[The Prostate Cancer Prevention Trial (PCPT). Relevance for clinical practice]. / Das "Prostate Cancer Prevention Trial" (PCPT). Die Bedeutung für den klinischen Alltag.

The Prostate Cancer Prevention Trial (PCPT) has been the first interventional trial directly aimed at the prevention of prostate cancer. A total of 18,882 men over 55 years with a PSA serum level less than 3.0 ng/ml were randomized to receive either the 5-alpha-reductase inhibitor finasteride 5 mg/day or placebo for 7 years. Despite a 25% reduction of prostate cancers in the treatment arm the results were discussed controversially. This criticism was mainly due to the observation of significantly more high-grade cancers in the finasteride group. Meanwhile, results of extensive follow-up analyses have been published suggesting that this finding is most likely due to optimized tumor detection in smaller glands. Further work-up demonstrated that PSA diagnosis and the histopathological examination were not compromised by finasteride. Furthermore, in addition to a decrease of prostate cancer the amount of prostatic intraepithelial dysplasia (PIN) was also reduced under finasteride. Future research must now aim at defining high-risk groups specifically profiting from chemoprevention with a 5-alpha-reductase inhibitor.

Production of a Human Antibody Library in the Phage-Display Vector pSEX81.

Human monoclonal antibodies (MAbs) are more suitable than MAbs of animal origin for clinical applications because of lower hypersensitivity reactions, less formation of circulating immune complexes and lower anti-immunoglobulin responses The classical production of human MAbs via the hybridoma technique or Epstein-Barr virus (EBV) transformation is limited by the instability of cell lines, low antibody production, and the problems of imununizing humans with certain antigens (1,2). A promising alternative 1s the production of human recombinant antibodies (3). Recombinant DNA technology has made it possible to clone human antibody genes in vectors and to generate antibody expression libraries (4-7). One approach has been to amplify and recombine the IgG repertoire of an "immunized" donor. This has been used to isolate several antibodies related to diseases (8,9). In order to obtain more universal antibody libraries the naive IgM repertoire of several "unimmunized" donors were pooled (10,12). The complexity of the combinatorial libraries has been further increased by creating the so-called "semisynthetic" antibody libraries (22-14).

Screening of phage-displayed antibody libraries.

The problem of amplifying a specific antibody in a population of millions of other antibodies has been solved by the immune system using the process of clonal selection Binding of an antigen to an IgM receptor on the surface of B-lymphocytes stimulates the proliferation and differentiation of the lymphocyte until it matures to an IgG-producing plasma cell. To mimic the first step of this process in bacteria, vectors have been constructed for the expression of antibodies on the surface of bacteria and phages (for review see Chapter 32 ).

Antibodies to steroids from a small human naive IgM library.

Human antibodies specific for digoxigenin, estradiol, testosterone and progesterone have been isolated from a small combinatorial IgM repertoire (4 x 10(7)) of single chain antibodies (scFv). The affinities of both the anti-estradiol and antiprogesterone scFv were approximately 10(8) M(-1). Naive IgM genes appeared to be highly represented, since only the heavy chain variable domain of the anti estradiol antibody contained differences to corresponding germline sequences. The light chain variable domain of the progesterone receptor was also identical to a germline sequence, showing that it is possible for completely naive antibodies to bind steroids with affinities comparable to those obtained after a secondary immune response.

The antigen-binding domain of a human IgG-anti-F(ab')2 autoantibody.

Recent studies revealed an immunoregulatory role of natural IgG-anti-F(ab')2 antibodies in both healthy individuals and patients with certain diseases. The implication of anti-F(ab')2 antibodies in the pathogenesis of diseases prompted us to study the gene segment structure of their antigen-binding domains and their binding characteristics. cDNA was prepared from the lymphocytes of a patient with a high IgG-anti-F(ab')2 serum titer. Variable heavy and light gene segments were amplified by PCR and inserted into a phagemid surface expression vector. Single-chain antibodies displayed on the phage surface were screened for binding to F(ab')2 fragments. The subsequent analysis of 95 single clones demonstrated that they all bound specifically to F(ab')2. Sequence analyses of 12 clones showed that 11 were identical and 1 contained a silent point mutation in the heavy chain and three amino acid exchanges in the light chain. The heavy chains belonged to the V(H)3 and the light chains to the V(kappa)2 gene family. The 11 identical light-chain genes were completely homologous to a germ-line sequence (DPK-15). Binding assays showed that the single-chain antibodies bind to F(ab')2, but not to Fab, Fc, or intact IgG. This binding pattern was confirmed by surface plasmon resonance studies, which revealed a relatively high affinity (Ka = 2.8 x 10(7) M(-1)). The strong binding capacity was further demonstrated by competitive inhibition of the serum anti-IgG antibody's interaction with antigen. The present study defines for the first time to our knowledge the gene segment structure of the antigen-binding domain of two human IgG-anti-F(ab')2 autoantibody clones and describes the binding kinetics of the purified monomeric fragments.

Cell surface display of a single-chain antibody for attaching polypeptides.

To provide an efficient means of coupling proteins, peptides and other suitable moieties to cells, we have constructed a retroviral expression vector for cell surface display of a single-chain antibody (scFv) against the hapten 4-ethoxymethylene-2-phenyl-oxazo-line-5-one (phOx). The hapten phOx can be easily conjugated to primary amino and sulfhydryl groups, thus providing points of attachment for the cell surface-bound anti-phOx scFv. This universal cell coupling system could prove to be particularly useful for anchoring monoclonal antibodies for tumor targeting and to present co-stimulatory molecules and other ligands (even mixtures) at the cell surface for gene therapy.

A single expression system for the display, purification and conjugation of single-chain antibodies.

To facilitate the purification and conjugation of single-chain antibodies (scFv) selected from a phage display library, we have incorporated His6, an amber stop codon and a C-terminal Cys into a surface expression vector. The vector also contains a lacIq gene for improving the efficiency of regulation and a sequence coding for a marker peptide.

Function of N-terminal import signals in trypanosome microbodies.

The glycosomes of trypanosomes are related to eukaryotic peroxisomes. For many glycosomal and peroxisomal proteins, a C-terminal SKL-like tripeptide known as PTS-1 serves as the targeting signal. For peroxisomes, a second N-terminal signal (PTS-2) was demonstrated on rat 3-ketoacyl-CoA thiolase. Several glycosomal proteins do not bear a PTS-1. One such protein, fructose bisphosphate aldolase, has a PTS-2 homology at its N-terminus. To find out whether the PTS-2 pathway exists in trypanosomes, we expressed chloramphenicol acetyltransferase fusion proteins bearing N-terminal segments of either rat thiolase or trypanosome aldolase. The mammalian PTS-2 clearly mediated glycosomal import. The aldolase N-terminus mediated import with variable efficiency depending on the length of the appended sequence. These results provide evidence for the existence of the PTS-2 pathway in trypanosomes.

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.

Glycosome assembly in trypanosomes: variations in the acceptable degeneracy of a COOH-terminal microbody targeting signal.

Trypanosomes compartmentalize most of their glycolytic enzymes in a peroxisome-like microbody, the glycosome. The specificity of glycosomal targeting was examined by expression of chloramphenicol acetyltransferase fusion proteins in trypanosomes and monkey cells. Compartmentalization was assessed by cell fractionation, differential detergent permeabilization, and immunofluorescence. The targeting signal of trypanosome phosphoglycerate kinase resides in the COOH-terminal hexapeptide, NRWSSL; a basic amino acid is not required. The minimal targeting signal is, as for mammalian cells, a COOH-terminal tripeptide related to -SKL. However, the acceptable degeneracy of the signal for glycosomal targeting in trypanosomes is considerably greater than that for peroxisomal targeting in mammals, with particularly relaxed requirements in the penultimate position.

Amino acid sequence of crayfish (Astacus fluviatilis) trypsin If.

The complete amino acid sequence of trypsin from the crayfish Astacus fluviatilis has been determined. The protein was fragmented with cyanogen bromide after S-carboxymethylation of the reduced disulfide bonds and by trypsin after S-carboxymethylation as well as after succinylation of lysine residues and aminoethylation of the reduced disulfide bonds. Peptides were purified by gel filtration and by reversed-phase high-performance liquid chromatography. Stepwise degradation was performed in a spinning cup sequencer. The enzyme contains 237 amino acid residues and has a molecular weight of 25 030. In contrast to bovine trypsin, it contains three rather than six disulfide bonds which are paired in the same fashion as those in trypsin from Streptomyces griseus. The constituents of the active site of bovine trypsin are present in corresponding positions in the crayfish enzyme. Crayfish trypsin shows 43.6% sequence identity with the bovine enzyme as compared to 40.0% identity with the S. griseus enzyme. The present analysis affords the first detailed view into the evolution of trypsins at the invertebrate level.