Preparation and characterization of conjugates of recombinant CD4 and deglycosylated ricin A chain using different cross-linkers.
In a previous study, we have demonstrated that conjugates containing soluble, recombinant human CD4 (rCD4) and the deglycosylated form of ricin A chain (dgA) (rCD4-dgA) effectively kill a human T cell line infected with the human immunodeficiency virus (HIV) in vitro. In contrast, such conjugates are 100-1000-fold less toxic to uninfected cells. In order to use a rCD4-dgA conjugate effectively in vivo, it was important to demonstrate that (1) it binds to and kills HIV-infected, but not uninfected, human cells, (2) it is stable in the circulation, and (3) it has an optimal therapeutic index (toxicity to animals versus toxicity to target cells). A major factor affecting the efficacy of such conjugates in vitro and in vivo is the nature of the cross-linker between the ligand (rCD4) and the toxin (dgA). In this report, we have prepared rCD4-dgA conjugates using three different cross-linkers. Different methods of purification have been compared by determining the optimal yield, purity, and retention of biological activity (i.e., binding to gp120 and dgA chain activity). The structure of these conjugates as well as their cytotoxicity to target cells in vitro has been analyzed. Finally, we have compared their pharmacokinetics, tissue localization, and toxicity in mice.
Ghetie
V
V
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Till
M A
MA
Ghetie
M A
MA
Tucker
T
T
Porter
J
J
Patzer
E J
EJ
Richardson
J A
JA
Uhr
J W
JW
Vitetta
E S
ES
D000818:Animals; D001667:Binding, Competitive; D015704:CD4 Antigens / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents; D006031:Glycosylation; D006678:HIV / Q000187:drug effects; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D051379:Mice; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D012276:Ricin / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity
D015704:CD4 Antigens; C069062:CD4-ricin A chain conjugate; D003432:Cross-Linking Reagents; D015699:HIV Envelope Protein gp120; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D012276:Ricin