EP3720470A1 - Il-2 muteine und deren verwendung - Google Patents

Il-2 muteine und deren verwendung

Info

Publication number
EP3720470A1
EP3720470A1 EP18887098.4A EP18887098A EP3720470A1 EP 3720470 A1 EP3720470 A1 EP 3720470A1 EP 18887098 A EP18887098 A EP 18887098A EP 3720470 A1 EP3720470 A1 EP 3720470A1
Authority
EP
European Patent Office
Prior art keywords
peptide
seq
juvenile
disease
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18887098.4A
Other languages
English (en)
French (fr)
Other versions
EP3720470A4 (de
Inventor
Nathan HIGGINSON-SCOTT
Joanne L. Viney
Jyothsna Visweswaraiah
Erik Robert Sampson
Kevin Lewis Otipoby
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pandion Operations Inc
Original Assignee
Pandion Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US16/109,897 external-priority patent/US10174092B1/en
Application filed by Pandion Therapeutics Inc filed Critical Pandion Therapeutics Inc
Publication of EP3720470A1 publication Critical patent/EP3720470A1/de
Publication of EP3720470A4 publication Critical patent/EP3720470A4/de
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • Embodiments provided herein relate to proteins referred to as IL-2 muteins, compositions comprising the same, and methods of using the same.
  • IL-2 binds three transmembrane receptor subunits: IL-2R.p and IL-2Ry, which together activate intracellular signaling events upon IL-2 binding, and CD25 (IL-2Roc) which serves to present IL-2 to the other 2 receptor subunits.
  • the signals delivered by IL-2RP7 include those of the PI3-kinase, Ras-MAP -kinase, and STAT5 pathways.
  • T cells require expression of CD25 to respond to the low concentrations of IL-2 that typically exist in tissues.
  • T cells that express CD25 include both CD4 + FOXP3 + regulatory T cells (T-reg cells)— which are essential for suppressing autoimmune inflammation— and FOXP3 T cells that have been activated to express CD25.
  • FOXP3 CD4 + T effector cells (T-eff) may be either CD4 + or CD8 + cells, both of which can be pro-inflammatory and may contribute to autoimmunity and other diseases where the subject’s immune system attacks an organ or other tissues.
  • IL-2-stimulated STAT5 signaling is crucial for normal T-reg cell growth and survival and for high FOXP3 expression.
  • peptides comprising an amino acid sequence of SEQ ID NO: 1, wherein the peptide comprises a mutation at position 73, 76, 100, or 138 are provided.
  • peptides comprising an amino acid sequence of SEQ ID NO: 2, wherein the peptide comprises a mutation at position 53, 56, 80, or 118 are provided.
  • peptides comprising an amino acid sequence of SEQ ID NO: 43, wherein at least one of Xi, X 2 , and X 3 and X 4 are I and the remainder are L or I.
  • compositions comprising the same and nucleic acid molecules encoding the proteins described herein.
  • vectors comprising the nucleic acid molecule encoding the proteins described herein.
  • plasmids comprising the nucleic acid encoding the proteins described herein are provided.
  • cells comprising the nucleic acid molecules, vectors, or plasmids, encoding the proteins described herein are provided.
  • methods of activating T regulatory cells comprise contacting a T regulatory cell with a peptide described herein or a pharmaceutical composition described herein.
  • methods of treating an inflammatory disorder in a subject comprise administering to a subject, including but not limited to a subject in need thereof, a peptide (e.g . a therapeutically effective amount of the peptide).
  • methods of promoting or stimulating STAT5 phosphorylation in T regulatory cells comprise administering to a subject a peptide (e.g. a therapeutically effective amount of the peptide).
  • FIG. 1 illustrates a non-limiting embodiment of a IL-2 mutein provided for herein.
  • the term“selective” refers to the therapeutic or protein modulating the activity in T-reg cells but has limited or lacks the ability to promote the activity in non-regulatory T cells.
  • the therapeutic is a mutant of IL-2.
  • a mutant of IL-2 can be referred to as an IL-2 mutein.
  • IL-2 can exist in two different forms, an immature form and a mature form. The mature form is where the leader sequence has been removed. This is done during a post-translational process.
  • the wild-type sequence of the immature IL-2 is as follows:
  • the wild-type sequence of the mature IL-2 is as follows:
  • An IL-2 mutein molecule can be prepared by mutating one or more of the residues of IL- 2.
  • Non-limiting examples of IL-2-muteins can be found in WO2016/164937, US9580486, US7105653, US9616105, US 9428567, US2017/0051029, US2014/0286898A1,
  • the alanine at position 1 of the sequence above is deleted.
  • the IL-2 mutein molecule comprises a serine substituted for cysteine at position 125 of the mature IL-2 sequence.
  • Other combinations of mutations and substitutions that are IL-2 mutein molecules are described in US20060269515, which is incorporated by reference in its entirety.
  • the cysteine at position 125 is also substituted with a valine or alanine.
  • the IL-2 mutein molecule comprises a V91K substitution.
  • the IL-2 mutein molecule comprises a N88D substitution.
  • the IL-2 mutein molecule comprises a N88R substitution. In some embodiments, the IL-2 mutein molecule comprises a substitution of H16E, D84K, V91N, N88D, V91K, or V91R, any combinations thereof. In some embodiments, these IL-2 mutein molecules also comprise a substitution at position 125 as described herein.
  • the IL-2 mutein molecule comprises one or more substitutions selected from the group consisting of: T3N, T3A, L12G, L12K, L12Q, L 12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20H, D20I, D20Y, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81 S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q D84R, D84S, D84T, S87R, N88A, N88D, N88E,
  • the amino acid sequence of the IL-2 mutein molecule differs from the amino acid sequence set forth in mature IL-2 sequence with a C125A or C125S substitution and with one substitution selected from T3N, T3A, L12G, L12K, L12Q L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81 S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N
  • the IL-2 mutein molecule differs from the amino acid sequence set forth in mature IL-2 sequence with a C125A or C125S substitution and with one substitution selected from D20H, D20I, D20Y, D20E, D20G, D20W, D84A, D84S, H16D, H16G, H16K, H16R, H16T, H16V, I92K, I92R, L12K, L19D, L19N, L19T, N88D, N88R, N88S, V91D, V91G, V91K, and V91S.
  • the IL-2 mutein comprises N88R and/or D20H mutations.
  • the IL-2 mutein molecule comprises a mutation in the polypeptide sequence at a position selected from the group consisting of amino acid 30, amino acid 31, amino acid 35, amino acid 69, and amino acid 74.
  • the mutation at position 30 is N30S.
  • the mutation at position 31 is Y31H.
  • the mutation at position 35 is K35R.
  • the mutation at position 69 is V69A.
  • the mutation at position 74 is Q74P.
  • the mutein does not comprise a mutation at position 30, 31, and/or 35.
  • the IL-2 mutein molecule comprises a substitution selected from the group consisting of: N88R, N88I, N88G, D20H, D109C, Q126L, Q126F, D84G, or D84I relative to mature human IL-2 sequence provided above.
  • the IL-2 mutein molecule comprises a substitution of D109C and one or both of a N88R substitution and a C125S substitution.
  • the cysteine that is in the IL-2 mutein molecule at position 109 is linked to a polyethylene glycol moiety, wherein the polyethylene glycol moiety has a molecular weight of from about 5 to about 40 kDa.
  • the mutein does not comprise a mutation at position 109, 126, or 84.
  • any of the substitutions described herein are combined with a substitution at position 125.
  • the substitution can be a C125S, C125A, or C125V substitution.
  • the mutein does not comprise a mutation at position 125.
  • the numbering referred to herein, unless indicated otherwise for the IL-2 muteins refers to the mature sequence. If a sequence or position refers to SEQ ID NO: 1 it is the immature sequence. However, to transpose the positions from the immature sequence (SEQ ID NO: 1) to the mature sequence (SEQ ID NO: 2) all that need be done is to subtract 20 from the position referred to in SEQ ID NO: 1 to get the corresponding position in SEQ ID NO: 2.
  • the IL-2 mutein has a substitution/mutation at one or more of positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 1 or positions at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 2.
  • the IL-2 mutein comprises a mutation at positions 73 and 76; 73 and 100; 73 and 138; 76 and 100; 76 and 138; 100 and 138; 73, 76, and 100; 73, 76, and 138; 73, 100, and 138; 76, 100 and 138; or at each of 73, 76, 100, and 138 that correspond to SEQ ID NO: 1.
  • the IL-2 mutein comprises a mutation at positions 53 and 56; 53 and 80; 53 and 118; 56 and 80; 56 and 118; 80 and 118; 53, 56, and 80; 53, 56, and 118; 53, 80, and 118; 56, 80 and 118; or at each of 53, 56, 80, and 118 that correspond to SEQ ID NO: 2.
  • the term corresponds to as reference to a SEQ ID NOs: 6 or 15 refer to how the sequences would align with default settings for alignment software, such as can be used with the NCBI website.
  • the mutation is leucine to isoleucine.
  • the IL-2 mutein can comprise one more isoleucines at positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 1 or positions at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 2.
  • the mutein comprises a mutation at L53 that correspond to SEQ ID NO: 2.
  • the mutein comprises a mutation at L56 that correspond to SEQ ID NO: 2.
  • the mutein comprises a mutation at L80 that correspond to SEQ ID NO: 2.
  • the mutein comprises a mutation at Ll 18 that correspond to SEQ ID NO: 2.
  • the mutation is leucine to isoleucine.
  • the mutein also comprises a mutation as position 69, 74, 88, 125, or any combination thereof in these muteins that correspond to SEQ ID NO: 2.
  • the mutation is a V69A mutation.
  • the mutation is a Q74P mutation.
  • the mutation is a N88D or N88R mutation.
  • the mutation is a C125A or C125S mutation.
  • the IL-2 mutein comprises a mutation at one more of positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145 that correspond to SEQ ID NO: 1 or one or more positions 29, 31, 35, 37, 48, 69, 71, 74, 88, and 125 that correspond to SEQ ID NO: 2.
  • the substitutions can be used alone or in combination with one another.
  • the IL-2 mutein comprises substitutions at 2, 3, 4, 5, 6, 7, 8, 9, or each of positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145.
  • Non-limiting examples such combinations include, but are not limited to, a mutation at positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145; 49, 51, 55, 57, 68, 89, 91, 94, and 108; 49, 51, 55, 57, 68, 89, 91, and 94; 49, 51, 55, 57, 68, 89, and 91; 49, 51, 55, 57, 68, and 89; 49, 51, 55, 57, and 68; 49, 51, 55, and 57; 49, 51, and 55; 49 and 51; 51, 55, 57, 68, 89, 91, 94, 108, and 145; 51, 55, 57, 68, 89, 91, 94, and 108; 51, 55, 57, 68, 89, 91, and 94; 51, 55, 57, 68, 89, 91, and 94; 51,
  • the IL-2 mutein comprises a mutation at one or more positions of 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 1 or the equivalent positions at SEQ ID NO: 2 ( e.g . positions 15, 16, 22, 84, 95, and 126). .
  • mutations can be combined with the other leucine to isoleucine mutations described herein or the mutation at positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 1 or at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 2.
  • the mutation is a E35Q, H36N, Q42E, D104N, E115Q, or Q146E, or any combination thereof.
  • one or more of these substitutions is wildtype.
  • the mutein comprises a wild-type residue at one or more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 1 or the equivalent positions at SEQ ID NO: 2 (e.g. positions 15, 16, 22, 84, 95, or 126).
  • the mutations at these positions can be combined with any of the other mutations described herein, including, but not limited to substitutions at positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 1 or positions at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 2 described herein and above.
  • the IL-2 mutein comprises a N49S mutation that corresponds to SEQ ID NO: 1.
  • the IL-2 mutein comprises a Y51S or a Y51H mutation that corresponds to SEQ ID NO: 1.
  • the IL-2 mutein comprises a K55R mutation that corresponds to SEQ ID NO: 1.
  • the IL-2 mutein comprises a T57A mutation that corresponds to SEQ ID NO: 1. In some embodiments, the IL-2 mutein comprises a K68E mutation that corresponds to SEQ ID NO: 1. In some embodiments, the IL-2 mutein comprises a V89A mutation that corresponds to SEQ ID NO: 1. In some embodiments, the IL-2 mutein comprises a N91R mutation that corresponds to SEQ ID NO: 1. In some embodiments, the IL-2 mutein comprises a Q94P mutation that corresponds to SEQ ID NO: 1. In some embodiments, the IL-2 mutein comprises a N108D or a N108R mutation that corresponds to SEQ ID NO: 1. In some embodiments, the IL-2 mutein comprises a C145A or C145S mutation that corresponds to SEQ ID NO: 1.
  • the mutein comprises each of these substitutions. In some embodiments, the mutein comprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein comprises a wild-type residue at one or more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 1 or the equivalent positions at SEQ ID NO: 2 (e.g. positions 15, 16, 22, 84, 95, 126, and 126).
  • the IL-2 mutein comprises a N29S mutation that corresponds to SEQ ID NO: 2. In some embodiments, the IL-2 mutein comprises a Y31S or a Y31H mutation that corresponds to SEQ ID NO: 2. In some embodiments, the IL-2 mutein comprises a K35R mutation that corresponds to SEQ ID NO: 2. In some embodiments, the IL-2 mutein comprises a T37A mutation that corresponds to SEQ ID NO: 2. In some embodiments, the IL-2 mutein comprises a K48E mutation that corresponds to SEQ ID NO: 2. In some embodiments, the IL-2 mutein comprises a V69A mutation that corresponds to SEQ ID NO: 2.
  • the IL-2 mutein comprises a N71R mutation that corresponds to SEQ ID NO: 2. In some embodiments, the IL-2 mutein comprises a Q74P mutation that corresponds to SEQ ID NO: 2. In some embodiments, the IL-2 mutein comprises a N88D or a N88R mutation that corresponds to SEQ ID NO: 2. In some embodiments, the IL-2 mutein comprises a C125A or C125S mutation that corresponds to SEQ ID NO: 2. These substitutions can be used alone or in combination with one another. In some embodiments, the mutein comprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein comprises each of these substitutions.
  • the mutein comprises a wild-type residue at one or more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 1 or the equivalent positions at SEQ ID NO: 2 (e.g. positions 15, 16, 22, 84, 95, and 126).
  • positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 1 or the equivalent positions at SEQ ID NO: 2 are wild-type (e.g. are as shown in SEQ ID NOs: 1 or 2).
  • 2, 3, 4, 5, 6, or each of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 1 or the equivalent positions at SEQ ID NO: 2 are wild-type.
  • the IL-2 mutein comprises a sequence of:
  • MYRMQLLSCIALSLALVTN S APTS S STKKTQLQLEHLLLDLQMILNGISNH KNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLR PRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT
  • the IL-2 mutein comprises a sequence of:
  • the IL-2 mutein comprises a sequence of:
  • the IL-2 mutein comprises a sequence of:
  • the IL-2 mutein sequences described herein do not comprise the IL-2 leader sequence.
  • the IL-2 leader sequence can be represented by the sequence of M YRMQLL S Cl AL SL AL VTN S (SEQ ID NO: 7). Therefore, in some embodiments, the sequences illustrated above can also encompass peptides without the leader sequence.
  • SEQ ID NOs; 3-6 are illustrated with only mutation at one of positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 1 or positions at one or more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 2, the peptides can comprise 1, 2, 3, or 4 of the mutations at these positions.
  • the substitution at each position is isoleucine or other type of conservative amino acid substitution.
  • the leucine at the recited positions are substituted with, independently, isoleucine, valine, methionine, or glycine, alanine, glutamine or glutamic acid.
  • the IL-2 protein of SEQ ID NO: 2 comprises the following mutations: V69A, Q74P, N88D, and C125S or C125A and one mutation selected from the group consisting of L53I, L56I, L80I, and L118I.
  • the IL-2 protein comprises two mutations selected from the group consisting of L53I, L56I, L80I, and L118I.
  • the IL-2 protein comprises three or each of the mutations selected from the group consisting of L53I, L56I, L80I, and L118I.
  • the IL-2 protein comprises L53I and L56I, L53I and L80I, L53I and L118I, L56I and L80I, L56I and L118I, L80I and Ll 181, L53I, L56I, and L80I, L53I, L56I, and Ll 181, L56I, L80I, and Ll 181 or L53I, L56I, L80I, and L118I.
  • the IL-2 mutein does not comprise L53I, L56I, L80I, or Ll 181 mutations.
  • the IL-2 mutein comprises a T3A mutation.
  • the IL-2 protein of SEQ ID NO: 2 comprises the following mutations: V69A, Q74P, N88D, and C125S or C125A and one or more mutations, such as but not limited to conservative substitutions, in regions of 45-55, 50-60, 52-57, 75-85, 100-130, 115- 125 of SEQ ID NO: 2.
  • the IL-2 mutein molecule is fused to a Fc Region or other linker region as described herein.
  • Fc Region or other linker region examples of such fusion proteins can be found in US9580486, US7105653, US9616105, US 9428567, US2017/0051029, WO2016/164937,
  • the Fc Region comprises what is known at the LALA mutations.
  • the Fc region comprises L234A and L235A mutations (EU numbering).
  • the Fc Region comprises a G237A (EU numbering).
  • the Fc Region does not comprise a mutation at position G237 (EU numbering) Using the Rabat numbering this would correspond to L247A, L248A, and/or G250A.
  • using the EU numbering system the Fc region comprises a L234A mutation, a L235A mutation, and/or a G237A mutation.
  • the Fc portion can comprise mutations that corresponds to one or more of these residues.
  • the Fc Region comprises N297G or N297A (kabat numbering) mutations.
  • the Kabat numbering is based upon a full-length sequence, but would be used in a fragment based upon a traditional alignment used by one of skill in the art for the Fc region (see, for example, Kabat et al. (“Sequence of proteins of immunological interest,” US Public Health Services, NIH Bethesda, MD, Publication No. 91, which is hereby incorporated by reference), which is hereby incorporated by reference.
  • the Fc Region comprises a sequence of:
  • the Fc Region comprises a sequence of:
  • the IL-2 mutein is linked to the Fc Region.
  • linkers are glycine/serine linkers.
  • a glycine/serine linker can be, or comprise, a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9) or be, or comprise a sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 16). This is simply a non-limiting example and the linker can have varying number of GGGGS (SEQ ID NO: 10) repeats.
  • the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the GGGGS (SEQ ID NO: 10) repeats.
  • the IL-2 mutein is linked to the Fc Region using a flexible, rigid or cleavable linker.
  • the linker can be as described herein or as illustrated in the following table:
  • the IL-2/Fc Fusion can be represented by the formula of Zi L. 2 M -L gs -Z Fc , wherein ZIL-2M is an IL-2 mutein as described herein, L gs is a linker sequence as described herein (e.g. glycine/serine linker) and Z Fc is a Fc region described herein or known to one of skill in the art.
  • the formula can be in the reverse orientation Z Fc -L gs -Zi L-2M ⁇
  • the IL-2/Fc fusion comprises a sequence of: M YRMQLL S Cl AL SL AL VTN S AP T S S S TKK TQL QLEHLLLDL QMILN GI SNH KNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLR PRDLI SDIN VIVLELKGSETTFMCE Y ADET ATI VEFLNRWITF S Q SII STLTG GGGS GGGGS GGGGS GGGGSDKTHT CPPCP APE A AGAP S VFLFPPKPKDTL MISRTPEVTC VVVD V SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVV S VLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQ VY TLPP SREEMTKN Q V SLTCL VKGF YP SDI AVEWE SN GQPENNYKTTPP VLD
  • the IL-2/Fc fusion comprises a sequence of:
  • the IL-2/Fc fusion comprises a sequence of:
  • the IL-2/Fc fusion comprises a sequence of: M YRMQLL S Cl AL SL AL VTN S AP T S S S TKK TQL QLEHLLLDL QMILN GI SNH
  • the Fc region of SEQ ID NO: 8 is replaced with SEQ ID NO: 15.
  • proteins described herein can also be fused to another protein, such as an antibody or other type of therapeutic molecule.
  • sequence of IL-2 mutein or IL-2/Fc fusion are as shown in the following table:
  • Each of the proteins may also be considered to have the C125S and the LALA and/or G237A mutations as provided for herein.
  • the C125 substitution can also be C125A as described throughout the present application.
  • the sequences shown in the table or throughout the present application comprise or don’t comprise one or more mutations that correspond to positions L53, L56, L80, and Ll 18. In some embodiments, the sequences shown in the table or throughout the present application comprise or don’t comprise one or more mutations that correspond to positions L59I, L63I, I24L, L94I, L96I or L132I or other substitutions at the same positions.
  • the mutation is leucine to isoleucine. In some embodiments, the mutein does not comprise another mutation other than as shown or described herein.
  • the peptide comprises a sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,
  • SEQ ID NO: 36 SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:
  • the Fc portion of the fusion is not included.
  • the peptide consists essentially of an IL-2 mutein provided for herein.
  • the protein is free of a Fc portion.
  • a polypeptide comprising SEQ ID NO: 43, wherein at least one of Xi, X 2 , X 3 , and X 4 is I and the remainder are L or I.
  • Xi, X 2 , and X 3 are L and X 4 is I.
  • Xi, X 2 , and X 4 are L and X 3 is I.
  • X 2 , X 3 , and X 4 are L and Xi is I.
  • Xi, X 3 , and X 4 are L and X 2 is I.
  • Xi and X 2 are L and X 3 and X 4 are I.
  • Xi and X 3 are L and X 2 and X are I.
  • Xi and X are L and X 2 and X 3 are I.
  • X 2 and X 3 are L and Xi and X 4 are I.
  • X 2 and X 4 are L and Xi and X 3 are I.
  • X 3 and X 4 are L and Xi and X 2 are I.
  • Xi, X 2 , and X 3 are L and X 4 is I.
  • X 2 , X 3 , and X 4 are L and Xi is I. In some embodiments, Xi, X 3 , and X 4 are L and X 2 is I. In some embodiments, Xi, X 2 , and X 4 are L and X 3 is I.
  • the IL-2 mutein can be in the format as illustrated in FIG. 1. But as described herein, the IL-2 mutein can, in some embodiments, be used without a Fc domain or the Fc-domain is linked to the N-terminus of the IL-2 mutein as opposed to the Fc domain being linked to the C-terminus of the IL-2 mutein.
  • the polypeptides described herein also encompass variants of the peptides described.
  • the IL-2 variants comprise a sequence of amino acids at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% substantially similar to the sequences provided for herein.
  • the variants include those that are described herein with the various substitutions described herein and above.
  • the variant has 1, 2, 3, 4, or 5 additional substitutions.
  • the substitution is G to A, L to I, G to S K to R, or other types of conservative substitutions.
  • the conservative substitution is selected based upon the following tables:
  • the percent identity of two amino acid or two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or for example, the comparison is done by comparing sequence information using a computer program.
  • An exemplary computer program is the Genetics Computer Group (GCG; Madison, Wis.) Wisconsin package version 10.0 program, GAP (Devereux et al. (1984), Nucleic Acids Res. 12: 387-95).
  • GAP Genetics Computer Group
  • the preferred default parameters for the GAP program includes: (1) The GCG implementation of a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted amino acid comparison matrix of Gribskov and Burgess, ((1986) Nucleic Acids Res.
  • the IL-2 muteins provided herein include proteins that have altered signaling through certain pathways activated by wild-type IL-2 via the IL-2R and result in preferential proliferation/survival/activation of T-regs.
  • the IL-2 muteins provided for herein can be produced using any suitable method known in the art, including those described in U.S. Pat. No. 6,955,807 for producing IL-2 variants, which is hereby incorporated by reference. Such methods include constructing a DNA sequence encoding the IL-2 variant and expressing those sequences in a suitably transformed host, such as a host cell. Utilizing these methods will produce recombinant proteins as provided herein. Proteins can also be produced synthetically or a combination of synthetic and recombinantly producing fragments in a cell and then combining the fragments to make the entire protein of interest.
  • a nucleic acid molecule (e.g . DNA or RNA) is prepared by isolating or synthesizing a nucleic acid molecule encoding the protein of interest.
  • the wild-type sequence of IL-2 can be isolated and the mutated using routine techniques, such as site-specific mutagenesis.
  • Another method of constructing a DNA sequence encoding the IL-2 variant would be chemical synthesis.
  • This for example includes direct synthesis of a peptide by chemical means of the protein sequence encoding for an IL-2 variant exhibiting the properties described herein.
  • This method may incorporate both natural and unnatural amino acids at various positions.
  • a nucleic acid molecule which encodes a desired protein may be synthesized by chemical means using an oligonucleotide synthesizer.
  • the oligonucleotides are designed based on the amino acid sequence of the desired protein, which can also be selected by using codons that are favored in the cell in which the recombinant variant will be produced.
  • nucleic acid molecule that encodes the proteins described herein.
  • the nucleic acid molecule can be DNA or RNA.
  • the nucleic acid molecule will encode a signal sequence.
  • a signal sequence can be chosen based upon the cell that will be expressed in.
  • the nucleic acid molecule does not comprise a signal sequence.
  • the signal sequence can be used.
  • the signal sequence is the IL-2 signal sequence.
  • “Recombinant” as it applies to polypeptides or proteins means that the production of the protein is dependent on at least one step in which nucleic acids, which may or may not encode the protein, are introduced into a cell in which they are not naturally found.
  • Suitable host cells include, but are not limited to, bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants.
  • these hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.
  • coli Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (Sf9), animal cells such as Chinese hamster ovary (CHO) and mouse cells such as NS/O, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BNT 10, and human cells, as well as plant cells in tissue culture.
  • CHO cells and COS 7 cells in cultures and particularly the CHO cell line CHO (DHFR-) or the HKB line may be used.
  • vectors and expression control sequences will function equally well to express the DNA sequences described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vectors copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
  • preferred vectors for use in this invention include those that allow the DNA encoding the IL-2 variants to be amplified in copy number. Such amplifiable vectors are well known in the art.
  • vectors encoding the proteins described herein are provided, as well as host cells transformed with such vectors.
  • Any nucleic acids encoding the proteins described herein may be contained in a vector, which can, for example, comprise a selectable marker and an origin of replication, for propagation in a host.
  • the vectors further include suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect genes, operably linked to the nucleic acid molecule encoding the protein. Examples of such regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences that control transcription and translation.
  • Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the target protein.
  • a promoter nucleotide sequence is operably linked to a nucleic acid molecule if the promoter nucleotide sequence directs the transcription of the nucleic acid molecule.
  • compositions e.g., pharmaceutically acceptable compositions, which include a therapeutic compound (IL-2 mutein) described herein, formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, local, topical, spinal or epidermal administration (e.g. by injection or infusion).
  • compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions e.g., dispersions or suspensions
  • liposomes e.g., liposomes and suppositories.
  • Typical compositions are in the form of injectable or infusible solutions.
  • the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the therapeutic molecule is administered by intravenous infusion or injection.
  • the therapeutic molecule is administered by intramuscular or subcutaneous injection.
  • the therapeutic molecule is administered locally, e.g., by injection, or topical application, to a target site.
  • parenteral administration and“administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • compositions typically should be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high therapeutic molecule concentration.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e., therapeutic molecule) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • a therapeutic compound can be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
  • the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • To administer a compound of the invention by other than parenteral administration it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
  • Therapeutic compositions can also be administered with medical devices known in the art.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a therapeutic compound is 0.1-30 mg/kg, more preferably 1-25 mg/kg. Dosages and therapeutic regimens of the therapeutic compound can be determined by a skilled artisan.
  • the therapeutic compound is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 40 mg/kg, e.g., 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, 1 to 10 mg/kg, 5 to 15 mg/kg, 10 to 20 mg/kg, 15 to 25 mg/kg, or about 3 mg/kg.
  • the dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks, or, in some embodiments, the dosing schedule can be, once every month, every 2 months, every 3 months, or every 6 months.
  • the therapeutic compound is administered at a dose from about 10 to 20 mg/kg every other week.
  • the therapeutic compound can be administered by intravenous infusion at a rate of more than 20 mg/min, e.g., 20-40 mg/min, and typically greater than or equal to 40 mg/min to reach a dose of about 35 to 440 mg/m2, typically about 70 to 310 mg/m2, and more typically, about 110 to 130 mg/m2.
  • the infusion rate of about 110 to 130 mg/m2 achieves a level of about 3 mg/kg.
  • the therapeutic compound can be administered by intravenous infusion at a rate of less than 10 mg/min, e.g., less than or equal to 5 mg/min to reach a dose of about 1 to 100 mg/m2, e.g., about 5 to 50 mg/m2, about 7 to 25 mg/m2, or, about 10 mg/m2.
  • the therapeutic compound is infused over a period of about 30 min. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated.
  • compositions of the invention may include a “therapeutically effective amount” or a“prophylactically effective amount” of a therapeutic molecule of the invention.
  • A“therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of a therapeutic molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic compound to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of a therapeutic molecule t is outweighed by the therapeutically beneficial effects.
  • A“therapeutically effective dosage” preferably inhibits a measurable parameter, e.g., immune attack at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
  • a measurable parameter e.g., immune attack
  • the ability of a compound to inhibit a measurable parameter, e.g., immune attack can be evaluated in an animal model system predictive of efficacy in transplant rejection or autoimmune disorders. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.
  • A“prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • kits comprising a therapeutic compound described herein.
  • the kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, a therapeutic molecule to a label or other therapeutic agent, or a radioprotective composition; devices or other materials for preparing the therapeutic molecule for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.
  • other reagents e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, a therapeutic molecule to a label or other therapeutic agent, or a radioprotective composition
  • devices or other materials for preparing the therapeutic molecule for administration e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, a therapeutic molecule to a label or other therapeutic agent, or a radioprotective composition
  • devices or other materials for preparing the therapeutic molecule for administration
  • the proteins described herein can also be administered in conjunction with other agents useful for treating the condition with which the patient is suffering from.
  • agents include both proteinaceous and non-proteinaceous drugs.
  • dosages may be adjusted accordingly, as is recognized in the pertinent art.
  • “Co- administration” and combination therapy are not limited to simultaneous administration, but also include treatment regimens in which a T-reg- selective IL-2 protein is administered at least once during a course of treatment that involves administering at least one other therapeutic agent to the patient.
  • a T-reg- selective IL-2 protein is administered in combination with an inhibitor of the PB-K/AKT/mTOR pathway, e.g., rapamycin (rapamune, sirolimus). Inhibitors of this pathway in combination with IL-2 favor T-reg enrichment.
  • the IL-2 protein is administered without another therapeutic that is not directly fused or attached to the IL-2 protein.
  • Treatment of any disease mentioned herein encompasses an alleviation of at least one symptom of the disease, a reduction in the severity of the disease, or the delay or prevention of disease progression to more serious symptoms that may, in some cases, accompany the disease or to at least one other disease. Treatment need not mean that the disease is totally cured.
  • a useful therapeutic agent needs only to reduce the severity of a disease, reduce the severity of symptom(s) associated with the disease or its treatment, or delay the onset of more serious symptoms or a more serious disease that can occur with some frequency following the treated condition.
  • a therapeutic agent may reduce the number of distinct sites of inflammation in the gut, the total extent of the gut affected, reduce pain and/or swelling, reduce symptoms such as diarrhea, constipation, or vomiting, and/or prevent perforation of the gut.
  • a patient's condition can be assessed by standard techniques such as an x-ray performed following a barium enema or enteroclysis, endoscopy, colonoscopy, and/or a biopsy. Suitable procedures vary according to the patient's condition and symptoms.
  • the proteins are used to treat inflammatory disorders.
  • the inflammatory disorder is inflammation, autoimmune disease, atopic diseases, paraneoplastic autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis (e.g.
  • juvenile arthritis juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), dermatomyositis, psoriatic arthritis, enter
  • myasthenia gravis myasthenia gravis
  • IBD inflammatory bowel disease
  • MS multiple sclerosis
  • asthma COPD
  • rhinosinusitis rhinosinusitis with polyps
  • eosinophilic esophogitis eosinophilic bronchitis
  • Guillain-Barre disease Type I diabetes mellitus
  • Type I diabetes mellitus thyroiditis(e.g., Graves' disease)
  • Addison's disease Raynaud's phenomenon
  • autoimmune hepatitis graft versus host disease
  • steroid refractory chronic graft versus host disease transplantation rejection(e.g.
  • the proteins are used to treat steroid refractory chronic graft versus host disease. In some embodiments, the proteins are used to treat active systemic lupus erythematosus. In some embodiments, the proteins are used to treat active rheumatoid arthritis.
  • the methods comprise administering a pharmaceutical composition comprising the proteins described herein to the subject.
  • the subject is a subject in need thereof.
  • Any of the above-described therapeutic proteins can be administered in the form of a compositions (e.g . pharmaceutical compositions) that are described herein.
  • a composition may comprise an IL-2 protein as described herein plus a buffer, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (such as those having less than 10 amino acids), a protein, amino acids, carbohydrates such as glucose, sucrose, or dextrins, chelating agent such as EDTA, glutathione, and/or other stabilizers, excipients, and/or preservatives.
  • composition may be formulated as a liquid or a lyophilizate.
  • Further examples of components that may be employed in pharmaceutical formulations are presented in Remington's Pharmaceutical Sciences, l6.sup.th Ed., Mack Publishing Company, Easton, Pa., (1980) and others as described herein.
  • compositions comprising therapeutic molecules described herein can be administered by any appropriate method including, but not limited to, parenteral, topical, oral, nasal, vaginal, rectal, or pulmonary (by inhalation) administration. If injected, the composition(s) can be administered intra-articularly, intravenously, intraarterially, intramuscularly, intraperitoneally, or subcutaneously by bolus injection or continuous infusion. Localized administration, that is, at the site of disease, is contemplated, as are transdermal delivery and sustained release from implants, skin patches, or suppositories.
  • Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation in aerosol form, and the like.
  • Administration via a suppository inserted into a body cavity can be accomplished, for example, by inserting a solid form of the composition in a chosen body cavity and allowing it to dissolve.
  • Other alternatives include eyedrops, oral preparations such as pills, lozenges, syrups, and chewing gum, and topical preparations such as lotions, gels, sprays, and ointments.
  • therapeutic molecules that are polypeptides can be administered topically or by injection or inhalation.
  • the therapeutic molecules described above can be administered as described herein and above.
  • the composition can be administered at any dosage, frequency, and duration that can be effective to treat the condition being treated.
  • the dosage depends on the molecular nature of the therapeutic molecule and the nature of the disorder being treated. Treatment may be continued as long as necessary to achieve the desired results.
  • Therapeutic molecules of the invention can be administered as a single dosage or as a series of dosages given periodically, including multiple times per day, daily, every other day, twice a week, three times per week, weekly, every other week, and monthly dosages, among other possible dosage regimens.
  • the periodicity of treatment may or may not be constant throughout the duration of the treatment. For example, treatment may initially occur at weekly intervals and later occur every other week. Treatments having durations of days, weeks, months, or years are encompassed by the invention. Treatment may be discontinued and then restarted. Maintenance doses may or may not be administered after an initial treatment.
  • Dosage may be measured as milligrams per kilogram of body weight (mg/kg) or as milligrams per square meter of skin surface (mg/m 2 ) or as a fixed dose, irrespective of height or weight. All of these are standard dosage units in the art. A person's skin surface area is calculated from her height and weight using a standard formula.
  • kits for promoting stimulating STAT5 phosphorylation in T regulatory cells comprise administering to a subject in need thereof a therapeutically effective amount of a peptide described herein or a pharmaceutical composition comprising the same.
  • the phrase“in need thereof’ means that the subject (animal or mammal) has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the animal or mammal can be in need thereof. In some embodiments, the animal or mammal is in an environment or will be traveling to an environment in which a particular disease, disorder, or condition is prevalent.
  • the term“individual” or“subject,” or“patient” used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans.
  • the terms“comprising” (and any form of comprising, such as“comprise”, “comprises”, and“comprised”),“having” (and any form of having, such as“have” and“has”), “including” (and any form of including, such as“includes” and“include”), or“containing” (and any form of containing, such as“contains” and“contain”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any step or composition that uses the transitional phrase of“comprise” or“comprising” can also be said to describe the same with the transitional phase of“consisting of’ or“consists.”
  • the term“contacting” means bringing together of two elements in an in vitro system or an in vivo system.
  • “contacting” a peptide or composition described herein with a T-reg cell or with an individual or patient or cell includes the administration of the compound to an individual or patient, such as a human, as well as, for example, introducing a compound into a sample containing a cellular or purified preparation containing the T-reg cell.
  • the term“fused” or“linked” when used in reference to a protein having different domains or heterologous sequences means that the protein domains are part of the same peptide chain that are connected to one another with either peptide bonds or other covalent bonding.
  • the domains or section can be linked or fused directly to one another or another domain or peptide sequence can be between the two domains or sequences and such sequences would still be considered to be fused or linked to one another.
  • the various domains or proteins provided for herein are linked or fused diretctly to one another or a linker sequences, such as the glycine/serine sequences described herein link the two domains together.
  • embodiments provided herein also include, but are not limited to:
  • a peptide comprising an amino acid sequence of SEQ ID NO: 1, wherein the peptide comprises a mutation at position 73, 76, 100, or 138.
  • a peptide comprising an amino acid sequence of SEQ. ID. NO: 27.
  • a pharmaceutical composition comprising a peptide of any one of the preceding embodiments.
  • a method of activating T regulatory cells comprising contacting a T regulatory cell with a peptide of any one of embodiments 1-57 or the pharmaceutical composition of embodiment 58.
  • a method of treating an inflammatory disorder in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of embodiments 1-57 or the pharmaceutical composition of embodiment 58.
  • inflammatory disorder is inflammation, autoimmune disease, atopic diseases, paraneoplastic autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis
  • kidney, lung, heart, skin, and the like kidney damage, hepatitis C-induced vasculitis, spontaneous loss of pregnancy, alopecia, vitiligo, focal segmental glomerulosclerosis (FSGS), Minimal Change Disease, Membranous Nephropathy, ANCA Associated Glomerulonephropathy, Membranoproliferative Glomerulonephritis, IgA Nephropathy, lupus nephritis, and the like.
  • a method of promoting or stimulating STAT5 phosphorylation in T regulatory cells comprising administering to a subject in need thereof a therapeutically effective amount of a peptide of any one of embodiments 1-57 or the pharmaceutical composition of embodiment 58.
  • inflammatory disorder is inflammation, autoimmune disease, atopic diseases, paraneoplastic autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis
  • kidney, lung, heart, skin, and the like kidney damage, hepatitis C-induced vasculitis, spontaneous loss of pregnancy, alopecia, vitiligo, focal segmental glomerulosclerosis (FSGS), Minimal Change Disease, Membranous Nephropathy, ANCA Associated Glomerulonephropathy, Membranoproliferative Glomerulonephritis, IgA Nephropathy, lupus nephritis, and the like.
  • FGS focal segmental glomerulosclerosis
  • Minimal Change Disease Membranous Nephropathy, ANCA Associated Glomerulonephropathy, Membranoproliferative Glomerulonephritis, IgA Nephropathy, lupus nephritis, and the like.
  • 65 A nucleic acid molecule encoding a peptide of any one of embodiments 1-57.
  • a vector comprising the nucleic acid molecule of embodiment 65.
  • a plasmid comprising the nucleic acid molecule of embodiment 65.
  • a cell comprising the nucleic acid molecule of embodiment 65.
  • a cell comprising the plasmid of embodiment 67.
  • a cell comprising the vector of embodiment 66.
  • a cell comprising or expressing a peptide of any one of embodiments 1-57 or a peptide as described herein.
  • a therapeutic composition comprising a protein of SEQ ID NOs: 11, 12, 13, or 14 is administered to a subject suffering from IBD.
  • the subject s immune system is down-regulated and the symptoms of the IBD are alleviated.
  • a therapeutic composition comprising a protein of SEQ ID NOs: 3, 4, 5, or 6, with or without the leader sequence, is administered to a subject suffering from IBD.
  • the subject s immune system is down-regulated and the symptoms of the IBD are alleviated.
  • a pTT5 vector containing the single gene encoding the human IL-2M polypeptide fused N-terminally (SEQ ID NO: 40) or C-terminally (SEQ ID NO: 41) to human IgGl Fc domain was transfected into HEK293 Expi cells. After 5-7 days, cell culture supernatants expressing IL-2Ms were harvested, and clarified by centrifugation and filtered through a 0.22um filtration device. IL-2Ms were captured on proA resin. The resin was washed with PBS pH 7.4 and the captured protein was eluted using 0.25% acetic acid pH 3.5, with neutralization using a tenth volume of 1M Tris pH 8.0.
  • the protein was buffer exchanged into 30mM HEPES l50mM NaCl pH 7, and analyzed by size exclusion chromatography on a Superdex 200 3.2/300 column. Analysis of 5ug of purified material by reducing and non-reducing SDS-PAGE on a Bis-Tris 4- 12% gel was conducted. The IL-2Ms were expressed at over lOmg/L, and were over 95% monodispersed after purification as shown by size exclusion chromatography and reducing/non reducing SDS-PAGE.
  • Example 4 IL-2M Molecules can bind CD25
  • An immunosorbent plate was coated with CD25 at a concentration of 0.5 mg/mL in PBS pH 7.4, 75 ul/well, and incubated overnight at 4°C. Wells were washed with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer) three times, and then blocked with 200ul/well 1% BSA in PBS pH 7.4 (block buffer) for two hours at room temperature. After three washes with wash buffer IL-2M molecules were diluted to eleven -two fold serial dilution in PBS containing 1% BSA and 0.05% Tween-20 (assay buffer) with 2nM being the highest concentration. The diluted material was added to the CD25 coated plate at 75ul/well for 1 hour at room temperature.
  • Example 5 In Vitro P-STAT5 Assay To Determine Potency and Selectivity of IL- 2M Molecules.
  • Peripheral blood mononuclear cells PBMCs
  • FICOLL- PAQEIE Premium and Sepmate tubes from freshly isolated heparinized human whole blood.
  • PBMCs were cultured in 10% fetal bovine serum RPMI medium in the presence of wild-type IL- 2 or IL-2M of Example 12 for 20 minutes and then fixed for 10 minutes with BD Cytofix.
  • Fixed cells were sequentially permeabilized with BD Perm III and then BioLegend FOXP3 permeabilization buffer.
  • IL- 2M of SEQ ID NO: 23 potently and selectively induces STAT5 phosphorylation in Tregs but not Teffs.
  • IL-2 Mutein Mutant sequences were analyzed using the NetMHCIIPan 3.2 software, which can be found at www“dot” cbs“dot” dtu“dot” dk/services/NetMHCIIpan/. Artificial neural networks were used to determine peptide affinity to MHC class II alleles. In that analysis, 9-residue peptides with potentially direct interaction with the MHC class II molecules were recognized as binding cores. Residues adjacent to binding cores, with potential to influence the binding indirectly, were also examined as masking residues. Peptides comprising both the binding cores and masking residues were marked as strong binders when their predicted K D to the MHC class II molecule was lower than 50 nM. Strong binders have a greater chance of introducing T cell immunogenicity.
  • MHCII alleles that are highly represented in North America and Europe were included in the in silico analysis.
  • the panel of IL-2M (IL- 2 muteins) molecules tested included the IL-2 Muteins with L53I, L56I, L80I, or Ll 181 mutations. Only MHCII alleles DRBl l 101, DRB1 1501, DRB1 0701, and DRB1 0101 yielded hits with any of the molecules assessed.
  • the peptide hits for DRB 1501 were identical between all constructs tested including wild-type IL-2 with the C125S mutation.
  • L80I removes 1 T cell epitope for DRB 1-0101 [ALNLAPSKNFHLRPR] and modestly reduces the affinity of two other T cell epitopes [EE ALNL AP SKNFHLR and EALNLAPSKNFHLRP].
  • L80I removes 1 T cell epitope [EEALNLAP SKNFHLR] Therefore, the data demonstrates that a IL-2 mutein comprising the L80I mutation should be less immunogenic, which is a surprising and unexpected result from the in silico analysis.
  • a pTT5 vector containing the single gene encoding the single IL-2M (IL-2 mutein) SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 (and IL-2M control; SEQ ID NO: 34) polypeptide with human IL-2M or IL-2M fused N-terminally of human IgGl Fc domain was transfected into HEK293 Expi cells.
  • cell culture supernatants expressing SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 (and IL-2M control; SEQ ID NO: 34) were harvested, and clarified by centrifugation and filtration through a 0.22um filtration device.
  • SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 (and IL-2M control; SEQ ID NO: 34) were captured on proA resin. The resin was washed with PBS pH 7.4 and the captured protein was eluted using 0.25% acetic acid pH 3.5, with neutralization using a tenth volume of 1M Tris pH 8.0.
  • the protein was buffer exchanged into 30mM HEPES l50mM NaCl pH 7, and analyzed by size exclusion chromatography on a Superdex 200 3.2/300 column. Analysis of 5ug of purified material by reducing and non reducing SDS-PAGE on a Bis-Tris 4-12% gel was conducted.
  • IL-2Ms SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 (and IL-2M control; SEQ ID NO: 34) expressed at over 45mg/L, and were over 95% monodispersed after purification as shown by size exclusion chromatography and reducing/non-reducing SDS- PAGE.
  • Example 8 IL-2Ms can bind CD25
  • An immunosorbent plate was coated with CD25 at a concentration of 0.5 mg/mL in PBS pH 7.4, 75 ul/well, and incubated overnight at 4°C. Wells were washed with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer) three times, and then blocked with 200ul/well 1% BSA in PBS pH 7.4 (block buffer) for two hours at room temperature. After three washes with wash buffer IL-2Ms SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 were diluted to eleven -two fold serial dilution in PBS containing 1% BSA and 0.05% Tween-20 (assay buffer) with 2nM being the highest concentration.
  • the diluted material was added to the CD25 coated plate at 75ul/well for 1 hour at room temperature.
  • a goat biotinylated anti-IL-2 polyclonal antibody diluted to 0.05 mg/mL in assay buffer, was added to the plate at 75ul/well for 1 hr at room temperature.
  • wash buffer high sensitivity streptavidin HRP diluted in assay buffer at 1 :5000 was added to the plate at 75ul / well for 15 minutes at room temperature.
  • wash buffer and 1 wash with wash buffer (with no tween-20)
  • the assay was developed with TMB, and stopped with 1N HCL. OD 450nm was measured.
  • the experiment included appropriate controls for non-specific binding of the molecules to the plate/block in the absence of CD25.
  • the results indicate that at concentrations of 2nM-l.9pM, the muteins of Example 7 were able to bind CD25 with sub nanomolar EC50s. Additionally, there was no detection of any compound at any concentration tested, when CD25 was not present on the plate surface, indicating none of the test compounds were interacting non-specifically with the plate surface. Thus, the muteins of Example 7 can bind to CD25.
  • Example 9 IL-2 Muteins of Example 7 are Potent and Selective
  • PBMCs Peripheral blood mononuclear cells
  • FICOLL- PAQEIE Premium and Sepmate tubes from freshly isolated heparinized human whole blood.
  • PBMCs were cultured in 10% fetal bovine serum RPMI medium in the presence of wild-type IL- 2 or the muteins of Example 7 for 20 minutes and then fixed for 10 minutes with BD Cytofix.
  • Fixed cells were sequentially permeabilized with BD Perm III and then BioLegend FOXP3 permeabilization buffer.
  • IL-2 muteins of Example 7 were found to be potent and have selectivity against Treg versus Teff.
  • the mutein comprising the L118I mutation was found to have increased activity and selectivity as compared to the other muteins.
  • Example 10 IL-2 muteins Expand Tregs in Humanized Mice
  • mice humanized with human CD34+ hematopoietic stem cells were purchased from Jackson Labs. On days 0 and 7, the mice were dosed subcutaneously with lug IL-2Mutein SEQ ID NO: 34 or other IL-2 muteins SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40. On Day 7, mice were euthanized and whole blood and spleens were collected. Whole blood was aliquoted into a 96 well deep well plate and fixed for 10 minutes using BD Fix Lyse. Splenocytes were isolated using 70um filters (BD) and red blood cells were lysed using RBC lysis buffer from BioLegend.
  • BD 70um filters
  • splenocytes were labeled with near infrared live dead stain (Invitrogen) for 20 minutes and then fixed for 20 minutes using BioLegend fixation buffer. Both whole blood cells and splenocytes were then permeabilized using BioLegend FOXP3 permeabilization buffer, blocked with human serum and stained for 30 minutes with antibodies against human CD8a FITC (BL), human CD25 PE (BD), human FOXP3 AF647 (BD) CD4 PerCP Cy5.5 (BD), human Siglec-8 PE Cy7 (BL), human CD3 BV421 (BL), human CD45 BV605 (BL), human CD56 BV785 (BL) and mouse CD45 (BV711) and acquired on an Attune NXT with plate loader.
  • BL human CD8a FITC
  • BD human CD25 PE
  • BD human FOXP3 AF647
  • CD4 PerCP Cy5.5 BD
  • Siglec-8 PE Cy7 BL
  • human CD3 BV421 BL
  • IL-2Ms SEQ ID NO: 37 and SEQ ID NO: 38 and
  • SEQ ID NO: 39 and SEQ ID NO: 40 selectively induced human Tregs in mouse spleens and whole blood in humanized mice. There were no significant changes in the frequencies of human CD56pos NK cells, CD3pos T cells, CD8pos cytotoxic T lymphocytes, CD4pos helper T cells or CD25lo/FOXP3neg T effectors. These results demonstrate that the IL-2 muteins increase the frequency of regulatory T cells.
  • Example 11 Durability of Signaling induced by IL-2 muteins
  • PBMCs Peripheral blood mononuclear cells
  • FICOLL- PAQUE Premium and Sepmate tubes from freshly isolated heparinized human whole blood.
  • PBMCs were cultured in 10% fetal bovine serum RPMI medium in the presence of IL-2M for 60 minutes. Cells were then wash 3 times and incubated for an additional 3 hours. Cells were then fixed for 10 minutes with BD Cytofix. Fixed cells were sequentially permeabilized with BD Perm III and then BioLegend FOXP3 permeabilization buffer.
  • IL-2 mutein can function to selectively and potently activate Tregs over Teffs, which demonstrates that the molecules can be used to treat or ameliorate the conditions described herein.
  • the IL-2 muteins, as provided for herein, can also be generated and used with or without being fused to a Fc domain or a linker as provided for herein.
  • Example 12 Muteins exhibit overall POI and lower aggregation.
  • IL-2 muteins with the mutations of V69A, Q74P, N88D, and C125S and one of the following mutations L53I, L56I, L80I, or L118I linked to a Fc region comprising L234A, L235A, and G237A mutations as provided for herein were expressed in a pTT5 vector by transfecting the vector into HEK293 Expi cells.
  • the IL-2 mutein was linked to the N-terminus of the Fc region with 4 GGGGS repeats. After 5-7 days, cell culture supernatants expressing the different IL-2 muteins were harvested, and clarified by centrifugation and filtration through a 0.22um filtration device.
  • the IL-2 muteins were captured on proA resin.
  • the resin was washed with PBS pH 7.4 and the captured protein was eluted using 0.25% acetic acid pH 3.5, with neutralization using a tenth volume of 1M Tris pH 8.0.
  • the protein was buffer exchanged into 30mM HEPES l50mM NaCl pH 7, and analyzed by size exclusion chromatography on an AdvanceBio SEC column for percent peak of interest (POI). The results demonstrated that the different muteins were expressed at over 60mg/L.
  • muteins with the L80I or L118I mutation were greater than 90% monodispersed while muteins with the L53I or L56I mutations were not as shown by size exclusion chromatography.
  • the muteins with the L80I or Ll 181 substitution had less aggregation.
  • the differences in aggregation amongst the four molecules were surprising due to the type of mutation that was being made. Therefore, the muteins with the L80I or the L118I mutation have a surprising advantage over other muteins in that it does not aggregate as much as other muteins.
  • Example 13 IL-2 Muteins Have Unexpected Increase in Potency
  • PBMCs were isolated from heparinized human whole blood and stimulated with the different muteins at a concentration for 30 min at 37 C. The stimulation was stopped by fixation. After permeabilization, PBMCs were stained for intracellular FoxP3 and phospho- STAT5 levels and surface CD4 and CD25 expression and analyzed by flow cytometry. Regulatory T cells (Tregs) and effector T cells (Teffs) were gated as CD4+CD25hiFoxP3+ or CD4+CD25loFoxP3-, respectively. The percent of cells that stained positive for phospho-STAT5 is shown. This assay measures the ability of the muteins to specifically stimulate Tregs without stimulating Teffs. A best-fit dose-response curve for each test article was used to calculate an EC 50 value.
  • the muteins with the mutations of L118I, L80I, L56I, or L53I had increased potency (stimulating Tregs) as compared to an IL-2 mutein without any of these mutations.
  • the IL-2 mutein without a mutation of L118I, L80I, L56I, or L53I, but having the V69A, Q74P, and N88D mutations was approximately 51 pM.
  • Each of the EC 50 s for the muteins comprising one of L 1181, L80I, L56I, or L53I had EC 50S of approximately 30, 40, 41, and 45, respectively.
  • the differences in EC50 for stimulating Tregs was surprising and would not have been predicted for the muteins having one of the mutations described in this example.
  • the data can also be evaluated by comparing the ratio of the parent IL-2 muteins (comprising V69A, Q74P, N88D, and C125S) to the muteins that also comprise one of L118I, L80I, L56I, or L53I mutations. Using this ratio normalizes for different cell populations that are used for different experiments. Using this ratio the L118I had an average increase of approximately 25% more potency (standard error of mean 0.16) as compared to the parent control, whereas the other mutations had a decrease in activity as compared to the parent control using this ratio.
  • Peripheral blood leukocytes were isolated by lysis of red blood cells and stained with antibodies reactive to the human markers CD45, CD3, CD8, CD4, FoxP3, CD25 and CD56.
  • the percent of human regulatory T cells (Tregs, CD45+CD3+CD4+CD25+FoxP3+), activated effector T cells (act Teff, CD45+CD3+CD4+CD25+FoxP3-) and NK cells (CD45+CD56+) was determined by flow cytometry. The frequency of total CD45+, total CD4+ and total CD8+ cells did not change. Similar results were observed in the spleens of mice.
  • the in vivo potency as measured in this assay of the IL-2 mutein with the L80I mutation was slightly increased as compared to a mutein without the L80I mutation and the in vivo potency as measured in this assay of the muteins with either the L56I or the L53I mutation was about the same as a mutein without the mutations.
  • the muteins were N-terminal linked to a Fc region as described herein with a 20 amino acid (GGGGS) 4 linker. That is the linker connected the C-terminus of the IL-2 mutein to the N- terminus of the Fc region.
  • Example 14 N-terminal Fc Orientation with a 20 Amino Acid Linker is Most Effective at Stimulating Tregs.
  • An IL-2 mutein with V69A, Q74P, and N88D was fused to a Fc region comprising the mutations of L234A, L235A and G237A mutation using different lengths of GGGGS repeats.
  • IL2-Mutein molecules fused via the c-terminus of the mutein to the n- terminus of human IgGl Fc with linkers comprising 3 and 4 GGGGS repeats were tested to determine if the length of the linker affected the potency of the IL-2 mutein.
  • mice reconstituted with human CD34+ hematopoietic stem cells were injected subcutaneously with 1 microgram of the different 11-2 muteins with different linker lengths or vehicle on day 0. On Day 7, mice were sacrificed and blood was collected by cardiac puncture into tubes containing heparin.
  • Peripheral blood leukocytes PBLs were isolated by lysis of red blood cells and stained with antibodies reactive to the human markers CD45, CD3, CD8, CD4, FoxP3, CD25 and CD56.
  • the percent of human regulatory T cells (Tregs, CD45+CD3+CD4+CD25+FoxP3+), activated effector T cells (act Teff, CD45+CD3+CD4+CD25+FoxP3-) and NK cells (CD45+CD56+) was determined by flow cytometry. The frequency of total CD45+, total CD4+ and total CD8+ cells did not change. Similar results were observed in the spleens of mice.
  • a pharmaceutical composition comprising a IL-2 mutein protein comprising a sequence of SEQ ID NO: 37, 38, 39, or 40 are administered to patients with active rheumatoid arthritis.
  • the IL-2 muteins are found to be effective in treating active rheumatoid arthritis in the patients.
  • Example 16 Treating Patients with Subjects With Active Systemic Lupus Erythematosus.
  • a pharmaceutical composition comprising a IL-2 mutein protein comprising a sequence of SEQ ID NO: 37, 38, 39, or 40 are administered to patients with active systemic lupus erythematosus.
  • the IL-2 muteins are found to be effective in treating active systemic lupus erythematosus.
  • Example 17 Treating Patients with Subjects With Steroid Refractory Chronic Graft Versus Host Disease.
  • a pharmaceutical composition comprising a IL-2 mutein protein comprising a sequence of SEQ ID NO: 37, 38, 39, or 40 are administered to patients with Steroid refractory chronic graft versus host disease.
  • the IL-2 muteins are found to be effective in treating steroid refractory chronic graft versus host disease.
  • Example 18 IL-2 muteins induce pSTAT5 in Human Tregs.
  • Purified PBMC from heparinized whole blood from six healthy donors were treated with serial dilutions of a IL- 2 mutein protein comprising a sequence of SEQ ID NO: 39 or 40 at 37 C for 30 minutes. Cells were fixed, washed, permeabilized and washed. Cells were stained with antibodies that detect both surface markers and intracellular/nuclear markers (pSTAT5 and FOXP3). Data was collected on Attune NxT cytometer. Tregs were gated as mononuclear, singlet, CD3pos, CD4pos, CD25hi, FoxP3pos.
  • the IL-2 comprising a sequence of SEQ ID NO: 40 is more potent than the IL-2 sequence comprising SEQ ID NO: 39, but both are active across multiple populations of cells.
  • Example 19 IL-2 muteins induce pSTAT5 in monkey PBMCs in vitro.
  • Purified PBMC from heparinized whole blood from three healthy monkeys were treated with serial dilutions a IL-2 mutein protein comprising a sequence of SEQ ID NO: 39 or 40 at 37 C for 60 minutes.
  • Fluorochrome conjugated Anti-CD25 and anti-CD4 were added for the final 30 min of of the IL-2 mutein treatment.
  • Cells were fixed, washed, permeabilized and washed. Cells were stained with remaining antibodies that detect both surface markers and intracellular/nuclear markers (pSTAT5 and FOXP3). Data was collected on Attune NxT cytometer.
  • Tregs were gated as mononuclear, singlet, CD4pos, CD25hi, FoxP3pos.
  • the % of gated Tregs that express phosphorylated STAT5 was measured.
  • the IL-2 muteins were found to induce pSTAT5 in monkeys.
  • Example 20 IL-2 muteins induce expand Treg cells and induce Treg proliferation in vivo.
  • Venous whole blood was collected in K2EDTA tubes from monkeys (cynomolgus) before dosing with IL-2 muteins of SEQ ID NO: 39 or 40 (2 timepoints/cyno, 5 cynos) and after dosing with either SEQ ID NO: 39 (5 timepoints/cyno, 2 cynos) or SEQ ID NO: 40 (5 timepoints/cyno, 3 cynos).
  • Samples were divided in two and stained for two FACS panels separately. One was a“Treg panel” and one was a general immunophenotyping panel.
  • RBCs were lysed and cells were stained for surface and intracellular markers after fixation and permeabilization.
  • FACs FACs analysis the number of total cells/ul was determined by AD VIA. The number of cells of a given subpopulation/ul was then calculated with the total number/ul and the % of total.
  • the average number of a given cell type/ul of the two pre-dose bleeds was averaged and used to normalize the post-dose bleeds, such that“fold-change from pre-dose” was determined.
  • plasma from K2EDTA whole blood was frozen until the end of the study.
  • IL-2 muteins function in an in vivo animal model that is similar to humans. It was also found that neither molecule significantly expanded Tconv cells, CD4 cells (Tnaive) or CD8 cells (Cytotoxic T), NK cells in the monkeys (non-human primate). It was also found that neither molecule significantly induced Serum chemokines. This data demonstrates that the IL-2 muteins can expand Treg cells and induce Treg cell proliferation without unwanted expansion or activation of other pathways. Thus, the IL-2 muteins are surprisingly potent, effective, and selective for Treg expansion and proliferation.
EP18887098.4A 2017-12-06 2018-11-28 Il-2 muteine und deren verwendung Pending EP3720470A4 (de)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US201762595357P 2017-12-06 2017-12-06
US201862675972P 2018-05-24 2018-05-24
US201862721644P 2018-08-23 2018-08-23
US16/109,897 US10174092B1 (en) 2017-12-06 2018-08-23 IL-2 muteins
US16/109,875 US10174091B1 (en) 2017-12-06 2018-08-23 IL-2 muteins
PCT/US2018/062808 WO2019112854A1 (en) 2017-12-06 2018-11-28 Il-2 muteins and uses thereof

Publications (2)

Publication Number Publication Date
EP3720470A1 true EP3720470A1 (de) 2020-10-14
EP3720470A4 EP3720470A4 (de) 2021-09-15

Family

ID=66751187

Family Applications (1)

Application Number Title Priority Date Filing Date
EP18887098.4A Pending EP3720470A4 (de) 2017-12-06 2018-11-28 Il-2 muteine und deren verwendung

Country Status (11)

Country Link
EP (1) EP3720470A4 (de)
JP (1) JP7250790B2 (de)
KR (2) KR20200097275A (de)
CN (1) CN111432831A (de)
AU (1) AU2018378078A1 (de)
BR (1) BR112020011343A2 (de)
CA (1) CA3083941A1 (de)
IL (1) IL274844A (de)
MX (1) MX2020005208A (de)
SG (1) SG11202003965VA (de)
WO (1) WO2019112854A1 (de)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3056630A1 (en) 2017-03-15 2018-09-20 Pandion Therapeutics, Inc. Targeted immunotolerance
CA3064435A1 (en) 2017-05-24 2018-11-29 Pandion Therapeutics, Inc. Targeted immunotolerance
US10946068B2 (en) 2017-12-06 2021-03-16 Pandion Operations, Inc. IL-2 muteins and uses thereof
US10174091B1 (en) 2017-12-06 2019-01-08 Pandion Therapeutics, Inc. IL-2 muteins
BR112021023345A2 (pt) 2019-05-20 2022-02-01 Pandion Operations Inc Imunotolerância com alvo em madcam
WO2021034890A1 (en) * 2019-08-19 2021-02-25 Pandion Therapeutics, Inc. Targeted immunotolerance with a pd-1 agonist
CN110642934B (zh) * 2019-09-10 2022-08-23 中国医学科学院北京协和医院 靶向调节t细胞的长效白介素-2及其在治疗自身免疫病中的应用
WO2021076805A1 (en) 2019-10-15 2021-04-22 Moderna TX, Inc. Mrnas encoding immune modulating polypeptides and uses thereof
CN112358540A (zh) * 2020-11-03 2021-02-12 安徽环球基因科技有限公司 一种重组il2在毕赤酵母中的生产方法
TW202345889A (zh) * 2022-03-03 2023-12-01 大陸商海南先聲藥業有限公司 ＩＬ２突變體－抗體Ｆｃ嵌段融合蛋白的藥物組合物及其應用

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1007907B (zh) * 1985-04-30 1990-05-09 弗·哈夫曼-拉罗彻有限公司 重组人类白细胞介素-2的提纯
WO2005007121A2 (en) * 2003-07-18 2005-01-27 Massachusetts Institute Of Technology Mutant interleukin-2(il-2) polypeptides
US9428567B2 (en) * 2010-12-22 2016-08-30 The Board Of Trustees Of The Leland Stanford Junior University Antagonists of interleukin-2 receptor
US20140170153A1 (en) * 2011-05-31 2014-06-19 Novo Nordisk A/S Il-21 epitope and il-21 ligands
US9546203B2 (en) * 2013-03-14 2017-01-17 Amgen Inc. Aglycosylated Fc-containing polypeptides with cysteine substitutions
SG10202010158TA (en) * 2014-07-21 2020-11-27 Delinia Inc Molecules that selectively activate regulatory t cells for the treatment of autoimmune diseases
KR20180133198A (ko) * 2016-05-04 2018-12-13 암젠 인크 T-조절 세포의 증식을 위한 인터류킨-2 뮤테인
WO2018184965A1 (en) * 2017-04-03 2018-10-11 F. Hoffmann-La Roche Ag Immunoconjugates of il-2 with an anti-pd-1 and tim-3 bispecific antibody
CA3064435A1 (en) * 2017-05-24 2018-11-29 Pandion Therapeutics, Inc. Targeted immunotolerance
WO2019112852A1 (en) * 2017-12-06 2019-06-13 Pandion Therapeutics, Inc. Targeted immunotolerance

Also Published As

Publication number Publication date
WO2019112854A1 (en) 2019-06-13
EP3720470A4 (de) 2021-09-15
CN111432831A (zh) 2020-07-17
AU2018378078A1 (en) 2020-06-18
JP2021507690A (ja) 2021-02-25
BR112020011343A2 (pt) 2020-11-17
MX2020005208A (es) 2020-08-20
KR20200097275A (ko) 2020-08-18
KR20240039201A (ko) 2024-03-26
IL274844A (en) 2020-07-30
SG11202003965VA (en) 2020-06-29
CA3083941A1 (en) 2019-06-13
JP7250790B2 (ja) 2023-04-03

Similar Documents

Publication Publication Date Title
US11945852B2 (en) IL-2 muteins and uses thereof
US11779632B2 (en) IL-2 muteins and uses thereof
EP3720470A1 (de) Il-2 muteine und deren verwendung
JP7227951B2 (ja) 制御性ｔ細胞の増殖のためのインターロイキン－２ムテイン
JP2016529215A (ja) Ｃｔｌａ−４およびｃｄ４０の両方に特異的に結合可能である二重特異性分子
KR20110112299A (ko) 자가면역 및 염증성 장애의 치료에 사용하기 위한 가용성 폴리펩티드
AU2013223801B2 (en) Tumour necrosis factor receptor fusion proteins and methods of using the same
WO2020119728A1 (zh) 抗人白细胞介素5(il-5)单克隆抗体及其应用
MXPA04003057A (es) Proteina 4 de enlace ul16.
US20230265145A1 (en) Il-10 muteins and fusion proteins thereof
EA042572B1 (ru) Мутеины il-2 и способы их применения
TW202322846A (zh) 新穎的il27受體促效劑及其使用方法
BR112015023145B1 (pt) Polipeptídeos contendo fc aglicosilados e anticorpo ou proteína de fusão fc

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20200605

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

RIN1 Information on inventor provided before grant (corrected)

Inventor name: VISWESWARAIAH, JYOTHSNA

Inventor name: SAMPSON, ERIK ROBERT

Inventor name: OTIPOBY, KEVIN LEWIS

Inventor name: VINEY, JOANNE L.

Inventor name: HIGGINSON-SCOTT, NATHAN

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: PANDION OPERATIONS, INC.

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40039617

Country of ref document: HK

A4 Supplementary search report drawn up and despatched

Effective date: 20210813

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 37/06 20060101ALI20210809BHEP

Ipc: A61P 37/04 20060101ALI20210809BHEP

Ipc: A61P 37/02 20060101ALI20210809BHEP

Ipc: A61P 29/00 20060101ALI20210809BHEP

Ipc: A61K 47/68 20170101ALI20210809BHEP

Ipc: A61K 38/20 20060101AFI20210809BHEP