TREATMENT OF MONOGENIC DISEASES WITH AN ANTI-CD45RC ANTIBODY

Abstract

Anti-CD45RC antibodies, for use in the treatment of monogenic diseases caused by genes not associated with immune function but whose deficiency is associated with inflammation and/or immune reactions (such as genes deficient in Duchenne muscular dystrophy (DMD), cystic fibrosis, lysosomal diseases and al-anti-trypsin deficiency); or caused by genes involved in the immune system and whose deficiency generates inflammation and/or autoimmune reactions (such as genes deficient in T-cell primary immunodeficiencies such as IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked syndrome), APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy), B cell primary immunodeficiencies, Muckle-Wells syndrome, mixed autoinflammatory and autoimmune syndrome, NLRP12-associated hereditary periodic fever syndrome, and tumor necrosis factor receptor 1 associated periodic syndrome).

Claims

1-14. (canceled)

15. A method of preventing and/or treating a monogenic disease in a subject in need thereof, comprising administering to said subject an anti-CD45RC antibody, wherein said monogenic disease is selected from the group consisting of: a. monogenic diseases caused by a gene which is not associated with immune function but whose deficiency is associated with inflammation and/or immune reactions, and selected from Duchenne muscular dystrophy (DMD), cystic fibrosis, lysosomal diseases and al-anti-trypsin deficiency; and/or b. monogenic diseases caused by a gene involved in the immune system and whose deficiency generates inflammation and/or autoimmune reactions, and selected from immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), B cell primary immunodeficiencies, Muckle-Wells syndrome, mixed autoinflammatory and autoimmune syndrome, NLRP12-associated hereditary periodic fever syndrome, and tumor necrosis factor receptor 1 associated periodic syndrome.

16. The method according to claim 15, wherein said anti-CD45RC antibody is a monoclonal antibody.

17. The method according to claim 15, wherein said anti-CD45RC antibody is an anti-human CD45RC monoclonal antibody.

18. The method according to claim 15, wherein said anti-CD45RC antibody is a chimeric antibody, a bispecific antibody, a humanized antibody or a fully human antibody.

19. The method according to claim 15, wherein prevention and/or treatment of monogenic diseases comprises reduction, alleviation, lessening and/or inhibition of symptoms or signs associated with said monogenic diseases, preferably of autoimmune and/or inflammatory symptoms or signs.

20. The method according to claim 15, wherein said anti-CD45RC antibody depletes T CD45RC.sup.high cells.

21. The method according to claim 15, wherein said anti-CD45RC antibody is a multispecific antibody comprising a first antigen binding site directed against. CDR45RC and at least one second antigen binding site directed against an effector cell able to mediate depletion of T CD45RC.sup.high cells through direct binding, antibody-dependent cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and/or antibody-dependent phagocytosis.

22. The method according to claim 15, wherein said anti-CD45RC antibody is conjugated to a cytotoxic moiety.

23. The method according to claim 15, in the form of a pharmaceutical composition comprising said anti-CD45RC antibody and a pharmaceutically acceptable carrier or excipient or vehicle.

24. The method according to claim 15, wherein said anti-CD45RC antibody is to be administered in combination with an immunosuppressive and/or anti-inflammatory drug.

25. The method according to claim 15, wherein said anti-CD45RC antibody is to be administered in combination with gene therapy or cell therapy.

26. The method according to claim 25, wherein said gene therapy or cell therapy is to be administered before or after administration of said anti-CD45RC antibody, preferentially before administration of said anti-CD45RC antibody.

27. The method according to claim 15, wherein said monogenic disease is selected from DMD, cystic fibrosis, lysosomal storage diseases and al-anti-trypsin deficiency, preferably said monogenic disease is DMD.

28. The method according to claim 15, wherein said monogenic disease is selected from IPEX, APECED, B cell primary immunodeficiencies, Muckle-Wells syndrome, mixed autoinflammatory and autoimmune syndrome, NLRP12-associated hereditary periodic fever syndrome, and tumor necrosis factor receptor 1 associated periodic syndrome, preferably said monogenic disease is APECED.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0280] FIG. 1 is a set of graphs showing the number of leukocytes in muscle and spleen of Dmd.sup.mdx rats. Hind limb skeletal muscles and spleen were harvested from littermate wild-type (WT) or Dmd.sup.mdx (KO) rats at the indicated time points of age. Muscle and spleen were digested with collagenase and mononuclear cells were isolated using a density gradient. (A) Representative dot-plot analysis of mononuclear cells muscle (left panel) and spleen (right panel) from animals at 8 weeks of age stained with a viability dye and a pan anti-leukocyte CD45 monoclonal antibody (OX1). (B) Number of CD45.sup.+ cells by gram of muscle tissue or by total spleen at different time points. WT, n=4, 5, 7, 7, 9 at 2, 4, 8, 12 and 16 weeks, respectively. KO, n=3, 6, 10, 11, 16 at 2, 4, 8, 12 and 16 weeks, respectively. * p<0.05, ***p<0.001, ****p<0.0001.

[0281] FIG. 2 is a set of two graphs showing the number of TCR cells in muscle and spleen of littermate WT and Dmd.sup.mdx rats. PBMCs from hind limbs (left panel) and spleen (right panel) were obtained as explained in the legend of FIG. 1. The number of TCR.sup.+ cells was determined by staining PBMCs with an anti-TCRab monoclonal antibody (clone R7/3) and calculating the number of TCR cells among viable CD45R.sup.+ cells. * p<0.05, **p<0.01.

[0282] FIG. 3 is a set of four graphs showing the numbers of T CD8.sup.+CD45RC.sup.high and CD45RC.sup.int/neg cells after treatment using an anti-CD45RC monoclonal antibody. Control littermate wild-type (WT) or Dmd.sup.mdx (KO) rats received from week 2 of age intraperitoneal injections of the mouse anti-rat CD45RC monoclonal antibody up to week 12, when the animals were sacrificed. Muscle and spleen mononuclear cells were isolated and analyzed for the presence of TCR.sup.+CD8.sup.+ cells expressing CD45RC at either high (CD45RC.sup.high) or intermediate/negative levels (CD45RC.sup.int/neg). Each point represents a single animal * p<0.05, ***p<0.001.

[0283] FIG. 4 is a graph showing the muscle strength in Dmd.sup.mdx rats after treatment with an anti-CD45RC monoclonal antibody. Wild type (WT) or Dmd.sup.mdx (KO) rats received intraperitoneal injections of the mouse anti-rat CD45RC or prednisolone from week 2 of age up to week 12, when the muscle strength was analyzed using a grip test. Each point represents a single animal. * p<0.05.

[0284] FIG. 5 is a set of four graphs showing the numbers of T CD8+CD45RC.sup.high and CD45RC.sup.int/neg cells after treatment using prednisolone. Control littermate wild-type (WT) or Dmd.sup.mdx (KO) rats received from week 2 of age intraperitoneal injections of prednisolone up to week 12, when the animals were sacrificed. Muscle and spleen mononuclear cells were isolated and analyzed for the presence of TCR.sup.+CD8.sup.+ cells expressing CD45RC at either high (CD45RC.sup.high) or intermediate/negative levels (CD45RC.sup.int/neg). Each point represents a single animal. * p<0.05.

[0285] FIG. 6 is a set of two graphs showing the weight of wild-type or Dmd.sup.mdx rats during treatment using prednisolone or anti-CD45RC monoclonal antibody. Control littermate wild-type (WT) or Dmd.sup.mdx (KO) rats received intraperitoneal injections of prednisolone or anti-CD45RC monoclonal antibody (OX22) from week 2 up to week 12 of age and weight was recorded at the indicated time points.

[0286] FIG. 7 is a set of photographs and a graph showing reduced autoimmune signs of disease in anti-CD45RC mAb-treated Aire.sup.−/− rats. Aire.sup.−/− rats received from week 2 of age intraperitoneal injections of the mouse anti-rat CD45RC MAb (clone 0X22, 1.5 mg/kg twice per week) up to week 20, when the animals were sacrificed. (A). Picture showing visual aspect of 20 weeks old Aire.sup.−/− rats treated with isotype control (top row) or anti-CD45RC mAb (bottom row). (B) Picture showing the size of the thymus from anti-CD45RC mAb or isotype control Aire.sup.−/− treated rats. (C) Weights of anti-CD45RC mAb or isotype control Aire.sup.−/− littermate SPD rats were measured every week during 20 weeks following birth. Results are shown as the % of initial body weight starting at week 2 after birth (mean 31.3 g)±SEM (n=3).

[0287] FIG. 8 is a set of graphs showing efficient depletion of CD45RC.sup.high T cells in spleen and mesenteric lymph nodes (MLN) following anti-CD45RC mAb administration. Representative dot-plot analysis of mononuclear cells (CD4.sup.+ and CD8.sup.+ T cells) from MLN and spleen from Aire.sup.−/− treated with anti-CD45RC or isotype control Mabs or WT untreated animals at 20 weeks of age and treated from week 2 stained with a viability dye and a pan anti-leukocyte CD45 MAb (OX1), CD3, TCR, CD4 and CD45RC MAb.

[0288] FIG. 9 is a graph defining 3 populations of CD8.sup.+ cells according to the different levels of CD45RC expression (CD45RC.sup.high, CD45RC.sup.int and CD45RC.sup.neg) in rat CD8+ T cells labeled with the anti-CD45RC Mab OX22.

[0289] FIG. 10 is a set of two SDS-PAGE gels showing the effect of anti-CD45RC MAb treatment on serum autoantibodies in Aire-deficient rats. Aire-deficient rats were treated with (A) an isotype control MAb or (B) an anti-CD45RC MAb (OX22) at 1.5 mg/kg twice per week from weeks 2 to 20 of age. Silver staining. β-actin was used as a control.

[0290] FIG. 11 is a set of eight photographs of tissue sections (thymus, pancreas, skin, kidney) stained with hematoxilin-eosine-safran, showing the tissue architecture and lymphocyte infiltrates in rats treated with an isotype antibody (left photographs) or with an anti-CD45RC antibody (right photographs). White arrows represent major regions of each organ differing between anti-CD45RC or isotype treated recipients.

EXAMPLES

[0291] Materials and Methods Related to Examples 1 to 4

Preparation of Muscle and Spleen Single-Cell Suspensions

[0292] Muscles of both hind limbs from WT or Dmd.sup.mdx rats were excised and weighed. Muscles were minced and placed in gentle MACS C tubes with collagenase D in the presence of FCS 2%, 1 mM EDTA. Two runs of 30 minutes each in a gentle MACS dissociator were performed with new collagenase added between each run. Cells were suspended in 30 mL of PBS FCS 2%, 1 mM EDTA, were then applied to 15 mL of Hystopaque and centrifuged at 1000 g for 30 minutes without a break. Mononuclear cells were collected from the Hystopaque and PBS interface, washed and suspended in PBS FCS 2%, 1 mM EDTA.

[0293] Spleen was harvested, perfused with PBS and digested by collagenase D for 15 minutes at 37° C. Cells were suspended in PBS FCS 2%, 1 mM EDTA and mononuclear cells were recovered as explained above.

Flow Cytometry Analysis

[0294] Mononuclear cells were stained with antibodies against the following antigens: CD45 (clone OX-1), T-cell receptor (TCR; clone R7/3), CD45RC (clone OX22), CD8 (clone OX8) and CD4 (W3/25), as well as with viability dyes eFluor506 or eFluor450, all from eBiosciences. Analysis was performed on a BD FACS Verse with FACSuite Software version 1.0.6. Post-acquisition analysis was performed with FlowJo software.

Treatment with Anti-CD45RC or Prednisolone

[0295] Wild-type (WT) or Dmd.sup.mdx (KO) rats received intraperitoneal injections of a mouse anti-rat CD45RC monoclonal antibody (clone OX22, 2 mg/kg, every 3.5 days) from week 2 of age up to week 12.

[0296] Prednisolone was administered by intraperitoneal injections (0.5 mg/kg, 5 days a week) from week 2 of age up to week 12.

[0297] At week 12 of age treated rats were analyzed for muscle strength using a grip test.

Grip Test

[0298] Rats were placed with their forepaws on a grid and were gently pulled backward until they released their grip. A grip meter (Bio-GT3, BIOSEB, France), attached to a force transducer, measured the peak force generated.

Example 1

[0299] Analysis of Total Keukocytes and T cells in Dmd.sup.mdx Rats

[0300] Leukocytes in the muscle and spleen of Dmd.sup.mdx rats were analyzed by flow cytometry (FIG. 1).

[0301] Total leukocytes in the muscle of littermate WT and Dmd.sup.mdx rats were comparable at 2 weeks of age, but at 4 weeks, Dmd.sup.mdx rats showed a sharp increase that was maintained until week 8 and then decreased at weeks 12 and 14, although still significantly higher than in littermate WT rats.

[0302] Spleen leukocyte numbers were comparable between WT and Dmd.sup.mdx rats at all-time points analyzed.

[0303] Analysis of the number of TCR cells in muscle and spleen of littermate WT and Dmd.sup.mdx rats showed a significant increase in the muscle of Dmd.sup.mdx rats at 4 and 12 weeks of age with an increased tendency at week 8 and 16 weeks (FIG. 2).

[0304] The numbers of TCR.sup.+ cells in spleen did not show differences between WT and Dmd.sup.mdx rats at any time point.

Example 2

[0305] Treatment with Anti-CD45RC Monoclonal Antibody Depletes CD45RC.sup.high T Cells

[0306] Administration of a mouse anti rat-CD45RC monoclonal antibody from week 2 of age resulted in partial depletion of T CD8.sup.+ CD45RC.sup.high cells analyzed at 12 weeks of age in muscle of Dmd.sup.mdx rats and in spleen of both littermate WT and Dmd.sup.mdx rats (FIG. 3).

[0307] T CD8.sup.+ or CD4.sup.+ CD45RC.sup.int/neg, including all CD8.sup.+ and CD4.sup.+T.sub.regs, were not modified in the spleen or muscle of neither WT nor Dmd.sup.mdx rats (FIG. 3).

[0308] All other major leukocyte populations (macrophages, B cells and NK cells) were unchanged (data not shown).

Example 3

[0309] Treatment with Anti-CD45RC Monoclonal Antibody Improves Muscle Strength in Dmd.sup.mdx Rats

[0310] To examine whether muscle function was improved by the anti-CD45RC monoclonal antibody treatment, muscle strength was analyzed by a grip test of forelimbs in WT and Dmd.sup.mdx rats at 12 weeks of age after initiation of treatment at 2 weeks of age.

[0311] A significant decrease in forelimb grip strength indicated a generalized alteration in the whole-body muscular performance. As previously described by Larcher et al. (2014. PLoS One. 9(10):e110371), a 30% weaker force was exerted by Dmd.sup.mdx rats compared to WT littermates.

[0312] After administration of the anti-CD45RC monoclonal antibody, muscle strength in Dmd.sup.mdx rats vs. untreated Dmd.sup.mdx rats was significantly increased and was indistinguishable from WT littermate controls (FIG. 4).

Example 4

[0313] Treatment with Prednisolone Improved Skeletal Muscle Strength Since corticoids are standard treatment in DMD patients (Alman, 2005. J Pediatr Orthop. 25(4):554-6), the effect of prednisolone was analyzed in the muscle strength of Dmd.sup.mdx rats.

[0314] Treatment of Dmd.sup.mdx rats with prednisolone, since 2 weeks of age, increased muscle strength at 12 weeks to levels identical to those of WT or anti-CD45RC-treated rats (FIG. 4).

[0315] Interestingly, prednisolone-treated rats also showed a specific decrease of CD8.sup.+CD45RC.sup.high cells in both muscle and spleen of Dmd.sup.mdx rats and in spleen of WT rats, whereas CD8.sup.+CD45RC.sup.int/neg cells were maintained (FIG. 5).

[0316] Similarly, CD4.sup.+CD45RC.sup.high cells were numerically reduced in both muscle and spleen of Dmd.sup.mdx rats and in spleen of WT rats whereas CD8.sup.+CD45RC.sup.int/neg cells were maintained (data not shown).

[0317] Prednisolone-treated Dmd.sup.mdx rats showed severe reduction in animal growth whereas anti-CD45RC monoclonal antibody-treated rats did not (FIG. 6).

Example 5

[0318] Treatment of APECED Disease

Materials and Methods

Cell Isolation

[0319] Spleen and lymph nodes were digested by collagenase D for 30 minutes at 37° C. The reaction was stopped by adding 0.01 mM EDTA.

[0320] Cells from blood and bone marrow were also isolated and red blood cells were lysed using a lysis solution (8,29 g NH.sub.4Cl, 1 g KHCO3, 37,2 mg EDTA qsp 1 L deionized water pH 7.2-7.4).

[0321] Antibodies and Flow Cytometry

[0322] Cellular phenotype was analyzed using monoclonal antibodies from BD pharmigen: against TCRαβ (R73), CD25 (Ox39), CD4 (Ox35), and CD45 (Ox1).

[0323] Abs against CD45RC (Ox22) and CD8 (Ox8) produced in our lab were used.

[0324] Antibodies were used to stain cells and fluorescence was measured with a FACSCanto II flow cytometer (BD Bioscience) and FlowJo software was used to analyze data. Cells were first gated on their morphology and then dead cells were excluded by staining with fixable viability dye efluor 506 (Ebioscience).

Treatment with Anti-CD45RC or Isotype Control

[0325] Aire.sup.−/− (KO) rats received intraperitoneal injections of a mouse anti-rat CD45RC monoclonal antibody (clone OX22, 1.5 mg/kg, every 3.5 days) from week 2 of age up to week 20 when the animals were sacrificed. Isotype control was administered similarly. The rats were weighted twice a week from the start of the treatment until the day of sacrifice. At week 20 of age, treated rats were sacrificed and analyzed.

Autoantibodies Detection

[0326] Serum from Aire-deficient rats treated with anti-CD45RC or treated with an isotype control at 20 weeks of age were incubated with membranes in which lysates from different organs (spleen, colon, kidney, eye, lung, testis, mesenteric lymph nodes [MLN] and ileum) from an Il2rg-deficient rat (Il2rg: interleukin 2 receptor subunit gamma), thus without endogenous immunoglobulins, were previously electrophoresed. Binding of rat autoantibodies was revealed using anti-rat immunoglobulin antibodies or with an anti-β-actin antibody coupled to peroxidase.

Histochemistry

[0327] Organs from Aire-deficient rats treated with anti-CD45RC or treated with an isotype control were harvested at 20 weeks of age. Tissue sections (thymus, pancreas, skin, kidney) were prepared and stained with hematoxilin-eosine-safran.

Results

[0328] We observed that 100% of Aire-deficient rats spontaneously developed alopecia and skin depigmentation in isotype control treated animals (FIG. 7A, top row), signs that correlate with a severe auto-immune disease and that are clinical manifestations regularly found in APECED patients.

[0329] In contrast, Aire-deficient rats treated with anti-CD45RC mAb did not develop alopecia and skin depigmentation (FIG. 7A, bottom row), had a bigger thymus size (FIG. 7B) and a normal body weight (FIG. 7C), indicating a normal growth, a preserve thymus structure and altogether a decreased auto-immune disease.

[0330] Administration of a mouse anti rat-CD45RC monoclonal antibody from week 2 of age resulted in strong depletion of T CD8.sup.+ and CD4.sup.+ CD45RC.sup.high cells in both spleen and lymph node in Aire−/− rats compared to rats treated with isotype control and untreated WT rats (FIG. 8).

[0331] Numbers of other major leukocyte populations (macrophages, B cells and NK cells) were unchanged (data not shown).

[0332] Analyses of autoantibodies directed against tissue antigens was also evaluated, by western blot, in tissue homogenates and serum.

[0333] In the absence of anti-rat-CD45RC monoclonal antibody treatment, many bands were detected in different organs, indicating the presence of antoantibodies (FIG. 10A).

[0334] By contrast, treatment with an anti-rat-CD45RC monoclonal antibody reduced the number of bands and thus, the number of autoantibodies (FIG. 10B).

[0335] At week 20 of treatment, tissue architecture was destroyed and lymphocyte infiltrates were present in many organs in the absence of treatment, while treatment with the anti-rat-CD45RC monoclonal antibody restored tissue integrity and reduced lymphocyte infiltrates (FIG. 11).

Conclusion

[0336] Altogether, the data presented hereinabove in Examples 1-5 provide a clear demonstration of reduced if not inhibited inflammation and autoimmune reactions typically associated with certain monogenic diseases through the use of anti-CD45RC antibodies. A disbalance between T.sub.eff and T.sub.regs with the end result of increased T responses, but also B cell-mediated responses through production of autoantibodies, is involved in other monogenic diseases, such as cystic fibrosis, lysosomal diseases, α1-anti-trypsin deficiency, IPEX, B cell primary immunodeficiencies, Muckle-Wells syndrome, mixed autoinflammatory and autoimmune syndrome, NLRP12-associated hereditary periodic fever syndrome, and tumor necrosis factor receptor 1 associated periodic syndrome.