Log Fluorescence Intensity

Figure 8. Stabilization of cell surface expression of HLA-B*2705 antigens on TAP-deficient lymphoblastoid cells T2 by a synthetic peptide identified with anti-anti-id mAb GH704. HLA-B*2705 transfected human lymphoblastoid cells T2 (T2-B*2705) were incubated with increasing concentrations (0.1-1 mg/ml) of peptides in RPMI 1640 medium supplemented with 10% FBS. Following an overnight incubation at 4oC, cells were harvested, washed twice with PBS-5% FBS-0.2% sodium azide (FACS buffer) and incubated for 30 min at 4°C with anti-HLA-B7 crossreacting group mAb KS4. Following washing with FACS buffer, cells were incubated for 30 min at 4°C with a 1:100 dilution of a fluorescein isothiocyanate conjugated goat antimouse IgG antibody solution. Cells were then washed with FACS buffer and resuspended in 0.5 ml FACS buffer. Cytofluorometry was carried out using a FACS IV flow cytometer (Becton Dickinson and Co., Mountain View, CA). Histograms shown represent stabilization of HLA-B*2705 antigens on T2-B*2705 cells at a peptide concentration of 0.5 mg/ml. The reference HLA-B*2705 binding peptide RRYQKSTEL was used as a positive control.

mology between anti-id mAb and nominal antigen to identify peptides to be used as immunogens. First, this approach can be applied to a very limited number of antigenic systems, since amino acid sequence homology between anti-id mAb and the nominal anitgen is rarely found (for a review, see [103]). Second, a peptide constructed on the basis of its homology with the nominal antigen [30, 31] is not likely to be very immunogenic in patients with melanoma since the corresponding clones are likely to have been deleted.

By panning two phage display peptide libraries with a panel of anti-HMW-MAA mAb, we have isolated a number of peptides with distinct sequences. Some display homology with the HMW-MAA core protein amino acid sequence. Furthermore some peptides have HLA class I antigen binding motifs. Representative examples are shown in Table 9, Figurea 7 and 8. The possibility that these peptide mimics of TAA may elicit HLA class I antigen restricted, TAA

specific CTL is supported by the recognition by CTL of an antibody defined TAA [104] and by the sharing of specificity between T-cell receptors and antibodies [105], If studies in progress prove the validity of our working hypothesis, then it is likely that the field of TAA mimicry will witness the replacement of anti-id mAb with peptide mimics. The latter will also have the advantages: (i) to be easily synthesized and standardized to meet the regulatory requirements for clinical trials; (ii) to avoid the induction of antimouse Ig antibodies; and (iii) to facilitate the monitoring of humoral and cellular anti-HMW-MAA immunity elicited by active specific immunotherapy.

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