Mechanism of Action of the Tumor Vessel Targeting Agent NGR-hTNF: Role of Both NGR Peptide and hTNF in Cell Binding and Signaling.

NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug's activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.

Molecular dynamics reveal that isoDGR-containing cyclopeptides are true αvß3 antagonists unable to promote integrin allostery and activation.

Ain't got that swing(-out): The cyclopeptide isoDGR is emerging as a new αvß3 integrin binding motif. Agreement between the results of computational and biochemical studies reveals that isoDGR-containing cyclopeptides are true αvß3 integrin antagonists that block αvß3 in its inactive conformation (see scheme). isoDGR-based ligands may give αvß3 antagonists without paradoxical effects.

Characterization and Functional Analysis of CD44v6.CAR T Cells Endowed with a New Low-Affinity Nerve Growth Factor Receptor-Based Spacer.

Effectiveness of adoptively transferred chimeric antigen receptor (CAR) T cells strongly depends on the quality of CAR-mediated interaction of the effector cells with the target antigen on tumor cells. A major role in this interaction is played by the affinity of the single-chain variable fragment (scFv) for the antigen, and by the CAR design. In particular, the spacer domain may impact on the CAR T cell function by affecting the length and flexibility of the resulting CAR. This study addresses the need to improve the manufacturing process and the antitumor activity of CD44v6-specific CAR T cells by defining the optimal structure of a spacer region derived from the extracellular domain of the human low-affinity nerve growth factor receptor (LNGFR). We tailored the LNGFR spacer to modulate CAR length to efficiently recognize distal or proximal epitopes and to allow selection of transduced CAR T cells by the use of clinical-grade validated manufacturing systems. The different LNGFR spacers investigated in this study are responsible for the generation of CAR T cells with a different memory phenotype, which is mainly related to the level of CAR expression and the extent of the associated tonic signaling. In particular, the CD44v6-NWN2.CAR T cells are enriched in central memory cells and show improved in vitro functions in terms of killing capability, and in vivo antitumor activity against hematological and solid tumors. Clinical Trial Registration numbers: clinicaltrial.gov NCT04097301; ClinicalTrials.gov, NCT00423124.

CAR T Cells Redirected to CD44v6 Control Tumor Growth in Lung and Ovary Adenocarcinoma Bearing Mice.

The main challenge of adoptive therapy with Chimeric Antigen Receptor modified T cells (CAR T) is the application to the field of solid tumors, where the identification of a proper antigen has emerged as one of the major drawbacks to CAR T cell treatment success. CD44 is a glycoprotein involved in cell-cell and cell-matrix interactions. The isoform containing the variant domain 6 of CD44 gene (CD44v6) has been implicated in tumorigenesis, tumor cell invasion and metastasis and represents an attractive target for CAR T cell therapies. Targeting CD44v6 antigen has been shown to control tumor growth in acute myeloid leukemia and multiple myeloma mouse models. While CAR T approach for the treatment of B cell malignancies has shown great success, response rates among patients with solid cancer are less favorable. The purpose of our study was to test the efficacy of CD44v6.CAR T cells, produced in compliance with Good Manufacturing Practice (GMP), in adenocarcinoma tumor models. We generated a bicistronic retroviral vector containing the CD44v6 CAR and the HSV-TK Mut2 suicide gene to enhance the safety of the proposed CAR T cell therapy. CD44v6 transduced CAR T cells were homogeneously positive for ΔLNGFR selection marker, were enriched in T central memory (TCM) and T memory stem cells (TSCM) and displayed a highly activated phenotype. In vitro assays revealed antigen-specific activation and cytotoxicity of human CD44v6.CAR T cells against CD44v6 expressing tumor cell lines. When infused in immunodeficient tumor bearing mice, human CD44v6.CAR T cells were able to reach, infiltrate and proliferate at tumor sites, finally resulting in tumor growth control. Next, we checked if cells produced in compliance with GMP grade standards retained the same antitumor activity of those produced with research grade materials and protocols. Noteworthy, no differences in the potency of the CAR T obtained with the two manufacturing processes were observed. In conclusion, our preclinical results suggest that CD44v6.CAR T based adoptive therapy could be a promising strategy in solid cancer treatment.

Human recombinant heat shock protein 70 affects the maturation pathways of dendritic cells in vitro and has an in vivo adjuvant activity.

Heat shock proteins (HSPs) are potent inducers of an antigen-specific immunological response. A role of chaperon of immunogenic peptides and a direct effect on APC activation and function have been described. However, the signal transduction events involved in the activation of human APCs are poorly characterized. We investigated, using human monocyte-derived dendritic cells (DCs), the signal transduction pathways activated by a human recombinant HSP70 (r)HSP70 purified from eukaryotic cells. rHSP70 effectively induced a partial maturation of DCs in vitro and a significant increase in the titers of antigen-specific IgG when used as a vaccine adjuvant in vivo. rHSP70 did not desensitize human DCs to LPS stimulation and retained its adjuvant properties in C3H/HeJ mice, which are LPS-resistant as a result of a mutation in TLR-4, ruling out the potential interference of LPS contamination. Effects on DC maturation and in vivo functions correlate to the ability of rHSP70 to activate IkappaB-alpha/NF-kappaB and ERK1/2 pathways in human DCs. No activation of p38 was induced in the same experimental conditions. Our data suggest that the IkappaB-alpha/NF-kappaB pathway has a critical role in the partial maturation of DCs induced by rHSP70.

Selected natural and synthetic retinoids impair CCR7- and CXCR4-dependent cell migration in vitro and in vivo.

Dendritic cell (DC) migration to secondary lymphoid organs is a crucial step to initiate adaptive immune responses. This step requires the expression of a functional CCR7 chemokine receptor on DC undergoing maturation. Here, we show that the natural retinoid 9-cis retinoic acid (9cRA) and the synthetic retinoid fenretinide (4-HPR) specifically inhibit the functional up-regulation of CCR7 on maturing human DCs, without affecting early steps of DC maturation. As a consequence, mature DCs do not migrate in vitro toward the chemokine CCL19. Importantly, 4-HPR and 9cRA by inhibiting the expression of CCR7 on bone marrow-derived murine DCs dampen their in vivo migration to draining lymph nodes. 4-HPR also inhibits the expression of the chemokine receptors CXCR4, therefore, impairing in vitro migration of human DCs to CXCL12 and inhibiting in vivo the CXCR4-dependent migration of the posterior lateral line primordium (PLLp) in zebrafish embryos. Taken together, these data highlight a novel function of retinoids and suggest the possibility of using retinoids to treat inflammatory or autoimmune diseases.

Succinimide-Based Conjugates Improve IsoDGR Cyclopeptide Affinity to αvß3 without Promoting Integrin Allosteric Activation.

The isoDGR sequence is an integrin-binding motif that has been successfully employed as a tumor-vasculature-homing molecule or for the targeted delivery of drugs and diagnostic agents to tumors. In this context, we previously demonstrated that cyclopeptide 2, the product of the conjugation of c(CGisoDGRG) (1) to 4-( N-maleimidomethyl)cyclohexane-1-carboxamide, can be successfully used as a tumor-homing ligand for nanodrug delivery to neoplastic tissues. Here, combining NMR, computational, and biochemical methods, we show that the succinimide ring contained in 2 contributes to stabilizing interactions with αvß3, an integrin overexpressed in the tumor vasculature. Furthermore, we demonstrate that various cyclopeptides containing the isoDGR sequence embedded in different molecular scaffolds do not induce αvß3 allosteric activation and work as pure integrin antagonists. These results could be profitably exploited for the rational design of novel isoDGR-based ligands and tumor-targeting molecules with improved αvß3-binding properties and devoid of adverse integrin-activating effects.

Anti-metastatic activity of the tumor vascular targeting agent NGR-TNF.

Tumor vessels are an attractive target for cancer therapy, including metastasis treatment. Angiogenesis inhibitors targeting the VEGF signalling pathway have proven to be efficacious in preclinical cancer models and in clinical trials. However, angiogenesis inhibition concomitantly elicits tumor adaptation and progression to stages of greater malignancy, with heightened invasiveness and in some cases increased distant metastasis. Here, we investigated whether NGR-TNF, a vascular targeting agent in phase III clinical development, coupling the CNGRCG angiogenic vessel-homing peptide with TNF-α, has an effect on metastasis in a model of murine breast cancer, which spontaneously metastasize to lungs, and on the growth of experimental melanoma lung metastasis. We report that NGR-TNF does not increase cancer invasiveness, as other antiangiogenics agents do, but controls metastatic growth in both models, both when administered as primary treatment and in adjuvant settings, improving the overall survival of metastasis-bearing mice.

The tumor vessel targeting agent NGR-TNF controls the different stages of the tumorigenic process in transgenic mice by distinct mechanisms.

NGR-TNF is a vascular targeting agent in advanced clinical development, coupling tumor necrosis factor-α (TNF) with the CNGRCG peptide, which targets a CD13 isoform specifically expressed by angiogenic vessels. Antitumor efficacy of NGR-TNF has been described in different transplantation tumor models. Nevertheless, the mechanism underlying its activity is not fully understood. In the wild type and in the immunodeficient (RAG-/-) RIP1-Tag2 models of multistage pancreatic carcinogenesis, we demonstrate that CD13 is highly expressed on endothelial cells of hyperplastic and angiogenic islets, whereas its expression is down regulated in tumors where it partially colocalize with pericytes. In vivo CNGRCG peptides coupled to fluorescent nanoparticles (quantum dots) bind to CD13 and colocalize with anti-CD31, in pancreatic islets. At early stage, low doses of NGR-murine (m)TNF have a direct cytotoxic effect inducing endothelial cell apoptosis, reducing vessel density and eventually inhibiting the development of angiogenic islets. At a later stage, NGR-mTNF is able to reduce tumor growth inducing vascular normalization, exclusively when treatment is carried out in the immunocompetent mice. Interestingly, NGR-mTNF-treated tumors from these mice are characterized by CD8+ T cell infiltration. At molecular level, overexpression of genes involved in vessels normalization was detected only in NGR-mTNF-treated tumors from immunocompetent mice. These findings identified a new mechanism of action of NGR-mTNF, providing support for the development of new therapeutic strategies combining chemotherapy or active/adoptive immunotherapies to low dose NGR-TNF treatment.

Tumor-mediated liver X receptor-alpha activation inhibits CC chemokine receptor-7 expression on dendritic cells and dampens antitumor responses.

Sterol metabolism has recently been linked to innate and adaptive immune responses through liver X receptor (LXR) signaling. Whether products of sterol metabolism interfere with antitumor responses is currently unknown. Dendritic cells (DCs) initiate immune responses, including antitumor activity after their CC chemokine receptor-7 (CCR7)-dependent migration to lymphoid organs. Here we report that human and mouse tumors produce LXR ligands that inhibit CCR7 expression on maturing DCs and, therefore, their migration to lymphoid organs. In agreement with this observation, we detected CD83(+)CCR7(-) DCs within human tumors. Mice injected with tumors expressing the LXR ligand-inactivating enzyme sulfotransferase 2B1b (SULT2B1b) successfully controlled tumor growth by regaining DC migration to tumor-draining lymph nodes and by developing overt inflammation within tumors. The control of tumor growth was also observed in chimeric mice transplanted with bone marrow from mice lacking the gene encoding LXR-alpha (Nr1h3(-/-) mice) Thus, we show a new mechanism of tumor immunoescape involving products of cholesterol metabolism. The manipulation of this pathway could restore antitumor immunity in individuals with cancer.

Direct effects of polymyxin B on human dendritic cells maturation. The role of IkappaB-alpha/NF-kappaB and ERK1/2 pathways and adhesion.

Polymyxin B is a lipopolysaccharide binding antibiotic used to inactivate potential lipopolysaccharide contaminations when evaluating the activity of different agents on innate immune cells. We report that polymyxin B is able to induce directly in monocyte-derived human dendritic cells (DCs) several functional and molecular modifications characteristic of DCs undergoing a maturation process. DCs incubated with polymyxin B up-regulate the expression of HLA class I and II, the co-stimulatory CD86 molecule, and show an increase in the fraction of adherent cells at short time, which persist at 48 h of incubation. Adhesion to the plate was required for the polymyxin B-induced DCs maturation. A transient activation of IkappaB-alpha/NF-kappaB and ERK1/2 pathways at short time and a further ERK1/2 activation at long term were also detected. Neither up-regulation of the maturation marker CD83 nor activation of p38 nor induction of cytokines secretion was observed in DCs treated with polymyxin B. We demonstrated that inhibition of IkappaB-alpha/NF-kappaB pathway abolishes polymyxin B effects. ERK1/2 inhibition instead allowed DCs treated with polymyxin B to progress in their maturation process as revealed by the increased up-regulation of the CD83 co-stimulatory molecules, the activation of p38, and the reduced adhesion to culture plates at 48 h of incubation. Our results indicate that polymyxin B induces a partial maturation of human DCs through increased adhesion to a substrate and activation of the IkappaB-alpha/NF-kappaB pathway. The increased ERK1/2 activation observed, even though correlating with the initial phases of the maturation process, actually inhibits the occurrence of full maturation.

Release of high mobility group box 1 by dendritic cells controls T cell activation via the receptor for advanced glycation end products.

High mobility group box 1 (HMGB1) is an abundant and conserved nuclear protein that is released by necrotic cells and acts in the extracellular environment as a primary proinflammatory signal. In this study we show that human dendritic cells, which are specialized in Ag presentation to T cells, actively release their own HMGB1 into the extracellular milieu upon activation. This secreted HMGB1 is necessary for the up-regulation of CD80, CD83, and CD86 surface markers of human dendritic cells and for IL-12 production. The HMGB1 secreted by dendritic cells is also required for the clonal expansion, survival, and functional polarization of naive T cells. Using neutralizing Abs and receptor for advanced glycation end product-deficient (RAGE(-/-)) cells, we demonstrate that RAGE is required for the effect of HMGB1 on dendritic cells. HMGB1/RAGE interaction results in downstream activation of MAPKs and NF-kappaB. The use of an ancient signal of necrosis, HMGB1, by dendritic cells to sustain their own maturation and for activation of T lymphocytes represents a profitable evolutionary mechanism.

Rhabdomyosarcomas are potential target of MAGE-specific immunotherapies.

The search for alternative strategies of therapy remains a major issue for most neoplastic diseases. The expression of several tumor antigens makes human rhabdomyosarcomas, which are the most frequent form of soft tissue tumor in children, a good candidate for tumor-specific immunotherapy. To assess the feasibility of this approach, we evaluated the ability of rhabdomyosarcoma cell lines to process and present the MAGE-A tumor antigens to effectors of the immune system. To this end, we investigated recognition of MAGE-A-positive rhabdomyosarcoma cells by HLA-B*3701-restricted T cells specific for a MAGE-A-derived peptide. Low level of HLA expression impaired recognition of the tumor cells. Therefore, to obtain HLA expression avoiding the use of IFN-gamma and TNF-alpha, which could affect the proteasome activity, a rhabdomyosarcoma line was transduced by a retroviral vector encoding the HLA-B*3701 allele. Recognition of the infected cells was then observed also in the absence of IFN-gamma and TNF-alpha treatment, thus demonstrating that rhabdomyosarcoma cells were indeed able to naturally process and present the MAGE-A antigens. These results demonstrate that rhabdomyosarcoma cells expressing MAGE-A can be targets of tumor-specific effectors, suggesting the feasibility of clinical protocols of specific immunotherapy also for the treatment of rhabdomyosarcoma.

HMGB1 is an endogenous immune adjuvant released by necrotic cells.

Immune responses against pathogens require that microbial components promote the activation of antigen-presenting cells (APCs). Autoimmune diseases and graft rejections occur in the absence of pathogens; in these conditions, endogenous molecules, the so-called 'innate adjuvants', activate APCs. Necrotic cells contain and release innate adjuvants; necrotic cells also release high-mobility group B1 protein (HMGB1), an abundant and conserved constituent of vertebrate nuclei. Here, we show that necrotic HMGB1(-/-) cells have a reduced ability to activate APCs, and HMGB1 blockade reduces the activation induced by necrotic wild-type cell supernatants. In vivo, HMGB1 enhances the primary antibody responses to soluble antigens and transforms poorly immunogenic apoptotic lymphoma cells into efficient vaccines.