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Anti-HIV Immunotoxin and Antibody-Drug Conjugate Targeted by the Same Anti-gp41 Antibody Display Distinct Modes of Cellular Killing

ABSTRACT: Background: We are developing cytotoxic immunoconjugates (CICs) to eradicate the persistent reservoir of HIV infection, the barrier to a cure. Having identified the most effective monoclonal antibodies (mAbs) for targeting CICs to HIV-infected cells, we investigated the efficacy and mode of killing of different forms of CICs targeted by the same mAb, an immunotoxin (IT) and antibody drug conjugate (ADC). Methods: We compared the in vitro effects of CICs made by conjugating anti-gp41 mAb 7B2 to either deglycosylated ricin A chain (7B2-dgA) or the anthracycline derivative PNU-159682 (7B2-PNU). Cytotoxicity was tested on cell lines stably expressing the HIV envelope. Metabolic and transcriptional analyses of treatment effects were performed on cells persistently infected with HIV. Results: 7B2-dgA was more potent and acted more rapidly to kill cells. 7B2-PNU induced bystander-cell killing but stimulated cell growth upon low dose or brief exposure. Six hr post treatment, 7B2-dgA elicited both metabolic and transcriptional alterations, whereas 7B2-PNU treated cells did not differ from untreated cells. At 24 hr, the profiles following both treatments differed from untreated cells and from each other. 7B2-dgA treated cells exhibited elevated intracellular levels of many AAs, and early activation of gene pathways for apoptosis and transcriptional control; later transcriptional changes included decreases in expression of enzymes involved in AA synthesis and carbon metabolism. Conclusions: An IT and ADC, each delivered by the same antibody and tested in the same cell lines showed substantial differences in their mode of killing, of potential clinical significance. The results of this side-by-side comparison of an ADC and IT have implications for the development of CICs to treat other conditions, including cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE289396 | GEO | 2026/04/07

REPOSITORIES: GEO

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Project description:We have developed a monoclonal antibody (mAb) C7 that reacts with Als3p and enolase present in Candida albicans cell wall and exerts three anti-Candida activities: candidacidal activity and inhibition of both adhesion and filamentation. To investigate the mode of action of mAb C7 on fungal viability, we examined changes in the genome-wide gene expression profile of C. albicans grown in presence of a subinhibitory concentration of mAb C7 (12.5 µg/ml) by using microarrays. A total of 49 genes were found to be differentially expressed upon treatment with mAb C7. Of these, 28 were found to be up-regulated and 21 down-regulated. The categories of up-regulated genes with the largest number of variations were those involved in iron uptake or related to iron homeostasis (42.86%), while the energy-related group accounted for 38.10% of the down-regulated genes (8/21). Results were validated by real Time PCR. Since these effects resembled those found under iron-limited conditions, the activity of mAb C7 on C. albicans mutants with deletions in key genes implicated in the three iron acquisition systems described in this yeast was also assessed. Only mutants lacking TPK1 and TPK2 genes were less sensitive to the candidacidal effect of mAb C7. FeCl3 or hemin at concentrations ≥ 7.8µM reversed the candidacidal effect of mAb C7 on C. albicans, on a concentration dependent manner. The results presented in this study provide evidence that the candidacidal effect of mAb C7 is related to the blockage of the reductive iron uptake pathway of C. albicans. A saturated culture of C. albicans grown overnight was diluted to an optical density at 600 nm of approximately 0.1 and divided in two aliquots. One of them was used untreated as control and the second one was treated with a subinhibitory concentration (12.5 µg/ml) of monoclonal antibody C7 . Both cultures were incubated for 18 h at 37ºC before harvesting cells. Antibody added and control samples were obtained each time. The experiment was repeated once. Dye-swap technique was used for hybridization and four arrays were analyzed to compare the expresion of over six thousands genes in response to antibody C7.

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Anti-HIV Immunotoxin and Antibody-Drug Conjugate Targeted by the Same Anti-gp41 Antibody Display Distinct Modes of Cellular Killing

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