Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment.

Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don't respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/ß2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development.

First-in-human phase I study of ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-ß2), in subjects with open-angle glaucoma undergoing glaucoma filtration surgery.

PURPOSE: To evaluate the safety and tolerability of intravitreal ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-ß2), in patients with primary open angle glaucoma (POAG) undergoing trabeculectomy (TE; glaucoma filtration surgery). METHODS: In this prospective phase I trial glaucoma patients scheduled for TE with mitomycin C (MMC) received a single intravitreal injection of ISTH0036 at the end of surgery in escalating total doses of 6.75 µg, 22.5 µg, 67.5 µg or 225 µg, resulting in calculated intraocular ISTH0036 concentrations in the vitreous humor of approximately 0.3 µM, 1 µM, 3 µM or 10 µM after injection, respectively. Outcomes assessed included: type and frequency of adverse events (AEs), intraocular pressure (IOP), numbers of interventions post trabeculectomy, bleb survival, visual acuity, visual field, electroretinogram (ERG), slit lamp biomicroscopy and optic disc assessment. RESULTS: In total, 12 patients were treated in the 4 dose groups. Main ocular AEs observed were corneal erosion, corneal epithelium defect, or too high or too low IOP, among others. No AE was reported to be related to ISTH0036. All other safety-related analyses did not reveal any toxicities of concern, either. The mean medicated preoperative IOP at decision time-point for surgery was 27.3 mmHg +/- 12.6 mmHg (SD). Mean IOP (±SD) for dose levels 1, 2, 3, and 4 were at Day 43 9.8 mmHg ± 1.0 mmHg, 11.3 mmHg ± 6.7 mmHg, 5.5 mmHg ± 3.0 mmHg and 7.5 mmHg ± 2.3 mmHg SD; and at Day 85 9.7 mmHg ± 3.3 mmHg, 14.2 mmHg ± 6.5 mmHg, 5.8 mmHg ± 1.8 mmHg and 7.8 mmHg ± 0.6 mmHg, respectively. In contrast to IOP values for dose levels 1 and 2, IOP values for dose levels 3 and 4 persistently remained below 10 mmHg throughout the observation period. CONCLUSION: This first-in-human trial demonstrates that intravitreal injection of ISTH0036 at the end of TE is safe. Regarding IOP control, single-dose ISTH0036 administration of 67.5 µg or 225 µg at the time of TE resulted in IOP values persistently < 10 mmHg over the three month postoperative observation period.

Bispecific T-Cell Engager (BiTE) Antibody Construct Blinatumomab for the Treatment of Patients With Relapsed/Refractory Non-Hodgkin Lymphoma: Final Results From a Phase I Study.

PURPOSE: Blinatumomab is a CD19/CD3 BiTE (bispecific T-cell engager) antibody construct for the treatment of Philadelphia chromosome-negative acute B-lymphoblastic leukemia. We evaluated blinatumomab in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL). PATIENTS AND METHODS: This 3 + 3 design, phase I dose-escalation study determined adverse events and the maximum tolerated dose (MTD) of continuous intravenous infusion blinatumomab in patients with relapsed/refractory NHL. Blinatumomab was administered over 4 or 8 weeks at seven different dose levels (0.5 to 90 µg/m(2)/day). End points were incidence of adverse events, pharmacokinetics, pharmacodynamics, and overall response rate. RESULTS: Between 2004 and 2011, 76 heavily pretreated patients with relapsed/refractory NHL, who included 14 with diffuse large B-cell lymphoma, were enrolled; 42 received treatment in the formal dose-escalation phase. Neurologic events were dose limiting, and 60 µg/m(2)/day was established as the MTD. Thirty-four additional patients were recruited to evaluate antilymphoma activity and strategies for mitigating neurologic events at a prespecified MTD. Stepwise dosing (5 to 60 µg/m(2)/day) plus pentosan polysulfate SP54 (n = 3) resulted in no treatment discontinuations; single-step (n = 5) and double-step (n = 24) dosing entailed two and seven treatment discontinuations due to neurologic events, respectively. Grade 3 neurologic events occurred in 22% of patients (no grade 4/5). Among patients treated at 60 µg/m(2)/day (target dose; n = 35), the overall response rate was 69% across NHL subtypes and 55% for diffuse large B-cell lymphoma (n = 11); median response duration was 404 days (95% CI, 207 to 1,129 days). CONCLUSION: In this phase I study of relapsed/refractory NHL, continuous infusion with CD19-targeted immunotherapy blinatumomab at various doses and schedules was feasible, with an MTD of 60 µg/m(2)/day. Single-agent blinatumomab showed antilymphoma activity.

TGF-ß isoforms in cancer: Immunohistochemical expression and Smad-pathway-activity-analysis in thirteen major tumor types with a critical appraisal of antibody specificity and immunohistochemistry assay validity.

The literature on TGF-ß in cancer including data on the expression or activation of TGF-ß pathway components in specific tumors types is steadily growing. However, no systematic and uniform analysis exists reporting expression levels of the main TGF-ß pathway components across the most frequent tumor types. We used a standardized immunohistochemical assay investigating TGF-ß isoform expression and pathway activation across 13 different tumor types and corresponding non-neoplastic tissues. The study was performed on tissue microarrays allowing for the parallel analysis of a total of 1638 human tumor samples. TGF-ß1, TGF-ß2 and p-Smad2/3 were substantially expressed in multiple cancers widening the options for TGF-ß isoform directed therapies. Of note, TGF-ß antigens appear to be expressed in an individual manner pointing towards a need for patient preselection for TGF-ß isoform specific treatment. Yet, a thorough investigation of antibody specificity and assay validity revealed that immunohistochemistry did not correlate with other detection methods on mRNA or protein level in all instances. As such, with the currently available means (i.e. antibodies tested) a stratification of patients within clinical trials for TGF-ß directed antisense therapies based upon TGF-ß immunohistochemistry alone has to be interpreted with caution and should be carefully evaluated in combination with other parameters.

Preclinical anti-myeloma activity of the novel HDAC-inhibitor JNJ-26481585.

Pharmacological inhibitors of histone deacetylases (HDACs) are currently being developed and tested as anti-cancer agents and may be useful to enhance the therapeutic efficiency of established anti-myeloma treatments. This study preclinically evaluated the effects of the 'second generation' pan-HDAC inhibitor JNJ-26481585 on human multiple myeloma (MM) cells from established cell lines and primary MM samples (n=42). Molecular responses in both groups of MM cells included histone acetylation, a shift in Bcl2-family members towards proapoptotic bias, attenuation of growth and survival pathway activity and Hsp72 induction. Mcl-1 depletion and Hsp72 induction were the most reliable features observed in JNJ-26481585-treated primary MM samples. The drug alone effectively induced myeloma cell death at low nanomolar concentrations. In vitro combination of JNJ-26481585 with anti-myeloma therapeutic agents generally resulted In effects close to additivity. In view of the favourable activity of this novel HDAC-inhibitor towards primary myeloma cells further evaluation in a clinical setting is warranted.

Tumor regression in cancer patients by very low doses of a T cell-engaging antibody.

Previous attempts have shown the potential of T cells in immunotherapy of cancer. Here, we report on the clinical activity of a bispecific antibody construct called blinatumomab, which has the potential to engage all cytotoxic T cells in patients for lysis of cancer cells. Doses as low as 0.005 milligrams per square meter per day in non-Hodgkin's lymphoma patients led to an elimination of target cells in blood. Partial and complete tumor regressions were first observed at a dose level of 0.015 milligrams, and all seven patients treated at a dose level of 0.06 milligrams experienced a tumor regression. Blinatumomab also led to clearance of tumor cells from bone marrow and liver. T cell-engaging antibodies appear to have therapeutic potential for the treatment of malignant diseases.

A phase I study with adecatumumab, a human antibody directed against epithelial cell adhesion molecule, in hormone refractory prostate cancer patients.

AIM OF THE STUDY: Adecatumumab (also known as MT201) is a human recombinant IgG1 monoclonal antibody binding with low affinity to epithelial cell adhesion molecule (EpCAM). To explore safety, pharmacokinetics and pharmacodynamics of adecatumumab, a phase I trial in patients with hormone refractory prostate cancer (HRPC) was performed. METHODS: Twenty patients were treated with two adecatumumab infusions on days 0 and 14 in cohorts with doses of ten up to 262 mg/m2. RESULTS: Adecatumumab was well tolerated at all doses tested, and no maximum tolerated dose reached. Most adverse events were mild or moderate with pyrexia and nausea being most frequent. The highest dose of adecatumumab induced shortly after infusion robust and transient increases of TNF-alpha serum levels. At all doses, significant transient declines of peripheral natural killer cells were observed shortly after antibody infusions. Adecatumumab had a serum half-life of 15 days, and immune responses to the antibody were not detected. CONCLUSIONS: A benign safety profile, long serum half-life and low immunogenicity do warrant further exploration of adecatumumab for treatment of EpCAM-expressing neoplasia.

One single dose of rituximab added to a standard regimen of CHOP in primary treatment of follicular lymphoma appears to result in a high clearance rate from circulating bcl-2/IgH positive cells: Is the end of molecular monitoring near?

Accurate monitoring of the t(14;18) translocation is regarded as highly desirable in patients with follicular lymphoma (FL) as absence of bcl-2/IgH positive cells has been correlated with a reduced risk of recurrence and, more recently, pre-treatment t(14;18) load with response to rituximab (R; Blood 2004;103:4416-23). With the arrival of R clinical and molecular remission rates for various lymphoma entities improved significantly, creating the need to carefully review and reassess the role of PCR negativity for clinical outcome, specifically when considering the prolonged presence of the drug as compared to chemotherapy. To determine the rate of molecular clearance achieved by the addition of different doses of R 16 newly diagnosed, t(14;18) positive patients with FL (Ann Arbor stages III/IV) were investigated before, during and after primary chemotherapy with six cycles of CHOP combined with varying (1, 3 or 6) cycle numbers of R (varR1-, varR3- or varR6-CHOP, respectively) regarding molecular remission status. For this purpose classic nested PCR as well as a newly established RQ-PCR employing juxtaposed hybridisation probes were employed to assess molecular remission prior, during and post therapy. Interestingly, administering just a single cycle of R (varR1-CHOP) in combination with a standard regimen of CHOP resulted in rapid and effective clearance of t(14;18) carrying cells from the peripheral blood of 4/5 patients in this treatment group. 6/8 (6/8) varR1-/varR3-CHOP patients were fully cleared and 8/8 (7/8) var6-CHOP patients as assessed by RQ- (nested) PCR. This indicates the high clearance capacity of this combination therapy approach even when adding a very low cycle number of R (1 and 3, respectively) to CHOP in primary therapy of FL. In summary, the relationship established between molecular clearance and minimal residual disease/risk of recurrence may have to be redefined in the times of R. Upcoming large prospective trials will have to elucidate to what degree the clearance of peripheral blood from t(14;18) positive cells can still be regarded as informative regarding absence of minimal residual disease, remission status and/or risk of recurrence.

T-cell activation and B-cell depletion in chimpanzees treated with a bispecific anti-CD19/anti-CD3 single-chain antibody construct.

BscCD19xCD3 is a bispecific single-chain antibody construct with exceptional cytotoxic potency in vitro and in vivo. Here, we have investigated the biological activity of bscCD19xCD3 in chimpanzee, the only animal species identified in which bscCD19xCD3 showed bispecific binding, redirected B-cell lysis and cytokine production comparable to human cells. Pharmacokinetic analysis following 2-h intravenous infusion of 0.06, 0.1 or 0.12 mug/kg of bscCD19xCD3 as part of a dose escalation study in a single female chimpanzee revealed a half-life of approximately 2 h and elimination of the bispecific antibody from circulation within approximately 8 h after the end of infusion. This short exposure to bscCD19xCD3 elicited a transient increase in serum levels of IFNgamma, IL-6, IL-2, soluble CD25, and transiently upregulated expression of CD69 and MHC class II on CD8-positive cells. Cytokine release and upregulation of T-cell activation markers were not observed with vehicle controls. A multiple-dose study using 5 weekly doses of 0.1 mug/kg in two animals also showed transient cytokine release and an activation of peripheral T cells with a first-dose effect, accompanied by a transient lymphopenia. While oscillations of T-cell counts were relatively even during repeated treatments, the amplitudes of peripheral B cells declined with every infusion, which was not observed in a vehicle control animal. Our data show that bscCD19xCD3 can be safely administered to chimpanzees at dose levels that cause fully reversible T-cell activation and, despite a very short exposure time, cumulative loss of peripheral B lymphocytes. A clinical trial testing prolonged administration of bscCD19xCD3 (MT103) for improving efficacy is currently ongoing.

Expression of epithelial cellular adhesion molecule (Ep-CAM) in chronic (necro-)inflammatory liver diseases and hepatocellular carcinoma.

Epithelial cell adhesion molecule (Ep-CAM) is expressed in a several epithelial tissues and carcinomas, but not on mature hepatocytes. Here, we analysed the expression of Ep-CAM in 230 patients suffering from various liver diseases like chronic hepatitis B and C (HBV and HCV infection), chronic autoimmune hepatitis (AIH), chronic alcoholic liver disease (ALD), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), hereditary hemochromatosis and dysplastic nodules (DNs) as well as hepatocellular carcinomas (HCCs) and cholangiocellular carcinomas (CCCs) by immunohistochemistry. De novo hepatocellular Ep-CAM expression was found in 75.9% of ALD (22/29), 63.6% of HCV (21/33) and 55.6% of each AIH and HBV cases (5/9 and 15/27, respectively). Lower Ep-CAM expression levels were observed for primary sclerosing liver diseases (PBC and PSC) with 25% (3/12) and 7.7% (1/13) of cases. Moreover, only 14.3% of HCCs (9/63) manifested expression, while all CCCs showed strong Ep-CAM expression (5/5). For DNs and hereditary hemochromatosis, Ep-CAM expression was found in 10 and 50% (3/30 and 2/4), respectively. In HBV and HCV, Ep-CAM expression correlated significantly with inflammatory activity as assessed by histological parameters and to the extent of fibrosis. In addition, for HCV also transaminase levels correlated significantly with Ep-CAM expression. Our results indicate that de novo Ep-CAM expression in hepatocytes is frequent in inflammatory liver diseases and is potentially linked to regenerative activity. CCCs and Ep-CAM positive HCCs may represent an attractive target group for Ep-CAM-directed immunotherapies, yet unwanted toxicity may limit the use of such strategies due to Ep-CAM expression in biliary epithelium and several chronic liver diseases such as HBV-and HCV-hepatitis.

The bcl-2/IgH rearrangement in a population of 204 healthy individuals: occurrence, age and gender distribution, breakpoints, and detection method validity.

This study assessed prevalence, frequency, age and gender distribution and breakpoint locations, and detection method validity for the bcl-2/IgH rearrangement in 204 healthy individuals. For this purpose, both classic two-step, nested, semi-quantitative PCR as well as a newly established sequence-specific, hybridization probe-based real-time quantitative PCR (RQ-PCR) were employed and tested for their sensitivity and specificity for detecting t(14;18) positive cells in healthy blood donors. Interestingly, almost a quarter (24%; 39/204) of all healthy individuals carried the translocation, confirming data of one large prior report [Summers KE, Goff LK, Wilson AG, Gupta RK, Lister TA, Fitzgibbon J. Frequency of the Bcl-2/IgH rearrangement in normal individuals: implications for the monitoring of disease in patients with follicular lymphoma. J Clin Oncol 2001;19(2):420-4]. Regarding presence as well as frequency of the translocation, no correlation to age (mean frequency 2.0:10(4), with a median of or=40 years) nor gender was detected. Comparing the two PCR approaches, a 95.1% concordance (194/204) regarding t(14;18) detection was determined for nested and RQ-PCR, with nested PCR being slightly more sensitive (reproducible detection limit l:10(5) cells versus 1:10(4); maximum detection limit l:10(6) versus 1:10(5)). Sequence analysis confirmed individual breakpoints for all samples analyzed (29/29), indicating detection validity for both PCR approaches and ruling out contamination. The breakpoint location distribution pattern appeared to be comparable to the pattern seen with follicular lymphoma (FL) patient collectives. In conclusion, clonal bcl-2/IgH rearrangements are indeed a very frequent observation in healthy individuals, and appear to be independent of age and gender in regard to presence and frequency. This represents a conflicting finding in context of potential biological significance, and presents a potential disruptive factor for minimal residual disease (MRD) monitoring in FL patients. Prospective future trials will have to clarify the biological significance of this important observation.

Significant thrombocytopenia associated with the addition of rituximab to a combination of fludarabine and cyclophosphamide in the treatment of relapsed follicular lymphoma.

Fludarabine in combination with cyclophosphamide is an effective treatment for newly diagnosed as well as relapsed follicular lymphoma. The anti-CD20 antibody rituximab has been employed successfully for the same indications. No such data were available on a combined use of these agents. Therefore, we conducted a phase II study to evaluate the safety and efficacy of a combination of rituximab (375 mg/m2), fludarabine (4 x 25 mg/m2) and cyclophosphamide (1 x 750 mg/m2), for the treatment of relapsed follicular lymphoma. An unexpected, severe hematologic toxicity with significant, prolonged thrombocytopenias WHO grade III/IV in 6 (35%) of 17 patients treated in total occurred, leading to early termination of the trial. Cytologic and serologic analyses point toward a direct toxic effect. Older patients (mean age 64.7 vs. 56.5 yr) were significantly (P = 0.02) more likely to suffer from this toxicity, whereas no other clinical or hematologic parameter differed statistically between the patients suffering from thrombocytopenia and those who did not. The addition of rituximab to fludarabine/cyclophosphamide employed at doses given above in relapsed follicular lymphoma may have led to this increase in thrombocytopenias. Therefore, caution should be exercised when combining these drugs for the treatment of patients with relapsed follicular lymphoma, especially when treating older patients.