Apoptosis of a human melanoma cell line specifically induced by membrane-bound single-chain antibodies.

CD28 is a key regulatory molecule in T cell responses. Ag-TCR/CD3 interactions without costimulatory signals provided by the binding of B7 ligands to the CD28R appear to be inadequate for an effective T cell activation. Indeed, the absence of B7 on the tumor cell surface is probably one of the factors contributing to the escape of tumors from immunological control and destruction. Therefore, to increase the immunogenicity of tumor cell vaccines, we have expressed anti-CD3 and anti-CD28 single-chain Abs (scFv) separately on the surface of a human melanoma SkMel63 cell line (HLA-A*0201). A mixture of cells expressing anti-CD3 with cells expressing anti-CD28 resulted in a marked activation of allogeneic human PBL in vitro. The apparent induction of a Th1 differentiation pathway was accompanied by the proliferation of MHC-independent NK cells and MHC-dependent CD8+ T cells. PBL that had been cultured together with transfected SkMel63 tumor cells were able to specifically induce apoptosis in untransfected SkMel63 cells. In contrast, three other tumor cell lines expressing HLA-A*0201, including two melanoma cell lines, showed no significant apoptosis. These results provide valuable information for both adoptive immunotherapy and the generation of autologous tumor vaccines.

T cell activation by monoclonal antibodies bound to tumor cells by a cell surface displayed single-chain antibody.

Tumor cells often lack the costimulatory molecules necessary for T cell activation. However, the transformation of cells with more than one stimulatory molecule is a difficult procedure. We therefore developed a retroviral vector for the expression of a cell membrane anchored single-chain antibody fragment (scFv) directed against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (phOx). Proteins and peptides can be readily modified with this hapten, thus, enabling them to be bound to cells with the cell surface displayed anti-phOx scFv. To test combinations of surface-bound stimulatory molecules on T cell activation, SK-Mel63 human melanoma cells expressing the membrane anchored anti-phOx scFv were incubated with phOx-labeled mAbs against CD3, CD28 and CD5. Cells presenting a given mixture of modified anti-CD3 and anti-CD28 molecules stimulated T cell activation better than any single antibody and a given mixture of anti-CD3, anti-CD28 and anti-CD5 provided a stimulatory response higher than the best double combination. However, the relative concentrations are very important and must be carefully chosen. Concentrations of antibodies giving good T cell responses when used alone can block synergistic effects.

A TNF receptor antagonistic scFv, which is not secreted in mammalian cells, is expressed as a soluble mono- and bivalent scFv derivative in insect cells.

Single chain antibodies (scFv) are usually produced in E. coli, but generation of certain scFv derivatives, such as complex fusion proteins or glycosylated forms of scFv is restricted to eukaryotic expression systems. We investigated the production of soluble mono- and bivalent single chain antibodies (scFv) in eukaryotic cells and describe a cassette vector system for mammalian and baculovirus expression which is compatible with an established vector system for bacterial expression and phage display selection of scFvs. The applied model scFv was derived from a murine antibody (H398) against human tumor necrosis factor receptor 1 (TNFR60), known to be a potent antagonist of TNF action in its monomeric form and a potential therapeutic agent for treatment of TNF-mediated diseases. Surprisingly, the monomeric scFv form of H398 (scFv H398) is expressed but not secreted in different mammalian cells. In contrast, in insect cells using recombinant baculovirus, a monovalent scFv H398 and a bivalent scFv fusion protein with an human IgG1 Fc region were expressed and secreted with correctly processed signal sequence. Concerning the influence of valency of the model Ab and its derivatives on antigen binding affinity and neutralisation of TNF activity, we found that the mono- and bivalent form of scFv H398 possesses the same characteristics as proteolytically produced Fab H398 and original mAb H398, respectively. Furthermore, fusion of the Ig Fc protein to scFv H398 increase the in vitro half-life at 37 degrees C. We conclude that the described cassette vectors readily allow the eukaryotic expression of mono- and bivalent scFv derivatives to analyse the influence of valency of scFv molecules on antigen binding and biological activity.

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.

Molecular characterization and determination of the coding capacity of the genome of equine herpesvirus type 2 between the genome coordinates 0.235 and 0.258 (the EcoRI DNA fragment N; 4.2 kbp).

The complete DNA nucleotide sequence of the EcoRI DNA fragment N (0.235 to 0.258 viral map units) of equine herpes virus type 2 (EHV-2) strain T400/3 was determined. This DNA fragment comprises 4237 bp with a base composition of 55.23% G+C and 44.77% A+T. Nineteen open reading frames (ORFs) of 50-287 amino acid (aa) residues were detected. ORF number 10 is located between the nucleotide position 2220 and 2756 coding for a protein of 179 amino acid residues. This protein shows significant homology to the cytokine synthesis inhibitory factor (CSIF; interleukin 10) of human (76.4%) and mouse (68.5%), and to the Epstein-Barr virus (EBV) protein BCRF1 (70.6%). The existence of an interleukin 10 (IL-10) analogous gene within the genome of the EHV-2 was confirmed by screening the genome of nine EHV-2 strains using specific oligonucleotide primers corresponding to the 5' and 3' region of this particular gene by polymerase chain reaction. In all experiments an 870 bp DNA product was amplified. The specifity of the amplified DNA fragments obtained from individual EHV-2 strains was confirmed by DNA-DNA hybridization experiments. The DNA sequence analysis of the amplified DNA products of the EHV-2 strain LK was carried out. This analysis revealed the identity of the corresponding IL-10 gene (540 bp) of this strain to the IL-10 gene of EHV-2 strain T400/3. The presented data indicate that the EHV-2 genome harbors a viral interleukin 10-like gene. This is further evidence that the IL-10 gene can be present in the genomes of members of the Herpesviridae family.

The genome of equine herpesvirus type 2 harbors an interleukin 10 (IL10)-like gene.

A gene was identified within the DNA sequences of the EcoRI DNA fragment N (4.3 kbp) of the genome of equine herpesvirus type 2 (EHV-2) coding for a protein (179 amino acid residues) homologous to the cytokine synthesis inhibitory factor (CSIF; interleukin 10) of the human and mouse, and to the Epstein-Barr virus (EBV) protein BCRF1. This finding is further significant evidence that the interleukin 10 (IL-10) and/or IL-10-like gene can indeed be present in the genomes of members of the herpesviral family.