TREATMENT OF DISEASES ASSSOCIATED WITH FAT ACCUMULATION

Abstract

The present invention is directed to immunmodulators in the form of compositions, compounds, proteins and/or fragments with RNase activity thereof for use in the treatment of diseases associated with fat accumulation, including obesity and obesity-related disorders and metabolic disorders.

Claims

1-19. (canceled)

20. A method for the treatment of diseases associated with fat accumulation, comprising the administration of a ribonuclease protein of the T2 family, or a fragment thereof that induces IL-33 release to initiate a type 2, optionally in adipose cells and/or tissues, to a subject in need thereof.

21. The method according to claim 20 wherein the ribonuclease protein is Omega-1 protein or a fragment thereof.

22. The method according to claim 20 wherein the diseases associated with fat accumulation include obesity, obesity-related disorders and metabolic disorders.

23. The method according to claim 20 wherein the ribonuclease protein or a fragment thereof induces IL-33 release to initiate a type 2 response in adipose cells and/or tissues,

24. The method according to claim 20 wherein the ribonuclease protein or a fragment thereof comprises at least one or more RNAase catalytic domains.

25. The method according to claim 20 wherein the ribonuclease protein or a fragment thereof comprises at least a first conserved amino acid sequence (CAS1) comprising amino acid residues FTIHGLWPT and/or a second conserved amino acid sequence (CAS2) comprising amino acid residues PSFWKHEFEKHGLCAV.

26. The method according to claim 20 wherein the ribonuclease protein or a fragment thereof is Omega-1 protein or a fragment thereof.

27. The method according to claim 26 wherein the Omega-1 protein fragment comprises at least part of amino acid residues 1 to 224.

28. The method according to claim 26 wherein the Omega-1 protein fragment comprises at least one or more RNAase catalytic domains.

29. The method according to claim 26 wherein the Omega-1 protein fragment comprises at least a first conserved amino acid sequence (CAS1) comprising amino acid residues FTIHGLWPT; and/or a second conserved amino acid sequence (CAS2) comprising amino acid residues PSFWKHEFEKHGLCAV.

30. The method according to claim 20 wherein the ribonuclease protein or fragment thereof further comprises a glycoprotein carrier.

31. The method according to claim 20 for the treatment of obesity and obesity-related disorders and/or inducing weight loss by decreasing the number of adipose cells after administration.

32. The method according to claim 20 wherein the obesity related disorders are selected from heart disease, stroke, high blood pressure/hypertension, glucose disorders including diabetes (type 1 and type 2 diabetes mellitus), cancer, gallbladder disease and gallstones, osteoarthritis, gout, breathing problems, such as sleep apnea and asthma.

33. The method according to claim 22 for the treatment of metabolic disorders by restoring glucose and insulin homeostasis after administration.

34. The method according to claim 22 wherein the metabolic disorders associated with fat accumulation include type 1 and type 2 diabetes mellitus, high blood pressure/hypertension, nonalcoholic fatty liver disease, atherosclerosis, cancers, breathing problems including sleep apnea and cardiovascular diseases.

35. The method according to claim 22 wherein the metabolic disorders associated with fat accumulation is nonalcoholic fatty liver disease.

36. The method according to claim 20 for the treatment of a liver disorder by decreasing the number of adipose cells in the liver after administration.

37. The method according to claim 20 wherein the fat accumulation disorder is fatty liver disease.

38. A pharmaceutical composition comprising a ribonuclease protein of the T2 family, or a fragment thereof that induces IL-33 release to initiate a type 2, preferably in adipose cells and/or tissues.

39. The pharmaceutical composition according to claim 38 wherein the ribonuclease protein is Omega-1 protein or a fragment thereof.

Description

FIGURE LEGENDS

[0065] FIG. 1: Expression of his-tagged recombinant omega-1 from HEK-293 cells and confirmation of RNase activity. Recombinant protein was Ni-Affinity and gel filtration chromatography purified and subjected to endotoxin removal by detergent-based methods. (A) SDS-PAGE of purified proteins stained with Coomassie Blue (1) recombinant ω1; (2) RNase null ω1. (B) RNA from murine bone-marrow derived macrophages was incubated with 500 ng/ml and 100 ng/ml of recombinant ω1 and RNase null ω1 (ω1Δ.sup.RNase) for 1 hour and RNA integrity analyzed on a 2% agarose gel. RNase A was used as a positive control.

[0066] FIG. 2: Nucleotide and amino acid sequence of Omega-1. N-glycosylation sites are bold; Conserved Amino acid Sequences (CAS-1 and CAS-2) are underlined.

[0067] FIG. 3: Recombinant ω1 induces weight loss and an improvement in glucose homeostasis in obese mice. (A) Weight gain, expressed as a percentage from starting weight, in WT mice on high fat diet (HFD) for 8 weeks, and treated with 25 μg ω1, or 25 μg OVA on days 0, 2 and 4. Weight was monitored for 21 days. (B) Weight of excised visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) in OVA and ω1 treated mice at 6 days post initial injection. (C) Immunohistochemistry depicting H&E staining from excised VAT from control diet (CD) fed animals, and HFD fed animals treated with OVA and ω1. VAT was excised at day 6 post initial injection. Adipocyte area was calculated from histological slides. Blood glucose was assessed basally in fasted mice (D) and glucose tolerance assessed after injection of 2 g/kg glucose i.p. at day 6 post initial injection of r-ω1 (E). Levels of triglyceride (F) were determined in the serum of OVA and ω1 treated mice. Data are representative of n=6 (+/−SEM) from 3 independent experimental replicates (*P<0.05, ** P<0.01, ***P<0.001, ****P<0.0001).

[0068] FIG. 4: Short-term, low dose treatment with recombinant omega-1 reduces liver damage in obese mice. Mice were fed CD and HFD and treated with 25 μg endotoxin-free ω1, or 25 μg endotoxin-free OVA on days 0, 2 and 4. On day 6 AST and ALT were quantified in the serum and expressed as a ratio of AST to ALT. Data are representative of n=6 (+/−SEM) from 3 independent experimental replicates (ns—not-significant, *P<0.05).

[0069] FIG. 5: Long-term treatment with recombinant ω1 maintains weight loss and glucose homeostasis in obese mice. (A) Weight gain, expressed as a percentage from starting weight, in WT mice on high fat diet (HFD) for 8 weeks, and treated with 25 μg endotoxin-free ω1, or 25 μg endotoxin-free OVA on days 0, 2 and 4 (short-term), or on days 0, 4, 8, 12, 16 and 20 (long-term). Weight was monitored for 21 days. (B) Weight of excised VAT in OVA and ω1 treated mice at 21 days post initial injection. Blood glucose was assessed basally in fasted mice (C) and glucose tolerance assessed after injection of 2 g/kg glucose i.p. at day 21 post initial injection of ω1 (D). Data are representative of n=3 (+/−SEM) from 2 independent experimental replicates (*P<0.05, **P<0.01).

[0070] FIG. 6: Recombinant ω1 induces a type 2 immune cell repertoire in the VAT of obese mice. Cellular infiltration into the VAT of obese mice treated with 25 μg endotoxin-free ω1, or 25 μg endotoxin-free OVA on days 0, 2 and 4 was assessed by flow cytometry on day 6 post initial injection. Alternatively activated macrophages (AAM) were identified as CD11b.sup.+F4/80.sup.+CD206.sup.lo and CD11b.sup.+F4/80.sup.hiCD206.sup.hi respectively. Eosinophils were identified as CD11b.sup.+SiglecF.sup.+, and ILC2 as Lineage.sup.−IL-7Rα.sup.+Sca-1.sup.+T1/ST2.sup.+KLRG1.sup.+. Data are representative of n=6 (+/−SEM) from 3 independent experimental replicates (*P<0.05, ** P<0.01, ***P<0.001).

[0071] FIG. 7: Recombinant ω1 induces the localized release of type 2 cytokines, including IL-33. Release of IL-4, IL-5, IL-13 (A) and IL-33 (B) were quantified in the peritoneal fluid at 1, 3, 6 and 24 hours post injection of 25 μg endotoxin-free ω1 (A; 6 hours post injection) by ELISA. Culture of mouse (C) and human (D) adipocytes in the presence of 500 ng/ml ω1 results in a peak of IL-33 production after 3 hours. Data are representative of n=4-6 (+/−SEM) from 2-3 independent experimental replicates (*P<0.05, ** P<0.01).

[0072] FIG. 8: RNase mutant ω1 does not induce significant weight loss or IL-33 induction in obese mice. (A) Weight gain, expressed as a percentage from starting weight, in WT mice on high fat diet (HFD) for 8 weeks, and treated with 25 μg endotoxin-free ω1 (WT), 25 μg endotoxin-free ω1Δ.sup.RNase or 25 μg endotoxin-free OVA on days 0, 2 and 4. Weight was monitored for 6 days. (B) Weight of excised visceral adipose tissue (VAT) in OVA and WT and ω1Δ.sup.RNase treated mice at 6 days post initial injection. (C) Glucose tolerance assessed after injection of 2 g/kg glucose i.p. at day 6 post initial injection of WT or ω1Δ.sup.RNase, blood glucose was measured at 30, 60 and 120 minutes after injection of glucose. (Data are representative of n=5 (+/−SEM) from 2 independent experimental replicates (*P<0.05, **P<0.01, ***P<0.001).

[0073] FIG. 9: Weight loss by ω1 is mediated by RNase activity. (A) Weight gain, expressed as a percentage from starting weight, in WT mice on high fat diet (HFD) for 8 weeks, and treated with 25 μg ω1 (WT), 25 μg ω1Δ.sup.RNase or 25 μg OVA on days 0, 2 and 4. Weight was monitored for 6 days. (B) Weight of E-WAT in OVA and WT and ω1Δ.sup.RNase treated mice at 6 days post initial injection. (C) Glucose tolerance assessed after injection of 2 g/kg glucose i.p. at day 6 post initial injection of WT or ω1Δ.sup.RNase. (D) Cellular infiltration into the E-WAT was assessed by flow cytometry 6 days after initial injection of OVA, ω1 or ω1Δ.sup.RNase. AAM were identified as CD11b.sup.+F4/80.sup.hiCD206.sup.hi, eosinophils were identified as CD11b.sup.+SiglecF.sup.+ and ILC2 as Lin.sup.−IL-7Rα.sup.+Sca-1.sup.+T1/ST2.sup.+KLRG1.sup.+. Data are representative of n=5-8 (+/−SEM) from 2 independent experimental replicates (ns—not significant, *P<0.05, **P<0.01, ***P<0.001).

[0074] FIG. 10: Effective binding to CD206 is essential for the functional activity of ω1. Weight gain, expressed as a percentage from starting weight, in WT mice on high fat diet (HFD) for 8 weeks, and treated with 25 μg ω1 (WT), 25 μg ω1Δ.sup.GLY or 25 μg OVA on days 0, 2 and 4. Data are representative of n=2-6 (+/−SEM) from 2 independent experimental replicates (ns—not significant, ** P<0.01).

[0075] FIG. 11: Hepatic steatosis assesment showing the ratio of AST to ALT in lean control mice and obese mice. Serum was recovered from lean (normal) mice or obese mice subjected to high fat diet for 8 weeks. Obese mice were treated with 25 μg OVA or omega-1 on days 0, 2 and 4. Serum was recovered on Day 6 and serum levels o AST and ALT analsyde by ELISA>f Data are representative of n=4-6 (+/−SEM) from 2 independent experiments. P<0.05 significant differences between obese mice treated with OVA or omega-1.

EXAMPLES

Example 1

Method

[0076] Recombinant omega-1 was expressed with a 6×His-tag in HEK293 cells that were transfected with the expression vector pSecTag2-omega-1. In addition a recombinant Omega-1 RNase mutant protein (ω1Δ.sup.RNase), was prepared by mutating the Histidine 58 in CAS1 (FIG. 2) to Phenylalanine. Recombinant proteins were purified from culture supernatants by nickel-affinity and gel-filtration chromatography. Purified protein was subjected to detergent extraction, with recombinant omega-1 preparations having <0.5 EU per mg protein. The resultant ˜31 kDa protein was checked for purity by SDS-PAGE and western blotting using an anti-His tagged mAb (FIG. 1).

[0077] For all studies diet-induced obesity was initiated and maintained in 7-9 week old C57BU6J strain mice by feeding a 60% fat diet ad libitum for <8 weeks, during which time mice gain approximately 20% additional body weight (termed HFD), as described [10]. As a control for diet-induced obesity, mice were fed a nutritionally balanced diet containing 20% fat ad libitum which maintains a normal body weight gain with age (termed CD).

[0078] For acute treatment HFD and CD mice were treated with endotoxin-free recombinant omega-1 (25 μg in PBS i.p.) on days 0, 2 and 4; as a glycoprotein control HFD and CD mice were treated with endotoxin-free ovalbumin (OVA) (25 μg in PBS i.p.) on days 0, 2 and 4. Weight and condition were monitored daily. Metabolic parameters and cellular accumulation in the VAT were assessed on day 6, 2 days after the final treatment with recombinant omega-1, or on day 21, 16 days after the final omega-1 treatment. Chronic treatment involved administration of endotoxin-free recombinant omega-1 (25 μg in PBS i.p.) on days 0, 4, 8, 12, 16 and 20 with metabolic studies conducted on days 21.

[0079] Blood glucose was measured using a glucometer in mice fasted for 16 hours. Glucose tolerance was determined after i.p. injection of 2 g/kg glucose and blood glucose measured 30, 60 and 120 minutes to determine clearance from the blood. Serum triglyceride and liver enzyme (ALT; alanine transaminase, AST; glutamic oxaloacetate transaminase) levels were determined using commercially available kits from Abnova and Abcam respectively. Histological analysis of formalin fixed adipose tissue stained with hematoxylin and eosin allowed calculation of adipocyte area. Oil red O staining was performed on cryopreserved liver sections to determine lipid deposition in the liver.

[0080] To determine the cellular composition of the VAT, flow cytometric analysis was performed on a single cell suspension generated from VAT, with data collection on a CyAn ADP cytometer and data analysed using FlowJo software. To identify ILC2 cells were stained with BD Biosciences mAbs; CD8-APC (Ly-2), B220-APC (RA3-6B2), F4/80-APC (BM8), ICOS-PE (7E.17G9), Siglec-F-APC (E50-2440); eBiosciences mAbs; CD4-APC (RM4-5), CD11b-APC (M1/70), Gr-1-APC (RB6-8CS), FcER1-APC (MAR-1) and T1/ST2-FITC mAb (DJ8: MD biosciences). To identify eosinophils and AAM cells were stained with BD Biosciences mAbs; Siglec-F-PE (E50-2440), F4/80-APC (BM8), eBiosciences mAb; CD11b-PerCP (M1/70) and BioLegend mAb; CD206-PECy7 (C068C2).

[0081] Murine adipocytes were isolated from VAT after mechanical shredding and incubation with 1 mg/ml Collagenase D for 1 hour at 37° C. Adipocytes were collected from the surface of the media, washed in PBS supplemented with 2% FCS and resuspended at a density of 2×10.sup.6 cells/ml in RPMI supplemented with 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Human adipocytes were isolated from omental adipose tissue biopsies from patients undergoing elective abdominal surgery. Omental samples were processed to isolate adipocytes as described for mouse VAT samples. Mouse and human adipocytes were incubated with 500 ng/ml endotoxin-free recombinant omega-1 for 3 and 24 hours.

[0082] ELISA techniques were used to determine IL-4, IL-5, IL-13 and IL-33 levels in the peritoneal cavity in response to recombinant omega 1, 3 and 6 hours after treatment. IL-33 release from adipocytes in response to omega 1 was also determined in the culture supernatant by ELISA. All ELISAs were performed using duoset kits from R&D Systems, following the manufacturer's protocols.

Results

[0083] Obese mice, ˜30 g after being maintained on a HFD for >8 weeks, were treated with recombinant ω1 (25 μg per mouse i.p. injections on days 0, 2 and 4) and had a significant (P<0.01-0.05) transient weight loss relative to obese mice injected with control protein (OVA) (FIG. 3A). The weight loss in obese mice treated with ω1 was specifically associated with decreased adiposity as determined by a significant decrease in both visceral and subcutaneous white adipose tissue weight (FIG. 3B). In addition, the size of adipocytes was reduced size in ω1-treated mice (FIG. 3C). Treatment with ω1 also significantly (P<0.05) decreased serum levels of free triglyceride in obese animals (FIG. 3D).

[0084] Importantly, treatment with ω1 did not induce anorexia or pyrexia in the mice (data not shown). In addition to weight loss and decreased adiposity, treatment with ω-1 significantly reduced fasting blood glucose levels in obese mice, to a point where blood glucose is no longer significantly elevated above levels seen in mice maintained on a 20% fat control diet (CD) (FIG. 3E). Furthermore, ω1-treated obese mice show significantly (P<0.05) improved glucose tolerance when compared to control OVA-treated mice (FIG. 3F). Obese mice develop liver steatosis, as indicated by elevated serum levels of ratios of hepatic enzymes glutamic oxaloacetate transaminase (AST) to alanine transaminase (ALT) in HFD fed mice relative to control diet fed mice (FIG. 4). Mice treated with ω1 [25 μg (1 mg/kg); 3 treatments day 0, 2, 4] had reductions in AST/ALT ratio (FIG. 4), indicating that following this short-term regimen ω1 improved the hepatic steatosis typically associated with obesity.

[0085] To assess the effects of ω1 treatment over time a long-term treatment regimen was employed. In mice treated with ω1 every 4 days for 20 days there was rapid weight loss, which was sustained throughout the treatment regimen (FIG. 5A), with decreased adiposity 21 days after commencement of treatment (FIG. 5B). Furthermore, long-term treatment sustained the decrease in basal blood glucose and improvement in glucose tolerance (FIG. 5C, D).

[0086] Studies have identified type 2 cells such as eosinophils, ILC2 and AAM as pivotal in promoting insulin sensitivity and improved glucose tolerance [3, 10, 11]. Treatment of obese mice with recombinant ω1 significantly increases accumulation of ‘anti-inflammatory’ type 2 cells in the adipose tissue of obese mice (FIG. 6).

[0087] The ability of w 1 to induce type 2 cells, including eosinophils, ILC2 and AAM is due to the localized induction of type 2 cytokines including IL-4, IL-5, IL-13 and IL-33 (FIG. 7A, B). Obese mice were treated with ω1 i.p. and peritoneal lavage fluid collected at 1, 3, 6 and 24 hours. Levels of IL-4, IL-5, IL-13 and IL-33 were all induced in response to ω1. Furthermore, we identify ω1 as a potent inducer of IL-33 from both mouse (FIG. 7C) and human adipocytes (FIG. 7D).

[0088] S. mansoni ω1 has been identified as a T2 RNase, a property shown to be integral to the ability of ω1 to induce a type 2 response [9]. Treating obese mice with ω1Δ.sup.RNase did not induce significant weight loss, or a significant reduction in adiposity (FIG. 8A, B). Furthermore, ω1Δ.sup.RNase did not improve glucose tolerance in obese mice (FIG. 8C). In contrast to the ω1Δ.sup.RNase protein the intact recombinant ω1 (FIG. 1B) was efficacious in modulating these parameters in obese mice (FIG. 8A-C).

[0089] It will be understood that the invention is not limited to the embodiment hereinbefore described, but may be varied in both construction and detail within the scope of the claims.

Example 2

Method

[0090] It is in public domain that S. mansoni ω1 has been identified as a T2 RNase, a property shown to be integral to the ability of ω1 to induce IL-4 and IL-5 release.

[0091] A recombinant ω1 RNase-null (ω1Δ.sup.RNase) mutant was generated, by substituting a phenylalanine residue in the RNase catalytic domain with a histidine residue (H58F) that was devoid of RNase activity.

Results

[0092] Treating obese mice with ω1Δ.sup.RNase did not induce significant weight loss, or a significant reduction in adiposity (FIG. 9A,B). Furthermore, ω1Δ.sup.RNase did not improve glucose tolerance in obese mice (FIG. 9C). The absence of functional RNase activity also diminished type 2 cell infiltration into the adipose tissue, with fewer ILC2 and AAM observed, although interestingly, eosinophil infiltration is still significantly (P<0.001) increased (FIG. 9D).

[0093] The function of ω1 is also known to be partly mediated through its binding to the surface of DCs via the mannose receptor (CD206). Using a recombinant ω1 with mutations in the sites responsible for glycosylation (N71/176Q; ω1Δ.sup.GLY) and thus unable to bind to CD206, we show no effect on weight gain in the absence of the ability to bind to CD206 (FIG. 10).

Conclusion

[0094] We have now confirmed that the weight loss induced by omega-1 is mediated by the known RNAse activity and glycosylation pattern of the molecule.

Example 3 Hepatic Steatosis Assessment

Method

[0095] The levels of the enzymes Alanine Transaminase (ALT) and Asparate Transaminase (AST) were quantified in the serum recovered from omega-1 or control (Ovalbumin; OVA) protein treated obese mice and lean mice to assess hepatic steatosis. The activity of both enzymes was quantified using kits from Abcam (Cambridge, UK), following the manufacturer's instructions.

Results

[0096] Results are displayed as a ratio of AST to ALT in lean control mice and obese mice and shown in FIG. 11.

Discussion

[0097] Omega-1 has been reported to be hepatotoxic, with hepatocyte microvesicular damage developing when the native molecule is released from eggs that are deposited in the liver of mice infected with Schistosoma mansoni.

[0098] In contrast, we found that when recombinant omega-1 was injected into the peritoneal cavity of obese mice in addition to inducing weight loss and improving glucose tolerance it also reduced the ratio (AST:ALT) of the enzyme markers of hepatic steatosis. Thus, intraperitoneal injection of recombinant omega-1 does not cause hepatoxicity.

Conclusion

[0099] One of the diseases that arise as part of the metabolic syndrome in man is non-alcoholic fatty liver disease with hepatic steatosis. In mice fed HFD-diet the obese state that develops is associated with hepatic steatosis, with hepatocyte microvesicular damage reflected by an elevated ratio of aspartate transaminase (AST) to alanine transaminase (ALT) enzymes in the serum. We found that obese mice treated with omega-1 had reduced hepatic steatosis as demonstrated by significantly (P<0.05) reduced circulating AST:ALT levels, comparable to non-obese mice, 6 days after initial treatment.

The Invention Will Now be Described by the Following Non-Limiting Statements:

[0100] 1. A compound or protein with ribonuclease activity or a fragment thereof that induces IL-33 release to initiate a type 2 response, preferably in adipose cells and/or tissues, for use in the treatment of diseases associated with fat accumulation.
2. The compound, protein or fragment thereof for use according to statement 1 which is a ribonuclease protein or a ribonuclease-like protein.
3. The protein or fragment thereof for use according to statement2 which is a ribonuclease protein of the T2 family.
4. The protein or fragment thereof for use according to any of the preceding statements which is a ribonuclease protein of the T2 family or fragment thereof comprising at least one or more RNAase catalytic domains.
5. The protein or fragment thereof for use according to any of the preceding statements which is a ribonuclease protein of the T2 family or fragment thereof and comprises at least a first conserved amino acid sequence (CAS1) comprising amino acid residues FTIHGLWPT and/or a second conserved amino acid sequence (CAS2) comprising amino acid residues PSFWKHEFEKHGLCAV.
6. The protein or fragment thereof for use according any of the preceding statements wherein the protein is Omega-1 protein or a fragment thereof.
7. The protein or fragment thereof for use according to statement6 wherein the protein is an Omega-1 protein or an Omega-1 protein fragment, preferably comprising at least part of amino acid residues 1 to 224, more preferably comprising at least one or more RNAase catalytic domains.
8. The protein or fragment thereof for use according to statement6 or 7 wherein the Omega-1 protein fragment comprises at least a first conserved amino acid sequence (CAS1) comprising amino acid residues FTIHGLWPT and/or a second conserved amino acid sequence (CAS2) comprising amino acid residues PSFWKHEFEKHGLCAV.
9. The protein or fragment thereof for use according any of the preceding statements with modified N-glycolysation sites or lacking N-glycolysation sites.
10. The protein or fragment thereof for use according any of the preceding statements further comprising a glycoprotein carrier.
11. The compound, protein or fragment thereof for use according to any of the preceding statements in the treatment of obesity and obesity-related disorders and/or inducing weight loss by decreasing the number of adipose cells after administration.
12. The compound, protein or fragment thereof for use according to any of the preceding statements in the treatment of metabolic disorders by restoring glucose and insulin homeostasis after administration.
13. The compound, protein or fragment thereof for use according to any of the preceding statements in the treatment of a liver disorder by decreasing the number of adipose cells in the liver after administration.
14. A pharmaceutical composition comprising the compound, protein or fragment thereof according to any of the preceding statements.
15. The compound, protein or fragment thereof or pharmaceutical composition according to any of the preceding statements for use as an adjuvant therapy.

REFERENCES

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