Cloning and cytotoxicity of a human pancreatic RNase immunofusion.

BACKGROUND: Immunotoxins based on plant and bacterial proteins are usually very immunogenic. Human ribonucleases could provide an alternative basis for the construction of less immunogenic reagents. Two members of the human RNase family, angiogenin and eosinophil-derived neurotoxin (EDN), have been fused to a single chain antibody against the transferrin receptor, which is known to be internalised by endocytosis. The fusion proteins proved to be very efficient inhibitors of protein synthesis using various cell lines. It is not yet known whether the side effects of angiogenin and EDN will compromise their potential use as immunotoxins. OBJECTIVES: The goal of this work was to construct a human immunotoxin with no harmful side effects. Bovine pancreatic ribonuclease has been shown to be as potent as ricin at abolishing protein synthesis on injection into oocytes. We therefore decided to clone its human analogue, which is fairly ubiquitous and per se non-toxic. An immunofusion of human pancreatic RNase with a single chain antibody against the transferrin receptor was tested for its ability to inhibit protein synthesis in three different human tumor cell lines. STUDY DESIGN: DNA coding for the human pancreatic RNase was cloned partially from a human fetal brain cDNA library and then completed by PCR using a human placental cDNA library as a template. The RNase gene was then fused with a DNA coding for an single chain antibody against the transferrin receptor (CD71). After expressing the fusion protein in E. coli, the gene product was isolated from inclusion bodies and tested for cytotoxicity. RESULTS: This fusion protein inhibited the protein synthesis of three human tumor cell lines derived from a melanoma, a renal carcinoma and a breast carcinoma, with IC50s of 8, 5 and 10 nM, respectively. These values were comparable with those using a similar fusion protein constructed with eosinophil derived neurotoxin (EDN) as the toxic moiety (IC50s of 8, 1.2 and 3 nM, respectively). The slightly lower activities of the human pancreatic RNase-scFv (pancRNase-scFv) with two of the cell lines suggests that fewer molecules are reaching the cytoplasmic compartment, since it was twice as active as EDN-scFv in inhibiting the protein synthesis of a rabbit reticulocyte lysate. CONCLUSION: These results demonstrate that the human pancreatic RNase, which is expected to have a very low immunogenic potential in humans with no inherent toxicity, may be a potent cytotoxin for tumor cells after antibody targeting.

Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes.

Previously described primers for PCR amplification of variable immunoglobulin (Ig) genes were based on gene sequences. To include the large number of amino acid sequences of antibodies whose DNA has not been sequenced and to ensure a maximal fit to rearranged human Ig variable region genes, we have made a comprehensive comparison of both protein and nucleotide sequences. The resulting set of 15 primers was able to amplify a wide range of rearranged antibody variable region genes. Restriction sites included in the primers facilitate cloning of the PCR products into various expression vectors. Sequence analyses of PCR-amplified cDNA derived from a polyclonal B cell population showed that maximal enrichment is obtained for highly represented variable Ig gene subgroups. Rarely occurring V kappa 4 and V lambda 5 subgroups were not detected. Rearranged Ig variable region genes from each of 19 human B cell lines were also amplified. Comparisons to germline sequences allowed the allocation of rearranged genes to the original Ig genes. This primer set should be very useful for generating large repertoires of rearranged V genes and for amplifying genes of individual B cell clones.

Isolation of IgG antibody Fv-DNA from various mouse and rat hybridoma cell lines using the polymerase chain reaction with a simple set of primers.

To facilitate the isolation of IgG antibody Fv-DNA sequences from hybridoma cell lines, we have established a polymerase chain reaction (PCR) procedure requiring only a small number of primers. The sense primers homologous to DNA coding for the first framework sequences were designed to hybridize to all the known antibody sequences under conditions that permit a high number of mismatches. The antisense primers were homologous to DNA coding for the beginning of the constant regions of the gamma and kappa chains. Restriction sites introduced by the primers enable the DNA to be cloned into bacterial expression vectors. Only three sense VH primers and two sense VL primers paired with one backward primer for the heavy and light chains, respectively, were necessary for the amplification of Fv-DNA from a total of 17 rodent cell lines that we have so far worked with. These consisted of 12 mouse cell lines and five rat cell lines. This procedure will therefore probably be sufficient to isolate the Fv-DNA from most mouse cell lines and possibly also from most rat cell lines.

Simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR.

A method for the facile simultaneous mutagenesis of complementary-determining regions (CDRs) in a single chain antibody (scFv) is described. Overlapping sets of oligonucleotides containing random sequences within the CDRs corresponding to the heavy chain variable region (VH) jointed to a linker peptide (J) and the light chain variable region (VL) were extended under PCR conditions to full-length genes. These gene products were then further amplified using short PCR primers containing complementary overlaps between the 3' and 5' ends of the VH-J and VL genes respectively. In a final step, the VH-J and VL gene products were mixed and assembled into scFv DNA products by overlap extension under standard PCR conditions. Sequence analyses indicated that the method is basically successful. However, some deletions were observed, which probably reflects difficulties in the automatic synthesis of long degenerate oligonucleotides.

Gene structure and chromosomal localization of the murine lamin B2 gene.

The structure of the murine lamin B2 gene has been analyzed by cloning, sequencing and hybridization techniques, including in situ hybridization. The gene exists in single copy on the distal arm of chromosome 10 and comprises at least 15 kb, containing 12 exons and 11 introns. The transcriptional start point, as determined by primer extension analysis and RNase protection, was mapped to the region -264 to -254 upstream the ATG start codon. The 5' upstream region does not reveal any classical TATA box elements but typical features of genes encoding "housekeeping" proteins. The intron pattern is strikingly similar to those of the Xenopus laevis lamin LIII (Döring, V., R. Stick, EMBO J. 9, 4073-4081 (1990)) and of the intermediate filament protein of the invertebrate Helix aspersa (Dodemont, H., D. Riemer, K. Weber, EMBO J. 9, 4083-4094 (1990], particularly in the central rod and in the tail domains. Moreover, this lamin gene contains an additional intron in the region encoding the rod domain. Our data are compatible with the evolutionary hypothesis that IF proteins have evolved from a lamin-like ancestor molecule.