Insertion of constant region domains of human IgG1 into CD4-PE40 increases its plasma half-life.
CD4-PE40 is a recombinant toxin containing the binding domain of CD4 and a mutant form of Pseudomonas exotoxin A from which the cell binding domain has been removed. To increase the serum half-life of CD4-PE40, we have inserted various portions of the constant domain of human IgG1 into CD4-PE40. The constructs made include CD4-CH2-PE40, CD4-CH3-PE40, CD4-CH1-CH2-PE40 and CD4-CH2-CH3-PE40. The fusion proteins were expressed and purified from E. coli. Insertion of various domains from the constant region of IgG1 did not alter the cytotoxic activity of CD4-PE40; all these molecules were equally cytotoxic to cells expressing gp120 on their surface. However, there was a marked increase in the serum mean residence time of CD4-CH2-PE40 which was 115 min as compared to 47 min for CD4-PE40. Insertion of other domains also increased the half-life of CD4-PE40, however, CD4-CH2-PE40 was found to have the longest mean residence time in the circulation. One possible explanation for the increase in plasma half-life is diminished susceptibility of proteins to proteolysis. It was found that CD4-CH2-PE40 was much more resistant to proteolysis by trypsin than CD4-PE40. We proposed that insertion of the CH2 domain into CD4-PE40 covers up the protease sensitive sites in the molecule, thereby making the molecule less susceptible to degradation. The increase in size and reduced sensitivity to proteases could both be responsible for the increased plasma half-life of CD4-CH2-PE40.
Batra
J K
JK
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Kasturi
S
S
Gallo
M G
MG
Voorman
R L
RL
Maio
S M
SM
Chaudhary
V K
VK
Pastan
I
I
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity; D001483:Base Sequence; D003601:Cytotoxicity Tests, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D005098:Exotoxins / Q000737:chemistry / Q000276:immunology / Q000493:pharmacokinetics* / Q000633:toxicity; D005341:Fibrinolysin / Q000494:pharmacology; D005822:Genetic Vectors; D006207:Half-Life; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000494:pharmacology*; D007074:Immunoglobulin G / Q000494:pharmacology*; D000922:Immunotoxins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000493:pharmacokinetics* / Q000633:toxicity; D013917:Thrombin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D001426:Bacterial Proteins; C057689:CD4-Pseudomonas toxin; D005098:Exotoxins; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D000922:Immunotoxins; D011994:Recombinant Proteins; D014357:Trypsin; D013917:Thrombin; D005341:Fibrinolysin