Characterization of syngeneic antiidiotypic monoclonal antibodies to murine anti-human high molecular weight melanoma-associated antigen monoclonal antibodies.

Abstract

Hybridization with murine myeloma cells P3-X63-Ag8.653 of splenocytes from BALB/c mice immunized with syngeneic anti-human high molecular weight melanoma-associated Ag (HMW-MAA) mAb 149.53, 225.28, 763.74, and TP41.2 has resulted in the formation of antiidiotypic antibody-secreting hybridomas with a frequency ranging between 1.2% and 5.2%. No marked difference was detected in the frequency of antibody secreting hybridomas in the fusions generated from mice immunized with the four anti-HMW-MAA mAb, suggesting that the idiotopes expressed by each of them display similar immunogenicity in a syngeneic combination. The number of antiidiotypic mAb that did not inhibit the binding of immunizing mAb to melanoma cells was higher than that of those that died, suggesting that idiotopes not associated with the Ag-combining site are more immunogenic than those that are. The idiotopes recognized by mAb were not detected on a large panel of anti-HLA Class I mAb, anti-HLA Class II mAb, and anti-human melanoma-associated Ag mAb. The latter included also mAb that cross-inhibit the immunizing anti-HMW-MAA mAb. The idiotopes recognized by mAb were not detected on the isolated H and L chain of the immunizing anti-HMW-MAA mAb. Cross-blocking experiments with a selected number of antiidiotypic mAb identified three distinct idiotopes on mAb 149.53, 225.28, and TP41.2 and two on mAb 763.74. Three, 5, 2, and 5 antiidiotypic mAb to idiotopes within the Ag-combining site of mAb 149.53, 225.28, 763.74, and TP41.2, respectively, were tested for their ability to induce anti-HMW-MAA antibodies. Serological and immunochemical assays detected anti-HMW-MAA antibodies only in sera from BALB/c mice immunized with mAb MK2-23. Therefore, mAb MK2-23 can be classified as beta, while the remaining 14 can be classified as gamma.

Turning anecdotal irradiation-induced anticancer immune responses into reproducible in situ cancer vaccines via disulfiram/copper-mediated enhanced immunogenic cell death of breast cancer cells

“…A stock solution of DSF (10 mM) was aliquoted and stored at −20 °C for up to 1 year and freshly diluted with cell culture medium (in vitro assays) or PBS (in vivo assays) prior to use. Antibodies for flow cytometry analyses were as follows: mouse anti-calreticulin antibody TO-11 and an isotype-matched mouse IgG1 MK2-23 were produced by our laboratory [ 30 , 31 ], APC-Cy7-conjugated hamster anti-mouse CD3e (557596), FITC-conjugated rat anti-mouse CD4 clone H129.19 (553650), PE-Cy7-conjugated rat anti-mouse CD8a (552877), BV421 rat anti-mouse CD25 clone 3C7 (564370), PE-conjugated rat anti-mouse Foxp3 clone MF23 (560408), fixable viability stain 620 [fvs620] (564996), transcription factor buffer set (562574), anti-rat and anti-hamster Igκ/negative control compensation particles set (552845), BV650-conjugated rat anti-CD11b (563402), BV605-conjugated rat anti-CD11c (563015), and APC-conjugated rat anti-mouse Gr-1 (553129) were purchased from BD Biosciences(Franklin Lakes, NJ, USA). APC-conjugated mouse anti-human CD47 monoclonal antibody [B6H12] (17-0479-42) and APC-conjugated mouse IgG1 kappa isotype control (17-4714-82) were purchased from ThermoFisher (Waltham, MA, USA).…”

Turning anecdotal irradiation-induced anti-cancer immune responses into reproducible in situ cancer vaccines via disulfiram/copper-mediated enhanced immunogenic cell death of breast cancer cells

“…Antibodies for flow cytometry analyses were as follows: mouse anti-calreticulin antibody TO-11 and an isotype-matched mouse IgG1 MK2–23 were produced by our laboratory[ 29 , 30 ], APC-Cy7-conjugated hamster anti-mouse CD3e (557596), FITC-conjugated rat anti-mouse CD4 clone H129.19 (553650), PE-Cy7-conjugated rat anti-mouse CD8a (552877), BV421 rat anti-mouse CD25 clone 3C7 (564370), PE rat anti-mouse Foxp3 clone MF23 (560408), fixable viability stain 620 [fvs620] (564996), transcription factor buffer set (562574), anti-rat and anti-hamster Igκ/negative control compensation particles set (552845), BV650 rat anti-CD11b (563402), BV605 rat anti-CD11c (563015), and APC rat anti-mouse Gr-1 (553129)were purchased from BD Biosciences(Franklin Lakes, NJ, USA).…”

rs822336 binding to C/EBPβ and NFIC modulates induction of PD-L1 expression and predicts anti-PD-1/PD-L1 therapy in advanced NSCLC

“…Nivolumab was purchased from Thermo Fisher (Waltham, MA, USA). mAb LGIII-147.4.1 which recognizes β2m-associated HLA-A heavy chain, excluding -A23, -A24, -A25, -A32 [42]; mAb B1.23.2 which recognizes β2m-associated HLA-B and -C heavy chains [43]; mAb TP25.99.8.4 which recognizes β2massociated HLA-A, -B and -C heavy chains [44]; mAb NAMB-1 which recognizes β2-microglobulin [45]; mAb LGII-612.14 which recognizes HLA-DR, DQ, DP [46] and anti-idiotypic (anti-id) mAb MK2-23 [47] were all kindly provided from Prof. Soldano Ferrone (Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA). Allophycocyanin (APC) anti-PD-L1 IgG2b Ab (catalog on.…”