Friederike Hesse

Separation of fast from slow anabolism by site-specific PEGylation of insulin-like growth factor I (IGF-I).

Insulin-like growth factor I (IGF-I) has important anabolic and homeostatic functions in tissues like skeletal muscle, and a decline in circulating levels is linked with catabolic conditions. Whereas IGF-I therapies for musculoskeletal disorders have been postulated, dosing issues and disruptions of the homeostasis have so far precluded clinical application. We have developed a novel IGF-I variant by site-specific addition of polyethylene glycol (PEG) to lysine 68 (PEG-IGF-I). In vitro, this modification decreased the affinity for the IGF-I and insulin receptors, presumably through decreased association rates, and slowed down the association to IGF-I-binding proteins, selectively limiting fast but maintaining sustained anabolic activity. Desirable in vivo effects of PEG-IGF-I included increased half-life and recruitment of IGF-binding proteins, thereby reducing risk of hypoglycemia. PEG-IGF-I was equipotent to IGF-I in ameliorating contraction-induced muscle injury in vivo without affecting muscle metabolism as IGF-I did. The data provide an important step in understanding the differences of IGF-I and insulin receptor contribution to the in vivo activity of IGF-I. In addition, PEG-IGF-I presents an innovative concept for IGF-I therapy in diseases with indicated muscle dysfunction.

Aggregation and Chemical Modification of Monoclonal Antibodies under Upstream Processing Conditions

PurposeTo investigate antibody stability and formation of modified species under upstream processing conditions.MethodsThe stability of 11 purified monoclonal human IgG1 and IgG4 antibodies, including an IgG1-based bispecific CrossMab, was compared in downscale mixing stress models. One of these molecules was further evaluated in realistic bioreactor stress models and in cell culture fermentations. Analytical techniques include size exclusion chromatography (SEC), turbidity measurements, cation exchange chromatography (cIEX), dynamic light scattering (DLS) and differential scanning calorimetry (DSC).ResultsSensitivity in downscale stress models varies among antibodies and results in formation of high molecular weight (HMW) aggregates. Stability is increased in cell culture medium and in bioreactors. Media components stabilizing the proteins were identified. Extensive chemical modifications were detected both in stress models as well as during production of antibodies in cell culture fermentations.ConclusionsProtective compounds must be present in chemically defined fermentation media in order to stabilize antibodies against the formation of HMW aggregates. An increase in chemical modifications is detectable in bioreactor stress models and over the course of cell culture fermentations; this increase is dependent on the expression rate, pH, temperature and fermentation time. Consequently, product heterogeneity increases during upstream processing, and this compromises the product quality.

Characterization of a re-engineered, mesothelin-targeted Pseudomonas exotoxin fusion protein for lung cancer therapy.

Mesothelin overexpression in lung adenocarcinomas correlates with the presence of activating KRAS mutations and poor prognosis. Hence SS1P, a mesothelin‐targeted immunotoxin, could offer valuable treatment options for these patients, but its use in solid tumor therapy is hampered by high immunogenicity and non‐specific toxicity. To overcome both obstacles we developed RG7787, a de‐immunized cytotoxic fusion protein comprising a humanized SS1 Fab fragment and a truncated, B‐cell epitope silenced, 24 kD fragment of Pseudomonas exotoxin A (PE24). Reactivity of RG7787 with sera from immunotoxin‐treated patients was >1000 fold reduced. In vitro RG7787 inhibited cell viability of lung cancer cell lines with picomolar potency. The pharmacokinetic properties of RG7787 in rodents were comparable to SS1P, yet it was tolerated up to 10 fold better without causing severe vascular leak syndrome or hepatotoxicity. A pharmacokinetic/pharmacodynamic model developed based on NCI‐H596 xenograft studies showed that for RG7787 and SS1P, their in vitro and in vivo potencies closely correlate. At optimal doses of 2–3 mg/kg RG7787 is more efficacious than SS1P. Even large, well established tumors (600 mm3) underwent remission during three treatment cycles with RG7787. Also in two patient‐derived lung cancer xenograft models, Lu7336 and Lu7187, RG7787 showed anti‐tumor efficacy. In monotherapy two treatment cycles were moderately efficacious in the Lu7336 model but showed good anti‐tumor activity in the KRAS mutant Lu7187 model (26% and 80% tumor growth inhibition, respectively). Combination of RG7787 with standard chemotherapies further enhanced efficacy in both models achieving near complete eradication of Lu7187 tumors.

Abstract 4510: RG7787 - a novel de-immunized PE based fusion protein for therapy of mesothelin-positive solid tumors

Attaching toxic payloads to antibodies has recently been established as a breakthrough in cancer therapy. Most of the currently developed antibody drug conjugate programs represent targeted chemotherapy with microtubule polymerization inhibiting drugs and hence share resistance mechanisms and side effects with classical chemotherapeutics like Taxol. Inhibition of protein synthesis by Pseudomonas Exotoxin (PE) is a more powerful mode of action that interferes with all hallmarks of cancer cells, not just with proliferation. However, its clinical use has been limited by immunogenicity as shown for SS1P, a mesothelin targeted immunotoxin. We have developed a novel de-immunized PE fusion protein (RG7787) for treatment of mesothelin-positive tumors. In order to de-immunize PE, point mutations have been introduced into domain III and the entire domain II has been deleted reducing the size of the effector moiety to approximately 24 kD. PE24 fusions with a disulfide-stabilized Fv fragment have previously been shown to be much better tolerated in rodents, but this could, at least in part, also be attributed to a much reduced serum half-life and exposure. In order to also de-immunize the targeting moiety of RG7787 and restore PK parameters similar to those of SS1P, we substituted the mouse dsFv moiety by a humanized Fab fragment. We show that RG7787 has indeed similar PK properties to SS1P in mouse and cyno. Cell viability assays show that RG7787 has similar cytotoxic potency as SS1P on different cell lines. In-vivo equipotency to SS1P in different xenograft models has been achieved at ∼3 fold higher doses indicating that tumor penetration of SS1P might be slightly better. However, RG7787 is up to tenfold better tolerated in rodents and cynomolgus monkeys, indicating that the therapeutic window is improved. RG7787 achieves potent tumor growth inhibition and even tumor regressions in several xenograft models. We also observed clearly synergistic efficacy with Taxol treatment in different tumor models making this a promising combination for clinical trials. Citation Format: Gerhard Niederfellner, Frieder Bauss, Sabine Imhof-Jung, Friederike Hesse, Sven Kronenberg, Roland Staak, Martin Lechmann, Ben Krippendorff, Wolfgang Richter, Rita Mateus, Gwendlyn Kollmorgen, Ulli Brinkmann, Masanori Onda, Ira Pastan, Klaus Bosslet. RG7787 - a novel de-immunized PE based fusion protein for therapy of mesothelin-positive solid tumors. [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 4510. doi:10.1158/1538-7445.AM2014-4510