Title:  Method for treatment of SLE

United States Patent:  6,342,218

Assignee:  Oklahoma Medical Research Foundation (Oklahoma City, OK)

Filed:  February 14, 1997

Treatments have been developed for lupus patients using either anti-ID antibodies to dsDNA to block anti-dsDNA antibodies and/or kill the B cells producing the anti-dsDNA antibodies or ribosomal protein S1 peptides immunoreactive with anti-dsDNA antibodies. Examples demonstrate that (1) anti-dsDNA antibodies are cross-reactive with ribosomal protein S1, (2) anti-dsDNA antibodies suppress protein synthesis, presumably through inhibition of mRNA translation initiation, and (3) a normal human sera contains an anti-idiotypic antibody (anti-Id) to anti-dsDNA antibodies isolated from SLE patients which blocked the interactions between the anti-Id antibody fragment (Fab₂) and various anti-dsDNA preparations.

DETAILED DESCRIPTION OF THE INVENTION

Therapeutic Applicants and Pharmaceutical Compositions

Based on the results in the examples, one can prepare anti-Id reagents (for anti-dsDNA) that can be used to downregulate the production of anti-dsDNA.

In one embodiment, reagents that are anti-idiotypic antibodies to anti-dsDNA could be used to down regulate or even curtail anti-dsDNA production by SLE patients.

In a second embodiment, free peptide or a conjugate of this peptide based on peptide sequence of human ribosomal protein S1 can be used in tolerance induction which could ablate anti-dsDNA.

Peptide or Protein-based Compositions

Attempts to influence anti-DNA production in mouse lupus in vivo or in human lymphocytes in vitro, are described by Borel, et al., Science, 182:76-77 (1973); Borel, et al., J. Clin. Invest., 61:276-286 (1978); Borel, Y. and Borel, H., J. Clin. Invest., 82:1901-1907 (1988). As described by Borel, et al., oligonucleotides or nucleosides are attached to isologous (same species) IgG and this is allegedly effective in (1) inhibiting the development of an immune response to DNA in murine lupus and decreasing disease severity, and (2) inhibiting human cells from producing anti-DNA in vitro. The "DNA" used by Borel is single stranded or denatured which is not optimal since the most important response in SLE is to native or double stranded DNA. Borel's work provides an appropriate "carrier" for the toleragen, isologous gamma globulin.

As described herein, peptide(s) that are immunoreactive with dsDNA and are derived from the human ribosomal protein S1 can be used to induce tolerance in a patient. Antibodies to dsDNA are the disease specific pathogenic autoantibodies of the greatest interest. There are two major possibilities: (1) inject free peptide, or (2) inject peptide-coupled to human IgG, for example, coupled using glutaraldehyde or carbodiimide. These two approaches should both induce T cell tolerance. They may also be effective in inducing B cell tolerance. Both approaches are attractive since there is little chance of "boosting" the anti-dsDNA response. Should the latter occur, it can be treated by standard immunosuppressive drugs, alone or in combination with anti-La/SSB or anti-U₁ RNP, as described below.

Behavior of the peptide or peptide conjugate is first studied in an appropriate animal model in order to determine efficacy and optimal dosages. There are several that could be used, but the most attractive is the Palmerston North Mouse. It has been shown that these mice, which all produce anti-dsDNA and develop nephritis, also develop anti-U₁ RNP and Sm responses in almost all the animals with a dominant immune response against the A protein of U₁ RNP measured in Western blot, as reported by Handwerger, et al., Clin. Res. 42:315A (1994). These mice have no detectable antibodies in the first three months of life but rapidly develop them after six months of age and experience a fulminant glomerulonephritis associated with anti-dsDNA antibodies. Dosage would range from 3 to 300 micrograms per mouse given weekly in the first experiments.

The same result obtained by administering peptide or a peptide conjugate can be achieved by coupling recombinant or isolated human ribosomal protein S1 to human IgG.

Although described herein with reference to the whole protein, it is preferable to use peptides of between a few amino acids up to about 100 amino acids, more preferably less than forty amino acids, still more preferably less than ten to twenty amino acids. These peptides can be easily ascertained by immobilizing the anti-dsDNA antibodies from a patient(s) and screening for binding of the peptides. Peptides can be prepared using standard techniques for amino acid synthesis or recombinantly, by engineering the cDNA (SEQ ID NO:1) encoding the protein.

Anti-Id Antibodies that are Immunoeactive with Anti-dsDNA Antibodies

Normal human sera contains anti-Id antibodies immunoreactive with anti-dsDNA antibodies present in many SLE patients. Antibodies for use in treating patients can be obtained using standard techniques to harvest antibodies from normal people, or, more preferably, antibody producing cells are isolated by binding of cells expressing antibody.  The antibody producing cells are then transformed with Eppstein-Barr virus (EBV), amplified in culture, the gene encoding the variable region of the anti-Id antibodies cloned, inserted into an appropriate vector, and expressed in bacteria or another appropriate expression system, using known techniques. Preliminary studies have yielded several clones.

In either case, antibody is administered to a patient in a dosage which decreases the amount of anti-dsDNA antibody. This is readily determined since SLE patients are routinely assayed for blood levels of anti-dsDNA. In most cases patients are expected to respond as they do to standard immunosuppressive therapy, by decreasing production of anti-dsDNA antibodies. In some cases, the antibodies will result in killing of the antibody producing cells in the patient. Treatments will be repeated as required.

An alternative approach is to screen recombinant libraries of Ig variable ("V") regions made from cDNA's reverse transcribed from mRNA extracted from peripheral blood lymphocytes from patients who produce anti-anti-dsDNA antibodies. A number of such libraries can be constructed and then screened for clones reactive with Fab anti-dsDNA but not normal Fab. These can then be used to produce any desired amount of anti-idiotype to anti-dsDNA. Alternatively, murine recombinant monoclonal anti-idiotypic antibodies directed against relevant idiotope(s) on anti-dsDNA can be produced.

This can be accomplished by the use of Pharmacia's (Pharmacia LKB Biotechnology, Sweden) "Recombinant Phage Antibody System" (RPAS), which generates a single-chain Fv fragment (ScFv) that incorporates the complete antigen-binding domain of the antibody. In the RPAS, antibody variable heavy and light chain genes are separately amplified from the hybridoma mRNA and cloned into an expression vector. The heavy and light chain domains are co-expressed on the same polypeptide chain after joining with a short linker DNA which codes for a flexible peptide. This assembly generates a single-chain Fv fragment (ScFv) which incorporates the complete antigen-binding domain of the antibody. Compared to the intact monoclonal antibody, the recombinant ScFv includes a considerably lower number of epitopes, and thereby presents a much weaker immunogenic stimulus when injected into humans. The murine ScFv molecules can be "humanized" to further reduce the immunogenic stimulus presented.

Methods for "humanizing" antibodies, or generating less immunogenic fragments of non-human antibodies, are well known. A humanized antibody is one in which only the antigen-recognized sites, or complementarily-determining hypervariable regions (CDRs) are of non-human origin, whereas all framework regions (FR) of variable domains are products of human genes.

These "humanized" antibodies present a lesser xenograft rejection stimulus when introduced to a human recipient.

To accomplish humanization of a selected mouse monoclonal antibody, the CDR grafting method described by Daugherty, et al., Nucl. Acids Res., 19:2471-2476, 1991, incorporated herein by reference, can be used. Briefly, the variable region DNA of a selected animal recombinant anti-idiotypic ScFv is sequenced by the method of Clackson, T., et al., Nature, 352:624-688, 1991, incorporated herein by reference. Using this sequence, animal CDRs are distinguished from animal framework regions (FR) based on locations of the CDRs in known sequences of animal variable genes. Kabat, H. A., et al.l, Sequences of Proteins of Immunological Interest, 4th Ed. (U.S. Dept. health and Human Services, Bethesda, Md., 1987). Once the animal CDRs and FR are identified, the CDRs are grafted onto human heavy chain variable region framework by the use of synthetic oligonucleotides and polymerase chain reaction (PCR) recombination. Codons for the animal heavy chain CRDs, as well as the available human heavy chain variable region framework, are built in four (each 100 bases long) oligonucleotides. Using PCR, a grafted DNA sequence of 400 bases is formed that encodes for the recombinant animal CDR/human heavy chain FR protection.

Claim 1 of 12 Claims

We claim:

1. A method for treating a lupus patient having anti-dsDNA antibodies comprising administering to the patient a therapeutic composition in a pharmaceutically acceptable carrier for administration to a patient selected from the group consisting of peptides between four and forty amino acids in length which have sequence identity with ribosomal protein SI and which are immunoreactive with anti-dsDNA antibodies, and anti-idiotypic monoclonal antibody or antibody fragments immunoreactive with anti-dsDNA antibodies which are cross-reactive with human ribosomal protein S1 and which bind with greater affinity to ribosomal protein S1.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.