Tumor Regression and Delayed Onset Toxicity Following B7-H4 CAR T Cell Therapy.

B7-H4 protein is frequently overexpressed in ovarian cancer. Here, we engineered T cells with novel B7-H4-specific chimeric antigen receptors (CARs) that recognized both human and murine B7-H4 to test the hypothesis that B7-H4 CAR T cell therapy can be applied safely in preclinical models. B7-H4 CAR T cells specifically secreted IFN-γ and lysed B7-H4(+) targets. In vivo, B7-H4 CAR T cells displayed antitumor reactivity against B7-H4(+) human ovarian tumor xenografts. Unexpectedly, B7-H4 CAR T cell treatment reproducibly showed delayed, lethal toxicity 6-8 weeks after therapy. Comprehensive assessment of murine B7-H4 protein distribution uncovered expression in ductal and mucosal epithelial cells in normal tissues. Postmortem analysis revealed the presence of widespread histologic lesions that correlated with B7-H4(+) expression, and were inconsistent with graft versus host disease. Lastly, expression patterns of B7-H4 protein in normal human tissue were comparable to distribution in mice, advancing our understanding of B7-H4. We conclude that B7-H4 CAR therapy mediates control of cancer outgrowth. However, long-term engraftment of B7-H4 CAR T cells mediates lethal, off-tumor toxicity that is likely due to wide expression of B7-H4 in healthy mouse organs. This model system provides a unique opportunity for preclinical evaluation of safety approaches that limit CAR-mediated toxicity after tumor destruction in vivo.

Optimized gene engineering of murine CAR-T cells reveals the beneficial effects of IL-15 coexpression.

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature-targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic.

Unsupervised Analysis of Flow Cytometry Data in a Clinical Setting Captures Cell Diversity and Allows Population Discovery.

Data obtained with cytometry are increasingly complex and their interrogation impacts the type and quality of knowledge gained. Conventional supervised analyses are limited to pre-defined cell populations and do not exploit the full potential of data. Here, in the context of a clinical trial of cancer patients treated with radiotherapy, we performed longitudinal flow cytometry analyses to identify multiple distinct cell populations in circulating whole blood. We cross-compared the results from state-of-the-art recommended supervised analyses with results from MegaClust, a high-performance data-driven clustering algorithm allowing fast and robust identification of cell-type populations. Ten distinct cell populations were accurately identified by supervised analyses, including main T, B, dendritic cell (DC), natural killer (NK) and monocytes subsets. While all ten subsets were also identified with MegaClust, additional cell populations were revealed (e.g. CD4+HLA-DR+ and NKT-like subsets), and DC profiling was enriched by the assignment of additional subset-specific markers. Comparison between transcriptomic profiles of purified DC populations and publicly available datasets confirmed the accuracy of the unsupervised clustering algorithm and demonstrated its potential to identify rare and scarcely described cell subsets. Our observations show that data-driven analyses of cytometry data significantly enrich the amount and quality of knowledge gained, representing an important step in refining the characterization of immune responses.

Rapid tumor vaccine using Toll-like receptor-activated ovarian cancer ascites monocytes.

BACKGROUND: Novel therapeutic strategies in ovarian cancer (OC) are needed as the survival rate remains dismally low. Although dendritic cell-based cancer vaccines are effective in eliciting therapeutic responses, their complex and costly manufacturing process hampers their full clinical utility outside specialized clinics. Here, we describe a novel approach of generating a rapid and effective cancer vaccine using ascites-derived monocytes for treating OC. METHODS: Using the ID8 mouse ovarian tumor model and OC patient samples, we isolated ascites monocytes and evaluated them with flow cytometry, Luminex cytokine and chemokine array analysis, ex vivo cocultures with T cells, in vivo tumor challenge and T cell transfer experiments, RNA-sequencing and mass spectrometry. RESULTS: We demonstrated the feasibility of isolating ascites monocytes and restoring their ability to function as bona fide antigen-presenting cells (APCs) with Toll-like receptor (TLR) 4 lipopolysaccharide and TLR9 CpG-oligonucleotides, and a blocking antibody to interleukin-10 receptor (IL-10R Ab) in the ID8 model. The ascites monocytes were laden with tumor antigens at a steady state in vivo. After a short 48 hours activation, they upregulated maturation markers (CD80, CD86 and MHC class I) and demonstrated strong ex vivo T cell stimulatory potential and effectively suppressed tumor and malignant ascites in vivo. They also induced protective long-term T cell memory responses. To evaluate the translational potential of this approach, we isolated ascites monocytes from stage III/IV chemotherapy-naïve OC patients. Similarly, the human ascites monocytes presented tumor-associated antigens (TAAs), including MUC1, ERBB2, mesothelin, MAGE, PRAME, GPC3, PMEL and TP53 at a steady state. After a 48-hour treatment with TLR4 and IL-10R Ab, they efficiently stimulated oligoclonal tumor-associated lymphocytes (TALs) with strong reactivity against TAAs. Importantly, the activated ascites monocytes retained their ability to activate TALs in the presence of ascitic fluid. CONCLUSIONS: Ascites monocytes are naturally loaded with tumor antigen and can perform as potent APCs following short ex vivo activation. This novel ascites APC vaccine can be rapidly prepared in 48 hours with a straightforward and affordable manufacturing process, and would be an attractive therapeutic vaccine for OC.

Tumor Landscapes: ß-Catenin Drives Immune Desertification.

Immune checkpoint blockade therapy requires a preestablished activated immune landscape. Understanding tumor-intrinsic mechanisms that lead to T-cell desertification is key to resensitizing them to such therapies. The WNT/ß-catenin tumor-intrinsic signaling is emerging as an immune exclusion pathway that holds high promise to counteract resistance to immunotherapy.See related article by Luke et al., p. 3074.

Cooperation between Constitutive and Inducible Chemokines Enables T Cell Engraftment and Immune Attack in Solid Tumors.

We investigated the role of chemokines in regulating T cell accumulation in solid tumors. CCL5 and CXCL9 overexpression was associated with CD8+ T cell infiltration in solid tumors. T cell infiltration required tumor cell-derived CCL5 and was amplified by IFN-γ-inducible, myeloid cell-secreted CXCL9. CCL5 and CXCL9 coexpression revealed immunoreactive tumors with prolonged survival and response to checkpoint blockade. Loss of CCL5 expression in human tumors was associated with epigenetic silencing through DNA methylation. Reduction of CCL5 expression caused tumor-infiltrating lymphocyte (TIL) desertification, whereas forced CCL5 expression prevented Cxcl9 expression and TILs loss, and attenuated tumor growth in mice through IFN-γ. The cooperation between tumor-derived CCL5 and IFN-γ-inducible CXCR3 ligands secreted by myeloid cells is key for orchestrating T cell infiltration in immunoreactive and immunoresponsive tumors.

Reactive oxygen species and HIF-1 signalling in cancer.

The heterodimeric transcription factor HIF-1 (hypoxia-inducible factor 1) represents the key mediator of hypoxia response. HIF-1 controls numerous genes of pivotal importance for cellular metabolism, angiogenesis, cell cycle regulation and inhibition of apoptosis. HIF-1 overexpression and enhanced transcriptional activity are linked to tumour initiation and progression. Malfunction of the HIF-1 signalling network has been associated with breast, ovarian and prostate cancers. Elevated reactive oxygen species (ROS), also observed in such tumours, have been implicated in HIF-1 signalling. Deciphering the role of ROS in cancer onset and their involvement in signalling networks should prove invaluable for the design of novel anticancer therapeutics.

Sensitive and frequent identification of high avidity neo-epitope specific CD8 + T cells in immunotherapy-naive ovarian cancer.

Immunotherapy directed against private tumor neo-antigens derived from non-synonymous somatic mutations is a promising strategy of personalized cancer immunotherapy. However, feasibility in low mutational load tumor types remains unknown. Comprehensive and deep analysis of circulating and tumor-infiltrating lymphocytes (TILs) for neo-epitope specific CD8+ T cells has allowed prompt identification of oligoclonal and polyfunctional such cells from most immunotherapy-naive patients with advanced epithelial ovarian cancer studied. Neo-epitope recognition is discordant between circulating T cells and TILs, and is more likely to be found among TILs, which display higher functional avidity and unique TCRs with higher predicted affinity than their blood counterparts. Our results imply that identification of neo-epitope specific CD8+ T cells is achievable even in tumors with relatively low number of somatic mutations, and neo-epitope validation in TILs extends opportunities for mutanome-based personalized immunotherapies to such tumors.

Personalized cancer vaccine effectively mobilizes antitumor T cell immunity in ovarian cancer.

We conducted a pilot clinical trial testing a personalized vaccine generated by autologous dendritic cells (DCs) pulsed with oxidized autologous whole-tumor cell lysate (OCDC), which was injected intranodally in platinum-treated, immunotherapy-naïve, recurrent ovarian cancer patients. OCDC was administered alone (cohort 1, n = 5), in combination with bevacizumab (cohort 2, n = 10), or bevacizumab plus low-dose intravenous cyclophosphamide (cohort 3, n = 10) until disease progression or vaccine exhaustion. A total of 392 vaccine doses were administered without serious adverse events. Vaccination induced T cell responses to autologous tumor antigen, which were associated with significantly prolonged survival. Vaccination also amplified T cell responses against mutated neoepitopes derived from nonsynonymous somatic tumor mutations, and this included priming of T cells against previously unrecognized neoepitopes, as well as novel T cell clones of markedly higher avidity against previously recognized neoepitopes. We conclude that the use of oxidized whole-tumor lysate DC vaccine is safe and effective in eliciting a broad antitumor immunity, including private neoantigens, and warrants further clinical testing.

Ovarian cancer chemokines may not be a significant barrier during whole tumor antigen dendritic-cell vaccine and adoptive T-cell immunotherapy.

Tumor barriers preventing T-cell homing and engraftment should be neutralized during cancer immunotherapy. We recently discovered that ovarian cancer expresses quasi-universal chemokines that can support T-cell homing. Furthermore, T cells elicited by whole tumor antigen dendritic-cell vaccines express cognate chemokine receptors which are upregulated by CD3/CD28 costimulation.

Isolation and Validation of Anti-B7-H4 scFvs from an Ovarian Cancer scFv Yeast-Display Library.

B7-H4 (VTCN1, B7x, B7s) is an inhibitory modulator of T-cell response implicated in antigen tolerization. As such, B7-H4 is an immune checkpoint of potential therapeutic interest. To generate anti-B7-H4 targeting reagents, we isolated antibodies by differential cell screening of a yeast-display library of recombinant antibodies (scFvs) derived from ovarian cancer patients and we screened for functional scFvs capable to interfere with B7-H4-mediated inhibition of antitumor responses. We found one antibody binding to B7-H4 that could restore antitumor T cell responses. This chapter gives an overview of the methods we developed to isolate a functional anti-B7-H4 antibody fragment.

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.

The Ovarian Cancer Chemokine Landscape Is Conducive to Homing of Vaccine-Primed and CD3/CD28-Costimulated T Cells Prepared for Adoptive Therapy.

PURPOSE: Chemokines are implicated in T-cell trafficking. We mapped the chemokine landscape in advanced stage ovarian cancer and characterized the expression of cognate receptors in autologous dendritic cell (DC)-vaccine primed T cells in the context of cell-based immunotherapy. EXPERIMENTAL DESIGN: The expression of all known human chemokines in patients with primary ovarian cancer was analyzed on two independent microarray datasets and validated on tissue microarray. Peripheral blood T cells from five HLA-A2 patients with recurrent ovarian cancer, who previously received autologous tumor DC vaccine, underwent CD3/CD28 costimulation and expansion ex vivo. Tumor-specific T cells were identified by HER2/neu pentamer staining and were evaluated for the expression and functionality of chemokine receptors important for homing to ovarian cancer. RESULTS: The chemokine landscape of ovarian cancer is heterogeneous with high expression of known lymphocyte-recruiting chemokines (CCL2, CCL4, and CCL5) in tumors with intraepithelial T cells, whereas CXCL10, CXCL12, and CXCL16 are expressed quasi-universally, including in tumors lacking tumor-infiltrating T cells. DC-vaccine primed T cells were found to express the cognate receptors for the above chemokines. Ex vivo CD3/CD28 costimulation and expansion of vaccine-primed Tcells upregulated CXCR3 and CXCR4, and enhanced their migration toward universally expressed chemokines in ovarian cancer. CONCLUSIONS: DC-primed tumor-specific T cells are armed with the appropriate receptors to migrate toward universal ovarian cancer chemokines, and these receptors are further upregulated by ex vivo CD3/CD28 costimulation, which render T cells more fit for migrating toward these chemokines. Clin Cancer Res; 21(12); 2840-50. ©2015 AACR.

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.

A human ErbB2-specific T-cell receptor confers potent antitumor effector functions in genetically engineered primary cytotoxic lymphocytes.

The ErbB2 protein is a member of the tyrosine kinase family of growth factor receptors that is overexpressed in cancers of the breast, ovary, stomach, kidney, colon, and lung, and therefore represents an attractive candidate antigen for targeted cancer immunotherapy. Cytotoxic T lymphocytes specific for various immunogenic ErbB2 peptides have been described, but they often exhibit both poor functional avidity and tumor reactivity. In order to generate potent CD8(+) T cells with specificity for the ErbB2(369-377) peptide, we performed one round of in vitro peptide stimulation of CD8(+) T cells isolated from an HLA-A2(+) patient who was previously vaccinated with autologous dendritic cells pulsed with HLA class I ErbB2 peptides. Using this approach, we enriched highly avid ErbB2-reactive T cells with strong ErbB2-specific, antitumor effector functions. We then stimulated these ErbB2-reactive T cells with ErbB2(+) HLA-A2(+) tumor cells in vitro and sorted tumor-activated ErbB2(369-377) peptide T cells, which allowed for the isolation of a novel T-cell receptor (TCR) with ErbB2(369-377) peptide specificity. Primary human CD8(+) T cells genetically modified to express this ErbB2-specific TCR specifically bound ErbB2(369-377) peptide containing HLA-A2 tetramers, and efficiently recognized target cells pulsed with low nanomolar concentrations of ErbB2(369-377) peptide as well as nonpulsed ErbB2(+) HLA-A2(+) tumor cell lines in vitro. In a novel xenograft model, ErbB2-redirected T cells also significantly delayed progression of ErbB2(+) HLA-A2(+) human tumor in vivo. Together, these results support the notion that redirection of normal T-cell specificity by TCR gene transfer can have potential applications in the adoptive immunotherapy of ErbB2-expressing malignancies.

Blocking the B7-H4 pathway with novel recombinant antibodies enhances T cell-mediated antitumor responses.

B7-H4 inhibits T-cell activation and is widely expressed by solid neoplasms. We have recently demonstrated that the expression of B7-H4 on the surface of malignant cells in vivo is inducible, and that novel anti-B7-H4 recombinant antibodies can reverse the inhibition of tumor-specific T cells. Thus, antibodies targeting the B7-H4 pathways may extend the survival of cancer patients by restoring T cell-mediated antitumor responses.

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.

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.

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.