EA040303B1 - THERAPEUTIC METHODS WHERE GIBSPLA2 INHIBITORS ARE USED TO TREAT DISEASES ASSOCIATED WITH CD4 T-CELL IMMUNODEFICIENCY AND COMPOSITIONS CONTAINING LIKE GIBSPLA2 INHIBITORS - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to compositions and methods for modulating the immune system in a subject in need thereof. In particular, the invention discloses the presence of a key endogenous immune response factor and characterizes it, and provides new therapeutic and diagnostic methods and compositions based on the modulation of this factor. In particular, the invention provides compositions and methods useful for stimulating or inhibiting a CD4 T cell-mediated immune response in a subject, as well as methods and compositions for monitoring immunodeficiency, including immunodeficiency associated with human immunodeficiency virus (HIV) infection. Also provided are methods and compositions for diagnosing and analyzing defects in CD4 T cells left after antiretroviral therapy, as well as methods for developing drugs that can specifically treat this immunodeficiency.

Introduction

CD4 T-lymphocytes play an exceptional role in the control of the immune system (both cellular and humoral response) and are critical in various pathological conditions.

In immunological disease associated with HIV pathogenesis, less than 0.5% of CD4 T cells are actually infected (as measured in peripheral blood), but the vast majority of CD4 T cells show significant regulatory dysfunction. Uninfected CD4 T lymphocytes progressively lose their function, become anergic, and decrease in number, leading to CD4 lymphopenia. Anergy and lymphopenia are key features of the immunodeficiency characteristic of HIV-infected patients. The mechanisms underlying this phenomenon have never been fully explained (1).

Immune activation and inflammation also play a critical role in the pathogenesis of HIV (2, 3). The inventors have previously shown that a decrease in interleukin-2 (IL-2) responsiveness leading to CD4 anergy (4) and a decrease in interleukin-7 (IL-7) responsiveness upon disruption of the IL-7/CD4 regulatory loop is involved in the mechanism leading to CD4 lymphopenia (5). The mechanisms involved are related to defects in the Janus kinase/transmitter and transcription activator (STAT) pathway (6, 7). Similar results have been obtained by other laboratories (8, 9). Therefore, compartmentalization of the IL-7 receptor (IL-7R) is required to initiate a normal CD4 T cell response (10). Upon binding of IL-7, two chains of IL-7R (IL-7R alpha and gamma-c) are first directed to membrane microdomains (MMDs). There are cellular compartments that, like lipid rafts, are rich in cholesterol and sphingomyelin, but they also contain significant amounts of structural and functional proteins (11) IL-7R complexes induce cytoskeletal reorganization, which then interacts with their networks. These two consecutive steps are required to initiate the Jak/STAT pathway (12).

The authors of the present invention investigated the mechanisms underlying the inability to respond CD4 T-lymphocytes in HIV-infected patients with viremia (VP). Experiments have demonstrated that chronic activation of CD4 T lymphocytes puts them into an aberrant state of activation/differentiation that renders them unresponsive to certain physiological signals, such as those provided by interleukin-7. The present invention further describes the identification, isolation from human plasma, and characterization of the protein responsible for this aberrant state of CD4 T cell activation. Thus, first of all, the present invention discloses that immunosuppression can be mediated by an endogenous circulating protein which, when expressed, is capable of inducing damage and inactivation of CD4 T cells and, when inhibited, can stimulate the subject's immune system.

Based in part on these significant observations, the invention provides novel methods, compositions and compounds for modulating the immune system, in particular for modulating the immune system in immunocompromised subjects (eg, immunosuppressed or abnormal immune responses). The invention also provides novel methods for treating immune disorders by modulating CD4 T cells. In particular, the invention is useful in the treatment of immunodeficiencies associated with CD4 T cell disorders such as immunodeficiency syndrome associated with HIV infection. The invention also provides reagents and methods for characterizing an aberrant state of activation, reactivity to IL-7, and/or for monitoring an immune response that is impaired in HIV-infected patients. The CD4 T cell response of untreated or antiretroviral treated patients can be assessed and their response to treatment qualified and their CD4 T cell competence assessed.

Statement of the Invention

The present invention relates to the use of a group IB secreted phospholipase A2 (GIBsPLA2) inhibitor for the treatment of a disease associated with CD4 T cell immunodeficiency in a subject in need thereof. The GIBsPLA2 inhibitor is selected from the group consisting of an anti-GIBsPLA2 antibody, an inhibitory nucleic acid molecule based on the GIBsPLA2 gene sequence, a soluble GIBsPLA2 receptor, and a peptide containing 3 to 20 amino acid residues, wherein said peptide is produced by a method comprising (i) bringing to contacting a test peptide with GIBsPLA2, (ii) selecting a test peptide that binds GIBsPLA2, and (iii) selecting that peptide

- 1 040303 from (ii) which inhibits the activity of GIBsPLA2.

The GIBsPLA2 protein may contain amino acid residues 23-148 of SEQ ID

NO: 2 or its natural version.

When the GIBsPLA2 inhibitor is an anti-GIBsPLA2 antibody, it can be selected from an anti-GIBsPLA2 polyclonal antibody, an anti-GIBsPLA2 monoclonal antibody, or GIBsPLA2 binding fragments selected from F(ab') ₂ fragments, Fab fragments, single chain variable fragments (scFvs), single domain antibodies (VHH or nanobodies), bivalent antibody fragments (diabodies). The antibody may also be human or humanized.

In turn, when the GIBsPLA2 inhibitor is an inhibitory nucleic acid based on the GIBsPLA2 gene sequence, it can be selected from an antisense nucleic acid, small interfering RNA, small hairpin RNA, miRNA, aptamers, or ribozymes that inhibit the GIBsPLA2 gene or GIBsPLA2 protein expression.

The GIBsPLA2 inhibitor can be used to induce CD4 T cell activation in a subject.

A subject in need of such treatment may be immunodeficient or immunosuppressed, have an infectious disease such as a viral infection, or have cancer. In particular, this invention will be useful for the treatment of AIDS in an HIV-infected subject. For example, it can be used to suppress or reverse an HIV-mediated immunodeficiency.

The GIBsPLA2 inhibitor may be administered by injection, preferably intramuscular, subcutaneous, transdermal, intravenous or intra-arterial injection; or by nasal, oral, mucosal, rectal administration, or by inhalation.

Another object of the present invention relates to a pharmaceutical composition in the form of an injection solution or suspension for injection for the treatment of a disease associated with CD4 T cell immunodeficiency in a subject in need thereof. This composition comprises (i) a GIBsPLA2 inhibitor and (ii) a pharmaceutically acceptable carrier or excipient suitable for injection or suspension for injection. The GIBsPLA2 inhibitor in this composition is an anti-GIBsPLA2 antibody selected from an anti-GIBsPLA2 polyclonal antibody, an anti-GIBsPLA2 monoclonal antibodies, or GIBsPLA2 binding fragments thereof, selected from F(ab') ₂ fragments, Fab fragments, single chain variable fragments (scFvs), single domain antibodies (VHH or nanobodies), divalent antibody fragments (diabodies). While GIBsPLA2 is a protein containing amino acid residues 23-148 of SEQ ID NO: 2 or its natural variant.

In another aspect of the invention, there is provided a method for treating a disease associated with CD4 T cell immunodeficiency in a subject in need thereof. The method comprises administering to said subject an effective amount of a GIBsPLA2 inhibitor selected from the group consisting of an anti-GIBsPLA2 antibody inhibiting a nucleic acid based on the GIBsPLA2 gene sequence, a soluble GIBsPLA2 receptor, and a peptide containing 3 to 20 amino acid residues, wherein said peptide is produced by a method comprising (i) contacting a test peptide with GIBsPLA2, (ii) selecting a test peptide that binds GIBsPLA2, and (iii) selecting that peptide from (ii) that inhibits GIBsPLA2 activity.

Brief description of the drawings

Fig. 1a-e show that prior to CD4 stimulation, VP-derived T cells exhibit an aberrant activation state with many large membrane microdomains unaffected by IL-7.

(a) Membrane microdomains (MMDs) were labeled with the B subunit of cholera toxin (CtxBAF488) and analyzed by STED microscopy (induced emission reduction microscopy). From top to bottom, purified CD4 T cells from FID, VP, and PHA-activated (40 μg/ml, 30 min) HD T cells. For each upper half group, exemplary CD4 T cells before and after IL-7 stimulation (2 nM, 15 min) are shown with a series of Z-axis slices. plus sphingomyelinase (CMase; 2.7 μM, 5 min) before IL-7 stimulation.

(b, c) MMD was counted over the entire surface of purified CD4 T cells. An average of 50 cells were evaluated, (b) HD cells before (HDc: NS) and after stimulation with IL-7 (HDc: IL-7). (c) VP cells before (VPc: NS) and after IL-7 stimulation VP (VPc: IL-7), PHA-activated HD cells before (HDc: PHA) and after IL-7 stimulation (HDc: PHA/IL- 7).

(d, e) MMD size was determined on the surface of purified CD4 T cells (d) IL-7 stimulated HD cells (HDc: IL-7), (e) IL-7 stimulated VP cells (VPc: IL-7) and IL -7-stimulated PHA-activated HD cells (HDc: IL-7).

Fig. 2a-c show that IL-7R chains from VP CD4 T cells are embedded in detergent-resistant microdomains (DRMs) that are unaffected by IL-7. Purified CD4 T lymphocytes were lysed (0.5% Triton X-100) and 200 μl of the lysate was loaded onto a 5-40% sucrose gradient. After 16 hours of centrifugation (50,000 rpm) at 4° C., 18 fractions were collected (#1 left = tube top = 5% sucrose; #18 right = tube bottom = 40% sucrose). Each fraction was analyzed on SDS-PAGE (7% acrylamide-bisacrylamide). Flottilin, IL-7R alpha and gamma-c were detected by immunoblotting (10).

- 2 040303 (a) Flottilin was used as a marker to indicate low density fractions corresponding to DRM and high density fractions outside the rafts.

(b) IL-7R-alpha and (c) gamma-c bands are shown for purified unstimulated HD CD4 T cells (HDc:NS), IL-7-stimulated HD cells (HDc:IL-7), unstimulated VP cells ( VPc:NS) and PHA-activated HD cells (HDc:PHA).

Fig. 3a-c show that IL-7R function is damaged in the membrane microdomains of VP CD4 T cells.

(a) Two-dimensional effective diffusion rates D _eff for IL-7R-alpha were measured as shown in FIG. 7. Diffusion rates were also measured after the addition of various drugs: Choase (31 µM, 30 min), plus SMase (2.7 µM, 5 min) (CO/SM), Col (10 µM, 30 min), plus CytD ( 20 μM, 30 min) (CytD/Col), or in the presence of all of these inhibitors (all). CD4 T cells from HD (HDc) and VP (VPc) as well as PHA-activated HD CD4 T cells (HDc: PHA) were studied. Bars indicate SD from 5 independent experiments. Additional experimental data is shown in Fig. 8.

(b) IL-7-induced STAT5 phosphorylation and nuclear translocation was performed using rabbit phospho-STAT5 labeled with goat anti-rabbit Atto642 and analyzed by flash STED microscopy (0.5 μm slices). Purified unstimulated HD CD4 T cells (HDc: NS), IL-7 stimulated HD CD4 T cells (HDc: IL-7), unstimulated VP CD4 T cells (VPc: NS), IL-7 stimulated VP CD4 T cells (VPc:IL-7), PHA-activated HD CD4 T cells (HDc:PHA), and PHA-activated HD CD4 T cells stimulated with IL-7 (HDc:PHA/IL-7). The effects of colchicine plus cytochalasin D are shown in the left panel.

(c, d, e) Following IL-7 stimulation, the kinetics of phospho-STAT5 appearance in the cytoplasm and accumulation in the nucleus was measured using ImageJ software. (c) HD CD4 T cells (black line) and HD CD4 T cells treated with Col plus CytD (blue line), (d) VP CD4 T cells (red line) and (f) PHA-activated HD CD4 T cells (green line).

Fig. 4a-d show that VP plasma induces an aberrant pattern of activation in HD CD4 T cells as measured by MMD number.

(a) Exemplary images of HD CD4 T cells treated with plasma (10%) from VP (HDc: VPp), HIC (HDc: HICp) or ART patients (HDc: ARTp) are shown. MMD was stained with cholera toxin (CtxBAF488). For each group, the upper half of the Z-axis images of exemplary CD4 T cells before (left) and after IL-7 stimulation (2 nM, 15 min) (right) are shown.

(b) MMD induced on the surface of CD4 T cells (HDc) by plasma (10%) from 5 different VPs (VPp1-VPp5). Results were obtained from analysis of 50 cells before (white) and after (blue) IL-7 stimulation. Means and quartiles are shown.

(c) Comparison of plasma effects from HD (HDp), VP (VPp), HIC (HICp), and ART patients (ARTp) after (blue) and before (white) IL-7 stimulation.

(d) Dose response (0.01-10%) obtained for the plasma described in (c). The number of MMD induced on the surface of HDc CD4 T cells is shown. The plasma VP effect is shown as a solid red line.

In FIG. 5a-d show that VP plasma inhibits IL-7-induced STAT5 phosphorylation and phospho-STAT5 nuclear translocation in HD CD4 T lymphocytes.

(a) Prior to IL-7 stimulation, purified HD CD4 T cells were pre-incubated with plasma (10%). IL-7-induced phosphorylation and nuclear translocation of phospho-STAT5 was examined by pulsed STED microscopy (sections 0.5 μm). The following plasma was examined: control (HDc: NS), VP (HDc: VPp), HIC (HDc: HICp) and ART patients (HDc: ARTp).

(b) Phospho-STAT5 analysis in the cytoplasm (blue) and nucleus (red) of IL-7-stimulated HD CD4 T cells pretreated with plasma from 5 different VPs (10%).

(c) Comparison of the effects of pre-incubation with plasma (10%) for IL-7-stimulated HD CD4 T cells. Plasma was taken from HD (HDp), VP (VPp), SH (HICp) and ART patients (ARTp).

(d) Dose-dependent response (0.01-10%) obtained with plasma as measured by inhibition of phospho-STAT5 nuclear translocation in IL-7 stimulated HD CD4 T cells. The plasma VP effect is shown as a solid red line.

In FIG. 6a-d show the molecular characterization of resistance inducing factor (RIF) extracted from VP plasma.

(a) Treatment of plasma VP with trypsin, DNase, RNase and PNase. RIF activity was examined by measuring the number of MMDs and the effect on IL-7-induced nuclear phospho-STAT5 in HD CD4 T cells.

(b) The molecular weight of RIF was determined by gel filtration on a Sephadex-G100 column. RIF activity on HD CD4 T cells was determined by measuring the number of MMDs induced by different fractions from the column (thick red curve). Each fraction was also tested for the presence of viral proteins by dot blotting using polyclonal antibodies from VP. The background obtained with HD plasma was subtracted. The experiments were repeated three times.

- 3 040303 (c) RIF molecular weight was also determined after gel filtration on a Sephadex column

G100 and its activity after inhibition of IL-7-induced phospho-STAT5 was determined by FACS. The percentage of maximum IL-7-induced phospho-STAT5 was recorded.

The amount of protein in each fraction was also recorded. The experiments were repeated twice.

(d) The isoelectric point was determined as follows. RIF eluted from the Sephadex G100 column was loaded onto an anion (MonoQ) or cation (MonoS) exchange column. RIF activity was eluted with buffers in a stepwise pH gradient. Built a curve depending on the number of MMD on HD CD4 T cells against pH.

In FIG. 7a-c show two-dimensional gel analysis of IL-7 signalosome on purified CD4 T cells from HD, VP and IL-7 stimulated HD cells.

(a) Unstimulated (NS) HD CD4 T cells.

(b) VP CD4 T cells.

(c) IL-7 stimulated HD CD4 T cells.

In FIG. 8a-g shows diffusion rate analysis of IL-7R-alpha on the surface of purified CD4 T cells from HD, VP and PHA-stimulated HD cells, (a, d) on the surface of HD CD4 T cells, (b, e) on the surface of VP CD4 T cells, (c, f) on the surface of HD CD4 T cells pre-activated with PHA (1 µg/mL).

(g) Schematic of the diffusion mechanism of IL-7R-alpha embedded in MMD before and after treatment with MMD inhibitors or cytoskeletal inhibitors.

In FIG. 9a-d show a schematic representation of the hypothetical mode of action of RIF on HD CD4 T cells, and the mechanism of non-response to IL-7. RIF induces abnormal MMD that are non-functional. Thus, the IL-7 signalosome is damaged and the cells remain unable to respond to the cytokine, as in VP CD4 T cells. The aberrant activation mode and signal transduction defects in RIF-induced HD CD4 T cells and in VP CD4 T cells are indistinguishable. The left side of the diagram illustrates the various steps in the mechanisms of IL-7 signaling in HD (10, 12).

(a) In resting CD4 T cells, prior to IL-7 recognition, the IL-7R chains are associated, but their intracytoplasmic domains are distant and Jak1 and Jak3 signaling molecules do not interact.

(b) In IL-7 activated CD4 T cells, IL-7R is compartmentalized into normal MMDs (90 nm in diameter) and the signalosome becomes functional. After cytoskeletal organization, STAT5A and STAT5B are phosphorylated in contact with the IL-7R/Jak1/Jak3 complexes, then migrate to the nucleus via microtubule migration, as discussed previously (12).

The right side of the diagram illustrates the hypothetical mechanism of action of RIF. The proposed mechanism of action is derived from preliminary data and comparison of RIF-induced defects with those characterizing purified CD4 T cells from VP (unpublished data).

(c) RIF induces many large anomalous MMDs. IL-7R is embedded in abnormal MMDs and its ability to induce a functional signalosome is impaired.

(d) RIF HD CD4 treated T cells did not respond to IL-7. Jak1 and Jak3 phosphorylate STAT5, although with reduced kinetics, but phospho-STAT5 does not migrate into the nucleus due to the lack of cytoskeletal and microtubule organization.

Panels a, b, c, and d show STED microscopy images of MMDs labeled CtxB:AF488 (Z-axis half bar from CW-STED). Panels b and d show tubulin stained with rabbit anti-tubulin/goat anti-rabbit Atto642; actin stained with mouse anti-actin/goat anti-mouse Chr494; and phosphoSTAT5 stained with rabbit anti-phospho-STAT5/goat anti-rabbit Atto642. Pulsed STED microscopy demonstrates a 0.5 μm section of methanol-treated CD4 T cells. Upon stimulation with IL-7, actin in the MMD cytoplasmic region of RIF-treated HD CD4 T lymphocytes was unable to concentrate into structured cushions and did not form a cortex surrounding the nucleus, unlike HD. Further, tubulin in these RIF-treated HD CD4 T cells, as in VP CD4 T cells, cannot form microtubules, which are hypothesized to play a key role in the formation of a bridge between the cytoplasm and the nuclear membrane and thus are required for STAT5 nuclear translocation. .

Summary of defects: circles numbered 1, 2, 3, and 4 indicate various defective steps associated with aberrant activation mode and failure to respond to IL-7 in RIF-treated HD T cells: (1) abnormal pattern of signaling complex proteins, as described by 2D gels, (2) abnormal membrane structures such as large MMDs as seen by STED microscopy, (3) abnormal organization of the cytoskeleton as determined by diffusion kinetics and STED microscopy, and (4) abnormal intermediate signal transducers and inhibition nuclear translocation of phospho-STAT5 as shown by STED microscopy.

Fig. 10. PLA2sGIB inhibits IL-2-induced nuclear translocation of PStat5 in healthy donor (HD) CD4 T cells. Resting CD4 T cells purified from 4 healthy donors were treated for 30 min at 37°C with 3% or 1% plasma from 5 VP (VP63, VP68, VP69, VP74 and VP75) and from 3

- 4 040303

HD used as control. When indicated, they were stimulated with 2nM IL-2 for 15 min at 37°C. Percentage of cells positive for nuclear PStat5, with mean and RSD, shown in all CD4 T cells (a) and CD4+CD25+ T cells (b), before (blue dots) and after IL-2 stimulation (red points). Intracellular localization of PStat5 was observed using laser scanning confocal microscopy (LSM 700, Zeiss) after indirect staining with rabbit anti-human PStat5 (pY694), followed by donkey anti-rabbit IgG with Die light 405. Total CD4 T cells were stained with goat antibodies. to human b-tubulin, and then donkey antibodies to goat IgG-AF555. CD25+ CD4 T cells were treated with mouse anti-human CD25 followed by donkey anti-mouse IgG-AF488.

Fig. 11. PLA2sGIB inhibits IL-4 induced nuclear translocation of PStat6 BCD4 T in healthy donor (HD) cells. Resting CD4 T cells purified from 4 healthy donors were treated for 30 min at 37°C with 3% or 1% plasma from 5 VP (VP63, VP68, VP69, VP74 and VP75) and from 3 HD used as control. When indicated, they were stimulated with 2 nM IL-4 for 15 min at 37°C. Shows the percentage of cells positive for the presence of nuclear PStat6, with mean and RSD, in all CD4 T cells, before (blue dots) and after IL-2 stimulation (red dots). Intracellular localization of PStat6 was observed using laser scanning confocal microscopy (LSM 700, Zeiss) after indirect staining with rabbit anti-human PStat6 (pY694), followed by goat anti-rabbit IgG-AF488. Total CD4 T cells were stained with mouse anti-human alpha-tubulin and then with goat anti-mouse IgG-AF647.

Fig. 12. No activity of mutant pPLA2GIB H48Q.

Fig. 13. Comparison of activity of cloned wild-type porcine PLA2 GIB and its H48Q mutant. A: induction of abnormal membrane microdomains (aMMD); B: effect on IL-7 induced phospho-STAT5 nuclear translocation (NT pSTAT5).

In FIG. 14 shows treatment of plasma from viraemic patients with goat anti-PLA2 G1B antibodies bound to sepharose beads. Green: VP68; pink: VP69; blue: VP LJT. After treatment (30 min at room temperature), the plasma was tested.

a) The percentage of CD4 T cells showing abnormal MMD was determined after staining with cholera toxin B (CtxB-AF488).

b) The nuclear translocation of pSTAT5 after IL-7 stimulation was determined and the percentage of positive nuclei was calculated.

Fig. 15. Effect of anti-PLA2 GIB antibodies on aMMD induction and NT pSTAT5 inhibition.

Fig. 16. Soluble PLA2G1B mouse receptor (sMR) inhibits the activity of human PLA2G1B (huPLA2GlB) on the IL-7 response of CD4 T cells from healthy donors, expressed as the percentage of cells positive for PStat5 nuclear translocation. Response recovery was calculated as

0x(% positive _huG1B+sMR cells - % positive _huG1B cells)/(% positive cells _{in culture medium} - % positive huG1B cells).

Fig. 17 shows that plasma from CD4 non-responding (CD4-NR) patients induces aberrant MMD in HD CD4 T cells.

(a) Images of HD CD4 T cells treated with plasma (1%) from a patient's CD4-NR obtained using structured illumination microscopy (SIM). MMD was stained with cholera toxin B (CtxB-AF488). A prominent portion of the Z-axis images of exemplary CD4 T cells is shown. After stimulation with IL-7 (2 nM, 15 min), no image change was noted (right).

(b) Dose response curve (0.0001%-1%) obtained from plasma from 5 CD4-NR patients (blue curve, mean and SD) and from an exemplary viraemic patient (red curve). Number of abnormal MMD induced on the surface of HD CD4 T cells.

Detailed description of the invention

The present invention relates to compositions and methods for modulating the immune system in a subject in need thereof. In particular, the invention discloses the identification of GIBsPLA2 as a key endogenous immune response factor and provides new therapeutic and diagnostic methods and compositions based on the modulation of this factor.

The hypothesis of the present invention was that chronic activation of the immune system in HIV-infected patients is abnormal and directs CD4 T cells into an aberrant activation/differentiation state in which there is no response to gamma-c cytokines involved in the control of many aspects of immune defense and homeostasis of the CD4 compartment. despite the fact that more than 99.5% of CD4 T cells from the peripheral compartment are uninfected. This hypothesis was evaluated by the authors of the present invention, and the present invention sheds light on the nature and significance of this aberrant activation state.

In particular, in the first aspect, the present invention demonstrates that the characteristics of this condition can be summarized as follows: 1) before any stimulation, all CD4 T cells

- 5 040303 HIV-infected viraemic patients (VP) have numerous large MMDs on their surface, 2) these abnormal MMDs sequester all cellular IL-7R-alpha and gamma-c chains, and 3) this sequestration of the chains in the abnormal MMDs disrupts their ability to induce the formation of a functional signalosome, 4) leading to a slowdown and decrease in STAT5 phosphorylation and 5) a decrease in nuclear import of phospho-STAT5. This abnormal pattern of pre-existing MMD on the surface of VP CD4 T lymphocytes has multiple implications and is a major mechanism that explains the various manifestations of immunodeficiency in HIV-infected patients. Loss of response to IL-7 is an important factor that partly explains the observed CD4 lymphopenia. The persistent loss of these cells in VP, due to their susceptibility to apoptosis and their destruction by low-level but persistent viral proliferation, cannot be compensated for despite increased levels of IL-7. In addition, since abnormal MMD sequester all gamma chains in a non-functional state, this blocks the function of other cytokines of this family.

The present invention further discloses the identification of key endogenous factors responsible for this abnormal state of the immune system in infected subjects and generally responsible for the intense modulation of the immune response in various pathophysiological conditions. Plasma samples from VP have been shown to indeed contain an activity designated as RIF, which is capable of inducing aberrant CD4 T lymphocytes from healthy (HD) donors. RIF was found in all VP plasma samples examined. The pathophysiological significance of this activity has been demonstrated by its absence in controlled HIV (HIC) patients in whom the IL-7/IL-7R system is normal and immune activation is favorable. RIF is also absent from the plasma of ART patients who have reduced immune activation, restored IL7R function, and restored CD4 counts >500/mm ³ (5).

Thus, RIF is the main factor that controls the immune response, in particular through the modulation of CD4 T-lymphocytes. Notably, RIF induces an aberrant pattern of HD CD4 T cell activation that is indistinguishable from that observed directly ex vivo in purified VP CD4 T cells. The invention further shows that RIF is a secreted phospholipase A2 from group IB (PLA2 GIB). The results disclosed in the present invention show that (i) overexpression of PLA2 GIB leads to potent immunosuppression, and that (ii) inhibition of PLA2 GIB leads to a significant increase in the stimulation of immune function. GIBsPLA2 inhibitors were capable of correcting the abnormal state of immune cells in the plasma of subjects, and thus could be used to treat (eg, prevent, correct) immunodeficiency or immune disorders in mammals. Inhibition of GIBsPLA2 can also induce, stimulate, or help maintain the number and function of CD4 T cells, and thus contribute to the stimulation of an effective immune response in patients. In particular, in HIV-infected patients, APT can be reduced or stopped, where a balance can be achieved between the patient's immune defenses and the virus. If APT given early after infection, as suggested by recent studies, is combined with RIF inhibitors, any RIF-induced impairment of the immune system can be prevented. Furthermore, in the context of some of the current APT failures, patients with low CD4 counts after long-term APT may benefit from these inhibitors. Accordingly, the invention provides methods for treating a subject by modulating the expression or activity of GIBsPLA2 in the subject. In particular, the invention provides a method for modulating an immune response in a subject in need thereof, comprising modulating the modulation of GIBsPLA2 expression or activity in said subject.

The data provided in the examples also demonstrate that the presence of RIF in a subject's plasma is indicative of an HIV-induced pathogenesis of the CD4 T cell condition. Accordingly, the present invention provides methods for monitoring and/or diagnosing HIV infection in a subject by determining the plasma level of RIF in the subject, among other things.

The data provided in the examples further demonstrate that the number and/or size of membrane microdomains (MMDs) on a subject's T cells are indicative of an HIV-induced pathogenesis of the CD4 T cell condition. Accordingly, this invention also provides methods for monitoring and/or diagnosing HIV infection in a subject by determining the number and/or size of membrane microdomains (MMDs) on the subject's T cells, among other things.

The data provided in the examples also indicate a role for RIF in creating and/or maintaining the pathological state of CD4 T cells in HIV-infected subjects. Accordingly, this invention also provides methods for identifying a potential therapeutic agent for HIV that involve comparing RIF-induced CD4 T cell activation in the presence of the agent with the level of RIF-induced CD4 T cell activation in the absence of the agent.

Definitions

The term sequence identity, as applied to a nucleic acid or protein sequence, refers to the quantitative expression (usually as a percentage) of nucleotides or amino acid residues that match in at least two sequences aligned using a standardized algorithm such as the Smith-Waterman alignment (Smith

- 6 040303 and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res

22:4673-4680), or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402). BLAST2 can be used in a standardized and reproducible manner to insert spaces into one of the sequences to optimize alignment and to achieve a more meaningful comparison between them.

As used in the present invention, treatment or treatment means a clinical intervention to change the natural course of the individual to be treated, and may be for prophylactic or curative purposes.

The desired effects of treatment include, but are not limited to, prevention of the development or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of progression of the disease, alleviation or amelioration of the disease state, and remission or improvement in prognosis. In some embodiments, the compositions and methods of the invention are used to slow the progression of a disease or disorder, or to slow the progression of a disease or disorder.

The term isolated, as used herein, refers to molecules (e.g., nucleic acid or amino acids) that are removed from a component from their natural environment, isolated, or separated, and which are at least 60% free, preferably 75% free, and most preferably 90% free of other components with which they are naturally associated. An isolated polypeptide (or protein) is, for example, a polypeptide isolated from a component from its natural environment and preferably purified to greater than 90% or 95% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF) , capillary electrophoresis) or chromatography (for example, ion exchange or reverse phase HPLC). Isolated nucleic acid means a nucleic acid molecule that has been separated from a component from its natural environment and/or assembled into a different construct (eg, vector, expression cassette, recombinant host, etc.).

Nucleic acid encoding an anti-GIBsPLA2 antibody means one or more nucleic acid molecules encoding heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) acids present at one or more sites on the host cell.

Subject means mammal. Examples of mammals include humans and non-human animals such as, but not limited to, pets (eg, cows, sheep, and dogs, and horses); non-human primates (such as monkeys); rabbits, and rodents (eg mice and rats).

Modulation of the immune response means, in the context of the present invention, any modification in the number or activity or ratio of immune cells, preferably white blood cells (eg T-lymphocytes, B-lymphocytes, NK, NKT cells, macrophages, dendritic cells). In a particular embodiment, modulating the immune response includes modulating the number or activity of T lymphocytes, preferably CD4 T lymphocytes.

Cell resistance inducing factor (RIF) or group IB phospholipase A2.

The term RIF is used interchangeably with the term group IB phospholipase A2, GIBsPLA2 (or PLA2 GIB). Group IB phospholipase A2 is a secreted protein having a molecular weight of about 15 kDa and an isoelectric point of about 6.5 to 8.0.

In the context of the present invention, the term GIBsPLA2 or group IB phospholipase A2 means any native GIBsPLA2 protein from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., rat mice), unless otherwise indicated. The term encompasses full-length, unprocessed GIBsPLA2, as well as any form of GIBsPLA2 produced by intracellular or extracellular processing. The term also encompasses natural variants of GIBsPLA2, such as splice variants or allelic variants.

The amino acid sequence of approximately human GIBsPLA2 is shown below (SEQ ID NO: 2).

MKLLVLAVLLTVAAADSGISPRAVWOFRKMIKCVIPGSDPFLEYNNYGCYCGLG

GSGTPVDELDKCCQTHDNCYDQAKKLDSCKFLLDNPYTHTYSYSCSGSAITCSSKNKEC

EAFICNCDRNAAICFSKAPYNKAHKNLDTKKYCQS

Amino acids 1-15 of SEQ ID NO: 2 (underlined) are the signal sequence, amino acids 16-22 of SEQ ID NO: 2 (bold) are the propeptide sequence. The mature protein corresponds to amino acid residues 23-148 of SEQ ID NO: 2, which is an example of a truncated human GIBsPLA2 protein.

Natural variants include any protein containing the sequence of SEQ ID NO: 2, or a sequence of amino acid residues 23-148 of SEQ ID NO: 2, with one or more amino acid substitutions, additions and/or deletions of one or more (typically 1, 2

- 7 040303 or 3) amino acid residues, preferably with no more than 10 separate amino acid substitutions, additions and/or deletions of one or more (usually 1, 2 or 3) amino acid residues. Representative natural variants retain the biological activity of SEQ ID NO: 2.

Therefore, in some embodiments, GIBsPLA2 has at least one activity selected from inducing the formation of membrane microdomains (MMDs) in CD4 T cells from healthy subjects, or rendering CD4 T cells of healthy subjects resistant to interleukin signaling, such as resistance to IL-2 signals or resistance to IL-7 signals.

In some embodiments, the induction of MMD formation includes increasing the number of MMD on CD4 T cells of healthy subjects to at least about 80 per cell, at least about 90 per cell, at least about 100 per cell, at least about up to 110 per cell, or at least up to about 120 per cell. In a non-limiting preferred embodiment, the induction of the formation of MMD includes increasing the number of MMD on CD4 T cells of healthy subjects to more than 100 MMD per cell.

In some embodiments, the implementation of the induction of the formation of MMD includes stimulation of the formation of larger MMD than those otherwise present on CD4 T cells. In some embodiments, inducing the formation of larger MMDs includes stimulating the formation of MMDs having a diameter of at least 100 nm, at least 110 nm, at least 120 nm, at least 130 nm, or at least 140 nm. In a non-limiting preferred embodiment, the induction of the formation of larger MMDs includes the stimulation of the formation of MMDs having a diameter greater than 120 nm.

In some embodiments, rendering CD4 T cells of healthy subjects resistant to interleukin-7 signals comprises reducing STAT5A and/or B phosphorylation in said cells by at least about 10%, at least about 20%, at least about 30%. % or at least about 40%. In some embodiments, rendering CD4 T cells of healthy subjects resistant to interleukin-7 signals comprises reducing the level of phospho-STAT5A and/or phospho-STAT5B nuclear translocation by at least about 20%, at least about 30%, at least about 40%, or at least about 50%.

GIBsPLA2 activity can be measured by any suitable method known in the art, as illustrated in the examples, or later developed. GIBsPLA2 activity can be measured in a plasma sample, such as, for example, a fractionated plasma sample, using, for example, ligand assisted assays, immunoassays, and/or enzyme assays.

In a particular embodiment, the term GIBsPLA2 means a human protein, in particular a protein comprising or having SEQ ID NO: 2, or a natural variant thereof.

GIBsPLA2 according to the present invention may be isolated, purified and/or recombinant. In some embodiments, the invention may use, instead of or in addition to the GIBsPLA2 protein, a nucleic acid encoding GIBsPLA2. The nucleic acid may be DNA or RNA, single or double stranded.

An exemplary nucleic acid sequence encoding GIBsPLA2 is shown in SEQ ID NO: 1 below.

ATGAAACTCCTTGTGCTAGCTGTGCTGCTCACAGTGGCCGCCGCCGACAGCG

GCATCAGCCCTCGGGCCGTGTGTGGCAGTTCCGCAAAATGATCAAGTGCGTGATCCCG

GGGAGTGACCCCTTCTTGGAATACAACAACTACGGCTGCTACTGTGGCTTGGGGGGC

TCAGGCACCCCCGTGGATGAACTGGACAAGTGCTGCCAGACACATGACAACTGCTA CGACCAGGCCAAGAAGCTGGACAGCTGTAAATTTCTGCTGGACAACCCGTACACCC ACACCTATTCATACTCGTGCTCTGGCTCGGCAATCACCTGTAGCAGCAAAAACAAAG AGTGTGAGGCCTTCATTTGCAACTGCGACCGCAACGCTGCCATCTGCTTTTCAAAAG CTCCATATAACAAGGCACACAAGAACCTGGACACCAAGAAGTATTGTCAGAGTTGA Альтернативные молекулы нуклеиновой кислоты, кодирующей GIBsPLA2, включают любой вариант SEQ ID NO: 1, который получен вследствие вырожденности генетического кода, а также любую последовательность, гибридизующуюся с SEQ ID NO: 1 в жестких условиях, более предпочтительно имеющую по меньшей least 80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO: 1, and encoding the GIBsPLA2 protein.

Method for obtaining GIBsPLA2

GIBsPLA2 can be produced by any of the usual known expression methods and protein purification methods. For example, (i) a peptide synthesis method can be used; (ii) a method for their purification and isolation from a living organism or cultured cells; (iii) a method for their preparation using genetic recombination techniques; and combinations thereof, or the like (e.g., the standard techniques described in Molecular Cloning (Sambrook, J., Fritsch, E. F., Maniatis, T., Cold Spring Harbor Laboratory Press) (1989) (Molecular Cloning) and Current Protocols in Molecular Biology (Ausubel, F. M., John

Wiley and Sons Inc. (1989) (Modern protocols of molecular biology)).

In a particular embodiment, the invention relates to a method for producing GIBsPLA2 by expressing an encoding nucleic acid in a host cell and harvesting or purifying GIBsPLA2. In this regard, the invention also describes recombinant host cells containing a nucleic acid encoding GIBsPLA2. Such cells may be prokaryotic (such as bacteria) or eukaryotic (such as yeast, insect, plant, or mammalian cells). The nucleic acid can be placed under the control of any suitable regulatory sequence, such as a promoter, terminator, or the like. Alternatively, the nucleic acid may be inserted into the host cell at a site where expression is driven by an endogenous promoter. Techniques for inserting nucleic acids into cells are well known in the art.

GIBsPLA2 modulation

The invention provides new methods involving the modulation of GIBsPLA2 in a subject in need thereof. The term modulation means any modification of the level (eg, expression) or activity of GIBsPLA2 in a subject. In addition, modulation means either an increase or decrease in the level or activity of GIBsPLA2. More preferably, modulation means a change of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more, compared to the unmodulated state. As a result, inhibition of GIBsPLA2 means a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the level or activity of GIBsPLA2, as well as complete blockade or suppression of the level or activity of GIBsPLA2. In contrast, GIBsPLA2 stimulation means an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of GIBsPLA2 level or activity. Depending on the situation, modulation can be temporary, continuous or continuous. Further, modulating activity includes modulating the amount of GIBsPLA2 in a subject, particularly in body fluids, modulating protein potency (eg, by modulating the level of cofactors or substrate in a subject), and modulating the level or activity of degradation products produced by GIBsPLA2.

GIBsPLA2 inhibition

In a particular embodiment, the invention provides compositions and methods for inhibiting GIBsPLA2 in a subject. Inhibition of GIBsPLA2 can be achieved by the use of GIBsPLA2 inhibitors, ie. any compound that inhibits the expression or activity of GIBsPLA2. GIBsPLA2 inhibitors include expression inhibitors, antagonists, sequestrators, or target-masking compounds. Preferred types of GIBsPLA2 inhibitors include GIBsPLA2 ligands (covalent or non-covalent), anti-GIBsPLA2 antibodies (and fragments and derivatives thereof), nucleic acids encoding anti-GIBsPLA2 antibodies (or fragments and derivatives thereof), inhibitory nucleic acids, peptides, or small molecular weight drugs, or a combination(s) thereof. Alternatively or in addition, inhibition of GIBsPLA2 can be achieved by vaccinating the subject against the GIBsPLA2 antigen such that antibodies are produced in the subject in need of GIBsPLA2 inhibition.

Antibodies against GIBsPLA2

Specific examples of GIBsPLA2 inhibitors are antibodies that specifically bind to GIBsPLA2.

Antibodies can be synthetic, monoclonal, or polyclonal, and can be obtained by methods well known in the art. Such antibodies specifically bind through the antigen-binding sites of the antibody (as opposed to non-specific binding). GIBsPLA2 polypeptides, fragments, variants, fusion proteins, etc. can be used as immunogens in obtaining antibodies that give an immune reaction with them. In particular, polypeptides, fragments, variants, fusion proteins, etc. contain antigenic determinants or epitopes that cause the formation of antibodies.

These antigenic determinants or epitopes may be linear or conformational (discontinuous). Linear epitopes consist of a single section of amino acids from a polypeptide, while conformational or discontinuous epitopes consist of amino acid sections with different regions of the polypeptide chain that come close together as the protein folds (C.A. Janeway, Jr. and P. Travers , Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded proteins have complex surfaces, the number of epitopes available is quite large; however, due to protein conformation and steric constraints, the number of antibodies that actually bind to epitopes is less than the number of available epitopes (C.A. Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed. 1996) ). Epitopes can be identified by any means known in the art. Both polyclonal and monoclonal antibodies can be prepared by standard techniques.

Preferred antibodies of the present invention are directed to a GIBsPLA2 epitope and/or generated by immunization with a polypeptide comprising a GIBsPLA2 epitope selected from a mature GIBsPLA2 protein, a GIBsPLA2 fragment comprising at least 8 contiguous amino acid sequences from SEQ ID NO: 2 (or corresponding residues of the natural variant SEQ ID

NO: 2), where the specified fragment includes at least amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof. Preferred antibodies of the present invention bind an epitope, including an epitope between amino acid residues 50-71 of SEQ ID NO: 2, or the corresponding residues from the natural variant of SEQ ID NO: 2.

The term antibodies is intended to include polyclonal antibodies, monoclonal antibodies, fragments thereof such as F(ab')2 and Fab fragments, single chain variable fragments (scFvs), single domain antibodies (VHH or nanobodies), bivalent antibody fragments (diabodies); as well as any recombinant and synthetically derived binding partners, human antibodies or humanized antibodies.

Antibodies are defined as specifically binding if they preferentially bind to GIBsPLA2 with a Ka value equal to or greater than about 10 ⁷ M' ¹ . Antibody affinities can be readily determined using conventional techniques such as those described by Scatchard et al, Ann. NY Acad. Sc. 51:660 (1949).

Polyclonal antibodies can be easily obtained from various sources, such as horses, cows, donkeys, goats, sheep, dogs, chickens, rabbits, mice or rats, using procedures well known in the art. In general, purified GIBsPLA2 or a suitably conjugated peptide based on the GIBsPLA2 amino acid sequence is administered to the acceptor animal, typically by parenteral injection. The immunogenicity of GIBsPLA2 can be enhanced by the use of an adjuvant, such as complete or incomplete Freund's adjuvant. After booster immunization, small serum samples are collected and tested for reactivity against the GIBsPLA2 polypeptide. Examples of various assays suitable for such determination include those described in Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988 (Antibodies: a laboratory manual); as well as procedures such as cross-sectional immunoelectrophoresis (EIEF), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blotting, and sandwich assays. See U.S. Patent Nos. 4,376,110 and 4,486,530.

Monoclonal antibodies can be easily obtained using well known procedures. See, for example, the procedures described in US Pat. Nos. RE 32011, 4902614, 4543439 and 4411993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyzes, Plenum Press, Kennett, McKeam, and Bechtol (eds.), 1980 (Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Assays).

For example, acceptor animals, such as mice, can be intraperitoneally administered at least once and preferably twice about 3 weeks apart with isolated and purified non-mutated or mutated GIBsPLA2 protein or GIBsPLA2 conjugated peptide, optionally in the presence of an adjuvant.

Mouse sera are then analyzed by conventional dot blot or antibody immobilization (ABC) techniques to determine which animal is best used. Approximately two to three weeks later, the mice receive an intravenous booster injection of the protein or peptide. The mice are then sacrificed and the spleen cells are fused with commercial myeloma cells such as Ag8.653 (ATCC) following established protocols. Briefly, myeloma cells are washed several times with medium and hybridized with mouse spleen cells at a ratio of about three spleen cells per myeloma cell. The hybridizing agent may be any suitable agent used in the art, such as polyethylene glycol (PEG). Hybridomas are placed on plates containing a medium that provides selective growth of hybrid cells. The hybrid cells can then be allowed to grow for about eight days. Supernatants from the resulting hybridomas are harvested and added to a plate which is first coated with goat anti-mouse immunoglobulins. After washing, a label, such as a GIBsPLA2 tagged polypeptide, is added to each well and then incubated. Positive wells can then be identified. Positive clones are then grown in mixed culture and then the supernatant is purified on a protein A column (Pharmacia).

The monoclonal antibodies of the invention can be prepared using alternative techniques such as those described by Alting-Mees et al., Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas, Strategies in Molecular Biology 3:1-9 (1990) (Expression Libraries monoclonal antibodies: a rapid alternative to hybridomas), incorporated by reference. Similarly, binding partners can be designed using recombinant DNA techniques to include different regions of a gene encoding a specific binding antibody. Such a technique is described, for example, in Larrick et al., Biotechnology, 7:394 (1989).

Antigen-binding fragments of such antibodies, which can be obtained by conventional techniques, are also covered by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab') ₂ fragments. Also provided are fragments and derivatives of antibodies obtained by genetic engineering techniques.

Monoclonal antibodies of the present invention include chimeric antibodies, such as humanized versions of mouse monoclonal antibodies. Such humanized antibodies can be prepared by known techniques and provide the advantage of reduced immunogenicity when administered to a human.

In one embodiment, the humanized monoclonal antibodies comprise the variable region of a mouse antibody (or simply its antigen-binding region) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise an antigen-binding region of a mouse monoclonal antibody and a variable region fragment (not containing the antigen-binding region) derived from a human antibody. Procedures for making chimeric and other engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, May, 1993). Procedures for transgenic production of antibodies can be found in GB 2272440, US Pat. Nos. 5,569,825 and 5,545,806.

Genetically engineered antibodies can be used, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, that can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be obtained by genetic engineering using standard DNA techniques known in the art, for example, using the methods described in Robinson et al., International Publication No. WO 87/02671; Akira, et al. European Patent Application 0184187; Taniguchi, M., European Patent Application 0171496; Morrison et al., European Patent Application 0173494; Neuberger et al., PCT International Patent Publication No. WO 86/01533; Cabilly et al., US Pat. No. 4,816,567; Cabilly et al., European Patent Application 0125023; Better et al. Science 240:1041 1043, 1988; Liu et al., PNAS 84:3439 3443, 1987; Liu et al., J. Immunol. 139:3521 3526, 1987; Sun et al. PNAS 84:214 218, 1987; Nishimura et al., Cane. Res. 47:999 1005, 1987; Wood et al., Nature 314:446 449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80:1553 1559, 1988); Morrison, S. L., Science 229:1202 1207, 1985; Oi et al., BioTechniques 4:214, 1986; Winter U.S. Pat. no. 5,225,539; Jones et al., Nature 321:552 525, 1986; Verhoeyan et al., Science 239:1534, 1988; n Beidler et al., J. Immunol. 141:4053 4060, 1988.

With respect to synthetic and semi-synthetic antibodies, such terms are intended to cover antibody fragments, isotype-switched antibodies, humanized antibodies (e.g., mouse-human, human-mouse), hybrids, multispecific antibodies, and fully synthetic antibody-like molecules, but not to limit them.

For therapeutic applications, human monoclonal antibodies having human constant and variable regions are often preferred in order to minimize the patient's immune response to the antibody. Such antibodies can be obtained by immunizing transgenic animals that contain human immunoglobulin genes. See Jacobovits et al. Ann NY Acad Sci 764:525-535 (1995).

Human monoclonal antibodies against GIBsPLA2 polypeptides can also be prepared by constructing a combinatorial immunoglobulin library, such as a Fab phage display library or scFv phage display library, using immunoglobulin light and heavy chain cDNA prepared from mRNA extracted from subject's lymphocytes. See, for example, McCafferty et al., PCT Publication WO 92/01047; Marks et al. (1991) J. Mol. Biol. 222:581 597; and Griffths et al. (1993) EMBO J 12:725-734. In addition, a combinatorial library of antibody variable regions can be generated by mutating a known human antibody. For example, the variable region of a human antibody known to bind to GIBsPLA2 can be mutated, eg using randomly altered mutant oligonucleotides, to generate a library of mutant variable regions, which can then be screened for binding to GIBsPLA2. Methods for inducing non-specific mutagenesis in the CDR regions of immunoglobulin heavy and/or light chains, methods for pairing randomized heavy and light chains, and screening methods can be found, for example, in Barbas et al., PCT publication WO 96/07754; Barbas et al. (1992) Proc. Nat'l Acad. sci. USA 89:4457-4461.

An immunoglobulin library can be expressed by a population of phage display, preferably selected from filamentous phage, to form an antibody display library. Examples of methods and reagents particularly useful in generating an antibody display library can be found, for example, in Ladner et al. U.S. Pat. no. 5,223,409; Kang et al., PCT publication WO 92/18619; Dower et al., PCT publication WO 91/17271; Winter et al., PCT publication WO 92/20791; Markland et al., PCT publication WO 92/15679; Breitling et al., PCT publication WO 93/01288; McCafferty et al., PCT publication WO 92/01047; Garrard et al., PCT publication WO 92/09690; Ladner et al., PCT publication WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81 85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) supra; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982. Once compounds are exposed on the surface of phages (eg, filamentous phage), the antibody library is screened to identify and isolate those expressing antibodies that bind the GIBsPLA2 polypeptide. In the preferred embodiment

- 11 040303 primary screening of the library involves panning with the immobilized GIBsPLA2 polypeptide and selecting those that express antibodies that bind the immobilized GIBsPLA2 polypeptide.

In a particular embodiment, the invention relates to a composition comprising an anti-GIBsPLA2 antibody (or fragment or derivative thereof) and a pharmaceutically acceptable excipient.

Existing monoclonal antibodies to phospholipase A2-GIB include Mab CH-7 (Labome), MAB5018 (Labome), EPR5186 (Genetex); LS-C138332 (Lifespan) or CABT-17153MH (Creative biomart). Examples of polyclonal antibodies include, for example, N1C3 from GeneTex. As stated above, preferred anti-GIBsPLA2 antibodies of the present invention bind mature GIBsPLA2, even more preferably an epitope contained in a domain from GIBsPLA2 comprising an amino acid selected from amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or their combinations. Preferred antibodies of the present invention bind an epitope containing amino acid residues 50-71 of SEQ ID NO: 2, or the corresponding residues from the natural variant of SEQ ID NO: 2.

In an alternative embodiment, the invention relates to a composition comprising a nucleic acid encoding an antibody to GIBsPLA2 (or a fragment or derivative thereof) and a pharmaceutically acceptable excipient, and its use.

inhibitory nucleic acids

In an alternative embodiment, the GIBsPLA2 inhibitor is an inhibitory nucleic acid, i. any nucleic acid molecule that inhibits the expression of the GIBsPLA2 gene or protein. Preferred inhibitory nucleic acids include antisense nucleic acids, small interfering RNA (siRNA), small hairpin RNA (shRNA), miRNA, aptamers or ribozymes. In a particular embodiment, the inhibitory nucleic acid is a small interfering RNA that prevents translation of the GIBsPLA2 mRNA. In another particular embodiment, the inhibitory nucleic acid is an antisense oligonucleotide that prevents translation of the GIBsPLA2 mRNA. In another particular embodiment, the inhibitory nucleic acid is a small hairpin RNA that prevents translation of the GIBsPLA2 mRNA.

miRNA contains the sense nucleic acid sequence and the antisense nucleic acid sequence of the polynucleotide of interest. miRNA is designed so that a single transcript (double-stranded RNA) has a sense and a complementary antisense sequence from the target gene. The siRNA nucleotide sequence can be designed using the siRNA design computer program available, for example, from the Ambion website on the Internet.

In some embodiments, the antisense oligonucleotide or siRNA is 10 nucleotides or less in length. In some embodiments, the length of the antisense oligonucleotides and siRNA is equal to the length of the natural transcript. In some embodiments, the implementation of antisense oligonucleotides and siRNA contain 18-30. In some embodiments, the antisense oligonucleotides and siRNAs are less than 25 nucleotides in length.

A preferred inhibitory nucleic acid molecule contains a domain having a nucleotide sequence fully complementary to the GIBsPLA2 gene or RNA region. Such a domain contains, as a rule, from 4 to 20 nucleotides, providing specific hybridization and optimal inhibition of gene transcription or RNA translation. The inhibitory nucleic acid sequence can be derived directly from the sequence of the gene encoding GIBsPLA2, such as SEQ ID NO: 1. Alternatively or in addition, inhibitory nucleic acids can hybridize to a regulatory element in the GIBsPLA2 gene or RNA, such as a promoter, splicing site, etc. .d., and prevent effective regulation.

Specific examples of inhibitory nucleic acid molecules of the present invention include isolated single-stranded nucleic acid molecules consisting of 10-50 consecutive nucleotides from SEQ ID NO: 1. Specific examples of inhibitory nucleic acid molecules of the present invention are antisense nucleic acids consisting of the following nucleotide sequence or chain completely complementary to it:

ATGAAACTCCTTGTGCTAG (SEQ ID NO: 3)

ACAGCGGCATCAGC (SEQ ID NO: 4)

TTCCGCAAAATGATCAA (SEQ ID NO: 5)

CCCGGGGAGTGACCCC (SEQ ID NO: 6)

TACGGCTGCTACTGTGGCTT (SEQ ID NO: 7)

- 12 040303

GACACATGACAACTGCTACGACC (SEQ ID NO: 8) ACCCACACCTATTCATACTCGT (SEQ ID NO: 9) ATCACCTGTAGCAGCA (SEQ ID NO: 10) AGCTCCATATAACAAGGCA (SEQ ID NO: 11) CAAGAAGTATTGTCAGAG (SEQ ID NO: 12) Peptide and small molecule drugs

In an alternative embodiment, the GIBsPLA2 inhibitor is a peptide or small molecule drug that inhibits GIBsPLA2 activity. The peptide or small molecule drug is typically a molecule that selectively binds to GIBsPLA2, or a GIBsPLA2 substrate, or a GIBsPLA2 cofactor, or a degradation product or metabolite of the GIBsPLA2 pathway.

Peptides preferably contain from 3 to 20 amino acid residues, and their sequence may be identical to the domain of GIBsPLA2 (bait peptide) or the domain of the substrate, cofactor, degradation product or metabolite of GIBsPLA2. Preferred peptides of the present invention contain from 4 to 30 consecutive amino acid residues from SEQ ID NO: 2 (or the corresponding sequence from the natural variant of SEQ ID NO: 2). The most preferred peptides of the present invention contain from 5 to 25 consecutive amino acid residues from SEQ ID NO: 2 (or the corresponding sequence from the natural variant of SEQ ID NO: 2) and additionally contain at least one of the following amino acid residues from SEQ ID NO: 2 (or the corresponding sequence from the natural variant of SEQ ID NO: 2): amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof. Specific examples of peptides of the present invention are peptides of less than 25 amino acids containing any of the following sequences:

NNYGCY (SEQ ID NO: 13)

CYCLG (SEQ ID NO: 14)

YNNYGCYCGLGGSG (SEQ ID NO: 15)

FLEYNNYGCYCGLGGSGTPV (SEQ ID NO: 16)

QTHDN (SEQ ID NO: 17)

CQTHDNC (SEQ ID NO: 18)

ECEAFICNC (SEQ ID NO: 19)

DRNAAI (SEQ ID NO: 20)

DRNAAICFSKAPYNKAHKNL (SEQ ID NO: 21)

The peptides of the present invention may include peptide, non-peptide and/or modified peptide bonds. In a particular embodiment, the peptides include at least one peptidomimetic bond selected from the intercalation of a methylene (-CH2-) or phosphate (-PO2-) group, a secondary amine (-NH-) or oxygen (-O-), alpha-azapeptides, alpha-alkyl peptides, N-alkyl peptides, phosphonamidates, depsipeptides, hydroxymethylenes, hydroxyethylenes, dihydroxyethylenes, hydroxyethylamines, retro-inverso peptides, methyleneoxy, cetomethylene, esters, phosphinates, phosphines, or phosphonamides. In addition, the peptides may contain a protected N and/or C-terminus, for example by acylation and/or amidation and/or esterification.

The peptides of the present invention can be obtained by methods known in the art, such as chemical, biological and/or genetic synthesis.

Each of these peptides in isolated form represents a specific subject of the present invention.

Preferred small molecule drugs are hydrocarbon compounds that selectively bind to GIBsPLA2.

Small molecule drugs and peptides are preferably prepared by a method comprising (i) contacting a test compound with GIBsPLA2 or a fragment thereof, (ii) selecting a test compound that binds to GIBsPLA2 or said fragment thereof, and (iii) selecting a compound from point (ii) , which inhibits GIBsPLA2 activity. Such a method is a specific object of the present invention.

Small molecule drugs and peptides are also preferably prepared by a method comprising (i) contacting a test compound with a substrate, cofactor, or degradation product of GIBsPLA2 or a fragment thereof, (ii) selecting a test compound that binds to said substrate, cofactor, or degradation product of GIBsPLA2 , or a fragment of it, and (iii)

- 13 040303 selecting a compound from point (ii) which inhibits the activity of GIBsPLA2. Such a method is a specific object of the present invention.

Soluble GIBsPLA2 receptors

In an alternative embodiment, the GIBsPLA2 inhibitor is a soluble form of the GIBsPLA2 receptor. Such compounds, which are soluble receptors, are able to bind GIBsPLA2, thus inhibiting its activity, acting as a bait or masking agent.

In a specific embodiment, such inhibitors are a soluble form of the human or mouse GIBsPLA2 receptor or GIBsPLA2 binding fragment thereof.

The amino acid sequences of mouse and human soluble receptors are shown in SEQ ID NOs: 22 and 23, respectively. Thus, the term soluble receptor encompasses any GIBsPLA2 binding polypeptide comprising the entire sequence of SEQ ID NOs: 22 and 23 or a fragment thereof.

GIBsPLA2-binding fragment means any fragment of such a polypeptide, preferably including at least 5 consecutive amino acid residues, more preferably at least 8, 10 or 12, which specifically binds PLA2GIB. Specific binding of the receptor molecule indicates that the receptor molecule binds to PLA2GIB with a higher affinity (eg, at least 5-fold) than to PLA2-IIA or IID. A fragment as defined above most preferably comprises less than 50 amino acid residues.

Examples of GIBsPLA2 binding polypeptides are, without limitation, polypeptides comprising at least one of the following amino acid sequences:

LSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKW (SEQ ID

NO: 24)

WEKDLNSHICYQFNLLS (SEQ ID NO: 25)

DCESTLPYICKKYLNHIDHEIVEK(SEQ ID NO: 26)

QYKVQVKSDNTVVARKQIHRWIAYTSSGGDICE(SEQ ID NO: 27)

LSYLNWSQEITPGPFVEHHCGTLEVVSA (SEQ ID NO: 28)

SRFEQAFITSLISSVAEKDSYFW (SEQ ID NO: 29)

WICRIPRDVRPKFPDWYQYDAPWLFYQNA (SEQ ID NO: 30)

AFHQAFLTVLLSRLGHTHWIGLSTTDNGQT (SEQ ID NO: 31)

SEQ ID NOs: 24-26 are derived from the sequence of the human soluble PLA2GIB receptor, while SEQ ID NOs: 27-31 are derived from the sequence of the mouse soluble PLA2GIB receptor.

Vaccination

In an alternative (or cumulative) embodiment, inhibition of GIBsPLA2 in a subject is achieved by vaccinating (or immunizing) the subject with GIBsPLA2 antigen. As a result of such vaccination or immunization, the subject produces antibodies (or cells) that inhibit GIBsPLA2. In particular, injection(s) of a GIBsPLA2 antigen (eg, an immunogenic GIBsPLA2 substantially devoid of biological activity) generates antibodies in the subject being treated. These antibodies will protect against overexpression of GIBsPLA2 and can be used as immunotherapy or vaccine prophylaxis.

Thus, it is an object of the present invention to provide a method for vaccinating a subject, comprising administering a GIBsPLA2 antigen to the subject.

Another object of the invention is a GIBsPLA2 antigen for use in vaccinating a subject in need thereof.

In a particular embodiment, the GIBsPLA2 antigen used for vaccination is an inactivated immunogenic molecule that induces an immune response against GIBsPLA2 in a subject. Inactivation can be achieved, for example, by chemical or physical alteration of GIBsPLA2, or by mutation with either a truncated protein or both; and immunogenicity can be achieved by inactivation and/or by additional conjugation of the protein with a suitable carrier or hapten such as KLH, HSA, polylysine, viral toxoid, or the like, and/or by polymerization, or the like. Thus, the antigen can be chemically or physically modified, for example to improve its immunogenicity.

In a preferred embodiment, the GIBsPLA2 antigen of the present invention comprises GIBsPLA2 or an epitope-containing fragment or mimotope thereof.

In a particular embodiment, the GIBsPLA2 antigen comprises the full length GIBsPLA2 protein. In another particular embodiment, the GIBsPLA2 antigen comprises a protein comprising SEQ ID

- 14 040303

NO: 2, or a sequence having at least 90% identity with SEQ ID NO: 2.

In an alternative embodiment, the GIBsPLA2 antigen comprises a GIBsPLA2 protein fragment comprising at least 6 consecutive amino acid residues and containing an immunogenic epitope or mimotope thereof. In a preferred embodiment, the GIBsPLA2 antigen comprises at least 6-20 amino acid residues. Preferred peptides of the present invention contain from 4 to 30 consecutive amino acid residues from SEQ ID NO: 2 (or the corresponding sequence from the natural variant of SEQ ID NO: 2). The most preferred peptides of the present invention comprise 5 to 25 consecutive amino acid residues from SEQ ID NO: 2 (or the corresponding sequence from the natural variant of SEQ ID NO: 2) and further contain at least one of the following amino acid residues from SEQ ID NO: 2 (or the corresponding sequence from the natural variant of SEQ ID NO: 2): amino acid 70, amino acid 121, amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof. Specific examples of peptides of the present invention are peptides of less than 50 amino acids containing any of the following sequences:

NNYGCY (SEQ ID NO: 13)

CYCLG (SEQ ID NO: 14)

YNNYGCYCGLGGSG (SEQ ID NO: 15)

FLEYNNYGCYCGLGGSGTPV (SEQ ID NO: 16)

QTHDN (SEQ ID NO: 17)

CQTHDNC (SEQ ID NO: 18)

ECEAFICNC (SEQ ID NO: 19)

DRNAAI (SEQ ID NO: 20)

DRNAAICFSKAPYNKAHKNL (SEQ ID NO: 21).

The GIBsPLA2 antigen can be in various forms, such as free form, polymerized, chemically or physically modified, and/or linked (ie linked) to a carrier molecule. Carrier linkage can increase immunogenicity and (additionally) inhibit the biological activity of the GIBsPLA2 polypeptide. In this regard, the carrier molecule can be any carrier molecule or protein commonly used in immunology, such as KLH (keyhole limpet hemocyanin), ovalbumin, bovine serum albumin (BSA), viral or bacterial toxoid, such as tetanus toxoid. , diphtheria toxin, cholera toxin B-subunit, their mutants such as diphtheria toxin CRM 197, vesicular outer membrane protein, polylysine molecule, or virus-like particle (VLP). In a preferred embodiment, the carrier is KLH or CRM197 or VLP.

Coupling of GIBsPLA2 to a carrier can be accomplished by covalent chemistry, using linking chemical groups or reactions such as glutaraldehyde, biotin, etc., for example. Preferably, the conjugate or GIBsPLA2 protein or fragment or mimotope is treated with formaldehyde to completely inactivate GIBsPLA2.

In a particular embodiment, the GIBsPLA2 antigen comprises a full length GIBsPLA2 protein optionally linked to a carrier protein. In a preferred embodiment, the GIBsPLA2 antigen comprises a protein comprising SEQ ID NO: 2 or a sequence having at least 90% identity with SEQ ID NO: 2 linked to a carrier protein.

In another particular embodiment, the GIBsPLA2 antigen comprises an immunogenic peptide or mimotope from GIBsPLA2 optionally linked to a carrier protein. In a more preferred embodiment, the GIBsPLA2 antigen comprises a polypeptide at least 10 amino acids long containing at least one of the following amino acid residues from SEQ ID NO: 2 (or from the corresponding sequence of the natural variant of SEQ ID NO: 2): amino acid 70, amino acid 121 , amino acid 50, amino acid 52, amino acid 54, amino acid 71, or a combination thereof optionally linked to a carrier molecule.

The immunogenicity of the GIBsPLA2 antigen can be tested in a variety of ways, such as by immunizing a non-human animal transplanted with human immune cells, followed by testing for the presence of antibodies, or by sandwich ELISA using human or humanized antibodies. The absence of biological activity can be confirmed by any of the activity assays described in the application. In a preferred embodiment, the GIBsPLA2 antigen has less than 20%, more preferably less than 15%, 10%, 5% or even 1% of the wild-type GIBsPLA2 protein activity in a method of (i) inducing MMD membrane microdomain formation in CD4 T cells or (ii) inducing resistance CD4 T cells to IL-2 signaling or resistance to IL-7 signaling.

- 15 040303

In a particular embodiment, the invention relates to an inactivated and immunogenic

GIBsPLA2.

In another particular embodiment, the invention relates to a GIBsPLA2 protein, or fragment or mimotope, conjugated to a carrier molecule, preferably KLH.

In another aspect, the invention relates to a vaccine comprising a GIBsPLA2 antigen, a suitable excipient, and optionally a suitable adjuvant.

Such molecules and conjugates and vaccines represent potent agents for use in immunizing subjects to provide long-term inhibition of GIBsPLA2. When repeated, such methods can be used to induce permanent inhibition of GIBsPLA2.

Another object of the present invention relates to a method of inducing the production of antibodies that neutralize the activity of endogenous GIBsPLA2 in a subject in need of it, where the method includes applying to said subject a therapeutically effective amount of GIBsPLA2 antigen or vaccine.

Administration of an antigen or vaccine of the present invention may be by any suitable route, such as by injection, preferably intramuscular, subcutaneous, transdermal, intravenous, or intra-arterial; or by intranasal, oral, mucosal or rectal administration.

GIBsPLA2 antigen or vaccine can be used to treat any disease associated with excessive production of GIBsPLA2. In particular, the present invention relates to a method of treating a disease associated with excessive production of GIBsPLA2 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a GIBsPLA2 antigen or a vaccine composition containing a GIBsPLA2 antigen.

GIBsPLA2 agonists or activators

The term GIBsPLA2 agonist in the context of the present invention encompasses any substance having, or mediating, or upregulating GIBsPLA2 activity, such as, without limitation, a peptide, polypeptide, recombinant protein, conjugate, natural or artificial ligand, degradation product, homologue, nucleic acid , DNA, RNA, aptamer, etc., or a combination thereof. The term agonist encompasses both full and partial agonists. A particular example of a GIBsPLA2 agonist is a GIBsPLA2 protein or a nucleic acid encoding a GIBsPLA2 protein.

In a particular embodiment, the invention provides methods for inhibiting an immune response in a subject, comprising administering to the subject a GIBsPLA2 protein or a nucleic acid encoding a GIBsPLA2 protein.

Compositions

The invention also relates to compositions comprising a GIBsPLA2 modulator or antigen as described herein as the active ingredient, and preferably a pharmaceutically acceptable carrier.

Pharmaceutical composition means a composition of a compound of the present invention (active ingredient) and a medium generally accepted in the art for delivering biologically active compounds to a subject in need thereof. Such a carrier includes all pharmaceutically acceptable carriers, diluents, vehicles or supports. Common pharmaceutical practice may be used to provide suitable formulations or compositions for a subject, eg in a dosage form.

The compounds or compositions according to the invention may be presented in the form of an ointment, paste, liquid solutions, suspensions, tablets, gelatin capsules, capsules, suppositories, powders, nasal drops or aerosol, preferably in the form of a solution or suspension for injection. For injection, the compounds are typically packaged in the form of liquid suspensions which can be administered, for example, by syringe or by perfusion. In this regard, the compounds are usually dissolved in saline, physiological, isotonic or buffer solutions compatible with pharmaceutical use and known to the person skilled in the art. Thus, the compositions may contain one or more agents or adjuvants selected from dispersants, solubilizers, stabilizers, preservatives, and the like. Agents or excipients which can be used in liquid and/or injectable formulations are in particular methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, etc. The carrier may also be selected from, for example, methyl-beta-cyclodextrin, an acrylic acid polymer (such as carbopol), a mixture of polyethylene glycol and propylene glycol, monoethanolamine and hydroxymethylcellulose.

The compositions typically contain an effective amount of a compound of the present invention, for example an amount that is effective to modulate GIBsPLA2. Typically, compositions in accordance with the present invention contain from about 1 μg to 1000 mg GIBsPLA2 modulator, such an amount as 0.001-0.01; 0.01-0.1; 0.05-100; 0.05-10; 0.05-5; 0.05-1; 0.1-100; 0.1-1.0; 0.1-5; 1.0-10; 5-10; 10-20; 20-50 and 50-100 mg, eg 0.05 to 100 mg, preferably 0.05 to 5 mg, eg 0.05; 0.1; 0.2; 0.3; 0.4; 0.5; 1; 2; 3; 4 or 5 mg. Dosage may be adjusted by one skilled in the art.

- 16 040303 technique, depending on modulator and disease.

Compositions of the present invention may further include one or more additional active compounds, for simultaneous or sequential use.

The invention also relates to a method for preparing a pharmaceutical composition, comprising mixing the GIBsPLA2 modulator as described above and a pharmaceutically acceptable excipient, and obtaining the composition in any suitable form or container (syringe, ampoule, vial, bottle, sachet, etc.).

The invention also relates to a kit comprising (i) a composition containing the GIBsPLA2 modulator as described above, (ii) at least one container, and optionally (iii) written instructions for use of the kit.

Diseases

The compounds and compositions of the present invention can be used to treat any disease associated with an inappropriate (e.g., defective or inappropriate) immune response, in particular inappropriate CD4 T cell activity, as well as any disease where increased immunity can alleviate the condition of the subject. These diseases are sometimes referred to as immune disorders in the present invention. These include immunodeficiency conditions (eg, caused by viral infection, pathogenic infection, cancer, etc.), autoimmune diseases, transplantation, diabetes, inflammatory diseases, cancer, allergies, asthma, psoriasis, urticaria, eczema, and the like.

Immunodeficiencies and related diseases

In a first aspect, the invention is based on the inhibition of GIBsPLA2 in a subject, with an increase or restoration of immune activity, in particular activity mediated by CD4 T cells.

Thus, in a particular embodiment, the invention is directed to methods of stimulating the immune response of a subject in need thereof, comprising inhibition of GIBsPLA2 in said subject.

In a particular embodiment, the invention is directed to methods for modulating white blood cells in a subject in need thereof, comprising inhibition of GIBsPLA2 in said subject.

Examples of diseases in which GIBsPLA2 inhibitors may be useful are all immunodeficiency diseases such as HIV-mediated immunodeficiency. Accordingly, in a particular embodiment, the invention is directed to methods for treating an immunodeficiency or related disorder in a subject in need thereof, comprising inhibition of GIBsPLA2 in said subject.

In another particular embodiment, the invention is directed to a GIBsPLA2 inhibitor for use in the treatment of an immunodeficiency or related disorder in a subject in need thereof.

Immunodeficiencies and related disorders means any condition or pathology characterized by and/or caused by a decrease in immune function or response in a subject. Immunodeficiencies can be caused, for example, by a viral infection (eg, HIV, hepatitis B, etc.), a bacterial infection, cancer, or other pathological conditions. Thus, the term immunodeficiency disorder means any disease caused by or associated with immunodeficiency. The invention is particularly useful in the treatment of CD4 T cell related immunodeficiencies and related diseases. The present application does demonstrate that the biological effects of GIBsPLA2 are involved in the CD4 T cell disease state. Accordingly, blockade of GIBsPLA2 activity has a therapeutic effect in immunodeficiency-induced cytokine dysregulatory subjects, as often seen in patients infected with HIV.

Accordingly, in a particular embodiment, the invention relates to methods of treating HIV infection in a subject by inhibiting GIBsPLA2 in the subject, preferably by administering a GIBsPLA2 inhibitor or vaccine to the subject. In some embodiments, the subject is an HIV-infected patient at an early stage of the disease, and the methods result in an increased likelihood that the patient will be able to control HIV. In some embodiments, the subject is a patient with poor immune reconstitution after antiretroviral treatment, and/or with severe idiopathic CD4 T lymphopenia (ICL). The invention also relates to a method for increasing CD4 T cell activity in an HIV-infected subject by inhibiting GIBsPLA2 in the subject, preferably by administering a GIBsPLA2 inhibitor or vaccine to the subject.

In another embodiment, the invention relates to methods for treating acute and/or chronic inflammation and the process resulting from inflammatory responses in a subject by administering GIBsPLA2 to the subject, directly or together with anti-inflammatory drugs.

The invention also provides methods for treating cancer by enhancing an immune response in a subject, comprising inhibiting GIBsPLA2 in the subject, preferably by administering a GIBsPLA2 inhibitor or vaccine to the subject. The invention also provides methods for treating CD4 T cell-associated immunodeficiency associated with cancer in a subject by inhibiting GIB-17 040303 sPLA2 in the subject, preferably by administering a GIBsPLA2 inhibitor or vaccine to the subject.

Pathological immune response and related diseases

The invention can be used to treat any disease associated with an inappropriate (eg pathological or inappropriate) immune response or with unwanted (hyper)activity or (hyper)activation of the immune system, in particular inappropriate activity of CD4 T cells. These diseases include, for example, autoimmune diseases, transplantation, diabetes, allergies, asthma, psoriasis, urticaria, eczema, and the like.

Thus, in another aspect, the invention is based on the activation or induction of GIBsPLA2 in a subject, with the inhibition of immune activity, in particular activity mediated by CD4 T cells.

Thus, in a particular embodiment, the invention is directed to methods for inhibiting an immune response in a subject in need thereof, comprising inducing or activating GIBsPLA2 in said subject.

In a particular embodiment, the invention is directed to methods for inhibiting white blood cells in a subject in need thereof, comprising inhibiting GIBsPLA2 in said subject.

In another particular embodiment, the invention is directed to methods of treating a disorder caused by an unwanted immune response in a subject in need thereof, comprising inducing or activating GIBsPLA2 in said subject.

Inducing or activating GIBsPLA2 in a subject preferably involves administering to the subject a GIBsPLA2 agonist, eg a GIBsPLA2 protein or functional fragment thereof.

In another particular embodiment, the invention is directed to a GIBsPLA2 agonist or activator for use in the treatment of a disorder caused by an unwanted immune response in a subject in need thereof.

Examples of diseases for which GIBsPLA2 agonists can be used are autoimmune disorders, cancer, viral diseases, bacterial infections, and so on.

In a particular embodiment, the invention is directed to methods for treating an autoimmune disorder in a subject in need thereof, comprising stimulating or inducing GIBsPLA2 in said subject.

In another particular embodiment, the invention is directed to a compound or composition of the present invention for use in the treatment of an autoimmune disorder in a subject in need thereof.

In a particular embodiment, the invention is directed to methods of treating cancer in a subject in need thereof, comprising stimulating or inducing GIBsPLA2 in said subject.

In another particular embodiment, the invention is directed to a compound or composition of the invention for use in the treatment of cancer in a subject in need thereof.

Another particular embodiment of the invention relates to a method of treating (e.g., reducing or preventing or inhibiting) graft rejection, for the treatment of graft versus host disease in a subject who has received a transplant, comprising stimulating or inducing GIBsPLA2 in said subject. Another aspect of the present invention is a method for improving tolerance to an allogeneic transplant in a subject, comprising stimulating or inducing GIBsPLA2 in said subject.

Antimicrobial activity

The present application also provides, in another aspect, a method for killing microorganisms using GIBsPLA2. By direct action on membranes, GIBsPLA2 can destroy or kill bacteria, enveloped viruses, parasites, and the like.

In acute infections or other infections, GIBsPLA2 can be used alone or together with antibiotics, antivirals, antiretrovirals, and antiparasitics. In the case of microbes resistant to known antimicrobial drugs, GlBsPLA2 may represent an alternative therapy. It can be used in very short-term treatment, for example in very dangerous and acute clinical situations.

Specific examples of diseases in which GlBsPLA2 treatment according to the invention may be beneficial are all clinical situations with immune system hyperactivity or chronic inflammation, such as multiple sclerosis, myasthenia gravis; autoimmune neuropathies such as Guillain-Barré syndrome; autoimmune uveitis, uveitis, autoimmune hemolytic anemia, pernicious anemia, autoimmune thrombocytopenia, temporal arteritis, antiphospholipid syndrome; vasculitis such as Wegener's granulomatosis; Behçet's disease, atherosclerosis, psoriasis, dermatitis herpetiformis, pemphigus vulgaris, vitiligo, mycosis fungoides, allergic contact dermatitis, atopic dermatitis, lichen planus, acute lichenoid and varioliform parapsoriasis, eczema, Crohn's disease, ulcerative colitis, primary biliary cirrhosis, autoimmune hepatitis, type 1 diabetes mellitus, Addison's disease, Graves' disease, Hashimoto's thyroiditis, autoimmune oophoritis and orchitis, autoimmune thyroiditis, rheumatoid arthritis, systemic lupus erythematosus,

- 18 040303 scleroderma, polymyositis, dermatomyositis; spondyloarthropathies such as ankylosing spondylitis or Sjögren's syndrome.

The duration, dosage and frequency of use of the compounds or compositions of the present invention may be adapted according to the subject and the disease. The treatment may be carried out alone or in combination with other active ingredients, simultaneously or separately or sequentially.

The compounds or compositions of the invention may be administered in a variety of ways or methods such as, but not limited to, systemic, intramuscular, intravenous, intraperitoneal, dermal, subcutaneous, dermal, transdermal, intrathecal, ocular (e.g., corneal), or rectal, or by topical application at the site of inflammation, and preferably by intramuscular or intravenous administration.

A typical regimen includes a single or repeated application of an effective amount of the GIBsPLA2 modulator over a period of one to several days, up to one year, and including a period of one week to about six months. It should be understood that the dosage of a pharmaceutical compound or composition of the present invention used in vivo will depend on the age, health, sex and body weight of the recipient (subject), the type of concomitant treatment, if any, the frequency of treatment, and the nature of the pharmaceutical effect desired. . The effective dose ranges provided by the present invention are not intended to be limiting and represent preferred dose ranges. However, the most preferred dosage will be tailored to the particular subject, as will be understood and can be determined by any person skilled in the art (see, e.g., Berkowet et al, eds., The Merck Manual, 16 ^th edition, Merck and Co., Rahway , NJ, 1992 (Merck manual, 16th edition); Goodmanetna., eds., Goodman and Cilman's The pharmacological Basis of Therapeutics, ^10th edition, Pergamon Press, Inc., Elmsford, NY, (2001) (Pharmacological basis of therapeutic funds)).

Diagnostics

The invention also provides methods for detecting an immune defect in a subject based on detection of the presence or amount or absence of GIBsPLA2 in a sample from the subject. The method of the present invention can be carried out using various detection technologies or platforms known per se in the art, such as, but not limited to, capture assay, sandwich assay, competitive assay, radioimmunoassay; enzymatic labels with substrates generating colored, fluorescent, chemiluminescent or electrochemically active products; fluorescence, fluorescent polarization, chemiluminescence, optical and colorimetric analysis, electrochemiluminescence, time-resolved fluorescence, surface plasmon resonance, evanescent wave analysis, multiwell plate analysis (ELISA), individual analysis, multichannel analysis, multichannel latex bead analysis, multichannel microarray analysis (on a flat surface), glass plate analysis, or strip-based analysis.

In a particular embodiment, the method includes determining the presence or amount or absence of a GIBsPLA2 gene, RNA or protein polymorphism. Our results showed that GIBsPLA2 has a high polymorphism and that this correlates with the subject's physiological status. Thus, the invention includes (i) determining the presence, amount, or absence of a particular GIBsPLA2 polymorphic isoform, and/or (ii) determining the overall level of GIBsPLA2 polymorphism in a subject, where these data correlate with the physiological status of the subject. In particular, specific isoforms may be indicative of the predisposition, presence, or development of a disease in a subject, as described above. Such a definition can also be used in personalized medicine to regulate treatment.

Methods for monitoring and/or diagnosing immunodeficiency associated with defects in CD4 T cells, including detection of GIBsPLA2

Methods for monitoring and/or diagnosing immunodeficiency associated with CD4 T cell defects, in particular human immunodeficiency virus (HIV) infection in a subject, are provided by the present invention. In some embodiments, the methods include (a) providing a sample containing body fluid, preferably plasma, from a subject, and (b) detecting a level of GIBsPLA2 in the sample above a threshold. The presence of GIBsPLA2 in a sample can be detected by any method known in the art, for example, by a method including enzymatic assay, ligand capture assay and/or immunoassay.

In some embodiments, the method includes obtaining a sample comprising plasma from a subject and determining whether the plasma has at least one activity selected from induction of abnormal membrane microdomain (MMD) formation in CD4 T cells from healthy subjects and induction of CD4 resistance. T cells from healthy subjects to interleukin-7 (IL7) signals. If the subject's plasma has such activity, then the subject is defined in some embodiments as having CD4 T cell-associated immunodeficiency, which is often seen in, but not limited to, HIV-infected patients. If the plasma fraction does not contain such activity, then the subject is defined in some embodiments as having a low susceptibility to immunodeficiency associated with CD4 T cell disruption in cytokine-regulated homeostasis.

In some embodiments, the subject is defined as having HIV infection. On the contrary, if the protein fraction does not contain such activity, then the subject in some embodiments is defined as not having an immunodeficiency associated with CD4 T cell defects, as disclosed in the present invention. In some embodiments, the implementation of the subject is defined as not having HIV infection.

In some embodiments, the methods include contacting a sample containing body fluid, preferably plasma, from a subject with antibodies specific for GIBsPLA2 and determining the presence or absence of an immune response. In some embodiments, the presence or absence of an immune response is determined by a method involving enzyme immunoassay (ELISA). The presence of an immune reaction between the GIBsPLA2 specific antibody and the sample indicates the presence of GIBsPLA2 in the sample, which in turn indicates that the subject has an immunodeficiency associated with CD4 T cell defects. In some embodiments, the subject is determined to have HIV infection. In contrast, the absence of an immune response between the GIBsPLA2 specific antibody and the sample indicates that the subject does not have an immunodeficiency associated with CD4 T cell defects as disclosed in the present invention. In some embodiments, the subject is determined not to have HIV infection.

In some embodiments, the analysis for the presence of GIBsPLA2 in a sample is qualitative. In some embodiments, the analysis for the presence of GIBsPLA2 in a sample is quantitative.

In some embodiments, the method includes comparing the results of the assay with the results of a similar assay of a control sample containing plasma from a subject that does not have an immunodeficiency associated with CD4 T cell defects. In some embodiments, the methods include comparing the results of the analysis with the results of a similar analysis of a sample containing plasma from the same subject previously collected.

Methods for monitoring and/or diagnosing immunodeficiency associated with a change in CD4 T cells, including the characterization of membrane microdomains on CD4 T cells

The data in the examples show that in HIV-infected patients, the formation of characteristic membrane microdomains (MMDs) on the surface of CD4 T cells is noted, although very few cells are actually infected with HIV.

Accordingly, the present invention also provides methods for diagnosing an immunodeficiency associated with a change in CD4 T cells, such as, for example, immunodeficiency caused by human immunodeficiency virus (HIV) infection in a subject. In some embodiments, the methods include (a) isolating CD4 T lymphocytes from a subject and (b) measuring the number and/or size of membrane microdomains (MMDs) on T cells. In some embodiments, the methods further comprise at least one of (c) measuring the amount of phospho-STAT5 in T cells, and (d) determining the proportion of nuclear import of phospho-STAT5 in T cells. In some embodiments, the number and/or size of MMD on T cells is measured in the absence of interleukin. In some embodiments, the number and/or size of MMD on T cells is measured in the absence of IL-2. In some embodiments, the number and/or size of MMD on T cells is measured in the absence of IL-7. In some embodiments, the number and/or size of MMD on T cells is measured in the presence of a subthreshold level of interleukin.

In some embodiments, if the number of MMD on T cells isolated from a subject is at least at a threshold level, this indicates that the subject has an immunodeficiency associated with a change in CD4 T cells. In some embodiments, this indicates that the subject has an HIV infection. In some embodiments, if the number of MMD on T cells isolated from a subject is not at least at a threshold level, this indicates that the subject does not have an immunodeficiency associated with a CD4 T cell change, as disclosed in the present invention. In some embodiments, this means that the subject does not have abnormal CD4 T cell responses to cytokine signals. In some embodiments, this means that the subject does not have an impaired CD4 T cell response to interleukin-7. In some embodiments, this indicates that the subject does not have HIV infection. In some embodiments, the threshold level is at least about 80 per cell, at least about 90 per cell, at least about 100 per cell, at least about 110 per cell, or at least about 120 per cell. In a non-limiting preferred embodiment, the threshold level is about 100 per cell. In some embodiments, if MMD on T cells isolated from a subject have a diameter of at least a threshold level, this indicates that the subject has HIV infection. In some embodiments, if MMDs on T cells isolated from a subject do not have a diameter of at least a threshold level, this indicates that the subject does not have an impaired response to interleukin-7 and more broadly to cytokines. In some embodiments, this indicates that the subject does not have HIV infection. In some embodiments, the threshold levels are a diameter of at least 100 nm, at least 110 nm, at least

120 nm, at least 130 nm, or at least 140 nm. In a non-limiting preferred embodiment, the threshold level is a diameter of at least 120 nm.

Since RIF can alter the ability of CD4 T cells to respond to IL-7 by aggregating membrane receptors into abnormally large MMDs, responses to other gamma-c and cytokines may be altered, and RIF may be associated with other pathologies in which an altered CD4 T cell response.

Methods for identifying potential therapeutic agents

The present invention also provides methods for identifying a potential therapeutic agent, comprising (a) contacting CD4 T lymphocytes with GIBsPLA2 in the presence of the agent, and (b) measuring GIBsPLA2-induced activation of CD4 T cells. In some embodiments, the methods include (c) comparing the level of GIBsPLA2-induced CD4 T cell activation in the presence of the agent with the level of GIBsPLA2-induced CD4 T cell activation in the absence of the agent. In some embodiments, if the level of GIBsPLA2-induced CD4 T cell activation in the presence of the agent is lower than the level of GIBsPLA2-induced CD4 T cell activation in the absence of the agent, then the agent is identified as a potential immunodeficiency treatment agent. In some embodiments, the agent is identified as a potential agent for the treatment of HIV. In some embodiments, if the level of GIBsPLA2-induced CD4 T cell activation in the presence of the agent is greater than the level of GIBsPLA2-induced CD4 T cell activation in the absence of the agent, then the agent is identified as a potential immunosuppressive therapeutic agent.

In some embodiments, the measurement of GIBsPLA2-induced activation of CD4 T cells includes determining the number of MMD per CD4 T cell.

In some embodiments, the measurement of GIBsPLA2-induced activation of CD4 T cells includes determining the average diameter of MMD on CD4 T cells.

In some embodiments, the measurement of GIBsPLA2-induced CD4 T cell activation comprises determining the ability of CD4 T cells to respond to IL-7 by assaying STAT5 phosphorylation and/or nuclear import.

As used in the present invention, the agent may be any chemical that is being evaluated as a potential therapeutic agent. In some embodiments, the agent is an organic molecule. In some embodiments, the agent contains 2 to 100 carbon atoms, such as 2 to 50 carbon atoms, 5 to 50 carbon atoms, or 10 to 50 carbon atoms. In some embodiments, the agent is a peptide, protein, glycoprotein, or lipoprotein. In some embodiments, the agent is an antibody.

In some embodiments, the agent has not previously been determined to have biological activity that would allow it to be used as a therapeutic agent for the treatment of immunodeficiency, such as that often associated with HIV infection. In some embodiments, the agent has previously been determined to have biological activity that allows it to be used as a therapeutic agent for the treatment of immunodeficiency, such as that often associated with HIV infection.

As used in the present invention, a potential agent for the treatment of immunodeficiency or a potential agent for the treatment of HIV is an agent that inhibits the ability of RIF to activate CD4 T cells in at least one assay. In accordance with the data provided in the present invention, the ability of an agent to suppress the ability of GIBsPLA2 to activate CD4 T cells in at least one assay is a suitable way to identify agents that are likely to be therapeutically useful for the treatment of immunodeficiencies, including immunodeficiencies associated with HIV- infection. Accordingly, it is also a suitable way to identify agents that are likely to be therapeutically useful in the treatment of HIV infection. Of course, as with all therapeutic molecules, additional characterization is needed. However, this does not diminish the utility of the potential HIV treatment agents of the present invention.

Additional aspects and advantages of the invention are disclosed in the following section on experiments; they are to be regarded as illustrative.

Examples

1. Materials and methods.

1.1. Patients.

The VPs included in the study were HIV positive for more than one year. They were not receiving any antiretroviral drugs and had a viral load >10,000 RNA copies/mL with a CD4 count >200/mkh at the time of blood collection (ANRS EP33 and EP20 studies). All blood samples from VP were collected at the Gonessa Hospital Center. HD blood was provided by the French Service

- 21 040303 blood (Centre Necker-Cabanel, Paris). Plasma samples from ART patients were taken from individuals receiving treatment for at least one year. Their viral load had been undetectable for at least 6 months, and their CD4 count was over 500/μl at the time of blood collection.

Plasma samples from HIC patients were taken from individuals with an undetectable viral load during the years after infection. Plasma samples were collected at the Necker-Pasteur Infection Center.

1.2. Analysis of membrane microdomains (MMDs), receptor diffusion rates, and cellular compartmentalization of phospho-STAT5 in purified CD4 T lymphocytes.

CD4 T cells were purified by negative selection as already described (10), then activated with 2 nM recombinant glycosylated human IL-7 (Cytheris) or 40 μg PHA (Sigma). Confocal and STED microscopy, used to study cell surface microdomains (MCDs) and the distribution of phospho-STAT5 in the cell compartment, have already been described (10, 12). FCS analysis of protein diffusion on the surface of living cells has also been described (10, 12).

1.3. Preparation and Analysis of Detergent-Resistant Microdomains (DRMs) Preparation with Triton-X100 of HD or VP CD4 T-lymphocyte lysates, followed by sucrose gradient centrifugation and Western blot analysis of collected fractions, has already been described (12). Monoclonal antibodies specific for flotillin, IL-7R-alpha and gamma-c, were used to detect the respective bands on Western blot (12).

1.4. Characterization of RIF from VP plasma 1.4.1.

Bioassays.

MMD induction was carried out as follows: VP plasma (5 or 10%) was first incubated (20 min) in medium with purified HD CD4 T cells. Cells were then placed on polylysine-coated glass slides for 10 min, then activated for 15 min with IL-7 (2 nM) or no control (NS), then fixed with PFA (PFA, 1.5%, 15 min at 37°C). C, then 15 min at room temperature), equilibrated for 1 h in PBS/SVF 5% before staining with cholera toxin B (CtxB-AF488). MMD was counted by STED microscopy.

STAT phosphorylation and nuclear translocation inhibition assay was performed as follows: VP plasma (5 or 10%) was first incubated with purified HD CD4 T cells (20 min) prior to IL-7 stimulation (2 nM, 15 min). Cells were then placed on polylysine-coated glass slides for 10 min, then activated for 15 min with IL-7 (2 nM) or not activated for control (NS), then fixed with PFA (PFA, 1.5%, 15 min at 37°C). C, then 15 min at room temperature) and permeabilized with methanol (90% at -20°C). Cells were equilibrated for 1 h in PBS/SVF 5%, then stained with phospho-STATS with rabbit anti-STAT5 labeled with goat anti-rabbit Atto642 and analyzed by FACS or STED microscopy.

1.4.2. Enzyme processing.

The effect of enzymatic digestion on RIF activity was assessed by VP treatment of plasma filtered on a 30 kDa membrane. Plasma compounds with a molecular weight less than 10 kDa were used as negative controls. The effects of porcine trypsin (1 U/ml for 30 min at 37°C, followed by PMSF inhibition and buffer exchange on Millipore 5 kDa membrane centrifuge filters) or DNase I (1 U/ml for 30 min at 37°C) were analyzed. ) or RNase (1 U/ml for 30 min at 37°C) or N-glycanase peptide (1 U/ml for 30 min at 37°C). All preparations were analyzed at a final concentration of 10%.

1.4.3. Molecular weight determination or RIF purification.

Gel permeation chromatography was performed by loading 1.6 ml of plasma onto an 85 ml Sephadex G-100 column previously equilibrated with ammonium carbonate (0.1 M) or PBS, then collecting 0.8 ml fractions of the eluate. The column was calibrated using a protein kit (GE-Healthcare). The protein concentration was measured by the Bradford method. VP plasma was pre-filtered on a 100 kDa membrane, total VP plasma was tested and identical results were obtained. Collected fractions of 13-17 kDa, which contained semi-purified RIF.

1.4.4. Determination of the isoelectric point.

Anion or cation exchange chromatography was performed on 1 ml MonoQ or MonoS columns (GE-Healthcare) eluting in successive pH steps (ammonium carbonate/ammonium acetate buffers). The pH of each eluted fraction was measured and then the pH was adjusted to 7.4 before biological effects were analyzed. RIF activity was measured in the respective fractions immediately after elution.

1.4.5. MS analysis.

Samples from gel filtration (G100) were lyophilized, then resuspended, collected and subjected to proteolysis with porcine trypsin, in accordance with methods known in the art. Peptides after proteolysis were separated into 12 fractions by chromatography on a C18 column, eluting with ammonium acetate. 12 fractions separated by C18 were eluted in reverse phase (with acetonitrile) and directly electrosprayed into Orbitrap Velos (Thermo Scientific) for MS analysis with secondary Ar fragmentation, followed by MS/MS for the 10 peaks with the highest intensity. 040303 intensity on MS scan.

The standard Mascot and X-Tandem programs were used. For each protein from the database subsets, 3 criteria were calculated:

i-index: calculation of the theoretical specificity of each of the peptides after trypsin digestion of an individual protein in the NextProt database enriched with mature proteins with signal sequence digestion (number of unique peptides/protein): number of specific peptides for all human sequences (all), sequences with N- terminal signal peptide (sec) per protein;

calculation of the theoretical frequency of peptides compatible with peaks from all series of MS scans (peaks coinciding with theoretical peptides/protein);

calculation of the theoretical coverage of the protein sequence with peptides matching the peaks.

For each protein, the p-index was determined from the results from all three indices.

Example 1. Aberrant activation of CD4 T-lymphocytes from VP according to the results of the analysis of the presence of abnormal membrane microdomains (MMD).

This example describes the study of new molecular and cellular parameters that explain some of the abnormal responses observed in CD4 T lymphocytes from patients with chronic HIV infection. Chronic activation of the immune system is usually determined by assessing the overexpression of cell surface molecules such as CD38, HLA-DR and CD25, which are considered major markers of CD4 dysfunction (15). However, despite many attempts, these data remain unclear, and the phenotype of CD4 T cells does not directly explain their immune defects.

STED microscopy and cholera toxin B (CtxB-AF488) labeling was used to detect the presence of MMD (12). It was found that before any stimulation, the surface of CD4 T lymphocytes isolated from VP carried much more MMD than resting CD4 T lymphocytes isolated from HD (Fig. 1a). And most importantly, all CD4 T cells from VP showed an increased number of MMD. This abnormal pattern was not due to IL-7 stimulation in VP plasma, since the mean IL-7 concentration in this plasma (0.4 pM) was only 100 Kd of IL-7R (13, 14). When purified HD CD4 T cells were stimulated with IL-7, a large amount of MMD was observed. In contrast, the pattern of MMD on CD4 T cells from VP was not affected by IL-7 (Fig. 1a). This abnormal activation, together with the absence of any response to IL-7, can be mimicked by a non-physiological stimulus such as phytohemagglutinin (PHA) (Fig. 1a).

These various anomalous MMDs were then counted. Approximately 150-200 MMD per CD4 T cell from VP were observed, as for PHA-stimulated HD CD4 T cells (Fig. 1c). Again, the results showed that all CD4 T cells from VP expressed MMD, including all major CD4 subsets (Fig. 1c). IL-7 did not cause an increase in the number of MMD in VP. In contrast, the number of MMDs on HD CD4 T cells increased from a baseline of about 10 MMD/cell to 300 after IL-7 stimulation. The MMD size was also studied (FIGS. 1d and 1e). This shows that MMD on CD4 T cells from VP and on PHA-stimulated HD CD4 cells were much greater (250 nm) than on HD CD4 T cells stimulated with IL-7 (90 nm).

Example 2 All IL-7R alpha and gamma c chains are sequestered in abnormal detergent-resistant membrane microdomains (DRMs) isolated from VP CD4 T cells.

Resting HD CD4 T cells were analyzed to confirm that IL-7R alpha and gamma c chains are located in high density fractions outside of MMD. When these HD CD4 T cells were stimulated with IL-7, these two chains were located in low density fractions corresponding to detergent-resistant MMD or DRM containing all proteins sequestered in MMD (FIG. 2).

When the study was repeated on CD4 T cells isolated from VP, the pattern was different (FIG. 2). Before any stimulation, all IL-7R alpha and gamma c chains were already sequestered into DRM; nothing was located in the high density fractions corresponding to free receptors outside of MMD. Further, pre-stimulation of CD4 T cells with IL-7 prior to receiving DRM does not affect this pattern (data not shown). Again, pre-stimulation of HD CD4 T cells with non-physiological PHA reproduces this pathological condition. This confirms the data in Fig. 1 and demonstrates that CD4 T cells in VP are subject to aberrant activation prior to any stimulation. In addition, these abnormal MMDs contain all of the IL-7R chains (FIG. 2).

Example 3 Two-dimensional gel analysis of IL-7 signalosome in purified CD4 T cells from HP, VP and IL-7-stimulated HD cells. Characterization of an aberrant activation state by the nature of the protein obtained after immunoprecipitation.

Two-dimensional electrophoresis was used to demonstrate that the composition of the IL-7 signalosome in VP was abnormal and different from that observed in resting and IL-7-activated CD4 T cells (Fig. 7a, b and c).

Proteins were immunoprecipitated with anti-IL-7R-alpha antibodies (mouse monoclonal antibodies 40131, R&D System) immobilized on protein G-Sepharose 4G from purified CD4 T cell lysate and separated by 2D PAGE (IEF on gel strips at pH 3- 10 followed by SDS electrophoresis in 12% acrylamide-bisacrylamide gel). 23 040303 pH and molecular weight (kDa) values are displayed. Gels were stained with Sypro-Ruby. The gels shown are examples of 8 NS/IL-7 pairs derived from HD and 3 gels from VP.

Fig. 7a. Unstimulated (NS) HD CD4 T cells.

Fig. 7b. VP CD4 T cells. More spots were observed in 2D gels stained with Sypro Ruby with samples from VP than from HD. In addition, we observed that overall stains were more intense when 2D gels were made with VP extracts.

Fig. 7s. IL-7-stimulated HD CD4 T cells. The pattern in HD CD4 T cells stimulated with IL-7 differs from that observed in VP CD4 T cells. This also supports the suggestion that the aberrant activation observed in VP is not a consequence of IL-7 stimulation, which can occur in organs with high levels of IL-7, such as IL-7-producing organs.

Based on this analysis, it can be concluded that the IL-7R chains in VP CD4 T cells are not part of the abnormal MMD, but also that they interact with protein complexes different from those found in the normal IL-7 signalosome.

Example 4 Diffusion rate of IL-7R-alpha on the surface of purified CD4 T cells from HP, VP and PHA-stimulated HD cells. IL-7R-alpha in VP CD4 T cells is embedded in lipid-rich abnormal MMDs, thus limiting their diffusion rates and preventing any interactions with the cytoskeleton and thus any signaling ability.

Two-dimensional diffusion of IL-7R-alpha stained with monoclonal antibodies AF488-anti-IL-7R-alpha was measured by FCS on the surface of live CD4 T cells. The results are shown in FIG. 8. Diffusion time tD (in 10 ^-3 s) was measured in the absence of IL-7 (O autocorrelation) or in the presence of IL-7-6uotuh-SAF633 (® cross-correlation) as described (10, 12). These time values were plotted against cell surface area ω ₀ ² (in 10 ³ nm ² ) interrupted by the confocal volume.

Diffusion curves are shown with or without pretreatment with or without MMD inhibitors (Choase 1 µg/mL plus SMase 0.1 µg/mL for 30 min) or cytoskeletal inhibitors (CytD 10 µM plus Col 10 µM for 30 min).

Bars indicate SD from 5 independent experiments. The slopes of the linear regression give the effective diffusion rates D _eff , and the y-intercepts extrapolate the retention time τ0 as we described earlier (12). D _eff are shown in the histogram in FIG. 3a.

Fig. 8a, 8d - on the surface of HD CD4 T cells.

Fig. 8b, 8e - on the surface of VP CD4 T cells.

Fig. 8c, 8f - on the surface of HD CD4 T cells pre-activated with PHA (1 μg/ml).

Fig. 8g. Schematic of the diffusion mechanism of IL-7R-alpha embedded in MMD before and after treatment with MMD inhibitors or cytoskeletal inhibitors. MMD are indicated by disks, receptors by rods, the cytoskeleton is shown as a network. Diffusion rates (fast, slow, very slow) are given to simplify data interpretation. This diagram illustrates the results, also shown in FIG. 3a.

Example 5 IL-7R chains sequestered in abnormal MMD VP CD4 T cells are non-functional.

IL-7R diffusion rates were measured on the surface of CD4 T cells as described previously (10, 12) and as detailed in Example 4. It was seen that prior to any stimulation, these diffusion rates were three times lower on VP, than on HD CD4 T cells (Fig. 3a). This further demonstrates that IL-7R-alpha chains are embedded in abnormal MMD on the surface of these CD4 T cells (Fig. 3a). Treatment with Choase plus SMase releases the receptor from its confining MMDs and thus increases its diffusion rate (Fig. 3a). In contrast, treatment with cytochalasin D (CytD) plus colchicine (Col), which disorganize the cytoskeleton, had no effect on the diffusion rate of the IL-7R chain on CD4 T cell VPs (Fig. 3a). Because cytoskeletal organization is absolutely essential for signal transduction, this absence of any functional or structural relationship between IL-7R-alpha and the cytoskeletal network suggests that signal transduction cannot occur when IL-7R complexes are sequestered in abnormal MMD, as is the case VP CD4 T cells.

Pulsed STED microscopy was then used to study STAT5 (phospho-STAT5) phosphorylation and phospho-STAT5 separation in the cytoplasm and nucleus of HD and VP CD4 T cells. Fig. 3b shows STED images of phospho-STAT5 distribution before and 15 minutes after IL-7 stimulation. We noted that phospho-STAT5 accumulated in the nucleus of HD CD4 T cells and this phenomenon was inhibited by cytoskeletal disorganization. In contrast, there was no translocation of phospho-STAT5 to the nucleus in VP CD4 T cells or in PHA-prestimulated HD CD4 T cells (Fig. 3b).

Then we studied the kinetics of the appearance of phospho-STAT5 in the cytoplasm and nucleus for 1 h (Fig. 3c, d, e). This shows that phospho-STAT5 in VP CD4 T cells accumulates mainly in the cytoplasm and does not migrate to the nucleus (Fig. 3d) as it does in PHA-stimulated HD CD4 T cells (Fig. 3e). This is especially clear when the results are compared with those obtained 5 min after IL-7 stimulation of HD CD4 T cells, where 50% of phospho-STAT5 was in the nucleus (Fig. 3c).

- 24 040303

Example 6 Plasma from VP induces abnormal MMD on the surface of purified HP CD4 T cells.

The source of aberrant activation of VP CD4 T cells was then studied. The fact that all CD4 T cells were involved and that a non-physiological signal such as PHA mimicked the results led to the study of VP plasma. Purified HD CD4 T cells were incubated with 10% VP plasma for 30 min and MMD was counted on the surface of CD4 T cells as determined by cholera toxin B labeling (CtxB-AF488). In FIG. 4 shows the resulting images. Plasma VP alone induced a large number of MMD on HD CD4 T cells. The addition of IL-7 did not affect the size or number of these MMDs (Fig. 4a). These results are shown for plasma from five different VPs (FIG. 4b) and were confirmed using more plasma samples from VPs (>15). The experiments were also repeated using CD4 T cells from various HDs (>5). Plasma samples from individuals with controlled HIV infection (HIC) and patients receiving antiretroviral therapy (ART) on purified HD CD4 T cells were used as controls. None of them induced MMD or inhibited IL-7 induction of MMD (Fig. 4c).

This was further confirmed by testing a large number of dilutions of different plasma samples (Fig. 4d). VP plasma to a dilution of 0.1% caused the formation of MMD, scattered over the cell surface. VP plasma, diluted 50-100 times, had 50% of the maximum activity. None of the plasma samples from HIC or ART patients induced MMD at any dilution.

Example 7 Plasma from VP inhibits IL-7-induced translocation of phospho-STAT5 to the nucleus.

IL-7R function in HD CD4 T cells treated with VP plasma was tested after phosphorylation and translocation to the STAT5 nucleus. As seen in FIG. 5a, pre-incubation of HD CD4 T cells with VP plasma (10% concentration) inhibited IL-7-induced STAT5 phosphorylation and nuclear translocation. In FIG. 5b shows the results obtained for five VP plasma samples. All of them, at a dilution of 10%, inhibited the translocation of phospho-STAT5 into the nucleus. These results were confirmed with plasma from different VPs (>15) and with different sources of HD CD4 T cells (>5).

The effect of plasma obtained from HIC and ART patients was also tested by pre-incubation with purified HD CD4 T cells (Fig. 5a and c). Again, only VP plasma was able to inhibit IL-7-induced translocation of phospho-STAT5 to the nucleus. It was also determined (FIG. 5d) that VP plasma was active up to a 0.1% dilution, and half-maximal activity was obtained at 50,100-fold dilution, thus correlating with the ability to induce abnormal MMD (FIG. 4d).

We also tested the effect of plasma from patients receiving antiretroviral therapy but not responding (CD4-NR) to treatment (low viral RNA and low CD4 T cells) by preincubating it with purified HD CD4 T cells. Again, only CD4-NR plasma was capable of inhibiting IL-7-induced translocation of phospho-STAT5 to the nucleus. It was also determined that CD4-NR plasma was active up to a 0.1% dilution, and half-maximal activity was obtained at 50-100-fold dilution, which correlated with the ability to induce abnormal MMD as observed with VP.

Example 8 Molecular characterization of the resistance inducing factor.

The chemical nature of RTF was investigated. The studies carried out showed (FIG. 6a) that RTF is a protein, since its activity was destroyed by trypsin. Treatment with peptide-I-glycanase (PGase) had no effect, indicating that N-glycosylation was not required for RTF activity.

The molecular weight of the RIF was then determined by gel permeation chromatography on Sephadex G-100. MMD induction (FIG. 6b) and inhibition of IL-7-induced nuclear translocation of phospho-STAT5 (FIG. 6c) were measured for all fractions eluted from the column. Two exemplary elution profiles are shown in FIG. 6. Both show that RTF is the only factor with a molecular weight between 10 and 15 kDa.

In FIG. 6d shows the densities of viral peptides or proteins measured by dot blot technique in each of 100 fractions collected from a Sephadex G100 column. The measurements were repeated three times with different polyclonal antibodies from plasma VP samples, characterized by high activity against viral proteins. For each experiment, the signals obtained with HD plasma samples were subtracted from these values. The picture shown in Fig. 6b demonstrates that no viral proteins or fragments are detected in the fraction containing RIF activity, while dot blot analysis shows the presence of viral proteins at higher molecular weights (190 to 32 kDa).

Active, enriched fractions of 10-15 kDa from a Sephadex G100 column were used to obtain the RIF isoelectric point by sorption on an anion (MonoQ) or cation exchange (MonoS) columns followed by elution in a pH gradient (increasing pH on MonoS or decreasing pH on MonoQ) (Fig. 6d). The MMD-inducing activity of the various pH fractions was then measured after adjusting the pH to 7.4. In general, from 25 to 30% of the initial activity was allocated in two fractions, the results corresponded to the isoelectric point of 6.5-8.0.

Thus, RIF is a secreted protein with a molecular weight of about 15 kDa, and pI

- 25 040303 about 7.5-8.0 containing a disulfide bridge. Following the above structural and functional characteristics, the identity of RIF was directly established. In particular, of all 36,853 known human proteins, only 62 have the above four RIF characteristics. Semi-purified material from three viraemic patients and three FIDs was analyzed by mass spectrometry using the standard Mascot software. The resulting proteins were arranged according to the p-scale described in Materials and Methods. The results shown in table. 1 directly indicate that the RIF is GIBsPLA2.

Table 1

Mnemocode ID pi Mm. i s p scale Description PA21B HUMAN P04054 7.95 14138.99 9 0.64 Phospholipase A2 group 1 TMEM9 HUMAN Q9P0T7 6.23 18568.37 5 0.29 Transmembrane protein 9 (TM) ESMI HUMAN Q9NQ30 6.83 18122.42 5 0.10 Endothelial cell specific molecule 1 CYTD HUMAN P28325 6.76 13858.6 3 0.08 C,statin-O SSRB HUMAN P43308 7.03 18273.74 7 0.05 Signal sequence receptor, subunit beta (TM) GPIX HUMAN P14770 6.14 17316.06 6 0.04 Platelet glycoprotein IX B2MG HUMAN P61769 7.67 18510.47 4 0.03 Beta-2-microglobulin EPGN HUMAN Q6UW09 7.72 14724.99 1 0.02 Epigen IL 19 HUMAN Q9UHD0 7.8 17812.56 5 0.02 Interleukin-19 IU3 HUMAN P08700 7.05 15091.38 3 0.02 Interleukin-3 GML HUMAN Q99445 6.67 15918.41 7 0.02 GPI-anchored protein CYTM HUMAN P05113 7.02 13149.22 4 0.017 C,statin-M

Thus, the protein found in the plasma of viraemic patients is the secreted form of GIBsPLA2. The mature protein contains 125 amino acids (MW 14138), has a pI of 7.95 and 7 disulfide bridges. Using commercial porcine GIBsPLA2, we were able to confirm in vitro that this protein induces abnormal MMDs that block translocation of IL-7 pSTAT5 to the nucleus in the plasma of viraemic patients, further confirming that RIF is GIBsPLA2, in particular its secreted form. The amino acid sequence of human GIBsPLA2 is given as SEQ ID NO: 2.

Example 9 PLA2sGIB induces inability to respond (anergy) of CD4 T-lymphocytes.

Example 7 shows that PLA2sGIB, through MMD induction, induces blockade of IL-7-induced translocation of phospho-STAT5 to the nucleus (NT pSTAT5). As a result, CD4 T lymphocytes do not respond to IL-7, and despite high levels of this cytokine in the plasma of HIV-infected patients, their number decreases, leading to CD4 lymphopenia, which is a key characteristic of HIV-infected patients.

Thus, we explored the possibility that PLA2sGIB is also involved in the induction of anergy, another characteristic of CD4 lymphocytes from HIV infection.

Bioanalysis.

MMD induction.

VP plasma containing PLA2sGIB was first incubated (20 min) in medium with purified HD CD4 T cells. Cells were then placed on polylysine-coated glass slides for an additional 10 min. They were then fixed with paraformaldehyde (PFA, 1.5%, 15 min at 37°C, then 15 min at room temperature) before staining with cholera toxin B (CtxB-AF488); MMD was calculated by CW-STED microscopy (microscopy based on the suppression of spontaneous emission with continuous pulse generation).

Inhibition of STAT phosphorylation and translocation to the nucleus.

VP plasma containing PLA2sGIB was first incubated with purified HD CD4 T cells (20 min) prior to IL-7 stimulation (2 nM, 15 min). Cells were then placed on polylysine-coated glass slides prior to PFA fixation (1.5%) and methanol permeabilization (90%, at -20°C). pSTAT5 was then stained with rabbit anti-STAT5 labeled with goat anti-rabbit Atto642 and analyzed by FACS (fluorescent cell sorting) or pulsed STED microscopy.

Results.

In FIG. 10a shows that after exposure to PLA2GIB (plasma from viraemic patients), CD4 lymphocytes from healthy donors (HD) become incapable of responding to IL-2, as determined by inhibition of IL-2-induced NT pSTAT5. This inhibition was complete with 3% plasma and was significantly pronounced with 1% plasma (p<0.0001).

We then studied the response of CD4+CD25+ T-lymphocytes to PLA2GIB. The results are shown in FIG. 10b. As shown, while 100% of healthy cells responded to IL-2 via NT pSTAT5, PLA2 GIB (1% plasma of viraemic patients) completely inhibited this signal transduction mechanism. Since CD4+CD25+ cells represent less than 5% of all CD4 T cells, they cannot significantly affect the data presented in FIG. 10a.

IL-7 and IL-2 are members of the γ-c family of cytokines. To confirm that the failure to respond to this could be due to γ-c, we tested their response to IL-4. The response to IL-4 was measured by IL-4-induced translocation to the pSTAT6 nucleus (Fig. 11). Our results clearly show that the response to

IL-4 is inhibited by PLA2 GIB (completely with 3% plasma and largely with 1% plasma).

Thus, these results indicate that the signal transduction mechanisms induced by the γο family cytokines are altered by PLA2 GIB. This is in complete agreement with our observation that the receptor gamma chain is completely sequestered in aMMD, spontaneously surfaced CD4 lymphocytes from HIV patients (data not shown).

Example 10 Activity of recombinant forms of PLA2 GIB.

In this example, the activity of various purified forms of the PLA2 GIB proteins was tested to further confirm the effect of this protein in the purified form on the immune system and to further confirm its specificity.

Enzymatic analysis.

The assay was performed on the Enz Check PLA2 assay kit from Life Technologies (ref.: E102147). This assay provides continuous, fast, real-time monitoring of PLA2 enzyme activity. PLA2 activity was determined by the increase in intensity at a single wavelength of 515 nm. PLA2 was detected by changes in the emission intensity ratio at 515/517 nm upon excitation at 460 nm. Specific activity was expressed as the amount of fluorescent substrate (U) per second and per μg of enzyme in solution.

Results.

The results are shown in table. 2 below.

Table 2. Activity of recombinant PLA2 GIB proteins

PLA2 Origin Initial concentration (mg/ml) Final concentration (mcg/ml) Quantity (mcg) Specific activity (Unit/µg/sec) ppPLA2 IB Purified from porcine pancreas 2.90 0.58 0.06 7694.31 pPLA2 IB Recombinant porcine (in E. coli) 1.40 2.80 0.14 10353.57 hPLA2 IB Recombinant human (in E.coli) 0.70 1.40 0.07 10694.57 hPLA2 IIA Recombinant human (in E.coli) 1.45 2.90 0.15 214.93 hPLA2 IID Recombinant human (in E.coli) 0.70 1.40 0.07 445.21 hPLA2 X Recombinant human (in E.coli) 0.68 1.36 0.07 3318.97

The results showed that the recombinant human PLA2 GIB produced in E. coli has potent enzymatic activity. Further, the results also showed that the recombinant porcine PLA2GIB produced in E. coli has a similar specific activity to that of the recombinant human PLA2GIB. In contrast, recombinant PLA2GIIA and PLA2GIID are not active, and PLA2GX has very limited activity.

Thus, recombinant PLA2 GIB is a powerful active agent for use in the present invention.

Example 11 Effect of PLA2 GIB on CD4 lymphocytes includes its enzymatic activity.

In this example, we examined whether the effect of PLA2 GIB on CD4 lymphocytes actually includes (eg, is a consequence of) the enzymatic (eg, catalytic) activity of PLA2 GIB. Such enzymatic activity can modify the membrane structure, leading to the formation of multiple aMMDs on the surface of CD4 lymphocytes.

In these experiments, we tested the PLA2 GIB mutant, where the critical histidine at position 48 was replaced by glutamine (the H48Q mutant). Using the enzymatic assay described in Example 10, we compared the enzymatic activity of the recombinant porcine PLA2 GIB generated in E. coli with that of the H48Q mutant also generated in E. coli. Each protein was used in an amount of 200 μM. As shown in FIG. 12, the mutant completely lost enzymatic activity, indicating a critical role for histidine at position 48 in PLA2 GIB. We then compared wild-type porcine PLA2 GIB activity with that of the H48Q mutant in a bioassay. The results shown in FIG. 13 show that the mutant has lost the ability of non-mutant PLA2 GIB to induce aMMD, or reduce or abolish IL-7-induced pSTAT5 nuclear translocation (NT pSTAT5).

These results thus demonstrate that enzymatic activity is involved in the pathogenetic effects of PL2 GIB on CD4 lymphocytes.

- 27 040303

Example 12 Anti-PL2 GIB Antibodies Restore CD4 T Cell Activity in Plasma of Patients with

HIV viremic.

This example shows that in the plasma of viraemic patients, GIBsPLA2 transforms CD4 lymphocytes from HD into diseased lymphocytes comparable to those found in the blood of HIV-infected patients. In addition, this example shows that antibodies to GIBsPLA2 effectively suppress pathogenic activity.

In the first series of experiments, plasma was treated with sepharose beads coated with either goat antibodies directed against human GIBsPLA2 or two control goat antibodies directed against mismatched antigens. In FIG. 14a clearly shows that anti-GIBsPLA2 antibodies completely abolish or remove plasma activity that becomes incapable of inducing abnormal MMD in HD-CD4 lymphocytes. Control antibodies I and II have no effect. These experiments were repeated three times for each plasma, and three different plasma samples from viraemic patients were studied.

Fig. 14b shows identical results. Here, plasma was treated as above but analyzed using a second bioassay. Plasma treated with sepharose beads coated with anti-GIBsPLA2 antibodies no longer inhibited IL-7-induced translocation of pSTAT5 to the nucleus. Control goat antibodies I and II did not affect the ability of plasma from viraemic patients to inhibit IL-7-induced translocation of pSTAT5 to the nucleus.

In a second series of experiments, we tested the effects of neutralizing rabbit antibodies specifically directed against human GIBsPLA2, -GIIA and -GIID. These antibodies were incubated with plasma and cells during bioassays. The results obtained showed that anti-GIBsPLA2 antibodies neutralized the effects of plasma viremic, as determined by the induction of abnormal MMD and by the inhibition of IL-7-induced translocation of pSTAT5 to the nucleus. Of note, antibodies directed against either secreted PLA2-GIIA or secreted PLA2-GIID, two phospholipases closely associated with GIBsPLA2, had no effect in this assay.

These results indicate that anti-GIBsPLA2 antibodies can reverse and prevent the immunosuppressive effect of viraemic plasma. These results indicate that anti-GIBsPLA2 antibodies can prevent immunodeficiency and re-stimulate an immune response in immunodeficient subjects.

In addition, these results demonstrate that the response is specific. GIBsPLA2 is the only effector involved in the studied pathogenic effect and characterizes the plasma of patients with viremia.

Example 13 Ahtu-PLA2GIB antibodies inhibit the effect of PLA2 GIB on CD4 cells.

Cloned and purified human PLA2G1B was used to immunize rabbits. Prepared immunoglobulin fractions from the respective sera. Their ability to inhibit PLA2G1B enzymatic activity was measured on radiolabeled E. coli membranes. Active immunoglobulin fractions were added to a bioassay containing CD4 lymphocytes purified from the blood of healthy donors. Then cloned and purified secreted PLA2 (GIB, GIIA, GIID and GX) were added to cultures. Immunoglobulin fractions prepared from rabbits immunized with various secreted PLA2 were used as controls.

Fig. 15 shows that different concentrations of polyclonal antibody inhibit aMMD induction (FIG. 15a) and block IL-7-induced NT pSTAT5 (FIG. 15b). This activity can be obtained with 1 µg/ml to 100 µg/ml Ig containing anti-PLA2 GIB antibodies. This activity is completely specific as antibodies directed against PLA2 GIIA, PLA2GIID or PLA2GX showed no effect in the bioassay (FIGS. 15a and b).

Thus, these results further demonstrate that inhibition of PLA2GIB can be used to treat immunodeficiencies and to restore CD4 activity.

Example 14 Soluble PLA2GIB receptor inhibits the effects of PLA2GIB on CD4 T cells.

As an additional demonstration that PLA2GIB inhibitors may have a therapeutic effect, we tested the soluble form of the PLA2GIB receptor.

In the first series of the experiment, we used a soluble mouse receptor specific for PLA2 GIB, having the following amino acid sequence (SEQ ID NO: 22):

MVQWLAMLQLLWLQQLLLLGIHQGIAQDLTHIQEPSLEWRDKGIFIIQSESLKTCI

- 28 040303

QAGK

SVLTLENCKQPNEHMLWKWVSDDHLFNVGGSGCLGLNISALEQPLKLYECDSTL ISLRWHCDRKMIEGPLQYKVQVKSDNTVVARKQIHRWIAYTSSGGDICEHPSRDLYTLK GNAHGMPCVFPFQFKGHWHHDCIREGQKEHLLWCATTSRYEEDEKWGFCPDPTSMKV FCDATWQRNGSSRICYQFNLLSSLSWNQAHSSCLMQGGALLSIADEDEEDFIRKHLSKV VKEVWIGLNQLDEKAGWQWSDGTPLSYLNWSQEITPGPFVEHHCGTLEVVSAAWRSR DCESTLPYICKRDLNHTAQGILEKDSWKYHATHCDPDWTPFNRKCYKLKKDRKSWLG ALHSCQSNDSVLMDVASLAEVEFLVSLLRDENASETWIGLSSNKIPVSFEWSSGSSVIFT NWYPLEPRILPNRRQLCVSAEESDGRWKVKDCKERLFYICKKAGQVPADEQSGCPAGW ERHGRFCYKIDTVLRSFEEASSGYYCSPALLTITSRFEQAFITSLISSVAEKDSYFWIALQD QNNTGEYTWKTVGQREPVQYTYWNTRQPSNRGGCVVVRGGSSLGRWEVKDCSDFKA MSLCKTPVKIWEKTELEERWPFHPCYMDWESATGLASCFKVFHSEKVLMKRSWREAE AFCEEFGAHLASFAHIEEENFVNELLHSKFNWTQERQFWIGFNRRNPLNAGSWAWSDG SPVVSSFLDNAYFEEDAKNCAVYKANKTLLPSNCASKHEWICRIPRDVRPKFPDWYQY DAPWLFYQNAEYLFHTHPAEWATFEFVCGWLRSDFLTIYSAQEQEFIHSKIKGLTKYGV KWWIGLEEGGARDQIQWSNGSPVIFQNWDKGREERVDSQRKRCVFISSITGLWGTENCS VPLPSICKRVKIWVIEKEKPPTQPGTCPKGWLYFNYKCFLVTIPKDPRELKTWTGAQEFC VAKGGTLVSIKSELEQAFITMNLFGQTTNVWIGLQSTNHEKWVNGKPLVYSNWS PSDII NIPSYNTTEFQKHIPLCALMSSNPNFHFTGKWYFDDCGKEGYGFVCEKMQDTLEHHVN VSDTSAIPSTLEYGNRTYKIIRGNMTWYAAGKSCRMHRAELASIPDAFHQAFLTVLLSRL GHTHWIGLSTTDNGQTFDWSDGTKSPFTYWKDEESAFLGDCAFADTNGRWHSTACESF LQGAICHVVTETKAFEHPGLCSETSVPWIKFKGNCYSFSTVLDSRSFEDAHEFCKSEGSN LLAIRDAAENSFLLEELLAFGSSVQMVWLNAQFDNNNKTLRWFDGTPTEQSNWGLRKP DMDHLKPHPCVVLRIPEGIWHFTPCEDKKGFICKMEAGIPAVTAQPEKGLSHSIVPVTVT LTLIIALGIFMLCFWIYKQKSDIFQRLTGSRGSYYPTLNFSTAHLEENILISDLEKNTNDEE VRDAPATESKRGHKGRPICISP

The inhibitor was tested in the bioassay described in Example 9 at a concentration of 100 nM. The results are shown in FIG. 16. They show that the recombinant soluble PLA2 receptor can be used as a potent antagonist and that such a molecule is also able to significantly block the negative effect of PLA2sGIB on NT pSTAT5 (FIG. 16).

Similar results can be obtained in additional series of experiments using PLA2-GIB binding polypeptides comprising the sequence of SEQ ID NO: 25 or 28.

Example 15 GIBsPLA2 Overexpression Induces Immunodeficiency.

Highly active antiretroviral therapy (HAART), which reduces viral load, has previously been shown to induce an increase in CD4 counts in most patients. However, in some patients, despite the fact that HIV becomes undetected, the CD4 count does not increase. We have previously studied this clinical situation and have shown that these patients, referred to as CD4 non-responders (CD4-NR), have severe and persistent defects in the CD4 T lymphocyte population.

Fig. 17 shows that plasma from CD4-NR patients contains more PLA2 GIB activity than plasma from viraemic patients as a control. This was first determined by the induction of abnormal MMD on the cells. These data were also confirmed by measuring the ability to inhibit IL-7-induced translocation of pSTAT5 to the nucleus.

Taken together, these results indicate that plasma from CD4-NR patients contains hundreds of times more PLA2 GIB activity than plasma from viraemic patients.

Discussion

Our results show that PLA2 GIB induces immunosuppression similar to that

- 29 040303 rai characterizes CD4 T cells from viraemic patients, including failure to respond to IL-2 (anergy) and IL-7 (the central mechanism leading to CD4 lymphopenia). Thus, GIBsPLA2 expression in HIV infection plays a central role in the pathophysiology of the immune disease that characterizes these patients. These defects are cell type specific because CD8 T lymphocytes from HIV patients do not show abnormal MMD and continue to respond to IL-7 (data not shown). This mode of action of PLA2 GIB is likely a consequence of its enzymatic activity. When the membrane of a CD4 lymphocyte is damaged, it modifies the fluidity of the membrane and probably ensures the formation of abnormal and very large MMD.

Inflammatory responses play an important role in HIV infection. However, their exact role in the pathogenesis of HIV has not yet been established. Based on our data, it can be hypothesized that HIV infection induces a very peculiar type of inflammation that includes GIBsPLA2. It can further be hypothesized that, after PLA2 GIB induction, its secretion escapes normal regulatory processes, thus leading to chronic production and immune disorders that are a direct consequence of CD4 T-lymphocyte dysfunction. Other defects may also be observed as side effects of CD4 T lymphocyte dysfunction. For example, a decrease in the production of interferon-gamma leads to a decrease in the functions of monocytes/macrophages and natural killers.

The correlation between the plasma yield of PLA2 GIB activity and the characteristics of different patient groups is also very informative. HIV controls are very rare patients who maintain an undetected viral load and near-normal CD4 count for many years. Our results showed that they do not express PLA2 GIB activity in plasma. On the contrary, in most patients this enzyme is expressed and represents the downside of inflammation, leading to immune disease. Taken together, this clearly shows that PLA2 GIB is a critical parameter in the pathophysiology of HIV infection.

Viral load reduction with HAART is accompanied by immune reconstitution, including an increase in CD4 count. During this treatment, the PLA2 GIB activity in the plasma of patients disappears. Thus, HAART is thought to reduce inflammatory responses, further suggesting that PLA2GIB is part of these inflammatory processes. More importantly, we have described a case of CD4-NR patients who remain at very low CD4 counts while HAART controls their viral load. The overproduction of PLA2 GIB found in these individuals may explain the persistence of the immune disorder that characterizes their clinical status. Thus, after HAART, there is a pronounced correlation between a decrease in the production of PLA2 GIB, leading to the restoration of immunity, or persistent excess production, leading to the irreversibility of the immune disease.

The therapeutic value and usefulness of this invention are enormous. Inhibition of PLA2 GIB can indeed be used to prevent or treat immune disease in HIV-infected patients, as well as, more broadly, immunocompromised individuals. When used early in infection, PLA2 GIB inhibitors lead to a controlled HIV status in patients. When used later, alone or in combination/alternate with HAART, they accelerate the recovery of CD4 T-lymphocyte function, and by enhancing host defense, PLA2 GIB inhibitors bring about an equilibrium between the virus and the immune system, as in many other chronic viral infections. Thus, PLA2 GIB inhibitors represent very potent agents for use, alone or in combination, in the treatment of disorders associated with an abnormal immune response or activity. They may also save on HAART and may also allow interruption of this therapy, which is known to be associated with severe side effects.

Further, since the lack of expression of GIBsPLA2 (as in mice with the corresponding knockout gene) is well tolerated, temporary or permanent suppression of GIBsPLA2 using inhibitors or through vaccination represents a powerful and reliable immunotherapy for immune diseases, in particular HIV patients.

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Claims (12)

CLAIM 1. Use of a group IB secreted phospholipase A2 (GIBsPLA2) inhibitor for the treatment of a disease associated with CD4 T cell immunodeficiency in a subject in need thereof, wherein the GIBsPLA2 inhibitor is selected from the group consisting of an anti-GIBsPLA2 antibody, an inhibitory nucleic acid molecule, based on the sequence of the GIBsPLA2 gene, the GIBsPLA2 soluble receptor, and a peptide containing 3 to 20 amino acid residues, wherein said peptide is produced by a method comprising (i) contacting a test peptide with GIBsPLA2, (ii) selecting a test peptide that binds GIBsPLA2, and (iii) selecting that peptide from (ii) that inhibits GIBsPLA2 activity. 2. Use according to claim 1, wherein GIBsPLA2 is a protein containing amino acid residues 23-148 of SEQ ID NO: 2 or a natural variant thereof. 3. Use according to claim 1 or 2, wherein the anti-GIBsPLA2 antibody is selected from an anti-GIBsPLA2 polyclonal antibody, an anti-GIBsPLA2 monoclonal antibody, or GIBsPLA2 binding fragments selected from F(ab') ₂ fragments, Fab fragments, single chain variable fragments ( scFvs), single domain antibodies (VHH or nanobodies), bivalent antibody fragments (diabodies). 4. Use according to claim 3, wherein the anti-GIBsPLA2 antibody is human or humanized. 5. Use according to claim 1, wherein the inhibitory nucleic acid based on the GIBsPLA2 gene sequence is selected from an antisense nucleic acid, small interfering RNA, small hairpin RNA, miRNA, aptamers, or ribozymes that inhibit the GIBsPLA2 gene or GIBsPLA2 protein expression. 6. Use according to any one of claims 1 to 4 to induce activation of CD4 T cells in a subject. 7. Use according to any one of claims 1 to 6, wherein the subject is immunodeficient or immunosuppressed, has an infectious disease such as a viral infection, or the subject has cancer. 8. Use according to any of the preceding claims for the treatment of AIDS in an HIV-infected subject. 9. Use according to claim 8 for the suppression or reversal of HIV-mediated immunodeficiency. 10. Use according to any of the preceding paragraphs, where the GIBsPLA2 inhibitor is administered by injection, preferably intramuscular, subcutaneous, transdermal, intravenous or intra-arterial injection; or by nasal, oral, mucosal, rectal administration, or by inhalation. 11. A pharmaceutical composition in the form of an injection solution or suspension for injection, comprising (i) a GIBsPLA2 inhibitor and (ii) a pharmaceutically acceptable carrier or excipient suitable for an injection solution or suspension for injection, for the treatment of a disease associated with CD4 T immunodeficiency cells, in a subject in need thereof, wherein the GIBsPLA2 inhibitor is an anti-GIBsPLA2 antibody selected from an anti-GIBsPLA2 polyclonal antibody. - 32 040303 la, aHTu-GIBsPLA2 monoclonal antibody, or their GIBsPLA2-binding fragments, selected from F(ab') ₂ fragments, Fab fragments, single-chain variable fragments (scFvs), single-domain antibodies (VHH or nanobodies), bivalent antibody fragments ( diatel), and where GIBsPLA2 is a protein containing amino acid residues 23-148 of SEQ ID NO: 2 or its natural variant. 12. A method of treating a disease associated with CD4 T cell immunodeficiency in a subject in need thereof, wherein the method comprises administering to said subject an effective amount of a GIBsPLA2 inhibitor selected from the group consisting of an anti-GIBsPLA2 antibody that inhibits a nucleic acid based on a gene sequence GIBsPLA2, a soluble GIBsPLA2 receptor, and a peptide containing 3 to 20 amino acid residues, wherein said peptide is produced by a method comprising (i) contacting a test peptide with GIBsPLA2, (ii) selecting a test peptide that binds GIBsPLA2, and (iii) selecting that peptide of (ii) that inhibits the activity of GIBsPLA2.