Molecules mimicking an autoantibody idiotype and compositions containing same

Abstract

Specific peptides have been discovered that mimic an idiotype of an autoantibody. Such peptides may be formed into polymers. The peptides may be used in pharmaceutical compositions for the treatment of an autoimmune disease together with a pharmaceutically acceptable excipient.

Claims

1. A molecule which mimics an idiotype of an autoantibody, wherein the molecule consists of a peptide selected from the group consisting of: amino acid sequence KHETTET (SEQ ID NO:16), amino acid sequence AGLKNSQ (SEQ ID NO:18), amino acid sequence ASTIRAG (SEQ ID NO:19), amino acid sequence SQLGMVS (SEQ ID NO:23), amino acid sequence SASFTMI (SEQ ID NO: 30), amino acid sequence VGALPLE (SEQ ID NO:41), amino acid sequence TQEPSPL (SEQ ID NO:42), amino acid sequence PPNHSHL (SEQ ID NO:17), amino acid sequence PLSSSLP (SEQ ID NO:20), amino acid sequence SLQRHPW (SEQ ID NO:36), amino acid sequence FEVASLP (SEQ ID NO:37), amino acid sequence GDSLRST (SEQ ID NO:38), amino acid sequence NSRDSSE (SEQ ID NO: 39), amino acid sequence PLPDWRV (SEQ ID NO:40), amino acid sequence DWLYSRS (SEQ ID NO:43), and amino acid sequence LRVSTTE (SEQ ID NO:44), or a dimer, oligomer or polymer of said peptide.

2. A molecule according to claim 1 in the form of a dimer, oligomer or polymer of said peptide.

3. A composition comprising an effective amount of a molecule according to claim 1, together with a pharmaceutically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to understand the invention and to see how it may be carried out in practice, preferred embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

(2) FIG. 1 is a flow chart showing one embodiment of a method according to the invention for identifying molecules which mimic idiotypes of an autoantibody;

(3) FIG. 2 is a flow chart showing one embodiment of a method according to the invention for preparation of anti-Id to autoantibodies;

(4) FIG. 3 is a graph illustrating specific binding of immunoglobulin peak elution to biotinylated peptides 706, 707 and R706 from a peptide column bearing the 706 peptide sequence; and

(5) FIG. 4 is a graph illustrating specific binding of immunoglobulin peak elution to biotinylated peptides 706, 707 and R706 from a peptide column bearing the 707 peptide sequence.

DETAILED DESCRIPTION OF AN EMBODIMENT

Example I

(6) One embodiment of a method for identifying molecules which mimic idiotypes of an autoimmune-a-a is illustrated in FIG. 1.

(7) The first step of the method, indicated in FIG. 1 by reference numeral 2, involves purifying autoantibodies from sera of one or more patients afflicted with the autoimmune disease. For example, anti-double-stranded DNA (anti-dsDNA) antibodies may be purified from several tens of patients suffering from SLE (lupus patients). In the next step 4, the autoantibodies are bound to a solid phase to form an affinity matrix. Following the above example, the anti-dsDNA antibodies are bound to a CNBr-activated Sepharose column.

(8) The following step 6 comprises contacting pooled plasma comprising immunoglobulins with the affinity matrix followed by the step 8 of removal of unbound plasma components. In the example, IVIG is loaded on the affinity column which is subsequently washed to remove unbound immunoglobulins. Only the immunoglobulins which bind the anti-dsDNA antibodies remain bound to the column.

(9) In the next step 10, the bound anti-Idiotype antibodies (=anti-Id) are eluted from the affinity matrix. In the example, these would be anti-anti-dsDNA anti-Idiotypes. The efficacy of the anti-Id may be confirmed 12, for example by in vitro tests and in vivo using a lupus experimental model.

(10) In the following step 14, a molecular library is provided comprising a plurality of molecule members, and the eluted anti-Id is brought into contact with the molecular library. Those molecules which are bound by the anti-Id are isolated and identified 16. These molecules mimic an idiotype of the autoantibodies. In the above example, the anti-dsDNA antibodies are introduced to a C7C-peptide phage display library, and the peptides bound by the antibodies are isolated and identified. These peptides mimic idiotypes of the anti-dsDNA antibodies. Mimicking peptides may then be synthesized 18.

Example II

(11) One embodiment of a method for preparation of anti-Id to autoantibodies is illustrated in FIG. 2.

(12) In the first step 22, an affinity matrix is prepared by binding the mimicking molecules obtained by the method illustrated in FIG. 1 to a solid phase to form a second affinity matrix. The mimicking molecules may be those isolated from the molecular library, or other molecules synthesized on the basis of the isolated molecules. Following the example given with respect to FIG. 1, these would be peptides which mimic idiotypes of the anti-dsDNA antibodies.

(13) In the next step 24, pooled plasma comprising immunoglobulins is contacted with this second affinity matrix followed by removal 26 of unbound plasma components. In the example, IVIG is loaded on the column which is subsequently washed to remove unbound immunoglobulins. In the following step 28, the anti-Id is eluted from the affinity matrix. The efficacy of the anti-Id may be confirmed 30, for example by in vitro tests and in vivo using a lupus experimental model. These second anti-Id are safe for use in the treatment of patients, not having been brought into contact with proteins derived from sera of autoimmune disease patients. For example, they may be used to treat lupus patients.

Example III

Identification of Molecules, which Mimic Idiotypes of an Autoantibody

(14) 1. Materials and Methods

(15) Anti-anti-dsDNA (anti-Id) isolated from IVIG as described above in Example I was employed to detect specific peptides presented by M13 phages. A commercial Ph.D.7? phage display library (cat. number E8100S, New England Biolabs Inc) was used according to the manufacturer's procedure, slightly modified by the inventors, as follows:

(16) Anti-Id in 50 mM NaHCO3 (Ph 8.5) was biotinylated by adding Sulfo-NHS-LC-Biotin (Pierce #21335), incubated 2 hrs on ice and dialyzed against 2% maltose. The efficacy of biotinylation was assayed by ELISA on neutravidine coated plates.

(17) Biotinylated (1 ?g) was introduced to the peptide phage display library (10 ?g-2?1011 pfu) overnight at 4? C. The mixture was subjected to streptavidin coated petri-dish (3 cm, Nunc) blocked with 3% BSA. Following 20 minutes of incubation, the non-specific low affinity binding between streptavidin and the phages was prevented by adding biotin 0.1 mM for 5 min. Following extensive washings with TBS/Tween, the bound phages were eluted with Glycine-HCl 0.2M pH 2.3 and immediately neutralized with Tris pH 9.

(18) The eluted phages were introduced to an ER2738 E. Coli host strain at a mid-log phase (OD600?0.5) and incubated at room temperature for 5 min to allow the phages to enter the bacteria; then incubated for 4.5 hrs for replication and the bacteria were sedimented by centrifugation. The phages were isolated from the supernatant by incubation with PEG-8000 1:5 at 4? C. The amplified phages were incubated again with the original biotinylated anti-Id for the second round. Finally, 5 rounds of amplification procedure was performed using the same procedure. Between each round, the phages again undergo panning on Fe coated plates to maximally delete the Fc binding phages.

(19) The eluted phages from the last round, at 10-fold serial dilutions, were mixed with top-agarose and plated on LB/IPTG/XGAL plates. The blue plaques were collected, and each plaque was grown separately for 4.5 hrs in 10 ml LB for phage collection and centrifuged. The phages were isolated from the cultures by incubating the supernatant with PEG-8000 1:5.

(20) The 847 colonies were screened for recognition by anti-Id by ELISA. Plates (96 wells) were coated with goat-anti-M13, blocked with 3% BSA and hemocyanin, and incubated with the phages. The binding was probed with biotinylated anti-Id or biotinylated individual IgG (from one person as negative control) followed by exposure to streptavidin-alkaline phosphatase and an appropriate substrate. Finally 33 clones were found to be significantly positive for anti dsDNA anti-Id and were not recognized by IgG affinity-purified from one healthy donor.

(21) The positive clones were introduced to ER2738 E. Coli host strain for amplifying the phages 5.5 hrs for DNA preparation, using QIAprep M13 (QIAGENE cat. no 27704) according to the manufacturer's procedure. Briefly, the supernatant was separated into 1.4 ml for small DNA preps and the rest for introduction to ER2537(1:100 diluted bacteria ER2537 from the overnight culture) in LB. Precipitation solution was added into the 1.4 ml supernatant tubes, vortexed and left for 7 min at RT. The mix was loaded onto minicolumn/2 ml tubes, in 2 rounds of 700 ul/minicolumn. The tubes were centrifuged for 15 sec at 8,000 rpm at RT, after the first and second loading. Lysis buffer was added to the columns (700 ul/column), incubated 1 min at RT and centrifuged. Washing buffer was added 700 ul/column and the columns were centrifuged 15 see, 8,000 rpm at RT, the liquid was discharged and the columns were centrifuged again. The columns were attached to sterile tubes. Prewarmed (50? C.) elution buffer was added onto the columns (50 ul/column). The columns were incubated 10 min at RT, and centrifuged 2 min at 14,000 rpm at RT.

(22) The DNA preparations were sequenced by mbc Company, Nes-Ziona, Israel.

(23) The peptides were synthesized as cyclic peptides at The Weizmann Institute, Rehovot, Israel. The peptide sequences are listed in Table 1:

(24) TABLE-US-00004 TABLE1 Listofsyntheticpeptidessynthesized: (a) KHETTET (SEQIDNO:16) (b) PPNHSHL (SEQIDNO:17) (c) AGLKNQ (SEQIDNO:18) (d) ASTIRAG (SEQIDNO:19) (e) PLSSSLP (SEQIDNO:20) (f) FLTLTEL (SEQIDNO:21) (g) VRVLLRS (SEQIDNO:22) (h) SQLGMS (SEQIDNO:23) (i) SEHTTVH (SEQIDNO:24) (j) TQPPELP (SEQIDNO:25) (k) LSQPERW (SEQIDNO:26) (l) PPPDLHA (SEQIDNO:27) (m) EESSYLV (SEQIDNO:28) (n) SNEQMY (SEQIDNO:29) (o) SASFTMI (SEQIDNO:30) (p) GTTQWL (SEQIDNO:31) (q) HSLTQPA (SEQIDNO:32) (r) QLALHST (SEQIDNO:33) (s) YGTPSSE (SEQIDNO:34) (t) KMHSVS (SEQIDNO:35) (u) SLQRHW (SEQIDNO:36) (v) FEVASLP (SEQIDNO:37) (w) GDSLRST (SEQIDNO:38) (x) NSRDSSE (SEQIDNO:39) (y) PLPDWV (SEQIDNO:40) (z) VGALPLE (SEQIDNO:41) (aa) TQEPSPL (SEQIDNO:42) (bb) DWLYSRS (SEQIDNO:43) (cc) LRVSTTE (SEQIDNO:44) (dd) PPQKHLL (SEQIDNO:45) (ee) EMTATVS (SEQIDNO:46) (ff) VRLEGLP (SEQIDNO:47) (gg) KYKRKP (SEQIDNO:48)
2. Direct Binding of Anti-Id to the Synthetic Peptides:

(25) The binding of anti-Id to the peptides was tested by ELISA.

(26) ELISA plates were coated by 1 Oug/ml of peptides in PBS overnight at 4? C. The plates were blocked with 3% BSA for 1 hr at 37? C. Anti-Id was added at different concentrations. IgG purified from one donor was used as negative control.

(27) The binding of the immunoglobulin was probed with goat anti-human IgG conjugated to alkaline phosphatase and the appropriate substrate. Between each step, extensive washings were performed with 0.05% PBS-Tween.

(28) The following peptides were used:

(29) TABLE-US-00005 (SEQIDNO:16) (1) KHETTET (SEQIDNO:17) (2) PPNHSHL (SEQIDNO:18) (3) AGLKNSQ (SEQIDNO:19) (4) ASTIRAG (SEQIDNO:22) (5) VRVLLRS (SEQIDNO:25) (6) TQPPELP (SEQIDNO:26) (7) LSQPERW (SEQIDNO:27) (8) PPPDLHA (SEQIDNO:31) (9) GTTQWVL (SEQIDNO:34) (10) YGTPSSE (SEQIDNO:36) (11) SLQRHPW (SEQIDNO:40) (12) PLPDWRV (SEQIDNO:43) (13) DWLYSRS (SEQIDNO:45) (14) PPQKHLL (SEQIDNO:48) (15) KYKRKYP

(30) The binding results (OD at 405 nm) are presented in Table 2.

(31) TABLE-US-00006 TABLE 2 control anti-Id IgG ?g/ml 10 5 2.5 1 5 1 Pep 1 1.815 1.329 0.269 0.046 0.016 0.013 Pep 2 2.063 1.296 0.256 0.041 0.009 0.009 Pep 3 1.444 0.804 0.155 0.042 0.011 0.011 Pep 4 1.648 0.926 0.184 0.041 0.011 0.013 Pep 5 2.036 1.356 0.284 0.039 0.008 0.009 Pep 6 2.051 1.182 0.228 0.049 0.009 0.007 Pep 7 1.457 0.316 0.093 0.045 0.048 0.045 Pep 8 1.319 0.258 0.074 0.05 0.046 0.043 Pep 9 1.125 0.235 0.067 0.043 0.044 0.045 Pep 10 2.539 0.695 0.14 0.048 0.048 0.06 Pep 11 1.798 0.487 0.148 0.066 0.068 0.068 Pep 12 1.356 0.273 0.072 0.042 0.045 0.043 Pep 13 0.748 0.387 0.099 0.112 0.078 0.069 Pep 14 0.987 0.611 0.234 0.113 0.083 0.079 Pep 15 0.287 0.117 0.067 0.049 0.068 0.037
3. Direct Binding Synthetic Peptides:

(32) The same protocol was used as above, but the peptides used in this study were taken from the literature except for peptide number 1 which is the first peptide in the previous experiment.

(33) The peptides used were:

(34) (1) KHETTET (SEQ ID NO:16)

(35) (2) TGYYMQWVKQSPEKSLEWIG (SEQ ID NO:11)

(36) (3) YYCARFLWEPYAMDYWGQGS (SEQ ID NO:12)

(37) (4) VAYISRGGVSTYYSDTVKGRFTRQKYNKRA (SEQ ID NO:2)

(38) (5) TGYYMQWVKQSPEKSLEWIG (SEQ ID NO:11)

(39) (6) YYCARFLWEPYAMDYWGQGS (SEQ ID NO:12)

(40) (7) TEKLRLRYFDYYG (SEQ ID NO:3)

(41) (8) LVKPGGSLKLSCAASGFT (SEQ ID NO:4)

(42) (9) MNWVKQSHGKSL (SEQ ID NO:7)

(43) (10) FYNQKFKGKATL (SEQ ID NO:8)

(44) (11) YYYGAGSYYKRGYFD (SEQ ID NO:10)

(45) TABLE-US-00007 TABLE 3 Control anti-Id IgG ?g/ml 10 5 2.5 1 5 1 Pep 1 1.21 0.216 0.074 0.041 0.043 0.044 Pep 2 1.701 0.357 0.105 0.047 0.041 0.04 Pep 3 0.965 0.164 0.065 0.041 0.043 0.043 Pep 4 0.518 0.092 0.0475 0.044 0.039 0.04 Pep 5 0.732 0.128 0.054 0.046 0.048 0.043 Pep 6 1.634 0.342 0.082 0.044 0.043 0.046 Pep 7 0.38 0.105 0.055 0.056 0.052 0.043 Pep 8 1.413 0.211 0.088 0.041 0.041 0.044 Pep 9 0.037 0.095 0.045 0.041 0.045 0.044 Pep 10 0.231 0.059 0.041 0.036 0.041 0.042 Pep 11 0.547 0.098 0.047 0.043 0.041 0.043
4. Percent Inhibition with Mix Cyclic Peptides

(46) The mix of 15 cyclic peptides given in Section 2 above (Direct binding of anti-Id to the synthetic peptides) was used to inhibit the binding of anti-Id to anti-dsDNA antibodies affinity purified from each of 7 Lupus patients. The mixture was made to increase the recognition probability of the anti-dsDNA Id(s) of the Lupus patient.

(47) ELISA plates were coated with anti-Fc 2 ug/ml in NaHCO3 0.05M pH 8.5 over night at 4? C. In this way the F(ab) portion of the immunoglobulin molecule of anti-dsDNA will present the idiotype more efficiently.

(48) The plates were blocked with 3% BSA for 1 hr at 37? C., and each human anti-dsDNA antibody solution was added at 10 ug/ml in PBS, and incubated overnight at 4? C. In separate tubes peptide mix 1 was added at different concentrations to biotinylated anti-dsDNA anti-Id from IVIG (at a concentration of 50% binding to anti-dsDNA) for overnight incubation at 4? C. The day after, the mixture of anti-Id and peptide mix was added to the anti-dsDNA coated plates for 4 hrs. The binding of unbound anti-Id which was not recognized by the peptide mix was probed with streptavidine conjugated with alkaline phosphatase and an appropriate substrate.

(49) The results presented in Table 4 show percentages of inhibition of IVIG specific fraction of anti-dsDNA anti-Id binding to anti-dsDNA from a particular Lupus patient by the peptide mixture.

(50) TABLE-US-00008 TABLE 4 Pat ?g/ml Pat #1 Pat #2 Pat #3 Pat #4 Pat #5 Pat #6 #7 2000 95.2 93.4 85.7 57.6 94.0 94.7 60.5 1000 92.2 91 74.3 12.8 90.1 92 34.6 500 87.4 94 49.1 5.3 85.4 81 12.6 250 45.3 69.1 23.5 2.8 51.3 56.4 5.4 125 21.2 32.4 11.7 1.1 27.1 21.7 1.9 620 9.3 12.9 2.6 0.6 11.5 11.7 0.6 31 4.3 5.3 1.1 0.9 4.3 4 0.2 15 3.8 2.1 0.7 0.3 2.1 0.1 0.4 7.5 4.1 1.9 0.4 0.5 1.6 3.3 0.5 3.25 2.3 0.6 0.2 0.2 1.1 1.1 0.1 1 2.1 0.3 0.3 0.1 0.6 0.7 0.2 0.5 1.9 0.7 0.6 0.1 0.5 0.6 0.1 0.25 0.6 0.1 0.1 0.1 0.2 0.5 0.1 0.1 0.5 0.1 0.5 0.1 0.1 0.1 0.4

Example IV

Using IVIG and/or Pooled Plasma as a Template for the Isolation of Idiotypic Mimicries of Autoimmune Disease

(51) 1. Introduction:

(52) The present example provides an experimental demonstration of direct selection in the conventional way which resulted in the identification of the common SLE idiotype, Id 16/6. It has been proven that one of the peptides based on the 16.6 CDRs, CDR3, can cause SLE symptoms in mice, whereas CDR1's predominate function is to ameliorate the disease in experimental SLE mice (1).

(53) Two peptides based on the sequences of the complementary-determining regions (CDR) of the pathogenic murine monoclonal anti-DNA Ab (5G12) that bears the 16/6 Id were synthesized. pCDR1 (CDR1-TGYYMQWVKQSPEKSLEWIG) and pCDR3 (CDR3-YYCARFLWEPYAMDYWGQGS) (the CDRs are underlined) were shown to be immunodominant T-cell epitopes in BALB/c and SJL mouse strains, respectively, and induced a mild SLE-like disease in responder mice (Kent, S. B. H., Hood, L. E., Beilan, H., Meister, S. & Geiser, T. (1984) in High Yield Chemical Synthesis of Biologically Active Peptides on an Automated Peptide Synthesizer of Novel Design, ed. Ragnarsson, U. (Almqvist & Wiksell, Stockholm), pp. 185-188). Further, the CDR-based peptides inhibited the priming of lymph-node cells (LNC) of mice immunized with the same peptides or with the monoclonal anti-DNA 16/6Id+Abs of either mouse or human origin. The CDR1-based peptide was also shown to prevent auto-Ab production in BALB/c neonatal mice that were immunized later with either pCDR1 or the pathogenic auto-Ab (Kent, op.cit.).

(54) 2. Materials and Method

(55) 2.1 Synthetic Peptides.

(56) The 16.6 monoclonal antibody CDR1-based peptide TGYYMQWVKQSPEKSLEWIG (pCDR1), designated 706, and the CDR3-based peptide YYCARFLWEPYAMDYWGQGS (pCDR3), designated 707, were prepared with an automated synthesizer (Applied Biosystem model 430A) using the company's protocols for t-butyloxycarbonyl (BOC) strategy (Kent, op.cit., Schnolzer, M., Alewood, P. F. & Kent, S. B. H. (1992) Int. J. Pept. Protein Res. 40, 180-193). The reverse order of CDR1 designated R706 GIWELSKEPSQKVWQMYYGT was used as a control. In another similar synthesis, the resulting peptides were labeled by biotin at their N-terminal, later to be used in the ELISA testing.

(57) 2.2 Affinity Chromatography: Preparation of the Peptide Column

(58) 2.2.1 Coupling of Peptide 707 by Reductive Amination

(59) 2.2.1.1 Activation Protocol

(60) a. Creation of Periodate-Oxidizable Matrix

(61) 10 ml of Toyopearl MH65F (Tosohass, Japan) were washed with 300 ml water on a sintered glass filter (porosity G3) followed by 5 successive (total 80 ml) washes with 1M NaOH. The resin was taken out of the sintered glass filter and suspended with 10 ml of 1M NaOH, 1 ml of glycidol (Sigma) and 0.01 g of Sodium Borohydrate (NaBH.sub.4). The reaction mixture was incubated at room temperature overnight with gentle rolling. In the morning, the resin was washed extensively with 200 ml of each of water, 1M NaCl and again with water. The glycidol-modified resin was now ready for periodate oxidation.

(62) b. Direct Periodate Oxidation of the Matrix

(63) The following protocol was designed to prepare periodate oxidation resin: 10 ml wet gel containing vicinal hydroxyl groups were resuspended in 10 ml 0.2M NaIO4 (4.28 g of sodium meta-periodate in 100 ml of water) and mixed well by gentle rolling. The reaction was continued for 90 min. at room temperature. The formyl resin was washed by 300 ml of water to stop the oxidation. The aldehydes created by this procedure are stable enough to allow the resin to be stored for long periods without a decrease in coupling potential.

(64) 2.2.1.2 Ligand Coupling Protocol

(65) 1 ml of phosphate buffer, pH 7.0 containing NaCNBH.sub.4 and the specific peptide was added to 2 ml of periodate-oxidized matrix. The concentration of the peptides in the phosphate buffer was 10 mg/ml (about 3.3 ?moles/ml). Therefore the ratio was 1.1 ?mole of 707 to ml resin. The reaction continued with stirring overnight at room temp. The coupled resin was washed extensively with water, 1M NaCl and again with water to remove unreacted ligand and sodium cyanoborohydride.

(66) The resin was stored in 20 Mm phosphate buffer pH 7.

(67) 2.2.2 Coupling of Peptide 706 to CNBr-Activated Matrix

(68) 3 g of freeze dried CNBr activated Sepharose 4 fast flow were suspended in 20 ml of 1 mM HCl (ice cold). The resin was washed intensely for 15 min. on a sintered glass filter (porosity G3) using 200 ml of acid. After the final wash, 2 ml of the washed resin were transferred to coupling solution, in which the peptide to be coupled had been dissolved. The coupling solution contained 900 ?l of 0.1-M sodium carbonate (NaHCO.sub.3), pH 8.5 and 1000 of 100 mg/ml of peptide 706. The ratio of peptide to resin was again 1.1 mole per ml of resin. The mixture was rolled overnight at 4? C. After coupling, the vial with the resin was left at a vertical position for sedimentation of the resin. The supernatant was discarded and 15 ml of 0.2M glycine, pH 8.0 were added for blocking of the remaining active groups on the resin. The blocking was performed at room temperature for 4.5 hours, while rolling.

(69) After blocking, the resin was washed with 10 column volumes of 0.1 M sodium carbonate (NaHCO.sub.3), pH 8.5 following by washing with 3 cycles of alternating pH. Each cycle consisted of a 15 ml wash of 0.1 M acetate buffer pH 4 containing 0.5 M NaCl followed by a wash with 0.1 M Tris HCl buffer pH 8 containing 0.5M NaCl.

(70) The resin was stored in 0.05 M Tris pH 7.4

(71) 2.3 Affinity Chromatography: Purification of Anti-Idiotypes from an Intravenous Immunoglobulin Solution Using the Peptide Column.

(72) The peptide columns were washed with at least 10 volumes of loading buffer (20 mM PB) until the absorbance measurement at 280 nm was stable (OD about 0.005).

(73) 100 ml of diluted IVIG (in process sample G-12 of the OmriGam, (Omrix Biopharmaceuticals Ltd, Israel) production process, high purified double inactivated IVIG) were loaded on top of a 2 ml peptide resin. The concentration of the starting material was 50 mg/ml. In order to reach this concentration, the IVIG was diluted with phosphate buffer, pH 7 to reach the desired molarity. The loading proceeded overnight at 4? C. at a flow rate of 100 ?l/min, approximately. The loading was performed twice. The column was then washed with loading buffer until the absorbance at 280 reached baseline. The elution was done with 0.1M glycine, HCl pH 2.7 into 1M Tris base buffer pH 9 to neutralize the eluate. Each elution consisted of a peak, usually of about 4-5 ml from a 1 ml column. Protein in the elution and the loading was measured by the Bradford method (Bradford (1976) Anal. Biochem. 72:248).

(74) 2.4 Efficacy of the Affinity Chromatography Step: ELISA

(75) To measure the binding efficacy of peptide column eluates (706 and 707) the following ELISA was performed:

(76) Microtiter plates (Costar, USA) were coated overnight at 2-8? C. with 10 ?g/ml NeutrAvidin (Pierce, USA) suspended in 0.1M, pH 8.8, of carbonate-bicarbonate buffer (Sigma USA). The coating solution was removed, washed three times with washing buffer (1?PBS) and 2000 of blocking solution (freshly prepared 1% I Block, Tropix, USA) per well was added and incubated for 1 hour at 37? C. Coated and blocked plates can be stored at ?20? C. for a month before use.

(77) Tested samples diluted in the blocking solution were mixed in a separate tube with 1 ?g/ml (final concentration) of the appropriate biotinylated peptide and the mixture was incubated for 1 hour at 37? C. 100 ?l from each reaction tube was then transferred to the blocked microtiter plate and incubated at 37? C. for ? hour. A 100 ?l of 1:5000 Alkaline Phosphatase conjugate goat anti human IgG (Heavy and light chain from Jackson, USA) diluted in the blocking buffer was applied into the microtiter well and incubated for 1 hour. The enzymatic activity of alkaline phosphatase is then revealed by an overnight incubation at room temperature with p-nitrophenyl phosphate substrate (Sigma USA). The measurement was done photometrically at 405 nm.

(78) 3. Results and Discussion.

(79) 200 ml of IVIG were loaded on each of the 2 ml peptide columns: one consisted of peptide 706 bind to a CNBr activated Sepharose matrix and the other a 707 peptide bind to the Polymeric matrix by reductive amination (see material and methods).

(80) The protein concentrations and the binding of biotinlyted peptides were tested, and the results are provided in table 5 and FIGS. 3 and 4.

(81) TABLE-US-00009 TABLE 5 Protein yields of 200 ml of intravenous immunoglobulin (50 mg/ml) loaded on 2 ml peptide columns. Total Protein protein in Protein concentration Volume of the the elution recovery in the elution elution peak peak from Column used (mg/ml) (ml) (mg) load (%) CNBr-706 0.60 3 1.80 0.018 Reductive 1.58 3 4.74 0.047 amination 707

(82) The elution of the bound peak resulted in different recoveries: around 0.018% for column peptide 706 versus 0.047 for column peptide 707. Thus, it can be concluded that peptide column 706 has the capacity of binding one out of 5555 molecules whereas peptide column 707 has the capacity of binding one out of 2237 immunoglobulin molecules found in the pooled IVIG. It therefore could be assumed that the 707 epitope is more abundant and can be found at higher frequency in the IVIg.

(83) The specificity of the elution peak was assessed by the specific binding to the peptide column elution peaks to three biotinylated peptides (706, 707 and R706) and the results are summarized in FIGS. 3 and 4.

(84) It can be noted that immunoglobulin eluted from a peptide column bearing the peptide 706 reacted specifically in a linear fashion only with the biotinylated peptide of 706 and not with peptide 707 or even with a peptide bearing the same sequence in a reverse order (FIG. 3). On the other hand the elution peak from a peptide column bound with peptide 707 reacted in a non-specific manner (FIG. 4). The resulting immunoglobulin reacted with biotinylated peptide 706 and even more interesting the immunoglobulin reacted even more strongly with the reversed sequence of 706. Indicating that the 707-immunoglobulin preparations may react non-specifically with peptide sequences of 707 and 706 and probably with numerous other sequences.

(85) The above results indicated that not all the CDR sequences found on the Id16.6 are specific and at least some of them react in a non specific manner with IVIg.

(86) These results support the notion that the use of monoclonal derived from SLE patients may result in non-specific selection of immunoglobulin.