04 Fakultät Energie-, Verfahrens- und Biotechnik
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Item Open Access Engineered antibodies for the therapy of cancer and inflammatory diseases(2010) Zettlitz, Kirstin Anja; Kontermann, Roland (Prof. Dr.)Detailed knowledge of the antibody structure and function allows researchers to engineer antibodies on a rational basis to design therapeutic antibodies dependent on the target antigen, therapeutic strategy and clinical setting. The development of therapeutic antibodies is a rapidly growing field with more than 30 antibodies approved over the past 25 years. Thereof, cancers as well as autoimmune and inflammatory diseases are the main indications. The purpose of the first part of this thesis was the cloning, chimerization and humanization of the monoclonal antibody (mAb) cmHsp70.1. This mouse mAb is specific for heat shock protein 70 (Hsp70) and able to bind the plasma membrane bound form (mHsp70), associated with various cancers including breast cancer, head-and-neck cancer, and acute myeloid leukemia. Humanization of cmHsp70.1 by grafting the complementarity-determining regions onto homologous human germline genes resulted in an antibody (humex) possessing a similar affinity (3 nM) as the parental antibody and an improved production and thermal stability. Epitope mapping confirmed that the parental, chimeric, and humanized antibodies recognize the same region including amino acids 473-504 of the Hsp70 substrate binding domain (SBD). Hence, this humanized antibody provides a basis for further development of a mHsp70-specific antibody therapy. The purpose of the second part of this thesis was to evaluate an appropriate format and an optimization strategy for a humanized antibody (IZI-06.1) specific for human tumor necrosis factor receptor 1 (TNFR1). Selective inhibition of TNFR1 provides the opportunity to neutralize the pro-inflammatory activity of TNF while maintaining the advantageous immunological responses mediated by TNFR2. Here, this humanized antibody was converted into an IgG1 molecule (ATROSAB) containing a modified human Fc region deficient in mediating effector functions. IgG ATROSAB was compared to monovalent antibody derivatives fused to human serum albumin (HSA). Using chimeric human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region (amino acid residues 1-70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain of CRD2. Purified ATROSAB, produced in CHO cells, inhibited in vitro typical TNF-mediated responses like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8 production. Moreover, ATROSAB showed strong binding to human and rhesus TNFR1-Fc fusion protein with an affinity identical to the parental mouse antibody H398. These findings open the way to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human primates. Furthermore, phage display technology was used for affinity maturation of the humanized variable domains by site directed mutagenesis of CDR1 and CDR2 of each VH and VL. Attempts towards affinity maturation resulted in a mutant (scFv IG11) showing a two-fold increase in antigen binding affinity which also translated into slightly improved inhibition of TNF-mediated cytotoxicity in vitro. The results of this study indicate that further engineering of ATROSAB could offer a number of benefits for its therapeutic efficacy. TNFR1-selective antagonist, such as ATROSAB, will permit new therapeutic options for diseases where anti-TNF therapeutics failed or even exacerbate disease progression and could be an especially useful therapeutic alternative in diseases already known to clinically respond to anti-TNF treatment and particularly in those diseases where specific blockage of TNFR1 and maintenance of TNFR2 function appears as a promising therapeutic approach.