Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-04-08
2001-11-27
Le, Long V. (Department: 1641)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C435S004000, C435S005000, C435S006120, C435S007800, C435S007210, C435S007100, C435S025000, C435S040500, C435S040510, C435S040520, C435S091500, C435S025000, C435S968000, C435S805000, C436S069000, C436S166000, C436S169000, C436S170000, C436S501000, C436S503000, C530S387300, C530S387700, C530S388150, C530S388260, C530S387900, C530S387100, C530S388850, C424S141100
Reexamination Certificate
active
06323325
ABSTRACT:
TECHNICAL FIELD
The present invention resides in the technical fields of immunology and enzymology.
BACKGROUND OF THE INVENTION
The cytochrome P450 family of enzymes is primarily responsible for the metabolism of xenobiotics such as drugs, carcinogens and environmental chemicals, as well as several classes of endobiotics such as steroids and prostaglandins. Members of the cytochrome P450 family are present in varying levels and their expression and activities are controlled by variables such as chemical environment, sex, developmental stage, nutrition and age.
More than 200 cytochrome P450 genes have been identified. There are multiple forms of these P450 and each of the individual forms exhibit degrees of specificity towards individual chemicals in the above classes of compounds. In some cases, a substrate, whether it be drug or carcinogen, is metabolized by more then one of the cytochromes P450.
Human CYP2A6 is an important member of the CYP superfamily and is present in liver up to 1% of the total CYP content (Yun et al., 1991). Human CYP2A6 metabolically activates the carcinogens aflatoxin B
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(Yun et al., 1991), a tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butone (Crespi et al., 1991), and N-nitrosodiethylamine (Fernandez-Salguero & Gonzalez, 1995). CYP2A6 also carries out coumarin metabolism by aromatic hydroxylation in humans (Pearce et al., 1992). Coumarin 7-hydroxylation has been used as a marker for CYP2A6 activity in vitro (Yamano et al., 1990) and the basis for measuring the in vivo expression of CYP2A6 (Cholerton et al., 1992; Rautio et al., 1992). A genetic polymorphism has been found in CYP2A6 (Fernandez-Salguero et al., 1995) that is due to three variant allelic forms, i.e., CYP2A6*1, 2A6*2, 2A6*3, respectively (Daly et al., 1996).
Genetic polymorphisms of cytochromes P450 result in phenotypically-distinct subpopulations that differ in their ability to perform biotransformations of particular drugs and other chemical compounds. These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to most humans may cause toxic side-effects in an individual suffering from a defect in an enzyme required for detoxification of the drug. Alternatively, a drug that is effective in most humans may be ineffective in a particular subpopulation because of lack of a enzyme required for conversion of the drug to a metabolically active form. Further, individuals lacking a biotransformation enzyme are often susceptible to cancers from environmental chemicals due to inability to detoxify the chemicals. Eichelbaum et al.,
Toxicology Letters
64/65, 155-122 (1992). Accordingly, it is important to identify individuals who are deficient in a particular P450 enzyme, so that drugs known or suspected of being metabolized by the enzyme are not used, or used only with special precautions (e.g., reduced dosage, close monitoring) in such individuals. Identification of such individuals may indicate that such individuals be monitored for the onset of cancers.
Existing methods of identifying deficiencies in patients are not entirely satisfactory. Patient metabolic profiles are often assessed with a bioassay after a probe drug administration. Individuals with below normal cytochrome P450 activity exhibit physiologic accumulation of unmodified drug and have a high metabolic ratio of probe drug to metabolite. This bioassay has a number of limitations: lack of patient cooperation, adverse reactions to probe drugs, and inaccuracy due to coadministration of other pharmacological agents or disease effects. See, e.g., Gonzalez et al.,
Clin. Pharmacokin.
26, 59-70 (1994). Genetic assays by RFLP (restriction fragment length polymorphism), ASO PCR (allele specific oligonucleotide hybridization to PCR products or PCR using mutant/wild-type specific oligo primers), SSCP (single stranded conformation polymorphism) and TGGE/DGGE (temperature or denaturing gradient gel electrophoresis), MDE (mutation detection electrophoresis) are time-consuming, technically demanding and limited in the number of gene mutation sites that can be tested at one time.
A complication in patient drug choice is that most drugs have not been characterized for their metabolism by P450 2A6 and other cytochromes P450. Without knowing which cytochrome(s) p450 is/are responsible for metabolizing an individual drug, an assessment cannot be made for the adequacy of a patient's P450 profile. For such drugs, there is a risk of adverse effects if the drugs are administered to deficient metabolizers.
Monoclonal antibodies that specifically bind to 2A6 and inhibit its activity, if available, could be used to screen drugs for their metabolism by 2A6 and/or identify 2A6 deficient metabolizers by simple bioassays, thereby overcoming the problems in prior complicated methods discussed above. However, such monoclonal antibodies represent, at best, a small subset of the total repertoire of antibodies to human cytochrome P450 2A6, and have not hitherto been isolated. Although in polyclonal sera, many classes of antibody may contribute to inhibition of enzyme activity of P450 2A6 as a result of multiple antibodies in sera binding to the same molecule of enzyme, only a small percentage of these, if any, can inhibit as a monoclonal. A monoclonal antibody can inhibit only by binding in such a manner that it alone block or otherwise perturb the active site of an enzyme. The existence and representation of monoclonal antibodies with inhibitory properties thus depend on many unpredictable factors. Among them are the size of the active site in an enzyme, whether the active site is immunogenic, and whether there are any sites distal to the active site that can exert inhibition due to stearic effects of antibody binding. The only means of obtaining antibodies with inhibitory properties is to screen large numbers of hybridoma until one either isolates the desired antibody or abandons the task through failure.
Notwithstanding these difficulties, the present invention provides inter alia monoclonal antibodies that specifically bind to human cytochrome P450 2A6 and inhibit its activity.
SUMMARY OF THE INVENTION
The invention provides isolated binding agents that competes with the monoclonal antibody MAb 151-45-4 for specific binding to human cytochrome p450 2A6, and that specifically inhibit 2A6-catalyzed metabolism of coumarin by at least 50%. Preferred binding agents are monoclonal antibodies. Some binding agents lacks specific binding to at least one cytochrome P450 selected from the group consisting of human cytochromes P450 1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 3A4 and 3A5. Some binding agents lack specific binding to each of human cytochromes P450 1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 3A4 and 3A5. Preferred binding agents are able to specifically inhibits the enzyme activity of human cytochrome p450 2A6 by at least 80%. Some binding agents are binding fragments, such as Fab fragments.
MAb 151-45-4 is an exemplified monoclonal antibody. Some other monoclonal antibodies are analogs of MAb 151-45-4 and comprises a light chain variable domain having at least 80% sequence identity with the light chain variable domain of MAb 151-45-4 wherein the percentage sequence identity is determined by aligning amino acids in the light chain variable domains by the Kabat numbering convention, and a heavy chain variable domain having at least 80% sequence identity with the heavy chain variable domain of MAb 151-45-4 wherein the percentage sequence identity is determined by aligning amino acids in the heavy chain variable domains by the Kabat numbering convention.
The invention further provides cell lines producing monoclonal antibodies as described above. Cells lines can be eucaryotic or procaryotic.
The invention further provides methods of determining whether cytochrome P450 2A6 metabolizes a compound. Such methods entail contacting the compound with cytochrome P450 2A6 in the presence of varying amounts of the binding agent of claim
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. Metabolism of the compound is then assayed as a funct
Gelboin Harry V.
Gonzalez Frank J.
Cook Lisa V.
Le Long V.
The United States of America as represented by the Department of
Townsend & Townsend & Crew LLP
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