Hanspeter Amstutz

Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity.

The glycosylation pattern of chCE7, an antineuroblastoma chimeric IgG1, was engineered in Chinese hamster ovary cells with tetracycline–regulated expression of β(1,4)–N–acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisected oligosaccharides that have been implicated in antibody–dependent cellular cytotoxicity (ADCC). Measurement of the ADCC activity of chCE7 produced at different tetracycline levels showed an optimal range of GnTIII expression for maximal chCE7 in vitro ADCC activity, and this activity correlated with the level of constant region–associated, bisected complex oligosaccharides determined by matrix–assisted laser desorption/ionization time–of–flight mass spectrometry. The new optimized variants of chCE7 exhibit substantial ADCC activity and, hence, may be useful for treatment of neuroblastoma. The strategy presented here should be applicable to optimize the ADCC activity of other therapeutic IgGs.

Anti‐neuroblastoma antibody chCE7 binds to an isoform of L1‐CAM present in renal carcinoma cells

Immunoprecipitation after cell surface labeling of human neuroblastoma cells showed that the anti‐neuroblastoma monoclonal antibody (mAb) chCE7 binds to a 200,000 Mr cell surface protein. The protein was partially purified by immuno‐affinity chromatography from a human renal carcinoma and a human neuroblastoma cell line, which both showed high levels of binding of MAb chCE7. NH2‐terminal sequences of 18 and 15 amino acid residues were determined. Both sequences isolated from the renal carcinoma and the neuroblastoma cells showed strong homology to human cell adhesion molecule L1 (L1‐CAM), and both were characterized by the NH2‐terminal deletion of 5 amino acids, comprising exon 2 of L1‐CAM. Reverse trancription‐polymerase chain reaction (RT‐PCR) analysis of the regions spanning exon 2 and exon 27 of L1‐CAM indicated that in neuroblastoma cells both transcripts for the full‐length and exon‐deleted forms are present, whereas in the renal carcinoma cell lines only the exon‐deleted L1‐CAM isoform were detected. Western blot analysis showed that 6 of 7 tested renal carcinoma cell lines and 5 of 15 renal carcinoma tissues expressed L1‐CAM. In normal adult kidney tissue, very low levels of protein expression were found. Northern blot analysis confirmed that in renal carcinoma and neuroblastoma cell lines L1‐CAM mRNA levels are correlated with protein expression. Int. J. Cancer 83: 401–408, 1999

Sequence and specificity analysis of recombinant human Fab anti-Rh D isolated by phage display.

Background and Objectives: Hyperimmune anti–Rh D serum is worldwide in short supply. As a first step to develop an alternative source of Rh D antibodies, we describe in this study the isolation and characterization of recombinant anti–Rh D Fab fragments. Materials and Methods: Peripheral blood mononuclear cells harvested from a hyperimmunized donor were used to construct two combinatorial Fab libraries. Phages expressing these Fab fragments were selected on whole red blood cells followed by testing of positive clones in an indirect hemagglutination assay. Results: Individual Fab clones are of high affinity and competitively inhibit the binding of a registered anti–D immunoglobulin. The Fab clones are also specific against the partial D phenotypes, Rh33, DIII, DIVa, DIVb, DVa, and DVII. The 13 different but highly homologous clones express preferentially VH3 segments. Conclusion: These Fab fragments show potential for the development of a new generation of therapeutic anti–Rh D reagents.

Fine specificity and heavy chain V gene usage of antibodies to the phosphorylcholine hapten

In the course of the immune response to PC-KLH in mice, two major groups of anti-PC antibodies are expressed which differ in fine specificity, Id expression and isotype preference. Group I antibodies react predominantly with the PC moiety of the hapten and display mostly the T15 Id. On the other hand, Group II antibodies recognize the PC hapten including the azophenyl spacer to the carrier and are T15 Id-. In order to investigate the relationship between the two antibody populations, we analyzed the composition of 20 anti-PC hybridomas of all isotypes with respect to their fine specificity and VH gene segment utilization. Characterization of the antibodies by hapten inhibition analyses with PC and p-nitro-phenyl-PC indicated that eight hybridomas secreted Group I and 12 Group II antibodies. Southern blot analysis of total genomic DNA using a germ-line JH probe demonstrated that, with the exception of aPC-111-1, all Group II cells utilized gene segments distinct from those of Group I. In contrast to the restricted VH gene usage in Group I hybridomas, a more pronounced heterogeneity in H chain V gene rearrangements in Group II cells was observed. We found three groups of hybridomas with identical VH gene rearrangements. Two hybridomas used a VH gene segment of the 7183 VH gene family. Three lambda 1 bearing Group II hybridomas shared a common hybridizing band. One of these, aPC-56-1, is known to utilize the same VH gene segment as MOPC 141. Finally, three cell lines, one of which (aPC-104-8) utilizes the VH1-DH-JH2 rearrangement, showed fragments of identical sizes. These results emphasize the independent origin of Group I and Group II anti-PC antibodies and demonstrate a larger germ-line repertoire of Group II antibodies as well as a less restricted use of particular VH genes relative to that of Group I.

Establishing and Developing Cho Cell Lines for the Commercial Productoin of Human Anti-Rhesus D IgG

Hemolytic Disease of the Newborn (HDN) can be a life threatening condition in Rhesus D positive babies born from Rhesus D negative mothers. Successful prophylaxis in mothers is commonly based on the use of plasma-derived anti-D immunoglobulin preparations, which are rapidly becoming a scarce resource. In consequence, the substitution of the human derived polyclonal anti-D is both a health and an ethical concern. Several groups are involved in the expression of immunoglobulins anti Rh D in different hosts, including fused EBV-transformed human lymphocytes with murine myeloma (only IgG1) (Olovnikova, Belkina et al. 1997) and EBV-transformed B-lymphoblastoid cell lines (IgG1 and IgG3 subclasses) (Kumpel 1997). Among all the eukaryotic expression systems, CHO cells are still the most popular cell host for the production of recombinant proteins as human therapeutic agents (Wurm 1997), because of safety considerations, as well as practical issues related to scale up.

The Influence of pH on Cell Growth and Specific Productivity of Two CHO Cell Lines Producing Human Anti Rh D IgG

The establishment of Chinese Hamster Ovary cells producing high affinity human recombinant anti RhD antibodies showing biological activity either by macrophage activation (chemiluminescence) and lysis of red blood cells by human lymphocytes (ADCC) has been published recently (1). To establish a process for the commercial production of human recombinant anti RhD antibodies, we studied pH as one of the most critical cell culture parameters. It was previously been reported the effects of lowering the pH on a generic fed-batch culture (3). In this study, two independent stable CHO cell lines expressing anti RhD antibodies (2) were compared with respect to the overall cell metabolism and productivity at pH values ranging from 6.9 up to 7.2 under batch conditions. The data we present confirms that addition of sodium ions in a form of NaCl raised product titers by 60 and 100% above those observed at respectively pH 7.2 and 6.9 for human recombinant Anti D producing CHO cell lines.

The SPP Biotech Module 1: Proteins for Medical Applications a Multidisciplinary Effort for the Development of a Recombinant Anti-RhD Antibody Preparation of Hemolytic Disease of the Newborn

A review with 0 refs. is presented regarding studies of a multidisciplinary team to establish the prophylactic principle and the manufg. process for anti-Rh D therapy based on recombinant antibody prepns. produced from Chinese hamster ovary cells. [on SciFinder (R)]

CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display: CHO Expression of Human Recombinant Anti-RhD Antibodies

Replacement of the hyperimmune anti‐Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti‐RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high‐affinity anti‐RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin‐treated red blood cells enabled the isolation of two high‐affinity Fab‐expressing phage clones, LD‐6‐3 and LD‐6‐33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants DIII, DIVa, DIVb, DVa, DVI types I and II, DVII, Rh33 and DFR. Full‐length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth‐adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti‐RhD antibody adapted to pilot‐scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody‐dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti‐RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.