Molecular insights into recognition of GUCY2C by T-cell engaging bispecific antibody anti-GUCY2CxCD3.

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.

HLAII peptide presentation of infliximab increases when complexed with TNF.

CD4+ T-cell activation through recognition of Human Leukocyte Antigen II (HLAII)-presented peptides is a key step in the development of unwanted immune response against biotherapeutics, such as the generation of anti-drug antibodies (ADA). Therefore, the identification of HLAII-presented peptides derived from biotherapeutics is a crucial part of immunogenicity risk assessment and mitigation strategies during drug development. To date, numerous CD4+ T-cell epitopes have been identified by HLAII immunopeptidomics in antibody-based biotherapeutics using either their native or aggregated form. Antibody-target immune complexes have been detected in patients with ADA and are thought to play a role in ADA development by enhancing the presentation of CD4+ T-cell epitopes at the surface of antigen presenting cells (APCs). The aim of this study was to investigate the effect of biotherapeutic antibody-target immune complexes on the HLAII peptide presentation of biotherapeutics in human primary monocyte-derived dendritic cells (DCs). The trimeric tumor necrosis factor (TNF) and its biotherapeutic antagonists infliximab (INFL), adalimumab (ADAL), and a single armed Fab' were used as a model system. The HLAII immunopeptidome of DCs loaded with antagonists or their immune complexes with TNF was analyzed by trapped ion mobility time-of-flight mass spectrometry (timsTOF MS) leading to the identification of ~ 12,000 unique HLAII-associated peptides per preparation. Anti-TNF sequences were detected at a median of 0.3% of the total immunopeptidome, against a majority background of peptides from endogenous and media-derived proteins. TNF antagonist presentation spanned the variable and constant regions in a widespread manner in both light and heavy chains, consistent with previously discovered HLAII peptides. This investigation extends the collection of observed HLAII peptides from anti-TNF biotherapeutics to include sequences that at least partially span the complementary determining regions (CDRs), such as the LCDR1 for both INFL and ADAL. Although antagonist presentation varied significantly across donors, peptides from both bivalent antagonists INFL and ADAL were more highly presented relative to the Fab'. While TNF immune complexes did not alter overall HLAII presentation, a moderate increase in presentation of a subset of peptide clusters was observed in the case of INFL-TNF, which included HCDR2, HCDR3 and LCDR2 sequences.

Dynamic changes in the microRNA expression profile reveal multiple regulatory mechanisms in the spinal nerve ligation model of neuropathic pain.

Neuropathic pain resulting from nerve lesions or dysfunction represents one of the most challenging neurological diseases to treat. A better understanding of the molecular mechanisms responsible for causing these maladaptive responses can help develop novel therapeutic strategies and biomarkers for neuropathic pain. We performed a miRNA expression profiling study of dorsal root ganglion (DRG) tissue from rats four weeks post spinal nerve ligation (SNL), a model of neuropathic pain. TaqMan low density arrays identified 63 miRNAs whose level of expression was significantly altered following SNL surgery. Of these, 59 were downregulated and the ipsilateral L4 DRG, not the injured L5 DRG, showed the most significant downregulation suggesting that miRNA changes in the uninjured afferents may underlie the development and maintenance of neuropathic pain. TargetScan was used to predict mRNA targets for these miRNAs and it was found that the transcripts with multiple predicted target sites belong to neurologically important pathways. By employing different bioinformatic approaches we identified neurite remodeling as a significantly regulated biological pathway, and some of these predictions were confirmed by siRNA knockdown for genes that regulate neurite growth in differentiated Neuro2A cells. In vitro validation for predicted target sites in the 3'-UTR of voltage-gated sodium channel Scn11a, alpha 2/delta1 subunit of voltage-dependent Ca-channel, and purinergic receptor P2rx ligand-gated ion channel 4 using luciferase reporter assays showed that identified miRNAs modulated gene expression significantly. Our results suggest the potential for miRNAs to play a direct role in neuropathic pain.

Phosphodiesterase 11A in brain is enriched in ventral hippocampus and deletion causes psychiatric disease-related phenotypes.

Phosphodiesterase 11A (PDE11A) is the most recently identified family of phosphodiesterases (PDEs), the only known enzymes to break down cyclic nucleotides. The tissue expression profile of this dual specificity PDE is controversial, and little is understood of its biological function, particularly in the brain. We seek here to determine if PDE11A is expressed in the brain and to understand its function, using PDE11A(-/-) knockout (KO) mice. We show that PDE11A mRNA and protein are largely restricted to hippocampus CA1, subiculum, and the amygdalohippocampal area, with a two- to threefold enrichment in the ventral vs. dorsal hippocampus, equal distribution between cytosolic and membrane fractions, and increasing levels of protein expression from postnatal day 7 through adulthood. Interestingly, PDE11A KO mice show subtle psychiatric-disease-related deficits, including hyperactivity in an open field, increased sensitivity to the glutamate N-methyl-D-aspartate receptor antagonist MK-801, as well as deficits in social behaviors (social odor recognition memory and social avoidance). In addition, PDE11A KO mice show enlarged lateral ventricles and increased activity in CA1 (as per increased Arc mRNA), phenotypes associated with psychiatric disease. The increased sensitivity to MK-801 exhibited by PDE11A KO mice may be explained by the biochemical dysregulation observed around the glutamate alpha-amino-3-hydroxy-5-methyl-4-isozazolepropionic (AMPA) receptor, including decreased levels of phosphorylated-GluR1 at Ser845 and the prototypical transmembrane AMPA-receptor-associated proteins stargazin (gamma2) and gamma8. Together, our data provide convincing evidence that PDE11A expression is restricted in the brain but plays a significant role in regulating brain function.

ADAMTS-8 exhibits aggrecanase activity and is expressed in human articular cartilage.

Members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family share common structural features including a disintegrin domain, a zinc metalloprotease domain, and at least one thrombospondin motif. Aberrant expression of several of these proteins has led to an understanding of their role in human disease; however, a link to function for many has not yet been made. One such uncharacterized family member, ADAMTS-8, shares significant protein sequence homology with a subgroup of ADAMTSs that includes ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMTS-15. Each of these proteases has been shown to cleave 'aggrecanase-susceptible' site(s) within the extracellular matrix (ECM) proteoglycan aggrecan, and ADAMTS-4 and ADAMTS-5 have been postulated to play a role in the depletion of articular cartilage in osteoarthritic disease. Based on sequence relationships, in the present study we examined the ability of ADAMTS-8 to exhibit 'aggrecanase' activity. A neoepitope monoclonal antibody (MAb; AGG-C1; anti-NITEGE373) was developed and used to demonstrate the ability of ADAMTS-8 to cleave aggrecan at the aggrecanase-susceptible Glu373-Ala374 peptide bond. In addition, expression analyses demonstrated the presence of ADAMTS-8 mRNA transcripts in normal and osteoarthritic human cartilage.