METHOD FOR TREATING IFNALPHA RELATED CONDITIONS

Abstract

An immunogenic product including IFN coupled to a carrier protein molecule is capable to induce in vivo anti-IFN antibodies and is useful in treating IFN related conditions.

Claims

1-3. (canceled)

4. A method for treating an IFN related condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an immunogenic product comprising IFN coupled to a carrier protein molecule, wherein said therapeutically effective amount is more than 30 mcg of immunogenic product per administration.

5. The method according to claim 4, wherein the administration of the therapeutically effective amount of the immunogenic product prevents the occurrence of symptoms of a disease linked to an over-production of IFN.

6. The method according to claim 4, wherein the administration of the therapeutically effective amount of the immunogenic product prevents the flare of a disease linked to an over-production of IFN.

7. The method according to claim 4, wherein the IFN related condition is selected from the group comprising systemic lupus erythematosus, rheumatoid arthritis, scleroderma, Sjgren syndrome, vasculitis, HIV, type I diabetes, autoimmune thyroiditis and myositis.

8. The method according to claim 4, wherein the therapeutically effective amount of the immunogenic product is from 35 mcg to 1000 mcg of immunogenic product per administration.

9. The method according to claim 4, wherein the immunogenic product is administrated to the subject at least twice in a month.

10. The method according to claim 9, wherein the immunogenic product is further administrated to the subject at least once every three months.

11. The method according to claim 9, wherein the immunogenic product is further administrated to the subject when, in a serum sample obtained from the subject, the amount of anti-IFN antibodies is undetectable.

12. The method according to claim 4, wherein the immunogenic product is strongly inactivated, which means that the product shows less than 5% of antiviral activity in the conditions of TEST B.

13. The method according to claim 4, wherein the immunogenic product is capable of neutralizing the antiviral activity of IFN in the conditions of TEST C.

14. The method according to claim 4, wherein the immunogenic product comprises at least one subtype of IFN.

15. The method according to claim 4, wherein the immunogenic product comprises the subtype IFN 2b of IFN and wherein the carrier protein molecule is KLH.

16. The method according to claim 4, wherein the immunogenic product is a vaccine, preferably in the form of an emulsion.

17. A unit dosage form comprising more than 30 mcg of an immunogenic product comprising IFN coupled to a carrier protein molecule.

18. A medical device comprising more than 30 mcg of an immunogenic product comprising IFN coupled to a carrier protein molecule.

19. A kit comprising at least one vial containing more than 30 mcg of an immunogenic product comprising IFN coupled to a carrier protein molecule, at least one vial containing adjuvant, and means for contacting said immunogenic product to the adjuvant, and for emulsifying the mixture with the adjuvant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0228] FIG. 1: Percentage of immunized patient serum samples showing IFN neutralizing activity during interim report.

[0229] FIG. 2: Differential evolution of IFN-induced genes in treatedversus placebo patients. Out of 11 patients displaying increased levels of IFN-induced gene expression at baseline, 8 were treated with the immunogenic product and 3 received placebo injections. The levels of 250 IFN-induced genes showing the highest levels of over-expression in SLE patients were evaluated using high-density microarrays. The results are depicted as the mean log 2(level of expression at V1)log 2(level of expression at V6). Thep value was calculated using a Student's t-test.

[0230] FIG. 3: Titers of IFN-binding antibodies in treated patients with positive or negative IFN-signature at baseline versus placebo receiving patients. Stars indicate p values<0.05.

[0231] FIG. 4: Differential evolution of IFN-induced genes in treated patients with positive or negative IFN-signature at baseline versus placebo patients, between V10 and V0 or between V11 and V0. The results are depicted as the mean Delta Log (Gene Expression). Stars indicate p values<0.05.

[0232] FIG. 5: Evolution of serum C3 values in treated patients with positive IFN-signature at baseline and in placebo receiving patients. Stars indicate p values<0.04.

EXAMPLES

Example 1

Preparation of the Immunogenic Product

[0233] Keyhole Limpet Hemocyanin (KLH) was extracted from the lymph of the marine gastropod mollusk Megathura crenulata and then purified under GMP condition. Results from stability assays performed in storage conditions at a temperature of 2-8 C. showed that the shelf life of the purified KLH is of 36 months at 2-8 C.

[0234] Recombinant human IFN 2b was produced in E. coli under GMP conditions.

[0235] Batches of the product of the invention at 350 mg IFN scale were produced using the manufacturing process developed below.

[0236] a) Conjugation with Glutaraldehyde

[0237] The filtered KLH is added to the IFN 2b solution (IFN 2b in 70 mM di-sodium hydrogen phosphate pH 7.8) with a IFN:KLH ratio of 20:1, (corresponding to a molar ratio of 20 monomer of IFN for 1 subunit of KLH) based on UV concentration. The conjugation is carried out with glutaraldehyde (added to reach 22.5 mM final concentration in the reaction medium) and borate pH 9 (added to reach 28.5 mM final concentration in the reaction medium), to obtain a pH of 8.5.

[0238] This solution at pH 8.5 is then mixed during 45 min at 232 C.

[0239] b) Quenching with Glycine

[0240] The reaction is quenched with Glycine 0.1 M during 30 min

[0241] c) First Tangential Flow Filtration (TFF 1)

[0242] The first TFF is performed with a Pall Minim II TFF system and a polyethersulfone membrane of 0.02 m.sup.2 with a molecular weight cut off of 10 kDa sanitized with 0.5 M NaOH and equilibrated with the working buffer (70 mM di-sodium hydrogen phosphate pH 7.8).

[0243] The quenched solution is then clarified by 0.22 m-filtration. The intermediate is diluted twice in the working buffer and then diafiltered by tangential flow filtration (TFF) and 12 volumes of working buffer. The retentate is harvested and is stored for less than 20 hours.

[0244] d) Inactivation with Formaldehyde

[0245] Formaldehyde is added to the retentate to reach a final concentration of 66.6 mM using a peristaltic pump. The inactivation reaction is performed during 40 hours in an incubator set to 372 C. with a daily mixing of the solution with a magnetic stirrer.

[0246] e) Quenching with Glycine

[0247] The reaction is then quenched with 0.1 M of Glycine during 30 min

[0248] f) Second Tangential Filtration (TFF 2)

[0249] The second TFF is performed with a Pall Minim II TFF system and a polyethersulfone membrane of 0.02 m.sup.2 with a molecular weight cut off of 100 kDa sanitized with 0.5 M NaOH and equilibrated with the formulation buffer (70 mM di-sodium hydrogen phosphate pH 7.8).

[0250] The quenched solution is clarified by 0.2 m filtration. The intermediate is concentrated to have a starting tangential volume of 900 mL and next filtrated by TFF with 12 volumes of formulation buffer (70 mM phosphate buffer) to eliminate the low molecular weight homopolymers of IFN and the non reactive reagents. The retentate is harvested and then diluted to a theoretical concentration of 300 g/mL based on concentration determination by Bradford protein assay and then 0.2m-filtered to obtain the immunogenic product of the invention.

Example 2

Antigenicity of the Product

[0251] A sandwich ELISA anti IFN/KLH was carried out as following. Briefly, a 96 wells plate was coated with the capture antibody: rabbit polyclonal anti-KLH antibody, and blocked with a blocking buffer (such as casein 2% in PBS for example) during 90 min at 37 C. The plate was incubated during 90 min at 37 C. the plate with a dilution series of the immunogenic product from 250 ng/ml to 8 two fold dilutions or with negative controls such as KLH and IFN. A detection antibody such as for example a biotinylated anti-IFN antibody was then added for 90 min. Finally the plate was incubated with streptavidin-HRP during 30 min at 37 C. and the complex developed with an o-phenylenediamine dihydrochloride (OPD) substrate solution during 30 min After stopping the enzymatic reaction, the intensity of the resulting color is determined by spectrophotometric methods at 490 nm.

[0252] This test confirmed that the product comprises IFN that is antigenic, i.e. recognized by anti-IFN antibody and that said IFN is coupled to KLH.

Example 3

Immunogenicity of the Product (TEST A)

[0253] 4 g of total proteins of the product as determined by Bradford protein assay were injected to 7 Balb/c mice of 6-8 weeks at day 0 and day 21.

[0254] At day 31, mice were bleeded and the sera were harvested.

[0255] An anti-IFN ELISA was carried out on preimmune and harvested sera as following: [0256] a 96 wells plate was coated with 100 ng of IFN-2b and incubated overnight at 2 C.-8 C., [0257] a blocking buffer was added during 90 min at 37 C., [0258] the immunogenic product was added at a dilution of 1/2500 up to at least 8 two fold dilutions and the plate was incubated during 90 min at 37 C., [0259] the plate was incubated with an anti-mouse immunoglobulin labeled antibody such as an HRP conjugated antibody during 90 min at 37 C., [0260] the ELISA was developed with an o-phenylenediamine dihydrochloride (OPD) substrate solution. After stopping the enzymatic reaction, the intensity of the resulting color was determined by spectrophotometric methods at 490 nm.

[0261] This test demonstrated that in the 7 mice, immunization with the immunogenic product led to the presence of anti-IFN antibodies titers.

Example 4

Residual Activity of the Product (TEST B)

[0262] This assay was based on the protective effect of IFN on the cytopathic effect (CPE) of Vesicular Stomatitis Virus (VSV) on Madin-Darby Bovine Kidney (MDBK) cells. The immunogenic product and the recombinant IFN 2b used for preparing the immunogenic product (positive control) were diluted at at least 500 ng/ml and at least 1000 U/ml respectively in Basal medium (RPMI supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 1 mM Hepes). 50 l of the immunogenic product and the positive control were plated in a 96 wells plate and diluted in a series of two fold dilutions in the Basal medium. 2 10.sup.4 MDBK cells were added in each well in 50 l of Cell medium (RPMI supplemented with 4% FBS, 2 mM glutamine, 1 mM sodium pyruvate and 1 min Hepes) and the plate was incubated overnight at 37 C., 5% CO.sub.2. The virus was then diluted in Basal medium to at least 10 TCID.sub.50 (Tissue Culture Infection Dose 50: 10 times the dilution to kill 50% of infected cells). The plate was emptied and 100 l of the diluted virus was added. The plate was then incubated overnight at 37 C., 5% CO.sub.2.

[0263] At the end of the culture, 20 l/well of a solution of MTS/PMS (100 l MTS/5 l PMS; Promega G5430) were added to the wells and the plate was incubated for another 4 h at 37 C. 5% CO2. The plate was then read at 490 nm on a spectrophotometer.

[0264] The percentage of antiviral activity of the immunogenic product was calculated and for the two batches of product tested, the antiviral activity was less than 1% of the antiviral activity of IFN.

Example 5

Neutralization Capacity of the Product (TEST C)

[0265] The neutralizing capacity of the product was assessed by evaluating the cell viability in presence of the vesicular stomatitis virus replicating in MDBK cells.

[0266] 4 g of total proteins (as determined by a Bradford protein assay) of the immunogenic product were injected in Balb/c mice of 6-8 weeks at day 0 and day 21. A serum sample was obtained before immunization (pre-immune serum sample) and at day 31 (test serum sample).

[0267] 25 l of pre-immune and test serum samples were plated in a 96-well plate at a dilution of 1/200 up to 8 dilutions from 1/200. The positive control (polyclonal anti-IFN from PBL, Piscataway, N.J., ref. 31100-1) was diluted from 3125 UI/well to 100 UI/well in Basal medium (RPMI supplemented with 2 mM glutamine, 1 mM sodium pyruvate and 1 mM hepes) and 25 l were also plated in the plate.

[0268] 25 U/well (final concentration) in 25 l of basal medium of IFN was added to each well and the plate is incubated for 60 min at room temperature.

[0269] 20000 MDBK cells in Assay medium (RPMI supplemented with 4% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1 mM hepes) were added to each well and the plate was incubated overnight at 37 C., 5% CO.sub.2.

[0270] The virus was diluted to at least 10 TCID.sub.50 (10 times the dilution to kill 50% of infected cells) in virus medium (RPMI supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 1 mM hepes). The plate was emptied and 100 l of virus was added to each well before incubation for 24 h at 37 C., 5% CO.sub.2.

[0271] At the end of the culture, 20 l/well of a solution of MTS/PMS (100 l MTS/5 l PMS; Promega G5430) were added to the wells and the plate was incubated for another 4 h at 37 C. 5% CO2. The plate was then read at 490 nm on a spectrophotometer.

[0272] The NC was calculated for all the 7 test samples: mean NC=253789 IU/ml (SEM=172526), demonstrating that all serum comprised antibodies anti-IFN capable of neutralizing the antiviral activity of IFN.

EXAMPLE 6

Examples of Compositions and Vaccine Comprising the Immunogenic Product

[0273] One illustrative composition comprising the immunogenic product is described in Table 1.

TABLE-US-00001 TABLE 1 Components Quantity Product of the invention 160 g di-sodium phosphate 8.95 mg Disodium dihydrogen phosphate 805 g Total volume 0.4 ml

[0274] One illustrative vaccine comprising the immunogenic product is described in Table 2.

TABLE-US-00002 TABLE 2 Emulsion Components Quantity Product of the invention 160 g di-sodium phosphate 8.95 mg Disodium dihydrogen phosphate 805 g Drakeol 6VR (mineral oil) 0.30 g Montanide 80 (mannide monooleate) 0.04 g Total volume 0.8 ml

Example 7

Clinical Trial

[0275] A clinical trial was carried out using the vaccine composition as described in Table 2.

[0276] Study Design:

[0277] 3 or 4 administrations of the product were performed at day 0, day 7 and day 28 or at day 0, day 7, day 28 and day 84 in adults subjected to SLE.

[0278] The following doses of the product were tested: 30 mcg, 60 mcg, 120 mcg and 240 mcg.

[0279] Study Population:

[0280] 28 male or female patients aged between 18 and 50 years, with mild to moderate SLE (SLEDAI 4-10), active disease despite receiving treatment. A normal control interferon gene signature was established in 48 healthy volunteers. PBMC of 18 out of the 48 healthy volunteers were stimulated in vitro with type I interferons in order to identify an interferon signature on the high-density arrays. A SLE signature was established by comparing the signatures between healthy volunteers and SLE patients at baseline.

[0281] An interim analysis was performed in the patients enrolled in the first three groups, i.e. having received the 30, 60 or 120 mcg doses or placebo.

TABLE-US-00003 TABLE 3 Demographics for enrolled patients (interim analysis) Summary 30 mcg 60 mcg 120 mcg Placebo Total Statistics (N = 3) (N = 6) (N = 6) (N = 5) (N = 20) Age (years) Mean (SD) 36.0 (9.85) 39.3 (3.98) 34.2 (12.12) 38.6 (11.52) 37.1 (9.28) Median 33.0 38.0 32.5 43.0 37.5 Sex, n(%) Male 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Female 3 (100.0) 6 (100.0) 6 (100.0) 5 (100.0) 20 (100.0) Race, n(%) White-Caucasian 3 (100.0) 6 (100.0) 6 (100.0) 5 (100.0) 20 (100.0) SLEDAI-2000 Mean 8.67 (1.15) 7.50 (2.81) 6.00 (2.19) 8.80 (1.09) Median 8.00 8.50 6.00 8.00 Anti-ds DNA ab Mean (SD) 53.93 (58.22) 61.25 (113.46) 140.55 (242.63) 88.70 (113.62) Median 33.10 15.45 23.60 40.90 DURATION OF DISEASE (YEARS) Mean (SD) 10.0 (2.18) 8.9 (8.82) 7.3 (5.99) 5.9 (4.75) 7.9 (6.11) Median 11.0 6.1 6.1 3.6 6.4 CONCOMMITANT 100% 66.7% 83.3% 100% CORTICOSTEROIDS indicates data missing or illegible when filed

TABLE-US-00004 TABLE 4 Demographics for enrolled patients (final analysis) 30 g 60 g 120 g 240 g Placebo Measure N = 3 N = 6 N = 6 N = 6 N = 7 Age (y) Mean SD 36.0 9.8 39.3 4.0 34.2 12.1 34.8 10.8 40.1 10.2 Median 33 38 33 36 43 Range 28-47 35-46 19-50 21-46 20-50 Sex Female n (%) 3 (100) 6 (100) 6 (100) 6 (100) 7 (100) Ethnicity White-Caucasian n (%) 3 (100) 6 (100) 6 (100) 6 (100) 6 (85.7) Asian n (%) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (14.3) Weight (kg) Mean SD 69.7 11.9 67.8 10.0 59.2 7.4 70.0 15.9 57.4 14.8 Median 75 63 59 65 55 Range 56-78 58-81 51-71 54-97 46-90 Height (cm) Mean SD 162.3 6.4 165.0 5.1 164.0 5.5 162.8 8.6 162.7 5.8 Median 165 166 165 163 163 Range 155-167 159-170 156-172 152-172 153-170 Body mass index Mean SD 26.6 6.0 25.0 4.3 22.1 3.5 26.7 7.2 21.7 5.2 (kg/m.sup.2) Median 27 24 21 25 20 Range 21-32 21-32 18-28 20-40 17-33 Disease duration (y) Mean SD 9.9 2.2 8.9 8.8 7.2 6.0 11.8 8.4 6.5 4.0 Range 7-11 1-23 0-18 2-21 1-11 SLEDAI 2000 Mean SD 8.7 1.2 7.5 2.8 6.0 2.2 6.0 1.8 8.4 1.1 index Range 8-10 4-10 4-10 4-8 7-10 Medications at baseline, n (%) Glucocorticoids n (%) 3 (100.0) 4 (66.7) 5 (83.3) 3 (50.0) 6 (85.7) Aminoquinolines n (%) 0 (0.0) 4 (66.7) 3 (50.0) 5 (83.3) 5 (71.4) Methotrexate n (%) 0 (0.0) 1 (16.7) 1 (16.7) 1 (16.7) 1 (14.3) Azathiopine n (%) 0 (0.0) 1 (16.7) 1 (16.7) 1 (16.7) 0 (0.0)

[0282] Results

[0283] Safety and Tolerability of the Vaccine

[0284] Two lupus flares have been reported as related SAEs. The first was in the placebo group. The other occurred after the first injection of IFN-K 240 mcg in a patient who had spontaneously stopped her corticosteroid therapy two days after injection. This abrupt stopping of corticosteroids treatment likely participated to the occurrence of the flare. Regular interim safety analyses were performed by an independent safety board. No clinically significant change in laboratory parameters has been detected (hematology, biochemistry, urine).

[0285] Immunogenicity of the Vaccine

[0286] Anti-IFN antibody titers were measured by ELISA from serum samples obtained from the patients.

[0287] An anti-IFN ELISA was carried out as described here above.

[0288] Results show that anti-IFN antibody titers were detected in all groups treated with the immunogenic product starting on day 28.

[0289] Neutralization Activity of the Vaccine

[0290] The neutralization activity was assessed in vitro using the following method: 50 l of serum samples obtained from the patients sera were plated in a 96-well plate at a dilution of 1/200 up to 8 dilutions from 1/200.

[0291] The positive control (polyclonal anti-IFN from PBL Piscataway, N.J., 31100-1) was diluted from 100 ng/well to 3.125 ng/well and 50 l were added to the plate. Dilutions were carried out in Basal medium (RPMI supplemented with 2 mM glutamine, 1 mM sodium pyruvate and 1 mM hepes).

[0292] 10 U/well (final concentration) of IFN 2b were added to each well and the plate was incubated for 60 min at room temperature.

[0293] 30000 MDBK cells in Assay medium (RPMI supplemented with 4% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1 mM hepes) were added to each well and the plate was incubated overnight at 37 C., 5% CO.sub.2.

[0294] The virus was diluted to at least 10 TCID.sub.50 (10 times the dilution to kill 50% of infected cells) in virus medium (RPMI supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 1 mM hepes). The plate was emptied and 100 l of virus was added to each well before incubation for 24 h at 37 C., 5% CO.sub.2.

[0295] At the end of the culture, 20 l/well of a solution of MTS/PMS (100 l MTS/5 l PMS; Promega G5430) were added to the wells and the plate was incubated for another 4 h at 37 C. 5% CO2. The plate was then read at 490 nm on a spectrophotometer.

[0296] The results of interim report showed that none of the sera from patients treated with 30 mcg of the immunogenic product presents anti-IFN antibodies having a neutralizing capacity at day 168 after immunization, whereas the sera from patients treated with 60 mcg of the immunogenic product present anti-IFN antibodies having a neutralizing capacity at day 168 (FIG. 1).

[0297] Moreover, the results of the final report showed that a neutralizing activity was detected in 50% of subject treated with 60 g or 120 g of the immunogenic product, and in 80% of subjects treated with 240 g of the immunogenic product (Table 5)

TABLE-US-00005 TABLE 5 Dose (mcg) Number of responder (%) NC50 median at peak (Dil-1) 30 0 0 60 50 390 120 50 733 240 80 316

[0298] These results demonstrated that treatment with more than 30 mcg of the immunogenic product is necessary for having an in vivo neutralization of IFN.

Example 8

Transcriptomic Analysis

[0299] PBMC were harvested at several time-points before and after injection of the immunogenic product. For this interim analysis, total RNA was extracted at V1 (day 0) and V6 (day 38 after the first injection) samples, labeled according to standard Affymetrix protocol, and hybridized on GENECHIP HGU133 Plus 2.0 arrays. Statistical analyses were performed on GENESPRING after RMA (Robust Microarray Analysis) normalization of the samples.

[0300] Unsupervized clustering algorithms were performed on the baseline samples, and grouped the patients in two categories: those with (n=11), and those without (n=7) the presence of an Interferon-signature at baseline, i.e. the spontaneous over-expression of genes induced by type I interferon (the IFN-induced genes were identified experimentally, based on microarray analyses of IFN-stimulated control PBMC). Not surprisingly, dsDNA titers were significantly higher in the patients with the signature (mean+/SEM: 131.1.+/50.1 UI/ml), compared to the patients without (mean+/SEM: 44.7+/33.3, p=0.006 by Mann-Whitney test). Measurable anti-IFN antibodies were found in 8 follow-up samples of the 8 patients with an IFN signature at baseline who received the immunogenic product, while this was only the case in 2 out of 6 patients without IFN signature treated with the immunogenic product, and none out of the 4 placebo-treated individuals (p=0.002 by Chi-squared test). Out of the 11 patients with a baseline IFN-signature, 2 received the 30 mcg dose, 1 received the 60 mcg dose, 5 received the 120 mcg dose and 3 were treated with a placebo injection. The changes observed in the expression of the IFN-induced genes between V1 and V6 were significantly different in the patients treated with the immunogenic product, as compared to the patients treated with the placebo (FIG. 2).

[0301] This result suggests that the immunogenic product has an effect on the expression of IFN-induced genes in vivo.

Example 9

IFN Signature and Response to Immunogenic Product-Treatment

[0302] The Interferon-signature at baseline was measured in the 28 patients with mild to moderate SLE of Example 7. The Interferon-signature (IFN-signature) corresponds to a score calculated using 21 IFN-induced genes, and was described in Yao et al, Arthritis & Rheumatism, 2009, 60 (6): 1785-1796. Measurement of the Interferon-signature was done as described in Example 8.

[0303] On the 28 SLE patients of Example 7, 19 showed a positive Interferon-signature and 9 a negative Interferon signature at baseline.

[0304] Interferon Signature and SLE Disease Activity

[0305] dsDNA antibody titers and serum levels of C3 were measured as indices of disease activity in both groups of patients.

[0306] dsDNA Antibody titers were determined using DPC Anti-DNA kit (PIKADD-4) from Diagnostic Products Corporation.

[0307] C3 serum levels were determined using Complement C3 kit (Kit #446450) from Beckman Coulter.

TABLE-US-00006 TABLE 6 Indices of SLE disease activity IFN signature IFN signature positive SLE negative SLE p value dsDNA Ab 147 57 44 93 <0.05 C3 834 55 1199 114 <0.005

[0308] As shown in the Table 6 above, SLE patients with positive Interferon-signature at baseline have biological indices of higher disease activity.

[0309] Interferon-Signature and Response to Treatment with the Immunogenic Product of the Invention

[0310] The effects of the treatment with the immunogenic product of the invention as described in Example 7 were compared in SLE patients with positive and negative IFN-signature at baseline.

[0311] Anti-IFN-Alpha Response

[0312] IFN-binding antibody titers were measured as described in Example 7 at V6 (day 38), V10 (day 112) and V11 (day 168 after immunization).

[0313] The results showed that SLE patients with positive Interferon-signature produce ten folds more IFN-binding antibodies in response to the immunogenic product of the invention than SLE patients with negative Interferon-signature at baseline (FIG. 3).

[0314] IFN-Induced Genes

[0315] The evolution of the expression of IFN-induced genes between V0 and V10 or V11 was measured in treated patients with positive or negative IFN signature at baseline and in patients treated with placebo.

[0316] The results showed that compared to placebo and IFN-signature negative patients, the effects of therapy with the immunogenic product of the invention on IFN-induced genes were strongly and significantly different at V10 and V11 in IFN-signature positive SLE patients (FIG. 4).

[0317] Complement C3

[0318] Serum C3 values were measured in treated patients and in placebo receiving patients at V-1 (30 days before immunization), V0 (day of immunization), V7 (day 56), V10 (day 112) and V11 (day 138 after immunization) as hereinabove described.

[0319] Results showed that there is a significant increase in C3 levels in treatedversus placebo patients. Moreover, there is a significant increase in C3 levels in IFN-signature positive SLE patients treated with the immunogenic product of the invention (FIG. 5).

Example 10

Determination of the Ratio IFN/KLH in the Product of the Invention

[0320] In order to assess the amount of IFN and KLH in the product of the invention, a method of quantification based on UV and fluorescence detection was developed. The products of the invention were manufactured with two fluorescent labels, each specific of IFN or KLH. After analysis by Size Exclusion Chromatography (SEC), quantification of IFN and KLH was determined by integration of the UV signal at 220 nm and fluorescent signal (FLD) specific for IFN label or KLH label. This method allowed calculating the ratio in weight of IFN/KLH.

[0321] a) Raw Materials Labeling:

[0322] Fluorescent tags were coupled on sulfhydryl groups in order to preserve amino groups used during the product manufacturing.

[0323] Labeling was conducted in 70 mM pH7 sodium phosphate buffer at room temperature during 3 h. KLH were labeled with 200 molar equivalent of Atto565-maleimide (18507, Sigma) and IFN with 100 molar equivalent of fluorescein maleimide (46130, Pierce). The labeled proteins (KLH-atto565 and IFN-Fluorescein) were then filtrated on Zeba column (cut off 7 kDa, Thermo Scientific, 89893) conditioned with 70 mM pH 7.8 phosphate buffer in order to eliminate unreacted tags.

[0324] b) Product Manufacturing:

[0325] The labeled raw materials were then used to manufacture labeled products with the same process as in Example 1 with dialysis filtration instead of tangential flow filtration.

[0326] c) KLH and IFN Homopolymers Standards Manufacturing

[0327] For the quantitative analytical method, homopolymers standards were manufactured. Labeled IFN homopolymers standard was manufactured with the same process as in Example 1 but with 70 mM phosphate buffer pH 7.8 instead of KLH and dialysis filtration instead of tangential flow filtration.

[0328] Labeled KLH homopolymers standard was manufactured with the same process as in Example 1 but with 70 mM phosphate buffer pH 7.8 instead of IFN and dialysis filtration instead of tangential flow filtration.

[0329] d) Method Analysis by Size Exclusion Chromatography

[0330] Batches were then analyzed by SEC with UV and specific fluorescent detection. 60 L of sample was injected on columns SEC5 (1000 A) SEC3 (300 A) connected in series (Agilent, 5190-2536, 5190-2511), elution was performed with PBS during 35 min with UV detection at 220 nm and specific fluorescent detection (for IFN-Fluorescein or KLH-Atto565), as described Table 7.

TABLE-US-00007 TABLE 7 Excitation and emission wavelength used for IFN-Fluorescein or KLH-Atto-565 Fluorescent specific wavelength nm detection Excitation Emission IFN.sub.-Fluorescein 490 520 KLH-Atto565 570 600

[0331] UV and fluorescent (FLD) signals were calculated by integrating the area under the chromatogram peaks between 0 and 20 min.

[0332] To validate this method, preliminary experiments were conducted to demonstrate: [0333] Fluorescent signal specificity (no signal overlapping was observed between the two labeled proteins), [0334] For each manufactured batch (product of the invention, labeled homopolymers of KLH and IFN), similar UV profiles by SE-HPLC were obtained, [0335] No quenching of the fluorescent signal due to the manufacturing was observed, [0336] FLD signals were linear and proportional to UV signals.

[0337] Labeled IFN UV contribution in the manufactured labeled kinoid was measured according to the curve Area by FLD.sub.IFN-Fluorescein=f(Area by UV) of labeled IFN homopolymers standard.

[0338] Labeled KLH UV contribution in the manufactured labeled kinoid was measured according to the curve Area by FLD.sub.KLH-Atto565=f(Area by UV) of labeled KLH homopolymers standard.

[0339] As UV area was checked to be a linear function of protein concentration, this method allowed assessing the percentage in weight of labeled IFN in the total manufactured labeled kinoid.

[0340] e) Batches Analysis

[0341] 3 batches of labeled kinoids were manufactured and analyzed by this method. Based on the proportionality of UV signal and concentration, and of FLD and UV signal, the ratio between the amount of IFN and KLH (mIFN/mKLH) was calculated for the three batches (Table 8).

TABLE-US-00008 TABLE 8 weight ratio of IFN/KLH in the three labeled kinoid manufactured Ratio m.sub.INF/m.sub.KLH Mean RSD % Batch 1 0.29 0.28 12 Batch 2 0.31 Batch 3 0.25

[0342] A mean ratio mIFN/mKLH of 0.28 was found with a relative standard deviation <15%.

Example 11

Anti-mIFN Antibodies Titers Produced and Neutralizing Capacities when Immunogenic Product of the Invention is Injected as an Emulsion with SWE or SWE-a

[0343] Manufacturing muIFN-K:

[0344] Briefly, murine IFNA (PBL Biomedical Laboratories) and native KLH (Sigma) were mixed at a 50:1 ratio and treated with 22.5 mM glutaraldehyde for 45 minutes. After dialysis against phosphate-buffered saline (PBS) to eliminate excess glutaraldehyde, the solution was incubated with 66 mM formaldehyde for 48 hours at 37 C. After quenching with glycine (0.1 M final) and subsequent dialysed against PBS using a 10-kDa cutoff membrane, the preparation was filter-sterilized using a 0.22-m membrane and stored at 4 C.

[0345] Immunization Protocol:

[0346] Mice were immunized i.m. twice at day 0 and day 21 with mIFN-K (10 g per injection) as an emulsion 1 to 1 with SE or SE-a adjuvant (100 l final volume).

[0347] Determination of Anti-muIFN and Anti-KLH Antibody Titers by ELISA

[0348] Sera were analyzed for antibodies against muIFN or KLH by ELISA. Briefly, 96-well Maxisorp plates (Nunc) were coated with 100 ng/well of muIFNA (PBL Biomedical Laboratories) to detect anti-muIFN antibodies or native KLH (Sigma) to detect anti-KLH antibodies.

[0349] Two-fold serial serum samples dilution (from 1:100 to 1:51,200) were added to the wells. Blank wells received 100 L of dilution buffer. After 1.5 hours at 37 C., antibodies were detected with 100 L of horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) and O-phenylenediamine, a colorimetric substrate for horseradish peroxidase. A pool of sera from muIFN-K immunized Balb/c mice was used as a positive control. The optical density (OD) was recorded at a wavelength of 490 nm. ELISA assays were performed in duplicate. In each plate, two wells were reserved for blanks; their mean value was subtracted from all wells.

[0350] Antibody titers were calculated by interpolating the maximum OD (ODmax)/2 on the x-axis. The equation used was y=ax+b for a straight line passing through two points surrounding the ODmax/2.

[0351] Determination of Neutralizing Capacity of Anti-muIFN Antibodies Induced after IFN-K Immunizations

[0352] Neutralizing capacity was determined using the classical antiviral cytopathic assay (EMCV/L929). In this assay, the antiviral activity titer of muIFN is determined regarding its capacity to inhibit the lethal effect of encephalomyocarditis virus (EMCV) on murine L-929 cells (ATCC).

[0353] Briefly, 25 L of diluted serum samples (or control antibody) were added to 96-well culture plates (Nunc) in two-fold serial dilutions (from 1:200 to 1:6400). A commercial rabbit polyclonal antibody anti-muIFN (from PBL, ref: 32100-1) was used as a positive control. After incubation with 25 IU/well of muIFN for 1 hour at room temperature, 20103 L-929 cells were seeded per well and incubated at 37 C. After overnight growth, plates were washed with PBS and 100 L/well of EMCV solution (100 times the dose needed to kill 50% of the cells) was added to each well. Plates were incubated during 48 hours at 37 C. Finally, 20 L per well of MTS/PMS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner sal/phenazine methosulfate) solution (Promega) was added and the plates were incubated for 4 h at 37 C., 5% CO2 in a humidified incubator (protected from light). Next, the OD at 490 nm was measured for each well. The OD of the blank (wells with 100 L of culture medium alone) was subtracted from the sample OD.

[0354] The neutralizing capacity of each sample was calculated as following:

Neutralizing capacity (%)=100[(OD testOD virus)/(OD cells)], where

[0355] ODtest is the OD for the tested sample (cells+IFN+serum+virus)

[0356] ODvirus is the OD for the virus control (cells+virus)

[0357] ODcells is the OD for 20,000 cells/well (cells+IFN+virus).

[0358] Neutralizing capacities were plotted as a function of serum dilution. The titer (number of serum dilution) neutralizing 50% of IFN activity values were determined by interpolation on the linear part of the curve.

[0359] Results showed that anti-muIFN titers and anti-KLH titers were present in mice sera collected at day 31 after first injection of muIFN-K emulsified in SWE or SWE-a; and that the anti-muIFN antibodies had neutralizing capacities (NC50>200).