Immunogenicity of an interferon-beta1a product.

In order to determine whether Blastoferon®, a biosimilar interferon (IFN)- beta 1a formulation, shares epitopes with other known IFN-beta products, a series of neutralization bioassays were performed with a set of well-characterized anti-IFN- beta monoclonal antibodies and human sera (World Health Organization Reference Reagents). The bioassay was the interferon-induced inhibition of virus cytopathic effect on human cells in culture (EMC virus and A-549 cells). Computer-calculated results were reported as Tenfold Reduction Units (TRU)/ml. To further assess Blastoferon® immunogenicity, in vivo production of anti-IFN beta antibodies was determined in sera of patients included in the pharmacovigilance plan of Blastoferon® by the level of IFN- beta 1a binding antibodies (by enzyme immunoassay -EIA) and neutralizing antibodies (in the Wish-VSV system). The highly characterized neutralizing monoclonal antibodies A1 and A5 that bind to specific regions of the IFN- beta molecule reacted positively with the three beta 1a IFNs: Blastoferon®, Rebif®, and the IFN- beta WHO Second International Standard 00/572. As expected, the non-neutralizing monoclonal antibodies B4 and B7 did not neutralize any of the IFN- beta preparations. The commercially available monoclonal antibody B-02 reacted essentially equally with Rebif® and Blastoferon®. The WHO Reference Reagent human serum anti-IFN- beta polyclonal antibody neutralized all the IFN- beta products, whereas the WHO Reference Reagent human serum anti-IFN-alpha polyclonal antibody G037-501-572 appropriately failed to react with any of the IFN- beta products. On the basis of in vitro reactivity with known, well-characterized monoclonal and polyclonal antibody preparations, Blastoferon® shares immunological determinants with other human interferon- beta products, especially IFN- beta 1a. In vivo antibodies were detected by EIA in 72.9% of 37 chronically treated multiple sclerosis patients, whereas neutralizing antibodies were found in 8.1% of them. Blastoferon® appears to have immunological characteristics comparable to other IFN- beta 1a products.

Whole genome analysis of the action of interferon-beta.

OBJECTIVES: To characterize the IFNbeta1a-regulated gene expression on leukocytes of Multiple Sclerosis (MS) patients using microarrays with whole human genome representation. METHODS: Genes differentially expressed by interferon-beta were identified by a microarray in vitro study performed in leukocytes obtained from 5 MS relapsing-remitting patients. RESULTS: Following the culture of peripheral blood mononuclear cells from MS relapsing-remitting patients for 24 hs with IFNbeta1a, the expression of 868 genes was modified: 545 increased (including CXCL11, CCL8, INDO, IFI27, CFB, CXCL10 and IFIT1) and 323 diminished (including RBP7, SEPT5, RNF8, ADORA2B and FOS). CONCLUSIONS: Since many of them were previously recognized as involved in MS pathogenesis, the IFNbeta1a mechanism of action could imply a compensatory regulation of systems deregulated in MS.

Conformational and sequential epitopes on the human granulocyte-colony stimulating factor molecule (hG-CSF) and their role in binding to human placenta receptors.

Monoclonal antibodies (mAbs) named 8C2 and 6E3, directed against the recombinant human granulocyte colony-stimulating factor (hG-CSF), were used as probes to study the cytokine orientation on its binding to receptors from human placenta. Competition enzyme linked immunoabsorbent assays (ELISA) revealed that mAb 8C2 would be directed to a linear epitope, whereas mAb 6E3 would delimit a more assembled epitope. Gel-filtration high performance liquid chromatography (HPLC) of the immune complexes formed by incubating [(125)I]hG-CSF with each mAb showed that epitope 8C2, but not 6E3, was altered after cytokine iodination. In addition, mAb 6E3 completely inhibited [(125)I]hG-CSF binding to human placental microsomes. Although [(125)I]mAb 6E3 was unable to bind to preformed hG-CSF-receptor complexes, [(125)I]mAb 8C2 did recognize hG-CSF previously bound to receptors, suggesting that epitope 8C2 would remain accessible in the hG-CSF-receptor complex. To identify the cytokine region defined by mAbs, hG-CSF was digested with different proteolytic enzymes: Arg-C, Glu-C, trypsin and alpha chymotrypsin. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation. Results showed that mAb 6E3 would be directed to a conformation-dependent epitope located close to the hG-CSF binding domain and included into the sequence 1-122/123, whereas mAb 8C2 recognized the region 41-58, which represents a linear epitope left exposed after cytokine binding to receptors from human placenta.

Identification of a linear epitope of interferon-alpha2b recognized by neutralizing monoclonal antibodies.

Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-alpha2b (IFN-alpha2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-alpha2b binding to immobilized mAbs was completely inhibited by IFN-alpha2b and IFN-alpha2a but neither IFNbeta nor IFNgamma showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-alpha2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN-alpha2b anti-viral and anti-proliferative activities as well as [125I]IFN-alpha2b binding to WISH cell-membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN-alpha2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107-132 or 107-146. As this zone includes the sequence 123-140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.

Proenkephalin system in human polymorphonuclear cells. Production and release of a novel 1.0-kD peptide derived from synenkephalin.

In the hematopoietic system a pluripotent stem cell generates precursors for lymphoid and myeloid lineages. Proenkephalin-derived peptides were previously detected in differentiated lymphoid cells. We have studied whether the proenkephalin system is expressed in a typical differentiated cell of the myeloid lineage, the neutrophil. Human peripheral polymorphonuclear cells contain and release proenkephalin-derived peptides. The opioid portion of proenkephalin (met-enkephalin-containing peptides) was incompletely processed, resulting in the absence of low molecular weight products. The nonopioid synenkephalin (proenkephalin 1-70) molecule was completely processed to a 1.0-kD peptide derived from the COOH-terminal. This molecule was characterized in neutrophils by biochemical and immunocytochemical methods. The chemotactic peptide FMLP and the calcium ionophore A23187 induced the release of the proenkephalin-derived peptides, and this effect was potentiated by cytochalasin B. The materials secreted were similar to those present in the cell, although in the supernatant a higher proportion corresponded to more processed products. The 1.0-kD peptide was detected in human, bovine, and rat neutrophils, but the chromatographic pattern of synenkephalin-derived peptides suggests a differential posttranslational processing among species. These findings demonstrate the existence of the proenkephalin system in human neutrophils and the production and release of a novel 1.0-kD peptide derived from the synenkephalin molecule. The presence of opioid peptides in neutrophils suggests their participation in the inflammatory process, including a local analgesic effect.