Metal dependent catalytic antibodies and method for producing the same

#1. Field of the Invention

#The present invention relates to catalytic antibodies, and more particularly to catalytic antibodies and a method for producing catalytic antibodies which are capable of hydrolyzing phosphoester bonds in a metal dependent manner.

#2. Description of the State of Art

#The accompanying drawings, which are incorporated in and form a part of the specifications, illustrate the preferred embodiments of the present invention, and together with the description serve to explain the principles of the invention.

#In the Drawings:

#FIG. 1a is a schematic representation of the definition of .DELTA.G.sup..dagger-dbl., the free energy of activation.

#In general, the catalytic antibodies and method for inducing catalytic antibodies according to this invention do not rely on the classical transition state analogue approach, but rather depend directly on eliciting antibodies to a hapten in the form of a stable derivative of a phosphodiester substrate capable of chelating metal ions. Such a hapten is not possible with normal phosphodiester bonds since their affinity for free metal ions is either low or the resulting complexes are hydrolytically unstable. Hence, the preferred embodiment of the present invention comprises a hapten having the two non-bridging oxygens of the phosphodiester bond replaced by sulfur thereby producing a phosphorodithioate analogue hapten. This phosphorodithioate hapten of the present invention is then attached to a carrier protein to produce an antigen prior to immunization.

#As discussed above, the process of the present invention and products produced thereby is grounded in the formation of a stable substrate metal ion chelate hapten. Hapten 10, shown in FIG. 2, is a schematic representation of the hapten having a stable phosphorous center used to induce catalytic antibodies according to the present invention wherein, L is a linker which facilitates attachment of hapten 10 to a carrier protein (not shown), P is phosphorus, O is oxygen, R is a chemical compound, and each of W, X and Z can be either a chemical compound or a chemical element; or R and Z are chemical compounds wherein the chemical compound of Z is attached to the phosphorus by oxygen and W and X are sulfur, (phosphorodithioate); or W is a sulfur and X is oxygen, (phosphorothioate); or W is methyl and X is oxygen, (methylphosphonate); or W is NR.sub.2 and X is oxygen, where R is alkyl or aryl substituents, (phosphoramidate).

#In the discussion which follows hapten or thymidine 3'-O-(7-hydroxy-4-methyl coumarin) phosphorodithioate 100, as shown in FIG. 3, was chosen to elicit catalytic antibodies. As this approach, eliciting catalytic antibodies to a stable ground state metal binding hapten, as opposed to a transition state hapten, is novel, it was necessary to choose chemical compounds which were easily assayed and chemically manipulated. While 7-hydroxy-4-methyl coumarin provides a fluorescent chromophore which can be easily assayed, a number of various coumarin derivatives could have been utilized (Pollack, S. J., et al., Science, 242:1038 (1988); Pollack, S. J., et al., J. Am Chem Soc., 111:2282 (1989); and Baldwin, et al., Science, 245:1104 (1989)). In addition to assayable chromophores, p-nitrophenyl esters are also easily assayed. The anion cleavage product, p-nitrophenolate, is yellow at basic pH, and its appearance can be followed spectrophotometrically at 405 nm. The chemical compounds discussed above and used in hapten 100 were helpful in tracking the catalytic activity of the resulting antibodies, however, other chemical compounds which can not be assayed may certainly be used. Thymidine was chosen as the nucleotide component because it can be easily manipulated chemically. The hapten described above was used to generate monoclonal antibodies that would hydrolyze a substrate having a natural phosphate diester bond, such as that present in DNA, shown in FIG. 4.