Sara Cingarlini

Derangement of immune responses by myeloid suppressor cells

In tumor-bearing mice and cancer patients, tumor progression is often associated with altered hematopoiesis leading to the accumulation of myeloid cells. Extensive studies in preclinical models indicate that these cells share the CD11b and the Gr-1 markers, possess a mixed mature-immature myeloid phenotype, and are responsible for the induction of T-cell dysfunctions, both tumor-specific and nonspecific. Moreover, CD11b+Gr-1+ myeloid cells are described under different unrelated situations associated with temporary impairment of the T-lymphocyte reactivity. This review examines recent findings on the nature, properties, and mechanisms of action of these myeloid suppressor cells (MSCs).

Leukocyte infiltration in cancer creates an unfavorable environment for antitumor immune responses: a novel target for therapeutic intervention.

The interaction between tumor cells and the nearby environment is being actively investigated to explore how this interplay affects the initiation and progression of cancer. Host-tumor relationship results in the production of pro-inflammatory cytokines and chemokines that promote the recruitment of leukocytes within and around developing neoplasms. Cancer cells, together with newly recruited tumor-infiltrating cells, can also activate fibroblast and vascular responses, thus resulting in a chronic microenvironment perturbation. In this complex scenario, interactions between innate and adaptive immune cells can be disturbed, leading to a failure of immune-mediated tumor recognition and destruction. On the basis of the recent awareness about tumor promotion and immune deregulation by immune/inflammatory cells, novel anti-cancer strategies can be exploited.

Effective Genetic Vaccination with a Widely Shared Endogenous Retroviral Tumor Antigen Requires CD40 Stimulation during Tumor Rejection Phase

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8+, but not CD4+, T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8+ T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8+ T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.

Therapeutic Effectiveness of Recombinant Cancer Vaccines Is Associated with a Prevalent T-Cell Receptor α Usage by Melanoma-specific CD8+ T Lymphocytes

Definition of immune variables that correlate with the antitumor activity of cancer vaccines is critical for monitoring immunotherapy protocols. To define surrogate end points predictive of the therapeutic efficacy of recombinant vaccines based on melanoma antigen tyrosinase-related protein (TRP)-2, we evaluated several properties of antigen-specific CD8+ T lymphocytes in single mice undergoing either prophylactic or therapeutic immunization. Predictive markers for the efficacy of genetic vaccination were identified in the prophylactic model used. Interestingly, the number of tetramer+ CD8+ T lymphocytes expanded in vitro after a single cycle of stimulation with the immunodominant TRP-2 peptide was of the highest predictive value. In the therapeutic model, no variable examined at a single mouse level predicted the long-term therapeutic effect. Mice that survived did not show the highest expansion of antigen-specific lymphocytes or the more functionally active effectors, ex vivo or after in vitro culture with the peptide antigen. Successful therapy correlated strictly with the skewing of the T-cell receptor repertoire of tetramer-sorted, TRP-2–specific CD8+ T lymphocytes, which showed a preferential α chain usage with a common CDR3 region.

PSMA-Specific CAR-Engineered T Cells Eradicate Disseminated Prostate Cancer in Preclinical Models

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affinity specific mAb. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter capable of robust and coordinated expression of the CAR molecule, and a bioluminescent reporter gene to allow the tracking of transgenic T cells after in vivo adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC, which in turn were readily stimulated to produce cytokines and to exert a relevant cytotoxic activity by engagement with PSMA+ prostate tumor cells. Upon in vivo transfer in tumor-bearing mice, CAR-transduced T cells were capable to completely eradicate a disseminated neoplasia in the majority of treated animals, thus supporting the translation of such approach in the clinical setting.

Apical Transport and Folding of Prostate-specific Membrane Antigen Occurs Independent of Glycan Processing

Prostate-specific membrane antigen (PSMA) is an integral cell-surface membrane glycoprotein that is overexpressed in prostate carcinomas rendering it an appropriate target for antibody-based therapeutic strategies. The biosynthesis of PSMA in transfected COS-1 cells reveals a slow conversion of mannose-rich to complex glycosylated PSMA compatible with slow transport kinetics from the endoplasmic reticulum to the Golgi. Importantly, mannose-rich PSMA persists as a trypsin-sensitive protein throughout its entire life cycle, and only Golgi-located PSMA glycoforms acquire trypsin resistance. This resistance, used here as a tool to examine correct folding, does not depend on the type of glycosylation, because different PSMA glycoforms generated in the presence of inhibitors of carbohydrate processing in the Golgi are also trypsin resistant. The conformational transition of PSMA to a correctly folded molecule is likely to occur in the Golgi and does not implicate ER molecular chaperones, such as BiP. We show here that PSMA is not only heavily N-but also O-glycosylated. The question arising is whether glycans, which do not play a role in folding of PSMA, are implicated in its transport to the cell surface. Neither the cell-surface expression of PSMA nor its efficient apical sorting in polarized Madin-Darby canine kidney cells are influenced by modulators of N- and O-glycosylation. The acquisition of folding determinants in the Golgi, therefore, is an essential prerequisite for protein trafficking and sorting of PSMA and suggests that altered or aberrant glycosylation often occurring during tumorigenesis has no regulatory effect on the cell-surface expression of PSMA.

Abstract LB-256: Impact of metformin on progression-free survival in diabetic patients with advanced pancreatic neuroendocrine tumors (pNETs) receiving everolimus and/or somatostatin analogues: A sensitivity analysis of the PRIME-NET (pancreatic multicentric, retrospective, italian metformin) study

Introduction: Several studies have correlated the diabetic status with increased cancer risk and worse cancer prognosis, while metformin (MET) use is associated with a better prognosis. MET could display antitumor effects by modifying systemic metabolism, e.g. by decreasing of blood glucose, insulin and IGF1 levels, or by affecting cancer cell metabolism and proliferation, e.g. through AMPK activation and inhibition of protein/lipid synthesis. Preliminary findings of the PRIME-net retrospective study, conducted on 445 Italian pts, suggested that the addition of MET to EVE and/or SSAs provides clinical benefit in diabetic patient with advanced pancreatic NETs. Methods: To exclude the possibility that the “time-on-treatment bias” could affect our results, with the risk that an early interruption of EVE or SSA-therapy due to early disease progression may result in lower pt exposure to these drugs and a consequently lower incidence of diabetes in poor responders, a sensitivity analysis on the PRIME-net study population was performed. The analysis considered only diabetics at baseline (BD), thus excluding pts who developed on-treatment diabetes as an adverse event (AE). All statistical tests were two-tailed and p-values Results: Out of 445 pts, 237 were diabetics. Of them, 179 had baseline diabetes (BD) while 57 developed on-treatment diabetes as an adverse event. Among pts with BD, 80 (44.7%) received MET, while 99 (55.3%) were not treated with MET, but received other treatments including insulin. mPFS was 24.7 mo in pts with BD and 15.1 mo in normoglycemic ones (HR 0.70, 95%CI 0.55-0.91; p=0.007). In pts on MET therapy, mPFS was 43.7 mo (HR vs normoglycemic pts 0.52, 95%CI 0.36-0.76, p=0.0006), while it was 20.8 mo in pts not receiving MET (HR vs normoglycemic pts 0.87, 95%CI 0.65-1.17, p=0.37). Conclusions: This sensitivity analysis confirms results emerged from the main analysis, thus demonstrating a mPFS advantage in diabetic patients with advanced pNETs patients receiving MET in combination with EVE and/or SSAs. Prospective investigations are ongoing to test the antitumor activity of MET in combination with everolimus and SSAs in normoglycaemic pts with advanced pNETs. Protocol number INT 85/15, approved by Ethical committee of fondazione IRCCS Istituto Tumori Milano on 15 June 2015. Citation Format: Sara Pusceddu, Claudio Vernieri, Massimo Di Maio, Femia Daniela, Natalie Prinzi, Riccardo Marconcini, Francesca Spada, Sara Massironi, Alberto Bongiovanni, Maria Pia Brizzi, Davide Campana, Antongiulio Faggiano, Dario Giuffrida, Gianfranco Delle Fave, Sara Cingarlini, Francesca Aroldi, Lorenzo Antonuzzo, Rossana Berardi, Laura Catena, Roberto Buzzoni, Filippo de Braud. Impact of metformin on progression-free survival in diabetic patients with advanced pancreatic neuroendocrine tumors (pNETs) receiving everolimus and/or somatostatin analogues: A sensitivity analysis of the PRIME-NET (pancreatic multicentric, retrospective, italian metformin) study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-256. doi:10.1158/1538-7445.AM2017-LB-256