Immunotoxins to Selectively Target Reservoirs of HIV-Infected Cells.

Edward A. Berger1, Paul E. Kennedy1, Tapan K. Bera2, Mark G. Lewis3, and Ira Pastan2

1Laboratory of Viral Diseases, NIAID and 2Laboratory of Molecular Biology, NCI, NIH, Bethesda, MD, and 3Southern Research Institute, Frederick, Maryland (USA)

We have developed immunotoxins targeted to the HIV-1 Env as an approach to deplete the infected cell reservoirs persisting after HAART. Each immunotoxin is a recombinant single-chain chimeric protein containing the translocation and cytotoxic domains of Pseudomonas aeruginosa exotoxin A linked to a specific protein that binds Env. The first agent (CD4-PE40) employs sCD4 as the Env-targeting moiety; a newer, more potent agent (B3-PE38) employs an SCFv of the high affinity 3B3 MAb against the highly conserved CD4 binding site. Each protein killed chronically HIV-infected cells with very high potency (IC50 ≈ 90 ng/ml for CD4-PE40, 2 ng/ml for 3B3-PE38), while showing negligible activity against the uninfected parental cells (IC50 » 1000 ng/ml). Each protein inhibited spreading infection by diverse primary HIV-1 isolates, in both PBMCs and primary macrophages; again 3B3-PE38 proved considerably more potent than CD4-PE40.  Parallel control experiments with free sCD4 or 3B3 Fab confirmed that the potency of the chimeric toxins reflected selective killing of the infected cells rather than simple neutralization by the targeting moiety. Combined treatment of acutely infected T cell cultures with toxin plus an RT inhibitor completely eliminated infectious virus, a result not achieved with either agent alone; similar results were obtained in the SCID-hu thy/liv murine model. These results highlight the special value of combining an anti-HIV agent that blocks the viral replication cycle with another agent that kills cells that are already infected. In rhesus macaques, high levels of CD4-PE40 displayed some hepatotoxicity, in keeping with the dose-limiting hepatotoxicity observed in earlier Phase I trials conducted in the pre-HAART era. By contrast, high levels of 3B3-PE38 displayed no hepatotoxicity in macaques, consistent with promising human clinical trial results with PE-based anti-cancer immunotoxins showing major remissions of leukemias and lymphomas without serious liver toxicity. Collaborative studies have led to the design of improved variants of 3B3-PE38 with enhanced potency, stability, and serum half-life, and have demonstrated immunotoxin efficacy in vitro and in vivo particularly when used in combination with HAART drugs plus activating agents that induce HIV expression from latently infected cells. These results suggest that Env-targeted immunotxins might prove highly valuable in efforts to deplete the HIV-infected cell reservoirs that persist in the face of effective antiretroviral therapy.