Improving the antitumor activity of R-CHOP with NGR-hTNF in primary CNS lymphoma: final results of a phase 2 trial.

Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) is the standard treatment of diffuse large B-cell lymphoma (DLBCL). Primary DLBCL of the central nervous system (CNS) (primary central nervous system lymphoma [PCNSL]) is an exception because of the low CNS bioavailability of related drugs. NGR-human tumor necrosis factor (NGR-hTNF) targets CD13+ vessels, enhances vascular permeability and CNS access of anticancer drugs, and provides the rationale for the treatment of PCNSL with R-CHOP. Herein, we report activity and safety of R-CHOP preceded by NGR-hTNF in patients with PCNSL relapsed/refractory to high-dose methotrexate-based chemotherapy enrolled in a phase 2 trial. Overall response rate (ORR) was the primary endpoint. A sample size of 28 patients was considered necessary to demonstrate improvement from 30% to 50% ORR. NGR-hTNF/R-CHOP would be declared active if ≥12 responses were recorded. Treatment was well tolerated; there were no cases of unexpected toxicities, dose reductions or interruptions. NGR-hTNF/R-CHOP was active, with confirmed tumor response in 21 patients (75%; 95% confidence interval, 59%-91%), which was complete in 11. Seventeen of the 21 patients with response to treatment received consolidation (ASCT, WBRT, and/or lenalidomide maintenance). At a median follow-up of 21 (range, 14-31) months, 5 patients remained relapse-free and 6 were alive. The activity of NGR-hTNF/R-CHOP is in line with the expression of CD13 in both pericytes and endothelial cells of tumor vessels. High plasma levels of chromogranin A, an NGR-hTNF inhibitor, were associated with proton pump inhibitor use and a lower remission rate, suggesting that these drugs should be avoided during TNF-based therapy. Further research on this innovative approach to CNS lymphomas is warranted. The trial was registered as EudraCT: 2014-001532-11.

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.

R-CHOP preceded by blood-brain barrier permeabilization with engineered tumor necrosis factor-α in primary CNS lymphoma.

Patients with primary central nervous system lymphoma (PCNSL) are treated with high-dose methotrexate-based chemotherapy, which requires hospitalization and extensive expertise to manage related toxicity. The use of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) could overcome these difficulties, but blood-brain barrier (BBB) penetration of related drugs is poor. Tumor necrosis factor-α coupled with NGR (NGR-hTNF), a peptide targeting CD13+ vessels, induces endothelial permeabilization and improves tumor access of cytostatics. We tested the hypothesis that NGR-hTNF can break the BBB, thereby improving penetration and activity of R-CHOP in patients with relapsed/refractory PCNSL (NCT03536039). Patients received six R-CHOP21 courses, alone at the first course and preceded by NGR-hTNF (0.8 µg/m2) afterward. This trial included 2 phases: an "explorative phase" addressing the effect of NGR-hTNF on drug pharmacokinetic parameters and on vessel permeability, assessed by dynamic contrast-enhanced magnetic resonance imaging and 99mTc-diethylene-triamine-pentacetic acid-single-photon emission computed tomography, and the expression of CD13 on tumor tissue; and an "expansion phase" with overall response rate as the primary end point, in which the 2-stage Simon Minimax design was used. At the first stage, if ≥4 responses were observed among 12 patients, the study accrual would have continued (sample size, 28). Herein, we report results of the explorative phase and the first-stage analysis (n = 12). CD13 was expressed in tumor vessels of all cases. NGR-hTNF selectively increased vascular permeability in tumoral/peritumoral areas, without interfering with drug plasma/cerebrospinal fluid concentrations. The NGR-hTNF/R-CHOP combination was well tolerated: there were only 2 serious adverse events, and grade 4 toxicity was almost exclusively hematological, which were resolved without dose reductions or interruptions. NGR-hTNF/R-CHOP was active, with 9 confirmed responses (75%; 95% confidence interval, 51-99), 8 of which were complete. In conclusion, NGR-hTNF/R-CHOP was safe in these heavily pretreated patients. NGR-hTNF enhanced vascular permeability specifically in tumoral/peritumoral areas, which resulted in fast and sustained responses.

The IL-1/IL-1 receptor axis and tumor cell released inflammasome adaptor ASC are key regulators of TSLP secretion by cancer associated fibroblasts in pancreatic cancer.

BACKGROUND: The thymic stromal lymphopoietin (TSLP), a key cytokine for development of Th2 immunity, is produced by cancer associated fibroblasts (CAFs) in pancreatic cancer where predominant tumor infiltrating Th2 over Th1 cells correlates with reduced patients' survival. Which cells and molecules are mostly relevant in driving TSLP secretion by CAFs in pancreatic cancer is not defined. METHODS: We performed in vitro, in vivo and ex-vivo analyses. For in vitro studies we used pancreatic cancer cell lines, primary CAFs cultures, and THP1 cells. TSLP secretion by CAFs was used as a read-out system to identify in vitro relevant tumor-derived inflammatory cytokines and molecules. For in vivo studies human pancreatic cancer cells and CAFs were orthotopically injected in immunodeficient mice. For ex-vivo studies immunohistochemistry was performed to detect ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) expression in surgical samples. Bioinformatics was applied to interrogate published data sets. RESULTS: We show in vitro that IL-1α and IL-1ß released by pancreatic cancer cells and tumor cell-conditioned macrophages are crucial for TSLP secretion by CAFs. Treatment of immunodeficient mice orthotopically injected with human IL-1 positive pancreatic cancer cells plus CAFs using the IL-1R antagonist anakinra significantly reduced TSLP expression in the tumor. Importantly, we found that pancreatic cancer cells release alarmins, among which ASC, able to induce IL-1ß secretion in macrophages. The relevance of ASC was confirmed ex-vivo by its expression in both tumor cells and tumor associated macrophages in pancreatic cancer surgical samples and survival data analyses showing statistically significant inverse correlation between ASC expression and survival in pancreatic cancer patients. CONCLUSIONS: Our findings indicate that tumor released IL-1α and IL-1ß and ASC are key regulators of TSLP secretion by CAFs and their targeting should ultimately dampen Th2 inflammation and improve overall survival in pancreatic cancer.

Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells.

In the clinic, chimeric antigen receptor-modified T (CAR T) cell therapy is frequently associated with life-threatening cytokine-release syndrome (CRS) and neurotoxicity. Understanding the nature of these pathologies and developing treatments for them are hampered by the lack of appropriate animal models. Herein, we describe a mouse model recapitulating key features of CRS and neurotoxicity. In humanized mice with high leukemia burden, CAR T cell-mediated clearance of cancer triggered high fever and elevated IL-6 levels, which are hallmarks of CRS. Human monocytes were the major source of IL-1 and IL-6 during CRS. Accordingly, the syndrome was prevented by monocyte depletion or by blocking IL-6 receptor with tocilizumab. Nonetheless, tocilizumab failed to protect mice from delayed lethal neurotoxicity, characterized by meningeal inflammation. Instead, the IL-1 receptor antagonist anakinra abolished both CRS and neurotoxicity, resulting in substantially extended leukemia-free survival. These findings offer a therapeutic strategy to tackle neurotoxicity and open new avenues to safer CAR T cell therapies.

Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene.

Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.

Immunomodulatory Drugs in the Context of Autologous Hematopoietic Stem Cell Transplantation Associate With Reduced Pro-tumor T Cell Subsets in Multiple Myeloma.

Immunomodulatory drugs (IMiDs) are effective therapeutics for multiple myeloma (MM), where in different clinical settings they exert their function both directly on MM cells and indirectly by modulating immune cell subsets, although with not completely defined mechanisms. Here we studied the role of IMiDs in the context of autologous hematopoietic stem cell transplantation on the T cell subset distribution in the bone marrow of newly diagnosed MM patients. We found that after transplantation pro-tumor Th17-Th1 and Th22 cells and their related cytokines were lower in patients treated with IMiDs during induction chemotherapy compared to untreated patients. Of note, lower levels of IL-17, IL-22, and related IL-6, TNF-α, IL-1ß, and IL-23 in the bone marrow sera correlated with treatment with IMiDs and favorable clinical outcome. Collectively, our results suggest a novel anti-inflammatory role for IMiDs in MM.

Vectofusin-1 Promotes RD114-TR-Pseudotyped Lentiviral Vector Transduction of Human HSPCs and T Lymphocytes.

Ex vivo transduction of human CD34+ hematopoietic stem/progenitor cells (hCD34+ HSPCs) and T lymphocytes is a key process that requires high efficiency and low toxicity to achieve effective clinical results. So far, several enhancers have been used to improve this process. Among them, Retronectin highly meliorates VSV-G and RD114-TR pseudotyped lentiviral vector delivery in hCD34+ HSPCs and T lymphocytes. However, Retronectin is expensive and requires pre-coating of culture dishes or bags before cell seeding, resulting in a cumbersome procedure. Recently, an alternative transduction adjuvant has been developed, named Vectofusin-1, whose effect has been demonstrated on gene delivery to cell lines and primary hCD34+ HSPCs by lentiviral vectors pseudotyped with different envelope glycoproteins. In this study, we have focused our analysis on the effect of Vectofusin-1 on the transduction of hCD34+ HSPCs and T lymphocytes by using mostly RD114-TR pseudotyped lentivectors and clinical transduction protocols. Here, we have proved that Vectofusin-1 reproducibly enhances gene delivery to hCD34+ HSPCs and activated T cells without cell toxicity and with efficacy comparable to that of Retronectin. The use of Vectofusin-1 will therefore help to shorten and simplify clinical cell manipulation, especially if automated systems are planned for transducing large-scale clinical lots.

Codon Optimization Leads to Functional Impairment of RD114-TR Envelope Glycoprotein.

Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies. Their optimization is therefore very important for the field of vectorology and gene therapy. A key molecule for LV function is the envelope because it guides cell entry. The most commonly used in transiently produced LVs is the vesicular stomatitis virus glycoprotein (VSV-G) envelope, whose continuous expression is, however, toxic for stable LV producer cells. In contrast, the feline endogenous retroviral RD114-TR envelope is suitable for stable LV manufacturing, being well tolerated by producer cells under constitutive expression. We have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34+ cells. To further improve RD114-TR-pseudotyped LV cell entry by increasing envelope expression, we codon-optimized the RD114-TR open reading frame (ORF). Here we show that, despite the RD114-TRco precursor being produced at a higher level than the wild-type counterpart, it is unexpectedly not duly glycosylated, exported to the cytosol, and processed. Correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in vivo, and, consequently, the unprocessed precursor is incorporated into LVs, making them inactive.

24-Hydroxycholesterol participates in pancreatic neuroendocrine tumor development.

Cells in the tumor microenvironment may be reprogrammed by tumor-derived metabolites. Cholesterol-oxidized products, namely oxysterols, have been shown to favor tumor growth directly by promoting tumor cell growth and indirectly by dampening antitumor immune responses. However, the cellular and molecular mechanisms governing oxysterol generation within tumor microenvironments remain elusive. We recently showed that tumor-derived oxysterols recruit neutrophils endowed with protumoral activities, such as neoangiogenesis. Here, we show that hypoxia inducible factor-1a (HIF-1α) controls the overexpression of the enzyme Cyp46a1, which generates the oxysterol 24-hydroxycholesterol (24S-HC) in a pancreatic neuroendocrine tumor (pNET) model commonly used to study neoangiogenesis. The activation of the HIF-1α-24S-HC axis ultimately leads to the induction of the angiogenic switch through the positioning of proangiogenic neutrophils in proximity to Cyp46a1+ islets. Pharmacologic blockade or genetic inactivation of oxysterols controls pNET tumorigenesis by dampening the 24S-HC-neutrophil axis. Finally, we show that in some human pNET samples Cyp46a1 transcripts are overexpressed, which correlate with the HIF-1α target VEGF and with tumor diameter. This study reveals a layer in the angiogenic switch of pNETs and identifies a therapeutic target for pNET patients.

The administration of drugs inhibiting cholesterol/oxysterol synthesis is safe and increases the efficacy of immunotherapeutic regimens in tumor-bearing mice.

Tumor-derived metabolites dampen tumor-infiltrating immune cells and antitumor immune responses. Among the various metabolites produced by tumors, we recently showed that cholesterol oxidized products, namely oxysterols, favor tumor growth through the inhibition of DC migration toward lymphoid organs and by promoting the recruitment of pro-tumor neutrophils within the tumor microenvironment. Here, we tested different drugs capable of blocking cholesterol/oxysterol formation. In particular, we tested efficacy and safety of different administration schedules, and of immunotherapy-based combination of a class of compounds, namely zaragozic acids, which inhibit cholesterol pathway downstream of mevalonate formation, thus leaving intact the formation of the isoprenoids, which are required for the maturation of proteins involved in the immune cell function. We show that zaragozic acids inhibit the in vivo growth of the RMA lymphoma and the Lewis lung carcinoma (LLC) without inducing side effects. Tumor growth inhibition requires an intact immune system, as immunodeficient tumor-bearing mice do not respond to zaragozic acid treatment. Of note, the effect of zaragozic acids is accompanied by a marked reduction in the LXR target genes Abcg1, Mertk, Scd1 and Srebp-1c in the tumor microenvironment. On the other hand, zoledronate, which blocks also isoprenoid formation, did not control the LLC tumor growth. Finally, we show that zaragozic acids potentiate the antitumor effects of active and adoptive immunotherapy, significantly prolonging the overall survival of tumor-bearing mice treated with the combo zaragozic acids and TAA-loaded DCs. This study identifies zaragozic acids as new antitumor compounds exploitable for the treatment of cancer patients.

RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope.

To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.

Tracking genetically engineered lymphocytes long-term reveals the dynamics of T cell immunological memory.

Long-lasting immune protection from pathogens and cancer requires the generation of memory T cells able to survive long-term. To unravel the immunological requirements for long-term persistence of human memory T cells, we characterized and traced, over several years, T lymphocytes genetically modified to express the thymidine kinase (TK) suicide gene that were infused in 10 patients after haploidentical hematopoietic stem cell transplantation (HSCT). At 2 to 14 years after infusion and in the presence of a broad and resting immune system, we could still detect effectors/effector memory (TEM/EFF), central memory (TCM), and stem memory (TSCM) TK(+) cells, circulating at low but stable levels in all patients. Longitudinal analysis of cytomegalovirus (CMV)- and Flu-specific TK(+) cells indicated that antigen recognition was dominant in driving in vivo expansion and persistence at detectable levels. The amount of infused TSCM cells positively correlated with early expansion and with the absolute counts of long-term persisting gene-marked cells. By combining T cell sorting with sequencing of integration (IS), TCRα and TCRß clonal markers, we showed that T cells retrieved long-term were enriched in clones originally shared in different memory T cell subsets, whereas dominant long-term clonotypes appeared to preferentially originate from infused TSCM and TCM clones. Together, these results indicate that long-term persistence of gene-modified memory T cells after haploidentical HSCT is influenced by antigen exposure and by the original phenotype of infused cells. Cancer adoptive immunotherapy might thus benefit from cellular products enriched in lymphocytes with an early-differentiated phenotype.

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.

Th22 cells increase in poor prognosis multiple myeloma and promote tumor cell growth and survival.

There is increased production of plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) of multiple myeloma (MM) patients and these favor Th22 cell differentiation. Here, we found that the frequency of interleukin (IL)-22+IL-17-IL-13+ T cells is significantly increased in peripheral blood (PB) and BM of stage III and relapsed/refractory MM patients compared with healthy donors and patients with asymptomatic or stage I/II disease. Th22 cells cloned from the BM of MM patients were CCR6+CXCR4+CCR4+CCR10- and produced IL-22 and IL-13 but not IL-17. Furthermore, polyfunctional Th22-Th2 and Th22-Th1 clones were identified based on the co-expression of additional chemokine receptors and cytokines (CRTh2 or CXCR3 and IL-5 or interferon gamma [IFNγ], respectively). A fraction of MM cell lines and primary tumors aberrantly expressed the IL-22RA1 and IL-22 induced STAT-3 phosphorylation, cell growth, and resistance to drug-induced cell death in MM cells. IL-13 treatment of normal BM mesenchymal stromal cells (MSCs) induced STAT-6 phosphorylation, adhesion molecule upregulation, and increased IL-6 production and significantly favored MM cell growth compared with untreated BM MSCs. Collectively, our data show that increased frequency of IL-22+IL-17-IL-13+ T cells correlates with poor prognosis in MM through IL-22 and IL-13 protumor activity and suggest that interference with IL-22 and IL-13 signaling pathways could be exploited for therapeutic intervention.

Improving the safety of cell therapy with the TK-suicide gene.

While opening new frontiers for the cure of malignant and non-malignant diseases, the increasing use of cell therapy poses also several new challenges related to the safety of a living drug. The most effective and consolidated cell therapy approach is allogeneic hematopoietic stem cell transplantation (HSCT), the only cure for several patients with high-risk hematological malignancies. The potential of allogeneic HSCT is strictly dependent on the donor immune system, particularly on alloreactive T lymphocytes, that promote the beneficial graft-versus-tumor effect (GvT), but may also trigger the detrimental graft-versus-host-disease (GvHD). Gene transfer technologies allow to manipulate donor T-cells to enforce GvT and foster immune reconstitution, while avoiding or controlling GvHD. The suicide gene approach is based on the transfer of a suicide gene into donor lymphocytes, for a safe infusion of a wide T-cell repertoire, that might be selectively controlled in vivo in case of GvHD. The herpes simplex virus thymidine kinase (HSV-TK) is the suicide gene most extensively tested in humans. Expression of HSV-TK in donor lymphocytes confers lethal sensitivity to the anti-herpes drug, ganciclovir. Progressive improvements in suicide genes, vector technology and transduction protocols have allowed to overcome the toxicity of GvHD while preserving the antitumor efficacy of allogeneic HSCT. Several phase I-II clinical trials in the last 20 years document the safety and the efficacy of HSV-TK approach, able to maintain its clear value over the last decades, in the rapidly progressing horizon of cancer cellular therapy.

Generation of human memory stem T cells after haploidentical T-replete hematopoietic stem cell transplantation.

Memory stem T cells (TSCM) have been proposed as key determinants of immunologic memory. However, their exact contribution to a mounting immune response, as well as the mechanisms and timing of their in vivo generation, are poorly understood. We longitudinally tracked TSCM dynamics in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), thereby providing novel hints on the contribution of this subset to posttransplant immune reconstitution in humans. We found that donor-derived TSCM are highly enriched early after HSCT. We showed at the antigen-specific and clonal level that TSCM lymphocytes can differentiate directly from naive precursors infused within the graft and that the extent of TSCM generation might correlate with interleukin 7 serum levels. In vivo fate mapping through T-cell receptor sequencing allowed defining the in vivo differentiation landscapes of human naive T cells, supporting the notion that progenies of single naive cells embrace disparate fates in vivo and highlighting TSCM as relevant novel players in the diversification of immunological memory after allogeneic HSCT.

In vivo tracking of T cells in humans unveils decade-long survival and activity of genetically modified T memory stem cells.

A definitive understanding of survival and differentiation potential in humans of T cell subpopulations is of paramount importance for the development of effective T cell therapies. In particular, uncovering the dynamics in vivo in humans of the recently described T memory stem cells (TSCM) would be crucial for therapeutic approaches that aim at taking advantage of a stable cellular vehicle with precursor potential. We exploited data derived from two gene therapy clinical trials for an inherited immunodeficiency, using either retrovirally engineered hematopoietic stem cells or mature lymphocytes to trace individual T cell clones directly in vivo in humans. We compared healthy donors and bone marrow-transplanted patients, studied long-term in vivo T cell composition under different clinical conditions, and specifically examined TSCM contribution according to age, conditioning regimen, disease background, cell source, long-term reconstitution, and ex vivo gene correction processing. High-throughput sequencing of retroviral vector integration sites (ISs) allowed tracing the fate of more than 1700 individual T cell clones in gene therapy patients after infusion of gene-corrected hematopoietic stem cells or mature lymphocytes. We shed light on long-term in vivo clonal relationships among different T cell subtypes, and we unveiled that TSCM are able to persist and to preserve their precursor potential in humans for up to 12 years after infusion of gene-corrected lymphocytes. Overall, this work provides high-resolution tracking of T cell fate and activity and validates, in humans, the safe and functional decade-long survival of engineered TSCM, paving the way for their future application in clinical settings.

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.