A. Lambiase

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 μg 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 μg m−2. One partial response (7%), at dose level 0.8 μg 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 μg m−2 NGR–hTNF plus doxorubicin 75 mg m−2 was selected for phase II development.

Defining the optimal biological dose of NGR-hTNF, a selective vascular targeting agent, in advanced solid tumours.

BACKGROUND NGR-hTNF consists of human tumour necrosis factor-alpha (hTNF-alpha) fused to the tumour-homing peptide NGR, a ligand of an aminopeptidase N/CD13 isoform, which is overexpressed on endothelial cells of newly formed tumour blood vessels. NGR-TNF showed a biphasic dose-response curve in preclinical models. This study exploring the low-dose range aimed to define safety and optimal biological dose of NGR-hTNF. PATIENTS AND METHODS Pharmacokinetics, plasma biomarkers and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) were evaluated at baseline and after each cycle in 16 patients enrolled at four doubling-dose levels (0.2-0.4-0.8-1.6 microg/m(2)). NGR-hTNF was given intravenously as 1-h infusion every 3 weeks (q3w). Tumour response was assessed q6w. RESULTS Eighty-three cycles (median, 2; range, 1-29) were administered. Most frequent treatment-related toxicity was grade 1-2 chills (69%), occurring during the first infusions. Only one patient treated at 1.6 microg/m(2) had a grade 3 drug-related toxicity (chills and dyspnoea). Both C(max) and AUC increased proportionally with dose. No shedding of soluble TNF-alpha receptors was observed up to 0.8 microg/m(2). Seventy-five percent of DCE-MRI assessed patients showed a decrease over time of K(trans), which was more pronounced at 0.8 microg/m(2). Seven patients (44%) had stable disease for a median time of 5.9 months, including a colon cancer patient who experienced an 18-month progression-free time. CONCLUSION Based on tolerability, soluble TNF-receptors kinetics, anti-vascular effect and disease control, NGR-hTNF 0.8 microg/m(2) will be further developed either as single-agent or with standard chemotherapy.

Phase I Study of NGR-hTNF, a Selective Vascular Targeting Agent, in Combination with Cisplatin in Refractory Solid Tumors

Purpose: NGR-hTNF exploits the tumor-homing peptide asparagine-glycine-arginine (NGR) for selectively targeting TNF-α to an aminopeptidase N overexpressed on cancer endothelial cells. Preclinical synergism with cisplatin was displayed even at low doses. This study primarily aimed to explore the safety of low-dose NGR-hTNF combined with cisplatin in resistant/refractory malignancies. Secondary aims included pharmacokinetics (PKs), pharmacodynamics, and activity. Experimental Design: NGR-hTNF was escalated using a doubling-dose scheme (0.2–0.4–0.8–1.6 μg/m2) in combination with fixed-dose of cisplatin (80 mg/m2), both given intravenously once every three weeks. PKs and circulating TNF-receptors (sTNF-Rs) were assessed over the first three cycles. Results: Globally, 22 patients (12 pretreated with platinum) received a range of one to ten cycles. Consistently with the low-dose range tested, maximum-tolerated dose was not reached. No dose-limiting toxicities (DLTs) were observed at 0.2 (n = 4) and 0.4 μg/m2 (n = 3). One DLT (grade 3 infusion-related reaction) was observed at 0.8 μg/m2. This dose cohort was expanded to six patients without further DLTs. No DLTs were noted also at 1.6 μg/m2 (n = 3). NGR-hTNF exposure increased dose-proportionally without apparent PK interactions with cisplatin. No shedding of sTNF-Rs was detected up to 0.8 μg/m2. At the dose level of 0.8 μg/m2, expanded to 12 patients for activity assessment, a platinum-pretreated lung cancer patient achieved a partial response lasting more than six months and five patients maintained stable disease for a median time of 5.9 months. Conclusions: The combination of NGR-hTNF 0.8 μg/m2 with cisplatin 80 mg/m2 showed favorable toxicity profile and promising antitumor activity. Clin Cancer Res; 17(7); 1964–72. ©2011 AACR.

Phase I Clinical and Magnetic Resonance Imaging Study of the Vascular Agent NGR-hTNF in Patients with Advanced Cancers (European Organization for Research and Treatment of Cancer Study 16041)

Purpose: This phase I trial investigating the vascular targeting agent NGR-hTNF aimed to determine the (a) dose-limiting toxicities, (b) maximum tolerated dose (MTD), (c) pharmacokinetics and pharmacodynamics, (d) vascular response by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and (e) preliminary clinical activity in solid tumors. Experimental Design: NGR-hTNF was administered once every 3 weeks by a 20- to 60-minute i.v. infusion to cohorts of three to six patients with solid tumors in escalating doses. Pharmacokinetic and pharmacodynamic analyses in blood were done during the first four cycles. DCE-MRI was done in cycle 1 at baseline and 2 hours after the start of the infusion. Results: Sixty-nine patients received a total of 201 cycles of NGR-hTNF (0.2-60 μg/m2). Rigors and fever were the most frequently observed toxicities. Four dose-limiting toxicities were observed (at doses of 1.3, 8.1, and 60 μg/m2), of which three were infusion related. The MTD was 45 μg/m2. The mean apparent terminal half-life ranged from 0.963 to 2.08 hours. DCE-MRI results of tumors showed a vascular response to NGR-hTNF. No objective responses were observed, but 27 patients showed stable disease with a median duration of 12 weeks. Conclusions: NGR-hTNF was well tolerated. The MTD was 45 μg/m2 administered in 1 hour once every 3 weeks. DCE-MRI results showed the antivascular effect of NGR-hTNF. These findings call for further research for defining the optimal biological dose and clinical activity of NGR-hTNF as a single agent or in combination with cytotoxic drugs. Clin Cancer Res; 16(4); 1315–23

Phase II study of NGR-hTNF, a selective vascular targeting agent, in patients with metastatic colorectal cancer after failure of standard therapy

BACKGROUND NGR-hTNF consists of human tumour necrosis factor (hTNF) fused with the tumour-homing peptide Asp-Gly-Arg (NGR), which is able to selectively bind an aminopeptidase N overexpressed on tumour blood vessels. Preclinical antitumour activity was observed even at low doses. We evaluated the activity and safety of low-dose NGR-hTNF in colorectal cancer (CRC) patients failing standard therapies. PATIENTS AND METHODS Thirty-three patients with progressive disease at study entry received NGR-hTNF 0.8 μg/m(2) given intravenously every 3 weeks. The median number of prior treatment regimens was three (range, 2-5). One-quarter of patients had previously received four or more regimens and two-thirds targeted agents. Progression-free survival (PFS) was the primary study objective. RESULTS NGR-hTNF was well tolerated. No treatment-related grade 3 to 4 toxicities were detected, most common grade 1 to 2 adverse events being short-lived, infusion-time related chills (50.0%). One partial response and 12 stable diseases were observed, yielding a disease control rate of 39.4% (95% CI, 22.9-57.8%). Median PFS and overall survival were 2.5 months (95% CI, 2.1-2.8) and 13.1 months (95% CI, 8.9-17.3), respectively; whereas in patients who achieved disease control the median PFS and overall survival were 3.8 and 15.4 months, respectively. In an additional cohort of 13 patients treated at same dose with a weekly schedule, there was no increased toxicity and 2 patients experienced PFS longer than 10 months. CONCLUSION Based on tolerability and preliminary evidence of disease control in heavily pretreated CRC patients, NGR-hTNF deserves further evaluation in combination with standard chemotherapy.

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

Antigen exposure and differentiation phenotype influence long-term persistence of memory T cells after hematopoietic stem cell transplant. Committing T cells to memory Adoptive cell transfer is an increasingly successful therapy for a variety of diseases; however, little is known about what regulates the survival of these cells in humans. Now, Oliveira et al. leverage patients who have received genetically modified hematopoietic stem cells to track T cells over time. They found labeled effector memory, central memory, and stem memory T cells 2 to 14 years after infusion in all patients. Antigen recognition was critical in driving persistence and expansion. The clones that survived long-term appeared to initiate preferentially from central and stem cell memory T cell populations. These data suggest that the original phenotype of infused cells may influence long-term persistence of adoptively transferred cells. 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 TCRβ 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.

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.

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 μg 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.

Clinical and immunologic responses in melanoma patients vaccinated with MAGE-A3-genetically modified lymphocytes.

Cancer vaccines have recently been shown to induce some clinical benefits. The relationship between clinical activity and anti‐vaccine T cell responses is somewhat controversial. Indeed, in many trials it has been documented that the induction of vaccine‐specific T cells exceeds the clinical responses observed. Here, we evaluate immunological and clinical responses in 23 MAGE‐A3+ melanoma patients treated with autologous lymphocytes genetically engineered to express the tumor antigen MAGE‐A3 and the viral gene product thymidine kinase of the herpes simplex virus (HSV‐TK). HSV‐TK was used as safety system in case of adverse events and as tracer antigen to monitor the immune competence of treated patients. The increase of anti‐TK and anti‐MAGE‐A3 T‐cells after vaccination was observed in 90 and 27% of patients, respectively. Among 19 patients with measurable disease, we observed a disease control rate of 26.3%, with one objective clinical response, and four durable, stable diseases. Three patients out of five with no evidence of disease (NED) at the time of vaccination remained NED after 73+, 70+ and 50+ months. Notably, we report that only patients experiencing MAGE‐A3‐specific immune responses showed a clinical benefit. Additionally, we report that responder and non‐responder patients activate and expand T cells against the tracer antigen TK in a similar way, suggesting that local rather than systemic immune suppression might be involved in limiting clinically relevant antitumor immune responses.

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 (Ktrans) 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 Cmax (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 Ktrans values (P<0.0001), which inversely correlated with NGR–hTNF Cmax (P=0.03) and baseline Ktrans values (P<0.0001). Lower sTNF-R2 levels and greater Ktrans 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.