Robert Weik

Characterization of molecular features, antigen-binding, and in vitro properties of IgG and IgM variants of 4E10, an anti-HIV type 1 neutralizing monoclonal antibody.

The human monoclonal antibody 4E10 has been generated previously by immortalization of peripheral blood cells from an HIV-1-infected individual. This antibody binds to the linear epitope NWFDIT on gp41 and exhibits exceptional neutralizing activity against a broad spectrum of primary HIV-1 isolates. In the present study, molecular features, immunoreactivity, and functional activity of 4E10 were studied. The original hybridoma-derived 4E10 was of subtype IgG(3). Analysis of the variable segment of the heavy chain (VH) demonstrated extensive somatic mutations compared to the closest homologous germline gene VH1-69. Most amino acid substitutions occurred in the complementarity-determining region (CDR) 2, characteristic for an antigen-driven somatic maturation. The heavy chain of the CDR3 (H3) is of unusual length and cannot be attributed with certainty to any specific D(H) locus. To enable mass production and to prolong the in vivo half-life, 4E10 was subsequently cloned as IgG(1) in Chinese hamster ovary (CHO) cells. In additional studies, 4E10 was class switched to the IgM isotype. Binding to the linear epitope NWFDIT was not significantly changed after the cloning procedures. However, in vitro studies revealed dramatic differences in the neutralizing potential. The antiviral activity could be greatly enhanced by change of IgG(3) to IgG(1). In contrast, the IgM isotype almost completely lost its neutralizing potential.

Anti-idiotypic antibody Ab2/3H6 mimics the epitope of the neutralizing anti-HIV-1 monoclonal antibody 2F5.

Anti-idiotypic antibodies directed against potentially neutralizing anti-HIV-1 antibodies may mimic epitopes on gp41 otherwise cryptic to the immune system. This study reports the generation of murine monoclonal antibody Ab2/3H6 blocking the binding of human Ab1 2F5 to the synthetic epitope and to gp160 in an enzyme-linked immunosorbent competition assay. Ab2/2H6 diminished the neutralizing potency of 2F5 in an in-vitro neutralization assay. Ab2/3H6 Fab fragments were capable of inducing neutralizing immune and 2F5-specific responses in B6D2F1 mice applying a simple prime-boost regimen of immunization.

Rational design of an improved induction scheme for recombinant Escherichia coli

In Escherichia coli, strong overexpression of a recombinant protein has been shown to be deleterious due to a heavy metabolic burden on the host cell, which may completely cease cell growth before maximum product accumulation has occurred. Aiming at a reduction of very high product formation rates, we engineered E. coli strains by mutating the Leloir pathway for galactose metabolization, so that galactose can be utilized to induce lac derived promoters. The induction with galactose was effective in every strain and expression construct tested, and it reduced the metabolic burden on a highly overproducing clone so that cell growth and product accumulation could be maintained for several generations.

Optimization of Recombinant Gene Expression in Escherichia colia

The major targets for improvement of recombinant expression efficiency in Escherichia coli are gene dosage, transcription and, to some extent, translation. In order to evaluate the relative importance of these factors, the kinetics of specific mRNA compared to product formation was studied for different widely used expression systems, producing recombinant human superoxide dismutase. For a system employing phage T7 RNA polymerase, where a high level of recombinant protein expression puts a high metabolic burden on the cells, it was shown that transcription is not the limiting factor. To improve the translation rate of a common vector based on the tac promoter, the Shine-Dalgarno (SD) sequence was mutated towards stronger homology to the anti-SD sequence of the E. coli 16S rRNA. A 12.2-fold increase in protein yield was accompanied by a 4.3-fold increase in specific mRNA, indicating that transcription of the recombinant gene is coupled to translation. As this coupling amplifies the detrimental effect of a low-efficiency ribosomal binding site, much attention should be paid to translation initiation when optimizing a recombinant protein production system. Finally, reasons for the high expression level before induction are discussed, and first results towards reducing it are presented.

Partial Humanization and Characterization of an Anti-idiotypic Antibody against Monoclonal Antibody 2F5, a Potential HIV Vaccine?

We recently developed a murine anti-idiotypic antibody (Ab2/3H6) versus the human monoclonal antibody 2F5, one of a few antibodies yet known to neutralize a broad range of HIV-1 primary isolates. Ab2/3H6 was not only able to bind to the paratope of mAb 2F5 but also significantly inhibited the binding of 2F5 to its synthetic epitope ELDKWA on gp41. In the present work we describe the partial humanization, expression, and characterization of Ab2/3H6 variants followed by several corresponding interaction studies with 2F5. The results of these studies support the high specificity of the recombinantly expressed Ab2s to the idiotype. Apparent affinities were designated by end point measurement and were similar compared to the murine Ab2/3H6. Moreover, the inhibition potency of chimeric Ab2/3H6 analyzed by in vitro studies could be shown to be the same as that detected for the hybridoma-derived murine Ab2/3H6.

Characterization of recombinant human diamine oxidase (rhDAO) produced in Chinese Hamster Ovary (CHO) cells

Human diamine oxidase (hDAO) efficiently degrades polyamines and histamine. Reduced enzyme activities might cause complications during pregnancy and be involved in histamine intolerance. So far hDAO has been characterized after isolation from either native sources or the heterologous production in insect cells. Accessibility to human enzyme is limited and insect cells produce non-human glycosylation patterns that may alter its biochemical properties. We present the heterologous expression of hDAO in Chinese Hamster Ovary (CHO) cells and a three step purification protocol. Analysis of metal content using ICP-MS revealed that 93% of the active sites were occupied by copper. Topaquinone (TPQ) cofactor content was determined using phenylhydrazine titration. Ninety-four percent of DAO molecules contained TPQ and therefore the copper content at the active site was indirectly confirmed. Mass spectrometric analysis was conducted to verify sequence integrity of the protein and to assess the glycosylation profile. Electronic circular dichroism and UV-vis spectra data were used to characterize structural properties. The substrate preference and kinetic parameters were in accordance with previous publications. The establishment of a recombinant production system for hDAO enables us to generate decent amounts of protein with negligible impurities to address new scientific questions.

Production of Recombinant Human Trypsinogen in Escherichia Coli and Pichia Pastoris

It is still common practice to find the most suitable expression system for a given protein by trial and error, as there are no clear rules available to make this decision on the basis of the properties of the desired protein. This work discusses several decision points between bacteria and yeasts, namely Escherichia coli and Pichia pastoris. Based on the expression of human trypsinogen 1, advantages and disadvantages of both expression systems are discussed, emphasizing yields and the efforts to obtain a functional product. Briefly, in E. coli a high yield of insoluble product was obtained which required substantial efforts for resolubilisation, whereas with P. pastoris a functional product was obtained at a comparatively low yield.

3D6 and 4B3: Recombinant expression of two anti-gp41 antibodies as dimeric and secretory IgA

Background Sexually transmitted diseases are predominantly acquired via mucosal membranes of the rectal or genital tract during sexual intercourse. This major port of virus entry is naturally defended by the humoral immune response, with immunoglobulin A (IgA) as the primary antibody class to elicit mucosal immunity. Dimeric IgA (dIgA) reaches the luminal side of mucosal tissues by transcytosis through epithelial cells lining the mucosa. In a first step, dIgAs specifically bind to the basolaterally expressed polymeric immunoglobulin receptor (pIgR) on epithelial cells. For release of IgAs on the luminal side the extracellular portion, termed secretory component (SC), remains attached to the antibody to form secretory IgA (sIgA) [1,2]. One example of a sexually transmitted disease is the human immunodeficiency virus (HIV) which annually infects several million individuals on a global scale and potentially leads to the acquired immunodeficiency syndrome (AIDS). Although current therapies can reduce disease progression in infected individuals, no cure is yet available or within reach in near future. As a consequence increased attention is now being paid to develop drugs that could prevent virus acquisition. 3D6 and 4B3 are two monoclonal antibodies (mAb) which have originally been isolated as IgG1 isotype from seroconverted HIV-1 patients and bind to the principal immunodominant domain of gp41. In the course of this project both mAbs were isotype switched to IgA1. Recombinant CHO cell lines were established for the production of 3D6 and 4B3 as dimeric as well as secretory IgA. While dIgA were expressed by a single cell line, sIgA are produced by a biochemical association of dIgA with SC. Both dIgA and sIgA variants were characterized and the contribution of the heavily glycosylated SC on IgA stability will be investigated.