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1.
Antimicrob Agents Chemother ; 44(5): 1397-403, 2000 May.
Article in English | MEDLINE | ID: mdl-10770790

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) produces two polyproteins, Pr55(Gag) and Pr160(Gag-Pol), that are cleaved into mature functional subunits by the virally encoded protease. Drugs that inhibit this protease are an important part of anti-HIV therapy. We studied the ordered accumulation of Gag and Gag-Pol processing intermediates by variably blocking the protease with HIV-1 protease inhibitors (PIs). Variable protease inhibition caused accumulation of a complex pattern of processing intermediates, which was the same after incubating HIV-1-infected cells with increasing concentrations of either one of the peptidomimetic inhibitors indinavir, saquinavir (SQV), ritonavir (RTV), nelfinavir, and SC-52151 or one of the nonpeptidomimetic inhibitors DMP450, DMP323, PNU-140135, and PNU-109112 for 3 days. The patterns of Gag and Gag-Pol processing intermediate accumulation were nearly identical when the following were compared: cell- versus virion-associated proteins, HIV-1-infected transformed cell lines versus primary human peripheral blood mononuclear cells (PBMCs) and HIV-1(MN) versus HIV-1(IIIB) virus strains. RTV was a more potent inhibitor of p24 production in PBMCs than SQV by approximately 7-fold, whereas SQV was a more potent inhibitor in transformed cells than RTV by approximately 30-fold. Although the antiretroviral potency of HIV-1 PIs may change as a function of cell type, the polyprotein intermediates that accumulate with increasing drug concentrations are the same. These results support sequential processing of Gag and Gag-Pol polyproteins by the HIV-1 protease and may have important implications for understanding common cross-resistance pathways.


Subject(s)
Gene Products, gag/metabolism , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , Protein Precursors/metabolism , Cell Transformation, Viral , Cells, Cultured , HIV-1/metabolism , Humans , Leukocytes, Mononuclear/metabolism , Protein Processing, Post-Translational , gag Gene Products, Human Immunodeficiency Virus , pol Gene Products, Human Immunodeficiency Virus
2.
Hybridoma ; 16(3): 243-8, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9219034

ABSTRACT

In radioimmunotherapy, the long circulation times of antibody radioconjugates correlate with high relative radiation doses to nontumor tissues. Tumor/normal tissue ratios can be significantly improved by using targeting molecules with shorter circulation times. IFabs are multimers of VH-CH1-linker-VK-CK monomers. The lack of the Fc region in IFabs should lead to circulation times that are shorter than those of IgG molecules. The monomers assemble into disulfide-bond-stabilized multimers, 90% of which are 100 kDa dimers (IFab2). IFab2s should not be rapidly eliminated through kidney filtration because their molecular weight is above the threshold for renal passage. We report the first experimental in vivo tests for 125I-IFab radioconjugates derived from a humanized version of the anti-breast mucin monoclonal antibody BrE-3. Biodistributions are reported for athymic nude mice carrying human mammary tumor MX-1 xenografts. The T1/2 beta's for the different tissues ranged from 13.3 h for blood to 19.9 h for tumor. Therefore, IFab radioconjugates cleared the body with a rate comparable to that of F(ab')2 fragments. Except for stomach, tumor/nontumor dose ratios were significantly better for IFabs than for the parent antibody (BrE-3)4 days after injection.


Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Immunoconjugates/pharmacokinetics , Immunoglobulin Fab Fragments/metabolism , Mammary Neoplasms, Experimental/metabolism , Mucins/immunology , Animals , Antibodies, Monoclonal/therapeutic use , Chromatography, High Pressure Liquid , Female , Humans , Immunoconjugates/therapeutic use , Immunoglobulin Fab Fragments/therapeutic use , Iodine Radioisotopes/pharmacokinetics , Iodine Radioisotopes/therapeutic use , Mammary Glands, Animal/immunology , Mammary Neoplasms, Experimental/therapy , Mice , Mice, Nude , Mucin-1/immunology , Neoplasm Transplantation , Radioimmunotherapy , Transplantation, Heterologous , Tumor Cells, Cultured
3.
Mol Immunol ; 33(14): 1095-102, 1996 Oct.
Article in English | MEDLINE | ID: mdl-9047375

ABSTRACT

The IFab2 molecule described is expressed by a single gene construct that encodes the VH-CH1 and the Vk-Ck modules of a humanized version of the anti-breast cancer antibody BrE-3, joined via a short peptide linker. It can be shown that this basic genetic construct can lead to the expression of monomers, dimers (IFab2s) and other multimer species, depending on the length of the linker. A 21-residue linker promotes the principal formation of the desired dimer IFab2 molecules. It was also shown that in binding competition assays, purified IFab2 completely retained the affinity and specificity of the original monoclonal antibody.


Subject(s)
Biopolymers/biosynthesis , Immunoglobulin Fab Fragments/biosynthesis , Immunoglobulin Fab Fragments/immunology , Protein Engineering/methods , Amino Acid Sequence , Antibody Affinity/immunology , B-Lymphocytes/metabolism , Base Sequence , Binding, Competitive/immunology , Humans , Molecular Sequence Data , Mucins/immunology
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