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Figure 6. SDS-PAGE analysis of antigens immunoprecipitated from 1251 labeled Colo 38 melanoma cells by sera from a rabbit sequentially immunized with the anti-id mAb MK2-23 and with melanoma cells Colo 38. Rabbit 97-6 was immunized on day 0, 14, 28 and 42 with KLH conjugated mouse anti-id mAb MK2-23 mixed with Freund's adjuvant and on day 56 with cultured human melanoma cells Colo 38 (2 x 106). Sera harvested before immunization (NRS) and on day 40 [(RS 97-6 (40)] did not immunoprecipitate any components from a Colo 38 cell extract immunodepleted with anti-96K MAA mAb 376.96. In contrast, serum harvested on day 63 [RS 97-6 (63)] immunoprecipitated the HMW-MAA (left panel). Furthermore, serum harvested on day 63 [RS 97-6 (63)] removed HMW-MAA recognized by mAb 763.74 from a Colo 38 cell extract (right panel). The anti-96 kD MAA mAb 376.96 and the anti-HMW-MAA mAb 763.74 were used as controls.

It is likely that the antibodies elicited by the booster with melanoma cells have high association constants since they could immunoprecipitate HMW-MAA from cultured melanoma cells (Figure 6).

To overcome the inability of anti-id mAb to elicit HLA class I antigen restricted, TAA specific CTL, we plan to replace them with peptide mimics of TAA isolated by panning phage display peptide libraries with anti-TAA mAb and with TAA binding anti-anti-id mAb. This strategy bypasses two potential limitations of the approach based on amino acid sequence ho-

Figure 7. Stabilization of cell surface expression of HLA-A*0201 antigens on TAP-deficient lymphoblastoid cells T2 by a synthetic peptide identified with anti-anti-id mAb GH786. Human lymphoblastoid T2 cells 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 4°C, 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-A2, A28 mAb CR11-351. 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 resus-pended 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-A*0201 on T2 cells at a peptide concentration of 1 mg/ml. The reference HLA-A*0201 binding peptide IMDQVPFSV was used as a positive control.

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