Thomas Jostock

Single chain Fab (scFab) fragment

BackgroundThe connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.ResultsHere, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised.ConclusionA new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection.

Human antibody RNase fusion protein targeting CD30 lymphomas

Targeted RNases (TRs) are immunoenzymes with ribonucleases as cytotoxic effector domains, which are less immunogenic as plant or bacterial toxin components of classical immunotoxins. In this study, we show the generation and production of the first entirely human TR (huTR) directed against CD30+ lymphomas. The scFv-Fc-RNase construct was produced in human embryonic kidney (HEK) 293T cells, yielding up to 4 mg/L soluble protein after purification by protein A affinity chromatography. Size exclusion chromatography revealed a homodimer of the predicted molecular mass. Surface plasmon resonance analysis revealed an affinity to CD30 of KD of less than 1 nM for both the scFv-Fc and the scFv-Fc-RNase proteins. Internalization of the scFv-Fc-RNase protein by CD30+ Karpas-299 cells was demonstrated by confocal microscopy. Proliferation of the CD30+ lymphoma cell line Karpas-299 was strongly inhibited by CD30-specific huTR protein (IC50=3.3 nM). The huTR is a promising candidate for the immunotherapy of CD30+ lymphomas because of its expected low immunogenicity, good production yields, and potent effector function upon target cell binding and internalization. Its modular design is set to target other internalizing tumor antigens using different antibody domains.

Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology

The availability of site-specific recombinases has revolutionized the rational construction of cell lines with predictable properties. Early efforts were directed to providing pre-characterized genomic loci with a single recombinase target site that served as an address for the integration of vectors carrying a compatible tag. Efficient procedures of this type had to await recombinases like ΦC31, which recombine attP and attB target sites in a one-way reaction — at least in the cellular environment of the higher eukaryotic cell. Still these procedures lead to the co-introduction of prokaryotic vector sequences that are known to cause epigenetic silencing. This review illuminates the actual status of the more advanced recombinase-mediated cassette exchange (RMCE) techniques that have been developed for the major members of site-specific recombinases (SR), Flp, Cre and ΦC31. In RMCE the genomic address consists of a set of heterospecific recombinase target (RT-) sites permitting the exchange of the intervening sequence for the gene of interest (GOI), as part of a similar cassette. This process locks the GOI in place and it is ‘clean’ in the sense that it does not co-introduce prokaryotic vector parts nor does it leave behind a selection marker.

Screening of molecular repertoires by microbial surface display.

A multitude of systems for the presentation of foreign peptides or proteins on the surface of microorganisms has been developed within the past two decades. However, the majority of the bacterial surface display systems are devoted to the presentation of heterologous antigens to the immune system (vaccine generation). Bacteria are the preferable hosts for the generation of vast genetic repertoires, and their genetic manipulation and cultivation is easy. As a consequence, they provide promising systems for large-scale functional screenings, e.g. for enzyme activity or protein-protein interactions. This review will focus on examples of microbial surface display used for the screening of combinatorial repertoires. Further, we discuss future opportunities and promising candidate proteins not yet employed for that task.

Combination of the 2A/furin technology with an animal component free cell line development platform process

The recently described 2A/furin technology combines both chains of the antibody in a single open reading frame. Upon translation and secretion, the peptide is processed by the cell to generate native fully functional IgG antibodies. Here, we describe the results of an evaluation study of this technology for an industrial CHO cell line development process. The 2A/furin expression cassette setup was combined with a Novartis vector system. A transfection, selection, and cloning procedure in chemically defined media was established at Novartis and applied for a monoclonal test antibody. The productivity of 2A/furin-vector-derived clones in non-optimized generic shake flask fed-batch models was in a comparable range with clones derived from the reference control vector. Higher clonal production stability was seen for the majority of clones generated with the 2A/furin technology compared to the clones generated with the reference control vector. Product quality was analyzed by SDS-PAGE and no significant difference was detected between the two systems. Thus, it was shown that the 2A/furin technology can be successfully combined with a Novartis CHO expression system and platform. Due to the single ORF setup, the 2A/furin technology may therefore offer a suitable approach to reduce vector size and complexity.

Functional knockdown of VCAM-1 at the posttranslational level with ER retained antibodies

Vascular cell adhesion molecule 1 (VCAM-1) is involved in the recruitment of leukocytes to inflammatory sites. In this study we present the first functional knockdown of VCAM-1 using an ER retained antibody construct. We generated a knockdown construct encoding the VCAM-1 specific single chain variable fragment scFv6C7.1 fused to the C-terminal ER retention sequence KDEL. HEK-293:VCAM-YFP cells stably expressing a VCAM-YFP fusion protein were transiently transfected with the knockdown construct and showed down-regulation of surface VCAM-1. Knockdown efficiency of the system is time-dependent due to used transient transfection of the intrabody construct. Furthermore, intrabody mediated knockdown of HEK-293:VCAM-YFP cells also impaired cell-cell interaction with Jurkat cells that are endogenously expressing VLA-4, the physiological partner of VCAM-1. Posttranslational knockdown with ER retained antibodies seems to be a promising technique, as shown here for VCAM-1.

Mammalian stable expression of biotherapeutics.

Many therapeutically relevant proteins, like IgG antibodies, are highly complex, multimeric glycoproteins that are difficult to express in microbial systems and thus usually produced in mammalian host cells. During the past two decades, stable mammalian expression technologies have made huge progress resulting in highly increased speed of cell line development and yield of manufacturing processes. Here, we give an overview of technologies that are applied at different stages of state-of-the-art cell line development processes for biomanufacturing.

Oligomeric forms of single chain immunoglobulin (scIgG)

Assembly of immunoglobulin G (IgG) molecules from two heavy and two light chains can be facilitated by connecting the light chain to the heavy chain by an oligopeptide linker. Production of the anti-lysozyme D1.3-single chain (sc) IgG1 in HEK293T cells yielded up to 8 mg/L functional scIgG polypeptide. Size exclusion chromatography of material purified by protein-A affinity chromatography revealed that the majority of the D1.3-scIgG1 molecules were 150kDa monomers, with a KD of 1.8x10-10M measured by surface plasmon resonance; however, significant fractions of scIgG dimers and oligomers with molecular masses of 300 kDa and >600 kDa, respectively, were identified. The oligomerization resulted in an increased avidity. The observed oligomerization capability may allow new approaches for the generation of bispecific / multivalent antibodies.

Expression of Antibody in Mammalian Cells

Despite having a reputation to be costly, mammalian cell culture processes are used to produce the majority of currently marketed recombinant biopharmaceuticals, many of which are antibodies. Historically, mammalian cells were mainly chosen for whole IgG manufacturing because of product quality requirements like e.g. glycosylation, folding and assembly of the individual chains, which make microbial expression difficult. However, during the past 2 decades, significant progress has been made in both: Speed of mammalian cell line generation and yield of the manufacturing processes, which makes mammalian systems also from a commercial point of view more and more competitive to microbial expression. The following chapter provides an overview of state of the art mammalian cell line technologies for antibody manufacturing starting from vector and selection systems over host cell lines to screening methods.

Deletion of a telomeric region on chromosome 8 correlates with higher productivity and stability of CHO cell lines

Chinese Hamster Ovary (CHO) cells are widely used for large scale production of recombinant biopharmaceuticals. Although these cells have been extensively used, a demand to further increase the performance, for example, to facilitate the process of clone selection to isolate the highest producing cell lines that maintain stability of production over time is still existing. We compared gene expression profiles of high versus low producing CHO clones to identify regulated genes which can be used as biomarkers during clone selection or for cell line engineering. We present evidence that increased production rates and cell line stability are correlated with the loss of the telomeric region of the chromosome 8. A new parental CHO cell line lacking this region was generated and its capability for protein production was assessed. The average volumetric productivity of cells after gene transfer and selection was found to be several fold improved, facilitating the supply of early drug substance material to determine for example, quality. In addition, significantly more cell clones with a higher average productivity and higher protein production stability were obtained with the new host cell line after single cell cloning. This allows reduced efforts in single cell sorting, screening of fewer clones and raises the opportunity to circumvent time and labor‐intensive stability studies. Biotechnol. Bioeng. 2016;113: 1084–1093.