Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab)

Heather Wanczyk,Michael Lynes,Daniel Zapata,Debra Rood,Lawrence Silbart,Tolga Barker,John Zinckgraf,Stewart Richardson,Gregory Marusov,Amy Howell

doi:10.3390/toxins5030568

Abstract

Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.

Highlights

Smokeless tobacco products have been listed as IARC Group 1 human carcinogens since 1987 based primarily upon human epidemiological data [1,2]
NNKB-carrier protein conjugates were characterized by UV absorption spectra, N-nitroso content by the Greiss reaction, and protein concentration, yielding conjugates with 7–12 NNKB substitutions per mole
Others have ameliorated the chronic toxicity associated with chemicals such as PCP, NNK and nicotine [17,41,42,43]

Summary

Introduction

Smokeless tobacco products have been listed as IARC Group 1 human carcinogens since 1987 based primarily upon human epidemiological data [1,2]. Among the most carcinogenic chemicals found in these products are polycyclic aromatic hydrocarbons such as benzo(a)pyrene, metals such as cadmium and lead, radioactive elements such as polonium-210, and a group of tobacco-specific nitrosamines (TSNAs) that include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosonornicotine (NNN), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (N-NAL). The mechanism of carcinogenicity for these compounds involves P-450 mediated α-hydroxylation reactions that bioactivate the parent molecule to an electrophile capable of reacting with DNA and other macromolecules. Extra-hepatic P-450 enzymes such as CYP2A13 and CYP2A6 have been shown to be involved in α-hydroxylation reactions of NNK resulting in the methylation or pyridyloxobutylation of deoxyguanosine residues, forming the O6-alkylguanosine and O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG) DNA adducts, respectively [3,5]. It is widely accepted that covalent modification of DNA leads to mutations being fixed in the genome following either aberrant repair processes or disruption of DNA replication in the S-phase of the cell cycle, thereby predisposing the affected cells to tumorigenesis

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Conclusion