Subcutaneous formulation of a peptide derived from chaperonin 60.1

Abstract

Described herein are stable formulations of a peptide derived from Chaperonin 60.1 suitable for subcutaneous administration. The formulation may have Chaperonin 60.1-related peptide with a concentration of concentration of 16‑150 mg / mL capable of delivering a dose of at least 40 mg in an injection volume of 2 mL or less, and a bioavailability of at least 50%.

Description

SUBCUTANEOUS FORMULATION OF A PEPTIDE DERIVED FROM CHAPERONIN 60.1FIELD OF THE INVENTION

[0001] The embodiments of the present invention relate to formulations of a peptide derived from Chaperonin 60.1 ; namely stable formulations suitable for subcutaneous administration.BACKGROUND OF THE INVENTION

[0002] Chaperonin polypeptides are a subgroup of heat shock polypeptides whose role in polypeptide folding is well known. There are two families of chaperonin polypeptide, the chaperonin 60 (approximately 60 kDa) and chaperonin 10 (approximately 10 kDa) families.1Some Chaperonin polypeptides have been shown to have a role in immune regulation. Mycobacterium tuberculosis (M. tuberculosis) produces Chaperonin 60.1 (Cpn60.1), a polypeptide that is named based on its amino acid sequence identity to other known chaperonins. International Patent Application WO 2002 / 0400372disclosed pharmaceutical compositions comprising Cpn60.1 from M. tuberculosis (MtCpn60.1) and its encoding nucleic acid molecules. A variety of therapeutic uses for these molecules is also disclosed, including the treatment and / or prevention of autoimmune disorders, allergic conditions, conditions typified by a Th2-type immune response and conditions associated with eosinophilia.

[0003] International Patent Application, Publication Number W02009 / 106819A2 disclosed a series of novel peptides derivable from MtCpn60.1 including a peptide (designated as "Peptide 4") having an amino acid sequence: DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1), which exhibited antiinflammatory activity and was shown to significantly reduce the recruitment of eosinophils in an animal model of allergic airway inflammation. This peptide (also known as IRL201104, PIN201104, and ‘1104) has been reported to have therapeutic efficacy in various diseases and conditions including, but not limited to, allergic airways diseases;3relapsing-remitting conditions, such as allergic inflammatory condition, such as, atopic dermatitis, rhinitis, rheumatoid arthritis, and inflammatory bowel disease;4food allergies and food intolerances;5and eosinophilic esophagitis.6In clinical studies conducted to date, ‘1104 was administered by intravenous (IV) infusion of reconstituted lyophilized powder containing 8 mg of ‘1104, with a reconstituted dose of either 0.8 mg / mL or 2.0 mg / mL strength.

[0004] However, IV administration requires access to a medical facility with trained medical professionals, which is prohibitive to many patients due to the distance or travel to medical facilities. Furthermore, IV administration typically requires a trained medicalprofessional to set-up an IV infusion, set and determine appropriate dosing / infusion rate, and time required before, during, and after the actual infusion for the preparation, infusion duration, and observation post-infusion. In addition to the risk of infection at the injection site, IV administration is also associated with the risk of systemic infection, potential exposure to IV particulate matter, and exposure to hospital-acquired infections.

[0005] Subcutaneous (SC) administration offers numerous advantages over IV infusion {e.g., self-administration by patient in home, superior dosing compliance, flexibility in the anatomical infusion site, smaller needle size decreasing pain during infusion, decreased risk of infusion site infection). However, SC administration has traditionally been limited by the volume which can be delivered (<1 to 2 mL) and dose range, which is quite challenging for low solubility peptides such as ‘1104. Accordingly, there is a need for a formulation of ‘1104 suitable for SC administration.BRIEF SUMMARY OF THE INVENTION

[0006] The embodiments of the present invention provide pharmaceutical formulations of ‘1104 (SEQ ID NO: 1) suitable for SC administration comprising a ‘1104 concentration of 4-150 mg / mL. The ‘1104 formulations are capable of delivering a high dose of 40 mg and optimally 64 mg in an injection volume of 3 mL or less, and optimally 2 mL or less. The ‘1104 formulations have a pH of 5.5-9.5, optimally a pH of 6-8; an osmolality of 210-350 mOs; and comprise United States Pharmacopeia (USP)-National Formulary (NF) excipients, i.e., USP-NF excipients, for parental formulations. The ‘1104 formulations can be stored frozen, at -20°C or lower, refrigerated a 2-8°C, or optimally at 15-25°C (controlled room temperature) and have a stability of at least six months, optimally at least 24 months. In some embodiments, the SC pharmaceutical formulations has a stability of at least 9 months, 12 months, 15 months, 18 months, 24 months or longer. In some embodiments the 4-150 mg / mL concentration of ‘1104 has a bioavailability of at least 50%. In alternative embodiments, the osmolality is within the range 240-320 mOs or 260-310 mOs. In alternative embodiments, the 4-150 mg / mL concentration of ‘1104 has a bioavailability of at least 55%, 60%, 65%, 70%, 75%, or 80%.

[0007] In some embodiments, the present invention provides a pharmaceutical formulation that comprises: (a) a peptide having SEQ ID NO: 1, at a concentration of from about 25 to about 40 mg / mL; (b) about 100 mM Tris buffer; (c) about 0.1% Polysorbate 80; and (d) a pH of about 8.5.

[0008] In some embodiments, the present invention provides a pharmaceutical formulation that comprises: (a) a peptide having SEQ ID NO: 1, at a concentration of from about 25 to about 40 mg / mL; (b) about 50 mM Tris buffer; (c) about 50 mM L-Arginine; and a pH of about 8.0.

[0009] In some embodiments, the pharmaceutical formulations present invention further comprises a tonicity enhancer to make the formulation isotonic. In some embodiments, the tonicity enhancer is mannitol.

[0010] Other implementations are also described and recited herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] For the purpose of illustration, certain embodiments of the present invention are shown in the drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions, and instruments shown. In the drawings:

[0012] FIG. 1 shows the hydropathy profile of the ‘1104 peptide, with hydrophobic residues above the line and hydrophobic residues below the line. Acidic residues are shown in pink (Asp); aromatic residues are shown in lime green (Tyr); basic residues are shown in blue (Lys, His); aliphatic residues are shown in grey (Vai, Leu, Ala, Leu); and polar residues are shown in dark green (Ser, Asn, Thr).

[0013] FIG. 2 shows a graph of the predicted solubility profile of the ‘1104 peptide as a function of pH with pH 4-5.5 providing the best pH range for stability but decreased solubility and pH 9-14 providing the best pH range for solubility but low stability for the ‘1104 peptide.

[0014] FIG. 3 shows a chromatogram of purification of the ‘1104 peptide at0.2 mg / mL.

[0015] FIG. 4 shows a diagrammatic representation of the protocol for the direct comparison of the administration of IV ‘1104 versus two SQ ‘1104 formulations in a model of allergic inflammation.

[0016] FIGS. 5A-B provides bar graphs showing the effect of ‘1104 on total number of cells in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) or vehicle (FIG. 5A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 5B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 5A) or day 30 (FIG. 5B). Each column represents the mean, and each bar represents the S.E.M. (standard error of the mean) of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. *** P<0.001.

[0017] FIGS. 6A-B provides bar graphs showing the effect of ‘1104 on eosinophil numbers in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15,16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 6A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 6B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 6A) or day 30 (FIG. 6B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. *** P<0.001.

[0018] FIGS. 7A-B provides bar graphs showing the effect of ‘1104 on neutrophil numbers in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 7A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 7B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 7A) or day 30 (FIG. 7B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. * P<0.05, ** P<0.01, and *** P<0.001.

[0019] FIGS. 8A-B provides bar graphs showing the effect of ‘1104 on macrophage numbers in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 8A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 8B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 8A) or day 30 (FIG. 8B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. * P<0.05, ** P<0.01, and *** P<0.001.

[0020] FIGS. 9A-B provides bar graphs showing the effect of ‘1104 on lymphocyte numbers in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 9A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge ondays 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 9B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 9A) or day 30 (FIG. 9B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. * P<0.05, ** P<0.01, and *** P<0.001.

[0021] FIGS. 10A-B provides bar graphs showing the effect of ‘1104 on IL-4 concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 10A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 10B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 10A) or day 30 (FIG. 10B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. *** P<0.001.

[0022] FIGS. 11A-B provides bar graphs showing the effect of ‘1104 on IL-5 concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 11A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 11B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 11 A) or day 30 (FIG. 11B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. *** P<0.001.

[0023] FIGS. 12A-B provides bar graphs showing the effect of ‘1104 on KC concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 12A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 12B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 12A) or day 30 (FIG. 12B). Each column represents themean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test.

[0024] FIGS. 13A-B provides bar graphs showing the effect of ‘1104 on IL-10 concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 13A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 13B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 13A) or day 30 (FIG. 13B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. ** P<0.01 and *** P<0.001.

[0025] FIGS. 14A-B provides bar graphs showing the effect of ‘1104 on IL-12 concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 14A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 14B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 14A) or day 30 (FIG. 14B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. * P<0.05, ** P<0.01, and *** P<0.001.

[0026] FIGS. 15A-B provides bar graphs showing the effect of ‘1104 on IL-13 concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 15A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 15B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 15A) or day 30 (FIG. 15B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared tovehicle matched treatment groups using ANOVA followed by Dunnett’s test. ** P<0.01 and *** P<0.001.

[0027] FIGS. 16A-B provides bar graphs showing the effect of ‘1104 on IL-17 concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 16A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 16B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 16A) or day 30 (FIG. 16B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. * P<0.05, ** P<0.01, and *** P<0.001.

[0028] FIGS. 17A-B provides bar graphs showing the effect of ‘1104 on Eotaxin concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 17A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 17B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 17A) or day 30 (FIG. 17B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. ** P<0.01 and *** P<0.001.

[0029] FIGS. 18A-B provides bar graphs showing the effect of ‘1104 on GM-CSF concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 18A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 18B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 18A) or day 30 (FIG. 18B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared tovehicle matched treatment groups using ANOVA followed by Dunnett’s test. * P<0.05, ** P<0.01, and *** P<0.001.

[0030] FIGS. 19A-B provides bar graphs showing the effect of ‘1104 on G-CSF concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 19A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 19B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 19A) or day 30 (FIG. 19B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. ** P<0.01 and *** P<0.001.

[0031] FIGS. 20A-B provides bar graphs showing the effect of ‘1104 on MCP-1 concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 20A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 20B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 20A) or day 30 (FIG. 20B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. * P<0.05, ** P<0.01, and *** P<0.001.

[0032] FIGS. 21A-B provides bar graphs showing the effect of ‘1104 on RANTES concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 21A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 21 B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 21 A) or day 30 (FIG. 21 B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared tovehicle matched treatment groups using ANOVA followed by Dunnett’s test. * P<0.05, ** P<0.01, and *** P<0.001.

[0033] FIGS. 22A-B provides bar graphs showing the effect of ‘1104 on Periostin concentration (pg / mL) in bronchoalveolar lavage fluid (BALF) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 22A) or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL- 201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (see FIG. 22B). BALF was collected 24 hours after the final OVA challenge at either day 18 (FIG. 22A) or day 30 (FIG. 22B). Each column represents the mean, and each bar represents the S.E.M. of n=8. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. ** P<0.01 and *** P<0.001.

[0034] FIGS. 23A-B provides bar graphs showing the effect of ‘1104 on CD3+FOXP3+ cells of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 23A) or vehicle or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (FIG. 23B). Blood was collected 24 hours after the final OVA challenge at either day 18 (FIG. 23A) or day 30 (FIG. 23B). Each column represents the mean, and each bar represents the S.E.M. of n=4. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test.

[0035] FIGS. 24A-B provides bar graphs showing the effect of ‘1104 on CD3+PD- L1+ cells (a subpopulation of Tregs) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 24A) or vehicle or treated on days 15, 16 and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (FIG. 24B). Blood was collected 24 hours after the final OVA challenge at either day 18 (FIG. 24A) or day 30 (FIG. 24B). Each column represents the mean, and each bar represents the S.E.M. of n=4. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test.

[0036] FIG. 25 provides a bar graph showing the effect of ‘1104 on CD3+IL-10+ blood cells (a subpopulation of Tregs) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) or vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment. Blood was collected 24 hours after the final OVA challenge at day 30. Each column represents the mean, and each bar represents the S.E.M. of n=4. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test.

[0037] FIG. 26 provides a bar graph showing the effect of ‘1104 on CD3+FOXP3+IL- 10+ blood cells (Tregs) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) or vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment. Blood was collected 24 hours after the final OVA challenge at day 30. Each column represents the mean, and each bar represents the S.E.M. of n=4. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test.

[0038] FIG. 27 shows that ‘1104 induces T regulatory cells in both preclinical data (FIG. 27, top panel) and in clinical data (FIG. 27, bottom panels) in human.

[0039] FIGS. 28A-B provides bar graphs showing the effect of ‘1104 on CD19+PD- L1+ blood cells (a subpopulation of Bregs) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) (FIG. 28A) or vehicle or treated on days 15, 16, and 17 prior to daily OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.), vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment (FIG. 28B). Blood was collected 24 hours after the final OVA challenge at either day 18 (FIG. 28A) or day 30 (FIG. 28B). Each column represents the mean, and each bar represents the S.E.M. of n=4. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test.

[0040] FIG. 29 provides a bar graph showing the effect of ‘1104 on CD19+IL-10+ blood cells (Bregs) of sensitized animals treated on days 15, 16, and 17 prior to daily PBS or OVA (1% w / v) aerosol challenge with IRL-201104 (80 pg / kg, s.c., vehicle 1 or vehicle 2), IRL-201104 (80 pg / kg, i.v.) or vehicle with further OVA challenge on days 27, 28 and 29 but without any additional IRL-201104 or vehicle treatment. Blood was collected 24 hours after the final OVA challenge at day 30. Each column represents the mean, and each bar represents the S.E.M. of n=4. OVA + vehicle groups were compared to vehicle matched treatment groups using ANOVA followed by Dunnett’s test. * P<0.05.

[0041] FIG. 30 shows that ‘1104 induces B regulatory cells in both preclinical data (FIG. 30, top panel) and in clinical data (FIG. 30, bottom panels) in human. * P<0.05, ** P<0.01, and *** P<0.001.

[0042] FIG. 31 provides a bar graph representing the dorsal skin thickness in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Skin thickness measurements were collected on days 35. Each symbol represents the mean, and each bar represents S.E.M. of n=4-8. Treatment groups were compared to their respective vehicle controls using ANOVA followed by Dunnett’s test. *** P<0.001.

[0043] FIGS. 32A-C provides bar graphs representing the skin pathology, erythema / hemorrhage (FIG. 32A), oozing / crust (FIG. 32B), erosion / excoriation (FIG. 32C), in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Skin pathological assessment was conducted on day 35. Each symbol represents the mean, and each bar represents S.E.M. of n=4-8. Naive mice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test. ** P<0.01 and *** P<0.001.

[0044] FIG. 33 provides a bar graph representing the serum IgE (pg / mL) in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Serum samples were collected on day 35. Each symbol represents the mean, and each bar represents SEM of n=4-8. naive mice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test.** P<0.01, *** P<0.001.

[0045] FIG. 34 provides a bar graph representing the serum TNF alpha (pg / mL) in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Serum samples were collected on day 35. Each symbol represents the mean, and each bar represents SEM of n=4-8. naive mice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test. ** P<0.01 and *** P<0.001.

[0046] FIG. 35 provides a bar graph representing the serum I L-1 p (pg / mL) in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Serum samples were collected on day 35. Each symbol represents the mean, and each bar represents SEM of n=4-8. naivemice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test.* P<0.05, ** P<0.01 , and *** P<0.001.

[0047] FIG. 36 provides a bar graph representing the serum IL-4 (pg / mL) in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Serum samples were collected on day 35. Each symbol represents the mean, and each bar represents SEM of n=4-8. naive mice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test.*** P<0.001.

[0048] FIG. 37 provides a bar graph representing the serum IL-5 (pg / mL) in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Serum samples were collected on day 35. Each symbol represents the mean, and each bar represents SEM of n=4-8. naive mice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test.*** P<0.001.

[0049] FIG. 38 provides a bar graph representing the serum IL-13 (pg / mL) in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Serum samples were collected on day 35. Each symbol represents the mean, and each bar represents SEM of n=4-8. naive mice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test.** P<0.01 and *** P<0.001.

[0050] FIG. 39 provides a bar graph representing the serum IL-17A (pg / mL) in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Serum samples were collected on day 35. Each symbol represents the mean, and each bar represents SEM of n=4-8. naive mice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test.* P<0.05.

[0051] FIG. 40 provides a bar graph representing the serum IFN-gamma (pg / mL) in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Serum samples were collected on day 35. Each symbol represents the mean, and each bar represents SEM of n=4-8. naive mice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test. * P<0.05, ** P<0.01 , and *** P<0.001.

[0052] FIG. 41 provides a bar graph representing the serum CCL-2 (pg / mL) in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Serum samples were collected on day 35. Each symbol represents the mean, and each bar represents SEM of n=4-8. naive mice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test.*** P<0.001.

[0053] FIG. 42 provides a bar graph representing the serum CCL-17 (pg / mL) in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Serum samples were collected on day 35. Each symbol represents the mean, and each bar represents SEM of n=4-8. naive mice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test.*** P<0.001.

[0054] FIG. 43 provides a bar graph representing the serum IL-31 (pg / mL) in mice treated with IRL201104 (2 mg / kg, i.v. or s.c.), vehicle or dexamethasone (3 mg / kg, i.p.) and sensitized to OVA (15 pg, s.c., day 1 and 7) and challenged (days 14-20 and 28-34) with OVA (300 pg, epicutaneously applied to the dorsal skin). Serum samples were collected on day 35. Each symbol represents the mean, and each bar represents SEM of n=4-8. naive mice were compared to OVA challenged mice using ANOVA followed by Dunnett’s test.*** P<0.001.

[0055] FIG. 44 provides representative images at day 35 of the clinical development of dermatitis in naive (left-most image) or OVA sensitized mice (15 pg, s.c., day 1 and 7 and 300 pg, epicutaneously applied to the dorsal skin, days 14-20 and 28-34) treated with vehicle (i.v.; center-left image), IRL201104 (2 mg / kg, iv., center-right image), or dexamethasone (3 mg / kg, i.p., right-most image).

[0056] FIG. 45 provides representative images at day 35 of the clinical development of dermatitis in naive (left-most image) or OVA sensitized mice (15 pg, s.c., day 1 and 7 and 300 pg, epicutaneously applied to the dorsal skin, days 14-20 and 28-34) treated with vehicle (s.c.; center-left image), IRL201104 (2 mg / kg, s.c., center-right image), or dexamethasone (3 mg / kg, i.p., right-most image).DETAILED DESCRIPTION OF THE INVENTION

[0057] The subject innovation is now described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It may be evident,however, that the present invention may be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form in order to facilitate describing the present invention. It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention.DEFINITIONS

[0058] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0059] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0060] As used herein, the term "approximately" or "about" in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0061] As used herein, the term “or” means “and / or.” The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0062] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0063] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0064] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0065] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0066] As used herein, the term "relapsing-remitting condition” refers to conditions such as an inflammatory condition, autoimmune disorders, and an allergic condition. In some embodiments, the relapsing-remitting condition is an inflammatory condition.Examples of inflammatory condition include, but is not limited to, asthma, Crohn’s disease, allergic inflammatory conditions such as atopic dermatitis and rhinitis, rheumatoid arthritis and inflammatory bowel disease. In some embodiments, the relapsing-remitting condition is an autoimmune disorder. Examples of autoimmune disorders include, but are not limited to, hemolytic anemia, thrombocytopenia, pernicious anemia, Addison’s disease, autoimmune diabetes, insulin dependent diabetes mellitus, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis, autoimmune encephalitis, connective tissue disease, multiple sclerosis (including relapsing multiple sclerosis), autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, graft-versus-host disease and autoimmune inflammatory eye disease. Preferred autoimmune disorders include rheumatoid arthritis, and systemic lupus erythematosus. In some embodiments, the relapsing-remitting condition is an allergic condition. Examples of allergic conditions and disorders, but is not limited to, eczema, atopic dermatitis, allergic rhinitis (hay fever), allergic airways diseases, hyper-eosinophilic syndrome, respiratory diseases characterized by eosinophilic airway inflammation and airway hyper-responsiveness, such as asthma, including allergic asthma and intrinsic asthma, allergic bronchopulmonary aspergillosis, eosinophilic pneumonia, allergic bronchitis bronchiectasis, interstitial lung disease, hyper- eosinophilic syndrome, urticaria, angioedema, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis and giant papillary conjunctivitis. Preferred allergic disorders and conditions include asthma, allergic rhinitis, and atopic dermatitis. In another aspect, the condition involves viral exacerbations of allergic conditions including asthma. In another aspect, the condition involves exacerbations associated with bacterial infections.

[0067] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition, refer to therapeutictreatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical parameters are improved. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or clinical parameters, but also a cessation or at least slowing down of progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of a relapsing-remitting condition.

[0068] As used herein, the terms "effective amount" and “therapeutically effective amount” include an amount sufficient to prevent or ameliorate a manifestation of a relapsingremitting condition. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds.

[0069] As used herein, the term "subject" refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets {e.g., dogs, cats), agricultural stock animals {e.g., cows, horses, pigs, chickens, etc.), laboratory animals {e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects include human subjects. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of either sex. The term subject is used interchangeably with the term patient.

[0070] The terms “decrease,” “reduced,” “reduction,” or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level {e.g., the absence of a given or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0071] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.PHARMACEUTICAL COMPOSITIONS

[0072] The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive corn, sunflower, grapeseed, vegetable, fish oil, or injectable organic esters. The excipients can be chosen, for example, to effectuate delayed release of an agent or to selectively target one or more cells, tissues or organs.

[0073] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, decrease degradation, rejection or clearance, increase solubility, increase immunogenicity, or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; adjuvants (e.g., Alum, MF59, AS01 / 03 / 04, CpG1018) or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends,for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0074] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, metals, radioactive isotope / radiation particles, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, rejection, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0075] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, vegetable oil, cottonseed oil, safflower oil, sesame oil, olive oil, sunflower oil, grapeseed oil, fish oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar, Matrigel or hydrogel; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; (21) nanoparticles such as liposomes, polymers, micelles, metal nanoparticles, carbon nanotubes, solid lipid nanoparticles, noisomes, and dendrimers; (22) extracellular vesicles; and (23) other non-toxic compatible substances employed in pharmaceutical formulations.

[0076] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0077] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0078] For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0079] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0080] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or clearance of the particular compound(s) being employed, the duration or frequency of the treatment, other drugs that may interact with or affect the metabolism or efficacy of the compound of the invention, compounds and / or materials {e.g., vaccines, antibodies and derivatives) used in combination with the particular compound(s) employed, other therapeutic approaches {e.g., surgery, cell-based therapy, chemotherapy, radiotherapy, interventional therapy), the age, sex, body weight, conditions or comorbidities, diet, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0081] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the body weight, sex, age,comorbidities, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, therapeutic approaches, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art. See, e.g., Isselbacher et al. (1996).7

[0082] In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent or combined with a therapeutic approach e.g., surgery, chemotherapy, radiotherapy, interventional therapy).

[0083] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, animal models, (engineered or genetically modified) cells, organoids, constructs, vectors, carriers, adjuvants, compounds, drug delivery system, antibodies and derivatives, vaccines, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy;8The Encyclopedia of Molecular Cell Biology and Molecular Medicine;9Molecular Biology and Biotechnology: a Comprehensive Desk Reference;10Immunology;11Janeway's Immunobiology;12Lewin's Genes XI;13Molecular Cloning: A Laboratory Manual.;14Basic Methods in Molecular Biology;15Laboratory Methods in Enzymology;16Current Protocols in Molecular Biology (CPMB);17Current Protocols in Protein Science (CPPS);18and Current Protocols in Immunology (CPI).19

[0084] In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0085] Other terms are defined herein within the description of the various aspects of the invention.CHAPERONIN 60.1 -RELATED PEPTIDES

[0086] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, andexperimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0087] Compositions useful in the methods and formulations of the present invention include, but are not limited to:DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD (SEQ ID NO: 1) (‘1104); DGSVVVNKVSELPAGH (SEQ ID NO: 2); GLNVNTLSYGDLAAD (SEQ ID NO: 3); SELPAGHGLNVNLTS (SEQ ID NO: 4); DGSVVVNKVS (SEQ ID NO: 5); ELPAGHGLNV (SEQ ID NO: 6); NTLSYGDLAAD (SEQ ID NO: 7); or a functionally equivalent fragment or variant thereof.

[0088] Other compositions useful in the methods and formulations of the present invention include, but are not limited to, Cpn60.1 -related peptides disclosed in United States Published Patent Application No. 2004013216320and United States Patent Nos.11,098, 090;219,320,791 ;229,085,632.23

[0089] In certain aspects, a Cpn60.1 -related peptide is composed of amino acid residues. As used herein, the term “amino acid residue” is used interchangeably with the terms “amino acid” or “aa” to refer to an amino acid which is part of a peptide or protein. In some such aspects, an agonist or ligand of the present invention is composed of amino acids with the standard structure NH2 — C(H)(R) — COOH, where R represents an individual amino acid side chain. In certain aspects, an agonist or ligand is composed of amino acid residues which are naturally occurring amino acids. In certain aspects, a naturally occurring amino acid includes one of the twenty standard amino acids found in naturally occurring peptides and proteins. In some such aspects, an agonist or ligand is composed of at least one naturally occurring amino acid residue which is alanine (“A”), arginine (“R”), asparagine (“N”), aspartic acid (“D”), cysteine (“C”), glutamine (“Q”), glutamic acid (“E”), glycine (“G”), histidine (“H”), isoleucine (“I”), leucine (“L”), lysine (“K”), methionine (“M”), phenylalanine (“F”), proline (“P”), serine (“S”), threonine (“T”), tryptophan (“W”), tyrosine (“Y”), or valine (“V”).

[0090] In other aspects a Cpn60.1 -related peptide used in the methods of the present invention is composed of at least one amino acid residue which is an unnatural or synthetic amino acid. In some such aspects, an unnatural or synthetic amino is a chemically modified amino acid including but not limited to amino acids which have been modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can change the physiochemical properties of a peptide. In further aspects, an unnatural or synthetic amino is a chemically modified amino acid which hasbeen modified with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, and the like).

[0091] In further aspects, a Cpn60.1 -related peptide used in the methods of the present invention is composed of at least one amino acid which has an L-configuration (the chirality of an L-amino acid). In additional aspects, a Cpn60.1 -related peptide used in the methods of the present invention is composed of at least one amino acid which has a D- configuration (the chirality of a D-amino acid).

[0092] In certain aspects, an agonist or ligand of the present invention is about 50 aa in length; 49 aa in length; 48 aa in length; 47 aa in length; 46 aa in length; 45 aa in length;44 aa in length; 43 aa in length; 42 aa in length; 41 aa in length; 40 aa in length; 39 aa in length; 39 aa in length; 38 aa in length; 37 aa in length; 36 aa in length; 35 aa in length; 34 aa in length; 33 aa in length; 32 aa in length; 31 aa in length; 30 aa in length; 29 aa in length; 28 aa in length; 27 aa in length; 26 aa in length; 25 aa in length; 24 aa in length; 23 aa in length; 22 aa in length; 21 aa in length; or 20 aa in length; 19 aa in length; 18 aa in length; 17 aa in length; 16 aa in length; 15 aa in length; 14 aa in length; 13 aa in length; 12 aa in length; 11 aa in length; 10 aa in length; 9 aa in length; 8 aa in length; 7 aa in length; 6 aa in length; or 5 aa in length.

[0093] In certain aspects, compositions of the present invention are administered to a patient by any appropriate route known and / or employed by those skilled in the art. In some aspects, compositions of the present invention are administered by oral (PO), intravenous (IV), intramuscular (IM), intra-arterial, intramedullary, intrathecal, subcutaneous (SQ), intraventricular, transdermal, interdermal, intradermal, rectal (PR), vaginal, intraperitoneal (IP), intragastric (IG), topical {e.g., by powders, ointments, creams, gels, lotions, and / or drops), mucosal, intranasal, buccal, enteral, intravitreal, sublingual, by intratracheal instillation, bronchial instillation, and / or inhalation, as an oral spray, nasal spray, aerosol, and / or through a portal vein catheter.

[0094] In certain aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof may be administered intravenously, for example, by intravenous infusion. In further aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof may be administered by intramuscular injection. In more aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof may be administered by intratumoral injection. In certain aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof may be administered by subcutaneous injection. In further aspects, a Cpn60.1 -related peptide usedin the methods of the present invention and / or pharmaceutical compositions thereof may be administered via portal vein catheter. In more aspects, the invention encompasses the delivery of a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof by any appropriate route taking into consideration likely advances in the art of drug delivery.

[0095] In some aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof may be administered at dosage levels sufficient to deliver from about 0.001 mg / kg to 100 mg / kg, from about 0.01 mg / kg to 50 mg / kg, from about 0.1 mg / kg to 40 mg / kg, from about 0.5 mg / kg to 30 mg / kg, from about 0.01 mg / kg to 10 mg / kg, from about 0.1 mg / kg to 10 mg / kg, or from about 1 mg / kg to 25 mg / kg of patient body weight per day to obtain the desired therapeutic effect. In certain aspects, the desired dosage may be delivered more than three times per day, three times per day, two times per day, once per day, once every other day, once every third day, once every week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once every twelve months. In certain aspects, the desired dosage may be delivered using multiple administrations {e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In certain aspects, the desired dosage may be delivered using one or more administrations during an initial period of time, followed by a period of time in which no dosage is administered.

[0096] In further aspects, a Cpn60.1 -related peptide used in the methods of the present invention may be utilized for prophylactic applications. In more aspects, prophylactic applications involve systems and methods for preventing, inhibiting progression of, and / or delaying the onset of a relapsing-remitting condition, in individuals susceptible to and / or displaying symptoms of a relapsing-remitting condition.

[0097] In certain aspects, a Cpn60.1 -related peptide used in the methods of the present invention is administered to a target cell in vivo. In other aspects, a Cpn60.1 -related peptide used in the methods of the present invention is administered to a target cell ex vivo. In additional aspects, a Cpn60.1 -related peptide used in the methods of the present invention is administered to a target cell ex vivo, then the target cell is re-introduced into an organism. In some such aspects, the target cell is cultured into multiple progeny cells ex vivo before being re-introduced in an organism. In more aspects, the organism is a human. In further aspects, the organism is a human patient. In certain aspects, the target cell was originally derived from the organism to which it is re-introduced. In other aspects, the target cell was originally derived from a different organism to which it is re-introduced.

[0098] In certain aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof are employed in combinationtherapies for treating or reducing the risk of a relapsing-remitting condition. In such aspects, administration can be in combination with one or more additional therapeutic agents. As used herein, the phrases “combination therapy,” “combined with,” “in combination,” and the like, refer to the use of more than one medication or treatment simultaneously to increase the response. In certain aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof are administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In certain aspects, a Cpn60.1 -related peptide used in the methods of the present invention and / or pharmaceutical compositions thereof are administered in combination together in a single composition or administered separately in different compositions.

[0099] In certain aspects, the particular combination of therapies to employ in a combination regimen will generally take into account the compatibility of the desired therapeutics and / or procedures, and the desired therapeutic effect to be achieved. In further aspects, the therapies employed may achieve a desired effect for the same purpose {e.g., a Cpn60.1 -related peptide used in the methods of the present invention which is useful for treating, preventing, and / or delaying the onset of a relapsing-remitting condition may be administered concurrently with another therapeutic agent which is also useful for treating, preventing, and / or delaying the onset of a relapsing-remitting condition symptoms), or they may achieve different effects. In further aspects, the combination of therapies employed may achieve the same or a substantially similar desired effect for the same disease, condition or disorder; may achieve the same or a substantially similar desired effect for one or more different diseases, conditions or disorders; may achieve different desired effects for the same disease, condition or disorder; or may achieve different desired effects for one or more different diseases, conditions or disorders.

[0100] In additional aspects, the delivery of a Cpn60.1 -related peptide used in the methods of the present invention as a pharmaceutical composition is in combination with one or more additional components that may improve the bioavailability of the Cpn60.1- related peptide used in the methods of the present invention, reduce and / or modify its metabolism, inhibit its excretion, and / or modify its distribution in the body.

[0101] In certain aspects, combination therapy may involve administrations of a plurality of Cpn60.1 -related peptides in accordance with the present invention. In further aspects, combination therapy may involve administrations of a plurality of a Cpn60.1 -related peptides that treat, prevent, improve, achieve remission of, and / or reduce the risk of a relapsingremitting condition. In more aspects, combination therapy can be a plurality of Cpn60.1- related peptides that treat, prevent, improve, achieve remission of, and / or reduce the risk of a relapsing-remitting condition.

[0102] In certain aspects, a Cpn60.1 -related peptide used in the methods of the present invention is combined with at least one pharmaceutically acceptable excipient, in the form of a pharmaceutical composition. As used herein, “pharmaceutical composition” refers to a formulation containing an active ingredient, and optionally a pharmaceutically acceptable carrier, diluent or excipient. The term “active ingredient” can interchangeably refer to an “effective ingredient,” and is meant to refer to any agent that is capable of inducing a sought- after effect upon administration. Examples of active ingredient include, but are not limited to, chemical compound, drug, therapeutic agent, small molecule, and the like.

[0103] In certain aspects of the present invention, the active ingredient is a Cpn60.1- related peptide as disclosed herein. In particular aspects, the active ingredient is PIN201104, or a derivative thereof. In certain aspects, the active ingredient is PIN201360, PIN201361, PIN201362, PIN201116, PI N201105 or a derivative thereof. In further aspects, the active ingredient is a peptide described in W02009 / 106819, or a derivative thereof.

[0104] In certain aspects, the pharmaceutical compositions are useful in medicine or the manufacture of medicaments. In further aspects, the pharmaceutical compositions are useful in one or more of the therapeutic applications disclosed herein, for example, in an individual suffering from a relapsing-remitting condition. In additional aspects, the pharmaceutical compositions are formulated for administration to a human patient.

[0105] In certain aspects, the pharmaceutical composition is in a sterile injectable form {e.g., a form that is suitable for subcutaneous injection or intravenous infusion). In more aspects, the pharmaceutical composition is in a liquid dosage form that is suitable for injection. In further aspects, the pharmaceutical composition is in a powder {e.g., lyophilized and / or sterilized), optionally under vacuum, which is reconstituted with an aqueous diluent e.g., water; buffer; salt solution, and the like) prior to injection. In additional aspects, the pharmaceutical composition is diluted and / or reconstituted in an aqueous diluent {e.g., water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, and the like). In certain aspects, the pharmaceutical composition is in a form that can be refrigerated and / or frozen. In further aspects, the pharmaceutical composition is in a form that cannot be refrigerated and / or frozen. In certain aspects, the pharmaceutical composition is a reconstituted solution and / or liquid dosage form which can be stored for a certain period of time after reconstitution {e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, a month, two months, or longer).

[0106] In certain aspects, preparatory methods for pharmaceutical compositions include the step of bringing the active ingredient {e.g., a Cpn60.1 -related peptide used in the methods of the present invention) into association with one or more pharmaceutically acceptable excipients and then shaping and / or packaging the product into a desired single- or multi-dose unit. A pharmaceutical composition in accordance with the invention may beprepared, packaged in bulk, packaged as a single unit dose, and / or packaged as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition including a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to a dose that would be administered to a subject and / or a convenient fraction of such a dose such as, for example, one-half or one- third of such a dose. The relative amounts of active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the invention may vary, depending upon the identity, size, and / or condition of the subject treated and / or depending upon the route by which the composition is to be administered. In certain aspects, for example, the composition may include between about 0.1% to 100% (w / w) of an active ingredient.

[0107] In another aspect, the present invention includes kits that are useful for carrying out the methods of the present invention. The components contained in the kit depend on a number of factors, including the particular application (e.g., the particular route of administration to be employed, or the particular disease, condition or disorder to be treated). In certain aspects, the present invention provides a kit for administering a Cpn60.1- related peptide in accordance with the present invention to treat a disease, condition or disorder disclosed herein. In some such aspects, the kit further includes instructions for administration. In certain aspects, the kit is for administering a Cpn60.1 -related peptide to treat a patient with a relapsing-remitting condition. In certain aspects, the kits contain one or more a Cpn60.1 -related peptides. In certain aspects, the kit includes a number of unit doses of a pharmaceutical composition containing a Cpn60.1 -related peptide. In additional aspects, kits for use in accordance with the present invention include instructions {e.g., for administration, for storage, and the like), buffers and / or other reagents. In some such aspects, the kit includes (i) at least one Cpn60.1 -related peptide, (ii) a syringe, needle, applicator, or the like for administration of the at least one Cpn60.1 -related peptide to a patient, and (iii) instructions for use. In further aspects, the kit includes a treatment schedule designating when the unit dosages are to be administered. In more aspects, placebo dosages, either in a form similar to or distinct from the dosages of the pharmaceutical compositions, are included. In certain aspects, kits include one or more containers so that certain of the individual components or reagents may be separately housed. In certain aspects, kits may include a means for enclosing the individual containers in relatively close confinement for commercial sale, e.g., a plastic box, in which instructions, packaging materials such as Styrofoam, and the like, may be enclosed.‘1104 (SEQ ID NO:1) FORMULATIONS FOR IV INFUSIONS

[0108] ‘1104 (SEQ ID NO:1, DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD) is a peptide derived from M. tuberculosis (mTB) bacterial chaperonin 60.1 which has shownimmunomodulatory and anti-inflammatory properties in a range of preclinical models of lung inflammation. Despite a very short half-life (about 10-15 min across species), ‘1104 shows a long-lasting effect in models of allergic inflammation of up to 14 days. ‘1104 appears to be effective in resetting the immune system from an inflammatory state and in inducing inflammatory disease remission in nonclinical models. Its short pharmacokinetic (PK) terminal elimination half-life (ti / 2) decreases the potential for the negative off-target effects associated with current therapies.

[0109] In clinical studies conducted to date, ‘1104 was administered by intravenous (IV) infusion of reconstituted lyophilized powder containing 8 mg of IRL201104. The reconstituted dose was prepared at either 0.8 mg / mL or 2.0 mg / mL strength. The qualitative and quantitative composition of IRL201104 reconstituted solution for injection is shown in Table 1.Table 1 : Composition of ‘1104 Formulation for IV AdministrationAbbreviations: Percent weight in volume (% w / v); Quantum satis (q.s. or Q.S.).

[0110] While the IV infusion delivery form is adequate for early clinical development, different delivery forms are preferred for the marketed product. For example, SC administration provide several advantages over IV infusion. SC administration facilitates patient self-administration in home or outpatient clinical environments, which enhances the convenience for the patient and provides a decrease in medical facility burden and costs. SC administration provides flexibility in the anatomical infusion site, and options include the stomach, thighs, and backs of the arms. SC infusion systems can be designed with smaller needle sizes, which may decrease pain during infusion. While the risk of infusion site infection still exists with SC administration, infusion site infections are generally limited to cellulitis and very rarely progress to systemic infections. Further, administration at home reduces the risk of exposure to hospital-acquired infections. Accordingly, research effortswere initiated to develop pharmaceutical ‘1104 formulations suitable for subcutaneous delivery.

[0111] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:1. A pharmaceutical formulation for subcutaneous injection of a peptide having SEQ ID NO: 1 , comprising:(a) a peptide having SEQ ID NO: 1 , at a concentration of from about 25 to about 40 mg / mL;(b) about 100 mM Tris buffer;(c) about 0.1 % Polysorbate 80; and(d) a pH of about 8.5.2. A pharmaceutical formulation for subcutaneous injection of a peptide having SEQ IDNO: 1 , comprising:(a) a peptide having SEQ ID NO: 1 , at a concentration of from about 25 to about 40 mg / mL;(b) about 50 mM Tris buffer;(c) about 50 mM L-Arginine; and(d) a pH of about 8.0.3. The pharmaceutical formulation according to paragraph 1 or 2, further comprising tonicity enhancer to make the formulation isotonic.4. The pharmaceutical formulation according to paragraph 3, wherein the tonicity enhancer is mannitol.5. The pharmaceutical formulation according to any one of paragraphs 1-4, wherein the formulation has a pH of 5.5-9.5.6. The pharmaceutical formulation according to any one of paragraphs 1-4, wherein the formulation has a pH of 6-8.7. The pharmaceutical formulation according to any one of paragraphs 1-6, wherein the formulation is stored at 15-25°C.8. The pharmaceutical formulation according to any one of paragraphs 1-6, wherein the formulation is stored at 2-8°C or colder.9. The pharmaceutical formulation according to any one of paragraphs 1-6, wherein the formulation is stored at -20°C or colder.10. The pharmaceutical formulation according to any one of paragraphs 1-9, wherein the formulation is stable for at least 6 months.11. The pharmaceutical formulation according to any one of paragraphs 1-9, wherein the formulation is stable for at least 12 months.12. The pharmaceutical formulation according to any one of paragraphs 1-9, wherein the formulation is stable for at least 24 months.

[0112] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0113] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0114] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.EXAMPLES

[0115] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.EXAMPLE 1 QUALITY TARGET PRODUCT PROFILE (QTPP) FOR SC ‘1104 FORMULATION

[0116] At the outset of developing a SC formulation for ‘1104, a quality target product profile (qTPP) was specified for the suitable formulation candidates, as shown in Table 2.Table 2: QTPP for ‘1104 Drug ProductEXAMPLE 2 INITIAL SCREENING OF EXCIPIENTS AND SOLVENTS

[0117] In an initial screen, 40 excipients and solvents were tested, none of which were remotely efficacious in dissolving ‘1104 (a full list is shown in the Table 3 below).Indeed, sodium hydroxide (1 N) was found to be the most effective solvents, which is not suitable for SC administration.Table 3: Excipients and Solvents Screened for Solubility

[0118] Previous work on ‘1104 has shown it to be a difficult peptide molecule to purify and solubilize. As described in International Patent Application WO 2023 / 156849, ‘1104 was difficult to purify by conventional methods for purifying a peptide molecules; it resulted in gelled fractions, which need to be re-dissolved by pH adjustment after visual inspection.24This initial screen confirmed that developing a stable SC formulation with ‘1104 would be challenging.EXAMPLE 3 PREDICTED SOLUBILITY PROFILE OF ‘1104 AS A FUNCTION OF PH

[0119] The physiochemical properties of ‘1104 were assessed in order to obtain a predicted solubility profile as a function of pH and assess the best strategy to develop a SC formulation with the properties falling within the above-described qTPP parameters.

[0120] Looking at the amino acid sequence of ‘1104, it consists of 31 amino acids with four acidic amino acids, two basic amino acids, eight neutral polar amino acids, and 17 neutral nonpolar amino acids, as follows:• Acidic - asp (3), glu (1);• Basic - his (1), lys (1);• Neutral polar - ser (3), thr (1), tyr (1), asn (3); and• Neutral nonpolar - gly (4) ala (3), val (5), leu (4), pro (1).A hydropathy profile of ‘1104 is provided in FIG. 1 depicting hydrophilic residues on top and hydrophobic residues on the bottom. The physiochemical properties of ‘1104 are summarized in Table 4 and this peptide is estimated to have poor water solubility.Table 4: Physiochemical Properties of the ‘1104 Peptide

[0121] FIG. 2 provides a graph of the net charge (y-axis) versus the pH for ‘1104, showing that (i) the pH range of 4-5.5 is best for solubility but stability of ‘1104 is expected to decrease, and (ii) the pH range of 9-14 is best for solubility, but ‘1104 is likely unstable. This leaves the pH range of 5.5-9 to develop a SC ‘1104 formulation with properties falling within the above-described qTPP parameters.

[0122] Accordingly, the physiochemical properties of ‘1104 confirmed that it has poor water solubility, and the development of an effective and stable SC formulation would be challenging.EXAMPLE 4 SUBCUTANEOUS FORMULATION DEVELOPMENT

[0123] Solubility assessments were conducted on ‘1104 prepared according to the methods described in International Patent Application WO 2023 / 156849. A chromatogram of the ‘1104 sample at 0.2 mg / mL (bulk) is shown in FIG. 3.

[0124] The initial SC formulation development focused on identifying stable formulation prototypes that can deliver a dose of up to 64 mg. Solubility of ‘1104 was evaluated in various organic solvents and in aqueous systems over the pH range of 5 to 9. Four formulation prototypes were then selected to assess their physical and chemical stability over a 2-week period when stored at 5°C or ambient conditions. The best two formulation candidates from the solubility and stability perspective were evaluated for stability up to 3 months at three storage conditions.Solubility

[0125] ‘1104 was mixed with 18 different vehicles to determine its solubility in each.Samples were prepared by weighing approximately 25 mg of ‘1104 into a vial and adding 2 mL of excipients and mixing. The samples were allowed to stand for several minutes before being filtered using a 25 mm 0.45 pm Nylon filter. The results are summarized in Table 5. The drug appeared insoluble or only slightly soluble in all vehicles except in dimethyl sulfoxide (DMSO), where the maximum concentration of ‘1104 was determined at 9.5 mg / mL.Table 5: Solubility Screening Summary

[0126] Next, ‘1104 solubility was evaluated in aqueous solvents over the pH range of 5-9. As described above in Example 3, the ‘1104 peptide consists of 31 amino acids, with four being acidic and two being basic. Its isoelectric point is at pH 3.8 and its charge at pH 7 is -2.9. As a result, the solubility is expected to increase with increasing pH.

[0127] As expected, the solubility at lower pH levels is very low. In fact, mixing the drug with buffered aqueous solvents below pH 7 resulted in immediate gelling, presumably due to the peptide aggregation. However, the peptide was soluble at or above pH 7. Maximum concentration data were generated for solutions ranging from pH 7 to pH 9 and are presented in Table 6.Table 6: ‘1104 Solubility vs. pH

[0128] In the studied pH range, the solubility data indicated strong correlation between pH and drug’s maximum concentration, which increased by approximately 50% with each increase of 0.5 pH unit. This suggests that a SC formulation will need to becontrolled to a narrow pH range in manufacture and on storage to minimize the risk of gelling or precipitation.Development of SC Formulations

[0129] Guided by the solubility results, the formulation screening experiments focused on DMSO and aqueous solvents in the pH range of 7.5 to 8.5. Two excipients were added to each aqueous formulation at varied concentrations: a buffer and a solubility enhancer. Tris buffer was selected as a preferred option to phosphate or carbonate buffers to minimize potential precipitation of corresponding salts. A surfactant or an amino acid was used to enhance solubilization, suppress peptide molecules interaction and aggregation and reduce viscosity of high concentration peptide formulations or, in case of glycine, as a potential buffer. Each formulation was prepared at target ‘1104 concentration of 45 mg / mL. Results of the formulation screen are summarized in Table 7.Table 7: Formulation Screening Summary for 45 mg / mL Target Strength

[0130] All formulations from the formulation ID series F-426 and F-427 series resulted in opalescent solutions, with some becoming viscous and milky in appearance within 24 hours. As a result, the last three formulation screens were made at the reduced excipient concentration and / or at the higher end of the pH range. Two of the screened formulations, F-428-1 and F-428-3, remained clear after 4-day storage at ambient conditions with no viscosity increase. The actual concentration was determined by the HPLC assay as 36 to 37 mg / mL. The F-428-2 formulation, which was prepared at pH 8, became opalescent with assayed concentration of 38 mg / mL.

[0131] The 2-week stability study was initiated for the F-428 series formulations stored at refrigerated (5°C) and ambient conditions. In addition, F-430-1 formulation, which is analogous to F-428-2 but with lower drug concentration, and F-429 formulation, which is the current IV formulation with drug concentration at the solubility limit, were added to the 2- week study. Results are summarized in Table 8.Table 8: Prototype Formulations for the 2-week Stability

[0132] The 1-week study showed that samples were physically stable, with no change in appearance for at least a week when stored at ambient and refrigerated conditions, except for the refrigerated F-429 (maximum solubility IV formulation), which was beginning to become opalescent at 1 week.

[0133] The formulations had no significant change in impurity profile over 1 week of ambient storage, with the % area of the ‘1104 peak at 99.5% area at initial for all formulations and ranging from 99.3 to 99.5% at week 1.Conclusion

[0134] Based on the formulation screening and 2-week stability data, formulations F-428-3 and F-430-1 were selected as the two SC formulation prototypes for further development. Both formulations contain Tris buffer, with F-428-3 at 100 mM and pH 8.5, and the F-430-1 at 50 mM and pH 8.0. The two formulations use different solubility enhancers, 0.1% Polysorbate 80 in F-428-3 and 50 mM L-arginine in F-430-1. The two prototype formulations were tested for stability for up to 12 weeks at three storage conditions, -20°C, 5°C, and ambient temperature, and were monitored for physical stability by visual appearance and drug content by HPLC assay. In addition, the formulation was evaluated for comparative efficacy signal against the IV formulation in a mouse OVA asthma rechallenge model.

[0135] When compared to the IV formulation, the two prototypes introduced critical modifications to enable SC delivery:• Increased drug concentration - the IV formulation is dosed at 0.8 or 2.0 mg / mL as 10 to 32 mL IV infusion. The SC formulations can be manufactured up to 36 mg / mL, which would require less than 2 mL SC injections for doses up to 64 mg.• Higher pH - the increased pH of 8.0 and 8.5 for the aqueous formulation maximizes the amount of dissolved drug while remaining within 1 pH unit of the biological environment (the pH of human SC tissue is approximately 7.4), which minimizes potential tolerability issues.• Buffer selection - Tris buffer used in the prototypes is preferred to the phosphate buffer for improved drug solubility and potentially better tolerability. Polysorbate 80 and L-arginine are effective in preventing peptide aggregation over time.• Osmolality - the IV formulation is hypertonic, which can impact tolerability when administered subcutaneously. The SC formulations are hypotonic, but addition of tonicity enhancer, such as mannitol, can be used to make the final formulation isotonic.EXAMPLE 5 EFFECTIVENESS OF SUBCUTANEOUS ‘1104 FORMULATIONS IN AN ANIMAL MODEL OF ALLERGIC INFLAMMATION

[0136] As described above, ‘1104 has shown immunomodulatory and antiinflammatory properties in a range of preclinical models of lung inflammation.25The aim of this study is to use an ovalbumin (OVA) driven re-challenge model of allergic inflammation tocompare the impact of ‘1140 through two different routes of administration (subcutaneous and intravenous) on inflammatory cell lung infiltration, relevant cytokines in bronchoalveolar lavage fluid (BALF), and OVA specific IgE in serum to confirm the effectiveness of the two SC formulations F-428-3 and F-430-1.MATERIALS AND METHODS

[0137] Animals: Male BalbC mice (20-30 g on arrival, Charles Rivers, UK) were used in the present study.

[0138] Animal welfare: On arrival from the supplier, animals were acclimatized for period of 7 days before start of experimental procedures. Mice were housed in cages of four on arrival based on weight (equal distribution of animal weights amongst each of the cages by the animal technician) with a 12 hour light dark cycle. Room temperature and humidity were maintained between 17-24°C and 40-70%, respectively. Environmental enrichment was provided in all cages. Mice had access to standard chow ad libitum and water was available from bottles ad libitum.

[0139] Protocol: The protocol for the study is provided in FIG. 4.

[0140] Blood sample collection: A blood sample was collected by venipuncture(via the lateral tail vein) and placed into a serum tube. Each serum sample was kept at room temperature for 45 minutes to allow coagulation, before being centrifuged (2000g, 15 min at 4°C) from which the resulting supernatant was extracted, aliquoted and stored at -80°C for analysis.

[0141] Bronchoalveolar Lavage (BAL) and Cell Counts: The trachea was cannulated to allow the lungs to be lavaged by aspirating 0.5 mL of phosphate buffered saline into the lungs. This procedure was repeated until the recovered volume is 1.6 mL. The isolated BALF was then centrifuged at 1500 rpm for 10 mins at 4°C and the supernatant was aliquoted (400 pL) at -80°C for cytokine analysis. The cell pellets were then resuspended in 1.6 mL of phosphate buffered saline and the BAL cells were then analyzed for total and differential numbers.

[0142] Total and differential cell counts of the BAL fluid samples were measured using a XT-2000iV analyzer (Sysmex). Results were expressed as cells / mL (total and differential). Cell types differentially classified were neutrophils, eosinophils, lymphocytes or macrophages.

[0143] OVA sensitization and challenge: Female Blab / C mice (~25 g) were sensitized with ovalbumin (15 pg, SC) and Imject Alum as an adjuvant on days 1 and 7.

[0144] In order to elicit a local inflammatory response in the lungs, mice were challenged on day 15, 16 and 17 with an aerosol of either 1% w / v ovalbumin in phosphatebuffered saline (PBS) or just PBS, generated with an ultrasonic nebulizer (Aerogen) for 20 min.

[0145] F-428-3 and F-430-1, or vehicle were administered SC (80 pg / kg) or F 429 or vehicle were administered IV (80 pg / kg) 15 minutes prior to the OVA or PBS challenges on days 15, 16 and 17. Then, 24 hrs after the final challenge to OVA or PBS (day 18), samples were collected.

[0146] In a separate cohort of animals, a second series of challenges on days 27, 28, and 29 with an aerosol of 1% w / v ovalbumin or PBS were carried out without further drug treatment. Samples were collected from these animals 24 hrs after the final challenge (day 30).BALF / Serum Collection and Differential Cell Counts

[0147] 24 hours after the last challenge (day 18 or 30), blood samples were taken by venipuncture (via the lateral tail vein) and placed into serum tubes. Each blood sample was kept at room temperature for 45 minutes to allow coagulation, before being centrifuged (2000g, 15 min at 4°C) and the resulting serum sample was stored at -80°C for cytokine analysis.

[0148] Animals were then overdosed with pentobarbitone and bronchoalveolar lavage was carried out using phosphate buffered saline. The isolated bronchoalveolar lavage fluid (BALF) was then centrifuged at 1500 rpm for 10 mins at 4°C and the supernatant was aliquoted (400 pL) and stored at -80°C for cytokine analysis.

[0149] Cell pellets were then re-suspended in 0.8 mL of 0.2% w / v NaCI to induce hemolysis of any erythrocytes. After isotonization with the same volume of 1.6% w / v NaCI, the BALF cells were analyzed for total and differential cell numbers using a XT-2000iV analyzer (Sysmex). Results were expressed as cells / mL.OVA specific IgE ELISA assay

[0150] Serum supernatant (days 14, 18, and 30) was evaluated for OVA specific IgE concentrations using ELISA kit (AssayGenie) as per the manufacturer’s instructions. Optical density was measured at 450 nM using a microplate reader (SpectraMax 340PC). Concentrations of IgE will be determined using SoftMax Pro v. 6.4 (Molecular Devices).Data were reported as OVA specific IgE (pg / mL), mean ±S.E.M. (standard error of the mean).Cytokine / chemokine Measurements

[0151] A 12-Plex cytokine / chemokine panel (IL-4, IL-5, KC, IL-10, I L-12(p70), IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, MCP-1, RANTES) was measured in BALF supernatant and serum using a magnetic multiplex assay as per the manufacturer’s instructions. Levelswere measured using a Magpix system (Luminex Corp). Results were expressed as concentration in pg / mL.Blood Immunophenotyping

[0152] 24 h after the final OVA challenges, blood samples were collected by venepuncture (via the lateral tail vein) and aliquoted with 100 pL being placed in a heparin tube and the rest placed into serum tubes. To increase the number of cells available, blood from individuals in each group were pooled in pairs (n=2), resulting in four samples per group ( / .e., 300-400 iL of blood). Immunophenotyping of T cells (specifically T-regitopes) and B cells (specifically B-regitopes) was then conducted on the blood samples. Reagents used in the immunophenotyping study are listed in Table 9.Table 9: Reagents Used for the Immunophenotyping

[0153] Whole Blood Flow Cytometry: Samples were transferred to a deep-well 96- well plate for red blood cell (RBC) lysis: two animals per group were pooled to give four samples per group. Samples were split for flow cytometry staining across two panels (protected from light throughout). RBC lysis buffer was added according to manufacturer’s instructions. Cells were incubated with a viability stain, an Fc blocking reagent and primary extracellular antibodies diluted to pre-determined optimal concentrations. Cells were fixed and stained for intracellular or nuclear antigens using a nuclear fixation / permeabilization buffer set. The flow cytometry panels are detailed below:Panel 1 : Tbet, Gata3, CD3, CD14, CD19, PD-L1, Granzyme B; andPanel 2: CD3, CD19, IFNy, IL-10, FoxP3.

[0154] The dosing groups analyzed at both day 18 and day 30 are detailed below:Formulation 1 (IV)• Group 1 : vehicle• Group 2: vehicle + OVA• Group 3: vehicle + OVA + ‘1104 (80 pg / kg)Formulation 2 (sub-cutaneous 1 , SC1)• Group 4: vehicle• Group 5: vehicle + OVA• Group 6: vehicle + OVA + ‘1104 (80 pg / kg) Formulation 3 (sub-cutaneous 2, SC2)• Group 7: vehicle• Group 8: vehicle + OVA• Group 9: vehicle + OVA + ‘1104 (80 pg / kg)For intracellular cytokine detection, cells were fixed and permeabilized prior to labelling with antibodies. Sample data was acquired on the Novocyte 3000, with compensation defined and applied using FMO controls.Statistical Analysis

[0155] Data are shown as mean ± S.E.M. (standard error of the mean). Inter-group deviations were statistically analyzed by a one-way analysis of variance (ANO A) followed by a Dunnett’s test. p< 0.05 is considered statistically significant.RESULTSOVA-lnduced Cell Infiltration

[0156] 24 hours post-challenge at day 18, exposure to OVA aerosol triggers inflammatory infiltration in the lung was measured by BALF differential cell counts.

[0157] F-428-3 (SC), F-430-1 (SC), and F 429 (IV) all significantly reduced OVA- induced lung infiltration of white blood cells (FIG. 5A), eosinophils (FIG. 6A), neutrophils (FIG. 7A), macrophages (FIG. 8A), and lymphocytes (FIG. 9A) in a dose response manner. The effects of F-428-3 (SC), F-430-1 (SC), and F 429 (IV) were maintained upon OVA rechallenge (day 30), 13 days after the last dose of the ‘1104 in F-428-3 (SC), F-430-1 (SC), and F 429 (IV). See FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, and FIG. 9B, respectively.

[0158] As such, these data indicates that 24 hours post-challenge at day 18, exposure to OVA aerosol triggered inflammatory infiltration in the lung as measured by BALFdifferential cell counts. IRL201104 significantly reduced OVA-induced lung infiltration of eosinophils, neutrophils, lymphocytes and macrophages through both routes of administration. The effect of IRL201104 was maintained upon OVA re-challenge (at day 30), 13 days after the last dose of the compound.OVA-lnduced Cytokine / Chemokine Release in BALF

[0159] At 24 hours post-challenge at day 18 and 30, exposure to OVA aerosol elicited the release of IL-4 (FIG. 10A), IL-5 (FIG. 11 A), KC (FIG. 12A), IL-10 (FIG. 13A), IL- 12(p70) (FIG. 14A), IL-13 (FIG. 15A), IL-17 (FIG. 16A), Eotaxin (FIG. 17A), GM-CSF (FIG. 18A), G-CSF (FIG. 19A), MCP-1 (FIG. 20A), RANTES (FIG. 21 A) and Periostin (FIG. 22A) in BALF supernatant.

[0160] F-428-3 (SC), F-430-1 (SC), and F 429 (IV) all significantly reduced the levels of all cytokines / chemokines at day 18 in a dose response manner. See FIG. 10A, FIG. 11 A, FIG. 12A, FIG. 13A, FIG. 14A, FIG. 15A, FIG. 16A, FIG. 17A, FIG. 18A, FIG. 19A,FIG. 20A, FIG. 21A, and FIG. 22A, respectively. The effects of F-428-3 (SC), F-430-1 (SC), and F 429 (IV) were maintained upon OVA re-rechallenge (day 30), 13 days after the last dose of the compound. See FIG. 10B, FIG. 11 B, FIG. 12B, FIG. 13B, FIG. 14B, FIG. 15B, FIG. 16B, FIG. 17B, FIG. 18B, FIG. 19B, FIG. 20B, FIG. 21B, and FIG. 22B, respectively.

[0161] As such, these data indicates that: (i) the OVA challenge elicited an increase in cytokine and chemokine release; (ii) IRL201104 treatment through both routes of administration significantly reduced cytokine and chemokine release at day 18; and (iii) the effect was maintained upon OVA re-challenge (day 30), 13 days after the last dose of the compound.Effect of ‘1104 Treatment on Tolerogenic Response Induction

[0162] IRL201104 treatment through both routes of administration seemed to drive an increase the percentage of T regulatory lymphocytes in the blood at day 30, 13 days after the last dose of the ‘1104 compound, as shown by the increased number of CD3+FOXP3+ cells (FIG. 23B), CD3+PD-L1+ cells, a subpopulation of Tregs (FIG. 24B), CD3+IL-10+ cells, a subpopulation of Tregs (FIG. 25), and CD3+FOXP3+IL-10+ (FIG. 26). These results are consistent with what has been previously observed in human clinical and ex vivo data (FIG. 27).

[0163] Similarly, IRL201104 treatment through both routes of administration seemed to drive an increase the percentage of B regulatory lymphocytes in the blood at day 30, 13 days after the last dose of the ‘1104 compound, as shown by the increased number of CD19+PD-L1+ cells, a subpopulation of Bregs (FIG. 28B) and CD19+IL-10+ cells (FIG. 29). Again, these results are consistent with what has been previously observed in human clinical and ex vivo data (FIG. 30).

[0164] As such, these data indicates that IRL201104 treatment through both routes of administration seemed to drive an increase of different phenotypes of T regulatory and B regulatory lymphocytes in the blood at day 30, 13 days after the last dose of the compound. This new observation is consistent with other preclinical data in humans, as well as clinical data from the human clinical trial. These data would support the hypothesis that the long pharmacodynamic effect of ‘1104 is driven through regulatory cell populations (Treg and B reg) and may require an ongoing inflammatory process (as present in EoE and the OVA rechallenge model).EXAMPLE 6 EFFECTIVENESS OF SUBCUTANEOUS ‘1104 FORMULATIONS IN AN ANIMAL MODEL OF ATOPIC DERMATITIS

[0165] The aim of this study is to use an ovalbumin (OVA) driven re-challenge model of local skin inflammation to compare the impact of ‘1140 through two different routes of administration (subcutaneous and intravenous) on skin thickness and pathology, relevant cytokines and OVA-specific IgE in serum to assess the effectiveness of the SC ‘1104 formulation, F-428-3, and the IV ‘1104 formulation, F-429 compared to the positive control dexamethasone.MATERIALS AND METHODS

[0166] Animals: Female BalbC mice (20-30 g on arrival, Charles Rivers, UK) were used in the present study.

[0167] Animal welfare: On arrival from the supplier, animals were acclimatized for period of 5-7 days before start of experimental procedures. Mice were housed in cages of four on arrival based on weight (equal distribution of animal weights amongst each of the cages by the animal technician) with a 12 hour light dark cycle. Room temperature and humidity were maintained between 17-24°C and 40-70%, respectively. Environmental enrichment was provided in all cages. Mice had access to standard chow ad libitum and water was available from bottles ad libitum.

[0168] Throughout the study, the following guidelines were used to assess nonspecific or unexpected adverse effects in animals undergoing regulated, relating to either the procedure or test compound dosing. Animals showing two or more of any of the limiting clinical signs in the category equivalent to the protocol severity limit were removed from the study. Limiting clinical signs included:• Body weight loss greater than 20% of the highest measured individual body weight.Marked piloerection with other signs of dehydration such as skin tenting.Unresponsive to activity and provocation.Hunched persistently (frozen).Distressed- persistent vocalization.Oculo-nasal discharge persistent and copious.Labored respiration.Persistent tremors.Persistent convulsions.

[0169] Animals also exposed to severe fight injuries were removed from the study. In this study, no animals were removed due to welfare concerns or due to fight injuries.

[0170] Protocol: The protocol for the study is provided in the Table 10 below:Table 10: Study DesignGrp N Sensitization Challenge Treatment Treatment Sample (i p ) (days 14-20) (the following Regime Collection (day 1 and and (days 28- phase B) (day 35) day 7) 34)G1 4 Blood serum & dorsal skinG2 8 OVA / alum (15 OVA (300 pg) Vehicle (i.v.) 21, 23, 25, 27, Blood serum & pg / 25ul) 32,33,34 dorsal skinG3 8 OVA / alum (15 OVA (300 pg) Vehicle (s.c.) 21, 23, 25, 27, Blood serum & pg / 25ul) 32,33,34 dorsal skinG4 8 OVA / alum (15 OVA (300 pg) IRL201104 21, 23, 25, 27, Blood serum & pg / 25ul) (2 mg / kg, i.v.) 32,33,34 dorsal skinG5 8 OVA / alum (15 OVA (300 pg) IRL201104 21, 23, 25, 27, Blood serum & pg / 25ul) (2 mg / kg, s.c.) 32,33,34 dorsal skinG6 8 OVA / alum (15 OVA (300 pg) Dexamethasone 21, 23, 25, 27, Blood serum & pg / 25ul) (3 mg / kg, i.p.) 32,33,34 dorsal skin pK satellite groupsG7 3 OVA / alum (15 OVA (300 pg) IRL201104 21,23, 25, 27, 32, Blood plasma pg / 25ul) (2 mg / kg, i.v.) 33, 34 (days 21 & 34) - 2, 15 60 min and 24 hours)G8 3 OVA / alum (15 OVA (300 pg) IRL201104 21,23, 25, 27, 32, Blood plasma pg / 25ul) (2 mg / kg, s.c.) 33, 34 (days 21 & 34) - 2, 15 60 min and 24 hours)

[0171] Treatment of animals with dexamethasone, IRL201104 or vehicle: N on- fasted mice were weighed and placed into a hotbox for five min before being placed in a whole body restrainer to receive an intravenous administration (5 mL / kg) via the tail vein of either IRL201104 or vehicle. Subcutaneous administration (10 mL / kg) were carried out on conscious animals. Animals receiving dexamethasone (3 mg / kg) treatment were administered doses intra-peritoneally (10 mL / kg). All treatments were administered on days 21 , 23, 25, 27, 32,33 and day 34.

[0172] Blood sample collection: On days 35, a terminal blood sample was collected via cardiac puncture and placed into a serum tube. Each serum sample was kept at room temperature for 45 minutes to allow coagulation, before being centrifuged (2000g, 15 min at 4°C) from which the resulting supernatant was extracted, aliquoted and stored at -80°C for analysis.

[0173] OVA sensitization and challenge: Mice were actively sensitized with ovalbumin (15 pg, s.c.) and 25 pL of Imject Alum on days 1 and 7. On day 14, mice were anaesthetized with isoflurane to allow for the dorsal skin to be shaved using clippers and hair removal cream followed by tape stripping which was conducted 6 consecutive times. After shaving, the mice were epicutaneously challenged with OVA patches. The OVA patches were prepared with 1 cm2sterile gauze moistened with 300 pg OVA in phosphate buffered saline and attached to the shaved and tape stripped dorsal skin with a transparent dressing for 7 days. These gauze dressings were changed daily. On day 28, a second 7 day cycle of daily OVA gauze patches were applied until day 34. From these animals samples were collected on day 35.Assessment of Skin Pathology

[0174] Immediately after blood collection the dermal area exposed to the OVA challenge was visually assessed for pathological features associated with atopic dermatitis. These included: erythema / hemorrhage, oozing / crust, erosion / excoriation, lichenification. Each pathology was scored based on the following scoring scheme:Dorsal Skin Sampling and Thickness Measurements

[0175] Immediately after blood collection via cardiac puncture, the mice were sacrificed by cervical dislocation and the dorsal skin excised very careful with a sharp scalpel so at to minimize damage to the skin during dissection. Randomly, three positions on the dorsal skin were measured doubled over by using digital calliper and the mean thickness (pm) calculated for each mouse. After measuring the dorsal skin thickness, the sample were divided into three parts, the middle section was placed for 48 hours in 10%formalin before being transferred into ethanol for future histopathology. Data are reported at skin thickness (pm) mean ± S.E.M. The other two sections were snap frozen and stored at -70°C for biomarker analysis. pK Plasma Collection (pK Satellite Groups)

[0176] Blood samples were collected by venipuncture, via the lateral tail vein, 2, 15, 60 min and 24 hours after dosing on day 21 (n=3 per administration route) and 2, 15, 60 min and 24 hours after dosing on day 34 (n=3 per administration route) of IRL201104 (group 7 and group 8). Each blood sample was placed into polypropylene tubes containing anticoagulant (heparin) before being mixed gently and centrifuged (2000g, 15 min at 4°C) from which the resulting plasma was extracted, aliquoted and stored at -80°C for bioanalysis.OVA specific IgE ELISA assay

[0177] Serum supernatant (days 14, 18, and 30) was evaluated for OVA specific IgE concentrations using ELISA kit (AssayGenie) as per the manufacturer’s instructions. Optical density was measured at 450 nM using a microplate reader (SpectraMax 340PC).Concentrations of IgE will be determined using SoftMax Pro v. 6.4 (Molecular Devices). Data were reported as OVA specific IgE (pg / mL), mean ±S.E.M. (standard error of the mean).Cytokine / Chemokine Analysis

[0178] Blood serum concentrations of eight cytokines (IL-4, IL-5, IL-13, IL-1 p, TNF-a, IFN-gamma, IL-17, CCL-22) were measured in duplicate in all groups using magnetic multiplex assays (Biotechne) as per the manufacturer’s instructions. Levels were measured using a Magpix system (Luminex Corp.). CCL-17 and IL-31 concentrations were measured using ELISA kit (Biotechne and Invitrogen respectively) as per the manufacturer’s instructions. Optical density was measured at 450 nM using a microplate reader (SpectraMax 340PC). Data will be reported as cytokine (pg / mL), mean ± S.E.M.Data Analysis

[0179] Inter-group deviations were statistically analyzed by a one-way analysis of variance (ANOVA). In the case of significant difference in the mean values among the different levels of treatment, comparisons versus the vehicle group will be carried out using the Dunnett’s test. p< 0.05 was considered statistically significant.Bioanalysis

[0180] Instrument and Method Set Up: The mass spectrometer was set up and the analytical conditions and sample preparation method established for the sensitive andselective analysis of IRL201104 over the appropriate concentration range for the matrices of mouse plasma.

[0181] Analysis: Quantitative bioanalysis by liquid chromatography-mass spectrometry was conducted for IRL2011104 of 48 plasma samples (50 pL volume) from animals in each of the treatment groups collected during the above detailed study.

[0182] Samples were prepared by protein precipitation and / or dilution. Analysis was carried out using a generic internal standard, with matrix-matched calibration standards bracketing the study samples. Batch acceptance was based primarily upon calibration curve data meeting criteria for quality as described in internal guidelines. Analyses was repeated where necessary.RESULTSSkin Thickness and Pathology

[0183] As shown in FIG. 31 , whether administered SC or IV, IRL201104 significantly reduced the dorsal skin thickness in the OVA-driven re-challenge model of local skin inflammation when compared to their respective vehicle controls. IRL201104, F-428-3 (SC) and F 429 (IV), significantly reduced the skin pathology, including a significant reduction in erythema / hemorrhage (FIG. 32A), oozing / crust (FIG. 32B), erosion / excoriation (FIG. 32C). The beneficial effects of IRL201104, F 429 (IV) and F-428-3 (SC), on the OVA-induced atopic dermatitis can be noted in FIG. 44 and FIG. 45, respectively.

[0184] These data indicates that the OVA challenge elicited an increase in dorsal skin thickness and IRL201104 treatments through both routes of administration significantly reduced dorsal skin thickness and dermal pathology similar to the positive control dexamethasone.Serum Biomarkers

[0185] OVA-specific IgE: As shown in FIG. 33, a significant increase in serum IgE was observed in mice sensitized to OVA. The SC ‘1104 formulation, F-428-3, and the IV ‘1104 formulation, F-429, both significantly reduced the serum IgE levels similar to the positive control dexamethasone (FIG. 33).

[0186] T1 Cytokines: The OVA challenge elicited an increase in serum TNF alpha (FIG. 34), IL-1 p (FIG. 35), and IFN-gamma (FIG. 40). The SC ‘1104 formulation, F-428-3, and the IV ‘1104 formulation, F-429, both significantly reduced the serum levels of these T1 cytokines, similar to the positive control dexamethasone.

[0187] T17 Cytokines: As shown in FIG. 39, the OVA challenge elicited an increase in serum IL-17A. The SC ‘1104 formulation, F-428-3, and the IV ‘1104 formulation, F-429, both significantly reduced the serum IL-17A levels, similar to the positive control dexamethasone.

[0188] T2 Cytokines: The OVA challenge elicited an increase in serum IL-4 (FIG. 36), IL-5 (FIG. 37), IL-13 (FIG. 38), and IL-31 (FIG. 43). The SC ‘1104 formulation, F-428-3, and the IV ‘1104 formulation, F-429, both significantly reduced the serum levels of these T2 cytokines, similar to the positive control dexamethasone.

[0189] Atopic Dermatitis Biomarkers: The OVA challenge elicited an increase in the serum levels of the atopic dermatitis biomarkers, CCL-17 (FIG. 42) and CCL-22 (FIG. 41). The SC ‘1104 formulation, F-428-3, and the IV ‘1104 formulation, F-429, both significantly reduced the serum levels of these atopic dermatitis biomarkers, similar to the positive control dexamethasone.

[0190] These data indicates that ‘1104, when dose therapeutically, has a significant beneficial therapeutic effects on skin thickness, skin pathology and serum biomarkers through both the IV and SC route comparable to positive control dexamethasone.CONCLUSIONS

[0191] In the current studies, the three ‘1104 formulations, F-428-3 (SC), F-430-1 (SC), and F 429 (IV) all showed a similar long lasting dose response immunomodulatory profile through two different routes of administration, intravenous and subcutaneous.

[0192] All three ‘1104 formulations, F-428-3 (SC), F-430-1 (SC), and F 429 (IV) did not only affect eosinophils, but also neutrophils, macrophages and lymphocytes, indicating a broad spectrum of action on inflammatory immune cells.

[0193] All three ‘1104 formulations, F-428-3 (SC), F-430-1 (SC), and F 429 (IV) also exhibited a significant long lasting effect in a range of relevant cytokines and chemokines such us IL-4, IL-5, IL-13 and Eotaxin, in BALF.

[0194] All three ‘1104 formulations, F-428-3 (SC), F-430-1 (SC), and F 429 (IV) also exhibited a significant long lasting increase of different phenotypes of T regulatory and B regulatory lymphocytes in the blood.

[0195] Despite the short half-life, IRL201104 showed a long pharmacodynamic effect through both routes of administration, which is consistent with the induction of tolerogenic T and B regulatory cell phenotypes

[0196] The present study support the potential of the two SC formulations, F-428-3 and F-430-1 , to be used in the clinical setting through the SC route of administration.REFERENCES:1Ranford, J.C., et al. (2000). “Chaperonins are cell signaling polypeptides:-the unfolding biology of molecular chaperones." Exp. Rev. Mol. Med., 15;2(8): 1 -17.2International Patent Application, Publication Number WO 2002 / 040037A2. “Biological Materials andUses Thereof.” Published 23 May 2002.United States Patent No. 9,085,632. “Biological Materials and Uses Thereof.” Issued July 21 , 2015. United States Patent No. 11 ,479,585. “Method for the Treatment of a Relapsing-RemittingCondition." Issued October 25, 2022. International Patent Application, Publication Number WO 2023 / 223015A1 . “Methods andCompositions for Preventing or Treating Food Allergies.” Published 23 November 2023. International Patent Application PCT / IB2024 / 25253. “Methods and Compositions for Preventing orTreating Eosinophilic Esophagitis.” Filed 18 April 2024. Isselbacher, et al. (1996). HARRISON’S PRINCIPLES OF INTERNAL MEDICINE, 13 ed., 1814-1882. THE MERCK MANUAL OF DIAGNOSIS AND THERAPY, (2011). 19thEdition, published by Merck Sharp &Dohme Corp., (ISBN 978-0-911910-19-3). THE ENCYCLOPEDIA OF MOLECULAR CELL BIOLOGY AND MOLECULAR MEDICINE, Robert S. Porter et al.(eds.), published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908). MOLECULAR BIOLOGY AND BIOTECHNOLOGY: A COMPREHENSIVE DESK REFERENCE, (1995). Robert A.Meyers (ed.), published by VCH Publishers, Inc. (ISBN 1-56081-569-8). IMMUNOLOGY, (2006). Werner Luttmann, published by Elsevier. JANEWAY'S IMMUNOBIOLOGY, (2014). Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor &Francis Limited, (ISBN 0815345305, 9780815345305). LEWIN'S GENES XI, (2014). published by Jones & Bartlett Publishers (ISBN-1449659055). Michael Richard Green and Joseph Sambrook, (2012). MOLECULAR CLONING: A LABORATORYMANUAL, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (ISBN 1936113414). Davis et al., (2012). BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier Science Publishing, Inc., NewYork, USA (ISBN 044460149X). LABORATORY METHODS IN ENZYMOLOGY: DNA, (2013). Jon Lorsch (ed.) Elsevier (ISBN 0124199542). CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (CPMB), (2014). Frederick M. Ausubel (ed.), JohnWiley and Sons (ISBN 047150338X, 9780471503385). CURRENT PROTOCOLS IN PROTEIN SCIENCE (CPPS), (2005). John E. Coligan (ed.), John Wiley andSons, Inc. CURRENT PROTOCOLS IN IMMUNOLOGY (CPI) (2003). John E. Coligan, ADA M Kruisbeek, David HMargulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc. (ISBN 0471142735, 9780471142737). United States Patent Published Patent Application No. 20040132163 entitled: “Biological materials and uses thereof.” Published: July 8, 2004. United States Patent No. 11 ,098,090 entitled: “Mycobacteria tuberculosis chaperonin 60.1 peptides and uses thereof." Issued: Aug. 24, 2021 . United States Patent No. 9,320,791 entitled: “Peptides from the polypeptide chaperonin 60.1, and their use in medicine for the treatment of inflammatory conditions are described.” Issued: Apr. 26, 2016. United States Patent No. 9,085,632 entitled: “Biological materials and uses thereof." Issued: Jul.21 , 2015. International Patent Application, Publication Number WO 2023 / 156849A2. “Methods of ProducingPeptide Derived from Chaperonin 60.1." Published 24 August 2023, at paragraph

[0060] . Riffo-Vasquez, Y., et al. (2020). “Modulation of allergic inflammation in the lung by a peptide derived from Mycobacteria tuberculosis chaperonin 60. 1.” Clin. Exp. Allergy, 50(4): 508-519.

[0197] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0198] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the present aspects and embodiments. The present aspects and embodiments are not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect and other functionally equivalent embodiments are within the scope of the disclosure. Various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects described herein are not necessarily encompassed by each embodiment. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A pharmaceutical formulation for subcutaneous injection of a peptide having SEQ ID NO: 1 , comprising a peptide having SEQ ID NO: 1 at a concentration of concentration of 16-150 mg / mL, and having the following properties:(a) capable of delivering a high dose of 40 mg and optimally 64 mg in an injection volume of 2 mL or less;(b) a pH of 5.5-9.5, optimally a pH of 6-8; and;(c) an osmolality of 210-350 mOs, optimally 260-310 mOs.

2. The pharmaceutical formulation according to claim 1, further comprising tonicity enhancer to make the formulation isotonic.

3. The pharmaceutical formulation according to claim 2, wherein the tonicity enhancer is mannitol.

4. The pharmaceutical formulation according to any one of claims 1-3, comprising (a) the peptide having SEQ ID NO: 1 at a concentration of from about 25 to about 40 mg / mL;(b) about 100 mM Tris buffer; and (c) about 0.1% Polysorbate 80; where the pharmaceutical formulation has a pH of about 8.5.

5. The pharmaceutical formulation according to any one of claims 1-3, comprising(a) the peptide having SEQ ID NO: 1 at a concentration of from about 25 to about 40 mg / mL;(b) about 50 mM Tris buffer; and (c) about 50 mM L-Arginine; where the pharmaceutical formulation has a pH of about 8.0.

6. The pharmaceutical formulation according to any one of claims 1-5, wherein the formulation is stored at 15-25°C.

7. The pharmaceutical formulation according to any one of claims 1-5, wherein the formulation is stored at 2-8°C or colder.

8. The pharmaceutical formulation according to any one of claims 1-5, wherein the formulation is stored at -20°C or colder.

9. The pharmaceutical formulation according to any one of claims 1-8, wherein the formulation is stable for at least 6 months.

10. The pharmaceutical formulation according to any one of claims 1-8, wherein the formulation is stable for at least 12 months.

11. The pharmaceutical formulation according to any one of claims 1-8, wherein the formulation is stable for at least 24 months.

12. The pharmaceutical formulation according to any one of claims 1-11 , wherein the concentration of 16-150 mg / mL of ‘1104 has a bioavailability of at least 50%.