Idiotypic mimicry and the assembly of a supramolecular structure: an anti-idiotypic antibody that mimics taxol in its tubulin-microtubule interactions.

Abstract

Taxol, rii y extrced fro the bark of the western yew, Taxus brenfolia, is reportedly the first of a new class of anti-cancer agent. It Taxol, reportedly the first of a new class of anti-cancer agents, was originally extracted from the bark of the western yew, Taxus brevifolia (1). In vivo, it prevents the disassembly of microtubules, causing the arrest of cells at the G2/M step of the cell cycle (2). Its mechanism differs from that of other anti-cancer agents that have tubulin as their target, such as colchicine, podophyllotoxin, or vinblastine, which inhibit tubulin polymerization (3). The site of action of taxol on tubulin is being investigated in several laboratories. Photoaffinity-labeling experiments implicate the N-terminal region of (3-tubulin as the site of attachment (4, 5).With the aim of using immunological techniques to investigate the action of taxol, we sought to prepare an anti-idiotypic antibody that mimics taxol, using an auto-anti-idiotypic strategy (6, 7). We have succeeded in obtaining an antibody, 82H, that, as intact antibody or Fab fiagment, mimics taxol in its tubulinmicrotubule interactions to the extent that it can cause tubulin to assemble into microtubules. MATERIALS AND METHODSPreparation of and Screening for Anti-Idiotypic Antibodies. The object was to screen for monoclonal antibodies that showed taxol-inhibitable binding to Fab fragments ofaffinitypurified rabbit anti-taxol antibody R585 (8).Preparation of Fab fragments of R585. Rabbit anti-taxol antiserum R585 (8) was affinity-purified as follows. Two affinity columns were prepared. Either 10 mg of bovine serum albumin (BSA) or 10 mg of taxol covalently linked to rabbit serum albumin (taxol-RSA) (8) was dissolved in 1 ml of 0.1 M 2-(N-morpholino)-ethanesulfonic acid (Mes) buffer (pH 4.8) and added to 2 ml of Affi-Gel 10 (Bio-Rad). The mixtures were rocked gently overnight at 40C. The gels were then washed with several column volumes of phosphatebuffered saline (PBS, pH 7.3) and with 0.2 M glycine hydrochloride buffer (pH 2.5) and then equilibrated with PBS. Six milliliters of R585 antiserum was first recirculated through the taxol-RSA-Affi-Gel 10 column overnight at 40C at a rate of0.2 ml/min. The column was then extensively washed with PBS at a rate of 1.0 ml/min to remove all nonspecifically bound proteins. The antibodies were then eluted with 0.2 M glycine hydrochloride (pH 2.5) at a rate of 0.5 ml/min. The eluted antibodies were immediately neutralized with 2 M Tris base and dialyzed against PBS at 40C overnight. On the following day, the eluted antibodies were recycled through the BSA-Affi-Gel 10 column at 40C overnight to remove BSA-binding antibodies. The effluent, which contained antitaxol antibodies in PBS, was collected and concentrated by a Centriprep-10 concentrator (Amicon) to a volume of 1-2 ml.Fab fragments were prepared from the affinity-purified antibodies as described (9).Screening for anti-idiotypic antibodies. A competitive ELISA was used to assay for taxol-inhibitable binding to the Fab fiagments of ...

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