Jennifer Kuehne, PhD 2001

Thesis Title: Design and characterization of a membrane-active GALA-OKT9 conjugate and its effect on transferrin-gelonin immunotoxin efficacy

Immunoconjugates are chimeric molecules with both targeting and toxic functionalities. Previously, our group developed a model relating protein synthesis and cellular processing characteristics of immunotoxins. From this model, it was determined that the rate-limiting step in immunoconjugate cellular intoxication is translocation to the cytosol. We hypothesized that translocation could be improved by adding a molecule that increased intracellular-membrane permeability. We chose to use a membrane-active peptide, GALA, to increase membrane permeability. The idea is to target both peptide and immunotoxin to cells to determine whether immunotoxin translocation was increased. Our delivery scheme involved co-internalization of two molecules, a transferrin-gelonin immunotoxin and a GALA-OKT9 conjugate.

We designed a GALA-OKT9 conjugate that retained membrane activity, as tested in a liposomal leakage assay. After investigating multiple constructs, we found that GALA-OKT9 conjugates with 3 GALA per OKT9 linked via SPDP were the most active. Conjugates were significantly less active than unconjugated GALA, due to reduced surface aggregation and partitioning as determined using a mathematical model of pore formation.

The specificity and cellular processing in HeLa cells of the most active GALA-OKT9 conjugate was investigated. GALA-OKT9 conjugates retained specificity for the transferrin receptor (TfR). Steady-state binding and internalization measurements showed equivalent numbers of surface, internal, and total cell-associated OKT9 and GALA-OKT9. Kinetics of GALA-OKT9 Internalization differed from OKT9 at early times; fewer GALA-OKT9 were internalized, measured by number of intracellular molecules. These data were analyzed using a mathematical model of cellular processing. Analysis showed that GALA-OKT9 had lower internalization and recycling rate constants than OKT9. Slower recycling could indicate a GALA-intracellular membrane interaction. At longer times, cell-associated OKT9 and GALA-OKT9 decreased, which we attributed to lower numbers of TfRs caused by degradation of TfRs along with bound OKT9 or GALA-OKT9.

GALA-OKT9 conjugates were incubated with transferrin-gelonin conjugates and their effect on HeLa cell protein synthesis was evaluated, Co-incubation of transferrin-gelonin and GALA-OKT9 or OKT9 resulted in decreased transferrin-gelonin efficacy, measured as increased protein synthesis. We attributed the reduction transferrin-gelonin efficacy with OKT9 and GALA-OKT9 to decreased numbers of cellular TfRs caused by degradation of TfRs bound to OKT9 or GALA-OKT9.