ANTI-LG3 ANTIBODY INHIBITION IN THE PREVENTION AND/OR TREATMENT OF VASCULAR REJECTION AND RELATED DISEASES AND CONDITIONS

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of United States Provisional Application serial No. 61/729,476, filed on November 23, 2012, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention generally relates to vascular damage and transplant rejection, and more specifically to the prevention and/or of vascular damage and/or acute vascular rejection and related diseases and conditions.

BACKGROUND ART

Rejection of transplanted organs is the main barrier of transplantation today. It occurs as a result of humoral and cell-mediated responses by the recipient to specific antigens present in the donor tissue. These antigens are known as major histocompatibility complex (MHC) molecules. In humans, this group of molecules is referred to as human leukocyte antigen (HLA) complex molecules.

Acute rejection usually occurs within the first weeks after transplantation. It is typically caused by mismatched HLA antigens that are present on all cells, which leads to activation of T cells in the host (or transplant recipient). HLA antigens are polymorphic therefore the chance of a perfect match is extremely rare. Endothelial cells in vascularized grafts such as kidneys are typically the earliest victims of acute rejection. Damage to the endothelial lining is often an early predictor of irreversible acute graft failure. The risk of acute rejection is highest in the first 3 months after transplantation, and is lowered by immunosuppressive agents in maintenance therapy.

Several antibodies against non-human leukocyte antigen (HLA) targets have been detected post- and/or pre-transplantation in renal transplantation and chronic renal allograft injury, and thus may be useful as diagnosis and/or prognosis markers (see, e.g., Li et a/., Proc. Natl. Acad. Sci. USA, 106(1 1): 4148-4153, 2009; Sigdel et a/., J Am Soc Nephrol 23: 750-763, 2012, PCT publication No. WO 201 1/109909). However, the role of these non-HLA antibodies in allograft rejection is unclear.

The incidence of acute cellular rejection of renal allografts has decreased over the past decade (USRDS Annual Data Report, 2009). This has been attributed at least in part to the use of new immunosuppressive agents with higher potency on T-cell mediated responses. However, the incidence of acute rejection with evidence of vascular injury (i.e., transplant arteritis or capillaritis and/or C4d deposition) has not been positively impacted (USRDS Annual Data Report, 2009). In most if not all forms of acute vascular rejection (AVR) of solid organ transplants, immune-mediated endothelial injury leading to a significant apoptotic response is a major characteristic (Solez, K., et a/., Am J Transplant, 2008. 8(4): p. 753-60; Shimizu, A., et a/., Kidney Int, 2000. 58: p. 2546-58; Shimizu, A., et a/., Lab Invest, 2002. 82(6): p. 673-86; Shimizu, A., et a/., Kidney Int, 2002. 61 : p. 1867-1879; Shimizu, A., et al., J Am Soc Nephrol, 2005. 16(9): p. 2732-45).

Immunosuppressive agents currently used to prevent or treat acute rejection of solid- organ transplant, such as calcineurin inhibitor (CNI) drugs (e.g., cyclosporine A, tacrolimus), corticosteroids (e.g., methylprednisolone), anti-proliferative agents (e.g., mycophenolate mofetil or MMF, azathioprine), and mammalian-target-of-rapamycin (mTOR) inhibitors (e.g., sirolimus), generally exhibit poor specificity and thus are associated with several and significant side effects.

There is a need for the development of novel strategies for the prevention and/or treatment of acute vascular rejection, or its associated effects.

The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention relates to the following items 1 to 37:

1. A method for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient, the method comprising decreasing the level of, or neutralizing, anti-LG3 antibodies in the transplant recipient.

2. The method of item 1 , wherein the method comprises decreasing the level of the anti-LG3 antibodies in the transplant recipient.

3. The method of item 2, wherein the method comprises treating a biological sample comprising anti-LG3 antibodies from the transplant recipient to remove anti-LG3 antibodies from the sample, and reintroducing the biological sample into the transplant recipient.

4. The method of item 3, wherein the treating comprises contacting the biological sample comprising anti-LG3 antibodies from the transplant recipient with a ligand that binds to the anti-LG3 antibodies, and removing the anti-LG3 antibody-ligand complexes from the biological sample.

5. The method of item 4, wherein the ligand is an antibody.

6. The method of item 5, wherein the antibody is an anti-human IgG antibody.

7. The method of item 4, wherein the ligand is an LG3 polypeptide. 8. The method of item 7, wherein the LG3 polypeptide comprises the sequence of SEQ ID NO:2 (FIG. 2C), or a sequence having at least 60% similarity or identity with the sequence of SEQ ID NO:2 (FIG. 2C).

9. The method of item 8, wherein the LG3 polypeptide comprises the sequence of SEQ ID NO:2 (FIG. 2C), or a sequence having at least 80% similarity or identity with the sequence of SEQ ID NO:2 (FIG. 2C).

10. The method of item 9, wherein the LG3 polypeptide comprises the sequence of SEQ ID NO:2 (FIG. 2C).

1 1. The method of any one of items 4 to 10, wherein the ligand is bound onto a solid support.

12. The method of item 1 1 , wherein the solid support is a chromatography resin or matrix.

13. The method of item 1 1 or 12, wherein the solid support is in a chromatography column.

14. The method of any one of items 3 to 13, wherein the biological sample is a blood- derived sample.

15. The method of any one of items 2 to 14, wherein the method is performed during plasmapheresis of the transplant recipient.

16. The method of item 2, wherein the method comprises administering to the transplant recipient an effective amount of autologous apoptotic bodies.

17. The method of item 16, wherein the apoptotic bodies derive from endothelial cells.

18. The method of item 1 , wherein the method comprises neutralizing the anti-LG3 antibodies in the transplant recipient.

19. The method of item 18, wherein the method comprises administering to the transplant recipient an effective amount of an agent that binds to the anti-LG3 antibodies and inhibits their interaction with LG3.

20. The method of item 19, wherein the agent is an antibody.

21. The method of any one of items 1 to 20, wherein the method is performed prior to the transplantation.

22. The method of any one of items 1 to 21 , wherein the method is performed after the transplantation.

23. The method of any one of items 1 to 22, wherein the allogeneic solid organ transplant is from a deceased donor.

24. The method of any one of items 1 to 23, wherein the solid organ is a kidney.

25. The method of any one of items 1 to 24, wherein the method prevents acute vascular rejection in the subject. 26. Use of an agent that neutralizes anti-LG3 antibodies for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient.

27. Use of an agent that neutralizes anti-LG3 antibodies for the preparation of a medicament for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient.

28. The use of item 26 or 27, wherein the agent is a ligand that binds to the anti-LG3 antibodies and inhibits their interaction with LG3.

29. The use of item 28, wherein the agent is an antibody.

30. A kit for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient, the kit comprising a ligand that binds to anti-LG3 antibodies bound to a solid support.

31. The kit of item 30, wherein the solid support is a chromatography resin or matrix.

32. The kit of item 30 or 31 , wherein the kit further comprises a chromatography column.

33. Use of the kit of any one of items 30 to 32 for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient.

34. An apheresis or plasmapheresis device comprising a chromatography column, the column comprising a ligand that binds to anti-LG3 antibodies bound to a solid support.

35. Use of autologous apoptotic bodies for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient.

36. Use of autologous apoptotic bodies for the preparation of a medicament for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient.

37. The use of item 35 or 36, wherein the apoptotic bodies are derived from endothelial cells.

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

In the appended drawings:

FIG. 1A shows the structure of perlecan;

FIG. 1 B shows the structure of Domain V/Endorepellin of perlecan, with the C-terminal LG3 domain circled;

FIGs. 2A and 2B show the amino acid sequence of human basement membrane- specific heparan sulfate proteoglycan core protein precursor (also known as perlecan, NCBI reference sequence No. NP_005520, SEQ ID NO:1), with the putative amino acids forming the LG3 domain (spanning about residues 4201-4389) depicted in bold;

FIG. 2C show the putative amino acid sequence of human LG3 (SEQ ID NO:2);

FIGs. 3A to D show (A) Hematoxylin and eosin (H&E) stained aortic sections of allografts exposed to warm ischemia for 0, 15 or 30 min pretransplantation and harvested 3 weeks post-surgery (magnification: 5X left panels and 20X right panels). (B) Intima/media ratio in allografts exposed to warm ischemia for 0, 15 or 30 min pre-transplantation and harvested 3 weeks post-surgery (n=8, 8 and 7 respectively). (C) Anti-donor IgG titers in sera from allografted mice 1 , 2 and 3 weeks post-surgery (n=8 for each time point) and in isografted mice 3 weeks post-surgery (n=6). Anti-donor antibodies are expressed in arbitrary unit (AU). (D) Anti-donor IgG titers in mice that received an allograft exposed to warm ischemia for 15 min assessed pretransplantation (pre), 10 days and 3 weeks post-surgery (n=8 for each time point). Donor- specific antibody expressed in arbitrary unit (AU);

FIG. 4 shows H&E stained section of adjacent isogenic aorta to an allograft exposed to warm ischemia for 30 min pre-transplantation and harvested 3 weeks post-surgery (magnification: 20X);

FIGs. 5A to 5D show (A) Anti-LG3 IgG titers in mice transplanted with a non-ischemic (no ischemia) or an ischemic allograft (15 min) and passively transferred with control IgG (white bars) or anti-LG3 IgG (black bars) (n=7 for each group) harvested at 1 , 2 or 3 weeks post- surgery. *p<0.01. (B) Anti-donor IgG titers assessed 3 weeks post-surgery in mice transplanted with a non-ischemic (no ischemia) or an ischemic allograft (15 min) and passively transferred with control IgG or anti-LG3 IgG (n=6 per group). (C) Intima/media ratio in mice transplanted with a non-ischemic allograft, passively transferred with control IgG or anti-LG3 IgG and sacrificed 3 weeks post-surgery (n=6 for each group). (D) H&E-stained aortic allografts from mice transplanted with a non-ischemic allograft and passively transferred with control IgG or anti-LG3 IgG (n=6 per group) and sacrificed 3 weeks post-surgery. (Top panels magnification: 5X). The square insert indicates the magnified area (Lower panels, magnification: 40X). The arrow indicates neointima formation;

FIG. 6 shows murine IgG subclasses levels (expressed as optical density X1 ,000) in anti-LG3 IgG used for passive transfer.

FIGs. 7A and 7B show (A) Upper panel: H/E staining and immunohistochemistry for C4d deposition in aortic allograft sections exposed to warm ischemia (15 min) in mice passively transferred with control IgG or anti-LG3 IgG. (magnification: 20X) In: intima. M: Media. The arrow indicates leukocyte infiltration. Lower panel: Intima/media ratio in mice transplanted with an ischemic allograft (15 min) and passively transferred with control IgG or anti-LG3 IgG (n=7 for each group) and sacrificed 3 weeks post-surgery. (B) Upper panels: Immunohistochemistry for the detection CD3 (T cells; top panels) or Asialo GM1 (NK cells: lower panels) positive cells in aortic allograft sections exposed to warm ischemia (15 min) in mice passively transferred with control IgG or anti-LG3 IgG. (magnification: 20X) In: intima. M: Media. The arrow indicates accumulation either T cells (top panel) or NK cells (lower panel). Lower panels: Average number of CD3 or Asialo GM 1 -positive cells per high power field in allografted mice passively transferred with control IgG or anti-LG3 IgG (n=3 for each group);

FIG. 8 shows H/E staining and immunohistochemistry for C4d deposition in adjacent isogenic aorta sections in allografted mice passively transferred with control IgG or anti-LG3 IgG;

FIG. 9: Left panel: H/E staining and immunohistochemistry for C4d deposition in aortic isograft sections exposed to warm ischemia (15 min) in mice passively transferred with control IgG or anti-LG3 IgG. (magnification: 20X) In: intima. M: Media; Right panel: Intima/media ratio in mice transplanted with an ischemic isograft (15 min) and passively transferred with control IgG or anti-LG3 IgG (n=4 for each group) and sacrificed 3 weeks post-surgery;

FIG. 10A shows that LG3 is released by apoptotic EC through caspase-3-dependent pathways. Media conditioned by wild type (WT) or Caspase 3 -/- (Casp3) murine primary EC cultured in pro-apoptotic conditions (serum-free medium for 4h) analyzed by WB for LG3 release. *p<0.05;

FIG. 10B shows electron micrographs of purified apoptotic nanovesicles and apoptotic bodies from apoptotic HUVEC. Note the difference in scale demonstrating size differences. Nanovesicles present a typical cup-shape morphology;

FIG. 10C shows that LG3 was identified within both apoptotic bodies and apoptotic nanovesicles produced by serum starved apoptotic EC by unbiased proteomic screen where the proteomes of endothelial apoptotic bodies and apoptotic nanovesicles were compared (in collaboration with Dr. Pierre Thibault, Universite de Montreal). Briefly, 12 ug of proteins from apoptotic bodies and nanovesicles were separated by SDS-PAGE and digested by trypsin. Peptides were extracted and separated on a nano-LC column coupled to an LTQ-Orbitrap™ mass spectrometer followed by three product ion scans (MS/MS). The table shows the different LG3 peptides identified.

FIG. 10D shows a confocal microscopy image confirming the presence of LG3 within membrane vesicles released by apoptotic EC. Immunofluorescence micrographs of purified membrane vesicles from apoptotic human EC (cultured in serum free medium for 4h) showing staining of LG3 (light grey) in 20 μΜ apoptotic body as well as in <1 μΜ (arrows) nanovesicles;

FIGs. 11A and 11 B show that in a murine model of vascular rejection, injections of nanovesicles from recipient endothelial apoptotic cells increase anti-LG3 antibody production and early neointima formation. In contrast, injections of apoptotic bodies alone or of combined apoptotic bodies and apoptotic nanovesicles did not increase anti-LG3 titers nor neointima formation. FIG. 11 A: Anti-LG3 titers in sera collected 3 weeks post-surgery from mice injected i.v. with purified nanovesicles, purified apoptotic bodies or nanovesicles and apoptotic bodies (Nano + apoptotic bodies) from apoptotic serum starved murine endothelial cells (mEC) or vehicle (n=8 for each group). FIG. 11 B: Intima/media ratio in aortic allograft from mice injected i.v. with purified nanovesicles, purified apoptotic bodies or nanovesicles and apoptotic bodies (Nano + apoptotic bodies) from apoptotic serum starved mEC or vehicle (n=8 for each group).

FIGs. 12A and 12B show the average number of Asialo GM 1 (NK cells, FIG. 12A) or F4/80 (macrophages, FIG. 12B) positive cells per high power field in aortic allograft sections from allograft recipients injected i.v. with vehicles, nanovesicles, or nanovesicles and apoptotic bodies (Nano + apoptotic bodies) from apoptotic serum starved mEC (n = 5-25 of high power field per condition, n=1 -5 mice in each group).

DISCLOSURE OF INVENTION

In the studies described herein, the present inventors have demonstrated that anti-LG3 antibodies increase vascular inflammation and obliterative remodeling in a murine model of vascular rejection. They have shown that mice receiving an allogeneic aortic graft exposed to ischemia prior to transplantation and in which anti-LG3 antibodies were passively transferred showed evidence of early neointima formation and increased infiltration of the allograft by mononuclear cells 3 weeks after transplantation.

Accordingly, in a first aspect, the present invention provides a method for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient, said method comprising decreasing the level of, or neutralizing, anti-LG3 antibodies in said transplant recipient.

Vascular inflammation following allogeneic solid organ transplant recipient may be measured, for example, by detection mononuclear cell (e.g., T cells, NK cells) infiltration, deposition of complement fragments (e.g., C4d, C1 q), and/or increased cytokine/chemokine secretion. Obliterative remodeling may be determined, for example, by detecting neointima formation (e.g., increased intima-media ratios). Vascular inflammation and/or obliterative remodeling typically occur, for example, during vascular rejection. Thus, in an embodiment, the above-mentioned method prevents or decreases vascular rejection, in a further embodiment acute or subacute vascular rejection, in the transplant recipient. In a further embodiment, the above-mentioned method prevents or decreases acute vascular rejection in the transplant recipient.

In an embodiment, the above-mentioned method comprises (selectively or nonselective^) decreasing the level of said anti-LG3 antibodies in said transplant recipient. In an embodiment, the method comprises treating a biological sample that comprises anti-LG3 antibodies from the transplant recipient to remove the anti-LG3 antibodies (i.e. decreasing the level/amount thereof), and reintroducing the treated biological sample (that contain no or less anti-LG3 antibodies) into the transplant recipient. "Selectively" decreasing the level of anti-LG3 antibodies means that the level of anti-LG3 antibodies is decreased preferentially relative to other antibodies (either in absolute and/or relative amounts).

In an embodiment, the above-mentioned biological sample is a biological fluid, e.g. , lymph, or a blood-derived sample. The term "blood-derived sample" as used herein refers to blood (e.g. , fresh blood, stored blood) or to a fraction thereof, such as serum, plasma and the like. It also refers to any sample that may be obtained following one or more purification, enrichment, and/or treatment steps using blood (obtained by venous puncture, for example) as starting material. In an embodiment, the above-mentioned blood-derived sample is blood or plasma.

Thus, in an embodiment, the method comprises collecting a biological sample (e.g., blood) from the subject, treating the sample to remove the anti-LG3 antibodies (i.e. decreasing the level/amount thereof), and reintroducing the treated biological sample.

In an embodiment, the treatment comprises contacting the biological sample comprising anti-LG3 antibodies with a ligand that binds to said anti-LG3 antibodies, removing the anti-LG3 antibodies-ligand complexes from the biological sample, and reintroducing the treated biological sample (that contain no or less anti-LG3 antibodies) into the transplant recipient (immunoadsorption).

In an embodiment, the above-mentioned ligand is an antibody. In an embodiment, the antibody non-specifically recognizes anti-LG3 antibodies, i.e. binds to all antibodies present in the sample. In an embodiment, the antibody is an anti-lgG antibody, in a further embodiment an anti-human IgG antibody.

In another embodiment, the antibody specifically recognizes anti-LG3 antibodies, i.e. binds preferentially to anti-LG3 antibodies relative to other antibodies present in the sample. Such a specific antibody may be generated by immunizing an animal with one or more of the complement determining regions (CDR1 , CDR2 and/or CDR3) of anti-LG3 antibodies. Methods to generate antibodies against an antigen are well known in the art.

In another embodiment, the ligand is an LG3 polypeptide. The term "LG3 polypeptide" refers to a polypeptide comprising a sequence corresponding to an LG3 polypeptide (an LG3 polypeptide domain), in a further embodiment a human LG3 polypeptide/protein. LG3 polypeptide/protein as used herein refers to a C-terminal domain of the perlecan polypeptide (FIGs. 1 B and 2A-2B, SEQ ID NO: 1), in an embodiment a domain comprising an amino acid sequence corresponding to about residues 4197 to about residue 4391 of the amino acid sequence of FIGs. 2A and 2B (SEQ ID NO: 1), for example from about residue 4201 to about residue 4389 (FIG. 2C, SEQ ID NO:2), in a further embodiment from about residue 4203 to about residue 4362 of the amino acid sequence of FIGs. 2A and 2B (SEQ ID NO: 1). In embodiments, the ligand may be the entire perlecan polypeptide, or any fragment or variant thereof that comprises the sequence of the LG3 region, or a sequence having at least 60, 65, 70, 75, 80, 85, 90, or 95% similarity or identity with the sequence of the LG3 region, and that retains the ability to bind to anti-LG3 antibodies present in a transplant recipient. Sequence identity refers to the degree of correspondence between two sequences, or the fraction of amino acids that are the same between two aligned sequences. Sequence similarity refers to the degree of resemblance between two sequences when they are compared, which is be calculated using a suitable similarity matrix, such as the PAM matrix or the BLOSUM matrix. Percent similarity and identity may be determined, for example, using well known algorithms and software. Optimal alignment of sequences for comparisons of identity/similarity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wl, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403- 10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information.

In an embodiment, the ligand is an LG3 polypeptide/protein fragment. The term "LG3 polypeptide/protein fragment" refers to a portion of the LG3 polypeptide/protein defined above and that is capable of binding to anti-LG3 antibodies present in biological samples from subjects, e.g. , a portion of the LG3 polypeptide/protein preferentially targeted by the anti-LG3 antibodies.

In an embodiment, the LG3 polypeptide comprises the sequence of SEQ ID NO:2 (FIG. 2C), or a sequence having at least 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 OR 99% similarity or identity with the sequence of SEQ ID NO:2 (FIG. 2C), and retains the ability to bind to anti-LG3 antibodies present in a transplant recipient. In an embodiment, the LG3 polypeptide comprises the sequence of SEQ ID NO:2 (FIG. 2C), or comprises a sequence having at least 60% similarity or identity with the sequence of SEQ ID NO:2 (FIG. 2C). In another embodiment, the LG3 polypeptide comprises the sequence of SEQ ID NO:2 (FIG. 2C), or comprises a sequence having at least 80% similarity or identity with the sequence of SEQ ID NO:2 (FIG. 2C). In another embodiment, the LG3 polypeptide comprises the sequence of SEQ ID NO:2 (FIG. 2C). In embodiments, the LG3 polypeptide may comprise, further to the LG3 polypeptide domain defined above, one more amino acids covalently linked to the amino- and/or carboxy-termini of said LG3 polypeptide domain. The above-mentioned LG3 polypeptide may thus be a chimeric or fusion peptide containing an LG3 polypeptide domain as described herein, linked at its amino- or carboxy-terminal end, or both, to an amino acid sequence of a different protein or peptide. In an embodiment, the above-mentioned LG3 polypeptide may comprise a peptide moiety, for example for facilitating its purification, detection and/or attachment to a solid support (a "tag", such as a "His-tag" or a GST-tag"). The above-mentioned LG3 polypeptide may comprise any other chemical moiety, which may be useful for example to stabilize, detect and/or purify the LG3 polypeptide.

In an embodiment, the LG3 polypeptide consists of the sequence of SEQ ID NO:2 (FIG. 2C).

In an embodiment, the ligand (e.g., LG3 polypeptide) is bound (covalently or non- covalently) to a solid support. In an embodiment, the ligand is covalently bound to, or immobilized on, a solid support. The ligand (e.g., LG3 polypeptide) may be immobilized to the solid support via a specific binding agent like a chemical group or a peptide group without significantly affecting the specific binding affinity.

In an embodiment, the above-mentioned solid support is a chromatography matrix/resin. The chromatography matrix/resin used according to the invention can be any material known in the art which is suitable for affinity separation, such as for example porous carrier materials, specifically porous solid phase carrier materials. Any conventional carrier material may be used, but is not limited to, agarose, Sepharose™, polysterene, controlled pore glass, dextrans, cellulose, synthetic polymers and copolymers like hydrophilic polymers, porous amorphous silica. The chromatography matrix/resin may be particulate like beads or granules generally used in columns or in sheet form like membranes or filters which may be flat, pleated, hollow fibers or tubes. In an embodiment, the above-mentioned matrix/resin is provided in the form of a column (chromatography column), e.g. , wherein the matrix is packed in a column. Where the resin is a bead, the beads may be provided in various sizes, depending, in part, on the nature of the sample being applied, where suitable bead sizes include from about 10 μηι to about 500 μηι, e.g. , from about 10 μηι to about 200 μηι, from about 20 μηι to about 150 μηι, from about 50 μηι to about 150 μηι. Non-limiting examples of formats in which a matrix is provided include a gravity-flow column; a fast protein liquid chromatographic (FPLC) column; a multi-well (e.g. , 96-well) column format; a spin column; and the like.

In embodiments, binding to the matrix may for example be achieved by column chromatography, batch treatment, or expanded bed absorption approaches. Column chromatography typically entails packing the solid matrix onto a chromatography column and passing the sample through the column to allow binding of the antibodies, the flow through being free or substantially free of antibodies, more particularly anti-LG3 antibodies. Batch treatment typically entails combining the sample (e.g., plasma) with the solid matrix in a vessel, mixing, separating the solid matrix (which contains the ligand/anti-LG3 complexes), and collecting the liquid phase that is free or substantially free of antibodies, more particularly anti- LG3 antibodies. In an embodiment, the above-mentioned methods are performed in vivo or in vitro, in a further embodiment in vitro.

In an embodiment, the above-mentioned method is performed through/during (extracorporeal) anapheresis or plasmapheresis.

Apheresis is a method wherein the therapeutic effects are based on the extracorporeal elimination of pathogenic proteins, protein-bound pathogenic substances, free pathogenic substances or pathogenic cells of the blood, in case of the present invention it is the removal of anti-LG3 antibodies. If the pathogenic protein can only be eliminated from cell-free plasma, plasma previously is separated from the blood cells by means of a membrane plasma separator (plasma separation) or by means of a haemocentrifuge. In selective whole blood apheresis methods, the anti-LG3 antibodies are specifically adsorbed directly from the non-pretreated blood without a previous plasma separation, whereby, in contrast to the plasma separation methods, both the plasma separation and the addition of a substitution solution can be omitted.

During plasmapheresis, blood is initially taken out of the body through a needle or previously implanted catheter. Plasma is then removed from the blood by a cell separator. Three procedures are commonly used to separate the plasma from the blood:

Discontinuous flow centrifugation: One venous catheter line is used. Typically, a 300 ml batch of blood is removed at a time and centrifuged to separate plasma from blood cells.

Continuous flow centrifugation: Two venous lines are used. This method requires slightly less blood volume to be out of the body at any one time as it is able to continuously spin out plasma.

Plasma filtration: Two venous lines are used. The plasma is filtered using standard hemodialysis equipment. This continuous process requires less than 100 ml of blood to be outside the body at one time.

During immunoadsorption, the blood of a patient is cleared from immunoglobulin (e.g., anti-LG3 antibodies) by an extracorporeal affinity purification step, chromatography column.

After plasma separation, the blood cells are returned to the person undergoing treatment, while the plasma, which contains the antibodies, is first treated and then returned to the patient in traditional plasmapheresis. Medication to keep the blood from clotting (e.g., an anticoagulant) is generally given to the patient during the procedure.

In embodiments, the above-mentioned method comprises: (a) obtaining a sample of blood from the transplant recipient; (b) isolating the plasma from the cellular components from said blood sample; (c) contacting said isolated plasma with a ligand that binds to the anti-LG3 antibodies as described above, whereby anti-LG3 antibodies are retained by the ligand (e.g. bound to a solid support such as a chromatography resin); (d) reintroducing the cellular components isolated from step (b) and the purified plasma from step (c) to the patient, e) and optionally repeating the steps c) and d) at least once. In embodiments, at least 50%, 60%, 70%, 80%, 85%, 90% or 95% of the anti-LG3 antibodies are removed from the biological sample (e.g., plasma).

In another embodiment, the above-mentioned method comprises administering an effective amount of apoptotic bodies, such as autologous apoptotic bodies, to the transplant recipient. In an embodiment, the apoptotic bodies are derived from endothelial cells. Apoptotic bodies (also called apobodies), are small sealed membrane vesicles that are produced from cells undergoing cell death by apoptosis. The formation of apoptotic bodies is a mechanism preventing leakage of potentially toxic or immunogenic cellular contents of dying cells and prevents inflammation or autoimmune reactions as well as tissue destruction.

Methods to produce/isolate apoptotic bodies or apoptotic bodies-rich medium (ABRM) are well known in art (see, e.g., Example 1 below). For example, to isolate endothelial cell- derived apoptotic bodies, isolated confluent human umbilical vein endothelial cells (HUVECs) may be incubated for a certain period of time (e.g., 12-36 hours, 24 hours) in endothelial basal medium without serum and growth factors to induce apoptosis (in the presence or not of TNFa). Apoptosis may be monitored/characterized by double staining with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI).

Medium from apoptotic HUVECs may be collected and clarified from dead cells and cell debris (e.g., by centrifugation). This HUVEC-derived apoptotic bodies-rich medium (ABRM) may be further centrifuged to isolate the apoptotic bodies. Apoptotic bodies may be characterized by flow cytometry after staining with annexin V-FITC and PI.

In an embodiment, the method comprises neutralizing the anti-LG3 antibodies in the transplant recipient. In an embodiment, the method comprises administering to the transplant recipient an effective amount of an agent that binds to anti-LG3 antibodies and inhibits their interaction with LG3. Without wishing to be bound by any theory, it is believed that allograft injury could foster intragraft expression of LG3 which in turn facilitates anti-LG3 antibody deposition, fuels further anti-LG3 antibody production, and enhances immune activation/inflammation at sites of allograft damages. Therefore, any agent that binds to anti- LG3 antibodies and inhibits their interaction with LG3, thus inhibiting anti-LG3 antibody deposition, would be suitable for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient. In an embodiment, the agent is an antibody, for example an antibody that binds to anti-LG3 antibodies and inhibits their interaction with LG3. Such a specific antibody may be generated by immunizing an animal with one or more of the complement determining regions (CDR1 , CDR2 and/or CDR3) of anti-LG3 antibodies. Methods to generate antibodies against an antigen are well known in the art.

In another embodiment, the agent is an LG3 polypeptide fused to a targeting moiety, to tether/attract the anti-LG3 antibodies away from the sites of allograft damages. The targeting moiety could target the LG3 polypeptide/anti-LG3 antibodies complexes to another organ or tissue (remote from the allograft), in which there is no inflammation.

The transplant may be from a living donor or a deceased donor (cadaveric). As shown in the Examples below, anti-LG3 antibodies appear to be more particularly detrimental in an ischemic microenvironment, which is typically sustained by organs from deceased donors (i.e. typically to a greater extent as compared to organs from living donors). Accordingly, in an embodiment, the transplant is from a deceased donor.

In embodiments, the above-mentioned method may be performed prior to and/or after the transplantation. In a further embodiment, the above-mentioned method is performed prior to the transplantation, so as to eliminate, or decrease the level of, anti-LG3 antibodies in the candidate transplant recipient prior to transplantation. Thus, in another aspect, the present invention provides a method for preventing or decreasing vascular inflammation and/or obliterative remodeling in a candidate allogeneic solid organ transplant recipient, said method comprising decreasing the level of, or neutralizing, anti-LG3 antibodies in said candidate transplant recipient (prior to transplantation).

In an embodiment, the above-mentioned method further comprises measuring or determining the levels of anti-LG3 antibodies in the transplant recipient (e.g., candidate transplant recipient). In another embodiment, the method further comprises selecting a transplant recipient (e.g., candidate transplant recipient) having elevated or increased levels of anti-LG3 antibodies, relative to a control level. Methods to measure the levels of anti-LG3 antibodies in a subject are described, for example, in PCT publication No. WO 201 1/109909.

The values for anti-LG3 levels can be absolute or relative values, e.g. , values provided in comparison to control levels. The values for expression levels can be raw values, or values that are optionally rescaled, filtered and/or normalized. The approach used will depend, for example, on the intended use for the data. The values for anti-LG3 levels may correspond to the intensity of a signal measured using a suitable device (e.g. , optical density (OD) values at a given wavelength measured using a spectrometer), or to an estimated anti-LG3 levels (based on a standard curve established using known concentrations of anti-LG3, for example).

"Control level" or "reference level" or "standard level" are used interchangeably herein and broadly refers to a separate baseline level measured in a comparable control sample, which is generally from a subject not suffering from vascular damage or acute vascular rejection or not at risk of suffering from vascular damage or acute vascular rejection. The corresponding control level may be a level corresponding to an average/mean or median level calculated based of the levels measured in several reference or control subjects (e.g. , a pre-determined or established standard level). The control level may be a pre-determined "cut-off value recognized in the art or established based on levels measured in one or a group of control subjects. The corresponding reference/control level may be adjusted or normalized for age, gender, race, or other parameters. The "control level" can thus be a single number/value, equally applicable to every patient individually, or the control level can vary, according to specific subpopulations of patients. Thus, for example, older men might have a different control level than younger men, and women might have a different control level than men. The predetermined standard level can be arranged, for example, where a tested population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group or into quadrants or quintiles, the lowest quadrant or quintile being individuals with the lowest amount of anti-LG3 antibodies (who are less likely to benefit from the treatment) and the highest quadrant or quintile being individuals with the highest amount of anti-LG3 antibodies (who are more likely to benefit from the treatment).

It will also be understood that the control levels according to the invention may be, in addition to predetermined levels or standards, anti-LG3 antibody levels measured in other samples (e.g. from healthy/normal subjects) tested in parallel with the experimental sample.

In an embodiment, the control level is a corresponding level or standard established based on anti-LG3 antibody levels in subjects not suffering from vascular damage or acute vascular rejection (AVR), or not at risk of suffering from vascular damage or AVR. In such a case, higher anti-LG3 antibody levels measured in a sample from subject relative to the control level is indicative that the subject is suffering from vascular damage or AVR, or is at risk (or is at high risk) of suffering from vascular damage or AVR (i.e. who is more likely to benefit from the treatment), whereas similar or lower anti-LG3 levels measured in a sample from subject relative to the control level is indicative that the subject is not suffering from vascular damage or AVR, or is not at risk (or is at low risk) of suffering from acute vascular rejection (i.e., who is less likely to benefit from the treatment).

In another embodiment, the control level is a corresponding level or standard established based on anti-LG3 levels in subjects known to suffer from vascular damage or AVR, or known to be at risk of suffering from AVR. In such a case, similar or higher anti-LG3 levels measured in a sample from the subject relative to the control level is indicative that the subject is suffering from vascular damage or AVR, or is at risk (or at high risk) of suffering from acute vascular rejection (i.e., who is more likely to benefit from the treatment), whereas lower anti-LG3 levels measured in a sample from subject relative to the control level is indicative that the subject is not suffering from vascular damage or AVR, or is not at risk (or is at low risk) of suffering from acute vascular rejection (i.e., who is less likely to benefit from the treatment).

As used herein the term "subject", "patient" or "recipient" is meant to refer to any animal, such as a mammal including human, mice, rat, dog, cat, pig, cow, monkey, horse, etc. In an embodiment, the above-mentioned "subject", "patient" or "recipient" is a mammal, in a further embodiment a human. The term "transplant recipient" as used herein refers to a subject who has already received solid organ transplantation, or who is a candidate for receiving solid organ transplantation (i.e. who will be undergoing solid organ transplantation because of a medical condition). In a further embodiment, the above-mentioned solid organ transplant is a kidney/renal transplant, a heart transplant recipient, a lung transplant or a pancreas transplant. In an embodiment, the transplant recipient suffers from acute vascular rejection or is at risk of (i.e., has a predisposition for) suffering from acute/active vascular rejection. In an embodiment, the above-mentioned acute vascular rejection is a Banff 97 classification grade I la, lib and/or III acute vascular rejection or an acute, antibody-mediated rejection. The Banff 97 classification is an internationally recognized classification system for the diagnosis of renal allograft pathology (Racusen et a/., Kidney International 55 (1999), pp. 713-723). Grade I Is typically defines cases with mild to moderate intimal arteritis (v1); grade lib typically defines cases with several intimal arteritis comprising > 25% of the luminal area (v2); and grade III typically defines cases with transmural arteritis and/or arterial fibrinoid change and necrosis of medial smooth muscle cells (v3 with accompanying lymphoctic inflammation). Antibody-mediated rejection is characterized by positive C4d staining in the graft peritubular capillaries, in the presence of anti-donor specific antibody (anti-HLA) in the circulation, a histologic appearance of acute tubular necrosis, peritubular capillaritis, glomerulitis or endarteritis. In an embodiment, the above-mentioned method prevents acute or subacute vascular rejection in said transplant recipient. In a further embodiment, the above-mentioned method prevents acute vascular rejection in said transplant recipient.

In another aspect, the present invention provides the use of a ligand that binds to anti-

LG3 antibodies in extracorporeal anapheresis or plasmapheresis for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient. The ligand that binds to anti-LG3 antibodies is as defined above. In an embodiment, the present invention provides the use of an LG3 polypeptide in extracorporeal anapheresis or plasmapheresis for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient.

In embodiments, the above-mentioned method/use may be combined with other treatments for solid organ graft rejection, for example immunosuppressive agents currently used to prevent or treat acute rejection of solid organ transplant, such as calcineurin inhibitor (CNI) drugs (e.g., cyclosporine A, tacrolimus), corticosteroids (e.g., methylprednisolone), antiproliferative agents (e.g., mycophenolate mofetil or MMF, azathioprine), mammalian-target- of-rapamycin (mTOR) inhibitors (e.g., sirolimus), and immunomodulatory agents (e.g., anti- CD20 antibodies that induces depletion of B-cells such as Rituximab™, antibodies directed against complement proteins such as Eculizumab™).

In another aspect, the present invention provides a kit for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient, said kit comprising a ligand that binds to anti-LG3 antibodies attached to, or immobilized on, a solid support. The ligand that binds to anti-LG3 antibodies and the solid support are as defined above. In an embodiment, the present invention provides a kit for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient, said kit comprising an LG3 polypeptide attached to, or immobilized on, a solid support. The kit may be used for partial or complete removal of anti-LG3 antibodies from a body fluid, specifically from blood, specifically from cell containing or cell-free blood fractions, more specifically from plasma. Such kit may further comprise, for example, instructions setting forth the above-mentioned methods (i.e., instructions for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient using the kit), containers (e.g., a column), reagents useful for performing the methods (e.g., buffers, enzymes, solutions, etc.), and the like. In an embodiment, the ligand that binds to anti-LG3 antibodies and the solid support are in a column. Accordingly, in another aspect, the present invention provides an affinity chromatography column comprising the ligand that binds to anti-LG3 antibodies and the solid support. Such a kit or affinity chromatography column may be incorporated into an apheresis or plasmapheresis device. In another aspect, the present invention provides an apheresis or plasmapheresis device comprising the above-noted kit or chromatography column.

In another aspect, the present invention provides the use of the above-mentioned kit or affinity chromatography column for preventing or decreasing vascular inflammation and/or obliterative remodeling in an allogeneic solid organ transplant recipient.

MODE(S) FOR CARRYING OUT THE INVENTION

The present invention is illustrated in further details by the following non-limiting examples. Example 1 : Materials and Methods

Animals and aorta transplantation procedures. Adult C57BI/6 and BALB/c mice (20-25 g) (Charles River, St-Constant, QC) were maintained on a 12-hour light-dark cycle and fed a normal diet ad libitum. Mice were anaesthetized using isoflurane (2%) by inhalation. Aortic transplantation was performed as described elsewhere with minor modifications (Koulack J et al., Microsurgery 1995; 16:1 10-1 13; Shimizu K, et al. Nat Med 2001 ; 7:738-741) In brief, 1 ml of heparinized saline (50 μΙ/ml) was injected into the vena cava to flush the aorta. A 6-mm segment of abdominal aorta from below the renal arteries to just above the aortic bifurcation was excised and soaked in ice-cold 0.9% normal saline. When mentioned, warm ischemia was induced by clamping the aorta for 15 or 30 min before the excision from the donor. The grafts were then excised and sutured orthotopically with end-to-end anastomoses, using 11-0 nylon interrupted sutures. Production and passive transfer of murine anti-LG3 IgG. Naive non-transplanted C57BI/6 mice were injected subcutaneously with either recombinant LG3 (50 μς) or PBS, emulsified in incomplete Freund's adjuvant (IFA), every 2 weeks for a total of 3 immunizations. Blood was harvested with cardiac puncture at sacrifice 12-14 days after the last immunization. Sera (diluted 1/100) were tested for the presence of anti-LG3 as described. IgG were isolated from pooled sera of either LG3-immunized mice (anti-LG3 IgG) or PBS-immunized mice (control IgG) using protein A Sepharose™ CL-4B (Sigma-Aldrich), and quantitated by Micro BCA assay (Pierce, Rockford, IL, USA). For passive transfer of anti-LG3 IgG, transplanted mice received tail vein intravenous injections of either 50 μg anti-LG3 IgG or 50 μg of control IgG. Each group received injections every other day during three weeks post-transplantation for a total of 8 doses.

Immunohistochemistry. Transplanted and adjacent native aortas were harvested at 3 weeks post-transplantation. Tissues were fixed with 10% neutral buffered formalin and paraffin embedded according to usual methods. Samples were cut into 4 μηι slices. Immunohistochemistry was assessed on an immunostainer (Discovery™ XT system, Ventana Medical Systems, Tucson, AZ) according to manufacturer's recommendations. Antigen retrieval was performed with proprietary reagents. When mentioned, samples were stained with haematoxylin-eosin (H&E). For the detection of NK cells, T cells or C4d deposition, indirect immunoperoxidase staining was performed using the primary anti-asialo GM 1 anti body (1/2000, Wako), anti-CD3 antibody (1/50, AbD Serotec™ MCA1477) or anti-C4d (1/50, Biomedica) respectively followed by incubation with specific secondary biotinylated antibodies. Streptavidin horseradish peroxidase, and 3,3-diaminobenzidine were used according to the manufacturer's instructions (DABmap™ detection kit, Ventana Medical Systems). Finally, sections were counterstained with haematoxylin. Digital images of stained tissues were captured by Leica™ DMLS microscope and Leica™ DFC420C camera (Leica Microsystems, Richmond Hill, ON). Intimal and medial areas grafts were outlined and quantified with a digital image analysis program (ImageJ™ 1 .42q, NIH, Bethesda, MD).

For the detection of NK cells or macrophages (FIGs. 12A and B), indirect immunoperoxidase staining was performed using the primary anti-asialo GM 1 antibody (1/2000, Wako) and anti-F4/80 antibody, respectively followed by incubation with specific secondary biotinylated antibodies. Streptavidin horseradish peroxidase, and 3,3-diaminobenzidine were used according to the manufacturer's instructions (DABmap® detection kit, Ventana Medical Systems). Finally, sections were counterstained with haematoxylin. Digital images of stained tissues were captured by Leica® DMLS microscope and Leica® DFC420C camera (Leica Microsystems, Richmond Hill, ON).

Measurement of murine anti-donor IgG. Sera were diluted 1 : 10 in FACS buffer and incubated with 1x 10 ⁶ BALB/c splenocyte targets for 30 minutes at 4°C. The samples were then washed three times and stained with PE goat anti-mouse IgG (1 : 100), Alexa™ 488 anti-mouse CD3e (1 : 100) (BD Biosciences) in FACS buffer for 30 minutes at 4°C, in the dark. Samples were run on a flow cytometer (FACScan™, Becton-Dickinson) and analyzed on computer software (FACS DIVA™, Becton-Dickinson). CD3 ⁺ parent gate was utilized to avoid nonspecific background signal due to Fc receptor-expressing cells.

Anti-LG3 measurements. Anti-LG3 titers were measured with a locally-developed ELISA. Recombinant mouse LG3 (10 nanograms/microliter) was first coated on 96-well Immulonll HB plates (Thermo Electron, Waltham, MA), for a total of 1 ,000 nanograms per well. The sera were diluted (1/100), and 100 microliters were added per well. The plates were washed, and bound IgGs were detected using horseradish peroxidase coupled anti-mouse IgG antibody (Amersham, Baie d'Urfe, QC). Reactions were revealed with 100 microliters of tetramethylbenzidine substrate (BD Biosciences) for 10 min, and stopped with 50 microliters of sulfuric acid (H ₂ S0 ₄ ). Spectrophotometric analysis was undertaken at 450 nanometers, and the results were expressed as optical density X1 ,000.

Conditioned medium fractionation and apoptotic bodies and nanovesicles isolation.

Conditioned media from serum starved HUVECs apoptotic was fractionated using sequential centrifugation 1) 1200 x g 15 min. to remove cellular debris 2) 50 000 x g, 15 min. to pellet apoptotic bodies, 3) 200 000 x g, 18h to pellet nanovesicles. Apoptotic bodies and nanovesicles pellets were collected and resuspended in a volume serum-free medium corresponding to the initial volume of conditioned medium.

Aortic transplantation and histology (apoptotic bodies and nanovesicles experiments). Adult C57BI/6 and BALB/c mice were purchased from Charles River. Aortic transplantation was performed as described previously (Soulez M. et al. Circ Res. 2012 Jan 6;110(1):p. 94-104). Very briefly, a 6-mm segment of flushed abdominal aorta from BALB/c mice were transplanted in C57BI/6 mice in the orthotopic position with end-to-end anastomoses. Mice were injected every other day post transplantation with nanovesicles and or apoptotic bodies (150 μΙ) up to 8 injections. Transplanted and adjacent native aortae were harvested at 3 weeks, embedded in paraffin, and processed for light microscopy with hematoxylin-eosin (H&E) staining. Digital images of stained tissues were captured by Leica™ DMLS microscope and Leica™ DFC420C camera (Leica Microsystems, Richmond Hill, ON). Intimal and medial areas of the aortic grafts were outlined and quantified with a digital image analysis program (ImageJ™ 1 .42q, NIH, Bethesda, MD).

Detection of anti-LG3 antibodies (apoptotic bodies and nanovesicles experiments). Murine anti-LG3 antibodies were detected by ELISA using recombinant LG3 as the antigen. Sera were diluted (1/100) to perform the assay. Specific antibodies to LG3 were detected with anti-mouse IgG antibody coupled with horseradish peroxidase (HRP), followed by revelation tetramethylbenzidine substrate which was stopped with sulfuric acid. Spectrophotometric analysis was performed at 450nm and titers are expressed as optical density (OD).

Cell culture and conditioned medium preparation. Murine EC (mEC) were isolated from the aorta of C57BI/6 mice, and grown in Dulbecco's Modified Eagle Medium (DMEM), low glucose culture media supplemented with endothelial cell growth supplements (ECGS), 10% FBS, 10% calf serum, 1 % penicillin-streptomycin and 1 % fungizone. To generate conditioned medium, cells were exposed to serum free medium for 8H. For all experiments, equal volumes of media conditioned by an equal number of cells were compared. We demonstrated in previous work that this system leads to the release of active mediators by apoptotic endothelial cells downstream of caspase-3 activation without cell membrane permeabilisation (Sirois I. et al. Cell Death Differ. 201 1 ; 18(3):p. 549-62).

Injection of murine apoptotic endothelial membrane vesicles. Transplanted mice received tail vein (150 μΙ_) IV injections of resuspended isolated vesicles. Each group received injections every other day during three weeks post-transplantation for a total of 8 doses.

Statistical analyses. When distributed normally, continuous variables are presented as means and standard deviations, otherwise as medians and interquartile ranges. Categorical variables are summarized as proportions. Between-group differences in categorical variables were assessed by chi-square tests (or Fisher's exact tests when expected frequencies were too small). Student's T tests were applied for continuous variables when the data was distributed normally. If such was not the case, Wilcoxon rank-sum tests were used. Associations between non-normally distributed continuous variables were analyzed by Spearman's correlation coefficients.

Example 2: Anti-LG3 antibodies increase obliterative remodeling in a murine model of vascular rejection

A murine model of pure vascular rejection, based on orthotopic transplantation of a fully MHC-mismatched aortic segment, was used to determine whether anti-LG3 behaves as an accelerator of allograft vascular injury. Transplantation of a fully MHC-mismatched aortic segment in absence of immunosuppression leads to subacute vascular inflammation and myointimal thickening progressing over 9 weeks (Soulez M, et al. Circ Res;110:94-104, 2012) Clamping the aortic allograft for 15 minutes before transplantation, aiming at reproducing the ischemic microenvironment sustained by organs from deceased donors, did not enhance vascular remodeling or leukocyte infiltration whereas prolonged ischemia for 30 minutes before transplantation, significantly increased neointima formation and allograft inflammation (FIGs. 3A and B, p=0.02). Vascular inflammation and neointima formation were specific to the allogeneic aorta, as the native aorta adjacent to the allograft did not show leukocyte infiltration or neointima formation (FIG. 4). Although largely described as a model of T-cell induced vascular injury, allografted mice also showed increasing donor-specific antibody (DSA) titers within the first 3 weeks post-transplantation (FIGs. 3C and D, p<0.01).

To evaluate the impact of anti-LG3 on the development of vascular rejection, murine anti-LG3 IgGs or control IgGs were passively transferred to recipients of an allogeneic aortic graft exposed, or not, to 15 minutes of ischemia prior to transplantation. Fifteen minutes of ischemic preconditioning was chosen as this condition does not induce vascular remodeling or inflammation per se. Purified anti-LG3 or control IgG were passively transferred intravenously to aortic allograft recipients every other day for three weeks post-transplantation. Anti-LG3 IgG titers (predominantly lgG1 and lgG2b, FIG. 6) were significantly higher in mice transferred with anti-LG3 antibodies as compared with mice transferred with mouse IgG (FIG. 5A, p<0.01). DSA titers were similar in both groups (FIG. 5B). Amongst the 6 recipients of a non-ischemic aortic allograft passively transferred with anti-LG3 antibodies, 5 showed evidence of early neointima formation 3 weeks after transplantation, whereas only 1 of the 6 mice transferred with control IgG developed neointima (FIGs. 5C and D). There was a trend for increased intima-media ratios in mice passively transferred with anti-LG3 antibodies but this did not reach statistical significance (FIG. 5C, p=0.17). Infiltration of the allograft by mononuclear cells was not noticeably increased by anti-LG3 injection (FIG. 5D).

In recipients of an ischemic allograft, anti-LG3 transfer led to a considerable increase in mononuclear cell infiltration, including CD3 T cells and NK cells (FIGs. 7A and B, p=0.03 and 0.003 respectively). Massive and transmural allograft C4d deposition was also observed and was specific to the allogeneic vasculature as C4d deposition was not enhanced in the adjacent isogenic aorta (FIGs. 7A and 8). Intima-media ratios were also significantly increased in recipients of an ischemic allograft transferred with anti-LG3 IgG (FIG. 7a, p=0.04, FIG. 9).

It was next assessed whether an acute ischemic insult was sufficient for anti-LG3- induced complement activation and vascular remodeling or whether both the initial ischemic insult and ongoing immune-mediated vascular injury were involved. To this end, anti-LG3 antibodies were passively transferred to recipients of an ischemic isograft. There was a trend towards increased neointima formation in recipients of an isograft transferred with anti-LG3 antibodies but this did not reach statistical significance and C4d deposition was not noticeably increased (FIG. 9). Collectively, these results demonstrate that anti-LG3 antibodies significantly increase vascular inflammation and obliterative remodeling in allografts exposed to an initial ischemic insult followed by ongoing immune-mediated vascular injury.

Example 3: Apoptotic bodies inhibit the production of anti-LG3 antibodies and neointima formation that is triggered by the injection of apoptotic membrane nanovesicles

LG3 is released by apoptotic EC through caspase-3-dependent pathways and murine caspase-3 -/- EC release significantly less LG3 when exposed to a pro-apoptotic stimulus as compared to wild-type EC (FIG. 10A). A novel type of membrane vesicles produced by apoptotic EC downstream of caspase-3 has been recently characterized (Sirois, I., et al., Cell Death Differ, 201 1. 18(3): p. 549-62). These membrane vesicles are functionally, structurally and biochemically distinct from classic apoptotic bodies and share common ultrastructural and biochemical markers with exosomes (FIG. 10B). Apoptotic bodies stem from blebbing of the cell membrane and their diameter ranges from 50 to 500 nm. Exosomes are secreted through fusion of multivesicular bodies with the cell membrane, range in size from 30 to 100 nm and present with a cup-shape morphology when evaluated by electron microscopy. (Sirois, I., et al., 201 1 , supra; Pallet, N., et al., Am J Transplant, 2012. 12(6): p. 1378-84; Thery, C, et al., Nat Rev Immunol, 2009. 9(8): p. 581-93). FIG. 10B shows membrane nanovesicles derived from apoptotic endothelial cells that share similarities with exosomes, such as size and morphology. To further characterize the distinct protein composition of apoptotic nanovesicles from apoptotic bodies, an unbiased proteomic screen was performed where the proteomes of endothelial apoptotic bodies and apoptotic nanovesicles was compared. LG3 was identified within both apoptotic bodies and apoptotic nanovesicles produced by serum starved apoptotic EC (FIG. 10C). Confocal microscopy confirmed the presence of LG3 within membrane vesicles released by apoptotic EC (FIG. 10D). As membrane vesicles are regulators of immune functions (Pallet, N., et al., 2012, supra; Thery, C, et al., 2009, supra), the possibility that membrane vesicles produced by apoptotic EC could prompt the production of anti-LG3 antibodies was considered. To test this hypothesis, mice transplanted with an allogeneic aortic graft were injected intravenously with either vehicle, apoptotic nanovesicles alone, apoptotic bodies alone, or medium containing both apoptotic bodies and membrane nanovesicles. In this murine model of aortic vascular rejection, mice injected with recipient membrane nanovesicles from endothelial apoptotic cells showed significant increases in anti-LG3 titers (p=0.001) and early neointima formation (p=0.01) compared to injections of vehicle (FIGs. 1 1A and B). Injections of a combination of nanovesicles and apoptotic bodies lead to decreased anti-LG3 titers (p=0.0003) and neointima formation (p=0.03) compared to the injection of nanovesicles alone (FIGs. 1 1A and B). Furthermore, infiltration of macrophages (F4/80) (FIG. 12A, p<0.05) and NK cells (Asialo) (FIG. 12B, p<0.05) was significantly increased in the aortic allograft recipients injected with apoptotic nanovesicles. Co-injection of apoptotic bodies and apoptotic nanovesicles blocked macrophage (FIG. 12A, p<0.05) and NK cell infiltration (FIG. 12B, p<0.05). These results provide evidence that LG3 is present in apoptotic nanovesicles from endothelial cells, is highly antigenic, and identify the release of apoptotic nanovesicles during endothelial death as a potential mechanism implicated in the production of anti-LG3 antibodies, neointima formation, and vascular injury and inflammation. Apoptotic bodies however tend to inhibit the production of anti-LG3 and vascular injury when mixed with apoptotic nanovesicles. This data provides evidence that apoptotic bodies, and more particularly apoptotic bodies derived from ECs, could be used for developing novel methods of inhibiting the production of anti-LG3 antibodies and decrease the severity of vascular injury in transplant recipients experiencing acute vascular rejection. Although the present invention has been described hereinabove by way of specific embodiments thereof, the scope of the claims should not be limited by the preferred embodiments, but should be given the broadest interpretation consistent with the description as a whole and as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise.