Change of antigenic and neutralizing specificity in substitutional epitope peptides of hemophilia B inhibitor.

We have previously described the epitope mapping and functional neutralization of three factor IX inhibitors in hemophilia B (HB-1, 3, and 7) by synthetic peptides (13). However, A concentration of synthetic peptide of about 1000 times the concentration of factor IX in plasma was essential to neutralize the purified antibodies. We now report that substitutional synthetic peptides of epitope are able to neutralize the factor IX inhibitor with a lower concentration. Using two major epitope peptides, 156Val-Asn-Ser-Thr-Glu-Ala-Glu-Thr-Ile164 and 168Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn176, we designed changes of antigenicity using the systematic substitution of different amino acids at each residue of the epitope peptides and neutralization rate of factor IX inhibitors by a lower concentration of substitutional synthetic peptides conjugated with bovine serum albumin (BSA). Twenty-five substitutional peptides for HB-1, twenty substitutional peptides for HB-3 and forty-four substitutional peptides for HB-7 reacted stronger than the native sequences. One of the peptides, 1.0 microM of 156Val-Asn-Ser-Thr-Glu-Tyr-Glu-Thr-Ile164 conjugated with BSA, neutralized 26.5% of inhibitor in HB-1's plasma maximally. Our data show that high antigenicity peptides conjugated with BSA ranging in concentration from 0.1 microM to 1.0 microM are able to neutralize factor IX inhibitors in plasma and there is a possibility of peptide neutralization inhibitor therapy.

A new murine lymphocytotoxic monoclonal antibody recognizing HLA-A2, -A28 and -A9.

Monoclonal antibodies recognizing polymorphic as well as monomorphic epitopes on HLA antigens are important tools for understanding the immunobiology of HLA molecules. We immunized BALB/c mice with a HLA-A2 transfectant and screened for hybridomas which reacted with a HLA-A2 transfectant but not with a HLA-B75 transfectant. After subcloning by limiting dilution four times, a hybridoma secreting a monoclonal antibody (mAb) (IgG 2a, kappa) designated 1-145 was established. 1-145 reacted with Epstein-Barr virus transformed B lymphoblastoid cell lines (B cell lines) which expressed HLA-A2, -A28, -A23 and -A24. The titer of 1-145 in culture supernatant against HLA-A2 and -A28 antigens was similar and the titer against HLA-A23 was lower. 1-145 reacted with cells expressing HLA-A24 but the titer against HLA-A24 antigens was even lower than that against HLA-A23 antigens. The HLA-A24 antigens on the peripheral blood lymphocytes were not detected by 1-145 possibly due to the lower expression compared to the B cell lines. These differences of the titers were reflected to microlymphocytotoxicity assay in which 1-145 culture supernatant lysed all PBLs expressing HLA-A2,-A28 and -A23 but did not lyse PBLs expressing HLA-A24. Published deduced amino acid sequence data of HLA class 1 molecules indicate that Lys in position 127 may be critical for 1-145 binding.