Evelyne Janzek

Human Recombinant ACE2 Reduces the Progression of Diabetic Nephropathy

OBJECTIVE Diabetic nephropathy is one of the most common causes of end-stage renal failure. Inhibition of ACE2 function accelerates diabetic kidney injury, whereas renal ACE2 is downregulated in diabetic nephropathy. We examined the ability of human recombinant ACE2 (hrACE2) to slow the progression of diabetic kidney injury. RESEARCH DESIGN AND METHODS Male 12-week-old diabetic Akita mice (Ins2WT/C96Y) and control C57BL/6J mice (Ins2WT/WT) were injected daily with placebo or with rhACE2 (2 mg/kg, i.p.) for 4 weeks. Albumin excretion, gene expression, histomorphometry, NADPH oxidase activity, and peptide levels were examined. The effect of hrACE2 on high glucose and angiotensin II (ANG II)–induced changes was also examined in cultured mesangial cells. RESULTS Treatment with hrACE2 increased plasma ACE2 activity, normalized blood pressure, and reduced the urinary albumin excretion in Akita Ins2WT/C96Y mice in association with a decreased glomerular mesangial matrix expansion and normalization of increased α-smooth muscle actin and collagen III expression. Human recombinant ACE2 increased ANG 1–7 levels, lowered ANG II levels, and reduced NADPH oxidase activity. mRNA levels for p47phox and NOX2 and protein levels for protein kinase Cα (PKCα) and PKCβ1 were also normalized by treatment with hrACE2. In vitro, hrACE2 attenuated both high glucose and ANG II–induced oxidative stress and NADPH oxidase activity. CONCLUSIONS Treatment with hrACE2 attenuates diabetic kidney injury in the Akita mouse in association with a reduction in blood pressure and a decrease in NADPH oxidase activity. In vitro studies show that the protective effect of hrACE2 is due to reduction in ANG II and an increase in ANG 1–7 signaling.

Recombinant angiotensin-converting enzyme 2 improves pulmonary blood flow and oxygenation in lipopolysaccharide-induced lung injury in piglets.

Objective: To study angiotensin-converting enzyme 2 in a piglet model with acute respiratory distress syndrome and to evaluate the therapeutic potential of this substance in a preclinical setting, as this model allows the assessment of the same parameters required for monitoring the disease in human intensive care medicine. The acute respiratory distress syndrome is the most severe form of acute lung injury with a high mortality rate. As yet, there is no specific therapy for improving the clinical outcome. Recently, angiotensin-converting enzyme 2, which inactivates angiotensin II, has been shown to ameliorate acute lung injury in mice. Design: Prospective, randomized, double-blinded animal study. Setting: Animal research laboratory. Subjects: Fifteen anesthetized and mechanically ventilated piglets. Interventions: Acute respiratory distress syndrome was induced by lipopolysaccharide infusion. Thereafter, six animals were assigned randomly into angiotensin-converting enzyme 2 group, whereas another six animals served as control. Three animals received angiotensin-converting enzyme 2 without lipopolysaccharide pretreatment. Measurements and Main Results: Systemic and pulmonary hemodynamics, blood gas exchange parameters, tumor necrosis factor-&agr;, and angiotensin II levels were examined before acute respiratory distress syndrome induction and at various time points after administering angiotensin-converting enzyme 2 or saline. In addition, ventilation-perfusion distribution of the lung tissue was assessed by the multiple inert gas elimination technique. Animals treated with angiotensin-converting enzyme 2 maintained significantly higher Pao2 than the control group, and pulmonary hypertension was less pronounced. Furthermore, angiotensin II and tumor necrosis factor-&agr; levels, both of which were substantially increased, returned to basal values. Multiple inert gas elimination technique revealed a more homogeneous pulmonary blood flow after treatment with angiotensin-converting enzyme 2. In intergroup comparisons, there were no differences in pulmonary blood flow to lung units with subnormal ventilation/perfusion ratios. Conclusions: Angiotensin-converting enzyme 2 attenuates arterial hypoxemia, pulmonary hypertension, and redistribution of pulmonary blood flow in a piglet model of acute respiratory distress syndrome, and may be a promising substance for clinical use.

Recombinant Expression and Characterization of Human and Murine ACE2: Species-Specific Activation of the Alternative Renin-Angiotensin-System

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase of the renin-angiotensin-system (RAS) which is known to cleave several substrates among vasoactive peptides. Its preferred substrate is Angiotensin II, which is tightly involved in the regulation of important physiological functions including fluid homeostasis and blood pressure. Ang 1–7, the main enzymatic product of ACE2, became increasingly important in the literature in recent years, as it was reported to counteract hypertensive and fibrotic actions of Angiotensin II via the MAS receptor. The functional connection of ACE2, Ang 1–7, and the MAS receptor is also referred to as the alternative axis of the RAS. In the present paper, we describe the recombinant expression and purification of human and murine ACE2 (rhACE2 and rmACE2). Furthermore, we determined the conversion rates of rhACE2 and rmACE2 for different natural peptide substrates in plasma samples and discovered species-specific differences in substrate specificities, probably leading to functional differences in the alternative axis of the RAS. In particular, conversion rates of Ang 1–10 to Ang 1–9 were found to be substantially different when applying rhACE2 or rmACE2 in vitro. In contrast to rhACE2, rm ACE2 is substantially less potent in transformation of Ang 1–10 to Ang 1–9.

Microheterogeneity of therapeutic monoclonal antibodies is governed by changes in the surface charge of the protein

It has previously been shown for individual antibodies, that the microheterogenity pattern can have a significant impact on various key characteristics of the product. The aim of this study to get a more generalized understanding of the importance of microheterogeneity. For that purpose, the charge variant pattern of various different commercially available therapeutic mAb products was compared using Cation‐Exchange Chromatography with linear pH gradient antigen affinity, Fc‐receptor affinity, antibody dependent cellular cytotoxicity (ADCC) and conformational stability. For three of the investigated antibodies, the basic charge variants showed a stronger binding affinity towards FcγRIIIa as well as an increased ADCC response. Differences in the conformational stability of antibody charge variants and the corresponding reference samples could not be detected by differential scanning calorimetry. The different biological properties of the mAb variants are therefore governed by changes in the surface charge of the protein and not by an altered structure. This can help to identify aspects of microheterogeneity that are critical for product quality and can lead to further improvements in the development and production of therapeutic antibody products.

Enhancement of retroviral infection in vitro by anti-Ley IgG: reversal by humanization of monoclonal mouse antibody

Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Ley antigen enhanced infection in vitro with HTLV‐1 and with HIV‐1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells, and did not use either Ley epitopes on target cells for cross‐linkage of virus to the cell or the Fc part of the antibody as a ligand for any cellular receptor. For enhancement to occur, binding of anti‐Ley antibody to virus was required to take place before virus binding to its specific receptor with no indication of any alternative pathway of infection, as evidenced by abrogation of enhancement by anti‐CD4 MAb or soluble recombinant CD4, and also the inability of anti‐Ley MAb to mediate HIV infection of HSB‐2 cells in which HTLV‐1/HIV pseudovirus infection was enhanced. While F(ab)2 fragments of ABL 364 also enhanced infection, a human/mouse chimeric antibody and a fully humanized antibody had no enhancing effect on free virus infection. We suggest that binding of anti‐Ley ABL 364 or its F(ab)2 fragment induced a conformational change in the gp120 oligomers facilitating the process of infection, and that this function was abrogated by the IgG1 Fc of the chimeric and the humanized antibodies. The observations indicate that the non‐paratope domains of antiviral antibodies can influence their function as neutralizing or enhancing for infection.

Tolerability, response and outcome of high-risk neuroblastoma patients treated with long-term infusion of anti-GD2 antibody ch14.18/CHO

ABSTRACT Immunotherapy with short term infusion (STI) of monoclonal anti-GD2 antibody (mAb) ch14.18 (4 × 25 mg/m2/d; 8–20 h) in combination with cytokines and 13-cis retinoic acid (RA) prolonged survival in high-risk neuroblastoma (NB) patients. Here, we investigated long-term infusion (LTI) of ch14.18 produced in Chinese hamster ovary cells (ch14.18/CHO; 10 × 10 mg/m2; 24 h) in combination with subcutaneous (s.c.) interleukin-2 (IL-2) in a single center program and report clinical response, toxicity and survival. Fifty-three high-risk NB patients received up to 6 cycles of 100 mg/m2 ch14.18/CHO (d8–17) as LTI combined with 6 × 106 IU/m2 s.c. IL-2 (d1–5; 8–12) and 160 mg/m2 oral RA (d19–32). Pain toxicity was documented with validated pain scores and intravenous (i.v.) morphine usage. Response was assessed in 37/53 evaluable patients following International Neuroblastoma Risk Group criteria. Progression-free (PFS) and overall survival (OS) was analyzed by the Kaplan-Meier method and compared to a matched historical control group from the database of AIEOP, the “Italian Pediatric Ematology and Oncology Association”. LTI of ch14.18/CHO showed acceptable toxicity profile indicated by low pain scores, reduced i.v. morphine usage and low frequency of Grade ≥3 adverse events that allowed outpatient treatment. We observed a best response rate of 40.5% (15/37; 5 CR, 10 PR), 4-year (4 y) PFS of 33.1% (observation 0.1- 4.9 y, mean: 2.2 y) and a 4 y OS of 47.7% (observation 0.27 – 5.20 y, mean: 3.6 y). Survival of the entire cohort (53/53) and the relapsed patients (29/53) was significantly improved compared to historical controls. LTI of ch14.18/CHO thus shows an acceptable toxicity profile, objective clinical responses and a strong signal of clinical efficacy in NB patients.

Long-term continuous infusion of anti-GD2 antibody CH14.18/CHO in relapsed/refractory neuroblastoma patients.

Purpose GD2 is highly expressed in neuroblastoma (NB) and specifically recognized by antibodies ch14.18. Immunotherapy with bolus infusion of ch14.18 in combination with cytokines prolonged survival in patients at risk for relapse but was associated with substantial toxicity, in particular severe pain, requiring i.v. morphine [EJM 2010]. In an attempt to reduce the pain side effect associated with anti-GD2 therapy and to optimize antibody exposure, we piloted a treatment using continuous infusion of ch14.18 produced in CHO cells (APN311) and report first results.

Abstract A032: Immunotherapy with ch14.18/CHO in combination with IL2 is active and effective in high-risk relapsed/refractory neuroblastoma patients

Background: Ganglioside GD 2 is a glycolipid highly expressed on neuro-ectodermal tumors including melanoma and neuroblastoma (NB). It is ranked on position 12 of the list of tumor associated antigens by the NCI. Anti-GD 2 antibody (Ab) ch14.18/CHO targets this antigen and effectively orchestrates innate immune effector mechanisms against GD 2 expressing malignancies. However, one on target effect of anti-GD 2 Abs is the induction of neuropathic pain when applied as short term infusion (STI) requiring co-medication with intravenous morphine. Here we investigated whether long-term infusion (LTI) of anti-GD 2 Ab ch14.18/CHO may have an improved pain toxicity profile and at the same time mediate an effective immune response in patients (pts) with high risk relapsed/refractory NB which translates to objective clinical responses and an improved survival. Methods: 97 pts received 6x10 6 IU/m 2 sc IL2 (d1-5; 8-12), LTI of 100 mg/m 2 ch14.18/CHO (d8-17) and 160 mg/m 2 oral 13-cis-RA (d19-32) in an ongoing SIOPEN Phase II study (APN311-202) (NCT01701479) (44 pts) and a closed single center program (53 pts) (APN311-303). Response assessments followed INRG criteria. Serum ch14.18/CHO concentration-time curves were determined by validated ELISA methods and pharmacokinetics parameter were analyzed using standard non-compartmental methods. Fcγ;-receptor polymorphisms FCGR2A (H131R), -3A (V158F) and -3B (NA1/NA2) and patient-specific antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and whole blood cytotoxicity (WBT) responses were determined. Results: LTI was associated with low pain scores and a decreasing morphine usage compared to STI. Clinical overall responses were 30% (APN311-303) and 31% (APN311-202). The survival update of the APN311-303 cohort revealed a 1-y & 4-y OS of 94.2±3.2% & 60.9±9.0% (median FU 2.9y [0.7-5.2y]) and a 1-y & 4-y PFS of 54.4±6.9% & 32.3%±6.9% (median FU 2.8y [0.7 - 4.9y]). Median TTP was 571d (95% CI: 232.7d). The comparator is the reported historical gold standard for relapsed refractory NB patients with 1-y & 4-y PFS of 19±2% & 8±3% and OS of 56±3% & 14±4% and a median TTP of 63 d (95% CI: 56.8d). NB pts with high affinity FCGR alleles and an increase in ADCC (cut off 15%) are associated with longer PFS and OS rates (p Parameters of immune modulation (CDC, and WBT) and PK of ch14.18/CHO were comparable between APN311-202 and -303 cohorts. PK of ch14.18/CHO was analyzed in cycle 1: Cmax=12.2±0.4μg/ml, t½=8.4±1.1 d, AUC=145.3± 5.8 μg*d/ml, Vd ss =9.3±0.5L/m 2 . A pro-inflammatory cytokine response (IL-2, IL-6, IL-8, IFNγ) translated into the expansion of effector NK- (3x) and T-cells (2x). We observed HACA in 17/97 pts (17.5%) of which only 9/97 (9.3%) were neutralizing with respect to the inhibition of CDC and WBT activity. In HACA negative patients, levels of ch14.18/CHO and functional parameters (CDC and WBT) analyzed before subsequent treatment cycles indicate persistent anti-NB activity for the entire treatment period. Conclusion: LTI of ch14.18/CHO has an improved pain toxicity profile and at the same time is active and effective in high-risk relapsed/refractory NB. Citation Format: Holger N. Lode, Dominique Valteau-Couanet, Alberto Garaventa, Juliet Gray, Victoria Castel, Isaac Yaniv, Nikolai Siebert, Christian Jensen, Stefanie Endres, Lena Pill, Christin Eger, Diana Seidel, Madlen Juttner, Silke Kietz, Karoline Ehlert, Evelyne Janzek, Carla Manzitti, Ina Muller, Hans Loibner, Ruth Ladenstein. Immunotherapy with ch14.18/CHO in combination with IL2 is active and effective in high-risk relapsed/refractory neuroblastoma patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A032.

Abstract CT410: Immune activation, clinical response and survival following long-term infusion of anti-GD2 antibody ch14.18/CHO in combination with interleukin-2 in high-risk neuroblastoma patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: Immunotherapy with monoclonal anti-GD2 antibody (mAb) ch14.18 in combination with cytokines effectively prolonged survival in high risk neuroblastoma (NB). We piloted a less toxic treatment using continuous infusion of ch14.18/CHO in combination with subcutaneous interleukin-2 (IL-2) and report initial clinical response and survival. Methods and Materials: 53 high risk NB patients received up to 6 cycles of 6x106 IU/m2 s.c. IL-2 (d1-5; 8-12), continuous infusion of 100 mg/m2 ch14.18/CHO (d8-17) and 160 mg/m2 oral 13-cis-RA (d19-32) in a single center compassionate use program. Killer immunoglobulin-like receptor (KIR)/KIR-ligand (KIRL) mismatch and Fcγ-receptor (FCGR) was analyzed with a validated PCR-based analysis of KIR, HLA, FCGR2A (H131R), -3A (V158F) and -3B (NA1/NA2) polymorphisms. Antibody levels and GD2 specific killing of neurpblastoma cells by ADCC, CDC, and whole blood were sequentially analyzed. Clinical response was assessed by mIBG, MRI/CT, bone marrow- and catecholamine- analysis before, after 2/3 and after 5/6 cycles of immunotherapy in patients with measurable disease. Results: : In the patients receiving ch14.18/CHO continuous infusions significantly less i.v. morphine was used compared to patients who receive ch14.18/CHO bolus infusions. In many patients it was possible to discontinue i.v. morphine and to treat pain with oral Gabapentin only. Response rates were 41.7 % in mIBG (15/36), 31.8 % MRI/CT (7/22), 28.6 % bone marrow- (6/21) and 38.1 % in catecholamine- (8/21) measurements. External review of mIBG responses of 28 patients confirmed a 32.1% response rate (9/28). The overall response following INRG guide lines was determined at 30% (12/40). The event-free-survival rate in the entire cohort was 32.4 % (observation 3.2 years, mean: 1.6 years) and an overall-survival rate of 66.8% (observation 3.9 years, mean: 3.1 years). Patients with KIR/KIRL mismatch and high affinity FCGR alleles showed a trend towards longer event-free survival (P = 0.08), which supports NK cell mediated antibody dependent cytotoxicity as the mechanism of action. Determination of antibody levels and functional immune parameters (ADCC, CDC and WBT) indicate persistent anti-neuroblastoma immune-activity measurable before susequent treatment cycles. Conclusion: Continuous infusion of anti-GD2 Ab ch14.18/CHO reduces the pain side effect, shows clinical activity in NB patients and improves the outcome. Analysis of KIR/KIRL-mismatch and FCGR-polymorphisms by genotyping may predict clinical outcome in patients receiving ch14.18/CHO. Functional immune parameters indicate anti-neuroblastoma activity over the entire treatment period. These results are subject to prospective confirmation in a SIOPEN Phase II study (EudraCT: 2009-018077-31). Citation Format: Holger Lode, Christian Jensen, Nikolai Siebert, Silke Kietz, Karoline Ehlert, Ina Muller, Ruth Ladenstein, Evelyne Janzek, Hans Loibner. Immune activation, clinical response and survival following long-term infusion of anti-GD2 antibody ch14.18/CHO in combination with interleukin-2 in high-risk neuroblastoma patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT410. doi:10.1158/1538-7445.AM2014-CT410

Abstract 2858: Binding characteristics of the immunocytokine hu14.18-IL2 and induction of human effector functions as anticipated mode of action.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Immunotherapy of neuroblastoma with anti-GD2 antibodies in combination with interleukin-2 (IL2) emerges as an important treatment option as shown in several clinical trials. In consequence, an antibody-IL2 fusion protein (hu14.18-IL2; immunocytokine) was designed that combines the targeting and effector functions of an anti-GD2 antibody and the immune modulation activity of IL2. In addition to mediation of typical effector functions such as CDC and ADCC this immunocytokine targets IL2 to GD2+ tumor cells and thereby links tumor cells to IL2-receptor bearing effector cells via an Fc-gamma independent mechanism. This concept has already shown encouraging results in Phase I/II trials in relapsed/refractory neuroblastoma patients. Here, we report the antigen binding characteristics and in vitro anti-neuroblastoma activity of a new GMP preparation of hu14.18-IL2 (APN301). Binding of APN301 to its nominal antigen GD2 was analyzed by solid phase ELISA and compared to other antibodies of the 14G2a family. Affinities of APN301 to GD2 and to anti-14.18 anti-idiotype antibodies were also determined by Biacore® analysis. GD2 specific ADCC, CDC and whole blood cytotoxicity were analyzed in cytotoxicity assays in vitro (calcein-release, 51Cr release) using GD2+ neuroblastoma target cell lines. We demonstrate similar binding characteristics of APN301 to GD2 compared to parent 14.18 antibodies (murine and chimeric). The anti-14.18 anti-id antibody ganglidiomab fully inhibits binding of APN301 to the nominal antigen GD2, thereby proving internal image properties. The dissociation constants of APN301 to GD2 and ganglidiomab as determined in Biacore analyses are in the 10-9M and 10-7M range, respectively, similar to those determined for the naked chimeric 14.18 antibody. Relevant for the prediction of clinical activity we demonstrate potent specific lysis of GD2+ human neuroblastoma cells mediated by APN301 in a whole blood cytotoxicity assay in vitro, utilizing whole blood of healthy donors as well as whole blood of high-risk neuroblastoma patients in advanced disease stage. Further clinical trials with APN301 in neuroblastoma patients are being planned, taking into account the found potent whole blood cytolytic activity and the pharmakokinetic properties of this immunocytokine. Citation Format: Hans Loibner, Manfred Schuster, Evelyne Janzek, Stefan Stranner, Bernhard Peball, Susanne Wiederkum, Oliver Mutschlechner, Nikolai Siebert, Holger Lode. Binding characteristics of the immunocytokine hu14.18-IL2 and induction of human effector functions as anticipated mode of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2858. doi:10.1158/1538-7445.AM2013-2858 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.