyl Cyclase C (GUCY2C) is a tumor-associated cell surface antigen expressed across gastrointestinal malignancies that can serve as an efficacious target for colorectal cancer immunotherapy. Here, we describe a yeast surface-display approach combined with an orthogonal peptide-based mapping strategy to identify the GUCY2C binding epitope of a novel anti-GUCY2CxCD3 bispecific antibody (BsAb) that recently advanced into the clinic for the treatment of cancer. The target epitope was localized to the N-terminal helix H2 of human GUCY2C, which enabled the determination of the crystal structure of the minimal GUCY2C epitope in complex with the anti-GUCY2C antibody domain. To understand if this minimal epitope covers the entire antibody binding region and to investigate the impact of epitope position on the antibody's activity, we further determined the structure of this interaction in the context of the full-length extracellular domain (ECD) of GUCY2C. We found that this epitope is positioned on the protruding membrane-distal helical region of GUCY2C and that its specific location on the surface of GUCY2C dictates the close spatial proximity of the two antigen arms in a diabody arrangement essential to the tumor killing activity of GUCY2CxCD3 BsAb., (© 2023. Springer Nature Limited.)

Administration of therapeutic antibodies can elicit adverse immune responses in patients through the generation of anti-drug antibodies that, in turn, reduce the efficacy of the therapeutic. Removal of foreign amino acid content by humanization can lower the immunogenic risk of the therapeutic mAb. We previously developed the ultra-humanization technology "Augmented Binary Substitution" (ABS) which enables single-step CDR germlining of antibodies. The application of ABS to a chicken anti-pTau antibody generated an ultra-humanized variant, anti-pTau C21-ABS, with increased human amino acid content in the CDRs and reduced in-silico predicted immunogenicity risk. Here, we report the high-resolution crystal structure of anti-pTau C21-ABS Fab in complex with the pTau peptide (7KQK). This study examines how ultra-humanization, via CDR germlining, is facilitated while maintaining near-identical antigen affinity (within 1.6-fold). The co-complex structure reveals that the ABS molecule targets the same antigenic epitope, accommodated by structurally-similar changes in the paratope. These findings confirm that ABS enables the germlining of amino acids within CDRs by exploiting CDR plasticity, to reduce non-human amino acid CDR content, with few alterations to the overall mechanism of binding., (© 2022. The Author(s).)

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

While we and our patients have many transferences to the modality of video or telephone therapy, these meanings are ultimately not intrinsic to the medium itself. Rather, they are symbolic meanings we make of the modality. This paper discusses the symbolic meanings that we attribute to teletherapy and the ways we may use these attributed meanings to dissociate the trauma of Covid-19. It also explores the clinical opportunities in identifying these dissociated countertransferential experiences and embodying them in our own and our patients' experiences.

Transgenesis has been a mainstay of mouse genetics for over 30 yr, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or an essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported, due in part to limitations in the discovery tools. Targeted locus amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments., (© 2019 Goodwin et al.; Published by Cold Spring Harbor Laboratory Press.)

In morphological terms, "form" is used to describe an object's shape and size. In dogs, facial form is stunningly diverse. Facial retrusion, the proximodistal shortening of the snout and widening of the hard palate is common to brachycephalic dogs and is a welfare concern, as the incidence of respiratory distress and ocular trauma observed in this class of dogs is highly correlated with their skull form. Progress to identify the molecular underpinnings of facial retrusion is limited to association of a missense mutation in BMP3 among small brachycephalic dogs. Here, we used morphometrics of skull isosurfaces derived from 374 pedigree and mixed-breed dogs to dissect the genetics of skull form. Through deconvolution of facial forms, we identified quantitative trait loci that are responsible for canine facial shapes and sizes. Our novel insights include recognition that the FGF4 retrogene insertion, previously associated with appendicular chondrodysplasia, also reduces neurocranium size. Focusing on facial shape, we resolved a quantitative trait locus on canine chromosome 1 to a 188-kb critical interval that encompasses SMOC2. An intronic, transposable element within SMOC2 promotes the utilization of cryptic splice sites, causing its incorporation into transcripts, and drastically reduces SMOC2 gene expression in brachycephalic dogs. SMOC2 disruption affects the facial skeleton in a dose-dependent manner. The size effects of the associated SMOC2 haplotype are profound, accounting for 36% of facial length variation in the dogs we tested. Our data bring new focus to SMOC2 by highlighting its clinical implications in both human and veterinary medicine., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted anti-myostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall "humanness" was increased for both the light and heavy chain variable regions.

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART ® ) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (-4.4 days in human FcRn knock-in mice), high stability (T m 1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.