Increased affinity and stability of an anti-HIV-1 envelope immunotoxin by structure-based mutagenesis.
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 approximately 10-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 Env-mediated cell fusion, and to limit spreading viral infection in peripheral blood mononuclear cells. Such site-directed mutants may increase the utility of immunotoxins for reducing or eradicating persistent HIV-1 infection in humans.
Laboratory of Biochemistry, National Cancer Institute/National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892, USA.
Louise
L
McHugh
Stella
S
Hu
B K
BK
Lee
Kenneth
K
Santora
Paul E
PE
Kennedy
Edward A
EA
Berger
Ira
I
Pastan
Dean H
DH
Hamer
D036002:ADP Ribose Transferases / Q000737:chemistry* / Q000378:metabolism; D019380:Anti-HIV Agents / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D004355:Drug Stability; D005098:Exotoxins / Q000737:chemistry* / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects*; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry* / Q000378:metabolism; D008561:Membrane Fusion; D016296:Mutagenesis; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000378:metabolism; D037521:Virulence Factors / Q000737:chemistry* / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D019380:Anti-HIV Agents; D001427:Bacterial Toxins; D005098:Exotoxins; D015699:HIV Envelope Protein gp120; D000922:Immunotoxins; D014759:Viral Envelope Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A