Combining SiRPα decoy-coengineered T cells and antibodies augments macrophage-mediated phagocytosis of tumor cells.

The adoptive transfer of T cell receptor-engineered (TCR-engineered) T cells (ACT) targeting the HLA-A2-restricted cancer-testis epitope NY-ESO-1157-165 (A2/NY) has yielded favorable clinical responses against several cancers. Two approaches to improve ACT are TCR affinity optimization and T cell coengineering to express immunomodulatory molecules that can exploit endogenous immunity. By computational design we previously developed a panel of binding-enhanced A2/NY-TCRs including A97L, which augmented the in vitro function of gene-modified T cells as compared with WT. Here, we demonstrated higher persistence and improved tumor control by A97L-T cells. In order to harness macrophages in tumors, we further coengineered A97L-T cells to secrete a high-affinity signal regulatory protein α (SiRPα) decoy (CV1) that blocks CD47. While CV1-Fc-coengineered A97L-T cells mediated significantly better control of tumor outgrowth and survival in Winn assays, in subcutaneous xenograft models the T cells, coated by CV1-Fc, were depleted. Importantly, there was no phagocytosis of CV1 monomer-coengineered T cells by human macrophages. Moreover, avelumab and cetuximab enhanced macrophage-mediated phagocytosis of tumor cells in vitro in the presence of CV1 and improved tumor control upon coadministration with A97L-T cells. Taken together, our study indicates important clinical promise for harnessing macrophages by combining CV1-coengineered TCR-T cells with targeted antibodies to direct phagocytosis against tumor cells.

Expression and purification of human interferon alpha 2a (IFNα2a) in the methylotrophic yeast Pichia pastoris.

Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous leukemia, advanced renal cell carcinoma, and metastatic malignant melanoma. So far, the pharmaceutical polypeptide of this cytokine is produced in prokaryotic expression systems (E. coli). Here we report the expression and purification of recombinant human IFNα2a in the methylotrophic yeast Pichia pastoris. The cDNA encoding for human IFNα2a, modified to bear the P. pastoris codon bias, was cloned into the pPinkα-HC vector in order to be expressed as a secreted protein upon induction. Proper expression and secretion of recombinant human IFNα2a (approximately 19 kDa) was confirmed by PCR-sequencing, SDS-PAGE and Western blot analysis following methanol-induced expression in a number of individual transformed strains. Purification of the recombinant protein was performed by affinity chromatography, achieving a robust yield of purified active form. The purified recombinant protein showed an impressive stability to thermal denaturation as observed by Differential Scanning Fluorimetry. The biological activity of the P. pastoris-produced IFNα2a was confirmed in A549 and HT29 cells by monitoring transcriptional up-regulation of a panel of known interferon-stimulated genes (ISGs). Our results document that the P. pastoris expression system is a suitable system for producing biologically functional IFNα2a in a secreted form.

TEM1-targeting PEGylated PLGA shikonin nanoformulation for immunomodulation and eradication of ovarian cancer.

Introduction: Tumor endothelial marker 1 (TEM1) is expressed by tumor vascular endothelial cells in various cancers. Methods: Here, we developed poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) PEGylated with polyethylene glycol (PEG) and functionalized with anti-TEM1 antibody fragment (78Fc) and loaded them with necroptosis-inducing agent shikonin (SHK) (78Fc-PLGA-SHK NPs). Results: The nanoformulation showed a smooth spherical shape (~120 nm; the ζ potential of -30 mV) with high drug entrapment and bioconjugation efficiencies (~92% and ~90%, respectively) and a sustained-release profile in serum. Having significant toxicity in vitro (e.g., MS1 and TC1 cells), the nanoformulation dramatically increased the cytotoxicity in the TC1 murine lung carcinoma subcutaneous and intravenous/metastatic models as aggressive tumor models. The injection of the 78Fc-PLGA-SHK NPs to the MS1-xenograft mice resulted in significantly higher accumulation and effects in the TEM1-positive tumor targets, while they were excreted via urine track without retaining in the liver/spleen. In the TC1 subcutaneous model, C57/BL6 mice treated with the 78Fc-PLGA-SHK NPs revealed a significant therapeutic effect. The mice, which were tumor-free after receiving the nanoformulation, were re-challenged with the TC1 cells to investigate the immune response. These animals became tumor-free a week after the injection of TC1 cells. Conclusion: Based on these findings, we propose the 78Fc-PLGA-SHK NPs as a highly effective immunostimulating nanomedicine against the TEM1-expressing cells for targeted therapy of solid tumors including ovarian cancer.

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Higher-order immunoglobulin repertoire restrictions in CLL: the illustrative case of stereotyped subsets 2 and 169.

Chronic lymphocytic leukemia (CLL) major stereotyped subset 2 (IGHV3-21/IGLV3-21, ∼2.5% of all cases of CLL) is an aggressive disease variant, irrespective of the somatic hypermutation (SHM) status of the clonotypic IGHV gene. Minor stereotyped subset 169 (IGHV3-48/IGLV3-21, ∼0.2% of all cases of CLL) is related to subset 2, as it displays a highly similar variable antigen-binding site. We further explored this relationship through next-generation sequencing and crystallographic analysis of the clonotypic B-cell receptor immunoglobulin. Branching evolution of the predominant clonotype through intraclonal diversification in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the 2 subsets were highlighted by the finding of shared SHMs within both the heavy and light chain genes in all analyzed cases at either the clonal or subclonal level. Particularly noteworthy in this respect was a ubiquitous SHM at the linker region between the variable and the constant domain of the IGLV3-21 light chains, previously reported as critical for immunoglobulin homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21-bearing CLL subset 169 immunoglobulin retains the same geometry and contact residues for the homotypic intermolecular interaction observed in subset 2, including the SHM at the linker region, and, from a molecular standpoint, belong to a common structural mode of autologous recognition. Collectively, our findings document that stereotyped subsets 2 and 169 are very closely related, displaying shared immunoglobulin features that can be explained only in the context of shared functional selection.

The homeodomain transcription factor MEIS1 triggers chemokine expression and is involved in CD8+ T-lymphocyte infiltration in early stage ovarian cancer.

CD8+ T-lymphocytes infiltration is a favorable prognostic marker in ovarian cancer. Recently we identified MEIS1 as a gene overexpressed in early stage ovarian tumors enriched for CD8+ T-cells. Here, we report the molecular mechanism of the homeodomain transcription factor MEIS1 in lymphocyte recruitment. We validated that MEIS1 expression is a positive predictor of CD8+ T cells in early stage ovarian cancer. We showed that MEIS1 induces the expression of CCL18, CCL4, CXCL7, CCL5, CXCL1, and IL8 chemokines in cancer cells followed by their secretion in the culture medium ultimately triggering CD8+ T-lymphocyte recruitment in vitro. Knock down of MEIS1 expression by siRNA resulted in downregulation of these chemokines. We verified that MEIS1 binds to the promoters of chemokine genes, both in vitro and in vivo. We also showed that the expression levels of MEIS1 correlated tightly with the mRNA levels of chemokines CCL4 and CCL18 in early stage ovarian cancer patient samples and served as a positive prognostic marker, as shown by Kaplan-Meyer survival analysis. In conclusion, we propose that MEIS1 plays a pivotal role in the regulatory circuitry governing T-cell chemo-attraction during the early stages of ovarian cancer.

Roles of NF-κB Signaling in the Regulation of miRNAs Impacting on Inflammation in Cancer.

The NF-κB family of transcription factors regulate the expression of genes encoding proteins and microRNAs (miRNA, miR) precursors that may either positively or negatively regulate a variety of biological processes such as cell cycle progression, cell survival, and cell differentiation. The NF-κB-miRNA transcriptional regulatory network has been implicated in the regulation of proinflammatory, immune, and stress-like responses. Gene regulation by miRNAs has emerged as an additional epigenetic mechanism at the post-transcriptional level. The expression of miRNAs can be regulated by specific transcription factors (TFs), including the NF-κB TF family, and vice versa. The interplay between TFs and miRNAs creates positive or negative feedback loops and also regulatory networks, which can control cell fate. In the current review, we discuss the impact of NF-κB-miRNA interplay and feedback loops and networks impacting on inflammation in cancer. We provide several paradigms of specific NF-κB-miRNA networks that can regulate inflammation linked to cancer. For example, the NF-κB-miR-146 and NF-κB-miR-155 networks fine-tune the activity, intensity, and duration of inflammation, while the NF-κB-miR-21 and NF-κB-miR-181b-1 amplifying loops link inflammation to cancer; and p53- or NF-κB-regulated miRNAs interconnect these pathways and may shift the balance to cancer development or tumor suppression. The availability of genomic data may be useful to verify and find novel interactions, and provide a catalogue of 162 miRNAs targeting and 40 miRNAs possibly regulated by NF-κB. We propose that studying active TF-miRNA transcriptional regulatory networks such as NF-κB-miRNA networks in specific cancer types can contribute to our further understanding of the regulatory interplay between inflammation and cancer, and also perhaps lead to the development of pharmacologically novel therapeutic approaches to combat cancer.

A step-by-step microRNA guide to cancer development and metastasis.

BACKGROUND: Cancer is one of the leading causes of mortality. The neoplastic transformation of normal cells to cancer cells is caused by a progressive accumulation of genetic and epigenetic alterations in oncogenes, tumor suppressor genes and epigenetic regulators, providing cells with new properties, collectively known as the hallmarks of cancer. During the process of neoplastic transformation cells progressively acquire novel characteristics such as unlimited growth potential, increased motility and the ability to migrate and invade adjacent tissues, the ability to spread from the tumor of origin to distant sites, and increased resistance to various types of stresses, mostly attributed to the activation of genetic stress-response programs. Accumulating evidence indicates a crucial role of microRNAs (miRNAs or miRs) in the initiation and progression of cancer, acting either as oncogenes (oncomirs) or as tumor suppressors via several molecular mechanisms. MiRNAs comprise a class of small ~22 bp long noncoding RNAs that play a key role in the regulation of gene expression at the post-transcriptional level, acting as negative regulators of mRNA translation and/or stability. MiRNAs are involved in the regulation of a variety of biological processes including cell cycle progression, DNA damage responses and apoptosis, epithelial-to-mesenchymal cell transitions, cell motility and stemness through complex and interactive transcription factor-miRNA regulatory networks. CONCLUSIONS: The impact and the dynamic potential of miRNAs with oncogenic or tumor suppressor properties in each stage of the multistep process of tumorigenesis, and in the adaptation of cancer cells to stress, are discussed. We propose that the balance between oncogenic versus tumor suppressive miRNAs acting within transcription factor-miRNA regulatory networks, influences both the multistage process of neoplastic transformation, whereby normal cells become cancerous, and their stress responses. The role of specific tumor-derived exosomes containing miRNAs and their use as biomarkers in diagnosis and prognosis, and as therapeutic targets, are also discussed.

Overexpression of GPC6 and TMEM132D in Early Stage Ovarian Cancer Correlates with CD8+ T-Lymphocyte Infiltration and Increased Patient Survival.

Infiltration of cytotoxic T-lymphocytes in ovarian cancer is a favorable prognostic factor. Employing a differential expression approach, we have recently identified a number of genes associated with CD8+ T-cell infiltration in early stage ovarian tumors. In the present study, we validated by qPCR the expression of two genes encoding the transmembrane proteins GPC6 and TMEM132D in a cohort of early stage ovarian cancer patients. The expression of both genes correlated positively with the mRNA levels of CD8A, a marker of T-lymphocyte infiltration [Pearson coefficient: 0.427 (p = 0.0067) and 0.861 (p < 0.0001), resp.]. GPC6 and TMEM132D expression was also documented in a variety of ovarian cancer cell lines. Importantly, Kaplan-Meier survival analysis revealed that high mRNA levels of GPC6 and/or TMEM132D correlated significantly with increased overall survival of early stage ovarian cancer patients (p = 0.032). Thus, GPC6 and TMEM132D may serve as predictors of CD8+ T-lymphocyte infiltration and as favorable prognostic markers in early stage ovarian cancer with important consequences for diagnosis, prognosis, and tumor immunobattling.

Overexpression of SMARCE1 is associated with CD8+ T-cell infiltration in early stage ovarian cancer.

T-lymphocyte infiltration in ovarian tumors has been linked to a favorable prognosis, hence, exploring the mechanism of T-cell recruitment in the tumor is warranted. We employed a differential expression analysis to identify genes over-expressed in early stage ovarian cancer samples that contained CD8 infiltrating T-lymphocytes. Among other genes, we discovered that TTF1, a regulator of ribosomal RNA gene expression, and SMARCE1, a factor associated with chromatin remodeling were overexpressed in first stage CD8+ ovarian tumors. TTF1 and SMARCE1 mRNA levels showed a strong correlation with the number of intra-tumoral CD8+ cells in ovarian tumors. Interestingly, forced overexpression of SMARCE1 in SKOV3 ovarian cancer cells resulted in secretion of IL8, MIP1b and RANTES chemokines in the supernatant and triggered chemotaxis of CD8+ lymphocytes in a cell culture assay. The potency of SMARCE1-mediated chemotaxis appeared comparable to that caused by the transfection of the CXCL9 gene, coding for a chemokine known to attract T-cells. Our analysis pinpoints TTF1 and SMARCE1 as genes potentially involved in cancer immunology. Since both TTF1 and SMARCE1 are involved in chromatin remodeling, our results imply an epigenetic regulatory mechanism for T-cell recruitment that invites deciphering.

Shikonin-loaded antibody-armed nanoparticles for targeted therapy of ovarian cancer.

Conventional chemotherapy of ovarian cancer often fails because of initiation of drug resistance and/or side effects and trace of untouched remaining cancerous cells. This highlights an urgent need for advanced targeted therapies for effective remediation of the disease using a cytotoxic agent with immunomodulatory effects, such as shikonin (SHK). Based on preliminary experiments, we found SHK to be profoundly toxic in ovarian epithelial cancer cells (OVCAR-5 and ID8 cells) as well as in normal ovarian IOSE-398 cells, endothelial MS1 cells, and lymphocytes. To limit its cytotoxic impact solely to tumor cells within the tumor microenvironment (TME), we aimed to engineer SHK as polymeric nanoparticles (NPs) with targeting moiety toward tumor microvasculature. To this end, using single/double emulsion solvent evaporation/diffusion technique with sonication, we formulated biodegradable NPs of poly(lactic-co-glycolic acid) (PLGA) loaded with SHK. The surface of NPs was further decorated with solubilizing agent polyethylene glycol (PEG) and tumor endothelial marker 1 (TEM1)/endosialin-targeting antibody (Ab) through carbodiimide/N-hydroxysuccinimide chemistry. Having characterized the physicochemical and morphological properties of NPs, we studied their drug-release profiles using various kinetic models. The biological impact of NPs was also evaluated in tumor-associated endothelial MS1 cells, primary lymphocytes, and epithelial ovarian cancer OVCAR-5 cells. Based on particle size analysis and electron microscopy, the engineered NPs showed a smooth spherical shape with size range of 120 to 250 nm and zeta potential value of -30 to -40 mV. Drug entrapment efficiency was ~80%-90%, which was reduced to ~50%-60% upon surface decoration with PEG and Ab. The liberation of SHK from NPs showed a sustained-release profile that was best fitted with Wagner log-probability model. Fluorescence microscopy and flow cytometry analysis showed active interaction of Ab-armed NPs with TEM1-positive MS1 cells, but not with TEM1-negative MS1 cells. While exposure of the PEGylated NPs for 2 hours was not toxic to lymphocytes, long-term exposure of the Ab-armed and PEGylated NPs was significantly toxic to TEM1-positive MS1 cells and OVCAR-5 cells. Based on these findings, we propose SHK-loaded Ab-armed PEGylated PLGA NPs as a novel nanomedicine for targeted therapy of solid tumors.

Novel recombinant human b7-h4 antibodies overcome tumoral immune escape to potentiate T-cell antitumor responses.

B7-H4 (VTCN1, B7x, B7s) is a ligand for inhibitory coreceptors on T cells implicated in antigenic tolerization. B7-H4 is expressed by tumor cells and tumor-associated macrophages (TAM), but its potential contributions to tumoral immune escape and therapeutic targeting have been less studied. To interrogate B7-H4 expression on tumor cells, we analyzed fresh primary ovarian cancer cells collected from patient ascites and solid tumors, and established cell lines before and after in vivo passaging. B7-H4 expression was detected on the surface of all fresh primary human tumors and tumor xenotransplants, but not on most established cell lines, and B7-H4 was lost rapidly by tumor xenograft cells after short-term in vitro culture. These results indicated an in vivo requirement for B7-H4 induction and defined conditions for targeting studies. To generate anti-B7-H4-targeting reagents, we isolated antibodies by differential cell screening of a yeast-display single-chain fragments variable (scFv) library derived from patients with ovarian cancer. We identified anti-B7-H4 scFv that reversed in vitro inhibition of CD3-stimulated T cells by B7-H4 protein. Notably, these reagents rescued tumor antigen-specific T-cell activation, which was otherwise inhibited by coculture with antigen-loaded B7-H4+ APCs, B7-H4+ tumor cells, or B7-H4- tumor cells mixed with B7-H4+ TAMs; peritoneal administration of anti-B7-H4 scFv delayed the growth of established tumors. Together, our findings showed that cell surface expression of B7-H4 occurs only in tumors in vivo and that antibody binding of B7-H4 could restore antitumor T-cell responses. We suggest that blocking of B7-H4/B7-H4 ligand interactions may represent a feasible therapeutic strategy for ovarian cancer.

Chimeric antigen receptor T Cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo.

Adoptive immunotherapy using T lymphocytes genetically modified to express a chimeric antigen receptor (CAR-T) holds considerable promise for the treatment of cancer. However, CAR-based therapies may involve on-target toxicity against normal tissues expressing low amounts of the targeted tumor-associated antigen (TAA). To specify T cells for robust effector function that is selective for tumor but not normal tissue, we developed a trans-signaling CAR strategy, whereby T-cell activation signal 1 (CD3z) is physically dissociated from costimulatory signal 2 (CD28) in two CARs of differing antigen specificity: mesothelin and a-folate receptor (FRa). Human T cells were genetically modified to coexpress signal 1 (anti-Meso scFv-CD3z) and signal 2 (anti-FRa scFv-CD28) CARs in trans. Trans-signaling CAR-T cells showed weak cytokine secretion against target cells expressing only one TAA in vitro, similar to first-generation CAR-T cells bearing CD3z only, but showed enhanced cytokine secretion upon encountering natural or engineered tumor cells coexpressing both antigens, equivalent to that of second-generation CAR-T cells with dual signaling in cis. CAR-T cells with dual specificity also showed potent anticancer activity and persistence in vivo, which was superior to first-generation CAR-T cells and equivalent to second-generation CARs. Importantly, second-generation CAR-T cells exhibited potent activity against cells expressing mesothelin alone, recapitulating normal tissue, whereas trans-signaling CAR-T cells did not. Thus, a dual specificity, trans-signaling CAR approach can potentiate the therapeutic efficacy of CAR-T cells against cancer while minimizing parallel reactivity against normal tissues bearing single antigen.

Primary human ovarian epithelial cancer cells broadly express HER2 at immunologically-detectable levels.

The breadth of HER2 expression by primary human ovarian cancers remains controversial, which questions its suitability as a universal antigen in this malignancy. To address these issues, we performed extensive HER2 expression analysis on a wide panel of primary tumors as well as established and short-term human ovarian cancer cell lines. Conventional immunohistochemical (IHC) analysis of multiple tumor sites in 50 cases of high-grade ovarian serous carcinomas revealed HER2 overexpression in 29% of evaluated sites. However, more sensitive detection methods including flow cytometry, western blot analysis and q-PCR revealed HER2 expression in all fresh tumor cells derived from primary ascites or solid tumors as well as all established and short-term cultured cancer cell lines. Cancer cells generally expressed HER2 at higher levels than that found in normal ovarian surface epithelial (OSE) cells. Accordingly, genetically-engineered human T cells expressing an HER2-specific chimeric antigen receptor (CAR) recognized and reacted against all established or primary ovarian cancer cells tested with minimal or no reactivity against normal OSE cells. In conclusion, all human ovarian cancers express immunologically-detectable levels of HER2, indicating that IHC measurement underestimates the true frequency of HER2-expressing ovarian cancers and may limit patient access to otherwise clinically meaningful HER2-targeted therapies.

Redirected antitumor activity of primary human lymphocytes transduced with a fully human anti-mesothelin chimeric receptor.

Cancer regression by gene-modified T cells bearing a chimeric antigen receptor (CAR) exodomain of mouse origin can be limited by the induction of transgene immunogenicity resulting in poor persistence and function in vivo. The development of functionally-active CAR of human origin can address this issue. Here, we constructed and evaluated fully human anti-mesothelin CARs comprised of a human mesothelin-specific single-chain antibody variable fragment (P4 scFv) coupled to T cell signaling domains. Primary human T cells expressing P4 CAR specifically produced proinflammatory cytokines, degranulated and exerted potent cytolytic functions when cultured with mesothelin-expressing tumors in vitro. P4 CAR T cells also mediated bystander killing of mesothelin-negative cancer cells during coculture. CAR reactivity was not abrogated by soluble tumor-secreted or recombinant mesothelin protein even at supraphysiological levels. Importantly, adoptive transfer of P4 CAR-expressing T cells mediated the regression of large, established tumor in the presence of soluble mesothelin in a xenogenic model of human ovarian cancer. Thus, primary human T cells expressing fully human anti-mesothelin CAR efficiently kill mesothelin-expressing tumors in vitro and in vivo and have the potential to overcome the issue of transgene immunogenicity that may limit CAR T cell trials that utilize scFvs of mouse origin.

Mannose receptor (MR) engagement by mesothelin GPI anchor polarizes tumor-associated macrophages and is blocked by anti-MR human recombinant antibody.

Tumor-infiltrating macrophages respond to microenvironmental signals by developing a tumor-associated phenotype characterized by high expression of mannose receptor (MR, CD206). Antibody cross-linking of CD206 triggers anergy in dendritic cells and CD206 engagement by tumoral mucins activates an immune suppressive phenotype in tumor-associated macrophages (TAMs). Many tumor antigens are heavily glycosylated, such as tumoral mucins, and/or attached to tumor cells by mannose residue-containing glycolipids (GPI anchors), as for example mesothelin and the family of carcinoembryonic antigen (CEA). However, the binding to mannose receptor of soluble tumor antigen GPI anchors via mannose residues has not been systematically studied. To address this question, we analyzed the binding of tumor-released mesothelin to ascites-infiltrating macrophages from ovarian cancer patients. We also modeled functional interactions between macrophages and soluble mesothelin using an in vitro system of co-culture in transwells of healthy donor macrophages with human ovarian cancer cell lines. We found that soluble mesothelin bound to human macrophages and that the binding depended on the presence of GPI anchor and of mannose receptor. We next challenged the system with antibodies directed against the mannose receptor domain 4 (CDR4-MR). We isolated three novel anti-CDR4-MR human recombinant antibodies (scFv) using a yeast-display library of human scFv. Anti-CDR4-MR scFv #G11 could block mesothelin binding to macrophages and prevent tumor-induced phenotype polarization of CD206(low) macrophages towards TAMs. Our findings indicate that tumor-released mesothelin is linked to GPI anchor, engages macrophage mannose receptor, and contributes to macrophage polarization towards TAMs. We propose that compounds able to block tumor antigen GPI anchor/CD206 interactions, such as our novel anti-CRD4-MR scFv, could prevent tumor-induced TAM polarization and have therapeutic potential against ovarian cancer, through polarization control of tumor-infiltrating innate immune cells.

Biochemical and molecular analysis of the interaction between ERK2 MAP kinase and hypoxia inducible factor-1α.

The mitogen activated protein kinase (MAPK) signaling pathways play significant roles in fundamental cellular processes, such as cell growth and differentiation. It has been shown that the specificity and efficacy of phosphorylation by MAP kinases rely upon distinct MAPK-docking domains (D-domains) found in a wide range of MAPK substrates including the ETS-transcription factor Elk-1. Importantly, the MAPK signaling cascade converges with the hypoxia-induced signaling pathway. The key regulator of hypoxia signaling is the heterodimeric transcription factor hypoxia inducible factor-1 (HIF-1). The α-subunit of HIF-1 (HIF-1α) is a substrate for the ERK2 MAP kinase. Unraveling the interplay of these main signaling systems is a prerequisite for understanding their role in tumor growth, a situation sustained by simultaneous mitogenic and hypoxic signals. In this work, we investigated the molecular cues that direct HIF-1α recognition and phosphorylation by ERK2. We showed that HIF-1α possesses a MAPK docking domain. Utilizing surface plasmon resonance (SPR) methodologies we demonstrated efficient binding between HIF-1α and ERK2, with a K(D) value in the low micromolar range. Although, the D-domain did not contribute to the above interaction significantly, it could act in trans by recruiting ERK2 and conferring responsiveness to poor ERK substrates. These results indicate that, via its conserved D-domain, HIF-1α could serve as a platform for ERK2 in the nucleus of the cell, thus potentially facilitating phosphorylation of other ERK2 substrates. The identification of an ERK2 recognition domain on HIF-1α opens new avenues for the analysis of HIF-1α-related ERK2 signaling and may allow designing of interfering compounds.

Novel surface targets and serum biomarkers from the ovarian cancer vasculature.

The molecular phenotype of tumor vasculature is different from normal vasculature, offering new opportunities for diagnosis and therapy of cancer, but the identification of tumor-restricted targets remains a challenge. We investigated 13 tumor vascular markers (TVMs) from 50 candidates identified through expression profiling of ovarian cancer vascular cells and selected to be either transmembrane or secreted, and to be either absent or expressed at low levels in normal tissues while overexpressed in tumors, based on analysis of 1,110 normal and tumor tissues from publicly available Affymetrix microarray data. Tumor-specific expression of each TVM was confirmed at the protein level in tumor tissue and/or in serum. Among the 13 TVMs, 11 were expressed on tumor vascular endothelium; the remaining 2 TVMs were expressed by tumor leukocytes. Our results demonstrate that certain transmembrane TVMs such as ADAM12 and CDCP1 are selectively expressed in tumor vasculature and represent promising targets for vascular imaging or anti-vascular therapy of epithelial ovarian cancer, while secreted or shed molecules such as TNFRSF21/DR6 can function as serum biomarkers. We have identified novel tumor-specific vasculature markers which appear promising for cancer serum diagnostics, molecular imaging and/or therapeutic targeting applications and warrant further clinical development.

Screening of the DNA mismatch repair genes MLH1, MSH2 and MSH6 in a Greek cohort of Lynch syndrome suspected families.

BACKGROUND: Germline mutations in the DNA mismatch repair genes predispose to Lynch syndrome, thus conferring a high relative risk of colorectal and endometrial cancer. The MLH1, MSH2 and MSH6 mutational spectrum reported so far involves minor alterations scattered throughout their coding regions as well as large genomic rearrangements. Therefore, a combination of complete sequencing and a specialized technique for the detection of genomic rearrangements should be conducted during a proper DNA-testing procedure. Our main goal was to successfully identify Lynch syndrome families and determine the spectrum of MLH1, MSH2 and MSH6 mutations in Greek Lynch families in order to develop an efficient screening protocol for the Greek colorectal cancer patients' cohort. METHODS: Forty-two samples from twenty-four families, out of which twenty two of Greek, one of Cypriot and one of Serbian origin, were screened for the presence of germline mutations in the major mismatch repair genes through direct sequencing and MLPA. Families were selected upon Amsterdam criteria or revised Bethesda guidelines. RESULTS: Ten deleterious alterations were detected in twelve out of the twenty-four families subjected to genetic testing, thus our detection rate is 50%. Four of the pathogenic point mutations, namely two nonsense, one missense and one splice site change, are novel, whereas the detected genomic deletion encompassing exon 6 of the MLH1 gene has been described repeatedly in the LOVD database. The average age of onset for the development of both colorectal and endometrial cancer among mutation positive families is 43.2 years. CONCLUSION: The mutational spectrum of the MMR genes investigated as it has been shaped by our analysis is quite heterogeneous without any strong indication for the presence of a founder effect.