Edgar Voss

Enzyme-catalyzed processes in pharmaceutical industry

Abstract Biocatalysis is enjoying an increasing interest not only in academia, but also in industry. It has been the topic of several excellent reviews or books [Bornscheuer and Kaszlauskas, Hydrolases in Organic Synthesis—Regio- and Stereoselective Biotransformations, Wiley-VCH, Weinheim, 1999; H.-J. Rehm, G. Reed, A. Puhler, P. Stadler (Eds.), Biotechnology, Biotransformations, Vol. 8, Wiley-VCH, Weinheim, 1998; Faber, Biotransformation, Organic Chemistry, Springer Berlin, 1996; K. Drauz, H. Waldmann, (Eds.), Enzyme Catalysis in Organic Synthesis, VCH Verlagsgesellschaft mbH, Weinheim, 1995; Wong and Whitesides, Enzymes in Synthetic Organic Chemistry, Tetrahedron Organic Chemistry, Vol. 12, Pergamon, Oxford, 1994; Sheldon, Chirotechnology, Marcel Dekker, New York, 1993; Chem. Today 13 (1995) 9; Drug Discovery Today 2 (1997) 513; Bioorg. Med. Chem. 7 (1999) 2253; J. Chem. Soc., Perkin Trans. 1 (1999) 1; U.T. Bornscheuer, in: Biotechnology—Biotransformations II, Vol. 8b, Wiley-VCH, Weinheim, 2000 pp. 277–294.] [1] , [2] , [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10] , [11] . Aim of this article is to present recent developments of biocatalysis in the pharmaceutical industry in a more didactical manner. An outlook on future aspects of biocatalysis will reflect the authors opinion. We will discuss the fundamental strength of biocatalysis but also some commonly held pre-conceptions, which we believe are superficial.

Global Quantitative Phosphoproteome Analysis of Human Tumor Xenografts Treated with a CD44 Antagonist

The cell surface glycoprotein CD44 plays an important role in the development and progression of various tumor types. RG7356 is a humanized antibody targeting the constant region of CD44 that shows antitumor efficacy in mice implanted with CD44-expressing tumors such as MDA-MB-231 breast cancer cells. CD44 receptor seems to function as the main receptor for hyaluronic acid and osteopontin, serving as coreceptor for growth factor pathways like cMet, EGFR, HER-2, and VEGFR and by cytoskeletal modulation via ERM and Rho kinase signaling. To assess the direct impact of RG7356 binding to the CD44 receptor, a global mass spectrometry-based phosphoproteomics approach was applied to freshly isolated MDA-MB-231 tumor xenografts. Results from a global phosphoproteomics screen were further corroborated by Western blot and ELISA analyses of tumor lysates from CD44-expressing tumors. Short-term treatment of tumor-bearing mice with RG7356 resulted in modifications of the MAPK pathway in the responsive model, although no effects on downstream phosphorylation were observed in a nonresponsive xenograft model. Taken together, our approach augments the value of other high throughput techniques to identify biomarkers for clinical development of targeted agents.

CD44 Isoform Status Predicts Response to Treatment with Anti-CD44 Antibody in Cancer Patients

Purpose: CD44, a cell surface glycoprotein, plays important roles in the development, progression, and metastasis of various tumor types. The aim of this study was to investigate how the expression of CD44 isoforms influences the interaction with hyaluronic acid (HA) and how differential isoform expression impacts antitumoral responses in vivo to treatment with RG7356, a humanized anti-CD44 antibody inhibiting CD44–HA interaction. Experimental Design: CD44 isoform expression on various tumor cell lines was analyzed by RNASeq while data on patients with different tumor types were obtained from the publicly available TCGA RNASeq dataset as well as a phase I clinical study (NCT01358903). We analyzed the link between HA production and CD44 isoform expression as well as the consequences of blocking the CD44-mediated cell adhesion to HA using RG7356. The correlation between CD44 isoform expression and antitumor response to RG7356 treatment was investigated in the corresponding murine xenograft in vivo models as well as in a subset of patients treated with RG7356 from a recently completed phase I clinical trial. Results: CD44 isoform expression, in particular expression of CD44s, is associated with HA production and predicts response to treatment with RG7356 in tumor xenograft models. Furthermore, patient data suggest that CD44 isoform status is a potential predictive biomarker for clinical response to treatment with RG7356. Conclusions: We provide new insights into the close interplay between CD44 and HA and a potential biomarker to enrich patient responses to RG7356 in the clinic. Clin Cancer Res; 21(12); 2753–62. ©2015 AACR.

Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model

CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages.

A re-engineered immunotoxin shows promising preclinical activity in ovarian cancer

RG7787 is a re-engineered mesothelin-targeted immunotoxin with reduced immunogenicity composed of a humanized anti-mesothelin Fab fragment and a B-cell epitope silenced 24 kD fragment of Pseudomonas exotoxin A. High prevalence of mesothelin-positive cases and a large unmet medical need make ovarian cancer a promising indication for the clinical development of RG7787. However, ovarian cancer patients also frequently have elevated serum levels of the cancer antigen 125 (CA-125). In principle this could pose a problem, since the binding sites for CA-125 and RG7787 on mesothelin were reported to overlap. However, we show here that RG7787 can readily displace even excess amounts of CA-125 in different cellular assays. Moreover when tested in-vitro on a panel of 12 ovarian cancer cell lines, RG7787 had high cytotoxic activity on COV644, Caov-4, and SNU-119 cells and fully inhibited growth of EFO-21, KURAMOCHI, OVSAHO, and Caov-3 cells with potency values ranging from 1 to 86 pM. Finally, we evaluated the in-vivo efficacy of RG7787 in OvCa6668, a patient-derived ovarian cancer model with high levels of CA-125 expression. RG7787 had moderate monotherapy efficacy but in combination with standard chemotherapies (cisplatin, paclitaxel) achieved pronounced tumor regressions. In summary our data support clinical testing of RG7787 in ovarian cancer.

Abstract 5321: Preclinical validation for treatment with RG7787 in ovarian cancer

RG7787 is composed of a Fab fragment from an anti-MSLN antibody fused to a de-immunized and truncated pseudomonas endotoxin A variant. Once RG7787 is bound and internalized by MSLN positive cells, the toxin is transported to the cytosol, where it inhibits protein synthesis, eventually causing tumor cell death. Mesothelin (MSLN) is a tumor specific differentiation antigen. On normal tissue, its expression is restricted to differentiated mesothelial cells that line as single cell layer body cavities and major organs (e.g. pleura, pericardium, and peritoneum). In cancer, MSLN is highly expressed not only on mesotheliomas but also on a number of other types of solid tumors like ovarian and pancreatic cancer. In both indications patients frequently also have significant serum levels of the cancer antigen-125 (CA-125), sometimes even >1000 U/ml. MSLN has been described to bind CA-125 and this interaction has been suggested to play a role for the ability of cancer cells to metastasize e.g. to the peritoneum. The region in MSLN that is responsible for its interaction with CA-125 has been reported to overlap with the binding epitope of RG7787 potentially resulting in competition between CA-125 and RG7787 for binding to MSLN. The high percentage of MSLN positive cases as well as a clear unmet medical need makes ovarian and pancreatic cancer promising indications for clinical development of RG7787. We investigated, whether abundance of CA-125 can negatively affect ability of RG7787 to bind and be taken up by tumor cells thereby antagonizing RG7787 treatment. We found that indeed RG7787 competes with CA-125 for binding to mesothelin. However, in SPR experiments, the interaction of MSLN with CA-125 was not strong enough to block binding of the anti-MSLN Fab moiety that targets RG7787 to tumor cells. At 20°C the absolute affinity (KD) of RG7787 for human MSLN was determined to be 12.5 pM using two orthogonal “affinity in solution” methods, ELISA and SPR. In agreement with such high affinity binding, we found that adding soluble CA-125 to cell viability assays did not reduce the cytotoxic potency of RG7787. In one set of assays, ascites fluid containing a 10 fold excess of CA-125 compared to the average levels observed in sera of ovarian patients was used for competition. In another set of assays, we used a 100 fold excess of a truncated recombinant CA-125 fragment, containing the domain that interacts with MSLN. Neither the ascites fluid, nor the recombinant CA-125 fragment had any effect on the in-vitro potency of RG7787, indicating that soluble CA-125 levels in patients will not antagonize RG7787 treatment. Finally we demonstrated in cell-cell interaction assays that RG7787 can efficiently block the attachment of MSLN positive cells to OVCAR3 cells that express high levels of CA-125 on their surface. Based on these encouraging data we are currently evaluating in-vivo efficacy of RG7787 in a patient-derived ovarian cancer model. Citation Format: Gwendlyn Kollmorgen, Klara Palme, Annette Seidl, Stefan Scheiblich, Christian Clemens, Edgar Voss, Martin Kaufmann, Klaus Hirzel, Pamela Wilfert, Moritz Marcinowski, Bernd Satzinger, Frank Herting, Gerhard Niederfellner. Preclinical validation for treatment with RG7787 in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5321. doi:10.1158/1538-7445.AM2015-5321

Simple, Short and Efficient Procedure for the Preparation of Hydroxyl- and Hydroxymethyl-Substituted 2,6-Dichlorobenzaldehydes

Hydroxyl- and hydroxymethyl-substituted 2,6-dichlorobenzaldehydes 1-4 have been obtained by lithiation of the corresponding TIPS-protected dichlorophenols or dichlorobenzylic alcohols followed by reaction with DMF and subsequent deprotection of the hydroxy group. Yields are high and formation of regioisomers is not observed.