ABSTRACT
PURPOSE: The present study aims to establish a method that provides fast, precise and reproducible pharmacokinetic (PK) parameters of antibody-calicheamicin conjugates. The method should discriminate between PK of the antibody moiety and PK of the conjugated calicheamicin (CM). METHODS: The conjugates gemtuzumab ozogamicin (CMA-676, Mylotarg) or inotuzumab ozogamicin (CMC-544) were injected in the tail vein of nude mice. At regular time intervals, 5 mul whole blood samples were taken from the tail artery. Concentrations of conjugated CMA-676 or CMC-544 as well as concentrations of their respective antibody moiety were determined by sandwich plasmon resonance. This detection system measures changes in the plasma resonance angle caused by the interaction of macromolecules on biosensor chips. We determined as a first measure the binding of CMA-676 or CMC-544 to their respective antigens, CD33 or CD22. As a second measure we determined the amount of CM on the antigen-bound conjugates. This was done by determination of changes in plasma resonance angle after binding of an anti-CM antibody. RESULTS: Sandwich plasmon resonance allowed detection of both conjugates in blood of mice in a range of 100-1,000 ng/ml protein. Due to the precision of the sampling and detection methods, PK values of each conjugate were determined in individual mice. Calicheamicin bound to antibody was eliminated faster than the antibody alone. The presence of a CD22-expressing tumour in mice reduced the plasma levels of the CD22-targeting conjugate but not of the CD33-targeting one. CONCLUSIONS: Using small blood samples from a mouse, the sandwich plasmon resonance method provided PK-values of CM-conjugates and information about the stability of the linkage in vivo. Comparison between the PK-values of CM-conjugates in tumour-bearing and tumour-free mice suggested that retention of the conjugate in tumour tissue due to antigen targeting could be deduced from the plasma levels.
Subject(s)
Aminoglycosides/pharmacokinetics , Antibodies, Monoclonal/pharmacokinetics , Aminoglycosides/administration & dosage , Aminoglycosides/blood , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/blood , Antibodies, Monoclonal, Humanized , Area Under Curve , Cell Line, Tumor , Gemtuzumab , Half-Life , Humans , Injections, Intraperitoneal , Injections, Intravenous , Inotuzumab Ozogamicin , Mice , Mice, Inbred BALB C , Mice, Nude , Rabbits , Surface Plasmon ResonanceABSTRACT
PURPOSE: Linking a cytotoxic anticancer drug to an antibody that recognizes a tumor-associated antigen can improve the therapeutic index of the drug. We asked whether a conjugate of the cytotoxic antibiotic N-acetyl gamma calicheamicin dimethyl hydrazide (CalichDMH) and an antibody recognizing Lewis(y) (Le(y)) antigen could eliminate carcinomas that express Le(y). Because Le(y) is highly expressed on carcinomas of colon, breast, lung, ovary, and prostate, a CalichDMH conjugate targeting Le(y) could provide a treatment option for various cancers. EXPERIMENTAL DESIGN: The humanized anti-Le(y) antibody hu3S193 was conjugated to CalichDMH via the bifunctional AcBut linker. Selectivity and avidity of the conjugate (hu3S193-CalichDMH) for Le(y)-BSA or Le(y+) cells was tested by BIAcore or flow cytometry. Cytotoxicity of hu3S193-CalichDMH was compared with toxicity of a control conjugate on monolayers of Le(y+) and Le(y-) carcinoma cells. Inhibition of tumor growth by hu3S193-CalichDMH was assessed on three types of s.c. xenografts. RESULTS: Hu3S193-CalichDMH had similar selectivity as hu3S193. The conjugate had lower affinity for Le(y)-BSA but not for Le(y+) cells. When tested on monolayers of human Le(y+) carcinoma cells, hu3S193-CalichDMH was more cytotoxic than a control conjugate. This difference in efficacy was not noted on Le(y-) cells. Efficacy of hu3S193-CalichDMH depended on the expression of Le(y) and on the sensitivity of the cells to CalichDMH. In vivo, hu3S193-CalichDMH inhibited growth of xenografted human gastric (N87), colon (LOVO), and prostate carcinomas (LNCaP). When used against N87 xenografts, hu3S193-CalichDMH arrested tumor growth for at least 100 days. CONCLUSION: Hu3S193-CalichDMH can specifically eliminate Le(y+) tumors. These results support development of this conjugate for treatment of carcinomas.
