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Novel immunotherapeutic approaches revolutionize cancer prognosis but the role of innate immunity in chemoresistance remains poorly understood. For example, toll-like receptor (TLR) ligands in combination with anticancer treatments yielded unpredictable effects, either beneficial or deleterious. We hypothesized that the function of tumor-infiltrating immune cells depends on constitutive TLR variants. We characterized healthy donor T cells, B cells, monocytes and natural killer (NK) for functional polymorphism after transwell co-culture with chemosensitive (OVCAR5) vs. chemoresistant (SKOV3TR) ovarian tumor cells +/- (1) medium only; (2) LPS; (3) PamCys3; (4) Paclitaxel (PTX); (5) OVCAR5; (6) OVCAR5+PTX; (7) SKOV3TR; (8) SKOV3TR+PTX. CD8+ T cell activation was measured by perforin staining, monocyte and B cell maturations by cytokine expression array, and NK-mediated cytotoxicity by PI staining of GFP-transfected K562 after 4 hour-incubation. We observed minor functional variations between donor T cells, B cells and monocytes. However, while all NK cells killed GFP-K562 cells as expected, in some donors NK cytotoxicity was specifically inhibited by SKOV3RT co-culture. NK genotyping and phenotyping are ongoing including tumor-dependent NKG2D expression, and PTX-dependent MICA release by tumor cells. These results support the hypothesis that chemoresistant tumors release soluble factors that modulate NK function though specific interactions with innate immune receptor variants.

We describe here a novel method for generation of yeast-secreted, in vivo biotinylated recombinant antibodies, or biobodies. Biobodies are secreted by diploid yeast resulting from the fusion of two haploid yeast of opposite mating type. One yeast carries a cDNA encoding an antibody recognition sequence fused to an IgA1 hinge and a biotin acceptor site (BCCP) at the C-terminus; the other carries a cDNA encoding an E. coli biotin ligase (BirA) fused to KEX2 golgi-localization sequences, so that BirA can catalyze the biotin transfer to the recognition sequence-fused BCCP within the yeast secretory compartment. We illustrate this technology with biobodies against HE4, a biomarker for ovarian carcinoma. Anti-HE4 biobodies were derived from clones or pools of anti-HE4-specific yeast-display scFv, constituting respectively monoclonal (mBb) or polyclonal (pBb) biobodies. Anti-HE4 biobodies were secreted directly biotinylated thus bound to labeled-streptavidin and streptavidin-coated surfaces without Ni-purification. Anti-HE4 biobodies demonstrated specificity and sensitivity by ELISA assays, flow cytometry analysis and Western blots prior to any maturation; dissociation equilibrium constants as measured by surface plasmon resonance sensor were of K(d)=4.8 x 10(-9) M and K(d)=5.1 x 10(-9) M before and after Ni-purification respectively. Thus, yeast mating permits cost-effective generation of biotinylated recombinant antibodies of high affinity.

Preventing peritoneal implantation of ovarian carcinoma cells could prolong patient remission and survival. CA125 is expressed on most ovarian cancer cells and was reported to be a ligand of mesothelin, a peritoneal protein. We developed a cell adhesion assay with CA125-expresser ovarian cancer cells and human mesothelin-transfected cells and we confirmed that CA125 and mesothelin mediate cell attachment. We also showed that this assay supplies a high-throughput screening system for reagents able to block CA125/mesothelin-dependent cell attachment with a sensitive quantitative readout. We finally demonstrated that a mesothelin chimeric protein and anti-CA125 antibodies block CA125/mesothelin-dependent cell attachment.

DNA vaccines are ideally suited for immunizing against tumor antigens because constructs can be formulated that not only encode the tumor antigen but also encode molecules chosen to improve the ability to elicit an antitumor response. Ligands expressed on antigen-presenting cells associated with stimulating a robust T-cell response are excellent candidates for inclusion in a DNA vaccine. Mice transgenic for the HER-2/neu homologue, rat neu, were immunized with full-length rat neu cDNA given alone or in combination with plasmids encoding costimulatory molecules CD80 or CD86 and the ligand for CD137 (CD137L). Intradermal injection of the plasmid constructs resulted in both plasmid transcript and antigen protein expression being detected in lymph nodes draining the injection site. Immunization with plasmids encoding the neu antigen along with plasmids encoding CD137L and either CD80 or CD86 resulted in the generation of neu-specific antibodies that induced phopshorylation of the neu tyrosine kinase and inhibited the growth of cultured tumor cells overexpressing neu. Survival of animals was significantly prolonged after immunization with vaccines encoding neu together with the costimulatory molecules. Although tumors eventually occurred in the vaccinated animals, they were markedly infiltrated with CD4+ T cells. DNA vaccines encoding neu, when given in combination with both CD137L and either CD80 or CD86, can induce cellular and humoral immunity and result in an antitumor effect.

Lymphocytes from blood or tumors of patients with advanced cancer did not proliferate and produced very low levels of tumor necrosis factor and IFN-γ when cultured with autologous tumor cells. Proliferation and lymphokine production dramatically increased in the presence of beads conjugated with mAbs to CD3 plus mAbs to CD28 and/or CD40, and the lymphocytes destroyed the tumor cells. Expression density of CD3 concomitantly increased from low to normal levels. Furthermore, beads providing a CD3 signal (in combination with CD28 or CD28 plus CD40) gave partial protection against the inhibitory effect of transforming growth factor type β1 on lymphocyte proliferation and production of tumor necrosis factor and IFN-γ. MHC class I-restricted cytolytic T cells lysing autologous tumor cells in a 4-h Cr51release assay were generated when peripheral blood leukocytes were activated in the presence of autologous tumor cells and anti-CD3/CD28 or anti-CD3/CD28/CD40 beads. Experiments performed in a model system using anti-V-β1 or anti-V-β2 mAbs to activate subsets of T cells expressing restricted T cell receptor showed that lymphocytes previously activated by anti-V-β can respond to CD3 stimulation with vigorous proliferation and lymphokine production while retaining their specificity, also in the presence of transforming growth factor type β1. Our results suggest that T lymphocytes from cancer patients can proliferate and form Th1 type lymphokines in the presence of autologous tumor cell when properly activated, and that antigen released from killed tumor cells and presented by antigen-presenting cells in the cultures facilitates the selective expansion of tumor-directed, CD8+cytolytic T cells.

mAb OV569 was made by immunizing mice with ovarian carcinoma cells. It binds to cells from ovarian carcinomas and, to a lesser extent, to cells from certain other carcinomas whereas the binding to normal tissues is low to nondetectable. It also binds to soluble molecule(s) in culture supernatants from antigen-positive carcinomas. OV569 recognizes a protein(s) of 42–45 kDa with the same N-terminal amino acid sequence as the membrane-bound portion of mesothelin and megakaryocyte potentiating factor (MPF). Binding assays with fusion proteins comprising either the N-terminal part of mesothelin/MPF (D1Ig), reported to be easily cleaved off, or a noncleavable, membrane-associated part (D2Ig) showed that OV569 only binds to D2hIg. A new member of the mesothelin/MPF family was discovered, which has an 82-bp insert in the membrane-associated part, leading to a frameshift of 212 bp, and whose predicted molecular structure indicates that it is soluble. To test patient sera for soluble tumor antigen, antigen was isolated from cell-free tumor culture supernatants via immunoadsorption with OV569 and used to generate murine mAbs to an epitope different from the one to which OV569 binds, after which mAbs to two different epitopes were used to develop a “sandwich ELISA.” Using this assay, the level of circulating antigen was elevated significantly in 23 of 30 sera from patients with ovarian carcinoma, as compared with 0 of 68 sera from healthy controls, 0 of 3 sera from patients with nonneoplastic diseases, and 25 of 75 sera from patients with other tumors. Soluble molecules of the mesothelin/MPF family may provide useful new marker(s) for diagnosis of ovarian carcinoma and/or monitoring its response to therapy.

Adoptive transfer of TH17 cells polarized and expanded in vitro is an attractive therapy for the treatment of cancer. CD278, the inducible costimulator (ICOS) has been shown to be critical for the sustained expansion of human TH17 cells after their primary activation. We hypothesized that the incorporation of the ICOS intracellular domain in a CAR can promote the Th17 phenotype after antigen priming and enhance the antitumor activity of engineered T cell therapies. Th17 polarized cells were engineered to express a CAR comprised of a single-chain variable fragment that binds mesothelin fused to the T cell receptor- ζ signal transduction domain in tandem with the CD28, CD137 (4-1BB) or CD278 (ICOS) intracellular domains. Upon antigen recognition, Th17 cells redirected with the CAR:ICOS maintained a core molecular signature of Th17 cells, secreted higher amounts of IL17A, IL17F and CCL20 and expressed higher levels of CD161, NKG2D, IL1R-1 when compared with CD28 and 4-1BB CARs. When transferred into NSG mice with large tumors, Th17/Tc17 cells redirected with the CAR: ICOS mediated enhanced anti tumor responses and showed increased persistence. Our studies indicate that redirection of Th17 cells with a CAR encoding the ICOS intracellular domain is critical for obtaining potent Th17 cells with enhanced function and persistence. The design of novel ICOS-based CARs has the potential to augment antitumor effects in clinical trials.

Tumor polarization of macrophages represents an essential immune escape mechanism thus targeting tumor-associated macrophages (TAMs) could boost anti-tumor immune response. To model tumor-induced macrophage polarization, we developed an in vitro system of transwell co-culture with human macrophages, T cells and ovarian cancer cells (OVCAR3) where in vitro-matured macrophages and CD3/CD28 subactivated T cells were separated from tumor cells; in vitro-generated TAMs overexpressed the immunosuppressive molecule B7-H4 and inhibited T cell functions. We then hypothesized that targeting B7-H4 could block T cell inhibition mediated by B7-H4+ TAMs. We isolated anti-human B7-H4 scFvs from a novel yeast-display scFv library derived from B cells of ovarian cancer patients’ ascites. T cell proliferation and activation were restored in presence of anti-B7-H4 scFv #56 during transwell co-culture, suggesting that scFv #56 could block B7-H4 inhibitory signals mediated by TAMs. To further address whether scFv #56 activity was TCR-dependent, we set up experiments with antigen-specific T cells (MART1) and wild type or B7-H4-transduced artificial APCs (T2) loaded with MART1 peptide. T cell cytokine response was downregulated by B7-H4 expression on T2 and partially restored in presence of scFv#56. Our results indicate that competing with B7-H4 inhibitory signals on TAMs can promote T cell activity. We propose that in vivo use of anti-B7-H4 scFv could increase anti-tumor T-cell immunity.

Suitable tumor xenograft models represent a significant improvement to the understanding of ovarian cancer. However phenotype and function of tumor-associated lymphocytes and macrophages in different tumor sites has not been reported. We characterized the ovarian tumor microenvironment in NSG mice reconstituted with human immune system using cord blood-derived CD34+ cells. Mice were injected with OVcar5 human ovarian tumor cells orthotopically (io), subcutaneously (sc) or both (io/sc) 12-16 weeks post CD34-transplant. Our findings indicated that 4 weeks after implantation, all human tumors were infiltrated with B cells, T cells and macrophages, regardless of the tumor site. However, sc tumors contained proportionally more B cells and macrophages than io tumors. Macrophages infiltrating sc and io tumors exhibited an IL10high immunosuppresive phenotype. Io macrophages expressed higher levels of the Fc receptor of high affinity (FcgRI) and of low affinity (FcgRII) and mannose receptor compared to macrophages infiltrating sc or io/sc tumors. Finally, io tumors were infiltrated by more B cells than all other tumors. B cells from tumor-bearing mice presented an altered phenotype compared to non tumor-bearing animals, characterized by the upregulation of plasma and activation markers and IgM and IgG production. Our results suggested that the location of the tumor implantation impacts the phenotype and function of human tumor infiltrate and may influence the tumor development.

Anti-cancer treatments are often highly effective in human tumor xenografts grown subcutaneously in immune deficient hosts such as athymic (nude) or severe combined immune deficient (SCID) mice. However, such preclinical results are frequently followed by failure or limited activity of the drug/therapy in clinical trial, prompting the development of more sophisticated animal models engrafted with fully human immune system (HIS). We reconstituted NOD-Scid-IL2Rγcnull (NSG) mice with cord blood-derived CD34+ cells and 12 to 16 weeks after transplantation NSG-HIS mice were injected with human ovarian tumor cells (OVCAR5) orthotopically (io), subcutaneously (sc), or both (io/sc). Although sc tumor growth was the fastest in the absence of human immune system, four weeks after tumor implantation the average sizes of sc, io and sc/io tumors were similar across all NSG-HIS groups (0.3cm3); regardless of the tumor site, all tumors were infiltrated with human B cells, T cells and macrophages. However, sc tumors contained proportionally more human B cells and macrophages than io tumors. Macrophages infiltrating sc and io, but not sc or sc/io tumors, exhibited an IL10high immunosuppressive phenotype. Io tumor-infiltrating macrophages expressed the highest levels of CD32 (FcγRII) and mannose receptor (CD206), and the lowest level of CD64 (FcγRI) compared to macrophages infiltrating sc or io/sc tumors. Finally, io tumors were infiltrated with less human B cells than all the other tumors; B cells from tumor-bearing mice presented an altered phenotype compared to non tumor-bearing animals, characterized by the upregulation of plasma and activation markers and the production of IgM and IgG. Our results support the hypothesis that the location of the tumor implantation impacts the phenotype and function of human tumor cell infiltrate. The novel NSG-HIS mouse model of ovarian cancer may help understanding the role of tumor microenvironment during tumor development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-117. doi:1538-7445.AM2012-LB-117

Background and Purpose: There is a clear logic to adjuvant immunotherapy to enhance conventional treatment of ovarian cancer. Mesothelin (MSLN) is a surface glycoprotein expressed at high levels by ovarian cancers and is already a clinical target for immunotherapy. We have created a novel immunotherapeutic agent that links a single-chain antibody variable fragment (scFv) targeting MSLN to Mycobacterium tuberculosis (MTB) heat shock protein 70 (Hsp70), which is known as a potent immune activator. This fusion protein binds MSLN on tumor cells and activates antigen presenting cells (APCs) through the interaction of MTBhsp70 with CD40 on APCs, thus promoting tumor antigen presentation and cross presentation and adjuvanting antitumor specific CD4 and CD8 T cell responses. We tested the hypothesis that the MSLN targeted MTBhsp70 fusion protein would improve survival and increase antitumor immune responses. Experimental Procedures: The efficacy of the MSLN targeted fusion protein was evaluated in a syngeneic mouse model of papillary ovarian cancer in immune competent FVB/NJ mice. Mice received 4 intraperitoneal (ip) treatments with experimental or control proteins from 7 days post ip injection of BR5FVB ovarian cancer cells. Survival time was compared from the day of tumor inoculation to that of the onset of disease (clinically evident ascites). In immunological studies, mice were sacrificed 26 days after tumor cell inoculation. Splenocytes were stained for CD3, CD4, CD8, CD25 and Foxp3, and examined by flow cytometry. Splenocytes were stimulated with MSLN or Her2/neu peptide, or mitomycin C-treated BR5FVB cells. IFNα-generated CD3+CD8+ T cells and CD3+CD8+ T Cell-surface exposure of CD107a/b were detected by flow cytometry. To examine the role of CD8+ T cells in the antitumor effect, we performed in vivo antibody depletion experiments using standard methodologies. Results: MSLN targeted MTBhsp70 fusion protein significantly delayed the onset of disease compared to saline (p=0.0025) or an unfused mixture of the component proteins, MTBhsp70 and scFv(MSLN) (p=0.0149). The survival advantage of the fusion protein treatment was abrogated by CD8 T cell depletion in tumor bearing mice. The fusion protein also reduced the proportion of CD4+CD25+Foxp3 Treg cells (p=0.0159) and increased the proportion of CD3+CD8+ T cells (p=0.0079) in the spleen compared to controls. In preliminary studies, significantly greater CD8 T cell responses were demonstrated in the spleen from the majority of fusion protein-treated mice compared to controls. The MSLN targeted fusion protein was also shown to evoke dendritic cell activation as well as antigen presentation and cross presentation in vitro. Conclusion: MSLN targeted MTBhsp70 fusion protein treatment of ovarian cancer significantly enhances survival and slows tumor growth while augmenting anti-tumor antigen responses in mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1578. doi:1538-7445.AM2012-1578

Current therapies efficiently treat most patients with gynecologic malignancies detected at an early stage. Thus, the identification of oncology biomarkers for screening and monitoring of occult tumors has been highly prioritized. Hyperglycosylated human chorionic gonadotropin (hCG) epitomizes oncologic biomarker, as the serum level of this hormone is elevated in virtually all cases of gestational trophoblastic diseases. On the other hand, despite the availability of various markers such as CA125, CA19.9, CA15.3, CA72-4, Inhibin, beta-hCG, AFP, CEA and many more biomarkers under investigation, fewer than 25 % of all ovarian cancers are currently detected in stage I. Large efforts have been undertaken to further identify composite markers for gynecologic malignancies that may exhibit greater specificity when studied over time, as well as to develop risk models and screening algorithms aimed at improving the specificity and sensitivity of diagnostic tests. In this review, we provide a comprehensive analysis of the biomarkers currently used in clinics for gynecologic malignancies, as well as an outlook of the most promising oncologic biomarkers currently under study.

Ovarian cancer is the eighth most common cancer in women and represents the most lethal gynecological cancer, where less than a third of the women survive longer than five years after diagnosis. The identification of novel therapeutic strategies is urgently needed to improve patient prognosis. Solid and hematologic malignant cells express a broad range of Toll-like receptors (TLR). Activation of TLR signaling through recognition of pathogen-associated molecular patterns leads to the transcriptional activation of genes encoding for pro-inflammatory cytokines, chemokines and co-stimulatory molecules. Solid tumor development has been associated with TLR engagement through both growth acceleration and immune escape, and epidemiological studies reported a link between TLR expression by ovarian tumor cells and chemoresistance. We previously observed a large proportion of B cells in the immune infiltrate of mouse ovarian tumors and in human ascites from ovarian cancer patients, but the role of TLR stimulation in ovarian tumor-infiltrating lymphocyte B (TIL-B) remains unclear. The objective of this study is to investigate the functional consequences of TLR expression and engagement on TIL-B in ovarian cancer. Our work has been carried in a transgenic mouse model (TgMISIIR-Tag) that spontaneously develops tumors through the expression of SV40 T antigen under the Mullerian Inhibitor Receptor type II (MISIIR) promoter. TgMISIIR-Tag tumors resemble human serous ovarian cancer, the most common type of ovarian cancer. Microarrays and flow cytometry analyzes were used to characterize the phenotype of TIL-B after CD19 magnetic sorting ex vivo or after in vitro culture. We first found that TIL-B significantly over-expressed transcripts encoding for inflammatory cytokines, growth factors, adhesion molecules and chemokines, compared to B cells from spleens and lymph nodes of healthy or tumor bearing mice. We also showed that B cells from TgMISIIR-TAg tumors and from ascites of ovarian cancer patients over-expressed TLR2, TLR4 and/or TLR9, to compare with mouse splenic or human peripheral B cells, respectively. In addition, five days co-culture of TIL-B with ovarian tumor cells and TLR4 and TLR 9 agonists further increased TLR4 and TLR9 expression, as well as the expression of CD5 and CD1d costimulatory molecules and IL10 production. Concomitant treatment with TLR antagonists reduced TLR expression and IL-10 production of TIL-B. In vivo studies aiming to interrogate the role of TLR engagement on TIL-B in ovarian cancer are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-292. doi:10.1158/1538-7445.AM2011-LB-292

IL-10, an immunosuppressive cytokine, is found in the tumor environment. While it is appreciated that Toll-Like Receptors (TLRs) in combination with signals through the B cell receptor and CD40 can modulate IL-10 production by B cells, the role that TLR stimulation plays on tumor-infiltrating lymphocytes B (TIL-B) remains unclear. To study this, we used a transgenic mouse model of serous ovarian cancer (Tg-MISIIR-TAg) and ascites of ovarian cancer patients; in vivo experiments in an IL-10 reporter mouse model are ongoing. We found that early tumors contained TIL-B phenotypically similar to B220hi CD21/35 med IgMmed follicular B cells, as demonstrated by flow cytometry. As the tumor progressed, phenotype evolved from fresh isolated TIL-B with up-regulation of CD1d and CD5, and overexpression of IL-10 compared to splenic B cells. In addition, freshly harvested TIL-B from Tg-MISIIR-TAg tumors and frozen B cells from ascites of ovarian cancer patients expressed high levels of TLR4, TLR2 and TLR9. Finally, TIL-B cultured in presence of TLR agonists increased further IL-10 production compared with fresh harvested TIL-B or splenic B cells cultured in the same conditions. Taken together, our findings indicate a direct link between the increased TIL-B production of IL-10 and stimulation through TLRs. We propose that chronic exposure of B cells to the tumor microenvironment may enhance their regulatory-like phenotype and increase their receptiveness to TLR-driven IL-10 production.

The use of stem cell-derived human immune system NSG mice and orthotopic tumors to recapitulate the human ovarian tumor microenvironment represents a significant improvement over existing mouse models of cancer. However, the careful phenotyping of a human ovarian tumor microenvironment in NSG mice has not been reported. We characterized the human ovarian tumor microenvironment in NSG mice reconstituted with a full human immune system using cord blood-derived CD34+ cell transplantation, and injected with human ovarian tumor cells. Our findings demonstrated human B cell and T cell infiltration in both orthotopic and subcutaneous tumors. Orthotopic tumors showed a 3 to 4-fold increase of human CD3+ cell infiltration in comparison with subcutaneous tumors or spleens from tumor bearing mice, however, the overall CD4/CD8 T cell ratio was similar in both tumor sites. Tumor-infiltrating T cells included effector memory TEM cells and Treg cells. Orthotopic tumor dissociates also contained significantly more human macrophages (TAMs) than subcutaneous tumors or spleens from tumor bearing mice. While all macrophages were found to have an M1-like phenotype, TAM subsets could be distinguished from splenic macrophages by CD40 or HLA-DR expression and TNFβ secretion. Furthermore, TAMs phenotypic differences observed in orthotopic tumors were associated with faster tumor growth kinetics and metastasis. Finally, we found that, in addition to the murine origin of the tumor stroma, a significant number of mouse neutrophils and macrophages were also present. We propose that humanized mice bearing human tumors could be a valuable tool for the preclinical evaluation of targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-286. doi:10.1158/1538-7445.AM2011-LB-286

Tumor-associated macrophages (TAMs) share properties with M2 macrophages (MΦ),including high IL10 and mannose receptor (MR) expression. MR cross-linking differentiates DCs into APCs inducing T-cell anergy. However,although MR is abundantly expressed on M2 MΦ,its role on TAMs remains poorly understood. To address this question,we isolated 3 novel recombinant antibodies(scFv) against the human mannose binding domain of MR (CRD4-MR),and set up transwell cocultures allowing chemical exchanges between MΦ and ovarian cancer cells. MΦ phenotype was assessed by FACS,qPCR and bead-based FACS. We also challenged wild type,MR+/- and MR-/- mice with ovarian cancer. We first confirmed that MΦ coculture with tumor cells triggers a tumor associated phenotype and showed that 1 anti-CRD4-MR scFv could block the phenotype switch. We further demonstrated that tumor-released mesothelin (MSLN), but not GPI-anchor truncated MSLN,bound to MR-expressing MΦ,and that MSLN binding could be blocked by all 3 anti-CRD4-MR scFvs. Finally, although tumor challenged MR-/- mice did not survive significantly longer than the wild type,MR+/- mice exhibited lower tumor burden and prolonged survival. MR+/- ascites MΦ secreted significantly higher levels of TNF-a consistent with a pro-inflammatory phenotype. Altogether,tumor-released mesothelin binds to MR and blocking of CRD4-MR binding inhibits TAM phenotype and promotes MΦ with proinflammatory proprieties. Blocking MR function could have therapeutic potential.

Tumors polarize their microenvironment to escape immune-mediated destruction and to promote their growth. Macrophages respond to microenvironmental signals and can be activated toward a classical (M1) or an alternative (M2) phenotype. Tumor-associated macrophages (TAM) resemble to M2 macrophages with a phenotype characterized by the overexpression of IL10, scavenger receptor SR-A, CD68 and MR phenotype but hampered levels of IL-12. M2, in contrast to M1 macrophages, promote TH2 immune responses as well as tissue remodelling and M2 infiltration in solid tumors has been shown to significantly correlate with poor prognosis. Recent evidences suggest that macrophage differentiation towards TAM involves a “chemical conversation” via exchange of unknown soluble extracellular mediators between tumor cells and macrophages. In addition, previous work showed that the cross-linking mannose receptor (MR) with an anti-MR mAb could cause dendritic cell differentiation into APCs promoting T-cell anergy. We hypothesized that MR expressed by macrophages could be bound by tumor-released GPI-anchored glycoproteins through the GPI-anchor mannose residues freed by cleavage. We also hypothesized that MR binding by GPI-anchor could trigger macrophage polarization toward TAMs. To test our hypotheses, we established an in vitro model system of cell co-culture in transwell allowing chemical exchanges between human monocyte-derived macrophages and human ovarian cancer cell line Ovcar3 that sheds mesothelin, a cancer biomarker and a GPI-anchored glycoprotein. Macrophage phenotype changes were monitored by flow cytometry and qRT-PCR. We also isolated three novel recombinant antibodies (scFv) against MR by magnetic and flow sortings from a novel yeast-display human scFv library. We describe here for the first time that tumor-released mesothelin bound to macrophage cell surface in presence of calcium, and that mesothelin binding to macrophages could be blocked by the anti-MR scFv. Furthermore, macrophage phenotype switch toward M2 during coculture with tumor cells could be prevented by one of the anti-MR scFv. Finally, we demonstrated that M1 phenotype could be rescued during coculture with tumor cells with one of the anti-MR scFv. To further characterize the role of MR in vivo, we have implanted mannose receptor knock-out mice intraperitoneally (IP) with luminescent-labeled ovarian cancer cells that overexpress mesothelin. We are now monitoring the tumor growth by in vivo imaging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3809.

Despite extensive studies of the role of tumor-infiltrating lymphocytes-T and macrophages, the role of tumor-infiltrating lymphocytes-B (TIL-B) during cancer progression remains poorly understood. To address this issue, we characterized the phenotype and function of TIL-B in a transgenic mouse model of epithelial ovarian cancer that resembles human serous ovarian cancer. We first observed that CD45+ CD19+ TIL-B cells are one of first populations to infiltrate ovarian tumors and their percentages decreased over time, from 32±5% at 13 weeks to 16±3% at 26 weeks. Our microarrays results revealed that TIL-B transcripts segregated with normal splenocytes, and significantly up-regulated Angiopoietin2, platelet derived growth factor (PDGF), Midkine, Insulin-like growth factor binding proteins and Transforming growth factors beta. In addition, multiplex cytokine assays showed that TIL-B spontaneously secreted PDGF-bb, CXCL9, Leukemia inhibitory factor and Macrophage-colony stimulating factor. Finally, we found that TIL-B secreted the suppressive cytokine IL-10 and attracted endothelial cells in vitro. Our results demonstrated for the first time that TIL-B beared different functional capabilities than normal B cells, in particular TIL-B synthesized angiogenic factors and attracted endothelial cells. These results suggest that TIL-B could promote angiogenesis and thus represent an attractive therapeutic target for solid tumor therapy.

Chronic inflammation is now clearly established as a key player in the process of carcinogenesis. Complement factors can be activated under various chronic inflammatory conditions, suggesting that they could contribute in promoting tumor development. However, the role of complement in ovarian cancer (OC) progression remains poorly investigated. OC is the second most common gynecologic cancer and the fourth leading cause of cancer death in women. Developing alternative therapies is required to decrease OC high mortality rate. To determine the role of complement in cancer establishment and progression, we used genetic and pharmaceutical approaches. We first crossed complement component 3 knockout (C3KO) mice with transgenic mice expressing the Simian Virus 40 TAg under the control of the Müllerian Inhibiting Substance type II Receptor promoter (Tg-MISIIR-TAg). Tg-MISIIR-TAg mice develop bilateral OC resembling human serous OC with ∼100% penetrance. We found that C3KO/MISIIR-TAg mice developed no or very small tumors at 16 weeks of age compared to control wild-type C3/MISIIR-TAg and C3/MISIIR-TAg heterozygote littermates. We also found that tumor-infiltrating leukocytes were functionally altered with changes of secretion profiles for cytokines and angiogenic factors. Second, we orthotopically implanted fluorescent ovarian carcinoma cells (MOV1Kat) in Tg-MISIIR-TAg mice treated with complement C5aR inhibitor peptide or control peptide. MOV1 cell line was isolated from Tg-MISIIR-Tag tumor and transducted with a Far-red fluorescent protein, Katushka, using TurboFP635 Lentivirus mammalian vector to permit in vivo optical imaging (IVIS). Our results showed that the pharmacological inhibition of C5a-binding to C5aR receptor promoted tumor regression. Altogether, our results indicated an important and understudied role of complement for ovarian cancer. We propose that complement inhibitors could be used as therapeutic agents for the treatment of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-163.

In our previous study we showed that scFv against tumor vasculature marker could be isolated from a yeast-display scFv library derived from an autoimmune patient (TTP). However, scFv library derived from cancer patients may yield more diverse and higher affinity reagents against tumor-associated markers. To address this question, we constructed a yeast display scFv library from PBMC and ascites B cells of 5 ovarian cancer (OC) patients. To clone the antibody repertoires into the yeast display vector, we designed a new set of primers capable of amplifying all antibody sub-families and we optimized a three-fragment gap repair method using VH, VL and linearized vector. While scFv assemblage by overlap PCR was not performing well, the recombination of VH, VL and linearized vector in yeast yielded 1x108 transformant per electroporation. Sequencing of 50 clones demonstrated that all of them contained different full-length scFv inserts. We are currently screening the OC scFv library with human TEM1 protein, to compare the frequency and affinity of the scFv against tumor markers when retrieved from OC scFv library vs. TTP scFv library. In conclusion, our findings so far indicate that three-fragment gap repair is a valid and cost-effective strategy to construct large yeast-display libraries. We anticipate that the comparative screening of the TTP and OC libraries using tumor markers may give indications about the repertoire and anti-tumoral specificity of cancer patient antibodies.

Objective Maternal morbidity and/or mortality (MM) is increased in pyelonephritis and influenza. Alterations in the immune response could account for the increase MM. We sought to determine whether the immune response is functionally different during pregnant and nonpregnant (NP) states. Study Design Mouse model of systemic and localized inflammation was used. Maternal serum was assessed for expression of T-helper cell type 1 and 2 cytokines. Maternal spleens were harvested for immunohistochemistry. Results Systemic administration of lipopolysaccharides resulted in no mortality to NP mice compared with 88% in preterm and 100% in term mice. A potent cytokine response was present in both NP and pregnancy. Systemic inflammation in pregnancy results in increased CD8 and CD11c expression in spleens. Conclusion Differences in cytokine response to systemic inflammation is unlikley to modulate the increased MM during pregnancy. Altered T-cell and dendritic cell responses in pregnancy may be responsible for the increase in MM.

The complement system plays a crucial role in inflammation, which is known to contribute to carcinogenesis and tumor progression. However, the exact role of complement in cancer development remains poorly understood. We investigated the contribution of complement to the development of ovarian carcinoma (OC). OC is the second most common gynecologic cancer and the fourth leading cause of cancer death in women. We assessed the influence of complement on angiogenesis and tumor microenvironment by crossing complement component 3 knockout (C3KO) with transgenic mice expressing the Simian Virus 40 TAg under the control of the Müllerian Inhibiting Substance type II Receptor promoter (MISIIR-TAg). MISIIR-TAg mice develop bilateral OC resembling human serous OC with ~100% penetrance. We found that C3KO/MISIIR-TAg mice exhibited reduced tumor volume and microvessel formation compared to control wild-type C3 and C3 heterozygote (C3HET) littermates. In addition, we found that tumor-infiltrating macrophages, B cells and T lymphocytes from C3KO/MISIIR-TAg mice produced significantly less cytokines after ex-vivo culture. Furthermore, the number of CD3+/CD4+/FoxP3+ T-cells was reduced in these mice. These results support the hypothesis that C3 plays an important role in tumor progression and vascularization, and that complement deficiency might compromise tumor-infiltrating leukocyte functions. This work was supported by P01 AI 068730 (NIH/NCI)

TEM1 is predominantly expressed on neovasculature, thus ideally suited for targeted imaging and therapy for cancer. Yeast has emerged as an alternative technology for display and selection of recombinant antibody fragments (scFv). Because autoimmune disorders may produce self-reactive specificities, an autoimmune repertoire derived from a patient with thrombotic thrombocytopenic purpura (TTP) was screened to isolate anti-TEM1 scFv. A yeast-display scFv library was constructed by homologous recombination of the TTP patient repertoire originally expressed on M13 bacteriophage. The library (10^9 members) was flow sorted with decreasing antigen concentration until a scFv pool detecting 2 nM of rhTEM1/GST was obtained. Secreted scFv were expressed and 470 scFv were screened by ELISA for binding to rhTEM1/GST. Nearly 50% of the scFv gave colorimetric signals higher than 3 standard deviations above background. No cross-reactivity with GST control protein was detected. Sequencing of 29 scFv identified 8 unique antibodies. Two of these clones exhibited Kd's in the 100 pM range and bound to both mouse and human TEM1. Such cross-reactive anti-mouse/human TEM will facilitate proof of concept for the development of novel anti-angiogenic targeted therapies. Our data suggest that the use of large scFv libraries derived from autoimmune patient repertoires may provide a rich source of high affinity antibody reagents to therapeutically relevant molecules. This work was supported by the Claneil Foundation

Background Glypican-3 (GPC3) is a heparan-sulfate proteoglycan frequently expressed on the cell membrane of malignant hepatocytes in hepatocellular carcinoma. The capacity for screening potential antibodies in vitro using human hepatocellular lines is critical to ensure binding to this highly post-translationally modified glycophosphatidylinositiol-linked protein. We hypothesized that we could utilize a recently described paired display/secretory yeast library to isolate human-derived scFv against glypican-3 for potential diagnostic and/or therapeutic application. Results Using two different biotinylated antigen targets, a synthesized 29mer fragment GPC3550-558 and a truncated GPC3368-548 fused with glutathione S-transferase (GST) we enriched the yeast display library to greater than 30% target-specific yeast with both positive selection and depletion of streptavidin- and GST-specific clones. After cloning of scFv cDNA from the enriched sub-library, scFv specificity was validated by ELISA for binding to recombinant protein from prokaryotic and eukaryotic sources and ultimately naturally presented human protein on the cell membrane of human hepatocellular cell lines. Specificity was confirmed using non-expressing cell lines and shRNA knockdown. Ultimately, five unique scFv with affinity EC50 ranging from 5.0-110.9nM were identified. Conclusions Using a paired display/secretory yeast library, five novel and unique scFvs for potential humoral or chimeric therapeutic development in human hepatocellular carcinoma were isolated and characterized.