Targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

Notch ligand delta-like1: X-ray crystal structure and binding affinity.

The Notch pathway is a fundamental signalling system in most multicellular animals. We have determined the X-ray crystal structure of the extracellular domain of the Notch ligand delta-like ligand-1 (Dll-1). The structure incorporates the N-terminal C2 domain, receptor-binding DSL domain and the first six (of eight) EGF (epidermal growth factor)-like repeats, which form a highly extended conformation, confirmed by analytical ultracentrifugation. Comparison of our structure with a fragment of Jagged1 ligand allows us to dissect the similarities and differences between the ligand families. Differences in the C2 domains of Dll-1 and Jagged1 suggest their lipid-binding properties are likely to differ. A conserved hydrophobic patch on the surface of both Dll-1 and Jagged1 provides a likely receptor-interaction site that is common to both ligands. We also explore the binding affinity of Dll-1 for a fragment of Notch1 using different techniques. Apparent binding affinities vary when different techniques are used, explaining discrepancies in the literature. Using analytical ultracentrifugation, we perform for the first time binding analyses where both receptor and ligand are in solution, which confirms a Kd of 10 µM for this interaction.

Identification of a Wnt-induced protein complex by affinity proteomics using an antibody that recognizes a sub-population of ß-catenin.

ß-catenin is a signaling protein with diverse functions in cell adhesion and Wnt signaling. Although ß-catenin has been shown to participate in many protein-protein interactions, it is not clear which combinations of ß-catenin-interacting proteins form discrete complexes. We have generated a novel antibody, termed 4B3, which recognizes only a small subset of total cellular ß-catenin. Affinity proteomics using 4B3, in combination with subcellular fractionation, has allowed us to define a discrete trimeric complex of ß-catenin, α-catenin and the tumor suppressor APC, which forms in the cytoplasm in response to Wnt signaling. Depletion of the limiting component of this complex, APC, implicates the complex in mediating Wnt-induced changes in cell-cell adhesion. APC is also essential for N-terminal phosphorylation of ß-catenin within this complex. Each component of ß-catenin/APC/α-catenin complex co-exists in other protein complexes, thus use of a selective antibody for affinity proteomics has allowed us to go beyond the generation of a list of potential ß-catenin-interacting proteins, and define when and where a specific complex forms.

Structural principles of peptide-centric chimeric antigen receptor recognition guide therapeutic expansion.

Peptide-centric chimeric antigen receptors (PC-CARs) recognize oncoprotein epitopes displayed by cell-surface human leukocyte antigens (HLAs) and offer a promising strategy for targeted cancer therapy. We have previously developed a PC-CAR targeting a neuroblastoma-associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes. Here, we determine the 2.1-angstrom crystal structure of the PC-CAR-PHOX2B-HLA-A*24:02-ß2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). This PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactive group, covering a combined global population frequency of up to 46.7%. Biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation, and CAR T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.

Structural principles of peptide-centric Chimeric Antigen Receptor recognition guide therapeutic expansion.

Peptide-Centric Chimeric Antigen Receptors (PC-CARs), which recognize oncoprotein epitopes displayed by human leukocyte antigens (HLAs) on the cell surface, offer a promising strategy for targeted cancer therapy 1 . We have previously developed a PC-CAR targeting a neuroblastoma- associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes 2 . Here, we determine the 2.1 Å structure of the PC-CAR:PHOX2B/HLA-A*24:02/ß2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). The PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactivity group, covering a combined American population frequency of up to 25.2%. Comprehensive characterization using biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation and CAR-T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.

Solubilisation of the armadillo-repeat protein ß-catenin using a zwitterionic detergent allows resolution of phosphorylated forms by 2DE.

ß-catenin is a member of the armadillo repeat family of proteins and has important functions in cell-cell adhesion and Wnt signalling. Different protein species of ß-catenin have been shown to exist in the cell and the relative proportions of these species are altered upon stimulation of cells with Wnt-3a (Gottardi and Gumbiner, 2004). In order to determine whether posttranslational modifications (PTMs) of ß-catenin underlie these different protein species, we have used 2DE separation and immunoblotting with an antibody specific for ß-catenin. High-resolution separation of differentially modified species of ß-catenin in 2DE required the addition of ASB-16, a zwitterionic detergent that can solubilise integral membrane proteins. ASB-16 was also necessary for focussing of other armadillo repeat proteins, such as γ-catenin and p120-catenin. 2DE using ASB-16 allowed detection of a previously unreported phosphorylation site in the transcriptionally active form of ß-catenin that binds to GST-Tcf in response to Wnt signalling.

Autophosphorylation of serine 608 in the p85 regulatory subunit of wild type or cancer-associated mutants of phosphoinositide 3-kinase does not affect its lipid kinase activity.

BACKGROUND: The α-isoform of the Type 1A Phosphoinositide 3-kinases (PI3Kα) has protein kinase activity as well as phosphoinositide lipid kinase activity. The best described substrate for its protein kinase activity is its regulatory subunit, p85α, which becomes phosphorylated on Serine 608. Phosphorylation of Serine 608 has been reported to down-regulate its lipid kinase activity. RESULTS: We have assessed whether oncogenic mutants of PI3Kα, which have up-regulated lipid kinase activity, have altered levels of Serine 608 phosphorylation compared to wild type PI3Kα, and whether differential phosphorylation of Serine 608 contributes to increased activity of oncogenic forms of PI3Kα with point mutations in the helical or the kinase domains. Despite markedly increased lipid kinase activity, protein kinase activity was not altered in oncogenic compared to wild type forms of PI3Kα. By manipulating levels of phosphorylation of Serine 608 in vitro, we found no evidence that the protein kinase activity of PI3Kα affects its phosphoinositide lipid kinase activity in either wild-type or oncogenic mutants of PI3Kα. CONCLUSIONS: Phosphorylation of p85α S608 is not a significant regulator of wild-type or oncogenic PI3Kα lipid kinase activity.

Characterization of a novel JNK (c-Jun N-terminal kinase) inhibitory peptide.

An improved understanding of the roles of protein kinases in intracellular signalling and disease progression has driven significant advances in protein kinase inhibitor discovery. Peptide inhibitors that target the kinase protein substrate-binding site have continued to attract attention. In the present paper, we describe a novel JNK (c-Jun N-terminal kinase) inhibitory peptide PYC71N, which inhibits JNK activity in vitro towards a range of recombinant protein substrates including the transcription factors c-Jun, ATF2 (activating trancription factor 2) and Elk1, and the microtubule regulatory protein DCX (doublecortin). Analysis of cell culture studies confirmed the actions of a cell-permeable version of PYC71 to inhibit c-Jun phosphorylation during acute hyperosmotic stress. The analysis of the in vitro data for the kinetics of this inhibition indicated a substrate-inhibitor complex-mediated inhibition of JNK by PYC71N. Alanine-scanning replacement studies revealed the importance of two residues (PYC71N Phe9 or Phe11 within an FXF motif) for JNK inhibition. The importance of these residues was confirmed through interaction studies showing that each change decreased interaction of the peptide with c-Jun. Furthermore, PYC71N interacted with both non-phosphorylated (inactive) JNK1 and the substrate c-Jun, but did not recognize active JNK1. In contrast, a previously characterized JNK-inhibitory peptide TIJIP [truncated inhibitory region of JIP (JNK-interacting protein)], showed stronger interaction with active JNK1. Competition binding analysis confirmed that PYC71N inhibited the interaction of c-Jun with JNK1. Taken together, the results of the present study define novel properties of the PYC71N peptide as well as differences from the characterized TIJIP, and highlight the value of these peptides to probe the biochemistry of JNK-mediated substrate interactions and phosphorylation.

Do dysfunctional cognitions mediate the relationship between risk factors and postnatal depression symptomatology?

BACKGROUND: This study investigated the mediating role of general and maternal-specific dysfunctional cognitions, in the relationship between non-cognitive risk factors and postnatal depressive symptomatology. METHODS: An Australian community sample comprising 406 postnatal women responded to the Dysfunctional Attitude Scale (DAS), the Maternal Attitudes Questionnaire (MAQ), the Vulnerable Personality Style Questionnaire (VPSQ) and the Edinburgh Postnatal Depression Scale (EPDS). They also responded to several questions related to perinatal and postnatal experiences. RESULTS: Path analysis demonstrated that different mediational pathways operated for different risk factors. The relationship between having a difficult baby and postnatal depression was fully mediated by maternal-specific dysfunctional cognitions (MAQ scores), whereas the relationship between past history of depression and postnatal depression was partially mediated by general dysfunctional cognitions (DAS scores). Finally, the relationship between a vulnerable personality and depressive symptomatology was mediated by both DAS and MAQ scores. LIMITATIONS: The study employed a correlational design. Thus, all inferences regarding possible causal pathways are tentative. In addition, the generalisability of these findings to other populations needs to be demonstrated in future research. CONCLUSIONS: The results of the study are consistent with the view that risk factors may influence postnatal depression indirectly through at least two distinct cognitive mediators (dysfunctional maternal and general cognitions). It may be possible to target therapies more effectively by identifying the relevant mediating mechanism(s) for individuals with different risk profiles.

Immunopurification of adenomatous polyposis coli (APC) proteins.

BACKGROUND: The adenomatous polyposis coli (APC) tumour suppressor gene encodes a 2843 residue (310 kDa) protein. APC is a multifunctional protein involved in the regulation of ß-catenin/Wnt signalling, cytoskeletal dynamics and cell adhesion. APC mutations occur in most colorectal cancers and typically result in truncation of the C-terminal half of the protein. RESULTS: In order to investigate the biophysical properties of APC, we have generated a set of monoclonal antibodies which enable purification of recombinant forms of APC. Here we describe the characterisation of these anti-APC monoclonal antibodies (APC-NT) that specifically recognise endogenous APC both in solution and in fixed cells. Full-length APC(1-2843) and cancer-associated, truncated APC proteins, APC(1-1638) and APC(1-1311) were produced in Sf9 insect cells. CONCLUSIONS: Recombinant APC proteins were purified using a two-step affinity approach using our APC-NT antibodies. The purification of APC proteins provides the basis for detailed structure/function analyses of full-length, cancer-truncated and endogenous forms of the protein.

The PI(3)P interactome from a colon cancer cell.

A comprehensive analysis of the phosphoinositide interactome has been performed using an ω-amino analogue of phosphatidylinositol 3-phosphate (PI(3)P immobilised onto Affi-10 beads for use as an affinity absorbent for cytosolic, membrane and nuclear extracts from the LIM1215 colonic carcinoma cell line. Affinity/LC/MS/MS experiments allowed the identification of 681 proteins/protein complexes which interact with PI(3)P. Protein domain enrichment analysis identified proteins possessing PI(3)P (e.g., FYVE, PX, PH), PIP and PIP/phospholipid binding domains along with small GTPases, GTPase regulators, kinases and SH2/SH3 containing proteins. Functional and pathway enrichment analyses highlighted the major role of PI(3)P in endocytosis dynamics and vesicular trafficking, intracellular cell signalling regulation, cell division and cytokinesis. BIOLOGICAL SIGNIFICANCE: This study provides an initial detailed assessment of the phosphatidylinositol 3-phosphate (PI(3)P) interactome, highlights the major role of PI(3)P in endocytosis dynamics and vesicular trafficking, cell intracellular regulation, signalling and cytokinesis and suggests potential PI(3)P specificity for further biochemical and biological characterisation.

PHLDA1 expression marks the putative epithelial stem cells and contributes to intestinal tumorigenesis.

Studies employing mouse models have identified crypt base and position +4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position +4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67-negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells.

In situ phosphorylation of immobilized receptors on biosensor surfaces: application to E-cadherin/beta-catenin interactions.

Phosphorylation is a key posttranslational modification for modulating biological interactions. Biosensor technology is ideally suited for examining in real time the role of phosphorylation on protein-protein interactions in signaling pathways. We have developed processes for on-chip phosphorylation of immobilized receptors on biosensor surfaces. These processes have been used to analyze E-cadherin/beta-catenin interactions. Phosphorylation of the intracellular domain (ICD) of E-cadherin modulates its affinity to beta-catenin and consequently the strength of cell-cell adhesion. We have phosphorylated immobilized E-cadherin ICD in situ using casein kinase 1 (CK1), casein kinase 2 (CK2), and src. On-chip phosphorylation of E-cadherin was confirmed using anti-phosphoserine and anti-phosphotyrosine antibodies. The binding of beta-catenin to E-cadherin was analyzed quantitatively. CK1 phosphorylation of E-cadherin increased the binding affinity to beta-catenin from approximately 230 to 4 nM. A similar increase in affinity, from 260 to 4 nM, was obtained with CK2 phosphorylation of E-cadherin. However, phosphorylation by src kinase decreased the affinity constant from approximately 260 nM to 4 microM. Interestingly, phosphorylation of E-cadherin by CK1 or CK2 prevented the inhibition of beta-catenin binding by src phosphorylation.