ABSTRACT
Antibody fragments are recognized as promising vehicles for delivery of imaging and therapeutic agents to tumor sites in vivo. The serum persistence of IgG1 and fragments with intact Fc region is controlled by the protective neonatal Fc receptor (FcRn) receptor. To modulate the half-life of engineered antibodies, we have mutated the Fc-FcRn binding site of chimeric anti-carcinoembryonic antigen (CEA) antibodies produced in a single-chain Fv-Fc format. The anti-CEA T84.66 single-chain Fv-Fc format wild-type and five mutants (I253A, H310A, H435Q, H435R, and H310A/H435Q, Kabat numbering system) expressed well in mammalian cell culture. After purification and characterization, effective in vitro antigen binding was shown by competition ELISA. Biodistribution studies in BALB/c mice using (125)I- and (131)I-labeled fragments revealed blood clearance rates from slowest to fastest as follows: wild-type > H435R > H435Q > I253A > H310A > H310A/H435Q. The terminal half-lives of the mutants ranged from 83.4 to 7.96 hours, whereas that of the wild-type was approximately 12 days. Additionally, (124)I-labeled wild-type, H435Q, I253A, H310A, and H310A/H435Q variants were evaluated in LS174T xenografted athymic mice by small animal positron emission tomography imaging, revealing localization to the CEA-positive xenografts. The slow clearing wild-type and H435Q constructs required longer to localize to the tumor and clear from the circulation. The I253A and H310A fragments showed intermediate behavior, whereas the H310A/H435Q variant quickly localized to the tumor site, rapidly cleared from the animal circulation and produced clear images. Thus, attenuating the Fc-FcRn interaction provides a way of controlling the antibody fragment serum half-life without compromising expression and tumor targeting.
Subject(s)
Carcinoembryonic Antigen/immunology , Immunoconjugates/pharmacokinetics , Immunoglobulin Fragments/metabolism , Iodine Radioisotopes/pharmacokinetics , Radiopharmaceuticals/pharmacokinetics , Animals , Carcinoembryonic Antigen/metabolism , Cell Line, Tumor , Colorectal Neoplasms/diagnostic imaging , Colorectal Neoplasms/metabolism , Female , Humans , Immunoglobulin Fragments/biosynthesis , Immunoglobulin Fragments/isolation & purification , Mice , Mice, Inbred BALB C , Mice, Nude , Multiple Myeloma/metabolism , Positron-Emission Tomography , Tissue Distribution , Transplantation, HeterologousABSTRACT
To evaluate the reliability of previously described morphologic, cytochemical and immunophenotypic criteria for the identification of acute myeloid leukemias (AMLs) with t(8;21), inv(16)/t(16;16) and t(15;17), 300 cases were reviewed retrospectively. Eighteen AMLs with features of t(8;21), 31 with features of inv(16)/t(16;16), and 22 with features of t(15;17) were identified. Cytogenetic studies were available for 228 cases and identified 15 cases of t(8;21), 30 cases of inv(16)/t(16;16), 18 cases of t(15;17) and 11 cases 11q23 AML. The true positive rate for pre-cytogenetic evaluation was 95% for t(15;17), 88% for inv(16)/t(16;16) and 87.5% for t(8;21). No difference in 5-year survival was identified in the precytogenetic and corresponding cytogenetic disease groups. No specific features to predict 11q23 abnormalities were identified. This study confirms the reliability of a combined morphologic, cytochemical and immunophenotypic approach to the initial classification of AML. Cytogenetic studies are still needed on all cases to identify the small proportion of cases that will be missed by these methods and to identify other significant cytogenetic abnormalities in AML.
Subject(s)
Bone Marrow/pathology , Leukemia, Myeloid/pathology , Acute Disease , Adolescent , Adult , Aged , Antigens, CD19/genetics , Antigens, CD19/metabolism , Child , Child, Preschool , Chromosome Aberrations , Female , Humans , Infant , Leukemia, Myeloid/classification , Leukemia, Myeloid/genetics , Leukemia, Myeloid/mortality , Male , Middle Aged , Prognosis , Retrospective Studies , Sensitivity and Specificity , Survival RateABSTRACT
To evaluate the prognostic impact of acute myeloid leukemia (AML) classifications, specimens from 300 patients with 20% or more bone marrow myeloblast cells were studied. Specimens were classified according to the French-American-British Cooperative Group (FAB), the World Health Organization (WHO), the Realistic Pathologic Classification, and a cytogenetic risk group scheme. Cases with fewer than 30% blast cells did not have a 5-year survival significantly different from cases with 30% or more blast cells, and survival was similar for the low blast cell count group and cases with multilineage dysplasia and 30% or more blasts. Categories of AML with recurrent cytogenetic abnormalities of t(15;17), t(8;21), inv(16)/t(16;16), and 11q23 showed significant differences in 5-year survival. No significant difference was identified between AMLs arising from myelodysplasia and de novo AMLs with multilineage dysplasia, but all cases with multilineage dysplasia had a worse survival than all other AMLs and other AMLs without favorable cytogenetics. FAB types M0, M3, and M4Eo showed differences in survival compared with all other FAB types, with M0 showing a significant association with high-risk cytogenetics and 11q23 abnormalities. Other FAB groups and WHO AML, not otherwise categorized subgroups did not show survival differences. These findings suggest that the detection of recurring cytogenetic abnormalities and multilineage dysplasia are the most significant features of current AML classification.
