Towards a ligand targeted enzyme prodrug therapy: Single round panning of a β‐lactamase scaffold library on human cancer cells

Abstract

A novel b-lactamase scaffold library in which the target-binding moiety is built into the enzyme was generated using phage display technology. The binding element is composed of a fully randomized 8 amino acid loop inserted at position between Y34 and K37 on the outer surface of Enterobacter cloacae P99 cephalosporinase (b-lactamase, E.C. 3.5.2.6) with all library members retaining catalytic activity. The frequency and diversity of amino acids distributions in peptide inserts from library clones were analyzed. The complexity of the randomized loop appears consistent with standards of other types of phage display library systems. The library was panned against SKBR3 human breast cancer cells in 1 round using rolling circle amplification of phage DNA to recover bound phage. Individual b-lactamase clones, independent of phage, were rapidly assessed for their binding to SKBR3 cells using a simple high throughput screen based on cell-bound b-lactamase activity. SKBR3 cell-binding b-lactamase enzymes were also shown to bind specifically using an immunochemical method. Selected b-lactamase clones were further studied for their protein expression, enzyme activity and binding to nontumor celllines. Overall, the approach outlined here offers the opportunity of rapidly selecting targeted b-lactamase ligands that may have a potential for their use in enzyme prodrug therapy with cephalosporin-based prodrugs. It is expected that a similar approach will be useful in developing tumor-targeting molecules of several other enzyme candidates of cancer prodrug therapy. ' 2007 Wiley-Liss, Inc.Key words: phage display; b-lactamase; screening; cancer; targeted enzyme; prodrug therapyThe major problems associated with cytotoxic cancer therapeutic drugs are lack of selectivity for tumor cells over normal cells, insufficient drug concentrations in tumors, systemic toxicity and development of drug resistant cancer cells.1,2 Developing a successful strategy for a targeted delivery of drugs to cancer cells without harming the rest of the body is one of the most important challenges for cancer researchers today. The ability to selectively concentrate or deliver chemotherapeutic drugs to cancer cells may make chemotherapy both more effective and less likely to cause side effects. A variety of strategies are being evaluated to achieve this goal. [3][4][5][6] The majority of cancer-targeting agents developed over the last 2 decades are based on the use of monoclonal antibodies (mAb) to selectively deliver toxic agents such as conventional cytotoxic drugs, radioisotopes, or plantand bacteria-derived toxins for a direct tumor-killing.7-9 These approaches can reduce systemic toxicity; however, the therapeutic results have generally been less than ideal at least in part because of inadequate conjugate uptake into the tumor. 10Another approach that shows promise is targeted prodrug therapy. This is based on the concept that a systemically administered nontoxic prodrug can be converted locally to high concentrations of a cytotoxic drug by an enzyme p...

Developing bifunctional β‐lactamase molecules with built‐in target‐recognizing module for prodrug therapy: identification of Enterobacter Cloacae P99 cephalosporinase loops suitable for randomization and phage‐display selection

“…The phagemid vector that we used in our earlier study (Shukla et al ., 2007) was modified to create different loop libraries. Our earlier vector expresses a truncated pIII representing only the CT domain of the protein.…”

“…Four P99 β -lactamase loops, namely the loop-2, loop-3, loop-4, and loop-5, were randomized with both the linear (X 7 ) and cysteine-constrained (CX 7 C) heptapeptides. The loop-1 of the P99 β -lactamase molecule was randomized with the CX 7 C only, as successful randomization of this loop with the linear octapeptides has been reported earlier (Shukla et al ., 2007). The vector expressing the pIII signal peptide was purified using Qiagen plasmid purification kit (Valencia, California).…”

“…The wells were washed eight times (5 min each), six times with PBST, and two times with PBS. The DNA of the bound phages was amplified by the rolling circle amplification using TempliPhi Amplification kit (GE Health Care), as described earlier (Shukla et al ., 2007). Briefly, the bound phages were eluted in 50 μl sample buffer by heating the samples at 95°C for 5 min.…”

“…Recently, we made an attempt to develop β -lactamase molecules that have target-recognition sites built within active enzyme molecules (Shukla et al ., 2007). Such bifunctional enzyme molecules are the smallest possible units that could be utilized in enzyme prodrug therapy.…”

“…Four classes of β -lactamase, namely A, B, C, and D, differing in their size, protein structure, inhibitors, or mechanism of action, have been reported (Majiduddin et al ., 2002; Jacoby and Munoz-Price, 2005). We developed bifunctional β -lactamase molecules by selecting a β -lactamase library, composed of randomized linear octapeptide-inserts (X 8 ) in an outer loop (loop-1) of the Enterobacter cloacae P99 cephalosporinase (P99 β -lactamase), on human cancer cells (Shukla et al ., 2007). P99 β -lactamase is a class C enzyme from E. cloacae strain P99.…”