Receptor binding domain

In the DT-based fusion toxins the native R domain of DT has been replaced with various targeting ligands (Table 2.1). The resulting fusion toxin proteins can be used for receptor distribution studies and structure-function studies of the targeting ligands. For example, a gastrin releasing peptide (GRP)-directed fusion toxin, DAB389GRP and substance P (SP) fusion toxin, DAB389SP were used to assess distribution of GRP and SP receptors on various small cell lung cancer cell lines (vanderSpek et al., 1997). The most studied DT-based fusion toxin is DAB389IL-2, which is the one now clinically available as Ontak. Bacha et al. (1988) demonstrated that cytotoxicity was dependent upon binding to the IL-2 receptor and that processing occurred in a manner analogous to native DT. Cytotoxicity was specific to cells that expressed IL-2 receptors and sensitivity was greatest for cells that expressed the high-affinity form of the IL-2 receptor (Waters et al., 1990; Re et al., 1996). The binding affinity of rIL-2 is approximately 10 pM and studies of DAB389IL-2 indicate approximately a 10-fold decreased binding affinity, probably due to steric hinderance by attachment of the DT portion (vanderSpek et al., 1996). Kiyokawa et al. (1991) used insertion mutagenesis to place a flexible linker between the end of the T domain and IL-2 and determined that binding was approximately 2-fold better than that of DAB389IL-2. Insertion of the linker also resulted in a 5-fold increase in cytotoxicity. The authors postulated that introduction of the linker resulted in more effective translocation of the A domain. Insertion of a vesicular stomatitis virus G protein epitope tag between the T domain and IL-2 also resulted in a more cytotoxic mutant (vanderSpek et al., 1994a).

The early events following binding of DAB486IL-2 were studied by Walz et al. (1989) They determined that within 7h of binding, mRNAs for IL-2, IL-2 receptor interferon y and c-myc were increased, as is the case following treatment with rIL-2. A rapid inhibition of protein synthesis followed.

Buchli and Ciardelli (1993) identified Gln126 of IL-2 as a residue involved with binding to the [3/y portion of the high affinity, afiy, IL-2 receptor using an IL-2 Q126D mutant. Chang et al. (1995) identified Thr51 as involved with receptor-ligand internalization using an IL-2 T51P mutant. The corresponding mutations were made in the IL-2 portion of DAB389IL-2. The Gln mutation resulted in a fusion protein that was not as cytotoxic, partially due to a decreased binding affinity (vanderSpek et al., 1996). Killeen et al. (1992) discovered that mutation of a Glu residue at position 148 of the C domain in DT (E149S in DAB389IL-2) to a Ser residue, resulted in a toxin that was 1000-fold less cytotoxic. The E149S mutation was introduced into DAB389IL-2 with and without the Gln mutation in the IL-2 portion. The inactive fusion proteins were studied to determine the effects of the Gln mutation on activation. DA(E149S)B389IL-2 activated CTLL-2 cells to 83% of rIL-2 activation. DA(E149S)B389IL-2(Q514D) activated CTLL-2 cells to 8%. Therefore, the Q154D mutation in IL-2 of the fusion toxin resulted in decreased activation. The authors concluded that the loss of binding affinity alone was not responsible for the loss in cytotoxicity. The decrease in activation activity also decreased cytotoxicity. Similar studies with the Thr51 mutation (T439P in the fusion toxin) indicated that Thr51 of IL-2 is involved with receptor binding and signaling internalization of the receptor-ligand complex.

The DT-based fusion toxins can also be used to determine the minimum requirements for binding and internalization of their respective ligands. Studies are currently underway using a DT-based fusion toxin with differently sized fragments from the C-terminus of Botulinum toxin A as targeting ligands (Ratts and Murphy, unpublished). Kreitman et al. (1994) mutated the IL-4 portion of a Pseudomonas exotoxin A-based fusion protein, such that the N-terminal 38 amino acid residues of IL-4 were moved to the C-terminus. The resulting construct bound to the IL-4 receptor with 10-fold better affinity. Permutations are currently being created in the IL-7 portion of DAB389IL-7 to determine if a protein with greater affinity for the IL-7 receptor can be produced (vanderSpek, unpublished).

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