Heterogeneity in the spontaneous induction of the promoter of the ColE9 operon in Escherichia coli.

Spontaneous production of E colicins is known to occur in only a small fraction of colicinogenic population. The current study aimed to determine if the same holds true for the production of colicin E9 in real time, by investigating the induction dynamics of the promoter of the ColE9 operon which results in the expression of the ColE9 activity and functional genes. A novel fluorescent reporter was constructed which carries the fusion of the ColE9 promoter and the gfpmut2 gene in a low copy number plasmid that was compatible with the native ColE9-J plasmid. Using the fluorescent reporter construct in the non colicinogenic E. coli cells, the induction of the ColE9 promoter was investigated. The current study demonstrates that the spontaneous induction of the ColE9 promoter occurs in a heterogenous manner and this heterogeneity is maintained in a bacterial population for several generations suggesting that it is unlikely due to any irreversible mutation in the bacterial culture. Furthermore, the same investigations were repeated using the colicin E9 producing E. coli cells. Flow cytometry analysis revealed that 7.1 ± 0.68% of the colicin E9 producing E. coli cells expressed GFP albeit only 2.45 ± 0.30% was observed from non colicinogenic E. coli cells. The considerable increase in the number of the fluorescent cells was likely due to the DNase activity of colicin E9 produced by their clonemates, resulting the auto-induction, which can be abolished with the inactivation of the DNase activity of the colicin E9.

The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody.

Cancer remains a leading cause of morbidity and mortality worldwide, requiring ongoing development of targeted therapeutics such as monoclonal antibodies. Carbohydrates on embryonic cells are often highly expressed in cancer and are therefore attractive targets for antibodies. Stage-specific embryonic antigen-4 (SSEA-4) is one such glycolipid target expressed in many cancers, including breast and ovarian carcinomas. Here, we defined the structural basis for recognition of SSEA-4 by a novel monospecific chimeric antibody (ch28/11). Five X-ray structures of ch28/11 Fab complexes with the SSEA-4 glycan headgroup, determined at 1.5-2.7 Å resolutions, displayed highly similar three-dimensional structures indicating a stable binding mode. The structures also revealed that by adopting a horseshoe-shaped conformation in a deep groove, the glycan headgroup likely sits flat against the membrane to allow the antibody to interact with SSEA-4 on cancer cells. Moreover, we found that the terminal sialic acid of SSEA-4 plays a dominant role in dictating the exquisite specificity of the ch28/11 antibody. This observation was further supported by molecular dynamics simulations of the ch28/11-glycan complex, which show that SSEA-4 is stabilized by its terminal sialic acid, unlike SSEA-3, which lacks this sialic acid modification. These high-resolution views of how a glycolipid interacts with an antibody may help to advance a new class of cancer-targeting immunotherapy.

Molecular and structural basis for Lewis glycan recognition by a cancer-targeting antibody.

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.

Detection of induced synthesis of colicin E9 using ColE9p::gfpmut2 based reporter system.

The majority of colicin operons are regulated by an SOS response inducible promoter (SOS promoter), located at upstream of the colicin operons. Therefore, colicin synthesis is induced by DNA damaging agents like mitomycin C (MMC) because the resulting DNA damage switches on the SOS response in bacteria. In this study, we have described the strategy for fusion of the SOS promoter of the colicin E9 operon (ColE9p) with a promoterless green fluorescent reporter gene (gfpmut2). We observed that the ColE9p-gfpmut2 is inducible by MMC which confirmed that the ColE9p-gfpmut2 is sensitive to SOS response inducing agents. The data implies that the ColE9p-gfpmut2 based reporter system is suitable for monitoring the ColE9 synthesis induced by SOS response inducing agents including antibiotics. Using green fluorescent protein expression from the ColE9p-gfpmut2 as an indicator of ColE9 synthesis; we have investigated, first time, the inducing effects of cephalexin antibiotic on ColE9 synthesis. Our data demonstrated that the cephalexin has potential to induce ColE9 synthesis from E. coli JM83 host cells albeit the level of this induction is very low hence its detection required a highly sensitive method.

Structural evidence that colicin A protein binds to a novel binding site of TolA protein in Escherichia coli periplasm.

The Tol assembly of proteins is an interacting network of proteins located in the Escherichia coli cell envelope that transduces energy and contributes to cell integrity. TolA is central to this network linking the inner and outer membranes by interactions with TolQ, TolR, TolB, and Pal. Group A colicins, such as ColA, parasitize the Tol network through interactions with TolA and/or TolB to facilitate translocation through the cell envelope to reach their cytotoxic site of action. We have determined the first structure of the C-terminal domain of TolA (TolAIII) bound to an N-terminal ColA polypeptide (TA(53-107)). The interface region of the TA(53-107)-TolAIII complex consists of polar contacts linking residues Arg-92 to Arg-96 of ColA with residues Leu-375-Pro-380 of TolA, which constitutes a ß-strand addition commonly seen in more promiscuous protein-protein contacts. The interface region also includes three cation-π interactions (Tyr-58-Lys-368, Tyr-90-Lys-379, Phe-94-Lys-396), which have not been observed in any other colicin-Tol protein complex. Mutagenesis of the interface residues of ColA or TolA revealed that the effect on the interaction was cumulative; single mutations of either partner had no effect on ColA activity, whereas mutations of three or more residues significantly reduced ColA activity. Mutagenesis of the aromatic ring component of the cation-π interacting residues showed Tyr-58 of ColA to be essential for the stability of complex formation. TA(53-107) binds on the opposite side of TolAIII to that used by g3p, ColN, or TolB, illustrating the flexible nature of TolA as a periplasmic hub protein.

Allosteric beta-propeller signalling in TolB and its manipulation by translocating colicins.

The Tol system is a five-protein assembly parasitized by colicins and bacteriophages that helps stabilize the Gram-negative outer membrane (OM). We show that allosteric signalling through the six-bladed beta-propeller protein TolB is central to Tol function in Escherichia coli and that this is subverted by colicins such as ColE9 to initiate their OM translocation. Protein-protein interactions with the TolB beta-propeller govern two conformational states that are adopted by the distal N-terminal 12 residues of TolB that bind TolA in the inner membrane. ColE9 promotes disorder of this 'TolA box' and recruitment of TolA. In contrast to ColE9, binding of the OM lipoprotein Pal to the same site induces conformational changes that sequester the TolA box to the TolB surface in which it exhibits little or no TolA binding. Our data suggest that Pal is an OFF switch for the Tol assembly, whereas colicins promote an ON state even though mimicking Pal. Comparison of the TolB mechanism to that of vertebrate guanine nucleotide exchange factor RCC1 suggests that allosteric signalling may be more prevalent in beta-propeller proteins than currently realized.

Prediction of Biomarker Expression on Primary Pancreatic Ductal Adenocarcinoma Tissues Using Fine-Needle Biopsies: Paving the Way for a Patient-Tailored Molecular Imaging Approach.

BACKGROUND: Targeted molecular imaging may improve tumor cell identification during diagnosis and resection of pancreatic ductal adenocarcinoma (PDAC). Although many molecular imaging biomarkers are (over)expressed in PDAC, intertumoral heterogeneity of biomarker expression hampers universal tracer administration. Preoperative, patient-specific screening and selection of the most optimal biomarker could therefore improve tumor delineation. OBJECTIVE: This study evaluated whether fine-needle biopsy (FNB) specimens could be used to preoperatively predict biomarker expression in the corresponding primary PDAC specimen. METHODS: Expression of previously identified PDAC biomarkers αvß6, CEACAM5, EGFR, mesothelin, Lea/c/x, and sdi-Lea on FNB and corresponding primary tumor (PT) specimens (n = 45) was evaluated using immunohistochemistry and quantified using a semi-automated image analysis workflow. RESULTS: Biomarker expression on FNB and PT tissues showed high concordance (∆H-score ≤ 50), i.e. was present in 62% of cases for αvß6, 61% for CEACAM5, 85% for EGFR, 69% for mesothelin, 76% for Lea/c/x, and 79% for sdi-Lea, indicating high concordance. Except for αvß6, biomarker expression on FNB tissues was positively correlated with PT expression for all biomarkers. Subgroup analyses showed that neoadjuvant therapy (NAT) had no major and/or significant effect on concordance, expression difference and, except for mesothelin, correlation of biomarker expression between FNB and PT tissues. CONCLUSION: This study demonstrated that biomarker expression in FNB tissues is predictive for PT expression, irrespective of the application of NAT. These findings thereby provide the foundation for the clinical application of an FNB-based biomarker-screening workflow, eventually facilitating a patient-specific approach of molecular imaging tracer administration in PDAC.

Membrane activities of colicin nuclease domains: analogies with antimicrobial peptides.

Nuclease colicins, such as colicin E9, are a class of Escherichia coli bacteriocins that kill E. coli and closely related Gram-negative bacteria through nucleolytic action in the cytoplasm. In order to accomplish this, their cytotoxic domains require transportation across two sets of membranes and the periplasmic space. Currently, little information is available concerning how the membrane translocation processes are achieved, and the present review summarizes our recent results on the in vitro membrane activities of the colicin nuclease domains. Using model membranes, we have analysed the cytotoxic domains of a number of DNase-type colicins and one rRNase colicin for their bilayer insertion depth and for their ability to induce vesicle aggregation, lipid mixing and increased bilayer permeability. We found that, by analogy with AMPs (antimicrobial peptides), the interplay between charge and hydrophobic character of the nuclease domains governs their pleiotropic membrane activities and these results form the basis of ongoing work to unravel the molecular mechanisms underlying their membrane translocation.

Colicin A binds to a novel binding site of TolA in the Escherichia coli periplasm.

Colicins are protein antibiotics produced by Escherichia coli to kill closely related non-identical competing species. They have taken advantage of the promiscuity of several proteins in the cell envelope for entry into the bacterial cell. The Tol-Pal system comprises one such ensemble of periplasmic and membrane-associated interacting proteins that links the IM (inner membrane) and OM (outer membrane) and provides the cell with a structural scaffold for cell division and energy transduction. Central to the Tol-Pal system is the TolA hub protein which forms protein-protein interactions with all other members and also with extrinsic proteins such as colicins A, E1, E2-E9 and N, and the coat proteins of the Ff family of filamentous bacteriophages. In the present paper, we review the role of TolA in the translocation of colicin A through the recently determined crystal structure of the complex of TolA with a translocation domain peptide of ColA (TA53-107), we demonstrate that TA53-107 binds to TolA at a novel binding site and compare the interactions of TolA with other colicins that use the Tol-Pal system for cell entry substantiating further the role of TolA as a periplasmic hub protein.

The crystal structure of the TolB box of colicin A in complex with TolB reveals important differences in the recruitment of the common TolB translocation portal used by group A colicins.

Interaction of the TolB box of Group A colicins with the TolB protein in the periplasm of Escherichia coli cells promotes transport of the cytotoxic domain of the colicin across the cell envelope. The crystal structure of a complex between a 107-residue peptide (TA(1-107)) of the translocation domain of colicin A (ColA) and TolB identified the TolB box as a 12-residue peptide that folded into a distorted hairpin within a central canyon of the beta-propeller domain of TolB. Comparison of this structure with that of the colicin E9 (ColE9) TolB box-TolB complex, together with site-directed mutagenesis of the ColA TolB box residues, revealed important differences in the interaction of the two TolB boxes with an overlapping binding site on TolB. Substitution of the TolB box residues of ColA with those of ColE9 conferred the ability to competitively recruit TolB from Pal but reduced the biological activity of the mutant ColA. This datum explains (i) the difference in binding affinities of ColA and ColE9 with TolB, and (ii) the inability of ColA, unlike ColE9, to competitively recruit TolB from Pal, allowing an understanding of how these two colicins interact in a different way with a common translocation portal in E. coli cells.

An Immunohistochemical Evaluation of Tumor-Associated Glycans and Mucins as Targets for Molecular Imaging of Pancreatic Ductal Adenocarcinoma.

Targeted molecular imaging may overcome current challenges in the preoperative and intraoperative delineation of pancreatic ductal adenocarcinoma (PDAC). Tumor-associated glycans Lea/c/x, sdi-Lea, sLea, sLex, sTn as well as mucin-1 (MUC1) and mucin-5AC (MU5AC) have gained significant interest as targets for PDAC imaging. To evaluate their PDAC molecular imaging potential, biomarker expression was determined using immunohistochemistry on PDAC, (surrounding) chronic pancreatitis (CP), healthy pancreatic, duodenum, positive (LN+) and negative lymph node (LN-) tissues, and quantified using a semi-automated digital image analysis workflow. Positive expression on PDAC tissues was found on 83% for Lea/c/x, 94% for sdi-Lea, 98% for sLea, 90% for sLex, 88% for sTn, 96% for MUC1 and 67% for MUC5AC, where all were not affected by the application of neoadjuvant therapy. Compared to PDAC, all biomarkers were significantly lower expressed on CP, healthy pancreatic and duodenal tissues, except for sTn and MUC1, which showed a strong expression on duodenum (sTn tumor:duodenum ratio: 0.6, p < 0.0001) and healthy pancreatic tissues (MUC1 tumor:pancreas ratio: 1.0, p > 0.9999), respectively. All biomarkers are suitable targets for correct identification of LN+, as well as the distinction of LN+ from LN- tissues. To conclude, this study paves the way for the development and evaluation of Lea/c/x-, sdi-Lea-, sLea-, sLex- and MUC5AC-specific tracers for molecular imaging of PDAC imaging and their subsequent introduction into the clinic.

Design of a polypeptide FRET substrate that facilitates study of the antimicrobial protease lysostaphin.

We have developed a polypeptide lysostaphin FRET (fluorescence resonance energy transfer) substrate (MV11F) for the endopeptidase activity of lysostaphin. Site-directed mutants of lysostaphin that abolished the killing activity against Staphylococcus aureus also completely inhibited the endopeptidase activity against the MV11 FRET substrate. Lysostaphin-producing staphylococci are resistant to killing by lysostaphin through incorporation of serine residues at positions 3 and 5 of the pentaglycine cross-bridge in their cell walls. The MV11 FRET substrate was engineered to introduce a serine residue at each of four positions of the pentaglycine target site and it was found that only a serine residue at position 3 completely inhibited cleavage. The introduction of random, natural amino acid substitutions at position 3 of the pentaglycine target site demonstrated that only a glycine residue at this position was compatible with lysostaphin cleavage of the MV11 FRET substrate. A second series of polypeptide substrates (decoys) was developed with the GFP (green fluorescent protein) domain of MV11 replaced with that of the DNase domain of colicin E9. Using a competition FRET assay, the lysostaphin endopeptidase was shown to bind to a decoy peptide containing a GGSGG cleavage site. The MV11 substrate provides a valuable system to facilitate structure/function studies of the endopeptidase activity of lysostaphin and its orthologues.

Glycan-Based Near-infrared Fluorescent (NIRF) Imaging of Gastrointestinal Tumors: a Preclinical Proof-of-Concept In Vivo Study.

PURPOSE: Aberrantly expressed glycans in cancer are of particular interest for tumor targeting. This proof-of-concept in vivo study aims to validate the use of aberrant Lewis glycans as target for antibody-based, real-time imaging of gastrointestinal cancers. PROCEDURES: Immunohistochemical (IHC) staining with monoclonal antibody FG88.2, targeting Lewisa/c/x, was performed on gastrointestinal tumors and their healthy counterparts. Then, FG88.2 and its chimeric human/mouse variant CH88.2 were conjugated with near-infrared fluorescent (NIRF) IRDye 800CW for real-time imaging. Specific binding was evaluated in vitro on human gastrointestinal cancer cell lines with cell-based plate assays, flow cytometry, and immune-fluorescence microscopy. Subsequently, mice bearing human colon and pancreatic subcutaneous tumors were imaged in vivo after intravenous administration of 1 nmol (150 µg) CH88.2-800CW with the clinical Artemis NIRF imaging system using the Pearl Trilogy small animal imager as reference. One week post-injection of the tracer, tumors and organs were resected and tracer uptake was analyzed ex vivo. RESULTS: IHC analysis showed strong FG88.2 staining on colonic, gastric, and pancreatic tumors, while staining on their normal tissue counterparts was limited. Next, human cancer cell lines HT-29 (colon) and BxPC-3 and PANC-1 (both pancreatic) were identified as respectively high, moderate, and low Lewisa/c/x-expressing. Using the clinical NIRF camera system for tumor-bearing mice, a mean tumor-to-background ratio (TBR) of 2.2 ± 0.3 (Pearl: 3.1 ± 0.8) was observed in the HT-29 tumors and a TBR of 1.8 ± 0.3 (Pearl: 1.9 ± 0.5) was achieved in the moderate expression BxPC-3 model. In both models, tumors could be adequately localized and delineated by NIRF for up to 1 week. Ex vivo analysis confirmed full tumor penetration of the tracer and low fluorescence signals in other organs. CONCLUSIONS: Using a novel chimeric Lewisa/c/x-targeting tracer in combination with a clinical NIRF imager, we demonstrate the potential of targeting Lewis glycans for fluorescence-guided surgery of gastrointestinal tumors.

Targeting Glycans and Heavily Glycosylated Proteins for Tumor Imaging.

Real-time tumor imaging techniques are increasingly used in oncological surgery, but still need to be supplemented with novel targeted tracers, providing specific tumor tissue detection based on intra-tumoral processes or protein expression. To maximize tumor/non-tumor contrast, targets should be highly and homogenously expressed on tumor tissue only, preferably from the earliest developmental stage onward. Unfortunately, most evaluated tumor-associated proteins appear not to meet all of these criteria. Thus, the quest for ideal targets continues. Aberrant glycosylation of proteins and lipids is a fundamental hallmark of almost all cancer types and contributes to tumor progression. Additionally, overexpression of glycoproteins that carry aberrant glycans, such as mucins and proteoglycans, is observed. Selected tumor-associated glyco-antigens are abundantly expressed and could, thus, be ideal candidates for targeted tumor imaging. Nevertheless, glycan-based tumor imaging is still in its infancy. In this review, we highlight the potential of glycans, and heavily glycosylated proteoglycans and mucins as targets for multimodal tumor imaging by discussing the preclinical and clinical accomplishments within this field. Additionally, we describe the major advantages and limitations of targeting glycans compared to cancer-associated proteins. Lastly, by providing a brief overview of the most attractive tumor-associated glycans and glycosylated proteins in association with their respective tumor types, we set out the way for implementing glycan-based imaging in a clinical practice.

Engineering the Human Fc Region Enables Direct Cell Killing by Cancer Glycan-Targeting Antibodies without the Need for Immune Effector Cells or Complement.

Murine IgG3 glycan-targeting mAb often induces direct cell killing in the absence of immune effector cells or complement via a proinflammatory mechanism resembling oncotic necrosis. This cancer cell killing is due to noncovalent association between Fc regions of neighboring antibodies, resulting in enhanced avidity. Human isotypes do not contain the residues underlying this cooperative binding mode; consequently, the direct cell killing of mouse IgG3 mAb is lost upon chimerization or humanization. Using the Lewisa/c/x -targeting 88mAb, we identified the murine IgG3 residues underlying the direct cell killing and increased avidity via a series of constant region shuffling and subdomain swapping approaches to create improved ("i") chimeric mAb with enhanced tumor killing in vitro and in vivo. Constant region shuffling identified a major CH3 and a minor CH2 contribution, which was further mapped to discontinuous regions among residues 286-306 and 339-378 that, when introduced in 88hIgG1, recapitulated the direct cell killing and avidity of 88mIgG3. Of greater interest was the creation of a sialyl-di-Lewisa-targeting i129G1 mAb via introduction of these selected residues into 129hIgG1, converting it into a direct cell killing mAb with enhanced avidity and significant in vivo tumor control. The human iG1 mAb, termed Avidimabs, retained effector functions, paving the way for the proinflammatory direct cell killing to promote antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity through relief of immunosuppression. Ultimately, Fc engineering of human glycan-targeting IgG1 mAb confers proinflammatory direct cell killing and enhanced avidity, an approach that could be used to improve the avidity of other mAb with therapeutic potential. SIGNIFICANCE: Fc engineering enhances avidity and direct cell killing of cancer-targeting anti-glycan antibodies to create superior clinical candidates for cancer immunotherapy.

Monoclonal Antibody Targeting Sialyl-di-Lewisa-Containing Internalizing and Noninternalizing Glycoproteins with Cancer Immunotherapy Development Potential.

Tumor glycans constitute attractive targets for therapeutic antibodies. The sialylated glycocalyx plays a prominent role in cancer progression and immune evasion. Here, we describe the characterization of the mAb, FG129, which targets tumor-associated sialylated glycan, and demonstrate its potential for multimodal cancer therapy. FG129, obtained through BALB/c mouse immunizations with liposomes containing membrane glycan extracts from the colorectal cancer cell line LS180, is an mIgG1κ that targets sialyl-di-Lewisa-containing glycoproteins. FG129, as well as its chimeric human IgG1 variant, CH129, binds with nanomolar functional affinity to a range of colorectal, pancreatic, and gastric cancer cell lines. FG129 targets 74% (135/182) of pancreatic, 50% (46/92) of gastric, 36% (100/281) of colorectal, 27% (89/327) of ovarian, and 21% (42/201) of non-small cell lung cancers, by IHC. In our pancreatic cancer cohort, high FG129 glyco-epitope expression was significantly associated with poor prognosis (P = 0.004). Crucially, the glyco-epitope displays limited normal tissue distribution, with FG129 binding weakly to a small percentage of cells within gallbladder, ileum, liver, esophagus, pancreas, and thyroid tissues. Owing to glyco-epitope internalization, we validated payload delivery by CH129 through monomethyl auristatin E (MMAE) or maytansinoid (DM1 and DM4) conjugation. All three CH129 drug conjugates killed high-binding colorectal and pancreatic cancer cell lines with (sub)nanomolar potency, coinciding with significant in vivo xenograft tumor control by CH129-vcMMAE. CH129, with its restricted normal tissue distribution, avid tumor binding, and efficient payload delivery, is a promising candidate for the treatment of sialyl-di-Lewisa-expressing solid tumors, as an antibody-drug conjugate or as an alternative cancer immunotherapy modality.

Combination vaccine based on citrullinated vimentin and enolase peptides induces potent CD4-mediated anti-tumor responses.

BACKGROUND: Stress-induced post-translational modifications occur during autophagy and can result in generation of new epitopes and immune recognition. One such modification is the conversion of arginine to citrulline by peptidylarginine deiminase enzymes. METHODS: We used Human leukocyte antigen (HLA) transgenic mouse models to assess the immunogenicity of citrullinated peptide vaccine by cytokine Enzyme linked immunosorbant spot (ELISpot) assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma and ID8 ovarian models expressing either constitutive or interferon-gamma (IFNγ) inducible Major Histocompatibility Complex (MHC) class II (MHC-II) as represented by most human tumors. To determine the importance of CD4 T cells in tumor therapy, we analyzed the immune cell infiltrate into murine tumors using flow cytometry and performed therapy studies in the presence of CD4 and CD8 T cell depletion. We assessed the T cell repertoire to citrullinated peptides in ovarian cancer patients and healthy donors using flow cytometry. RESULTS: The combination of citrullinated vimentin and enolase peptides (Modi-1) stimulated strong CD4 T cell responses in mice. Responses resulted in a potent anti-tumor therapy against established tumors and generated immunological memory which protected against tumor rechallenge. Depletion of CD4, but not CD8 T cells, abrogated the primary anti-tumor response as well as the memory response to tumor rechallenge. This was further reinforced by successful tumor regression being associated with an increase in tumor-infiltrating CD4 T cells and a reduction in tumor-associated myeloid suppressor cells. The anti-tumor response also relied on direct CD4 T cell recognition as only tumors expressing MHC-II were rejected. A comparison of different Toll-like receptor (TLR)-stimulating adjuvants showed that Modi-1 induced strong Th1 responses when combined with granulocyte-macrophage colony-stimulating factor (GMCSF), TLR9/TLR4, TLR9, TLR3, TLR1/2 and TLR7 agonists. Direct linkage of the TLR1/2 agonist to the peptides allowed the vaccine dose to be reduced by 10-fold to 100-fold without loss of anti-tumor activity. Furthermore, a CD4 Th1 response to the citrullinated peptides was seen in ovarian cancer patients. CONCLUSIONS: Modi-1 citrullinated peptide vaccine induces potent CD4-mediated anti-tumor responses in mouse models and a CD4 T cell repertoire is present in ovarian cancer patients to the citrullinated peptides suggesting that Modi-1 could be an effective vaccine for ovarian cancer patients.

Investigating early events in receptor binding and translocation of colicin E9 using synchronized cell killing and proteolytic cleavage.

Enzymatic colicins such as colicin E9 (ColE9) bind to BtuB on the cell surface of Escherichia coli and rapidly recruit a second coreceptor, either OmpF or OmpC, through which the N-terminal natively disordered region (NDR) of their translocation domain gains entry into the cell periplasm and interacts with TolB. Previously, we constructed an inactive disulfide-locked mutant ColE9 (ColE9(s-s)) that binds to BtuB and can be reduced with dithiothreitol (DTT) to synchronize cell killing. By introducing unique enterokinase (EK) cleavage sites in ColE9(s-s), we showed that the first 61 residues of the NDR were inaccessible to cleavage when bound to BtuB, whereas an EK cleavage site inserted at residue 82 of the NDR remained accessible. This suggests that most of the NDR is occluded by OmpF shortly after binding to BtuB, whereas the extreme distal region of the NDR is surface exposed before unfolding of the receptor-binding domain occurs. EK cleavage of unique cleavage sites located in the ordered region of the translocation domain or in the distal region of the receptor-binding domain confirmed that these regions of ColE9 remained accessible at the E. coli cell surface. Lack of EK cleavage of the DNase domain of the cell-bound, oxidized ColE9/Im9 complex, and the rapid detection of Alexa Fluor 594-labeled Im9 (Im9(AF)) in the cell supernatant following treatment of cells with DTT, suggested that immunity release occurred immediately after unfolding of the colicin and was not driven by binding to BtuB.

ADAMTS9-Regulated Pericellular Matrix Dynamics Governs Focal Adhesion-Dependent Smooth Muscle Differentiation.

Focal adhesions anchor cells to extracellular matrix (ECM) and direct assembly of a pre-stressed actin cytoskeleton. They act as a cellular sensor and regulator, linking ECM to the nucleus. Here, we identify proteolytic turnover of the anti-adhesive proteoglycan versican as a requirement for maintenance of smooth muscle cell (SMC) focal adhesions. Using conditional deletion in mice, we show that ADAMTS9, a secreted metalloprotease, is required for myometrial activation during late gestation and for parturition. Through knockdown of ADAMTS9 in uterine SMC, and manipulation of pericellular versican via knockdown or proteolysis, we demonstrate that regulated pericellular matrix dynamics is essential for focal adhesion maintenance. By influencing focal adhesion formation, pericellular versican acts upstream of cytoskeletal assembly and SMC differentiation. Thus, pericellular versican proteolysis by ADAMTS9 balances pro- and anti-adhesive forces to maintain an SMC phenotype, providing a concrete example of the dynamic reciprocity of cells and their ECM.

Cancer cell associated glycans as targets for immunotherapy.

Therapeutic anti-glycan antibodies for cancer treatment are limited, in spite of the differential glycophenotype of cancer cells and associated biology. We have generated anti-Le glycan antibodies with clinical potential and multifaceted functionality. Increased understanding of all aspects of glycomic research will ensure the continued development of glycan-targeted immunotherapeutics.