Increased Affinity and Stability of an Anti-HIV-1 Envelope Immunotoxin by Structure-based Mutagenesis

Abstract

HIV-infected cells are selectively killed by an immunotoxin in which a truncated form of Pseudomonas exotoxin A is joined to the variable region of a broadly neutralizing antibody (3B3) that recognizes the viral envelope glycoprotein (Env). To improve the efficacy of this molecule, we used three-dimensional structural information and phage selection data to design 23 single and multiple point mutations in the antibody variable region sequences that contact Env. Substituting an aromatic residue for an aspartate in the third complementarity-determining region of V H increased the potency of the immunotoxin by ϳ10-fold in a cell-killing assay. Detailed analysis of one such mutant, N31H/Q100eY, revealed both a higher affinity for monomeric and cell surface Env and an increased stability against aggregation compared with the starting immunotoxin. Conversion to a disulfide-linked two-chain format further stabilized the protein. N31H/Q100eY retained the ability to bind to Env from multiple viral isolates, to inhibit Envmediated cell fusion, and to limit spreading viral infection in peripheral blood mononuclear cells. Such sitedirected mutants may increase the utility of immunotoxins for reducing or eradicating persistent HIV-1 infection in humans. Human immunodeficiency virus type 1 (HIV-1)1 is a persistent virus. Although highly active antiretroviral treatment (HAART) can reduce circulating virus to below the limits of detection and partially restore immune function in many individuals, the suppression of viral replication is not permanent. When HAART is ceased, the virus almost invariably rebounds to high levels due to the existence of long-lived viral reservoirs including persistently infected macrophages, latently infected CD4 ϩ memory T-cells, thymocytes, microglia, seminal cells, and natural killer cells (1-5). In view of the cumulative toxicity and cost of HAART, as well as the potential for the development of drug-resistant virus, it is important to develop strategies to reduce or eliminate such viral reservoirs. Immunotoxins that recognize and kill cells expressing the viral envelope glycoprotein (Env), which is displayed on the surface of many HIV-1-infected cell types, are logical candidates for this purpose (6 -10).For such immunotoxins to be clinically useful, they must bind with high affinity and specificity to a region of Env that is conserved between different viral isolates. They should also display suitable pharmacokinetic properties including stability in the circulation and minimal nonspecific toxicity. Recently Bera et al. (11) described a recombinant fusion protein that begins to meet these criteria. This molecule, termed 3B3(scFv)-PE, consists of a single-chain variable region construct (scFv) of human Fab 3B3 joined to a truncated version of Pseudomonas exotoxin A. The 3B3 Fab recognizes the highly conserved CD4-binding site of gp120, the external subunit of Env (12). 3B3 was derived by mutagenesis of b12, a Fab isolated from a combinatorial phage display library constructed from bone marrow RNA...

Molecular Features of the Broadly Neutralizing Immunoglobulin G1 b12 Required for Recognition of Human Immunodeficiency Virus Type 1 gp120

“…Another potentially loop-stabilizing hydrogen bond exists between Q100e and A93, and we found that changing Q100e to Ala, Asn, or Phe resulted in diminished binding to gp120. We note that in a recent and related study by McHugh et al (47), it was shown by mutational analysis of a single chain Fv fragment corresponding to a b12 variant, 3B3-PE, that gp120 recognition was enhanced Ϸ2-fold by a Q100eY mutation. Thus, it appears that an aromatic residue at Q100e is compatible with strong gp120 binding in certain contexts.…”

HER2-targeting recombinant protein with truncated Pseudomonas Exotoxin: A translocation domain efficiently kills breast cancer cells

“…13,23 The PEA translocation domain has been successfully engineered as a vehicle to deliver therapeutic molecules into cells. [15][16][17][18][19] We have previously constructed a series of genes containing different truncated PEA translocation domains (PEA 253-412aa, PEA 253-364aa and PEA253-358aa) fused with the anti-HER2 single-chain antibody (e23sFv) at the 5'-end, and genes encoding diverse apoptosis-inducing molecules such as caspase-3, 20 caspase-6, 21 truncated AIF 22 and granzyme B 13 at the 3'-end. 14 In this study, we attempted to identify the minimal sequence of the PEA translocation domain for further development in therapeutic drugs.…”

Pseudomonas aeruginosa Exotoxin A Induces Human Mast Cell Apoptosis by a Caspase-8 and -3-dependent Mechanism

“…Because of its potent cytotoxicity, ETA has been widely used to generate fusion proteins to kill target cells. For example, chimeric cytotoxins have been constructed by the fusion of growth factors or antibodies with the enzymatic region of ETA to specifically target and eliminate cancer cells, virally infected cells, or mast cells (7)(8)(9)(10)(11)(12). It is generally accepted that ETA is internalized by the cell surface receptor CD91 (the ␣2-macroglobulin receptor/low density lipoprotein receptor-related protein) (13) and asserts its cellular toxicity by blocking protein synthesis through ADP ribosylation of translation elongation factor 2 (14 -16).…”