Preclinical efficacy of a HER2 synNotch/CEA-CAR combinatorial immunotherapy against colorectal cancer with HER2 amplification.

HER2 amplification occurs in approximately 5% of colorectal cancer (CRC) cases and is associated only partially with clinical response to combined human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR)-targeted treatment. An alternative approach based on adoptive cell therapy using T cells engineered with anti-HER2 chimeric antigen receptor (CAR) proved to be toxic due to on-target/off-tumor activity. Here we describe a combinatorial strategy to safely target HER2 amplification and carcinoembryonic antigen (CEA) expression in CRC using a synNotch-CAR-based artificial regulatory network. The natural killer (NK) cell line NK-92 was engineered with an anti-HER2 synNotch receptor driving the expression of a CAR against CEA only when engaged. After being transduced and sorted for HER2-driven CAR expression, cells were cloned. The clone with optimal performances in terms of specificity and amplitude of CAR induction demonstrated significant activity in vitro and in vivo specifically against HER2-amplified (HER2amp)/CEA+ CRC models, with no effects on cells with physiological HER2 levels. The HER2-synNotch/CEA-CAR-NK system provides an innovative, scalable, and safe off-the-shelf cell therapy approach with potential against HER2amp CRC resistant or partially responsive to HER2/EGFR blockade.

Three-dimensional dynamics of mesothelin-targeted CAR.CIK lymphocytes against ovarian cancer peritoneal carcinomatosis.

Intraperitoneal cellular immunotherapy with CAR-redirected lymphocytes is an intriguing approach to target peritoneal carcinomatosis (PC) from ovarian cancer (OC), which is currently evaluated in clinical trials. PC displays a composite structure with floating tumor cells within ascites and solid-like masses invading the peritoneum. Therefore, a comprehensive experimental model is crucial to optimize CAR-cell therapies in such a peculiar environment. Here, we explored the activity of cytokine-induced killer lymphocytes (CIK), redirected by CAR against mesothelin (MSLN-CAR.CIK), within reductionistic 3D models resembling the structural complexity of both liquid and solid components of PC. MSLN-CAR.CIK effectively killed and were functionally efficient against OC targets. In a "floating-like" 3D context with floating OC spheroids, both tumor localization and killing by MSLN-CAR.CIK were significantly boosted by fluid flow. In a "solid-like" context, MSLN-CAR.CIK were recruited through the extracellular matrix on embedded tumor aggregates, with variable kinetics depending on the effector-target distance. Furthermore, MSLN-CAR.CIK penetrated the inner levels of OC spheroids exerting effective tumor killing. Our findings provide currently unknown therapeutically relevant information on intraperitoneal approaches with CAR.CIK, supporting further developments and improvements for clinical studies in the context of locoregional cell therapy approaches for patients with PC from OC.

Unravelling the threads: understanding the interplay of Cultural values, female workforce engagement, human development index and suicide rates.

PURPOSE: Suicide is a major public health problem across the world. Extensive research on the field shows that suicide is affected by several sociological, economic, and cultural risk factors. Over the last century, social changes have driven the reshaping of traditional gender roles, often in an uneven fashion, strongly depending on context. This study proposes updated findings on the impact that changes in traditional gender roles could have on suicide rates METHODS: It will do so by examining the correlation between female labor force participation (FLPR) and sex-specific suicide rates. Moreover, it will examine this association depending on human development (HDI) and Hofstede's individualism index. To do so, data from 2010 to 2019 from 47 countries is collected from the WHO, ILOSTAT and UN agencies' websites. RESULTS: Analysis show a significant interaction between FLPR, HDI and individualism index scores on male suicide rates (p = 0.002). There is a negative association between FLPR and male suicide rates in relatively lower HDI countries, while in very high HDI countries an increase in FLPR is correlated with an increase in male suicide rates. Similar trends but no significant interaction is observed for female suicide rates. CONCLUSION: This study suggests that female participation is beneficial for male population as it reduces male suicide rates. However, this association appears to be context dependent. In countries where institutional adjustment is already established, and human development is very high, other factors might be of interest in examining the trends of suicide rates among men and women.

MET Oncogene Targeting for Cancer Immunotherapy.

The MET receptor is one of the main drivers of 'invasive growth', a multifaceted biological response essential during embryonic development and tissue repair that is usurped by cancer cells to induce and sustain the malignant phenotype. MET stands out as one of the most important oncogenes activated in cancer and its inhibition has been explored since the initial era of cancer-targeted therapy. Different approaches have been developed to hamper MET signaling and/or reduce MET (over)expression as a hallmark of transformation. Considering the great interest gained by cancer immunotherapy, this review evaluates the opportunity of targeting MET within therapeutic approaches based on the exploitation of immune functions, either in those cases where MET impairment is crucial to induce an effective response (i.e., when MET is the driver of the malignancy), or when blocking MET represents a way for potentiating the treatment (i.e., when MET is an adjuvant of tumor fitness).

The impact of COVID-19 pandemic on jobs for people with intellectual disabilities and autism in Wales.

BACKGROUND: The study reports the impact of the Covid-19 pandemic and lockdowns on jobs for people with intellectual disabilities and autism. The study focuses on the impact of the first and the fire-break lockdowns and the actions taken to support young people. METHOD: Data was collected from the cohort of young people currently working in Wales, and that received job coach support from the Engage to Change Project, on furlough arrangements, job retainment and job losses. Innovative initiatives to support young people are described. RESULTS: Review of the working situation during the pandemic was conducted for 184 jobs, evaluating the proportion of young people being furloughed or working remotely and compared with the general population in Wales. CONCLUSIONS: Supported employment agencies adapted their practice during the COVID-19 pandemic, offering new and innovative ways to support young people and facilitate their return to work.

Supported employment, quality of jobs and employment typicalness: The experience of the Engage to Change project.

BACKGROUND: Paid employment represents a challenge for people with an intellectual disability and/or autism. This paper analyses the quality of jobs offered by the Engage to Change project and their relationship to the 'typicalness' of the employment offered. METHOD: Data on the quality of 384 paid jobs were collected, including hours worked and wages earned, and reported social integration at work. The typicalness of the employment experience was assessed for 141 young people, using the Index of Typicalness of Placement Questionnaire. RESULTS: The Engage to Change project offered a wide range of jobs. There is no difference in the typicalness of the employment experience in relation to the interactions in the workplace, but there are some differences in the recruitment process for men and women. CONCLUSION: Job coaches should make sure that, despite the procedures being 'atypical' for the workplace, the outcome is 'a typical employment experience' for each employee.

A demographic and qualitative analysis of the determinants of success in a National Supported Employment project.

BACKGROUND: People with an intellectual disability and/or autism experience low employment rates compared to the general population. This study shows what the determinants of success are in getting this group of young people into paid employment. METHOD: The research is based on data collected on 1008 young people, aged 16-25, participating in the Engage to Change project across Wales, to support young people to achieve employment. A real-time data collection system was used from their engagement and throughout their journey to employment. RESULTS: This research indicates an overall employment rate of 23% for the project to date, based on total referral. Young people who engaged in 'significant work experience', such as paid placement or supported internship, had a greater employment rate of 37%. CONCLUSION: Previous and current real work experiences increased the chances of young people of becoming employed, above the effect of supported employment and job coach support.

Met inhibition revokes IFNγ-induction of PD-1 ligands in MET-amplified tumours.

BACKGROUND: Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immune-evasion. Patients featuring MET amplification, a genetic lesion driving transformation, may benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-γ modulation of PD-L1/PD-L2 in MET-amplified tumours. METHODS: PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-γ were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-γ stimulation, and after anti-MET therapy. RESULTS: PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-γ treatment. This induction was impaired by JNJ-605, a selective inhibitor of MET kinase activity, and MvDN30, an antibody inducing MET proteolytic cleavage. We found that activation of JAKs/ STAT1, signal transducers downstream of the Interferon-γ receptor, was neutralised by MET inhibitors. Moreover, JAK2 and MET associated in the same signalling complex depending on MET phosphorylation. Results were confirmed in MET-amplified organoids derived from human colorectal tumours, where JNJ-605 treatment revoked Interferon-γ induced PD-L1 expression. CONCLUSIONS: These data show that in MET-amplified cancers, treatment with MET inhibitors counteracts the induction of PD-1 ligands by Interferon-γ. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene.

Targeting the MET oncogene by concomitant inhibition of receptor and ligand via an antibody-"decoy" strategy.

MET, a master gene sustaining "invasive growth," is a relevant target for cancer precision therapy. In the vast majority of tumors, wild-type MET behaves as a "stress-response" gene and relies on the ligand (HGF) to sustain cell "scattering," invasive growth and apoptosis protection (oncogene "expedience"). In this context, concomitant targeting of MET and HGF could be crucial to reach effective inhibition. To test this hypothesis, we combined an anti-MET antibody (MvDN30) inducing "shedding" (i.e., removal of MET from the cell surface), with a "decoy" (i.e., the soluble extracellular domain of the MET receptor) endowed with HGF-sequestering ability. To avoid antibody/decoy interaction-and subsequent neutralization-we identified a single aminoacid in the extracellular domain of MET-lysine 842-that is critical for MvDN30 binding and engineered the corresponding recombinant decoyMET (K842E). DecoyMETK842E retains the ability to bind HGF with high affinity and inhibits HGF-induced MET phosphorylation. In HGF-dependent cellular models, MvDN30 antibody and decoyMETK842E used in combination cooperate in restraining invasive growth, and synergize in blocking cancer cell "scattering." The antibody and the decoy unbridle apoptosis of colon cancer stem cells grown in vitro as spheroids. In a preclinical model, built by orthotopic transplantation of a human pancreatic carcinoma in SCID mice engineered to express human HGF, concomitant treatment with antibody and decoy significantly reduces metastatic spread. The data reported indicate that vertical targeting of the MET/HGF axis results in powerful inhibition of ligand-dependent MET activation, providing proof of concept in favor of combined target therapy of MET "expedience."

MET/HGF Co-Targeting in Pancreatic Cancer: A Tool to Provide Insight into the Tumor/Stroma Crosstalk.

The 'onco-receptor' MET (Hepatocyte Growth Factor Receptor) is involved in the activation of the invasive growth program that is essential during embryonic development and critical for wound healing and organ regeneration during adult life. When aberrantly activated, MET and its stroma-secreted ligand HGF (Hepatocyte Growth Factor) concur to tumor onset, progression, and metastasis in solid tumors, thus representing a relevant target for cancer precision medicine. In the vast majority of tumors, wild-type MET behaves as a 'stress-response' gene, and relies on ligand stimulation to sustain cancer cell 'scattering', invasion, and protection form apoptosis. Moreover, the MET/HGF axis is involved in the crosstalk between cancer cells and the surrounding microenvironment. Pancreatic cancer (namely, pancreatic ductal adenocarcinoma, PDAC) is an aggressive malignancy characterized by an abundant stromal compartment that is associated with early metastases and resistance to conventional and targeted therapies. Here, we discuss the role of the MET/HGF axis in tumor progression and dissemination considering as a model pancreatic cancer, and provide a proof of concept for the application of dual MET/HGF inhibition as an adjuvant therapy in pancreatic cancer patients.

Quiescent neuronal progenitors are activated in the juvenile guinea pig lateral striatum and give rise to transient neurons.

In the adult brain, active stem cells are a subset of astrocytes residing in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. Whether quiescent neuronal progenitors occur in other brain regions is unclear. Here, we describe a novel neurogenic system in the external capsule and lateral striatum (EC-LS) of the juvenile guinea pig that is quiescent at birth but becomes active around weaning. Activation of neurogenesis in this region was accompanied by the emergence of a neurogenic-like niche in the ventral EC characterized by chains of neuroblasts, intermediate-like progenitors and glial cells expressing markers of immature astrocytes. Like neurogenic astrocytes of the SVZ and DG, these latter cells showed a slow rate of proliferation and retained BrdU labeling for up to 65 days, suggesting that they are the primary progenitors of the EC-LS neurogenic system. Injections of GFP-tagged lentiviral vectors into the SVZ and the EC-LS of newborn animals confirmed that new LS neuroblasts originate from the activation of local progenitors and further supported their astroglial nature. Newborn EC-LS neurons existed transiently and did not contribute to neuronal addition or replacement. Nevertheless, they expressed Sp8 and showed strong tropism for white matter tracts, wherein they acquired complex morphologies. For these reasons, we propose that EC-LS neuroblasts represent a novel striatal cell type, possibly related to those populations of transient interneurons that regulate the development of fiber tracts during embryonic life.

Cardiac concentric hypertrophy promoted by activated Met receptor is mitigated in vivo by inhibition of Erk1,2 signalling with Pimasertib.

Cardiac hypertrophy is a major risk factor for heart failure. Hence, its attenuation represents an important clinical goal. Erk1,2 signalling is pivotal in the cardiac response to stress, suggesting that its inhibition may be a good strategy to revert heart hypertrophy. In this work, we unveiled the events associated with cardiac hypertrophy by means of a transgenic model expressing activated Met receptor. c-Met proto-oncogene encodes for the tyrosine kinase receptor of Hepatocyte growth factor and is a strong inducer of Ras-Raf-Mek-Erk1,2 pathway. We showed that three weeks after the induction of activated Met, the heart presents a remarkable concentric hypertrophy, with no signs of congestive failure and preserved contractility. Cardiac enlargement is accompanied by upregulation of growth-regulating transcription factors, natriuretic peptides, cytoskeletal proteins, and Extracellular Matrix remodelling factors (Timp1 and Pai1). At a later stage, cardiac hypertrophic remodelling results into heart failure with preserved systolic function. Prevention trial by suppressing activated Met showed that cardiac hypertrophy is reversible, and progression to heart failure is prevented. Notably, treatment with Pimasertib, Mek1 inhibitor, attenuates cardiac hypertrophy and remodelling. Our results suggest that modulation of Erk1.2 signalling may constitute a new therapeutic approach for treating cardiac hypertrophies.

Sniffing out a solution: How emotional body odors can improve mindfulness therapy for social anxiety.

BACKGROUND: Human body odors (BOs) serve as an effective means of social communication, with individuals exposed to emotional BOs experiencing a partial replication of the sender's affective state. This phenomenon may be particularly relevant in conditions where social interactions are impaired, such as social anxiety. Our study aimed to investigate if emotional human BOs could augment the benefits of mindfulness-based interventions. METHODS: We enrolled 48 women with social anxiety symptoms and assigned them to groups exposed to happiness BO, fear BO, or clean air. Participants engaged in mindfulness practice over two consecutive days, which included breathing, meditation, and relaxation exercises. During these interventions, the odor specific to each group was presented. Affective symptoms were assessed at the beginning and end of each day, with heart rate variability (HRV) and skin conductance level (SCL) recorded during the intervention. RESULTS: Self-reported anxiety level revealed a significant reduction in anxiety on the second day for both happiness and fear conditions, but not for the clean air group. However, on a physiological level, fear BO exposure compared to clean air led to decreased HRV, indicating that fear BO may induce a less physiological relaxed state. No significant differences were observed in SCL between odor conditions. CONCLUSIONS: These findings suggest that exposure to BOs triggers the perception of a "social presence", improving the ecological validity of a psychological treatment. If replicated and expanded, these findings could pave the way for using BOs as catalysts in existing therapies.

Genetic Ablation of the MET Oncogene Defines a Crucial Role of the HGF/MET Axis in Cell-Autonomous Functions Driving Tumor Dissemination.

Cancer cell dissemination is sustained by cell-autonomous and non-cell-autonomous functions. To disentangle the role of HGF (Hepatocyte Growth Factor) and MET ligand/receptor axis in this complex process, we genetically knocked out the MET gene in cancer cells in which MET is not the oncogenic driver. In this way, we evaluated the contribution of the HGF/MET axis to cancer cell dissemination independently of its direct activities in cells of the tumor microenvironment. The lack of MET expression in MET-/- cells has been proved by molecular characterization. From a functional point of view, HGF stimulation of MET-/- cancer cells was ineffective in eliciting intracellular signaling and in sustaining biological functions predictive of malignancy in vitro (i.e., anchorage-independent growth, invasion, and survival in the absence of matrix adhesion). Cancer cell dissemination was assessed in vivo, evaluating: (i) the ability of MET-/- lung carcinoma cells to colonize the lungs following intravenous injection and (ii) the spontaneous dissemination to distant organs of MET-/- pancreatic carcinoma cells upon orthotopic injection. In both experimental models, MET ablation affects the time of onset, the number, and the size of metastatic lesions. These results define a crucial contribution of the HGF/MET axis to cell-autonomous functions driving the metastatic process.

CSPG4 CAR-redirected Cytokine Induced Killer lymphocytes (CIK) as effective cellular immunotherapy for HLA class I defective melanoma.

BACKGROUND: Even acknowledging the game-changing results achieved in the treatment of metastatic melanoma with the use of immune checkpoint inhibitors (ICI), a large proportion of patients (40-60%) still fail to respond or relapse due to the development of resistance. Alterations in the expression of Human Leukocyte Antigen class I (HLA-I) molecules are considered to play a major role in clinical resistance to ICI. Cellular immunotherapy with HLA-independent CAR-redirected lymphocytes is a promising alternative in this challenging setting and dedicated translational models are needed. METHODS: In this study, we propose an HLA-independent therapeutic strategy with Cytokine Induced Killer lymphocytes (CIK) genetically engineered with a Chimeric Antigen Receptor (CAR) targeting the tumor antigen CSPG4 as effector mechanism. We investigated the preclinical antitumor activity of CSPG4-CAR.CIK in vitro and in a xenograft murine model focusing on patient-derived melanoma cell lines (Mel) with defective expression of HLA-I molecules. RESULTS: We successfully generated CSPG4-CAR.CIK from patients with metastatic melanoma and reported their intense activity in vitro against a panel of CSPG4-expressing patient-derived Mel. The melanoma killing activity was intense, even at very low effector to target ratios, and not influenced by the expression level (high, low, defective) of HLA-I molecules on target cells. Furthermore, CAR.CIK conditioned medium was capable of upregulating the expression of HLA-I molecules on melanoma cells. A comparable immunomodulatory effect was replicated by treatment of Mel cells with exogenous IFN-γ and IFN-α. The antimelanoma activity of CSPG4-CAR.CIK was successfully confirmed in vivo, obtaining a significant tumor growth inhibition of an HLA-defective Mel xenograft in immunodeficient mice. CONCLUSIONS: In this study we reported the intense preclinical activity of CSPG4-CAR.CIK against melanoma, including those with low or defective HLA-I expression. Our findings support CSPG4 as a valuable CAR target in melanoma and provide translational rationale for clinical studies exploring CAR-CIK cellular immunotherapies within the challenging setting of patients not responsive or relapsing to immune checkpoint inhibitors.

Novel Lymphocyte-Independent Antitumor Activity by PD-1 Blocking Antibody against PD-1+ Chemoresistant Lung Cancer Cells.

PURPOSE: Antibodies against the lymphocyte PD-1 (aPD-1) receptor are cornerstone agents for advanced non-small cell lung cancer (NSCLC), based on their ability to restore the exhausted antitumor immune response. Our study reports a novel, lymphocyte-independent, therapeutic activity of aPD-1 against NSCLC, blocking the tumor-intrinsic PD-1 receptors on chemoresistant cells. EXPERIMENTAL DESIGN: PD-1 in NSCLC cells was explored in vitro at baseline, including stem-like pneumospheres, and following treatment with cisplatin both at transcriptional and protein levels. PD-1 signaling and RNA sequencing were assessed. The lymphocyte-independent antitumor activity of aPD-1 was explored in vitro, by PD-1 blockade and stimulation with soluble ligand (PD-L1s), and in vivo within NSCLC xenograft models. RESULTS: We showed the existence of PD-1+ NSCLC cell subsets in cell lines and large in silico datasets (Cancer Cell Line Encyclopedia and The Cancer Genome Atlas). Cisplatin significantly increased PD-1 expression on chemo-surviving NSCLC cells (2.5-fold P = 0.0014), while the sequential treatment with anti-PD-1 Ab impaired their recovery after chemotherapy. PD-1 was found to be associated with tumor stemness features. PD-1 expression was enhanced in NSCLC stem-like pneumospheres (P < 0.0001), significantly promoted by stimulation with soluble PD-L1 (+27% ± 4, P < 0.0001) and inhibited by PD-1 blockade (-30% ± 3, P < 0.0001). The intravenous monotherapy with anti-PD-1 significantly inhibited tumor growth of NSCLC xenografts in immunodeficient mice, without the contribution of the immune system, and delayed the occurrence of chemoresistance when combined with cisplatin. CONCLUSIONS: We report first evidence of a novel lymphocyte-independent activity of anti-PD-1 antibodies in NSCLC, capable of inhibiting chemo-surviving NSCLC cells and exploitable to contrast disease relapses following chemotherapy. See related commentary by Augustin et al., p. 505.

hOA-DN30: a highly effective humanized single-arm MET antibody inducing remission of 'MET-addicted' cancers.

BACKGROUND: The tyrosine kinase receptor encoded by the MET oncogene is a major player in cancer. When MET is responsible for the onset and progression of the transformed phenotype (MET-addicted cancers), an efficient block of its oncogenic activation results in potent tumor growth inhibition. METHODS: Here we describe a molecular engineered MET antibody (hOA-DN30) and validate its pharmacological activity in MET-addicted cancer models in vitro and in vivo. Pharmacokinetics and safety profile in non-human primates have also been assessed. RESULTS: hOA-DN30 efficiently impaired MET activation and the intracellular signalling cascade by dose and time dependent removal of the receptor from the cell surface (shedding). In vitro, the antibody suppressed cell growth by blocking cell proliferation and by concomitantly inducing cell death in multiple MET-addicted human tumor cell lines. In mice xenografts, hOA-DN30 induced an impressive reduction of tumor masses, with a wide therapeutic window. Moreover, the antibody showed high therapeutic efficacy against patient-derived xenografts generated from MET-addicted gastric tumors, leading to complete tumor regression and long-lasting effects after treatment discontinuation. Finally, hOA-DN30 showed a highly favorable pharmacokinetic profile and substantial tolerability in Cynomolgus monkeys. CONCLUSIONS: hOA-DN30 unique ability to simultaneously erase cell surface MET and release the 'decoy' receptor extracellular region results in a paramount MET blocking action. Its remarkable efficacy in a large number of pre-clinical models, as well as its pharmacological features and safety profile in non-human primates, strongly envisage a successful clinical application of this novel single-arm MET therapeutic antibody for the therapy of MET-addicted cancers.

Efficacy of CAR-T immunotherapy in MET overexpressing tumors not eligible for anti-MET targeted therapy.

BACKGROUND: Aberrant activation of the MET receptor in cancer is sustained by genetic alterations or, more frequently, by transcriptional upregulations. A fraction of MET-amplified or mutated tumors are sensible to MET targeting agents, but their responsiveness is typically short-lasting, as secondary resistance eventually occurs. Since in the absence of genetic alterations MET is usually not a tumor driver, MET overexpressing tumors are not/poorly responsive to MET targeted therapies. Consequently, the vast majority of tumors exhibiting MET activation still represent an unmet medical need. METHODS: Here we propose an immunotherapy strategy based on T lymphocytes expressing a Chimeric Antigen Receptor (CAR) targeting MET overexpressing tumors of different histotypes. We engineered two different MET-CAR constructs and tested MET-CAR-T cell cytotoxic activity against different MET overexpressing models, including tumor cell lines, primary cancer cells, organoids, and xenografts in immune-deficient mice. RESULTS: We proved that MET-CAR-T exerted a specific cytotoxic activity against MET expressing cells. Cell killing was proportional to the level of MET expressed on the cell surface. While CAR-T cytotoxicity was minimal versus cells carrying MET at physiological levels, essentially sparing normal cells, the activity versus MET overexpressing tumors was robust, significantly controlling tumor cell growth in vitro and in vivo. Notably, MET-CAR-T cells were also able to brake acquired resistance to MET targeting agents in MET amplified cancer cells carrying secondary mutations in downstream signal transducers. CONCLUSIONS: We set and validated at the pre-clinical level a MET-CAR immunotherapy strategy potentially beneficial for cancers not eligible for MET targeted therapy with inhibitory molecules, including those exhibiting primary or secondary resistance.

Monovalency unleashes the full therapeutic potential of the DN-30 anti-Met antibody.

Met, the high affinity receptor for hepatocyte growth factor, is one of the most frequently activated tyrosine kinases in human cancer and a validated target for cancer therapy. We previously developed a mouse monoclonal antibody directed against the extracellular portion of Met (DN-30) that induces Met proteolytic cleavage (receptor "shedding") followed by proteasome-mediated receptor degradation. This translates into inhibition of hepatocyte growth factor/Met-mediated biological activities. However, DN-30 binding to Met also results in partial activation of the Met kinase due to antibody-mediated receptor homodimerization. To safely harness the therapeutic potential of DN-30, its shedding activity must be disassociated from its agonistic activity. Here we show that the DN-30 Fab fragment maintains high affinity Met binding, elicits efficient receptor shedding and down-regulation, and does not promote kinase activation. In Met-addicted tumor cell lines, DN-30 Fab displays potent cytostatic and cytotoxic activity in a dose-dependent fashion. DN-30 Fab also inhibits anchorage-independent growth of several tumor cell lines. In mouse tumorigenesis assays using Met-addicted carcinoma cells, intratumor administration of DN-30 Fab or systemic delivery of a chemically stabilized form of the same molecule results in reduction of Met phosphorylation and inhibition of tumor growth. These data provide proof of concept that monovalency unleashes the full therapeutic potential of the DN-30 antibody and point at DN-30 Fab as a promising tool for Met-targeted therapy.

Human cord blood CD34+ progenitor cells acquire functional cardiac properties through a cell fusion process.

The efficacy of cardiac repair by stem cell administration relies on a successful functional integration of injected cells into the host myocardium. Safety concerns have been raised about the possibility that stem cells may induce foci of arrhythmia in the ischemic myocardium. In a previous work (36), we showed that human cord blood CD34(+) cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34(+) cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process. Umbilical cord blood CD34(+) cells were isolated by a magnetic cell sorting method, transduced with a lentiviral vector carrying the enhanced green fluorescent protein (EGFP) gene, and seeded onto primary cultures of spontaneously beating rat neonatal cardiomyocytes. Cocultured EGFP(+)/CD34(+)-derived cells were analyzed for their electrophysiological features at different time points. After 1 wk in coculture, EGFP(+) cells, in contact with cardiomyocytes, were spontaneously contracting and had a maximum diastolic potential (MDP) of -53.1 mV, while those that remained isolated from the surrounding myocytes did not contract and had a depolarized resting potential of -11.4 mV. Cells were then resuspended and cultured at low density to identify EGFP(+) progenitor cell derivatives. Under these conditions, we observed single EGFP(+) beating cells that had acquired an hyperpolarization-activated current typical of neonatal cardiomyocytes (EGFP(+) cells, -2.24 ± 0.89 pA/pF; myocytes, -1.99 ± 0.63 pA/pF, at -125 mV). To discriminate between cell autonomous differentiation and fusion, EGFP(+)/CD34(+) cells were cocultured with cardiac myocytes infected with a red fluorescence protein-lentiviral vector; under these conditions we found that 100% of EGFP(+) cells were also red fluorescent protein positive, suggesting cell fusion as the mechanism by which cardiac functional features are acquired.