Serum-derived carcinoembryonic antigen (CEA) activates fibroblasts to induce a local re-modeling of the extracellular matrix that favors the engraftment of CEA-expressing tumor cells.

Elevated levels of the carcinoembryonic antigen (CEA; CEACAM5) in the serum of colorectal cancer (CRC) patients represent a clinical biomarker that correlates with disease recurrence. However, a mechanistic role for soluble CEA (sCEA) in tumor progression and metastasis remains to be established. In our study, we report that sCEA acts as a paracrine factor, activating human fibroblasts by signaling through both the STAT3 and AKT1-mTORC1 pathways, promoting their transition to a cancer-associated fibroblast (CaF) phenotype. sCEA-activated fibroblasts express and secrete higher levels of fibronectin, including cellular EDA+ -fibronectin (Fn-EDA) that selectively promote the implantation and adherence of CEA-expressing cancer cells. Immunohistochemical analyses of liver tissues derived from CRC patients with elevated levels of sCEA reveal that the expression of cellular Fn-EDA co-registers with CEA-expressing liver metastases. Taken together, these findings indicate a direct role for sCEA as a human fibroblast activation factor, in priming target tissues for the engraftment of CEA-expressing cancer cells, through the differentiation of tissue-resident fibroblasts, resulting in a local change in composition of the extracellular matrix.

Induction of antigen-specific TH 9 immunity accompanied by mast cell activation blocks tumor cell engraftment.

The engraftment of circulating cancer cells at distal sites represents a key step in the metastatic cascade, yet remains an unexplored target for therapeutic intervention. In this study, we establish that a vaccination strategy yielding an antigen-specific TH 9 response induces long term host surveillance and prevents the engraftment of circulating cancer cells. Specifically, we show that vaccination with a recombinant CEA IgV-like N domain, formulated with the TLR3 ligand poly I:C, elicits a CEA-specific TH 9 response, wherein IL-9 secreting TH cells act in concert with CEA N domain-specific antibodies as well as activated mast cells in preventing tumor cell engraftment. The development of this immune response was dependent on TLR3, since interference with the TLR3-dsRNA complex formation led to a reduction in vaccine-imparted protection and a shift in the resulting immune response toward a TH 2 response. These findings point to the existence of an alternate tumor targeting immune mechanism that can be exploited for the purpose of developing vaccine therapies targeting tumor dissemination and engraftment.

Slow binding kinetics of secreted protein, acidic, rich in cysteine-VEGF interaction limit VEGF activation of VEGF receptor 2 and attenuate angiogenesis.

VEGF-A (VEGF) drives angiogenesis through activation of downstream effectors to promote endothelial cell proliferation and migration. Although VEGF binds both VEGF receptor 1 (R1) and receptor 2 (R2), its proangiogenic effects are attributed to R2. Secreted protein, acidic, rich in cysteine (SPARC) is a matricellular glycoprotein thought to inhibit angiogenesis by preventing VEGF from activating R1, but not R2. Because R2 rather than R1 mediates proangiogenic activities of VEGF, the role of human SPARC in angiogenesis was reevaluated. We confirm that association of SPARC with VEGF inhibits VEGF-induced HUVEC adherence, motility, and proliferation in vitro and blocks VEGF-induced blood vessel formation ex vivo. SPARC decreases VEGF-induced phosphorylation of R2 and downstream effectors ERK, Akt, and p38 MAPK as shown by Western blot and/or phosphoflow analysis. Surface plasmon resonance indicates that SPARC binds slowly to VEGF (0.865 ± 0.02 × 10(4) M(-1) s(-1)) with a Kd of 150 nM, forming a stable complex that dissociates slowly (1.26 ± 0.003 × 10(-3) s(-1)). Only domain III of SPARC binds VEGF, exhibiting a 15-fold higher affinity than full-length SPARC. These findings support a model whereby SPARC regulates angiogenesis by sequestering VEGF, thus restricting the activation of R2 and the subsequent activation of downstream targets critical for endothelial cell functions.

Cyclodimerization of immunosuppressive fragment of HLA-DR molecule. Design, synthesis and ESI-MS/MS analysis.

The nonapeptide fragment of the HLA-DR molecule, located in the exposed loop of the alpha-chain (164-172), having the VPRSGEVYT sequence, suppresses the immune response. Based on the three-dimensional structure of the HLA-DR superdimer, we designed a new cyclodimeric analog in which the two parallel peptide chains of VPRSGEVYT sequence are linked through their C-termini by spacer of (Gly5 )2 -Lys-NH2 and the N-termini are also linked by poly(ethylene glycol). The (VPRSGEVYTG5 )2 K-resin analog was synthesized using solid-phase peptide synthesis protocols. The cyclization was achieved by cross-linking the N-terminal positions of the dimeric peptide, attached to a MBHA resin, with alpha, omega-bis (acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the cyclodimerization of VPRSGEVYT results in enhanced immunosuppressive activity of the peptide. Mass spectrometry fragmentation analysis of the obtained cyclodimeric peptide is also presented. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Dimeric analogs of immunosuppressive decapeptide fragment of ubiquitin.

Our previous studies revealed that ubiquitin and its decapeptide fragment with the LEDGRTLSDY sequence, located on the exposed molecule loop, strongly suppressed the immune response. This suggested that the loop may serve as a functional epitope of ubiquitin molecule and that a possible mechanism of biological action of the synthesized peptides is associated with interfering in interactions of ubiquitin with other molecules. Ubiquitin is known to exist in oligomeric forms, which can interact with various oligomeric receptors. We designed and synthesized new dimeric analogs of the ubiquitin fragment, to probe whether dimeric peptides may have higher affinity towards the ubiquitin receptors responsible for immunosuppression, which are believed to form oligomeric structures. Three dimerization strategies, N-terminus to N-terminus, C-terminus to C-terminus, and N-terminus to C-terminus (head-to-tail) via PEG derivatives were used to synthesize the dimeric peptides on solid support. In the course of our research, we developed a new and straightforward procedure of dimerization where α-amino groups of the C-terminal lysine residues of two peptide fragments were linked by PEG spacer directly on solid support. The effect of dimeric analogs on the immunological response was tested in the AFC in vitro experiment. The immunological tests showed that the head-to-tail dimerization caused a more profound increase in the biological activity than other tested dimerization methods. Our results suggest that such orientation of peptide components may correspond to orientation of the hypothetic ubiquitin receptors responsible for the immunomodulatory activity.

Sensitive electrospray mass spectrometry analysis of one-bead-one-compound peptide libraries labeled by quaternary ammonium salts.

A rapid and straightforward method for high-throughput analysis of single resin beads from one-bead-one-compound combinatorial libraries with high resolution electrospray ionization tandem mass spectrometry (HR ESI-MS/MS) is presented. The application of an efficient method of peptide derivatization by quaternary ammonium salts (QAS) formation increases ionization efficiency and reduces the detection limit, allowing analysis of trace amounts of compounds by ESI-MS. Peptides, synthesized on solid support, contain a new cleavable linker composed of a Peg spacer (9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid), lysine with ɛ-amino group marked by the N,N,N-triethylglycine salt, and methionine, which makes possible the selective cleavage by cyanogen bromide. Even a small portion of peptides derivatized by QAS cleaved from a single resin bead is sufficient for sequencing by HR ESI-MS/MS experiments. The developed strategy was applied to a small training library of α chymotrypsin substrates. The obtained results confirm the applicability of the proposed method in combinatorial chemistry.

Derivatization of peptides as quaternary ammonium salts for sensitive detection by ESI-MS.

A series of model peptides in the form of quaternary ammonium salts at the N-terminus was efficiently prepared by the solid-phase synthesis. Tandem mass spectrometric analysis of the peptide quaternary ammonium derivatives was shown to provide sequence confirmation and enhanced detection. We designed the 2-(1,4-diazabicyclo[2.2.2] octylammonium)acetyl quaternary ammonium group which does not suffer from neutral losses during MS/MS experiments. The presented quaternization of 1,4-diazabicyclo[2.2.2]octane (DABCO) by iodoacetylated peptides is relatively easy and compatible with standard solid-phase peptide synthesis. This methodology offers a novel sensitive approach to analyze peptides and other compounds.

Glypican-6 stimulates intestinal elongation by simultaneously regulating Hedgehog and non-canonical Wnt signaling.

We report here that Glypican-6 (GPC6)-null mice display at birth small intestines that are 75% shorter than those of normal littermates. Notably, we demonstrate that the role of GPC6 in intestinal elongation is mediated by both Hedgehog (Hh) and non-canonical Wnt signaling. Based on results from in vitro experiments, we had previously proposed that GPC6 stimulates Hh signaling by interacting with Hh and Patched1 (Ptc1), and facilitating/stabilizing their interaction. Here we provide strong support to this hypothesis by showing that GPC6 binds to Ptc1 in the mesenchymal layer of embryonic intestines. This study also provides experimental evidence that strongly suggests that GPC6 inhibits the activity of Wnt5a on the intestinal epithelium by binding to this growth factor, and reducing its release from the surrounding mesenchymal cells. Finally, we show that whereas the mesenchymal layer of GPC6-null intestines displays reduced cell proliferation and a thinner smooth muscle layer, epithelial cell differentiation is not altered in the mutant gut.

A Synthetic Cross-Species CD200R1 Agonist Suppresses Inflammatory Immune Responses In Vivo.

Functional aptamers displaying agonistic or antagonistic properties are showing great promise as modulators of immune responses. Here, we report the development of a polyethylene glycol-modified (PEGylated) DNA aptamer as a cross-species (murine and human) CD200R1 agonist that modulates inflammatory responses in vivo. Specifically, DNA aptamers were discovered by performing independent SELEX searches on recombinant murine and human CD200R1. Aptamer motifs identified by next generation sequencing (NGS) were subsequently compared, leading to the discovery of motifs common to both targets. The CD200R1 DNA aptamer CCS13 displayed the highest agonistic activity toward CD200R1 in terms of suppressing the induction of cytotoxic T-lymphocytes (CTLs) in both human and murine allogeneic-mixed lymphocyte cultures (allo-MLCs). A 20-kDa polyethylene glycol (PEG) chain was covalently attached to the 5' end of this aptamer, and the resulting conjugate was shown to block inflammatory responses in murine models of skin graft rejection and house-dust-mite-induced allergic airway inflammation. Importantly, this agonistic aptamer does not suppress CTL induction in 5-day allo-MLCs with responder cells derived from CD200R1-/- mice, indicating that its mode of action is directly linked to CD200R1 activation. This study suggests that one can derive agonistic DNA aptamers that can be verified as immuno-modulators in murine models with outcomes potentially translatable to the treatment of human conditions.

Glypican-6 promotes the growth of developing long bones by stimulating Hedgehog signaling.

Autosomal-recessive omodysplasia (OMOD1) is a genetic condition characterized by short stature, shortened limbs, and facial dysmorphism. OMOD1 is caused by loss-of-function mutations of glypican 6 (GPC6). In this study, we show that GPC6-null embryos display most of the abnormalities found in OMOD1 patients and that Hedgehog (Hh) signaling is significantly reduced in the long bones of these embryos. The Hh-stimulatory activity of GPC6 was also observed in cultured cells, where this GPC increased the binding of Hh to Patched 1 (Ptc1). Consistent with this, GPC6 interacts with Hh through its core protein and with Ptc1 through its glycosaminoglycan chains. Hh signaling is triggered at the primary cilium. In the absence of Hh, we observed that GPC6 is localized outside of the cilium but moves into the cilium upon the addition of Hh. We conclude that GPC6 stimulates Hh signaling by binding to Hh and Ptc1 at the cilium and increasing the interaction of the receptor and ligand.

VISTA.COMP - an engineered checkpoint receptor agonist that potently suppresses T cell-mediated immune responses.

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a recently discovered immune checkpoint ligand that functions to suppress T cell activity. The therapeutic potential of activating this immune checkpoint pathway to reduce inflammatory responses remains untapped, largely due to the inability to derive agonists targeting its unknown receptor. A dimeric construct of the IgV domain of VISTA (VISTA-Fc) was shown to suppress the activation of T cells in vitro. However, this effect required its immobilization on a solid surface, suggesting that VISTA-Fc may display limited efficacy as a VISTA-receptor agonist in vivo. Herein, we have designed a stable pentameric VISTA construct (VISTA.COMP) by genetically fusing its IgV domain to the pentamerization domain from the cartilage oligomeric matrix protein (COMP). In contrast to VISTA-Fc, VISTA.COMP does not require immobilization to inhibit the proliferation of CD4+ T cells undergoing polyclonal activation. Furthermore, we show that VISTA.COMP, but not VISTA-Fc, functions as an immunosuppressive agonist in vivo capable of prolonging the survival of skin allografts in a mouse transplant model as well as rescuing mice from acute concanavalin-A-induced hepatitis. Collectively, we believe our data demonstrate that VISTA.COMP is a checkpoint receptor agonist and the first agent to our knowledge targeting the putative VISTA-receptor to suppress T cell-mediated immune responses.

Targeting the PD-1/PD-L1 Immune Evasion Axis With DNA Aptamers as a Novel Therapeutic Strategy for the Treatment of Disseminated Cancers.

Blocking the immunoinhibitory PD-1:PD-L1 pathway using monoclonal antibodies has led to dramatic clinical responses by reversing tumor immune evasion and provoking robust and durable antitumor responses. Anti-PD-1 antibodies have now been approved for the treatment of melanoma, and are being clinically tested in a number of other tumor types as both a monotherapy and as part of combination regimens. Here, we report the development of DNA aptamers as synthetic, nonimmunogenic antibody mimics, which bind specifically to the murine extracellular domain of PD-1 and block the PD-1:PD-L1 interaction. One such aptamer, MP7, functionally inhibits the PD-L1-mediated suppression of IL-2 secretion in primary T-cells. A PEGylated form of MP7 retains the ability to block the PD-1:PD-L1 interaction, and significantly suppresses the growth of PD-L1+ colon carcinoma cells in vivo with a potency equivalent to an antagonistic anti-PD-1 antibody. Importantly, the anti-PD-1 DNA aptamer treatment was not associated with off-target TLR-9-related immune responses. Due to the inherent advantages of aptamers including their lack of immunogenicity, low cost, long shelf life, and ease of synthesis, PD-1 antagonistic aptamers may represent an attractive alternative over antibody-based anti PD-1 therapeutics.

Agonistic CD200R1 DNA Aptamers Are Potent Immunosuppressants That Prolong Allogeneic Skin Graft Survival.

CD200R1 expressed on the surface of myeloid and lymphoid cells delivers immune inhibitory signals to modulate inflammation when engaged with its ligand CD200. Signalling through CD200/CD200R1 has been implicated in a number of immune-related diseases including allergy, infection, cancer and transplantation, as well as several autoimmune disorders including arthritis, systemic lupus erythematosus, and multiple sclerosis. We report the development and characterization of DNA aptamers, which bind to murine CD200R1 and act as potent signalling molecules in the absence of exogenous CD200. These agonistic aptamers suppress cytotoxic T-lymphocyte induction in 5-day allogeneic mixed leukocyte culture and induce rapid phosphorylation of the CD200R1 cytoplasmic tail thereby initiating immune inhibitory signalling. PEGylated conjugates of these aptamers show significant in vivo immunosuppression and enhance survival of allogeneic skin grafts as effectively as soluble CD200Fc. As DNA aptamers exhibit inherent advantages over conventional protein-based therapeutics including low immunogenicity, ease of synthesis, low cost, and long shelf life, such CD200R1 agonistic aptamers may emerge as useful and safe nonsteroidal anti-inflammatory therapeutic agents.

The carcinoembryonic antigen IgV-like N domain plays a critical role in the implantation of metastatic tumor cells.

The human carcinoembryonic antigen (CEA) is a cell adhesion molecule involved in both homotypic and heterotypic interactions. The aberrant overexpression of CEA on adenocarcinoma cells correlates with their increased metastatic potential. Yet, the mechanism(s) by which its adhesive properties can lead to the implantation of circulating tumor cells and expansion of metastatic foci remains to be established. In this study, we demonstrate that the IgV-like N terminal domain of CEA directly participates in the implantation of cancer cells through its homotypic and heterotypic binding properties. Specifically, we determined that the recombinant N terminal domain of CEA directly binds to fibronectin (Fn) with a dissociation constant in the nanomolar range (K(D) 16 ± 3 nM) and interacts with itself (K(D) 100 ± 17 nM) and more tightly to the IgC-like A(3) domain (K(D) 18 ± 3 nM). Disruption of these molecular associations through the addition of antibodies specific to the CEA N or A(3)B(3) domains, or by adding soluble recombinant forms of the CEA N, A(3) or A(3)B(3) domains or a peptide corresponding to residues 108-115 of CEA resulted in the inhibition of CEA-mediated intercellular aggregation and adherence events in vitro. Finally, pretreating CEA-expressing murine colonic carcinoma cells (MC38.CEA) with rCEA N, A3 or A(3)B(3) modules blocked their implantation and the establishment of tumor foci in vivo. Together, these results suggest a new mechanistic insight into how the CEA IgV-like N domain participates in cellular events that can have a macroscopic impact in terms of cancer progression and metastasis.

The Pseudomonas aeruginosa exotoxin A translocation domain facilitates the routing of CPP-protein cargos to the cytosol of eukaryotic cells.

The use of cell-penetrating peptides (CPPs), such as polyarginine, has been shown to facilitate the import of drugs and other cargos into cells. However, a major obstacle limiting their use as delivery agents is their entrapment following internalization into endocytic vesicles, leading to either their recycling out of cells or their degradation in lysosomes. To address this challenge, we fused a CPP sequence to the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA) to facilitate the endosomal escape of imported CPP-containing protein constructs. Specifically, a fusion protein incorporating ten arginines linked to residues 253 to 412 of ETA (ETA(253-412)) was tested for its ability to effectively route a protein cargo (enhanced green fluorescent protein, eGFP) to the cytosol of cells. Using flow cytometry and fluorescence live-cell imaging, we observed a 5-fold improvement of cellular uptake as well as a 40-fold increase in cytosolic delivery of the CPP-ETA(253-412)-eGFP construct in relation to CPP-eGFP. Furthermore, analysis of intracellular routing events indicated that the incorporation of ETA(253-412) within the CPP-containing protein fusion construct avoided lysosomal degradation by re-directing the construct from early endosomes to the ER lumen and finally to the cytosol. Studies using inhibitors of vesicular transport confirmed that the ER lumen is a key compartment reached by the CPP-ETA(253-412)-eGFP construct before accessing the cytosol. Together, these findings suggest that incorporating a CPP motif and the ETA translocation domain into protein constructs can facilitate their cytosolic delivery.

The competition of charge remote and charge directed fragmentation mechanisms in quaternary ammonium salt derivatized peptides--an isotopic exchange study.

Derivatization of peptides as quaternary ammonium salts (QAS) is a promising method for sensitive detection by electrospray ionization tandem mass spectrometry (Cydzik et al. J. Pept. Sci. 2011, 17, 445-453). The peptides derivatized by QAS at their N-termini undergo fragmentation according to the two competing mechanisms - charge remote (ChR) and charge directed (ChD). The absence of mobile proton in the quaternary salt ion results in ChR dissociation of a peptide bond. However, Hofmann elimination of quaternary salt creates an ion with one mobile proton leading to the ChD fragmentation. The experiments on the quaternary ammonium salts with deuterated N-alkyl groups or amide NH bonds revealed that QAS derivatized peptides dissociate according to the mixed ChR-ChD mechanism. The isotopic labeling allows differentiation of fragments formed according to ChR and ChD mechanisms.

Ubiquitin fragments: their known biological activities and putative roles.

Ubiquitin (Ub) is involved in many key processes of cell biology. Identification of compounds that could interfere in the ubiquitination process is of importance. It could be expected that peptides derived from the Ub-binding regions might be able to interact with Ub receptors themselves and modify an ability of the Ub receptors interactions. This review summarizes current knowledge about known Ub-derived peptides and discusses putative activity of unexplored Ub fragments. Among identified biologically active Ub-derived peptides, its decapeptide fragment of the LEDGRTLSDY sequence was found to exhibit strong immunosuppressive effects on the cellular and humoral immune responses, comparable to that of cyclosporine. Some of the Ub fragments possess strong antibacterial and antifungal potency. In the search for new peptides that could interfere in the interaction of Ub with other proteins, we investigated the pentapeptide Ub sequences present in non-ubiquitin proteins. Based on examination of the Swiss-Prot database, we postulated that sequences of some Ub fragments often exist in other protein molecules. However, some of those motives are represented more frequently than others and could be involved in regulation of cellular processes related to Ub.

Inhibitory activity of double-sequence analogues of trypsin inhibitor SFTI-1 from sunflower seeds: an example of peptide splicing.

Four 28-amino acid peptides were synthesized whose sequences comprised two molecules of trypsin inhibitor sunflower trypsin inhibitor 1 (SFTI-1) bound through a peptide bond. The peptides in their reactive positions (5 and 19 of the peptide chain) contain two Lys ([KK]BiSFTI-1) and two Phe ([FF]BiSFTI-1) residues, along with a combination of the amino acid residues named thereafter [KF]BiSFTI-1 and [FK]BiSFTI-1. Association constants of the analogues determined with trypsin and chymotrypsin, respectively, indicated that they were potent inhibitors of cognate proteinases. An MS study of the associates revealed that incubation of the compounds with the proteinases resulted in cutting out a fragment of the peptide chain to restore the native monocyclic molecule of SFTI-1 or its analogue [Phe(5)]SFTI-1. This process, analogous to that of the DNA and protein splicing, can be referred to as 'peptide splicing'.