WO1991005258A1

WO1991005258A1 - Rheumatoid factor activity encoded by variant immunoglobulin variable region

Info

Publication number: WO1991005258A1
Application number: PCT/US1990/005311
Authority: WO
Inventors: Richard H. Weisbart
Original assignee: The Regents Of The University Of California
Priority date: 1989-09-29
Filing date: 1990-09-18
Publication date: 1991-04-18
Also published as: EP0494265A4; EP0494265A1; CA2066674A1; JPH05500670A

Abstract

Diagnosis and therapy of inflammatory diseases is provided by detecting the presence of mutations in the framework regions of light chains of immunoglobulins. Particularly, a multiplicity of mutations in the framework region (3) at conserved sites is found to be associated with erosive arthritis. By detecting the presence of mutations in the nucleic acid or resulting encoded light chain, propensity for inflammatory diseases and prophylactic and therapeutic treatments is provided.

Description

RHEUMATOID FACTOR ACTIVITY ENCODED BY

VARIANT IMMUNOGLOBULIN VARIABLE REGION

INTRODUCTION

Technical Field

The technical field of the subject invention is the diagnosis and therapy of erosive arthritis. Background

Erosive arthritis is a chronic destructive inflammatory joint disease, which includes rheumatoid arthritis as one type of the disease. Rheumatoid factors are antiglobulin antibodies that bind the Fc portion of IgG immunoglobulins and are implicated in the pathogenesis of rheumatoid arthritis. Rheumatoid factors in patients with efosive arthritis, however, may be different from rheumatoid factor in individuals without rheumatoid arthritis, as indicated by studies of rheumatoid factor specificity and idiotype. In contrast to rheumatoid factor in patients without erosive arthritis, the genetic and molecular basis for rheumatoid factor autoantibodies in patients with erosive arthritis is completely unknown.

There is substantial interest in understanding the basis for the occurrence of erosive arthritis. The understanding of the characteristics of the

compositions in the immune system which lead to erosive arthritis could provide means for diagnosis and treatment of the disease. Relevant Literature

Waaler, Acta Pathol. Microbiol. Scand. 17;172- 188 (1940); Rose et al., Proc. Soc. Exp. Biol. Med. 68 :1-6 (1948); and Natvig et al., Clin. Exp. Immunol. 12:177-183 (1972) report that rheumatoid factors are antiglobulin antibodies that bond the Fc portion of IgG immunoglobulins and are implicated in the pathogenesis of rheumatoid arthritis. Allen and Kunkel, Arthritis Ragum. 9:758-768 (1966) and Fong et al., J. Immunol. 137:122-128 (1986) suggest that rheumatoid factors in patients with rheumatoid arthritis may be different from these factors in individuals without rheumatoid arthritis as indicated by studies of rheumatoid factor specificity and idiotype. See also Jirik et al., Proc. Natl. Acad. Sci. USA 83:2195-2199 (1986). The human lymphoblastoid cell line hRF-1 is reported by Weisbart et al., J. Immunol. 139:2925-2928 (1987). SUMMARY OF THE INVENTION

Methods and compositions are provided for the diagnosis and treatment of rheumatoid arthritis, where peptides comprising mutants of conserved framework regions encoded by germline genes for antibody light chains are found to be diagnostic of rheumatoid factor. The oligopeptides comprising the mutant framework region sequence or compositions inhibiting the binding of such mutant sequences to other

antibodies find use as therapeutics to inhibit the rheumatoid factor activity of the mutant light chain.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Methods and compositions are provided for the diagnosis and treatment of erosive arthritis, which includes about 70% of patients with rheumatoid

arthritis and 26% of patients with psoriasis and polyarthritis (Waaler, Acta Pathol. Microbiol. Scand. 17:172-188 (1940); Rose et al., Proc. Soc. Exp. Biol. Med. 68:1-6 (1948); Natvig et al., Clin. Exp. Immunol. 12:177-183 (1972); and Wright "Psoriatic Arthritis" in Textbook of Rheumatology (EDS Kelley, W. ; Harris, E.D.; Ruddy, S.; Sledge, C.B. (1051) W.B. Saunders,

Philadelphia, 1981). It is found that the light chain of immunoglobulin is mutated at a conserved site in the framework region, having at least one mutation, frequently having two or more mutations, usually not more than three mutations. The framework regions are the 1, 2 and 3 framework regions (FR-1, -2, and -3) where the mutations of interest are particularly in the FR-3 region and more particularly at sites 62 and 65. Furthermore, the light chains are divided into kappa and lambda, where the mutations are of particular interest in the kappa light chain, and where the kappa light chains are divided into subgroups I-IV, of particular interest is kappa II.

By conserved region is intended that at least about 80%, preferably at least about 85% and more preferably at least about 90% of known light chain sequences have the same amino acid or a conservative mutation at the particular site. Conserved amino acids of the third framework region include the following sequence: G-V-P-R-F-S-S-G-S-T-F-T-L-I-R-V-V-G-V-Y-Y-C (57-88). For example, at residue 62, phenylalanine occurs in all four of the kappa subgroups and is present in 78/80 of reported kappa light chains; only one light chain (a V_I) contains a change to valine while the other chain was a different amino acid.

Similarly, serine is present at residue 65 in 75/80 reported kappa chain sequences but only one light chain (a V_III) contained a change to arginine (Rabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services) (1987). None of the 80 reported kappa light chain sequences had changes at both residue 62 and 65. The mutation in the conserved region may be a conservative or non-conservative change, that is , it may be an exchange of one polar amino acid for another polar amino acid, or a nonpolar amino acid for a polar amino acid, an aliphatic amino acid for an aromatic amino acid, a charged polar amino acid for a non- charged polar amino acid, vice versa, or the like. In one kappa chain, at residue 62, phenylalanine was substituted by valine and at residue 65, serine was substitued by arginine. Therefore, the substitutions may be conservative or non-conservative, where the following table indicates conservative substitutions, where the same amino acids are found on the same line.

Desirably, the substitution may have the same charge or neutrality as the wild type amino acid.

In addition, of interest will be the particular J chain associated with the mutation. Of particular interest is the presence of a J5 segment associated with the mutant light chain.

The mutations will normally be associated with the germline gene, so that the variable region exon will include the mutation.

Oligopeptides may be provided which are of at least eight amino acids, and, furthermore, encompass the mutant framework region. The oligopeptides will generally be fewer than about 60 amino acids, more usually fewer than about 36 amino acids, although large oligopeptides may be employed to inhibit binding of the mutant framework region to other antibodies. In addition, using the procedure of PCT applications

WO 84/03564, WO 84/03506 and WO 86/00991, oligopeptides may be prepared synthetically which are specific for the binding site of the mutant antibody, so as to be able to bind to the mutant antibody and prevent its binding to an homologous ligand other than the

synthesized peptide. These peptides can be readily prepared in large number and screened for binding affinity to the particular antibody. The peptides may be used individually or in combination, blocking binding of the mutant antibody to other antibodies or proteins. If desired, the entire mutant light chain may be employed, but since this is not necessary, it will be frequently convenient to use a fragment of the light chain. The entire light chain may be used in purified form, generally greater than about 90%, usually greater than about 95% pure, in place of the oligopeptide.

Besides using oligopeptide sequences which are crossreactive with the mutant framework region, one may use other compositions which will bind to the mutant framework region. Of particular interest are

antibodies in a monovalent form, such as Fab, Fab', or Fv or an antiidiotype, where the variable region of the antiidiotype may compete with the mutant framework region and the antiidiotype is monovalent. Thus, only a single variable region of the antiidiotype antibody may be employed to inhibit binding.

A sequence of particular interest is an eight amino acid sequence coming within the sequence

GVPDRVSGRGSGTDF, where additional amino acids from the framework region of the kappa light chain particularly the kappa II light chain may be present, but the eight or greater amino acid sequence includes the sequence VSGR.

Where oligo- or polypeptides are employed, they may be modified in a variety of ways to enhance their stability, such as using an unnatural amino acid, such as the D- amino acid, at other than the tetrad indicated above, by functionalizing the amino or carboxy terminus, e.g., for the amino group, acylation or alkylation, and for the carboxy group, esterifi- cation or amidification, or the like. Other methods of stabilization may include liposome formation, other forms of encapsulation, etc.

The presence of a mutant light chain may be detected in a variety of ways. For example, one may use PCR (polymerase chain reaction), using either the mutated or the natural sequence or both as primers to detect the presence of the mutation in germline DNA of a human host. Alternatively, one may use primers which are based on conserved regions which flank the mutated region of interest and sequence the resulting DNA to determine the presence of mutations. Alternatively, one may provide for antibodies specific for the mutated framework region, which can distinguish the mutated framework region from the wildtype framework region, and detect the presence of antibodies which bind to the antibody for the mutated framework region. If

necessary, one may isolate antisera and identify the light chain and sequence the light chain or portions thereof including the framework region of interest and determine the presence of mutations.

A wide variety of protocols are available for performing immunoassays, sequencing nucleic acids and peptide sequences, and any or all of these may be employed, depending upon the particular situation.

Immunoassays include ELISA, EMIT, CEDIA, SLIFA, and the like. A variety of patents have issued describing a number of diagnostic procedures, which include U.S. Patent Nos. 3,791,932; 3,817,837; 3,398,943, and references cited therein.

The subject peptides may also be used for therapy, in inhibiting rheumatoid factor activity of the mutated framework region antibodies. These

compositions may be administered by any convenient way, preferably intravascularly or peritoneally, conveniently in a physiologically acceptable carrier, e.g., phosphate buffered saline, saline, deionized water, or the like. Generally, the amount administered will be in the range of about 100 to 1000 μg/kg of the

recipient. A single bolus may be employed or repetitive administrations may be employed, generally not more frequently than once weekly. The concentration of the peptide will generally be in the range of about 100 to 500 yg/ml in the dose administered. Other additives may be included, such as stabilizers, bacteriocides, etc. These additives will be present in conventional amounts.

The subject peptides may be prepared in a variety of ways. Peptides under about 60 amino acids can be readily synthesized today using conventional commercially available automatic synthesizers.

Alternatively, DNA sequences may be prepared encoding the desired peptide and inserted into an appropriate expression vector for expression in a prokaryotic or eukaryotic host. A wide variety of expression vectors are available today and may be used in conventional ways for transformation of a competent host for expression and isolation. If desired, the open reading frame encoding the desired peptide may be joined to a signal sequence for secretion, so as to permit

isolation from the culture medium. Methods for preparing the desired sequence, inserting the sequence into an expression vector, transforming a competent host, and growing the host in culture for production of the product may be found in U.S. Patent Nos. 4,710,473; 4,711,843 and 4,713,339.

The following examples are offered by way of illustration. and not by way of limitation.

EXAMPLES Preparation of Immortalized Cell Line Secreting IgG Rheumatoid Factor

Human synovium from rheumatoid arthritis patients was obtained at the time of surgery for joint replacement. The inflammatory cell population

containing 10⁷ to 10⁸ lymphoid cells was freed from stroma by digesting synovial tissue with DNAse and collagenase. The B-lymphocytes in the cell population were transformed with Epstein-Barr virus according to the method of Cole et al., Molecular and Cellular Biochemistry (1984) 62:109. The culture supernatant from one of ten transformed lymphoblastoid synovial primary cell cultures, hRF-1, produced detectable levels of IgG RF autoantibody by two weeks after EBV transformation. The IgG RF was detected by an enzyme- linked immunosorbent assay (ELISA) using purified human IgG Fc fragments bound to 96 well microtiter plates (Weisbart et al., J. Immunology (1984) 132:2909- 2912). Bound IgG RF was identified with peroxidase labeled rabbit antibodies specific for the F(ab')₂ of human IgG. After repeated cloning, these hRF-1 B- lymphoblastoid cells secreted only IgG (1-2 μg/ml), but not IgA (less than 20 mg/ml), or IgM (less than 10 mg/ml).

Isotype testing with subclass specific antiglobulins identified only human IgG₄ subclass gamma heavy chains. Anti-light chain specific antiglobulins detected only kappa, but not lambda, light chains.

Initial specificity of this IgG₄ RF for human

immunoglobulin was demonstrated by its binding to purified Fc fragments of human IgG, but not human serum albumin, bovine collagen, histone 2A, or denatured DNA, as shown in the following table.

* The human monoclonal antibody hRF-1 was bound to 96-well microtiter plastic plates at 1 μg/100 μL and characterized by ELISA using peroxidase conjugated antisera specific for human immunoglobulin isotypes, IgG subclasses, and light chains. The antigenic specificity of hRF-1 monoclonal was measured with the 5 antigens bound to 96-well microtiter plates at 1 μg/100 μL by adding hRF-1 at 2 μg/ml from culture media and detecting bound hRF-1 with affinity purified peroxidase-conjugated sheep antibodies specific for F(ab')₂ of human IgG. Results were recorded as absorbancy at 414 nm corrected for background values using control ELISA wells coated only with poly-L-lysine.

The hRF-1 human monoclonal IgG₄ RF was tested for binding to different species' IgGs and shown to bind to human, rabbit, mouse and guinea pig IgG more strongly than to horse, goat, and sheep IgG. The monoclonal IgG₄ RF did not bind at all to chicken

(nonmammalian) IgG. The observed binding pattern is analogous to the species-specific binding of Staphylo- coccal protein-A (SpA). SpA at concentrations as low as lμg/ml in direct competetive binding assays with hRF-1 monoclonal IgG RF for the ability to react with human IgG Fc resulted in a 30% inhibition of this low concentration, whereas equimolar concentrations of another Fc binding protein, complement factor Clq did not inhibit binding of the hRF-1 monoclonal IgG to human IgG Fc.

Purification of heavy and light chains of mAb hRF-1

HRF-1 lymphoblast cells were grown in serum free medium, and 2.0 mg of mAb hRF-1 was purified from 40 liters of serum free supernatant by absorption to 4 gm of Protein A Sepharose CL-4B (Pharmacia,

Piscataway, NJ), washed with 0.10 M phosphate buffer containing 0.5 M NaCl, and eluted with 0.10 M glycine, pH 2.8 into tubes containing carbonate buffer to produce a final pH of 4.0. MAb hRF-1 (1 mg/ml), (see U.S. application serial no. 562,781, filed March 30, 1987, whose disclosure is incorporated herein by reference), was reduced in a nitrogen environment using 2 mM dithioerythritol for 1 hr at room temperature, pH 8.3, and then alkylated with 22 mM iodoacetamide for 2 hr on ice. After dialysis against 1.0 M propionic acid containing 4.5 M urea and 50 mM sodium chloride (PUS) for 1 hr, the imrounoglobulin chains were separated by size permeation chromatography on a Waters HPLC system using a Protein pak SW300 (7.5 X 300 mm) and SW125 (7.8 X 300 mm) column in series equilibrated in PUS

buffer. The column flow rate was 1.0 ml/min, and 0.5 ml fractions were collected. The protein fractions were identified spectrophotometrically (A₂₈₀).

Comparison of rheumatoid factor binding activity of isolated light and heavy chains with intact mAb hRF-1.

Purified mAb hRF-1 and isolated light and heavy chains (20 μg/ml) were biotinylated in a molar ratio of 1:30 with NHS-LC-Biotin (Pierce Chemical Co., Rockford, IL) overnight at 4°C in N,N-dimethylformamide adjusted to pH 8.5 with 5% Na₂CO₃. Biotinylated peptides were dialyzed against PBS and assayed for binding human IgG and other mammalian IgG immunoglob- ulins by ELISA. Binding was measured by adding

streptavidin conjugated with alkaline phosphatase, and bound phosphatase was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenol (A₄₀₅). The binding of mAb Gm607 to human IgG and other mammalian IgG immunoglobulins was evaluated for comparison and was measured by adding supernatant from cultures of the Gm607 cell line and detecting mAb Gm607 ( IgM) with affinity purified alkaline phosphatase labeled goat antisera specific for human IgM. The supernatant from Gm607 was shown to contain IgM using the same

preparation of goat antibodies to human IgM.

The amino acid sequence of the variable region of mAb hRF-1 light chain predicted from the nucleotide sequence

The amino acid sequence is compared to Gm607, a prototype V_κII light chain produced by a lymphoblast cell line derived from a normal individual (Klobeck et al., Nature 309:73-76 (1984). The Gm607 amino acid sequence was also predicted from the nucleotide sequence. Six other reported V_κII light chains are shown for comparison. The amino acid sequence of hRF-1 is identical to Gm607 with the exception of two amino acid changes, corresponding to a change from phenyl- alanine to valine at residue 62 and a change from serine to arginine at residue 65. These amino acid changes occur in a relatively invariable region of FR3.

The gene for the immunoglobulin light chain of hRF-1 cells was cloned from a cDNA library expressed in Lambda Zap (Stratagene, La Jolla, CA), and the cDNA library was screened with a V_kJ chain oligonucleotide probe labeled with ³²P. Lambda Zap-hRF-1 was converted to Bluescript, from which double stranded and single stranded DNA were isolated (Stratagene, La Jolla, CA), and dideoxy sequencing was performed using an Applied BioScience Sequenator.

Two new restriction endonuclease cleavage sites are produced as a result of two nucleotide changes in FR3 of hRF-1 V_kII

The change from thymidine to guanine produced a Tthllll site, and the change from adenosine to cytidine produced a Haelll site in hRF-1 V_κII compared to the prototypic V_κII of Gm607.

Identification of the Tthllll and Haelll endonuclease sites in hRF-1 cDNA by PCR

A 156 bp fragment is amplified in hRF-1 genomic DNA, hRF-1 cDNA, and normal PBL DNA. The 156 bp fragment amplified from DNA from -normal peripheral blood lymphocytes (PBL) did not contain a Tthllll site. In contrast, a Tthllll site was present in hRF-1 cDNA. There is one Haelll site in the 156 bp fragment amplified from hRF-1 genomic DNA, hRF-1 cDNA, and normal PBL DNA which produces a fragment immediately beneath the 156 bp fragment. A second Haelll site is present in hRF-1 cDNA which produces an additional fragment.

Identification of the Tthllll and Haelll endonuclease sites in hRF-1 genomic DNA by PCR

A nucleotide sequence of 156 bp was amplified following 25 cycles of PCR with hRF-1 genomic DNA, cDNA prepared from hRF-1 polyadenylated mRNA by reverse transcriptase, and DNA isolated from normal PBL.

Oligonucleotide primers (100 ng) were used flanking the sequences in question. The primers used for PCR were TCTCCACAGCTCCTGATCT (sense) located at amino acids 43-48 and TTGTAGAGCTTGCATGCA (antisense) located at amino acids 88-93 in the 5' to 3' orientation. The antisense primer was labeled with [γ³²P]ATP. Reactions were performed in 25 mM tris-HCl, pH 8.0, 5 mM MgCl₂, 50 mM NaCl, and 0.25 mM each of deoxyadenosine

triphosphate, deoxycytidine triphosphate, deoxythymidine triphosphate, and deoxyguanosine triphosphate in a total volume of 25 μl, cycling at 65° for 2 min and at 91° for 1 min. After amplification, 5 μl of the reaction were used for further analysis by 8%

polyacrylamide gel electrophoresis, and the PCR

products were visualized by autoradiography after 2 and 12 hours of exposure.

The autoradiograph was overexposed to enhance the Tthllll and Haelll fragments in genomic DNA where the V_κII of interest is diluted by multiple other V_kII genes compared to amplification of the single hRF-1 V_κII in the hRF-1 cDNA.

The autoradiograph showed the absence of bands in normal DNA from PBL uncut or cut with Haelll

comparable to bands of hRF-1 genomic DNA uncut or cut by Haelll. In addition, both the cDNA and genomic DNA cut with Tthllll showed a comparable band.

It is evident from the above results that one can diagnose and treat specific diseases associated with erosive arthritis, psoriasis and polyarthritis by detecting the presence of mutations of conserved amino acids in the framework regions of immunoglobulin light chains. Thus, opportunities exist to diagnose the propensity for these diseases and act prophylactically or therapeutically in the treatment of such diseases.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A purified peptide composition comprising a sequence of at least about 8 amino acids comprising at least one framework region of a human light k chain and having a total of at least two mutations in conserved sites of the framework regions of said human light k chain, said sequence including said two

mutations.

2. A peptide composition according to

Claim 1, wherein said two mutations are in framework region 3.

3. A peptide composition according to

Claim 2, wherein the sites of said two mutations are 62 and 65.

4. A peptide composition according to

Claim 1, wherein said light k chain is a V_κII chain.

5. A peptide composition according to Claim 1, wherein said protein comprises the sequence VSGR in a framework region.

6. An antibody composition specific for the mutated site of a framework region of a human light k chain having a total of at least two mutations in conserved sites of the framework regions of said human light k chain.

7. An antibody composition according to Claim 6, wherein said antibody composition is

polyclonal.

8. An antibody composition according to Claim 6 , wherein said antibody composition is monoclonal.

9. An antibody composition according to Claim 6 wherein said two mutations are in framework region 3.

10. An antibody composition according to Claim 9, wherein the sites of said two mutations are 62 and 65.

11. An antibody composition according to Claim 6, wherein said human light k chain is a V_κII chain.

12. An antibody composition according to

Claim 6, wherein said human light k chain comprises the sequence VSGR in a framework region.

13. A method of diagnosing susceptibility to erosive arthritis comprising:

determining the presence of a human light k chain having a total of at least two mutations at conserved sites in the framework regions of said human light k chain, as indicative of said susceptibility.

14. A method according to Claim 13, wherein said human light k chain is a V_κII chain.

15. A method according to Claim 13, wherein antibody specific for said mutations is used for said determining.

16. A method of inhibiting an erosive arthritis forming complex in blood, said method comprising:

adding to said blood a complex-inhibiting amount of a peptide composition comprising a sequence of at least about eight amino acids of a human light k chain and having a total of at least two mutations in conserved sites of the framework regions of said human light k chain, wherein said sequence includes said mutations.

17. A pharmacologically acceptable composition comprising a purified peptide composition comprising a sequence of at least about 8 amino acids comprising at least one framework region of a human light k chain and having a total of at least two mutations in conserved sites of the framework regions of said human light k chain, said sequence including said two mutations.

18. A composition according to Claim 17, wherein said peptide comprises at least eight amino acids coming within the sequence GVPDRVSGRGSGTDF.