Method of designing an agent that mimics a functional epitope

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The present invention provides a new use of antibodies (Abs) as the basis for pharmaceutical compound and vaccine development. Antibodies raised against the functional epitope of a biological molecule, when selected using the appropriate criteria, will provide three-dimensional (3D) information of the binding site on the biological molecule. These antibodies will, therefore, act as 3D surrogates of the biological molecules. The surrogate antibody will be used for the rational design of small-molecule agonists, antagonists and vaccine antigens based on the 3D structure of the surrogate antibody combining site that binds the biologically functional epitope. This approach will be applicable to a wide range of biological molecules including, but not limited to, nucleic acids, proteins, lipids, carbohydrates, small molecular weight physiological ligands, pathogenic organisms and tumor cells. The use of the antibody 3D structure as the basis for drug development circumvents technical difficulties associated with biological molecules for which a 3D structure is not available. The present invention also provides an approach for high-specificity, high-throughput binding assay methods for the screening of chemical compounds including, but not limited to, those from combinatorial chemistry methodologies and natural products.

12 3D information in the monoclonal antibodies will readily allow for the application of rational design approaches such as, but not limited to, database searching and de novo design, to previously inaccessible biological molecules. The surrogate 3D structural information in the monoclonal antibody can be for the biological antigen itself or for the biological target with which the biological antigen interacts. Both types of information are useful for rational agonist and antagonist design.

14 The ascites fluid may then be collected and said monoclonal antibody purified via methods known to those skilled in the art. Fab fragments from said monoclonal antibodies may be generated and purified via methods known to the skilled in the art. Application of the 3D information in the selected monoclonal primary requires experimental determination of the 3D structure of said antibody. This may be performed on the full antibody, its Fab fragment and /or its Fv domain via, but not limited to, X-ray crystallography or NMR spectroscopy . These techniques, as known to those skilled in the art, require preparation of the antibody, its FAB fragment or its Fv domain to obtain crystals for X-ray crystallography or obtain a solution of adequate concentration for NMR studies. Application of standard approaches, known to those skilled in the art, allow for determination of the 3D structures of the antibody. Primary sequence information required for the 3D structure determination of said monoclonal antibody may be determined via polymerase chain reaction (PCR) approaches, known to those skilled in the art . These 3D structures may be obtained for the antibody alone, for the antibody complexed with said antigen or for the antibody complexed with the anti-idiotypic monoclonal antibody used in the selection of said antibody.

Availability of the 3D structure of said monoclonal antibody that is acting as a 3D surrogate of a biological receptor allows for the rational design of chemical compounds, including, but not limited to, novel therapeutic agents, that will specifically interact with the antigen combining site (combining site hereafter) of the monoclonal antibody. Rational design approaches include, but are not limited to, i) selecting chemical compounds from a database of compounds or ii) building novel chemical compounds that have 3D structures that are structurally complementary to the binding site. Approaches for database searching and building of novel chemical compounds referred to as de novo design , based on the 3D structures of biological macromolecules, are known to those skilled in the art. In the present embodiment, the combining site on the 3D structure of the monoclonal antibody will used be for the database selection or de novo design of chemical compounds. The location of the combining site can be identified based on either i) its interaction with the bound 15 antigen or the bound anti-idiotype monoclonal antibody in the 3D structure or ii) based on the known variable regions of the antibodies.

Once the combining site has been determined, chemical compounds with the potential to bind specifically and tightly to that site can be identified. In database searching approaches the 3D structures of the chemical compounds are overlaid onto the combining site in a variety of orientations. Chemical compounds with the best fit are selected. This fit can be based on, but is not limited to, shape complementarity or the energy of interaction between the chemical compound and the combining site. Chemical compounds can be built into the combining site via de novo design using, but not limited to, the following procedure.

Lead compound optimization is performed by determining the 3D structure of the antibody acting as the 3D receptor surrogate with one of the initial chemical compounds bound to the combining site via, but not limited to, X-ray crystallography or NMR spectroscopy. Information on the interactions between the chemical compound and said antibody can then be used to optimize the chemical structure of said chemical compound to enhance the binding and /or selectivity of said compound to said antibody combining site. Such enhancement may be obtained via, but not limited to, addition of hydrogen bonding groups to said chemical compound to complement hydrogen bonding groups on said antibody combining site. Lead optimization is often performed in a number of iterative steps, as follows. Initially, the 3D structure of said antibody-said chemical complex is obtained and used to identify modifications in said chemical to enhance binding. Chemical synthesis of the newly designed chemical(s) is performed followed by binding and/or biological assays of said newly designed chemicals. From the newly designed chemical those with enhanced binding and /or biological activity are subjected to 3D structure determination of said antibody-said newly designed chemical complex(es), and so on, in an iterative fashion. Application of this approach to enhance specificity can be performed via, but not limited to, inclusion of two or more antibodies acting as 3D receptor surrogates of different receptors. For example, monoclonal antibodies that are selective for different species of the receptor for insulin-like growth factor or monoclonal antibodies that act as agonists or antagonists of insulin activity have been identified. An alternative embodiment of the present invention is the use of the 3D structure of the combining site of said anti-idiotypic monoclonal antibody. Selection of said anti-idiotypic monoclonal antibody by the same biological assay used for said antigen assures that the 3D structure of the combining site of said anti-idiotypic monoclonal antibody mimics the biological properties of said antigen and, therefore, mimics the 3D structure of the functional epitope of said antigen. In essence, said anti-idiotypic monoclonal antibody is an antigen 3D structural surrogate. The 3D structure of the anti-idiotypic 17 monoclonal antibody combining site can be determined in a manner analogous to that of said primary monoclonal antibody. That 3D structure may then be used to identify chemical compounds that mimic the 3D structure of said antigen, thereby, mimicking its biological function. Identification of said chemical compounds can be performed via the_rational design approaches presented above. In this embodiment, chemical compounds would be selected from a database or created via de novo design based on their ability to reproduce the 3D structural properties of the combining site of said anti-idiotypic monoclonal antibody. Use of the 3D structure of the combining site of said anti-idiotypic monoclonal antibody may also be use for lead optimization procedures.

EXAMPLES

The following examples are provided for illustrative purposes only, and are in no way intended to limit the scope of the present invention.

Example 1: Rational Drug Design of a Peptide Hormone Agonist According to one embodiment of the present invention, one can develop a novel small molecule drug that agonizes a peptide hormone, such as insulin, following the approach outlined in Figure 1. Specifically, one starts by raising a large number of monoclonal antibodies (1° mAbs) against human insulin. Anti-idiotypic antibodies are then raised against the 1° mAbs. The anti-idiotypic antibodies are assayed for biological activity identical to that of insulin using, for example, cells expressing the human insulin receptor. The 1° mAb that acts as the epitope for the development of the biologically active anti-idiotypic antibody is identified. The selected 1° mAb is then produced in gram quantities, allowing for crystallization of the 1° mAb to elucidate its 3D structure via x-ray crystallography. The 3D structure of the 1° mAb combining site acts as a 3D structural surrogate of the human insulin receptor binding site.

Crystallization in the presence of the insulin allows for identification of the 1° mAb combining site. Knowledge of the location of the variable regions

18 in antibodies, however, does not make this step essential. 3D information on the antigen combining site of the 1° mAb acts as the basis for selection of lead compounds from chemical databases or for the de novo drug design of novel lead compounds. Compounds selected can then be synthesized and tested in real time for binding affinity in a high-throughput assay system based on use of the 1° mAb. An iterative approach, as shown in the bottom of Figure 1, can then be applied to refine the identified lead compound(s) to develop insulin agonists including, but not limited to, novel therapeutic agents.