NGR-TNF, a novel vascular-targeting agent, does not induce cytokine recruitment of proangiogenic bone marrow-derived cells.

BACKGROUND: Administration of certain chemotherapy drugs at the maximum tolerated dose, vascular-disrupting agents (VDAs) and irradiation can induce mobilisation and tumour homing of proangiogenic bone marrow-derived cells (BMDCs). Increases in cytokines and chemokines contribute to such mobilisation that eventually promotes tumour (re)growth. NGR-TNF is a vascular-targeting agent in advanced clinical development, coupling the CNGRCG angiogenic vessel-homing peptide with tumour necrosis factor-alpha (TNF). We investigated whether NGR-TNF mobilises host BMDCs and growth factors. METHODS: Blood was obtained from Lewis lung carcinoma and 4T1 tumour-bearing mice at different time points following NGR-TNF, VDA or anti-VEGFR2/flk-1 antibody treatment. Levels of circulating growth factors were assessed by ELISAs. BMDCs were characterised by FACS. Circulating endothelial progenitor cells were defined as CD45(-)/CD13(+)/flk-1(+)/CD117(+)/7AAD(-), Tie2-expressing monocytes as CD45(+)/CD11b(+)/Tie2(+) and myeloid-derived suppressor cells as CD45(+)/CD11b(+)/Gr1(+) cells. RESULTS: NGR-TNF decreases tumour blood vessel density-inducing apoptosis of tumour and tumour endothelial cells. Unlike VDAs, or high-dose NGR-TNF, lower doses of NGR-TNF, comparable to those used in clinical trials, neither mobilise nor recruit to the tumour site proangiogenic BMDCs or induce host growth factors. CONCLUSION: Low-dose NGR-TNF exerts antitumour activity without inducing proangiogenic host responses, conceivably important for preventing/overcoming resistance and designing combination therapeutic strategies.

Phase I and pharmacodynamic study of high-dose NGR-hTNF in patients with refractory solid tumours.

BACKGROUND: NGR-hTNF exploits the peptide asparagine-glycine-arginine (NGR) for selectively targeting tumour necrosis factor (TNF) to CD13-overexpressing tumour vessels. Maximum-tolerated dose (MTD) of NGR-hTNF was previously established at 45 µg m(-2) as 1-h infusion, with dose-limiting toxicity being grade 3 infusion-related reactions. We explored further dose escalation by slowing infusion rate (2-h) and using premedication (paracetamol). METHODS: Four patients entered each of 12 dose levels (n=48; 60-325 µg m(-2)). Pharmacokinetics, soluble TNF receptors (sTNF-R1/sTNF-R2), and volume transfer constant (K(trans)) by dynamic imaging (dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI)) were assessed pre- and post-treatment. RESULTS: Common related toxicity included grade 1/2 chills (58%). Maximum-tolerated dose was not reached. Both C(max) (P<0.0001) and area under the plasma concentration-time curve (P=0.0001) increased proportionally with dose. Post-treatment levels of sTNF-R2 peaked significantly higher than sTNF-R1 (P<0.0001). Changes in sTNF-Rs, however, did not differ across dose levels, suggesting a plateau effect in shedding kinetics. As best response, 12/41 evaluable patients (29%) had stable disease. By DCE-MRI, 28/37 assessed patients (76%) had reduced post-treatment K(trans) values (P<0.0001), which inversely correlated with NGR-hTNF C(max) (P=0.03) and baseline K(trans) values (P<0.0001). Lower sTNF-R2 levels and greater K(trans) decreases after first cycle were associated with improved survival. CONCLUSION: asparagine-glycine-arginine-hTNF can be safely escalated at doses higher than MTD and induces low receptors shedding and early antivascular effects.

Phase II study of NGR-hTNF in combination with doxorubicin in relapsed ovarian cancer patients.

BACKGROUND: The NGR-hTNF (asparagine-glycine-arginine-human tumour necrosis factor) is able to promote antitumour immune responses and to improve the intratumoural doxorubicin uptake by selectively damaging tumour blood vessels. METHODS: Patients progressing after ≥ 1 platinum/taxane-based regimen received NGR-hTNF 0.8 µg m(-2) and doxorubicin 60 mg m(-2) every 3 weeks. Primary endpoint was a Response Evaluation Criteria in Solid Tumors-defined response rate with a target of more than 6 out of 37 responding patients. RESULTS: A total of 37 patients with platinum-free interval lower than 6 months (PFI<6; n=25), or between 6 and 12 months (PFI=6-12; n=12) were enrolled. Median baseline peripheral blood lymphocyte count (PBLC) was 1.6 per ml (interquartile range, 1.2-2.1). In all, 18 patients (49%) received more than 6 cycles. Febrile neutropaenia was registered in one patient (3%). Among 35 assessable patients, 8 (23%; 95% CI 12-39%) had partial response (2 with PFI<6; 6 with PFI=6-12) and 15 (43%) had stable disease (10 with PFI<6; 5 with PFI=6-12). Median progression-free survival (PFS) was 5.0 months for all patients, 3.8 months for patients with PFI<6, and 7.8 months for patients with PFI=6-12. Median overall survival (OS) was 17.0 months. Patients with baseline PBLC higher than the first quartile had improved PFS (P=0.01) and OS (P=0.001). CONCLUSION: Tolerability and activity of this combination warrant further randomised testing in patients with PFI<6. The role of PBLC as a blood-based biomarker deserves further investigation.

Two doses of NGR-hTNF in combination with capecitabine plus oxaliplatin in colorectal cancer patients failing standard therapies.

BACKGROUND: asparagine-glycine-arginine-human tumour necrosis factor (NGR-hTNF), an agent selectively damaging the tumour vasculature, showed a biphasic dose-response curve in preclinical models. Previous phase I trials of NGR-hTNF indicated 0.8 and 45 µg/m(2) as optimal biological and maximum-tolerated dose, respectively. PATIENTS AND METHODS: Two sequential cohorts of 12 colorectal cancer (CRC) patients who had failed standard therapies received NGR-hTNF 0.8 or 45 µg/m(2) in combination with capecitabine-oxaliplatin (XELOX). RESULTS: Median number of prior treatment lines was 3 in the low-dose and 2 in the high-dose cohort. Overall, 21 patients had been pretreated with oxaliplatin-based regimens. No grade 3-4 NGR-hTNF-related toxicities were observed. Grade 1-2 chills were reported in 43% and 40% of cycles in the low-dose and high-dose cohorts, respectively. In the low-dose cohort, one patient achieved a partial response and five had stable disease for a median of 4.6 months. In the high-dose cohort, six patients had stable disease for a median of 3.6 months. Three-month progression-free survival (PFS) rates were 50% and 33% in the low-dose and high-dose cohort, respectively. Three patients in low-dose cohort experienced PFS longer than PFS on last prior therapy. CONCLUSIONS: Both NGR-hTNF doses were safely combined with XELOX in pretreated CRC patients. Hint of activity was apparent only with low-dose NGR-hTNF.

In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice.

Metachromatic leukodystrophy (MLD) is a lipidosis caused by deficiency of arylsulfatase A (ARSA). Although the genetics of MLD are known, its pathophysiology is not understood. The disease leads to progressive demyelination and early death and no effective treatment is available. We used lentiviral vectors to deliver a functional ARSA gene (human ARSA) into the brain of adult mice with germ-line inactivation of the mouse gene encoding ARSA, As2. We report sustained expression of active enzyme throughout a large portion of the brain, with long-term protection from development of neuropathology and hippocampal-related learning impairments. We show that selective degeneration of hippocampal neurons is a central step in disease pathogenesis, and provide evidence that in vivo transfer of ARSA by lentiviral vectors reverts the disease phenotype in all investigated areas. Therefore, in vivo gene therapy offers a unique option for MLD and other storage diseases affecting the central nervous system.

Progress and prospects: graft-versus-host disease.

Graft-versus-host disease (GvHD) is one of the major complications of allogeneic hematopoietic stem cell transplantation, an otherwise highly effective therapeutic modality for patients affected by hematological diseases. The main inducers of GvHD are alloreactive donor T cells, which recognize host antigens presented by recipient cells. The critical role of lymphocytes in GvHD is well documented by the observation that T-cell depletion from the graft prevents GvHD. Unfortunately, the removal of donor lymphocytes from the graft increases the incidence of disease relapse and life-threatening infectious complications. Gene transfer technologies are promising tools to manipulate donor T-cell immunity to enforce graft-versus-tumor/graft-versus-infection while preventing or controlling GvHD. For this purpose, several cell and gene transfer approaches have been investigated at the preclinical level and implemented in clinical trials.

Translation of a retained intron in tyrosinase-related protein (TRP) 2 mRNA generates a new cytotoxic T lymphocyte (CTL)-defined and shared human melanoma antigen not expressed in normal cells of the melanocytic lineage.

We report here the identification of a new shared human melanoma antigen recognized by a human leukocyte antigen (HLA)-A*68011-restricted cytotoxic T lymphocyte clone (CTL 128). The cDNA encoding this antigen is composed of a partially spliced form of the melanocyte differentiation antigen tyrosinase-related protein (TRP)-2, containing exons 1-4 with retention of intron 2 and part of intron 4 (TRP-2-INT2). The sequence coding for the antigenic epitope is located at the 5' end of intron 2 and is available for translation in the same open reading frame of the fully spliced TRP-2 mRNA. This peptide is also recognized by CTL 128 when presented by the HLA-A*3301, a member of the HLA-A3-like supertype that includes the HLA-A*68011. Quantitative reverse transcription PCR analysis carried out on total and/or cytoplasmic mRNA demonstrated that, in contrast to the fully spliced TRP-2 mRNA expressed in melanomas, normal skin melanocytes, and retina, the TRP-2-INT2 mRNA could be detected at significant levels in melanomas but not in normal cells of the melanocytic lineage. Instead, in these normal samples, both the spliced and the unspliced transcript of gp100 were expressed at high levels. Absence of endogenous TRP-2-INT2 expression in melanocytes was also confirmed by lack of recognition of HLA-A*68011-transduced, TRP-2(+) melanocyte lines by CTL 128. These results indicate that a partially spliced form of a differentiation antigen mRNA, present in the cytoplasmic compartment of neoplastic but not normal cells of the melanocytic lineage, can be the source of a melanoma-restricted T cell epitope.

Expression of human adenosine deaminase in nonhuman primates after retrovirus-mediated gene transfer.

Primate bone marrow cells were infected with a retroviral vector carrying the genes for human adenosine deaminase (h-ADA) and bacterial neomycin resistance (neor). The infected cells were infused back into the lethally irradiated donor animals. Several monkeys fully reconstituted and were shown to express the h-ADA and neor genes at low levels in their recirculating hematopoietic cells for short periods of time.

Activity and safety of NGR-hTNF, a selective vascular-targeting agent, in previously treated patients with advanced hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly vascularised and poor-prognosis tumour. NGR-hTNF is a vascular-targeting agent consisting of human tumour necrosis factor-alpha fused to the tumour-homing peptide NGR, which is able to selectively bind an aminopeptidase N overexpressed on tumour blood vessels. METHODS: Twenty-seven patients with advanced-stage disease resistant to either locoregional (59%; range, 1-3), systemic treatments (52%; range, 1-3) or both (33%) received NGR-hTNF 0.8 microg m(-2) once every 3 weeks. The primary aim of the study was progression-free survival (PFS). RESULTS: No grade 3-4 treatment-related toxicities were noted. Common toxicity included mild-to-moderate, short-lived chills (63%). Median PFS was 2.3 months (95% CI: 1.7-2.9). A complete response ongoing after 20 months was observed in a sorafenib-refractory patient and a partial response in a Child-Pugh class-B patient, yielding a response rate of 7%. Six patients (22%) experienced stable disease. The disease control rate (DCR) was 30% and was maintained for a median PFS time of 4.3 months. Median survival was 8.9 months (95% CI: 7.5-10.2). In a subset of 12 sorafenib-resistant patients, the response rate was 8% and the median survival was 9.5 months. CONCLUSION: NGR-hTNF was well tolerated and showed single-agent activity in HCC. Further investigation in HCC is of interest.

Phase Ib study of NGR-hTNF, a selective vascular targeting agent, administered at low doses in combination with doxorubicin to patients with advanced solid tumours.

BACKGROUND: Asparagine-glycine-arginine-human tumour necrosis factor (NGR-hTNF) is a vascular targeting agent exploiting a tumour-homing peptide (NGR) that selectively binds to aminopeptidase N/CD13, overexpressed on tumour blood vessels. Significant preclinical synergy was shown between low doses of NGR-TNF and doxorubicin. METHODS: The primary aim of this phase I trial was to verify the safety of low-dose NGR-hTNF combined with doxorubicin in treating refractory/resistant solid tumours. Secondary objectives included pharmacokinetics (PKs), pharmacodynamics, and clinical activity. In all 15 patients received NGR-hTNF (0.2-0.4-0.8-1.6 microg m(-2)) and doxorubicin (60-75 mg m(-2)), both given intravenously every 3 weeks. RESULTS: No dose-limiting toxicity occurred and the combination was well tolerated. Around two cases of neutropenic fevers, lasting 2 days, and two cases of cardiac ejection-fraction drops, one asymptomatic and the other symptomatic, were registered. Only 11% of the adverse events were related to NGR-hTNF and were short-lasting and mild-to-moderate in severity. There was no apparent PK interaction and the shedding of soluble TNF-receptors did not increase to 0.8 microg m(-2). One partial response (7%), at dose level 0.8 microg m(-2), and 10 stable diseases (66%), lasting for a median duration of 5.6 months, were observed. CONCLUSIONS: NGR-hTNF plus doxorubicin was administered safely and showed promising activity in patients pre-treated with anthracyclines. The dose level of 0.8 microg m(-2) NGR-hTNF plus doxorubicin 75 mg m(-2) was selected for phase II development.

Hematopoietic histoincompatibility reactions by NK cells in vitro: model for genetic resistance to marrow grafts.

In certain strains of mice, bone marrow grafts from parental donors fail to grow in first-generation hybrid mice. This "hybrid resistance" of nonsensitized F1 hybrid mice to the engraftment of parental hematopoietic transplants contradicts the classical laws of transplantation and is dependent on a radioresistant but immunogenetically specific effector mechanism. Studies in a new in vitro model reveal that committed hematopoietic precursors of parental origin can be inactivated by direct contact with natural killer-like splenic effectors from F1 mice. The reaction requires genetically restricted recognition, since only parental competitors syngeneic to the target bone marrow cells partially reversed this inactivation. Models of this type may be useful in studying the possible role of natural resistance in bone marrow transplantation in humans.

An in vivo model of somatic cell gene therapy for human severe combined immunodeficiency.

Deficiency of adenosine deaminase (ADA) results in severe combined immunodeficiency (SCID), a candidate genetic disorder for somatic cell gene therapy. Peripheral blood lymphocytes from patients affected by ADA- SCID were transduced with a retroviral vector for human ADA and injected into immunodeficient mice. Long-term survival of vector-transduced human cells was demonstrated in recipient animals. Expression of vector-derived ADA restored immune functions, as indicated by the presence in reconstituted animals of human immunoglobulin and antigen-specific T cells. Retroviral vector gene transfer, therefore, is necessary and sufficient for development of specific immune functions in vivo and has therapeutic potential to correct this lethal immunodeficiency.

Gene therapy in peripheral blood lymphocytes and bone marrow for ADA- immunodeficient patients.

Adenosine deaminase (ADA) deficiency results in severe combined immunodeficiency, the first genetic disorder treated by gene therapy. Two different retroviral vectors were used to transfer ex vivo the human ADA minigene into bone marrow cells and peripheral blood lymphocytes from two patients undergoing exogenous enzyme replacement therapy. After 2 years of treatment, long-term survival of T and B lymphocytes, marrow cells, and granulocytes expressing the transferred ADA gene was demonstrated and resulted in normalization of the immune repertoire and restoration of cellular and humoral immunity. After discontinuation of treatment, T lymphocytes, derived from transduced peripheral blood lymphocytes, were progressively replaced by marrow-derived T cells in both patients. These results indicate successful gene transfer into long-lasting progenitor cells, producing a functional multilineage progeny.

HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia.

In allogeneic bone marrow transplantation (allo-BMT), donor lymphocytes play a central therapeutic role in both graft-versus-leukemia (GvL) and immune reconstitution. However, their use is limited by the risk of severe graft-versus-host disease (GvHD). Eight patients who relapsed or developed Epstein-Barr virus-induced lymphoma after T cell-depleted BMT were then treated with donor lymphocytes transduced with the herpes simplex virus thymidine kinase (HSV-TK) suicide gene. The transduced lymphocytes survived for up to 12 months, resulting in antitumor activity in five patients. Three patients developed GvHD, which could be effectively controlled by ganciclovir-induced elimination of the transduced cells. These data show that genetic manipulation of donor lymphocytes may increase the efficacy and safety of allo-BMT and expand its application to a larger number of patients.

Identification of a promiscuous T-cell epitope encoded by multiple members of the MAGE family.

One of the major limitations of tumor-specific vaccination is the generation of antigen-loss variants that are able to escape the immune response elicited by a monoantigenic peptide epitope. Here, we report the identification of a new HLA-B*3701-restricted epitope shared by four different members of the MAGE family. Peripheral blood lymphocytes isolated from a melanoma patient were stimulated in vitro with the autologous HLA-negative melanoma line transfected with autologous HLA B*3701 molecule. This protocol led to the induction of tumor-specific, B*3701-restricted CTLs specific for a peptide epitope encoded by codons 127-136 of the gene MAGE-1. The same epitope is also encoded by the homologous region of three other members of the MAGE family, MAGE-2, -3, and -6. Consistent with the notion that the peptide encoded by MAGE-1 codons 127-136 is, indeed, processed from the proteins encoded by all four MAGE family members, the CTLs were able to specifically recognize Cos-7 cells cotransfected with HLA-B*3701 and any of these MAGE genes. Moreover, the CTLs also recognized a MAGE-6-positive melanoma line transfected with the B*3701 molecule. These findings allow the inclusion of a new set of tumor patients into clinical cancer vaccination trials. Furthermore, they suggest that some promiscuous peptide epitopes shared by different members of the MAGE family might be less prone to escape the immune response by generation of MAGE antigen loss variants.

The DAM gene family encodes a new group of tumor-specific antigens recognized by human leukocyte antigen A2-restricted cytotoxic T lymphocytes.

The DAM family of genes has a high degree of homology with MAGE, both in nucleotide sequence and in neoplastic tissue-specific expression. This study describes, for the first time, the identification of CTLs specific for a peptide epitope encoded by DAM genes. A human leukocyte antigen (HLA)-A2-restricted CTL clone was raised against a peptide, D10/6-271, encoded by codons 271-279 in the DAM cDNA. The corresponding peptide in the MAGE-3 sequence, M3-271, has been shown previously to be a natural T-cell epitope for HLA-A2-restricted CTLs recognizing the MAGE-3 protein. The D10/6-271-specific CTL clone required approximately 3 nM exogenous peptide for half-maximal lysis of target cells and was able to specifically recognize endogenous DAM antigen on HLA-A2+ melanoma cells infected with a vaccinia vector recombinant for gene DAM-6. These data suggest that DAM genes might encode a new group of tumor-specific antigens useful for the design of specific antitumor vaccines.

Human proinsulin production in primary rat hepatocytes after retroviral vector gene transfer.

The development of autologous somatic cells, engineered for the synthesis and release of human insulin under physiological stimuli, would certainly represent a major breakthrough in the therapy of insulin-dependent diabetes mellitus. We generated a retroviral vector containing the human proinsulin cDNA and the gene coding for the human nerve growth factor receptor for quantitative analysis of transduced cells. Primary rat hepatocytes were selected as target cells because of the constitutive expression of the pancreatic beta-cell glucose transporter GLUT-2 and the glycolitic enzyme glucokinase. Appropriate conditions for culture and retroviral transduction are described. The highest transduction efficiency, evaluated as percentage of LNGFr expressing cells was obtained by repeated infection cycles (40+/-10%). Human proinsulin accumulated in the culture medium of transduced rat hepatocytes (mean+/-SD): 18.1+/-7.9 (range 8.7-36.4) ng/24h/10(6) cells. Primary rat hepatocytes can be efficiently transduced by a retroviral vector and the de novo synthesis of human proinsulin can be induced. Primary cultured hepatocytes represent an useful model to test retroviral constructs engineered for the glucose-inducible expression of insulin under the control of liver-specific promoters.