Annexin 1 antibody

Abstract

The present invention provides a specific binding molecule raised against the human Anx-A1 protein having the amino acid sequence shown in FIG. 2A. The present invention also relates to the sue of such a specific binding molecule in the treatment of T cell-mediated disease.

Claims

1. An antibody or fragment thereof that binds human Anx-A1 having the amino acid sequence of SEQ ID NO:8, .[.said antibody or fragment thereof.]. comprising Complementarity Determining Regions (CDRs) VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3.[., each having a respective amino acid sequence as follows in which
VLCDR1 is KASENVVTYVS(SEQ ID NO:2)
VLCDR2 is GASNRYT(SEQ ID NO:3)
VLCDR3 is GQGYSYPYT(SEQ ID NO:4)
VHCDR1 is GYTFTNYWIG(SEQ ID NO:5)
VHCDR2 is DIYPGGDYTNYNEKFKG(SEQ ID NO:6)
VHCDR3 is WGLGYYFDY(SEQ ID NO:7).]. .Iadd.of an antibody produced by the hybridoma cell line deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301.Iaddend..

2. An antibody or fragment thereof as claimed in claim 1 wherein the antibody is a monoclonal antibody.

3. An antibody or fragment thereof as claimed in claim 2 wherein the monoclonal antibody is humanized.

4. An antibody or fragment thereof as claimed in claim 1 wherein the fragment is a Fab, F(ab).sub.2 or Fv fragment or an scFv molecule.

5. An antibody or fragment thereof as claimed in claim 1 comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:19 and/or a light chain variable region having the amino acid sequence of SEQ ID NO:15.

6. An antibody or fragment thereof as claimed in claim 1 produced by the hybridoma cell line deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301.Iadd., wherein the antibody is a humanized monoclonal antibody.Iaddend..

7. A hybridoma cell line which produces an antibody .[.or fragment thereof.]. that binds the human Anx-A1 protein having the amino acid sequence of SEQ ID NO:8, .[.said antibody or fragment thereof comprising.]. .Iadd.wherein the antibody comprises .Iaddend.Complementarity Determining Regions (CDRs) VLCDR1, VLCDR2, VLCDR3, VHCDR1, VHCDR2 and VHCDR3.[., each having a respective amino acid sequence as follows in which
VLCDR1 is KASENVVTYVS,(SEQ ID NO:2)
VLCDR2 is GASNRYT,(SEQ ID NO:3)
VLCDR3 is GQGYSYPYT,(SEQ ID NO:4)
VHCDR1 is GYTFTNYWIG,(SEQ ID NO:5)
VHCDR2 is DIYPGGDYTNYNEKFKG, and(SEQ ID NO:6)
VHCDR3 is WGLGYYFDY(SEQ ID NO:7).]. .Iadd.of an antibody produced by the hybridoma cell line deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301.Iaddend..

.[.8. A hybridoma cell line as claimed in claim 7 deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301..].

9. A pharmaceutical composition comprising an antibody or fragment thereof as claimed in claim 1.

10. A pharmaceutical composition as claimed in claim 9, further comprising another therapeutically active agent.

.Iadd.11. An antibody or fragment thereof as claimed in claim 1, wherein the antibody comprises: a light chain variable region produced by the hybridoma cell line deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301; and a heavy chain variable region comprising Complementarity Determining Regions (CDRs) VHCDR1, VHCDR2 and VHCDR3, each having a respective amino acid sequence as follows in which: VHCDR1 is GYTFTNYWIG (SEQ ID NO:5), VHCDR2 is DIYPGGDYTNYNEKFKG (SEQ ID NO:6), and VHCDR3 is WGLGYYFDY (SEQ ID NO:7)..Iaddend.

.Iadd.12. A humanized antibody comprising the fragment of the antibody as claimed in claim 11, wherein the fragment comprises the CDRs of the antibody defined in claim 11..Iaddend.

.Iadd.13. A hybridoma cell line as claimed in claim 7, wherein the antibody comprises: a light chain variable region produced by the hybridoma cell line deposited with the European Collection of Cell Cultures (ECACC) on 3 Jun. 2010 as Accession No. 10060301; and a heavy chain variable region comprising Complementarity Determining Regions (CDRs) VHCDR1, VHCDR2 and VHCDR3, each having a respective amino acid sequence as follows in which: VHCDR1 is GYTFTNYWIG (SEQ ID NO:5), VHCDR2 is DIYPGGDYTNYNEKFKG (SEQ ID NO:6), and VHCDR3 is WGLGYYFDY (SEQ ID NO:7)..Iaddend.

Description

(1) The present invention will now be further described by way of reference to the following Examples which are present for the purposes of illustration only. In the Examples, reference is made to a number of Figures in which:

(2) FIG. 1A is a ribbon diagram of annexin-1 structure showing the four annexin repeats and the N-terminal domain. FIG. 1B is a schematic representation of the annexin repeats and the location of the bioactive sequence, Annexin-1 peptide Ac,2-26. FIG. 1C shows the amino acid sequence of peptide Ac.2-26, which is an acetylated N-terminal peptide fragment of Anx-A1.

(3) FIG. 2A shows (i) the amino acid sequence (SEQ ID NO:8) and (ii) the nucleotide sequence (SEQ ID NO:9) of human Annexin-1 (Anx-A1), isoform ANXA1-003. FIG. 2B shows the amino acid sequence of human Annexin-1 (Anx-A1), isoform ANXA1-002 (SEQ ID NO:10). FIG. 2C shows the amino acid sequence of human Annexin-1 (Anx-A1), isoform ANXA1-004 (SEQ ID NO:11). FIG. 2D shows the amino acid sequence of human Annexin-1 (Anx-A1), isoform ANXA1-006 (SEQ ID NO:12).

(4) FIG. 3 shows the generation of VJ-4B6. (A) Schematic representation of the strategy used to isolate and produce VJ-4B6. (B) The histogram shows the staining of cell lines stably transfected with Annexin-1 cDNA (green line; right-hand peak) or an irrelevant control cDNA (red line; left-hand peak) with VJ-4B6.

(5) FIG. 4 shows validation of VJ-4B6. (A) Staining of permeabilised human Peripheral Blood Mononuclear Cells (PBMC) or Polymorph Mononuclear Cells (PMN) with VJ-4B6 (50 ng/ml). (B) Staining of permeabilized murine splenocytes or resident peritoneal macrophages with VJ-4B6 (50 ng/ml). In all tests, cells were fixed with 4% paraformaldehyde and then permeabilised with 0.02% saponin in FACS buffer. Cells were incubated with biotinylated VJ-4B6 overnight at 4 C. and then stained for 1 hour using a streptavidin-FITC conjugated antibody (dil. 1:200). In all graphs, left-hand peak represents control and right-hand peak represents VJ-4B6. The data shown are from a single experiment and are representative of other two experiments with similar results.

(6) FIG. 5 shows that VJ-4B6 inhibits anti-CD3-induced T cell proliferation. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1/ (B) mice were stimulated with plate-bound anti-CD3(1 g/ml) in presence of the indicated concentrations of VJ-4B6. After 18-20 hours, cells were pulsed with 3H-thymidine (1 Ci) for 12 hours and then processed by cell-harvester to measure the levels of 3H-thymidine incorporation. The data shown are from a single experiment and are representative of other two experiments with similar results.

(7) FIG. 6 shows that VJ-4B6 inhibits anti-CD3/CD28-induced T cell proliferation. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1/ (B) mice were stimulated with plate-bound anti-CD3(1 g/ml) and anti-CD28 (1 g/ml) in presence of the indicated concentrations of VJ-4B6. After 18-20 hours, cells were pulsed with 3H-thymidine (1 Ci) for 12 hours and then processed by cell-harvester to measure the levels of 3H-thymidine incorporation. The data shown are from a single experiment and are representative of other two experiments with similar results.

(8) FIG. 7 shows that VJ-4B6 inhibits anti-CD3-induced IL-2 production. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1/ (B) mice were stimulated with plate-bound anti-CD3 (1 g/ml) in presence of the indicated concentrations of VJ-4B6. After 20-24 hours, cell supernatants were collected and analyzed for the levels of IL-2 by ELISA. The data shown are from a single experiment and are representative of other two experiments with similar results.

(9) FIG. 8 shows that VJ-4B6 inhibits anti-CD3/CD28-induced IL-2 production. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1/ (B) mice were stimulated with plate-bound anti-CD3 (1 g/ml) and anti-CD28 (1 g/ml) in presence of the indicated concentrations of VJ-4B6. After 20-24 hours, cell supernatants were collected and analyzed for the levels of IL-2 by ELISA. The data shown are from a single experiment and are representative of other two experiments with similar results.

(10) FIG. 9 shows that VJ-4B6 inhibits anti-CD3-induced CD25/CD69 upregulation. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1/ (B) mice were stimulated with plate-bound anti-CD3 (1 g/ml) in presence of the indicated concentrations of VJ-4B6. After 18-20 hours, cells were collected and stained with anti-CD25 FITC plus anti-CD69 PE. Samples were acquired by FACScalibur and analyzed by FlowJo software. The data shown are from a single experiment and are representative of other two experiments with similar results.

(11) FIG. 10 shows that VJ-4136 inhibits anti-CD3/CD28-induced CD25/CD69 upregulation. T cells from spleen and lymph nodes of AnxA1+/+ (A) and AnxA1/ (B) mice were stimulated with plate-bound anti-CD3(1 g/ml) and anti-CD28 (1 g/ml) in presence of the indicated concentrations of VJ-4B6. After 18-20 hours, cells were collected and stained with anti-CD25 FITC plus anti-CD69 PE. Samples were acquired by FACScalibur and analyzed by FlowJo software. The data shown are from a single experiment and are representative of other two experiments with similar results.

(12) FIG. 11 shows the DNA (SEQ ID NO:13 and SEQ ID NO:14) and amino acid sequence (SEQ ID NO:15) of the light chain variable region of VJ-4B6.

(13) FIG. 12 shows the amino acid sequence (SEQ ID NO:16) of the light chain variable region of VJ-4B6 with the CDRs annotated. CDR1, CDR2, CDR3 and the beginning of the constant region are highlighted. Numbering and CDRs according to Kabat.

(14) FIG. 13 shows the DNA (SEQ ID NO:17 and SEQ ID NO:18) and amino acid sequence (SEQ ID NO:19) of the heavy chain variable region of VJ-4B6.

(15) FIG. 14 shows the amino acid sequence (SEQ ID NO:20) of the heavy chain variable region of VJ-4B6 with the CDRs annotated. CDR1, CDR2, CDR3 and the beginning of the constant region are highlighted. Numbering and CDRs according to Kabat. In the heavy chain variable region residues 26 to 29, although not part of the hypervariable region as defined by Kabat, are part of the CDR loop defined by Chothia (Chothia and Lesk, 1987). Positions at insertions 52, 52a, 82, 82a, 82b, 82c, 100 and 100a, are indicated as 52a, 82abc, 100a.

(16) FIG. 15 shows that VJ-4B6 inhibits the development of EAE. C57BL/6 mice were immunized with MOG.sub.35-55 in CFA and monitored daily for signs of EAE for 22 days. At day 6 after the immunization, mice received an i.p. injection of 100 ng of IgG (A) or 5 (B), 50 (C) 100 ng (D) of VJ-4B6 (in 100 l of PBS) every six days. Results are meansSEM (n=6/group). *p<0.05, ***p<0.001.

(17) FIG. 16 shows that VJ-4B6 reduces the weight loss associated with the development of EAE. C57BL/6 mice were immunized with MOG.sub.35-55 in CFA and monitored daily for weight gain/loss for 22 days. At day 6 after the immunization, mice received an i.p. injection of 100 ng of IgG (A) or 5 (B), 50 (C) 100 ng (D) of VJ-4B6 (in 100 l of PBS) every six days. Results are meansSEM (n=6/group). *p<0.05, ***p<0.001.

(18) FIG. 17 shows that VJ-4B6 reduces the development of CIA. DBA/1 mice were immunized with bovine type II collagen in CFA. At 21 days after the primary immunization, mice were boosted (s.c.) with 200 g type II in complete CFA and monitored daily for signs of disease for 20 days. The day of the boosting mice received an i.p. injection of 100 ng of VJ-4B6 (in 100 l of PBS) every six days. Results are meansSEM (n=6/group). ***p<0.001.

EXAMPLE 1

VJ-4B6 Specifically Inhibits T Cell Activation in Annexin-1 Containing T Cells

(19) Materials and Methods

(20) Mice (mice used in FIGS. 4B, 5, 6, 7, 8, 9 and 10)

(21) Balb/C mice were obtained from B&K Universal (Grimston, England). AnxA1.sup./ mice were generated in the inventors' lab and bred in pathogen free conditions at B&K Universal. All mice used in these studies were aged between 6 and 8 weeks. Animal work was performed according to United Kingdom Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures Act 1986) and regulations of the European Union directives.

(22) Murine T Cell Extraction (Cells Used in FIGS. 4B, 5, 6, 7, 8, 9 and 10)

(23) Spleen and lymph nodes (axillary, inguinal and intestinal) were removed from 6 to 8 week old mice and prepared by gentle disaggregation of tissue through a 50 m cell strainer (BD), with a syringe plunger as previously described. Cell suspensions were layered over Ficoll to obtain the mononuclear cells and then collected and washed with RPMI medium (Gibco).

(24) T Cell Proliferation Assay (Data in FIGS. 5 and 6)

(25) Purified lymph node T cells (10.sup.5 cells/ml) were stimulated by plate-bound anti-CD3 (clone 145-2C11; eBioscience) or anti-CD3 plus anti-CD28 (clone 37.51; eBioscience) in 96 well plates. After 18-20 h, cultures were pulsed for 12 h with 1 Ci of [.sup.3H]-thymidine (Amersham Pharmacia Biotech) and incorporated radioactivity was measured by automated scintillation counter (Packard).

(26) IL-2 Production (Data in FIGS. 7 and 8)

(27) Purified lymph node T cells (10.sup.5 cells/ml) were stimulated by plate-bound anti-CD3 (clone 145-2C11; eBioscience) or anti-CD3 plus anti-CD28 (clone 37.51; eBioscience) in 96 well plates. After 20-24 h, culture supernatants were collected and analyzed for IL-2 content using mouse IL-2 ELISA kit (eBioscience) according to the manufacturer's instructions.

(28) Flow Cytometric Analysis (FIGS. 9 and 10)

(29) Cells were resuspended in FACS buffer (PBS containing 1% FCS and 0.02% NaN.sub.2). Lymphocytes were stained at 110.sup.6/ml in 100 l of FACS buffer and acquired on a FACScalibur with the CellQuest software (Becton Dickinson). The antibodies used were anti-CD25 FITC (clone PC61, eBioscience) and PE-conjugated anti-CD69 (clone H1.2F3). Cells were preincubated in FACS buffer containing anti-CD16/32 for 30 min at 4 C. to avoid non-specific binding and then labeled with the appropriate concentration of conjugated antibodies for 30 minutes at 4 C. After labeling, cells were washed and analyzed. Forward and side scatters were set to exclude erythrocytes and dead cells, and at least 210.sup.4 lymphocytes were analyzed per sample. In all the experiments stained cells were acquired with a FACScalibur flow cytometer and analyzed using FlowJo software.

(30) Validation of VJ-4B6 by Flow Cytometric Analysis (FIG. 4)

(31) Human PBMC and PMN or murine splenocytes and peritoneal macrophages were resuspended first in FACS buffer containing 4% paraformaldehyde for 10 minutes and thereafter in FACS buffer containing 4% paraformaldehyde and 0.02% saponin for 15 minutes. Cells were incubated with 50 ng/ml of biotinylated VJ-4B6 for 12 hours at 4 C. Thereafter, cells were incubated with streptavidin-FITC (dil.1:200; eBioscience) for 1 hour at room temperature. After labeling, cells were washed and analyzed. Forward and side scatters were set to exclude erythrocytes and dead cells, and at least 210.sup.4 lymphocytes were analyzed per sample. In all the experiments stained cells were acquired with a FACScalibur flow cytometer and analyzed using FlowJo software.

(32) Human Peripheral Blood Leukocytes (Cells Used in FIG. 4A)

(33) Blood donors were 20- to 35-year-old healthy men and women who were tested to be negative for HIV, hepatitis B virus, and hepatitis C virus. Further exclusion criteria were manifest infections during the last 4 weeks, fever, symptomatic allergies, abnormal blood cell counts, increased liver enzymes, or medication of any kind. Fresh venous blood was collected from healthy volunteers and immediately transferred to a tube containing 3.2% sodium citrate (1:10 dilution). Cells were then separated using the density gradient method: 3 ml of Ficoll Histopaque-10771 was layered on top of 3 ml of Ficoll Histopaque-11911 (both from Sigma) to create discrete layers and 6 ml of blood (diluted 1:1 with RPMI medium) layered on top of the Histopaques. After centrifugation at 1500 RPM at room temperature for 30 minutes, PBMCs and PMNs were aspirated from their appropriate layer using a sterile Pasteur pipette and washed with RPMI three times.

(34) Statistical Analysis

(35) All statistical analysis was performed with Prism software (GraphPad software). All values are expressed as meanSE. Statistical analysis was assessed either by Student's t test or one-way ANOVA where appropriate. A probability of P<0.05 was considered significant.

(36) Results

(37) A novel anti-AnxA1 antibody was generated by genetic immunisation as indicated in the scheme in FIG. 3A (Genovac GmbH, Germany). Serum from several immunized mice were tested and three resulted positive for IgG recognizing cells transfected with AnxA1 cDNA. Splenocytes from these mice were fused to myeloma cells to generate hybridoma cells. Only one of the three hybridoma cell clones were successfully subcloned and expanded. These hybridoma cells are called VJ-4B6-E5-B10-D4. Purified IgG2b fraction from the hybridoma cells recognizes cells transfected with AnxA1 cDNA (FIG. 3B, green line; right-hand peak) but not cell transfected with an irrelevant cDNA (FIG. 3B, red line; left-hand peak).

(38) To validate the specificity of VJ-4B6, the expression of AnxA1 in permeabilised human and murine cells known to express different levels of protein was analysed by FACS. As shown in FIG. 4A, human PMN were highly positive for VJ-4B6 staining compared with human PBMC (FIG. 4A, right-hand peak, right and left panel, respectively). This is consistent with previous observations (D'Acquisto et al, unpublished results) that PMN express significantly higher levels of AnxA1 compared to PBMC. Similarly, staining of murine macrophages with VJ-4B6 showed higher levels of expression of AnxA1 compared to splenocytes (FIG. 4B, right-hand peak, right and left panel, respectively). Collectively, these results show that VJ-4B6 recognize both human and murine AnxA1.

(39) Next, the effects and the specificity of VJ-4B6 on T cell activation were tested. To this aim, first the effects of VJ-4B6 on anti-CD3-induced cell proliferation (as mean of .sup.3H-thymidine incorporation) were measured using T cells from AnxA1.sup.+/+ or AnxA1.sup./ mice. FIG. 5 shows an increased incorporation of .sup.3H-thymidine in both AnxA1.sup.+/+ and AnxA1.sup./ T cells following stimulation with anti-CD3 (FIGS. 5A and 5B, respectively). Addition of VJ-4B6 to the stimulated cultures, caused a concentration-dependent inhibition of .sup.3H-thymidine incorporation in AnxA1.sup.+/+ but not AnxA1.sup./ T cells. Similar results were obtained with T cells stimulated with anti-CD3 plus anti-CD28 (FIGS. 6A and 6B, respectively).

(40) To further confirm these results, the effect of VJ-4B6 on other classical markers of T cell activation, i.e. interleukin-2 (IL-2) production and CD25/CD69 upregulation, were tested. T cells stimulated with either anti-CD3 alone or anti-CD3 plus anti-CD28 produced large amount of IL-2 (FIGS. 7 and 8, respectively). Addition of VJ-4B6 inhibited significantly and in a concentration-dependent manner IL-2 production in AnxA1.sup.+/+ but not AnxA1.sup./ T cells. Similar results were obtained on CD25/CD69 upregulation. Activation of T cells with either anti-CD3 alone or anti-CD3 plus anti-CD28 induced a marked increase in the percentage of CD25/CD69 double positive T cells (second panels from the left, FIGS. 9 and 10, respectively). Stimulation in presence of VJ-4B6 inhibited significantly and in a concentration-dependent manner CD25/CD69 induction (and expression) in AnxA1.sup.+/+ but not AnxA1.sup./ T cells.

(41) Together these results show that VJ-4B6 significantly inhibits T cell proliferation, IL-2 production and CD25/CD69 upregulation induced by signalling elicited by either anti-CD3 or anti-CD3 plus anti-CD28. In addition this effect is specifically due to the neutralization of AnxA1 by VJ-4B6 since its effect is lost in AnxA1.sup./ T cells. This is consistent with the inventors' previous observations that activated T cells release endogenous AnxA1 that would bein turnrequired for proper T cell activation (D'Acquisto et al., Blood 109: 1095-1102, 2007; D'Acquisto et a, Eur. J. Immunol. 37: 3131-3142, 2007).

(42) In summary, these data show that VJ-4B6 inhibits anti-CD3-induced upregulation of CD25 and CD69 (markers of T cell activation) in a concentration dependent manner. Similar inhibitory effect was observed on IL-2 production and on T cell proliferation. Most importantly, this effect was not observed in Annexin-1-deficient T cells, demonstrating that the inhibition is specific and that the antibody does not cause any adverse cytotoxic effects.

EXAMPLE 2

Sequencing of VJ-4B6

(43) The aim of this Example was to clone the antibody heavy and light chain variable region genes from the hybridoma cells and to determine the DNA sequence and location of the complementarity determining regions (CDRs) and other features.

(44) Cloning and Sequencing of Antibody Variable Regions

(45) Total RNA was prepared from 1 vial of hybridoma cells using the Qiagen RNeasy mini kit (Cat No: 74104). RNA was eluted in 504, water and checked on a 1.2% agarose gel.

(46) V.sub.H and V.sub.K (variable kappa light chain) cDNAs were prepared using reverse transcriptase with IgG and kappa constant region primers. The first strand cDNAs were amplified by PCR using a large set of signal sequence primers. The amplified DNAs were gel-purified and cloned into the vector pGem T Easy (Promega). The V.sub.H and V.sub.K clones obtained were screened for inserts of the expected size. The DNA sequence of selected clones was determined in both directions by automated DNA sequencing. The locations of the complementarity determining regions (CDRs) in the sequences were determined with reference to other antibody sequences (Kabat E A et al., 1991).

(47) Results

(48) VJ-4B6 Light Chain

(49) A single V.sub.K sequence was identified. The DNA sequence and deduced amino acid sequence for the VJ-4B6 V.sub.K is shown in FIG. 11. The deduced protein sequence with CDRs annotated is shown in FIG. 12, Nine clones (seven independent) from two separate amplification steps gave identical V region sequence. The non-productive aberrant V.sub.K sequence that arises from the hybridoma fusion partner was also present in a number of clones and there was one clone with a deletion within the sequence.

(50) VJ-4B6 Heavy Chain

(51) A single V.sub.H sequence was identified. The DNA sequence and deduced amino acid sequence for the VJ-4B6 V.sub.H is shown in FIG. 13. The same V region sequence was found in nine independent clones. Two clones had a single base pair change, one clone had a single base pair deletion and a single base pair change, and one clone had two single base pair changes. Each of the five single base pair changes occurred in only one clone. The remaining five clones had identical sequence. The deduced protein sequence with CDRs annotated is shown in FIG. 14.

REFERENCES

(52) Chothia C and Lesk A M. Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol. 196: 901-17, 1987. Kabat E A, Wu T T, Perry H M, Gottesman K S, Foeller C. Sequences of proteins of Immunological Interest, US Department of Health and Human Services, 1991

EXAMPLE 3

Immunosuppressive Effects of VJ-4B6 In Vivo

(53) To test the immunosuppressive effects of VJ-4B6 in vivo, we chose two classical models of autoimmune diseases: the MOG.sub.33-55-induced experimental autoimmune encephalomyelitis (EAE; mouse model of multiple sclerosis) and the collagen-induced arthritis (CIA; mouse model of rheumatoid arthritis).

(54) Methods

(55) Experimental autoimmune encephalomyelitis. Mice were immunized subcutaneously on day 0 with 300 l of emulsion consisting of 300 g of MOG.sub.35-55 in PBS combined with an equal volume of CFA containing 300 g heat-killed M. tuberculosis H37Ra. The emulsion was injected in both flanks and followed by an intraperitoneal injection of B. pertussis toxin (500 ng/100 l) in 100 l of saline on days 0 and 2. Mice were observed daily for signs of EAE and weight loss. Disease severity was scored on a 6-point scale: 0=no disease; 1=partial flaccid tail; 2=complete flaccid tail; 3=impaired righting reflex; 4=partial hind limb paralysis; 5=complete hind limb paralysis; 6=moribund or dead animal.

(56) Collagen Induced Arthritis. Six to eight male DBA/1 mice (8-12 weeks old) were injected intradermally at the base of the tail with 200 g collagen type II emulsified in complete Freund's adjuvant (CFA; Hooke labs). At 21 days after the primary immunization, mice were boosted (s.c.) with 200 g type II in IFA (Hooke labs). Mice were monitored for signs of arthritis onset using two clinical parameters: paw swelling and clinical score. Paw swelling was assessed by measuring thickness of the affected hind paws with plethysmometer. Clinical arthritis was assessed as recommended by the manufacturer (http://hookelabs.com/protocols/ciaInductionDBA1/ciaInduction_DBA1.html). Each limb was graded, giving a maximum possible score of 12 per animal

(57) Results

(58) Male C57/BL6 mice were immunized with MOG.sub.33-55/CFA as previously described (Paschalidis et al., J Neuroinflammation. 2009; 6:33). Mice received an intraperitoneal (i.p.) administration of VJ-4B6 (5, 50 and 100 ng/100 l), IgG control (100 ng/l) or PBS vehicle (control) every six days starting at day 6 after the immunization with MOG.sub.33-55/CFA. As shown in FIG. 15, mice treated with 5, 50 and 100 ng of VJ-4B6 showed a statistically significant dose-dependent reduction of signs of disease compared with control mice (FIGS. 15B, C and D, respectively) while administration of IgG control had no effects (FIG. 15A). The area under the curve (AUC) for each treatment and the percentage of inhibition versus the control (27.29 AUC) were the following: IgG100 ng, 24.17 AUC, 11.4%; VJ-4B6 5 ng, 15.04 AUC, 44.8%; VJ-4B6 50 ng, 5.87 AUC, 78.5%; VJ-4B6 100 ng, 7.04 AUC, 74.2%.

(59) Studies on animal models of EAE have demonstrated that the acute phase of the disease coincides with weight loss, probably due to anorexia and deficient fluid uptake. Weight measurement of treated mice correlated with the severity of the clinical score and showed a dose-dependent reduced weight lossfrom day 18 onwardsin the VJ-4B6-treated but not IgG-treated mice compared to controls (FIG. 16).

(60) To confirm the therapeutic potential of VJ-4B6 as immunosuppressant in vivo, we tested its effects in the CIA model. Male DBA/1 mice were immunized with bovine type II collagen in CFA as previously described (D'Acquisto et al., Blood. 2007; 109(3):1095-102). Mice received an i.p. injection of VJ-4B6 (100 ng/100 l) or PBS vehicle (control) every six days starting at day 0 after the boost with collagen. Consistent with the data obtained on the EAE, administration of VJ-4B6 significantly reduced (AUC 26.75; 67.9%) the development of sign of disease compared to control mice (AUC 83.50) (FIG. 17).