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Methods for modulating the efficacy of nucleic acid based therapies

This application is a continuation-in-part application of U.S. patent application Ser. No. 11/058,728, filed Feb. 15, 2005, now abandoned, which in turn is a continuation of U.S. patent application Ser. No. 09/862,849, filed May 22, 2001, which is a divisional application of U.S. patent application Ser. No. 09/046,373, filed Mar. 23, 1998, now U.S. Pat. No. 6,235,714. This application also claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application 60/940,610, filed on May 29, 2007. The foregoing applications are incorporated by reference herein.

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

This invention relates to the fields of immunology, molecular biology and medicine. More specifically, the invention provides novel methods and compositions for stimulating the production of novel catalytic antibodies and inhibitors thereof. The invention also provides methods for identifying and isolating naturally occurring catalytic antibodies expressed from germline genes. Finally, the invention provides methods for synthesizing covalently reactive antigenic analogs which stimulate the production of catalytic antibodies and/or irreversibly inhibit the activity thereof.

Several publications are referenced in this application by numerals in brackets in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.

The observation that vasoactive intestinal peptide (VIP) is cleaved by antibodies (Abs) from asthma patients provided early evidence that Abs may possess peptidase activity [1,2]. This observation has been reproduced independently by Suzuki et al [3]. Autoantibody catalysis is not restricted to catalysis of VIP. Autoantibodies in Hashimoto's thyroiditis catalyze the cleavage of thyroglobulin [4]. Further evidence for autoantibody catalysis has been provided by reports of DNase activity in Abs from lupus patients [5,6]. The bias towards catalytic antibody (Ab) synthesis in autoimmune disease is supported by observations that mouse strains with a genetic predisposition to autoimmune disease produce esterase Abs at higher levels when compared to control mouse strains in response to immunization with a transition state analog [7].

Like noncatalytic Abs, peptidase Abs are capable of binding antigens (Ags) with high specificity mediated by contacts at residues from the VL and VH domains. The purified H and L subunits are known to be independently capable of binding Ags, albeit with lower affinity than the parent Ab. X-ray crystallography of Ab-Ag complexes have shown that the VL and VH domains are both involved in binding the antigen (Ag) [8]. The precise contribution of the two V domains varies in individual Ab-Ag complexes, but the VH domain may contribute at a somewhat greater level, because CDRH3 tends to be longer and more variable in sequence compared to CDRL3.

The initial complexation of a polypeptide Ag by a peptidase Ab is followed by cleavage of one or more peptide bonds. Just prior to cleavage, contacts with the catalytic residues of the antibody are established with the peptide bond in the transition state. The ability to hydrolyze peptide bonds appears to reside in the VL domain. This conclusion is based on the cleavage of VIP by polyclonal autoantibody L chains, monoclonal L chains isolated from multiple myeloma patients and their recombinant VL domains, and recombinant L chains raised by immunization with VIP. The H chains of polyclonal and monoclonal Abs to VIP are capable of VIP binding but are devoid of the catalytic activity [9]. The VH domain can nevertheless influence the peptidase activity by “remote control”, because in binding to VIP remote from the cleavage site, it can influence the conformation of the binding site as shown by the peptidase activity of Fv constructs composed of the catalytic anti-VIP VL domain linked to its VH domain. The anti-VIP VH domain exerted beneficial effects and an irrelevant VH domain exerted detrimental effects on the catalytic activity, as evaluated by the values of VIP binding affinity and catalytic efficiency. The proposed existence of distinct catalytic and antigen binding subsites in catalytic Abs is consistent with data that Abs generally contain large combining sites, capable of accommodating 15-22 amino acids of polypeptide substrates [8], and that substrate regions distant from the cleavage site are recognized by the Abs. Thus, the VH domain offers a means to control the specificity of the catalytic site.

Molecular modeling of the L chain suggested that its Asp1, Ser27a and His93 are appropriately positioned to serve as the catalytic triad [10]. The hydrolysis of VIP was reduced by >90% by substitution of Ala residues for Ser27a, His93 or Asp1 by site-directed mutagenesis [12]. The catalytic activity of the wild type protein was inhibited selectively by diisopropylfluorophosphate (DFP), a serine protease inhibitor, but the residual activity of the Ser27a mutant was refractory to DFP. The Km of the wild type L chain for VIP (130 nM) was unaffected by mutations at Ser27a, His93 and Asp1. In contrast, mutagenesis at residues forming the extended active site of the L chain (Ser26, H27d/Asp28) produced increases in the Km values (by 10-fold) and increases in turnover (by 10-fold). These results can be explained as arising from diminished ground state stabilization. The consequent decrease of ΔG554 cat produces an increase in turnover. Thus, two types of residues participating in catalysis by the L chain have been identified. Ser27a and His93 are essential for catalysis but not for initial high affinity complexation with the ground state of VIP. Ser26 and His27d/Asp28 participate in VIP ground state binding and limit turnover indirectly. See FIG. 1.

The VIPase L chain displayed burst kinetics in the early phase of the reaction, suggesting the formation of a covalent acyl-L chain intermediate, as occurs during peptide bond cleavage by serine proteases. The fluorescence intensity was monitored as a function of time after mixing the L chain with the substrate Pro-Phe-Arg-MCA. There was an immediate increase in fluorescence, corresponding to formation of the covalent intermediate, followed by a slower increase, corresponding to establishment of the steady rate. The number of active sites was computed from the magnitude of the burst by comparison with the fluorescence yield of standard aminomethylcoumarin. The concentration of catalytic sites was estimated at 114 nM, representing about 90% of the L chain concentration estimated by the Bradford method (125 nM).

The catalytic residues (Ser27a, His93, Asp1) in the anti-VIP VL domain are also present in its germline VL domain counterpart (GenBank accession number of the germline VL gene, Z72384). The anti-VIP VL domain contains 4 amino acid replacements compared to its germline sequence. These are His27d:Asp, Thr28e:Ser, Ile34:Asn and Gln96:Trp. The germline configuration protein of the anti-VIP L chain was constructed by introducing the required 4 mutations as described previously [12]. The purified germline protein expressed catalytic activity as detected by cleavage of the Pro-Phe-Arg-MCA substrate at about 3.5 fold lower level than the mature L chain (330±23 FU/0.4 μM L chain/20 min; substrate conc. 50 μM). The data suggest that remote effects due to the somatically mutated residues are not essential for expression of the catalytic activity.

The present invention provides novel compositions and methods for stimulating production of catalytic antibodies and fragments thereof. Catalytic antibodies with specificity for predetermined disease-associated antigens provide a valuable therapeutic tool for clinical use. Provided herein are methods for identifying, isolating and refining naturally occurring catalytic antibodies for the treatment of a variety of medical diseases and disorders, including but not limited to infectious, autoimmune and neoplastic disease. Such catalytic antibodies will also have applications in the fields of veterinary medicine, industrial and clinical research and dermatology.

According to one aspect of the invention, methods and compositions are provided herein for stimulating catalytic antibody production to predetermined target antigens, including but not limited to those involved in pathogenic and neoplastic processes. Covalently reactive antigen analogs (CRAAs) are described which stimulate the production of catalytic antibodies with therapeutic value in the treatment of a variety of medical conditions, including autoimmunity disorders, microbial diseases, lymphoproliferative disorders and cancer. The catalytic antibodies of the invention may also be used prophylatically to prevent certain medical disorders, including but not limited to septic shock, systemic inflammatory disease and acute respiratory distress syndrome.

The covalently reactive antigen analogs, (CRAAs) of the present invention contain three essential elements and have the following formula: X1-Y-E-X2. E is an electrophilic reaction center designed to react covalently with nucleophilic side chains of certain amino acids; Y is a basic residue (Arg or Lys) at the P1 position (first amino acid on the N-terminal side of the reaction center); and X1 and X2 comprise three to ten flanking amino acids on the N-terminal and C-terminal side of the reaction center. The resultant CRAA represents a novel combination of individual structural elements which act in concert to (a) bind chemically reactive serine residues encoded by the germline genes for certain serine protease types of catalytic antibodies (as well as residues such as Thr and Cys that might acquire their chemical reactivity via somatic sequence diversification of the germline genes); (b) utilize ion pairing and noncovalent forces to bind structures such as positively charged Asp/Glu residues that are responsible for the basic residue cleavage specificity of the germline encoded catalytic sites; and (c) bind antibody combining sites at multiple amino acids via ion pairing and noncovalent forces.

In one aspect of the invention, CRAAs are administered to a living organism under conditions whereby the CRAAs stimulate production of specific catalytic antibodies. The catalytic antibodies are then purified. Antibodies so purified are then adminstered to a patient in need of such treatment in an amount sufficient to inactivate antigens associated with a predetermined medical disorder.

According to another aspect of the present invention, methods and compositions are disclosed for administering immunogenic amounts of CRAAs combined with an immunogenic amount of a conventional transition state analog (TSA) to further stimulate catalytic antibody production.

According to another aspect of the present invention, a method is provided for treating a pathological condition related to the presence of endogenously expressed catalytic antibodies. Examples of such abnormal pathological conditions are certain autoimmune disorders as well as lymphoproliferative disorders. The method comprises administering to a patient having such a pathological condition a pharmaceutical preparation comprising covalently reactive antigen analog capable of irreversibly binding the endogenously produced catalytic antibodies, in an amount sufficient to inhibit the activity of the antibodies, thereby alleviating the pathological condition. In this embodiment, the CRAA contains a minimal B epitope only to minimize the immunogenicity of the CRAA.

According to another aspect of this invention, a pharmaceutical preparation is provided for treating a pathological condition related to the presence of endogenously produced catalytic antibodies. This pharmaceutical preparation comprises a CRAA in a biologically compatible medium. Endogenously produced catalytic antibodies are irreversibly bound and inactivated upon exposure to the CRAA. The preparation is administered an amount sufficient to inhibit the activity of the catalytic antibodies.

In another aspect of the invention, methods for passively immunizing a patient with a catalytic antibody preparation are provided. Catalytic antibodies are infused into the patient which act to inactivate targeted disease associated antigens.