Biomedical subjects
M J Embleton
Publications and source records attributed to M J Embleton.
Synthesis, conformation, biodistribution, and in vitro cytotoxicity of daunomycin-branched polypeptide conjugates.
Daunomycin has been attached to various structurally related synthetic branched polypeptides with a polylysine backbone, using its acid-labile cis-aconityl derivative (cAD). Due to the importance of the side-chain structure in alpha-helix formation and immunological and pharmacological properties of branched polypeptides, we have investigated the conformation, biodistribution, and in vitro cytotoxicity of cAD-carrier conjugates with polypeptides containing amino acid residues of different identity and/or configuration at the side-chain end (XAK type) or at the position next to the polylysine backbone (AXK type), where X = Leu, D-Leu, Pro, Glu, or D-Glu. According to CD studies, polycationic conjugates with hydrophobic Leu in the side chains could assume a highly ordered conformation, while amphoteric conjugates containing Glu proved to be unordered in PBS. The reduction of in vitro cytotoxic activity of cAD by conjugation to carriers and the biodistribution profile of the conjugates were found to be dependent predominantly on the charge properties and on the side-chain sequence of the carrier polypeptide. It was demonstrated that by proper combination of structural elements of the carrier molecule, it is feasible to construct a cAD-branched polypeptide conjugate with significantly prolonged blood survival and with no reduction in in vitro cytotoxicity of the drug.
Influence of carrier on biodistribution and in vitro cytotoxicity of methotrexate-branched polypeptide conjugates.
Methotrexate (MTX) has been conjugated to various structurally related, synthetic, branched polypeptides containing a poly(L-Lys) backbone by the aid of water-soluble carbodiimide. The average degree of MTX incorporated was found to be dependent on the size of the polymer and on the identity of the terminal amino acid residue of the side chains. Consequently the average molar substitution ratio was in the range of 4.9-72.0 MTX per carrier molecule. CD spectra of conjugates showed significant differences in solution conformation correlating with the identity of the side-chain-terminating amino acid. Polycationic conjugates XAK-MTX (X = Leu or D-Leu) assumed essentially ordered (helical) secondary structure, while the CD spectrum of the amphoteric conjugate (X = Glu) corresponded to only a partially ordered conformation in PBS. The covalent attachment of MTX to branched polypeptides results in a reduction of drug in vitro cytotoxicity influenced by the carrier structure. Conjugation to amphoteric polymers, depending on the configuration and position of glutamic acid (XAK-MTX vs AXK-MTX type conjugates) resulted in a decrease of anti-791T cell activity. However polycationic conjugates bearing L-Leu at the side chain terminal position (LAK-MTX) produced a compound with cytotoxicity only about 60 times less effective than free MTX. The biodistribution in mice has been characterized by blood clearance, whole-body retention, and tissue distribution 24 h after iv administration. Blood clearance of MTX-branched polypeptides could be significantly prolonged by incorporation of glutamic acid into the side chain.(ABSTRACT TRUNCATED AT 250 WORDS)
Antigenic changes associated with liver carcinogenesis.
Carcinogen-induced experimental hepatomas are often characterized by new individually distinct antigens capable of inducing tumor immunity in syngeneic hosts. These antigens arise as a consequence of cell-carcinogen interaction and may result from modification or replacement of normal cell-surface components. Their role in immunosurveillance is not established, but they offer a target for tumor immunotherapy. Reexpressed fetal antigens have also been detected, either as secretory products (alpha 1-fetoprotein) or as common cell-surface components on hepatoma cells. The role of fetal antigens in therapy is doubtful, but they may be important diagnostic indicators of neoplastic change. Possibly associated with these are common antigens initiated early after carcinogen treatment, before malignant cells are detected. Together, the antigens associated with liver carcinogenesis may prove to be powerful tools in understanding the process of liver neoplasia.
The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens.
Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3zeta chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.