US20230303649A1 - Modified il-2 polypeptides for treatment of inflammatory and autoimmune diseases - Google Patents

Modified il-2 polypeptides for treatment of inflammatory and autoimmune diseases Download PDF

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US20230303649A1
US20230303649A1 US17/861,180 US202217861180A US2023303649A1 US 20230303649 A1 US20230303649 A1 US 20230303649A1 US 202217861180 A US202217861180 A US 202217861180A US 2023303649 A1 US2023303649 A1 US 2023303649A1
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modified
polypeptide
amino acid
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Bertolt Kreft
Vijaya Raghavan PATTABIRAMAN
Rubén ALVAREZ SANCHEZ
Magali MULLER
Jean-Philippe CARRALOT
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Bright Peak Therapeutics AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • 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
    • 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
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • Interleukin-2 is a cytokine signaling molecule important in regulating the immune system. IL-2 is implicated in helping the immune system differentiate between foreign and endogenous cell types, thereby preventing the immune system from attacking a subject’s own cells. IL-2 accomplishes its activity through interactions with IL-2 receptors (IL-2R) expressed by lymphocytes. Through these binding interactions, IL-2 can modulate a subject’s populations of T-effector (T eff ) cells, natural killer (NK) cells, and regulatory T-cells (T reg ).
  • T eff T-effector
  • NK natural killer
  • T reg regulatory T-cells
  • IL-2′s ability to regulate the immune system is driven at least partially by its different affinities for the IL-2R ⁇ subunit (CD25) and the IL-2R ⁇ subunit (CD122).
  • Native IL-2 acts on resting lymphocytes via intermediate-affinity receptors consisting of IL-2R ⁇ and IL-2R ⁇ subunits.
  • Activated lymphocytes and T reg cells additionally express the IL-2R ⁇ subunit, which combines with the ⁇ and ⁇ subunits to form a receptor with high affinity for IL-2.
  • IL-2 can enhance the activation and proliferation of T reg cells, thus regulating the subject’s immune response.
  • IL-2 has been used in the treatment of various diseases involving the immune system, both alone and in combination with other therapies.
  • use of IL-2 as a treatment has been limited by toxicities, which include life threatening and sometimes fatal vascular leak syndrome, as well as by its short half-life, requiring dosing three times per day over eight days.
  • IL-2 polypeptides with different selectivity for various IL-2 receptor subunits for example, enhanced binding of the IL-2R ⁇ to enhance therapeutic potential and minimize the risk of side effects of IL-2 therapies.
  • a modified interleukin-2 (IL-2) polypeptide comprising: a modified IL-2 polypeptide, wherein the modified IL-2 polypeptide comprises up to seven natural amino acid substitutions, wherein the seven natural amino acid substitutions comprise amino acid substitutions at residues Y31, K35, and Q74; and wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence.
  • IL-2 interleukin-2
  • a modified IL-2 polypeptide comprising: a modified IL-2 polypeptide, wherein the modified IL-2 polypeptide exhibits a binding affinity for the IL-2 receptor alpha subunit (IL-2R ⁇ ) which is between about 0.1 nM and about 100 nM, and wherein the modified IL-2 polypeptide exhibits a binding affinity for the IL-2 receptor beta subunit (IL-2R ⁇ ) which is at least about 1000 nM.
  • IL-2R ⁇ IL-2 receptor alpha subunit
  • IL-2R ⁇ IL-2 receptor beta subunit
  • FIG. 1 shows a synthetic scheme used to synthesize a modified IL-2 polypeptide as provided herein as a linear depsipeptide.
  • FIG. 2 shows a scheme for rearranging and folding a linear depsipeptide to provide a folded modified IL-2 polypeptide as provided herein.
  • FIG. 3 shows a scheme for producing a PEGylated modified IL-2 polypeptide as provided herein.
  • FIG. 4 A shows mean fluorescence intensity (MFI) of STAT5 phosphorylation in T eff cells by aldesleukin, composition A, and composition A1 at various concentrations.
  • FIG. 4 B shows MFI of STAT5 phosphorylation in T reg cells by aldesleukin, composition A, and composition A1 at various concentrations.
  • FIG. 4 C shows EC50 values of STAT5 phosphorylation of a variety of T cell subtypes by modified IL-2 polypeptides provided herein.
  • FIG. 4 D shows the EC50 values of STAT5 phosphorylation of a variety of T cell subtypes by modified IL-2 polypeptides compositions provided herein.
  • FIG. 5 shows binding affinities of composition A1 and aldesleukin to the IL-2R ⁇ and IL-2R ⁇ subunits as determined by biolayer interferometry (BLI).
  • FIG. 6 shows pharmacokinetics of composition A1 administered subcutaneously to mice at 0.1 mg/kg or 0.3 mg/kg.
  • FIG. 7 shows the immuno-pharmacodynamic effect of composition A1 or aldesleukin on various lymphocyte populations at various time points after administration of the indicated doses.
  • Top left graph shows T reg %pSTAT5 positive cells;
  • Top center graph shows T eff %pSTAT5 positive cells;
  • Top right graph shows NK %pSTAT5 positive cells;
  • Middle left graph shows T reg %Ki67 positive cells;
  • Middle center graph shows T eff %Ki67 positive cells;
  • Middle right graph shows NK %Ki67 positive cells;
  • Bottom left graph shows T reg counts fold change versus baseline;
  • Bottom center graph shows T eff counts fold change versus baseline;
  • Bottom right graph shows NK counts fold change versus baseline.
  • FIG. 8 A shows an experimental design to assess composition A1′s ability to delay hypersensitivity to keyhole limpet hemocyanin in mice.
  • FIG. 8 B shows ear thickness difference between the right ear (challenged with KLH) and the contralateral ear (injected with saline) reported in mm as a measure of swelling at 24, 48, 72 and 96 hrs.
  • FIG. 8 C shows ear thickness difference between the right ear (challenged with KLH) and the contralateral ear (injected with saline) reported as area under the curve (AUC) as a measure of overall swelling after challenge.
  • AUC area under the curve
  • modified interleukin-2 (IL-2) polypeptides useful as therapeutic agents.
  • Modified IL-2 polypeptides provided herein can be used as treatments for various diseases and disorders, including inflammatory or other autoimmune diseases.
  • Such modified IL-2 polypeptides may display binding characteristics for the IL-2 receptor (IL-2R) that differ from wild-type IL-2 (SEQ ID NO:1) or aldesleukin (SEQ ID NO: 2).
  • modified IL-2 polypeptides described herein have increased affinity for the IL-2R ⁇ complex.
  • the modified IL-2 polypeptides have an unmodulated affinity for the IL-2R ⁇ complex.
  • the modified IL-2 polypeptides have a reduced affinity for the IL-2R ⁇ complex.
  • the modified IL-2 polypeptides provided herein may comprise amino acid substitutions that enhance the binding affinity for the IL-2R ⁇ receptor subunit.
  • the modified IL-2 polypeptides provided herein comprise amino acid substitutions that lower the modified IL-2 polypeptides affinity for the IL-2R ⁇ receptor subunit.
  • the modified IL-2 polypeptides have a biological activity of inducing fewer T-effector (T eff ) cells when administered in vivo compared to a wild type IL-2 or aldesleukin.
  • the modified IL-2 polypeptides provided herein have comparable ability (e.g., have an EC 50 no more than 10x greater, no more than 100x greater) to induce regulatory T-cells (T reg ) when administered in vivo compared to a wild type IL-2 or aldesleukin.
  • the modified IL-2 polypeptides described herein contain modified amino acid residues. Such modifications can take the form of amino acid substitutions of a wild type IL-2 polypeptide such as the amino acid sequence of SEQ ID NO: 1, addition or deletion of amino acids from the sequence of SEQ ID NO: 1, or the addition of moieties to amino acid residues.
  • the modified IL-2 polypeptide described herein contains a deletion of the first amino acid from the sequence of SEQ ID NO: 1.
  • the modified IL-2 polypeptide described herein comprises a C125S substitution, using the sequence of SEQ ID NO: 1 as a reference sequence.
  • the modified IL-2 polypeptide described herein comprises substitutions at one or more residues selected from Y31, K35, Q74, and/or N88, wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence. These substitutions may be in combination with the C125S substitution and/or an N-terminal deletion, such as a deletion of the first amino acids from the sequence of SEQ ID NO:1.
  • the Y31 substitution is a Y31H substitution.
  • the K35 substitutions is a K35R substitution.
  • the Q74 substitution is a Q74P substitutions.
  • the N88 substitution is an N88D substitution.
  • the modified IL-2 polypeptide comprises a Y31H substitution, a K35R substitution, and a Q74P substitution. In some embodiments, the modified IL-2 polypeptide comprises a Y31H substitution, a K35R substitution, a Q74P substitution, and an N88D substitution. In some embodiments, the modified IL-2 polypeptide comprises a Y31H substitution, a K35S substitution, a Q74P substitution, and a C125S substitution. In some embodiments, the modified IL-2 polypeptide comprises a Y31H substitution, a K35S substitution, a Q74P substitution, a N88D substitution, and a C125S substitution.
  • the modified IL-2 polypeptide is a synthetic polypeptide.
  • the modified IL-2 polypeptide is synthesized by ⁇ -ketoacid-hydroxylamine (KAHA) amide-forming ligation.
  • the modified IL-2 polypeptide comprises unnatural amino acids, such as homoserine, which are used during the KAHA ligation reaction to join multiple polypeptide fragments to synthesize the full-length modified IL-2 polypeptide. In some embodiments, these are the only unnatural amino acids in the modified IL-2 polypeptide.
  • the modified IL-2 polypeptide comprises norleucine (Nle) residue substitutions at one or more methionine residues present in wild type IL-2 or aldesleukin. In some embodiments, the modified IL-2 polypeptide comprises norleucine residues at positions 23, 39, and 46.
  • a modified IL-2 polypeptide as described herein can comprise one or more non-canonical amino acids (also referred to herein as “unnatural amino acids”).
  • “Non-canonical” amino acids can refer to amino acid residues in D- or L-form that are not among the 20 canonical amino acids generally incorporated into naturally occurring proteins.
  • one or more amino acids of the modified IL-2 polypeptides are substituted with one or more non-canonical amino acids.
  • Non-canonical amino acids include, but are not limited to N-alpha-(9-Fluorenylmethyloxycarbonyl)-L-azidolysine (Fmoc-L-Lys(N 3 )-OH), N-alpha-(9-Fluorenylmethyloxycarbonyl)-L-biphenylalanine (Fmoc-L-Bip-OH), and N-alpha-(9-Fluorenylmethyloxycarbonyl)-O-benzyl-L-tyrosine (Fmoc-L-Tyr(Bzl)-OH, or their unprotected analogs.
  • polymers may be added to modified IL-2 polypeptides.
  • the polymers are added in order to increase the half-life of the polypeptides.
  • Such half-life extending polymers can be added to the N-terminus of the modified IL-2 polypeptides.
  • the half-life extending polymers may be of any size, including up to about 6 kDa, up to about 30 kDa, or up to about 50 kDa.
  • the half-life extending polymers are PEG polymers.
  • the modified IL-2 polypeptide comprises one or more amino acid substitutions or deletions selected from Table 1.
  • a modified IL-2 polypeptide provided herein comprises one or more amino acid substitutions selected from Table 2.
  • a modified IL-2 polypeptide provided herein comprises one or more polymers selected from Table 3. In some embodiments, the one or more polymer is covalently attached the N-terminus of the modified IL-2 polypeptide.
  • the modified IL-2 polypeptides described herein may also be synthesized chemically rather than expressed as recombinant polypeptides.
  • the modified IL-2 polypeptides can be made by synthesizing one or more fragments of the full-length modified IL-2 polypeptides, ligating the fragments together, and folding the ligated full-length polypeptide.
  • the modified IL-2 polypeptide comprises Y31H, K35R, Q74P, and C125S substitutions and optionally a PEG polymer covalently attached to the N-terminus of the modified IL-2 polypeptide.
  • the modified IL-2 polypeptide comprises Y31H, K35R, Q74P, N88D, and C125S substitutions and optionally a PEG polymer covalently attached to the N-terminus of the modified IL-2 polypeptide.
  • the modified IL-2 polypeptides enhance regulatory T-cell (T reg ) cell proliferation or activation when administered to a subject. In some embodiments, the modified IL-2 polypeptides enhance T reg proliferation or activation while sparing T-effector cells (T eff ) and/or natural killer (NK) cells when administered to a subject. In some embodiments, the modified IL-2 polypeptides increase Treg cells without substantially increasing CD8+ T cells and NK cells when administered to a subject.
  • the term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “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. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.
  • polymers which are “attached” or “covalently attached” to residues of IL-2 polypeptides.
  • “attached” or “covalently attached” means that the polymer is tethered to the indicated residue, and such tethering can include a linking group (i.e., a linker).
  • a linking group i.e., a linker
  • Binding affinity refers to the strength of a binding interaction between a single molecule and its ligand/binding partner. A higher binding affinity refers to a higher strength bond than a lower binding affinity. In some instances, binding affinity is measured by the dissociation constant (K D ) between the two relevant molecules. When comparing K D values, a binding interaction with a lower value will have a higher binding affinity than a binding interaction with a higher value. For a protein-ligand interaction, K D is calculated according to the following formula:
  • [L] is the concentration of the ligand
  • [P] is the concentration of the protein
  • [LP] is the concentration of the ligand/protein complex.
  • amino acid sequences e.g., polypeptide sequences
  • Sequence identity is measured by protein-protein BLAST algorithm using parameters of Matrix BLOSUM62, Gap Costs Existence:11, Extension:1, and Compositional Adjustments Conditional Compositional Score Matrix Adjustment. This alignment algorithm is also used to assess if a residue is at a “corresponding” position through an analysis of the alignment of the two sequences being compared.
  • pharmaceutically acceptable refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • a “pharmaceutically acceptable excipient, carrier or diluent” refers to an excipient, carrier or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.
  • a “pharmaceutically acceptable salt” suitable for the disclosure may be an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication.
  • Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids.
  • Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2-hydroxyethyl sulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC-(CH 2 ) n -COOH where n is 0-4, and the like.
  • acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfur
  • pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium.
  • pharmaceutically acceptable salts include those listed by Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 ( 1985).
  • a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9.
  • a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.
  • subject refers to an animal which is the object of treatment, observation, or experiment.
  • a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.
  • moiety refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.
  • an N-terminus with glutaric acid and 0.5 kDa azido PEG refers to a modification to an N-terminal amine of an IL-2 polypeptide provided herein with a structure of
  • a modified IL-2 polypeptide comprises the N-terminus with glutaric acid and 0.5 kDa azido PEG.
  • Composition A refers to a modified IL-2 polypeptide of SEQ ID NO: 3 which comprises an N-terminus with glutaric acid and 0.5 kDa azido PEG.
  • composition A1 refers to the reaction product formed between composition A and a DBCO containing PEG having a molecular weight of about 30 kDa.
  • Composition B refers to a modified IL-2 polypeptide of SEQ ID NO: 4 which comprises an N-terminus with glutaric acid and 0.5 kDa azido PEG.
  • composition B1 refers to the reaction product formed between composition B and a DBCO containing PEG having a molecular weight of about 30 kDa..
  • Composition C refers to a modified IL-2 polypeptide of SEQ ID NO: 5 which comprises an N-terminus with glutaric acid and 0.5 kDa azido PEG.
  • composition C1 refers to the reaction product formed between composition B and a DBCO containing PEG having a molecular weight of about 30 kDa.
  • Composition D refers to a modified IL-2 polypeptide of SEQ ID NO: 6 which comprises an N-terminus with glutaric acid and 0.5 kDa azido PEG.
  • composition D1 refers to the reaction product formed between composition D and a DBCO containing PEG having a molecular weight of about 30 kDa.
  • Composition E refers to a modified IL-2 polypeptide of SEQ ID NO: 7 which comprises an N-terminus with glutaric acid and 0.5 kDa azido PEG.
  • composition E1 refers to the reaction product formed between composition E and a DBCO containing PEG having a molecular weight of about 30 kDa.
  • Composition F refers to a modified IL-2 polypeptide of SEQ ID NO: 8 which comprises an N-terminus with glutaric acid and 0.5 kDa azido PEG.
  • conjugation handle refers to a reactive group capable of forming a bond upon contacting a complementary reactive group.
  • a conjugation handle preferably does not have a substantial reactivity with other molecules which do not comprise the intended complementary reactive group.
  • conjugation handles, their respective complementary conjugation handles, and corresponding reaction products can be found in the table below. While table headings place certain reactive groups under the title “conjugation handle” or “complementary conjugation handle,” it is intended that any reference to a conjugation handle can instead encompass the complementary conjugation handles listed in the table (e.g., a trans-cyclooctene can be a conjugation handle, in which case tetrazine would be the complementary conjugation handle).
  • amine conjugation handles and conjugation handles complementary to amines are less preferable for use in biological systems owing to the ubiquitous presence of amines in biological systems and the increased likelihood for off-target conjugation.
  • number average molecular weight means the statistical average molecular weight of all the individual units in a sample, and is defined by Formula (1):
  • M i is the molecular weight of a unit and N i is the number of units of that molecular weight.
  • weight average molecular weight means the number defined by Formula (2):
  • M i is the molecular weight of a unit and N i is the number of units of that molecular weight.
  • peak molecular weight means the molecular weight of the highest peak in a given analytical method (e.g. mass spectrometry, size exclusion chromatography, dynamic light scattering, analytical centrifugation, etc.).
  • a modified IL-2 polypeptide which is biased in favor of activation of T reg cells compared to T eff cells.
  • a modified polypeptide that comprises a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide comprises one or more amino acid substitutions.
  • the modified IL-2 polypeptide comprises at least one amino acid substitutions at residues selected from Y31, K35, Q74, and N88, wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence.
  • the modified IL-2 polypeptide comprises amino acid substitutions at each of residues Y31, K35, and Q74, wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence.
  • the modified IL-2 polypeptide comprises the amino acid substitutions of Y31H, K35R, and Q74P.
  • the modified IL-2 polypeptide comprises amino acid substitutions at each of residues Y31, K35, Q74, and N88, wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence.
  • the modified IL-2 polypeptide comprises the amino acid substitutions of Y31H, K35R, Q74P, and N88D. In some embodiments, the modified IL-2 polypeptide does not comprise any additional substitutions that have a substantial impact on the binding of the modified IL-2 polypeptide to the IL-2R ⁇ receptor.
  • a modified polypeptide comprising: a modified interleukin-2 (IL-2) polypeptide, wherein the modified IL-2 polypeptide exhibits substantially lower ability to activate T eff cells than an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the modified IL-2 polypeptide retains the ability to activate T reg cells.
  • the modified IL-2 polypeptide exhibits an enhanced ability to activate T reg cells compared to an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2 In some embodiments, the modified IL-2 polypeptide exhibits at least about 4x lower dissociation constant (K d ) of IL-2R ⁇ than an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2. In some embodiments, the modified IL-2 polypeptide exhibits a 2-fold to 10-fold lower dissociation constant (K d ) of IL-2R ⁇ than an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • a modified IL-2 polypeptide that exhibits a greater affinity for IL-2 receptor ⁇ subunit than an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the affinity to IL-2 receptor a subunit is measured by dissociation constant (K d ).
  • K d dissociation constant
  • the phrase “the K d of the modified IL-2 polypeptide/IL-2 receptor ⁇ subunit” means the dissociation constant of the binding interaction of the modified IL-2 polypeptide and CD25.
  • the K d of the modified IL-2 polypeptide/IL-2 receptor ⁇ subunit is less than 10 nM. In some embodiments the K d of the modified IL-2 polypeptide/IL-2 receptor ⁇ subunit is less than 10 nM, less than 7.5 nM, less than 5 nM, less than 4 nM, or less than 3 nM. In some embodiments, the K d of the modified IL-2 polypeptide/IL-2 receptor ⁇ subunit between about 1 nM and 0.1 nM. In some embodiments, the K d of the modified IL-2 polypeptide/IL-2 receptor ⁇ subunit between about 10 nM and about 0.1 nM.
  • the K d of the modified IL-2 polypeptide/IL-2 receptor ⁇ subunit between about 10 nM and about 1 nM. In some embodiments, the K d of the modified IL-2 polypeptide/IL-2 receptor ⁇ subunit between about 7.5 nM and about 0.1 nM. In some embodiments, the K d of the modified IL-2 polypeptide/IL-2 receptor ⁇ subunit between about 7.5 nM and about 1 nM. In some embodiments, the K d of the modified IL-2 polypeptide/IL-2 receptor ⁇ subunit between about 5 nM and about 0.1 nM. In some embodiments, the K d of the modified IL-2 polypeptide/IL-2 receptor ⁇ subunit between about 5 nM and about 1 nM. In some embodiments, the K d is measured by surface plasmon resonance.
  • the modified IL-2 polypeptide that exhibits at least about a 10%, 50%, 100%, 250%, or 500% greater affinity for IL-2 receptor ⁇ subunit than an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2. In some embodiments, the modified IL-2 polypeptide exhibits at most about a 500%, 750%, or 1000% greater affinity for IL-2 receptor ⁇ subunit than an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the modified IL-2 polypeptide exhibits about 1.5-fold to about 10-fold greater affinity for IL-2 receptor ⁇ subunit than an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the modified IL-2 polypeptide exhibits substantially the same binding affinity for the IL-2R ⁇ as compared to an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2. In some embodiments, the modified IL-2 polypeptide exhibits a K d with IL-2R ⁇ that is within about 2-fold, about 4-fold, about 6-fold, about 8-fold, or about 10-fold of the K d between an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2 and IL-2R ⁇ .
  • the modified IL-2 polypeptide exhibits reduced affinity for the IL-2 receptor ⁇ subunit (IL-2R ⁇ ) as compared to an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the modified IL-2 polypeptide exhibits at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or at least about 500-fold fold lower affinity for the IL-2R ⁇ .
  • the modified IL-2 polypeptide exhibits at least about 100-fold lower affinity for IL-2R ⁇ .
  • the modified IL-2 polypeptide exhibits substantially no affinity for IL-2R ⁇ .
  • the affinity is measured as the dissociation constant K d (e.g., a lower affinity correlating with a higher dissociation constant).
  • the modified IL-2 polypeptide exhibits a binding affinity for IL-2R ⁇ which is at least 500 nM, at least 1000 nM, at least 5000 nM, at least 10000 nM, at least 50000 nM, or at least 100000 nM. In some embodiments, the modified IL-2 polypeptide exhibits substantially no binding affinity for IL-2R ⁇ .
  • the modified IL-2 polypeptide exhibits an affinity for IL-2R ⁇ which is at least about 30-fold greater, at least about 50-fold grater, at least about 75-fold greater, at least about 100-fold greater, at least about 500-fold greater, or at least about 1000-fold greater than for IL-2R ⁇ . In some embodiments, the modified IL-2 polypeptide exhibits an affinity for IL-2R ⁇ which is at least about 100-fold greater than for IL-2R ⁇ . In some embodiments, the modified IL-2 polypeptide exhibits an affinity for IL-2R ⁇ which is at least about 1000-fold greater than for IL-2R ⁇ .
  • a modified IL-2 polypeptide described herein is capable of expanding a regulatory T-cell (T reg ) cell population. In some embodiments, a modified IL-2 polypeptide described herein spares expansion of effector T-cells (T eff ).
  • a modified IL-2 polypeptide has a half maximal effective concentration (EC 50 ) for activation of T reg cells that at most moderately reduced compared to an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • activation of T reg cells is measured by assessing change in STAT5 phosphorylation in a population of T cells when in contact with the modified IL-2 polypeptide.
  • a T reg cell is identified by being CD4 + , CD25+ and FoxP3 + .
  • a T reg cell is identified by also showing elevated expression of CD25 (CD25 Hi ).
  • the modified IL-2 polypeptide has an EC 50 for activation of T reg cells of at most about 100 nM, at most about 75 nM, at most about 50 nM, at most about 40 nM, at most about 35 nM, at most about 30 nM, or at most about 25 nM. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T reg cells of at most about 50 nM, at most about 40 nM, at most about 35 nM, at most about 30 nM, or at most about 25 nM, at most about 20 nM, at most about 15 nM, at most about 10 nM, or at most about 5 nM.
  • the modified IL-2 polypeptide has an EC 50 for activation of T reg cells of at most about 100 nM. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T reg cells of at most about 50 nM. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T reg cells of at most about 25 nM.
  • the modified IL-2 polypeptide has an EC 50 for activation of T reg cells of from about 0.1 nM to about 100 nM, from about 1 nM to about 100 nM, from about 0.1 nM to about 50 nM, from about 1 nM to about 50 nM, from about 0.1 nM to about 25 nM, from about 1 nM to about 25 nM, from about 0.1 nM to about 10 nM, or from about 1 nM to about 10 nM.
  • the modified IL-2 polypeptide has an EC 50 for activation of T reg cells that is at most 2-fold, at most 5-fold, at most 10-fold, at most 20-fold, at most 50-fold, at most 100-fold, at most 200-fold, at most 500-fold, or at most 1000-fold greater compared to an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the modified IL-2 polypeptide has an EC 50 for activation of T reg cells that is at most 2-fold greater.
  • the modified IL-2 polypeptide has an EC 50 for activation of T reg cells that is at most 5-fold greater.
  • the modified IL-2 polypeptide has an EC 50 for activation of T reg cells that is at most 10-fold greater. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T reg cells that is at most 50-fold greater. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T reg cells that is at most 100-fold greater. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T reg cells that is at most 200-fold greater. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T reg cells that is at most 500-fold greater. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T reg cells that is at most 1000-fold greater.
  • a modified IL-2 polypeptide has a half maximal effective concentration (EC 50 ) for activation of T eff cells that is substantially greater compared to an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2.
  • the T eff cell is 1, 2, or 3 of a CD8 T eff cell (e.g., CD8 + ), a Naive CD8 cell (e.g., CD8 + , CD45RA+), or a CD4 Con cell (e.g., CD4 + , FoxP3 - ), or any combination thereof.
  • activation of cells is measured by assessing change in STAT5 phosphorylation in a population of T cells when in contact with the modified IL-2 polypeptide.
  • the modified IL-2 polypeptide has an EC 50 for activation of T eff cells of at least about 10 nM, at least about 50 nM, at least about 100 nM, at least about 500 nM, at least about 1000 nM, at least about 2000 nM, at least about 3000 nM, at least about 4000 nM, or at least about 5000 nM. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T eff cells of at least about 100 nM. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T eff cells of at least about 500 nM.
  • the modified IL-2 polypeptide has an EC 50 for activation of T eff cells of at least about 1000 nM. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T eff cells of at least about 5000 nM. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T eff cells of at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold greater compared to an IL-2 polypeptide of SEQ ID NO: 1 and/or SEQ ID NO: 2. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T eff cells of at least 10-fold greater.
  • the modified IL-2 polypeptide has an EC 50 for activation of T eff cells of at least 50-fold greater. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T eff cells of at least 100-fold greater. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T eff cells of at least 500-fold greater. In some embodiments, the modified IL-2 polypeptide has an EC 50 for activation of T eff cells of at least 1000-fold greater.
  • the modified IL-2 polypeptide exhibits a substantially greater ability to activate T reg cells compared to T eff cells.
  • a ratio of EC50 for activation of a T eff cell type over EC50 for activation of a T reg cell type is at least 10, at least 20, at least 50, at least 100, at least 150, or at least 200.
  • a ratio of EC50 for activation of a T eff cell type over EC50 for activation of a T reg cell type is at least 100.
  • a ratio of EC50 for activation of a T eff cell type over EC50 for activation of a T reg cell type is at least 200.
  • a ratio of EC50 for activation of a T eff cell type over EC50 for activation of a T reg cell type is at least 300. In some embodiments, a ratio of EC50 for activation of a T eff cell type over EC50 for activation of a T reg cell type is at least 500. In some embodiments, a ratio of EC50 for activation of a T eff cell type over EC50 for activation of a T reg cell type is at least 1000.
  • the level of activation is measured after about 0.5 h to about 1 h after incubation with the modified IL-2 polypeptide (e.g., 0.5 h to 1h before fixing the cells for in in vitro experiment).
  • a modified IL-2 polypeptide described herein comprises a covalently attached polymer for half-life extension.
  • the modified IL-2 polypeptide comprises a covalently attached polymer for plasma or serum half-life extension.
  • a plasma or serum half-life of the modified IL-2 polypeptide with polymer attached is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold longer compared to a plasma or serum half-life of a wild-type IL-2 polypeptide (SEQ ID NO 1) or aldesleukin (SEQ ID NO: 2) without a polymer attached.
  • a plasma or serum half-life of a modified IL-2 polypeptide described herein is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold longer compared to a plasma or serum half-life of the modified IL-2 polypeptide without the half-life extending polymer.
  • a modified IL-2 polypeptide described herein comprises one or more modifications at one or more amino acid residues.
  • the residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence.
  • the residue position numbering of the modified IL-2 polypeptide is based on a wild-type human IL-2 polypeptide as a reference sequence.
  • Modifications to the polypeptides described herein encompass amino acid substitutions, addition of various functionalities, deletion of amino acids, addition of amino acids, or any other alteration of the wild-type version of the protein or protein fragment.
  • Functionalities which may be added to polypeptides include polymers, linkers, alkyl groups, detectable molecules such as chromophores or fluorophores, reactive functional groups, or any combination thereof.
  • functionalities are added to individual amino acids of the polypeptides.
  • functionalities are added site-specifically to the polypeptides.
  • a modified IL-2 polypeptide comprising one or more amino acid substitutions.
  • the amino acid substitutions affect the binding properties of the modified IL-2 polypeptide to IL-2 receptor subunits (e.g. alpha, beta, or gamma subunits) or to IL-2 receptor complexes (e.g. IL-2 receptor ⁇ complex or ⁇ complex).
  • the amino acid substitutions are at positions on the interface of binding interactions between the modified IL-2 polypeptide and an IL-2 receptor subunit or an IL-2 receptor complex.
  • the amino acid substitutions cause an increase in affinity for the IL-2 receptor ⁇ complex or alpha subunit.
  • the amino acid substitutions cause a decrease in affinity for the IL-2 receptor ⁇ complex or beta subunit.
  • a modified IL-2 polypeptide comprising: a modified IL-2 polypeptide comprising natural amino acid substitutions relative to WT IL-2 (SEQ ID NO: 1).
  • the modified IL-2 polypeptide comprises up to seven natural amino acid substitutions.
  • the modified IL-2 polypeptide comprises up to six amino acid substitutions.
  • the modified IL-2 polypeptide comprises up to five amino acid substitutions.
  • the modified IL-2 polypeptide comprises up to four amino acid substitutions.
  • the modified IL-2 polypeptide comprises up to three amino acid substitutions.
  • the modified IL-2 polypeptide comprises from three to seven, three to six, three to five, three to four, four to seven, four to six, four to five, five to seven, five to six, or six to seven natural amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises at least one, at least two, at least three, at least four, at least five, or at least six amino acid substitutions.
  • a modified IL-2 polypeptide provided herein comprises natural amino acid substitutions at at least one of Y31, K35, Q74, and N88D wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO: 1 as a reference sequence.
  • the modified IL-2 polypeptide comprises natural amino acid substitutions at at least two of Y31, K35, Q74, and N88.
  • the modified IL-2 polypeptide comprises natural amino acid substitutions at at least three of Y31, K35, Q74, and N88.
  • the modified IL-2 polypeptide comprises natural amino acid substitutions at at least three of Y31, K35, Q74, and N88.
  • the modified IL-2 polypeptide comprises natural amino acid substitutions at each of Y31, K35, Q74, and N88. In some embodiments, the modified IL-2 polypeptide comprises the amino acid substitutions Y31H, K35R, Q74P, and N88D. In some embodiments, the modified IL-2 polypeptide further comprises an optional C125 substitution (e.g., C125S or C125A). In some embodiments, the modified IL-2 polypeptide further comprises an optional A1 deletion or substitution of residue A1. In some embodiments, the modified IL-2 polypeptide further comprises an optional A1 deletion.
  • C125 substitution e.g., C125S or C125A
  • the modified IL-2 polypeptide further comprises an optional A1 deletion or substitution of residue A1. In some embodiments, the modified IL-2 polypeptide further comprises an optional A1 deletion.
  • a modified IL-2 polypeptide provided herein comprises a Y31 substitution wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence.
  • the Y31 substitution is for an aromatic amino acid.
  • the Y31 substitution is for a basic amino acid.
  • the basic amino acid is weakly basic.
  • the Y31 substitution is selected from Y31F, Y31H, Y31W, Y31R, and Y31K.
  • the Y31 substitution is Y31H.
  • a modified IL-2 polypeptide provided herein comprises a K35 substitution, wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence.
  • the K35 substitution is for a basic amino acid.
  • the K35 substitution is for a positively charged amino acid.
  • the K35 substitution is K35R, K35E, K35D, or K35Q.
  • the K35 substitution is K35R.
  • a modified IL-2 polypeptide provided herein comprises a Q74 substitution, wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence.
  • the Q74 substitution is a cyclic amino acid.
  • the cyclic amino acid comprises a cyclic group covalently attached to the alpha carbon and the nitrogen attached to the alpha carbon.
  • the Q74 substitution is Q74P.
  • a modified IL-2 polypeptide provided herein comprises a N88 substitution, wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence.
  • the N88 substitution is a charged amino acid residue.
  • the N88 substitution is a negatively charged amino acid residue.
  • the N88 substitution is N88D or N88E.
  • the N88 substitution is N88D or N88E.
  • the N88 substitution is N88D.
  • a modified IL-2 polypeptide comprises a C125 substitution, wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence.
  • the C125 substitution stabilizes the modified IL-2 polypeptide.
  • the C125 substitution does not substantially alter the activity of the modified IL-2 polypeptide.
  • the modified IL-2 polypeptide comprises a C125S substitution.
  • the modified IL-2 polypeptide comprises a C125A substitution.
  • a modified IL-2 polypeptide comprises a modification at residue A1, wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ I DNO: 1 as a reference sequence.
  • the modification is an A1 deletion.
  • the modified IL-2 polypeptide comprises additional amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises an additional amino acid substitution that has an effect on binding to the IL-2 receptor alpha subunit or ⁇ complex. In some embodiments, the modified IL-2 polypeptide comprises an additional amino acid substitution that has an effect on binding to the IL-2 receptor beta subunit or ⁇ complex. In some embodiments, the modified IL-2 polypeptide comprises at least one additional amino acid substitution selected from Table 1. In some embodiments, the modified IL-2 polypeptide comprises at least one amino acid substitution at residue E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92.
  • the modified IL-2 polypeptide comprises at least one amino acid substitution at residue E15, N29, N30, T37, K48, V69, N71, I89, or I92. In some embodiments, the modified IL-2 polypeptide comprises 1, 2, 3, or 4 natural amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92. n some embodiments, the modified IL-2 polypeptide comprises 1, 2, 3, or 4 natural amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, I89, or I92.
  • the modified IL-2 polypeptide comprises 1 natural amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92. In some embodiments, the modified IL-2 polypeptide comprises 2 In some embodiments, the modified IL-2 polypeptide comprises up to 2 natural amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92. In some embodiments, the modified IL-2 polypeptide comprises up to 3 natural amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92.
  • the additional amino acid substitution comprises E15A, E15G, or E15S. In some embodiments, the additional amino acid substitution comprises N29S. In some embodiments, the additional amino acid substitution comprises N30S. In some embodiments, the additional amino acid substitution comprises T37A or T37R. In some embodiments, the additional amino acid substitution comprises K48E. In some embodiments, the additional amino acid substitution comprises V69A. In some embodiments, the additional amino acid substitution comprises N71R. In some embodiments, the additional amino acid substitution comprises N88A, N88D, N88E, N88F, N88G, N88H, N88I, N88M, N88Q, N88R, N88S, N88T, N88V, or N88W. In some embodiments, the additional amino acid substitution comprises N88D. In some embodiments, the additional amino acid substitution comprises I89V. In some embodiments, the additional amino acid substitution comprises I92K or I92R.
  • a modified IL-2 polypeptide provided herein comprises substitutions at Y31, K35, Q74, and optionally C125S.
  • the modified IL-2 polypeptide does not comprise any additional substitutions which substantially affect binding to the IL-2 receptor alpha subunit or ⁇ complex.
  • the modified IL-2 polypeptide does not comprise an additional amino acid substitution that has an effect on binding to the IL-2 receptor beta subunit or ⁇ complex
  • the modified IL-2 polypeptide does not comprise any additional natural amino acid substitutions selected from positions identified in Table 1.
  • the modified IL-2 polypeptide does not comprise any additional amino acid substitutions selected from Table 1.
  • the modified IL-2 polypeptide does not comprise any additional natural amino acid substitutions at residues E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92. In some embodiments, the modified IL-2 polypeptide does not comprise any additional amino acid substitutions at residues E15, N29, N30, T37, K48, V69, N71, N88, I89, or I92. In some embodiments, the modified IL-2 polypeptide does not have a V69 substitution. In some embodiments, the modified IL-2 polypeptide does not have a V69A substitution. In some embodiments, the modified IL-2 polypeptide does not have a K48 substitution.
  • the modified IL-2 polypeptide does not have a K48E substitution. In some embodiments, the modified IL-2 polypeptide does not comprise a substitution at V69 or K48. In some embodiments, the modified IL-2 polypeptide does not comprise a substitution at either of V69 or K48. In some embodiments, the modified IL-2 polypeptide does not comprise a V69A or K48E substitution. In some embodiments, the modified IL-2 polypeptide does not comprise either a V69A or K48E substitution.
  • a modified IL-2 polypeptide provided herein comprises substitutions at Y31, K35, Q74, N88, and optionally C125S.
  • the modified IL-2 polypeptide does not comprise any additional substitutions which substantially affect binding to the IL-2 receptor alpha subunit or ⁇ complex.
  • the modified IL-2 polypeptide does not comprise an additional amino acid substitution that has an effect on binding to the IL-2 receptor beta subunit or ⁇ complex.
  • the modified IL-2 polypeptide does not comprise any additional natural amino acid substitutions selected from positions identified in Table 1.
  • the modified IL-2 polypeptide does not comprise any additional amino acid substitutions selected from Table 1.
  • the modified IL-2 polypeptide does not comprise any additional natural amino acid substitutions at residues E15, N29, N30, T37, K48, V69, N71, I89, or I92. In some embodiments, the modified IL-2 polypeptide does not comprise any additional amino acid substitutions at residues E15, N29, N30, T37, K48, V69, N71, I89, or I92. In some embodiments, the modified IL-2 polypeptide does not have a V69 substitution. In some embodiments, the modified IL-2 polypeptide does not have a V69A substitution. In some embodiments, the modified IL-2 polypeptide does not have a K48 substitution.
  • the modified IL-2 polypeptide does not have a K48E substitution. In some embodiments, the modified IL-2 polypeptide does not comprise a substitution at V69 or K48. In some embodiments, the modified IL-2 polypeptide does not comprise a substitution at either of V69 or K48. In some embodiments, the modified IL-2 polypeptide does not comprise a V69A or K48E substitution. In some embodiments, the modified IL-2 polypeptide does not comprise either a V69A or K48E substitution.
  • a modified IL-2 polypeptide provided herein comprises an N-terminal deletion.
  • the N-terminal deletion is of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acids.
  • the N-terminal deletion is of at least 1 amino acid.
  • the N-terminal deletion is of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • the N-terminal deletion is from 1 to 15 amino acids.
  • the N-terminal deletion is a deletion of a single amino acid (e.g., an A1 deletion of SEQ ID NO: 1).
  • a modified IL-2 polypeptide as described herein can comprise one or more unnatural amino acids.
  • “Unnatural” amino acids can refer to amino acid residues in D- or L-form that are not among the 20 canonical amino acids generally incorporated into naturally occurring proteins.
  • one or more amino acids of the modified IL-2 polypeptides are substituted with one or more unnatural amino acids.
  • Unnatural amino acids include, but are not limited to L-azidolysine and L-biphenylalanine.
  • Exemplary unnatural amino acids also include homoserine, norleucine, p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl) alanine, 3-methyl-phenylalanine, tri-O-acetyl-GlcNAcp-serine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-Boronophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalan
  • the unnatural amino acids are selected from ⁇ -amino acids, homoamino acids, and cyclic amino acids.
  • the unnatural amino acids comprise ⁇ -alanine, ⁇ -aminopropionic acid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N ⁇ -ethylglycine, N ⁇ -ethylaspargine, isodesmosine, allo-isoleucine, N ⁇ -methylglycine, N ⁇ -methylisoleucine, N ⁇ -methylvaline, ⁇ -carboxyglutamate, N ⁇ -acetylserine, N ⁇ -formylmethionine, 3-methylhistidine, and/or other similar amino acids.
  • the unnatural amino acid substitutions provided herein can be incorporated into an IL-2 polypeptide in addition to any combination of natural amino acid substitutions provided herein, unless otherwise specified.
  • a modified IL-2 polypeptide comprising, for example, Y31H, K35R, and Q74P natural amino acid substitutions is described, it is expressly contemplated that the modified IL-2 polypeptide can also comprise unnatural amino acid substitutions (e.g., Hse41, Hse71, Hse104, Nle23, Nle39, and Nle46).
  • modified IL-2 polypeptide provided herein is described as having Y31H, K35R, Q74P, and N88D natural amino acid substitutions
  • the modified IL-2 polypeptide can further comprise unnatural amino acid substitutions (e.g., Hse41, Hse71, Hse104, Nle23, Nle39, and Nle46).
  • any combination of natural amino acid substitutions present in a recombinant modified IL-2 polypeptide provided herein can also be incorporated into a synthetic version of the modified IL-2 polypeptide (e.g., the corresponding modified IL-2 polypeptide containing, for example, Hse41, Hse71, Hse104, Nle23, Nle39, and Nle46).
  • a modified IL-2 polypeptide comprising one or more unnatural amino acid substitutions.
  • the modified IL-2 polypeptide comprises at least two unnatural amino acid substitutions.
  • the modified IL-2 polypeptide comprises at least one amino acid substitution at a residue selected from Y31, K35, Q74, and N88, wherein residue position numbering of the modified IL-2 polypeptide is based on SEQ ID NO:1 as a reference sequence.
  • the modified IL-2 polypeptide comprises a homoserine (Hse) residue located in any one of residues 36-45.
  • the modified IL-2 polypeptide comprises a Hse residue located in any one of residues 61-81. In some embodiments, the modified IL-2 polypeptide comprises a Hse residue located in any one of residues 94-114. In some embodiments, the modified IL-2 polypeptide comprises 1, 2, 3, or more Hse residues. In some embodiments, the modified IL-2 polypeptide comprises Hse41, Hse71, Hse104, or a combination thereof. In some embodiments, the modified IL-2 polypeptide comprises Hse41, Hse71, and Hse104.
  • the modified IL-2 polypeptide comprises at least two amino acid substitutions, wherein the at least two amino acid substitutions are selected from (a) a homoserine (Hse) residue located in any one of residues 36-45; (b) a homoserine residue located in any one of residues 61-81; and (c) a homoserine residue located in any one of residues 94-114.
  • the modified IL-2 polypeptide comprises Hse41 and Hse71.
  • the modified IL-2 polypeptide comprises Hse41 and Hse104.
  • the modified IL-2 polypeptide comprises Hse71 and Hse104.
  • the modified IL-2 polypeptide comprises Hse41. In some embodiments, the modified IL-2 polypeptide comprises Hse71. In some embodiments, the modified IL-2 polypeptide comprises Hse104. In some embodiments, the modified IL-2 polypeptide comprises 1, 2, 3, or more norleucine (Nle) residues. In some embodiments, the modified IL-2 polypeptide comprises a Nle residue located in any one of residues 18-28. In some embodiments, the modified IL-2 polypeptide comprises one or more Nle residues located in any one of residues 34-50. In some embodiments, the modified IL-2 polypeptide comprises a Nle residue located in any one of residues 20-60.
  • Nle norleucine
  • the modified IL-2 polypeptide comprises three Nle substitutions. In some embodiments, the modified IL-2 polypeptide comprises Nle23, Nle39, and Nle46. In some embodiments, the modified IL-2 polypeptide comprises SEQ ID NO: 3. In some embodiments, the modified IL-2 polypeptide comprises SEQ ID NO: 3 with an A1 deletion.
  • a modified IL-2 polypeptide provided herein comprises an amino acid sequence of any one of SEQ ID NOs: 3-43 provided in Table 7. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of any one of SEQ ID NOs: 3-43. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of any one of SEQ ID NOs: 3-43, wherein each residue in the reference amino sequence which is substituted relative to SEQ ID NO: 1 is retained. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO: 3.
  • the modified IL-2 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 3. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 85%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 3, wherein each residue which is substituted in SEQ ID NO: 3 relative to SEQ ID NO: 1 is retained. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO: 4. In some embodiments, the modified IL-2 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 4, wherein each residue which is substituted in SEQ ID NO: 4 relative to SEQ ID NO: 1 is retained.
  • a modified IL-2 polypeptide described herein comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 3.
  • a modified IL-2 polypeptide described herein comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 4.
  • the sequence identity is measured by protein-protein BLAST algorithm using parameters of Matrix BLOSUM62, Gap Costs Existence:11, Extension:1, and Compositional Adjustments Conditional Compositional Score Matrix Adjustment.
  • a modified IL-2 polypeptide described herein comprises at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 amino acid substitutions. In some embodiments, the modified IL-2 polypeptide comprises 3 to 9 amino acid substitutions.
  • the modified IL-2 polypeptide comprises 3 or 4 amino acid substitutions, 3 to 5 amino acid substitutions, 3 to 6 amino acid substitutions, 3 to 7 amino acid substitutions, 3 to 9 amino acid substitutions, 4 or 5 amino acid substitutions, 4 to 6 amino acid substitutions, 4 to 7 amino acid substitutions, 4 to 9 amino acid substitutions, 5 or 6 amino acid substitutions, 5 to 7 amino acid substitutions, 5 to 9 amino acid substitutions, 6 or 7 amino acid substitutions, 6 to 9 amino acid substitutions, or 7 to 9 amino acid substitutions.
  • the modified IL-2 polypeptide comprises 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions.
  • the modified IL-2 polypeptide comprises at most 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions. In some embodiments, one or more of the amino acid substitutions are selected from Table 1. In some embodiments, one or more of the amino acid substitutions are selected from Table 2.
  • the modified IL-2 polypeptide contains a substitution for modified natural amino acid residues which can be used for attachment of additional functional groups which can be used to facilitate conjugation reaction or attachment of various payloads to the modified IL-2 polypeptide (e.g., polymers).
  • the substitution can be for a naturally occurring amino acid which is more amenable to attachment of additional functional groups (e.g., aspartic acid/asparagine cysteine, glutamic acid/glutamine, lysine, serine, threonine, or tyrosine), a derivative of a modified version of any naturally occurring amino acid, or any unnatural amino acid (e.g., an amino acid containing a desired reactive group, such as a CLICK chemistry reagent such as an azide, alkyne, etc.).
  • additional functional groups e.g., aspartic acid/asparagine cysteine, glutamic acid/glutamine, lysine, serine, threonine, or tyrosine
  • a derivative of a modified version of any naturally occurring amino acid e.g., an amino acid containing a desired reactive group, such as a CLICK chemistry reagent such as an azide, alkyne, etc.
  • n is an integer from 1-30.
  • natural amino acids which can be similarly modified include those with heteroatoms capable of easily forming a bond with a suitable group to link the polymeric group to the amino acid (e.g., tyrosine, serine, threonine).
  • modified amino acid residues can be used at any location at which it is desirable to add an additional functionality (e.g., a polymer or additional polypeptide) to the modified IL-2 polypeptide.
  • the modified IL-2 polypeptide comprises a modification of a terminal residue (e.g., the N-terminal residue or the C-terminal residue) which comprises a polymer.
  • the modification to the terminal residue comprises the attachment of a conjugation handle to the terminal residue of the modified IL-2 polypeptide.
  • the conjugation handle is attached to the modified IL-2 polypeptide through the N-terminal amino group or the C-terminal carboxyl group of the modified IL-2 polypeptide.
  • the conjugation handle is attached to the modified IL-2 polypeptide through the N-terminal amino group of the modified IL-2 polypeptide.
  • the conjugation handle is attached to the N-terminal amino group of the modified IL-2 polypeptide through a glutaryl-amino-PEG linker. In some embodiments, the conjugation handle is attached to the N-terminal amino group of the modified IL-2 polypeptide through an adduct having a structure
  • each n is independently an integer from 1-30 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30), and wherein X is a conjugation handle (e.g., an azide or other conjugation handle provided herein, such as a DBCO group).
  • the modified IL-2 polypeptide will comprise the adduct above, but the conjugation handle X is replaced with a reaction product of the conjugation handle and a complementary conjugation handle (e.g., a 1,2,3 triazole) linking the modified IL-2 polypeptide to an additional moiety (e.g., a larger polymer or an additional polypeptide).
  • the N-terminal amino group of the modified IL-2 polypeptide comprises an adduct having a structure
  • a modified IL-2 polypeptide is linked with an additional polypeptide.
  • the modified IL-2 polypeptide and the additional polypeptide form a fusion polypeptide.
  • the modified IL-2 polypeptide and the additional polypeptide are conjugated together.
  • the additional polypeptide comprises an antibody or binding fragment thereof.
  • the antibody comprises a humanized antibody, a murine antibody, a chimeric antibody, a bispecific antibody, any fragment thereof, or any combination thereof.
  • the antibody is a monoclonal antibody or any fragment thereof (e.g., an antigen binding fragment).
  • the modified IL-2 polypeptide is not conjugated to an additional polypeptide. In some embodiments, the modified IL-2 polypeptide is not conjugated to an antibody. In some embodiments, the modified IL-2 polypeptide is not conjugated to an anti-TNF ⁇ antibody.
  • a herein described modified IL-2 polypeptide comprises one or more polymers covalently attached thereon.
  • the described modified IL-2 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polymers covalently attached to the modified IL-2 polypeptide.
  • the described modified IL-2 polypeptide comprises a polymer covalently attached to the N-terminus of the IL-2 polypeptide.
  • the polymers provided herein may attached directly to a residue of the modified IL-2 polypeptide, may be attached through a small linking group (e.g., attached through a reaction with a conjugation handle incorporated into the modified IL-2 polypeptide).
  • the polymer as provided herein can be attached at any desired residue of the IL-2 polypeptide. In some embodiments, it is preferable that the polymer be attached at a residue which does not impact binding of the IL-2 polypeptide with the IL-2 receptor or a specific IL-2 receptor subunit (e.g., the IL-2 receptor alpha subunit). In some embodiments, the polymer is attached at or near the N-terminus of the modified IL-2 polypeptide. In some embodiments, the polymer is attached to the N-terminus of the modified IL-2 polypeptide. In some embodiments, the N-terminus is residue A1 of the IL-2 polypeptide, wherein residue position numbering is based on SEQ ID NO: 1 as a reference sequence.
  • the N-terminus is residue P2 of the IL-2 polypeptide, wherein residue position numbering is based on SEQ ID NO: 1 as a reference sequence (e.g., the modified IL-2 polypeptide comprises a deletion of residue A1 from the sequence).
  • the polymer is attached at a residue position which blocks or diminished binding of the modified IL-2 polypeptide with the IL-2 receptor beta subunit. Such residues positions are provided in U.S. Pat. Publication No.
  • 20200231644A1 which is hereby incorporated by reference as if set forth herein in its entirety, and include, for example, residue positions K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126.
  • the polymer comprises a water-soluble polymer.
  • the water-soluble polymer comprises poly(alkylene oxide), polysaccharide, poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), or a combination thereof.
  • the water-soluble polymer is poly(alkylene oxide).
  • the water-soluble polymer is polysaccharide.
  • the water-soluble polymer is poly(ethylene oxide).
  • a modified IL-2 polypeptide described herein comprises a polymer covalently attached to the N-terminus of the IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises a second polymer covalently attached thereto. In some embodiments, the modified IL-2 polypeptide comprises a second and a third polymer covalently attached thereto.
  • the attached polymer such as the polymer has a weight average molecular weight of about 6,000 Daltons to about 50,000 Daltons. In some embodiments, the polymer has a weight average molecular weight of about 6,000 Daltons to about 10,000 Daltons, about 6,000 Daltons to about 30,000 Daltons, about 6,000 Daltons to about 50,000 Daltons, about 10,000 Daltons to about 30,000 Daltons, about 10,000 Daltons to about 50,000 Daltons, or about 30,000 Daltons to about 50,000 Daltons. In some embodiments, the polymer has a weight average molecular weight of about 6,000 Daltons, about 10,000 Daltons, about 30,000 Daltons, or about 50,000 Daltons.
  • the polymer has a weight average molecular weight of at least about 6,000 Daltons, about 10,000 Daltons, or about 30,000 Daltons. In some embodiments, the polymer has a weight average molecular weight of at most about 10,000 Daltons, about 30,000 Daltons, or about 50,000 Daltons.
  • the attached polymer such as the polymer attached to the N-terminus has a weight average molecular weight of about 120 Daltons to about 1,000 Daltons. In some embodiments, the polymer has a weight average molecular weight of about 120 Daltons to about 250 Daltons, about 120 Daltons to about 300 Daltons, about 120 Daltons to about 400 Daltons, about 120 Daltons to about 500 Daltons, about 120 Daltons to about 1,000 Daltons, about 250 Daltons to about 300 Daltons, about 250 Daltons to about 400 Daltons, about 250 Daltons to about 500 Daltons, about 250 Daltons to about 1,000 Daltons, about 300 Daltons to about 400 Daltons, about 300 Daltons to about 500 Daltons, about 300 Daltons to about 1,000 Daltons, about 400 Daltons to about 500 Daltons, about 400 Daltons to about 1,000 Daltons, or about 500 Daltons to about 1,000 Daltons.
  • the polymer has a weight average molecular weight of about 120 Daltons, about 250 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, or about 1,000 Daltons. In some embodiments, the polymer has a weight average molecular weight of at least about 120 Daltons, about 250 Daltons, about 300 Daltons, about 400 Daltons, or about 500 Daltons. In some embodiments, the polymer has a weight average molecular weight of at most about 250 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, or about 1,000 Daltons.
  • the attached polymer comprises a water-soluble polymer.
  • the water-soluble polymer comprises poly(alkylene oxide), polysaccharide, poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), or a combination thereof.
  • the water-soluble polymer is poly(alkylene oxide) such as polyethylene glycol (i.e., polyethylene oxide).
  • the water-soluble polymer is polyethylene glycol.
  • the water-soluble polymer comprises modified poly(alkylene oxide).
  • the modified poly(alkylene oxide) comprises one or more linker groups.
  • the one or more linker groups comprise bifunctional linkers such as an amide group, an ester group, an ether group, a thioether group, a carbonyl group and alike.
  • the one or more linker groups comprise an amide linker group.
  • the modified poly(alkylene oxide) comprises one or more spacer groups.
  • the spacer groups comprise a substituted or unsubstituted C 1 -C 6 alkylene group.
  • the spacer groups comprise —CH 2 —, —CH 2 CH 2 —, or —CH 2 CH 2 CH 2 —.
  • the linker group is the product of a biorthogonal reaction (e.g., biocompatible and selective reactions).
  • the bioorthogonal reaction is a Cu(I)-catalyzed or “copper-free” alkyne-azide triazole-forming reaction, the Staudinger ligation, inverse-electron-demand Diels-Alder (IEDDA) reaction, “photo-click” chemistry, or a metal-mediated process such as olefin metathesis and Suzuki- Miyaura or Sonogashira cross-coupling.
  • the polymer is attached to the IL-2 polypeptide via click chemistry.
  • a modified IL-2 polypeptide provided herein comprises a reaction group that facilitates the conjugation of the modified IL-2 polypeptide with a derivatized molecule or moiety such as an antibody and a polymer (e.g., an additional larger polymer).
  • the reaction group comprises one or more of: carboxylic acid derived active esters, mixed anhydrides, acyl halides, acyl azides, alkyl halides, N-maleimides, imino esters, isocyanates, and isothiocyanates.
  • the reaction group comprises azides.
  • the reaction group comprises alkynes.
  • the polymer comprises a conjugation handle which can be used to further attach an additional moiety to the modified IL-2 polypeptide (e.g., the addition of an additional polypeptide, such as an antibody).
  • an additional moiety e.g., the addition of an additional polypeptide, such as an antibody.
  • Any suitable reactive group capable of reacting with a complementary reactive group attached to another moiety can be used as the conjugation handle.
  • the polymer comprises a conjugation handle or a reaction product of a conjugation handle with a complementary conjugation handle.
  • the reaction product of the conjugation handle with the complementary conjugation handle results from a KAT ligation (reaction of potassium acyltrifluoroborate with hydroxylamine), a Staudinger ligation (reaction of an azide with a phosphine), a tetrazine cycloaddition (reaction of a tetrazine with a trans-cyclooctene), or a Huisgen cycloaddition (reaction of an alkyne with an azide).
  • KAT ligation reaction of potassium acyltrifluoroborate with hydroxylamine
  • Staudinger ligation reaction of an azide with a phosphine
  • tetrazine cycloaddition reaction of a tetrazine with a trans-cyclooctene
  • Huisgen cycloaddition reaction of an al
  • the polymer comprises a reaction product of a conjugation handle with a complementary conjugation handle which was used to attach the polymer to the modified IL-2 polypeptide.
  • the polymer comprises an azide moiety.
  • the polymer comprises an alkyne moiety.
  • the polymer comprises an azide moiety, an alkyne moiety, or reaction product of an azide-alkyne cycloaddition reaction.
  • the reaction product of the azide-alkyne cycloaddition reaction is a 1,2,3-triazole.
  • the polymer is attached to the modified IL-2 polypeptide through use of a bifunctional linker.
  • the bifunctional linker reacts with a reactive group of an amino acid residue on the modified IL-2 polypeptide (e.g., a cysteine sulfhydryl) to form a covalent bond.
  • the second reactive group of the bifunctional a linker e.g., a conjugation handle such as an azide or alkyne
  • a second moiety such as the polymer.
  • the polymer comprises a linker comprising a structure of Formula (X)
  • the polymer comprises a linker comprising a structure of Formula (X′)
  • a modified IL-2 polypeptide provided herein comprises a polymer which includes a linker selected from Table 4.
  • a linker selected from Table 4.
  • modified IL-2 polypeptide e.g., an amino group of the modified IL-2 polypeptide
  • polymeric portion of the polymer e.g., an amino group of the modified IL-2 polypeptide
  • the water-soluble polymer comprises from 1 to 10 polyethylene glycol chains. In some embodiments, the water-soluble polymer comprises 1 polyethylene glycol chains to 10 polyethylene glycol chains. In some embodiments, the first water-soluble polymer comprises 1 polyethylene glycol chains to 2 polyethylene glycol chains, 1 polyethylene glycol chains to 4 polyethylene glycol chains, 1 polyethylene glycol chains to 6 polyethylene glycol chains, 1 polyethylene glycol chains to 10 polyethylene glycol chains, 2 polyethylene glycol chains to 4 polyethylene glycol chains, 2 polyethylene glycol chains to 6 polyethylene glycol chains, 2 polyethylene glycol chains to 10 polyethylene glycol chains, 4 polyethylene glycol chains to 6 polyethylene glycol chains, 4 polyethylene glycol chains to 6 polyethylene glycol chains, 4 polyethylene glycol chains to 6 polyethylene glycol chains, 4 polyethylene glycol chains to 10 polyethylene glycol chains, or 6 polyethylene glycol chains to 10 polyethylene glycol chains.
  • the water-soluble polymer comprises 1 polyethylene glycol chains, 2 polyethylene glycol chains, 4 polyethylene glycol chains, 6 polyethylene glycol chains, or 10 polyethylene glycol chains. In some embodiments, the water-soluble polymer comprises at least 1 polyethylene glycol chains, 2 polyethylene glycol chains, 4 polyethylene glycol chains, or 6 polyethylene glycol chains. In some embodiments, the first water-soluble polymer comprises at most 2 polyethylene glycol chains, 4 polyethylene glycol chains, 6 polyethylene glycol chains, or 10 polyethylene glycol chains. In some embodiments, the water-soluble polymer comprises 4 polyethylene glycol chains. In some embodiments, the water-soluble polymer comprises a structure of Formula (I) wherein each m is independently an integer from 4-30. In some embodiments, at least one polyethylene glycol chain of the water-soluble polymer comprises the structure of Formula (II)
  • each polyethylene glycol chain of the water-soluble polymer comprises the structure of Formula (II).
  • m is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.
  • n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • each of the polyethylene glycol chains independently comprises from about 5 to about 300, from about 10 to about 200, from about 20 to about 100, or from about 25 to about 50 ethylene glycol units. In some embodiments, each of the polyethylene glycol chains independently comprises 5 ethylene glycol units to 300 ethylene glycol units.
  • each of the polyethylene glycol chains independently comprises 5 ethylene glycol units to 10 ethylene glycol units, 5 ethylene glycol units to 20 ethylene glycol units, 5 ethylene glycol units to 25 ethylene glycol units, 5 ethylene glycol units to 50 ethylene glycol units, 5 ethylene glycol units to 100 ethylene glycol units, 5 ethylene glycol units to 200 ethylene glycol units, 5 ethylene glycol units to 300 ethylene glycol units, 10 ethylene glycol units to 20 ethylene glycol units, 10 ethylene glycol units to 25 ethylene glycol units, 10 ethylene glycol units to 50 ethylene glycol units, 10 ethylene glycol units to 100 ethylene glycol units, 10 ethylene glycol units to 200 ethylene glycol units, 10 ethylene glycol units to 300 ethylene glycol units, 20 ethylene glycol units to 25 ethylene glycol units, 20 ethylene glycol units to 50 ethylene glycol units, 20 ethylene glycol units to 100 ethylene glycol units, 20 ethylene glycol units to 200 ethylene glycol units, 10 ethylene glycol units
  • each of the polyethylene glycol chains independently comprises 5 ethylene glycol units, 10 ethylene glycol units, 20 ethylene glycol units, 25 ethylene glycol units, 50 ethylene glycol units, 100 ethylene glycol units, 200 ethylene glycol units, or 300 ethylene glycol units. In some embodiments, each of the polyethylene glycol chains independently comprises at least 5 ethylene glycol units, 10 ethylene glycol units, 20 ethylene glycol units, 25 ethylene glycol units, 50 ethylene glycol units, 100 ethylene glycol units, or 200 ethylene glycol units.
  • each of the polyethylene glycol chains independently comprises at most 10 ethylene glycol units, 20 ethylene glycol units, 25 ethylene glycol units, 50 ethylene glycol units, 100 ethylene glycol units, 200 ethylene glycol units, or 300 ethylene glycol units.
  • each of the polyethylene glycol chains is independently linear or branched. In some embodiments, each of the polyethylene glycol chains is a linear polyethylene glycol. In some embodiments, each of the polyethylene glycol chains is a branched polyethylene glycol. For example, in some embodiments, each of the first and the second polymers comprises a linear polyethylene glycol chain.
  • each of the polyethylene glycol chains is independently terminally capped with a hydroxy, an alkyl, an alkoxy, an amido, or an amino group. In some embodiments, each of the polyethylene glycol chains is independently terminally capped with an amino group. In some embodiments, each of the polyethylene glycol chains is independently terminally capped with an amido group. In some embodiments, each of the polyethylene glycol chains is independently terminally capped with an alkoxy group. In some embodiments, each of the polyethylene glycol chains is independently terminally capped with an alkyl group. In some embodiments, each of the polyethylene glycol chains is independently terminally capped with a hydroxy group. In some embodiments, one or more of the polyethylene glycol chains independently has the structure
  • n is an integer from 4-30.
  • one or more of the polyethylene glycol chains independently has the structure
  • n is an integer from 4-30.
  • the modified IL-2 polypeptide comprises multiple polymers covalently attached thereto.
  • each polymer comprises a water-soluble polymer.
  • the water-soluble polymer comprises poly(alkylene oxide), polysaccharide, poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), or a combination thereof.
  • each water-soluble polymer is poly(alkylene oxide).
  • each water-soluble polymer is polyethylene glycol.
  • the modified IL-2 polypeptide comprises from 1 to 10 covalently attached water-soluble polymers. In some embodiments, the modified IL-2 polypeptide comprises 1 to 10 covalently attached water-soluble polymers. In some embodiments, the modified IL-2 polypeptide comprises 1 or 2 covalently attached water-soluble polymers, 1 to 3 covalently attached water-soluble polymers, 1 to 4 covalently attached water-soluble polymers, 1 to 6 covalently attached water-soluble polymers, 1 to 8 covalently attached water-soluble polymers, 1 to 10 covalently attached water-soluble polymers, 2 or 3 covalently attached water-soluble polymers, 2 to 4 covalently attached water-soluble polymers, 2 to 6 covalently attached water-soluble polymers, 2 to 8 covalently attached water-soluble polymers, 2 to 10 covalently attached water-soluble polymers, 3 or 4 covalently attached water-soluble polymers, 3 to 6 covalently attached water-soluble polymers, 3 to 8 covalently attached water-soluble polymers, 3 to 10 covalently attached water
  • a water-soluble polymer that can be attached to a modified IL-2 polypeptide comprises a structure of Formula (A):
  • a water-soluble polymer that can be attached to a modified IL-2 polypeptide comprises a structure of Formula (B):
  • a water-soluble polymer that can be attached to a modified IL-2 polypeptide comprises a structure of Formula (C):
  • a water-soluble polymer that can be attached to a modified IL-2 polypeptide comprises a structure of Formula (D):
  • a water-soluble polymer that can be attached to a modified IL-2 polypeptide comprises a structure of Formula (E):
  • the water-soluble polymer attached to the modified IL-2 polypeptide comprises one or more linkers and/or spacers. In some embodiments, the one or more linkers comprise one or more amide groups. In some embodiments, the one or more linkers comprise one or more lysine groups. .In some embodiment, the water-soluble polymer attached to the modified IL-2 polypeptide comprises a structure of Formula (I), Formula (II), Formula (III), or a combination thereof. In some embodiments, the water-soluble polymer attached to the modified IL-2 polypeptide comprises a structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E) or a combination thereof. In some embodiments, the water-soluble polymer attached to the modified IL-2 polypeptide comprises a structure of
  • the water-soluble polymer attached at the N-terminus comprises one or more linkers and/or spacers. In some embodiments, the one or more linkers comprise one or more amide groups. In some embodiments, the one or more linkers comprise one or more lysine groups. In some embodiment, the water-soluble polymer attached at the N-terminus comprises a structure of Formula (I), Formula (II), Formula (III), or a combination thereof. In some embodiments, the water-soluble polymer attached at the N-terminus comprises a structure of Formula (A), Formula (B), Formula (C), Formula (D), Formula (E), or a combination thereof. In some embodiments, the water-soluble polymer attached comprises a structure of
  • the polymers are synthesized from suitable precursor materials. In some embodiments, the polymers are synthesized from the precursor materials of, Structure 5, Structure 6, Structure 7, or Structure 8, wherein Structure 5 is Structure 6 is Structure 7 is and Structure 8 is
  • a pharmaceutical formulation comprising: a modified IL-2 polypeptide described herein; and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical formulation comprises a plurality of the modified IL-2 polypeptides.
  • the pharmaceutical formulations further comprises one or more excipient selected from a carbohydrate, an inorganic salt, an antioxidant, a surfactant, or a buffer.
  • the pharmaceutical formulation further comprises a carbohydrate.
  • the carbohydrate is selected from the group consisting of fructose, maltose, galactose, glucose, D-mannose, sorbose, lactose, sucrose, trehalose, cellobiose raffinose, melezitose, maltodextrins, dextrans, starches, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, cyclodextrins, and combinations thereof.
  • the pharmaceutical formulation comprises an inorganic salt.
  • the inoragnic salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium sulfate, or combinations thereof.
  • the pharmaceutical formulation comprises an antioxidant.
  • the antioxidant is selected from the group consisting of ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, potassium metabisulfite, propyl gallate, sodium metabisulfite, sodium thiosulfate, vitamin E, 3,4-dihydroxybenzoic acid, and combinations thereof.
  • the pharmaceutical formulation comprises a surfactant.
  • the surfactant is selected from the group consisting of polysorbates, sorbitan esters, lipids, phospholipids, phosphatidylethanolamines, fatty acids, fatty acid esters, steroids, EDTA, zinc, and combinations thereof.
  • the pharmaceutical formulation comprises a buffer.
  • the buffer is selected from the group consisting of citric acid, sodium phosphate, potassium phosphate, acetic acid, ethanolamine, histidine, amino acids, tartaric acid, succinic acid, fumaric acid, lactic acid, tris, HEPES, or combinations thereof.
  • the pharmaceutical formulation is prepared for parenteral or enteral administration. In some embodiments, the pharmaceutical formulation is formulated for intravenous or subcutaneous administration. In some embodiments, the pharmaceutical formulation is in a lyophilized form.
  • a liquid or lyophilized formulation that comprises a described modified IL-2 polypeptide.
  • the modified IL-2 polypeptide is a lyophilized powder.
  • the lyophilized powder is resuspended in a buffer solution.
  • the buffer solution comprises a buffer, a sugar, a salt, a surfactant, or any combination thereof.
  • the buffer solution comprises a phosphate salt.
  • the phosphate salt is sodium Na 2 HPO 4 .
  • the salt is sodium chloride.
  • the buffer solution comprises phosphate buffered saline.
  • the buffer solution comprises mannitol.
  • the lyophilized powder is suspended in a solution comprising 10 mM Na 2 HPO 4 buffer pH 7.5, 0.022% SDS and 50 mg/mL mannitol.
  • the modified IL-2 polypeptides described herein can be in a variety of dosage forms.
  • the modified IL-2 polypeptide is dosed as a lyophilized powder.
  • the modified IL-2 polypeptide is dosed as a suspension.
  • the modified IL-2 polypeptide is dosed as a solution.
  • the modified IL-2 polypeptide is dosed as an injectable solution.
  • the modified IL-2 polypeptides is dosed as an IV solution.
  • a method of treating an autoimmune disease or disorder in a subject in need thereof comprising: administering to the subject an effective amount of a modified IL-2 polypeptide or a pharmaceutical composition as described herein.
  • a method of treating an inflammatory disease or disorder in a subject in need thereof comprising: administering to the subject an effective amount of a modified IL-2 polypeptide or a pharmaceutical composition as described herein.
  • the autoimmune disease is a T cell mediated autoimmune disease.
  • the inflammatory disease or disorder comprises inflammation (e.g., cartilage inflammation), an autoimmune disease, an atopic disease, a paraneoplastic autoimmune disease, arthritis, rheumatoid arthritis (e.g., active), juvenile arthritis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, pauciarticular rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile psoriatic arthritis, psoriatic arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, reactive arthritis, Reiter’s syndrome, juvenile Reiter’s syndrome, juvenile dermatomyositis, juvenile scleroderma, juvenile vas
  • inflammation e.g.
  • kidney, lung, heart, skin, and the like kidney damage, hepatitis C-induced vasculitis, spontaneous loss of pregnancy, vitiligo, focal segmental glomerulosclerosis (FSGS), minimal change disease, membranous nephropathy, ANCA-associated Glomerulonephropathy, Membranoproliferative Glomerulonephritis, IgA nephropathy, lupus nephritis, or a combination thereof.
  • FGS focal segmental glomerulosclerosis
  • the inflammatory disease or disorder is a neuroinflammatory disorder.
  • the neuroinflammatory disorder is neuromyelitis optica spectrum disorder, multiple sclerosis, anti-myelin oligodendrocyte glycoprotein antibody disorder, autoimmune encepahlitis, transverse myelitis, optic neuritis, or neurosarcoidosis.
  • the diesease or disorder is amyotrophic lateral sclerosis.
  • a method of making a modified IL-2 polypeptide in one aspect, described herein, is a method of making a modified IL-2 polypeptide. In another aspect, described herein, is a method of making a modified IL-2 polypeptide comprising synthesizing two or more fragments of the modified IL-2 polypeptide and ligating the fragments. In another aspect, described herein, is a method of making a modified IL-2 polypeptide comprising a. synthesizing two or more fragments of the modified IL-2 polypeptide, b. ligating the fragments; and c. folding the ligated fragments.
  • Examples of methods synthesizing IL-2 polypeptides can also be found in, for example, at least PCT Publication No WO2021140416A2, US Patent Application Publication No US20190023760A1, and Asahina et al., Angew. Chem. Int. Ed. 2015, 54, 8226-8230, each of which is incorporated by reference as if set forth herein in its entirety.
  • the two or more fragments of the modified IL-2 polypeptide are synthesized chemically. In some embodiments, the two or more fragments of the modified IL-2 polypeptide are synthesized by solid phase peptide synthesis. In some embodiments, the two or more fragments of the modified IL-2 polypeptide are synthesized on an automated peptide synthesizer.
  • the modified IL-2 polypeptide is ligated from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more peptide fragments. In some embodiments, the modified peptide is ligated from 2 peptide fragments. In some embodiments, the modified IL-2 polypeptide is ligated from 3 peptide fragments. In some embodiments, the modified IL-2 polypeptide is ligated from 4 peptide fragments. In some embodiments, the modified IL-2 polypeptide is ligated from 2 to 10 peptide fragments.
  • the two or more fragments of the modified IL-2 polypeptide are ligated together. In some embodiments, three or more fragments of the modified IL-2 polypeptide are ligated in a sequential fashion. In some embodiments, three or more fragments of the modified IL-2 polypeptide are ligated in a one-pot reaction.
  • ligated fragments are folded. In some embodiments, folding comprises forming one or more disulfide bonds within the modified IL-2 polypeptide. In some embodiments, the ligated fragments are subjected to a folding process. In some embodiments, the ligated fragments are folding using methods well known in the art. In some embodiments, the ligated polypeptide or the folded polypeptide are further modified by attaching one or more polymers thereto. In some embodiments, the ligated polypeptide or the folded polypeptide are further modified by PEGylation.
  • the modified IL-2 polypeptide is synthetic.
  • the modified IL-2 polypeptide is recombinant.
  • a host cell comprising a modified IL-2 polypeptide.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the host cell is a mammalian cell, an avian cell, and an insect cell.
  • the host cell is a CHO cell, a COS cell, or a yeast cell.
  • a method of producing a modified IL-2 polypeptide comprising expressing the modified IL-2 polypeptide in a host cell.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the host cell is a mammalian cell, an avian cell, and an insect cell.
  • the host cell is a CHO cell, a COS cell, or a yeast cell.
  • Nle is a norleucine residue and Hse is a homoserine residue.
  • a modified IL-2 polypeptide as described herein such as a modified IL-2 polypeptide having an amino acid sequence of, for example, SEQ ID NO: 3, or any of SEQ ID NOs: 3-8 or 40-43, or a synthetic version of any one of SEQ ID NOs: 9-39, or a modified IL-2 polypeptide otherwise described herein, can be synthesized by ligating individual peptide segments prepared by solid phase peptide synthesis (SPPS). Individual peptides are synthesized on an automated peptide synthesizer using the methods described below.
  • SPPS solid phase peptide synthesis
  • Fmoc-amino acids with suitable side chain protecting groups for Fmoc-SPPS, resins polyethylene glycol derivatives used for peptide functionalization and reagents were commercially available and were used without further purification.
  • HPLC grade CH 3 CN was used for analytical and preparative RP-HPLC purification.
  • Fmoc-AA-protected- ⁇ -ketoacid (1.8 mmol, 1.00 equiv.) was dissolved in 20 mL DMF and pre-activated with HATU (650 mg, 1.71 mmol, 0.95 equiv.) and DIPEA (396 ⁇ L, 3.6 mmol, 2.00 equiv.).
  • the reaction mixture was added to the swollen resin. It was let to react for 6 h at r.t. under gentle agitation. The resin was rinsed thoroughly with DMF. Capping of unreacted amines on the resin was performed by addition of a solution of acetic anhydride (1.17 mL) and DIPEA (2.34 mL) in DMF (20 mL).
  • Pre-activation was performed at r.t. for 3 min by addition of DIPEA (585 ⁇ L, 3.36 mmol, 1.5 equiv).
  • the reaction mixture was added to the swollen resin. It was let to react overnight at r.t. under gentle agitation. The resin was rinsed thoroughly with DMF.
  • Capping of unreacted amines on the resin was initiated by addition of a solution of acetic anhydride (1.27 mL) and DIPEA (2.34 mL) in DMF (12 mL). It was let to react at r.t. for 15 min under gentle agitation. The resin was rinsed thoroughly with DCM and dried. The loading of the resin was measured (0.34 mmol/g).
  • Solid-phase peptide synthesis SPPS: The peptide segments were synthesized on an automated peptide synthesizer using Fmoc-SPPS chemistry. The following Fmoc-amino acids with side-chain protecting groups were used: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Nle-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, F
  • Fmoc-pseudoproline dipeptides were incorporated in the synthesis if necessary. Fmoc deprotections were performed with 20% piperidine in DMF (2 ⁇ 8 min) or 25% piperidine in DMF containing 0.1 M Cl-HOBt (2 ⁇ 8 min) or 20% piperidine in DMF containing 0.1 M Cl-HOBt (2 ⁇ 8 min) and monitored by UV at 304 nm with a feedback loop to ensure complete Fmoc removal.
  • Couplings were performed with Fmoc-amino acid (3.0 - 5.0 equiv to resin substitution), HCTU or HATU (2.9 - 4.9 equiv) as coupling reagents and DIPEA or NMM (6 - 10 equiv) in DMF at r.t. or at 50° C. After pre-activation for 3 min, the solution containing the reagents was added to the resin and allowed to react for 30 min or 2 h depending on the amino acid. In some cases, double couplings were required. In some cases, the resin was treated with 20% acetic anhydride in DMF for capping any unreacted free amine.
  • IL-2 Seg1 (1.2 equiv) and IL-2 Seg2 (1 equiv) were dissolved in DMSO:H 2 O (9:1, v/v) containing 0.1 M oxalic acid (20 mM peptide concentration) and allowed to react at 60° C. for 22 h.
  • the ligation vial was protected from light by wrapping it in aluminum foil.
  • the progress of the KAHA ligation was monitored by HPLC using a C18 column (4.6 ⁇ 150 mm) at 60° C. with CH 3 CN/H 2 O containing 0.1% TFA as mobile phase, with a gradient of 5 to 95% CH 3 CN in 7 min.
  • IL2-Seg3 (1.2 equiv) and IL2-Seg4 (1 equiv) were dissolved in DMSO/H 2 O (9.8:0.2) containing 0.1 M oxalic acid (15 mM) and allowed to react for 20 h at 60° C.
  • the progress of the KAHA ligation was monitored by HPLC using a C18 column (4.6 ⁇ 150 mm) at 60° C. using CH 3 CN/H 2 O containing 0.1 %TFA as mobile phase, with a gradient of 30 to 70 % CH 3 CN in 7 min.
  • reaction mixture was diluted with DMSO (6 mL), 5% of diethylamine (300 ⁇ L) was added and the reaction mixture was shaken for 7 min at room temperature. To prepare the sample for purification, it was diluted with DMSO (4 mL) containing TFA (300 ⁇ L).
  • IL2-Seg12 (1.2 equiv) and IL2-Seg34 (1 equiv) were dissolved in DMSO/H 2 O (9:1) or (9.8:0.2) containing 0.1 M oxalic acid (15 mM peptide concentration) and the ligation was allowed to proceed for 24 h at 60° C.
  • the progress of the KAHA ligation was monitored by analytical HPLC using a C18 column (4.6 ⁇ 250 mm) at 60° C. and CH 3 CN/H 2 O containing 0.1 %TFA as mobile phase, with a gradient of 30 to 95 % CH 3 CN in 14 min.
  • reaction mixture was diluted with DMSO followed by further dilution with a mixture of (1:1) CH 3 CN:H 2 O containing 0.1 % TFA (7 mL).
  • the sample was purified by injecting on a preparative HPLC.
  • Peptide segments, ligated peptides and linear proteins were purified by RP-HPLC. Different gradients were applied for the different peptides.
  • the mobile phase was MilliQ-H 2 O with 0.1% TFA (v/v) (Buffer A) and HPLC grade CH 3 CN with 0.1% TFA (v/v) (Buffer B).
  • Preparative HPLC was performed on a (50 ⁇ 250 mm) or on a C18 column (50 ⁇ 250 mm) at a flow rate of 40 mL/min at 40° C. or 60° C.
  • FTMS Fourier-transform mass spectrometry
  • SolariX (9.4 T magnet) spectrometer
  • HCCA 4-hydroxy- ⁇ -cyanocinnamic acid
  • the mass of the dried peptidyl resin was 1.6 g.
  • the crude peptide was precipitated following the procedure “Resin cleavage and side-chain deprotection of the peptides” using a cocktail of 95:2.5:2.5 TFA/DODT/H 2 O v/v/v (10 mL/g resin) at r.t. for 2.0 hours.
  • Opr-IL2(42-69)-Leu-photoprotected- ⁇ -ketoacid segment 2A (see 41-70 of SEQ ID NO: 3) was synthesized on a 0.2 mmol scale on Rink-Amide MBHA resin pre-loaded with Fmoc-Leu-photoprotected- ⁇ -ketoacid (0.8 g) with a substitution capacity of ⁇ 0.25 mmol/g.
  • Automated Fmoc-SPPS of segment 2A was performed following the general procedure “Solid-phase peptide synthesis (SPPS)”. The resin was washed with DCM and dried under vacuum. The mass of the dried peptidyl resin was 1.8 g.
  • the crude peptide was precipitated following the procedure “Resin cleavage and side-chain deprotection of the peptides” using a cocktail of 95:2.5:2.5 TFA/DODT/H 2 O v/v/v (15 mL/g resin) at r.t. for 2.0 hours.
  • Fmoc-Opr IL2 72-102
  • Phe- ⁇ -ketoacid segment 3A See residues 71-103 of SEQ ID NO: 3
  • Fmoc-Opr IL2 72-102
  • Phe- ⁇ -ketoacid segment 3A See residues 71-103 of SEQ ID NO: 3
  • Automated Fmoc-SPPS of segment 3A was performed following the general procedure “Solid-phase peptide synthesis (SPPS)”. The resin was washed with DCM and dried under vacuum. The mass of the dried peptidyl resin was 2.17 g.
  • the crude peptide was precipitated following the procedure “Resin cleavage and side-chain deprotection of the peptides” using a cocktail of 95:2.5:2.5 TFA/DODT/H 2 O v/v/v (10 mL/g resin) at r.t. for 2.0 hours.
  • Opr-IL2(105-133) segment 4A (See residues 104-133 of SEQ ID NO: 3) was synthesized on a 0.1 mmol scale on Wang resin pre-loaded with Fmoc-Thr-OH (0.294 g) with a substitution capacity of -0.34 mmol/g.
  • Automated Fmoc-SPPS of segment 4A was performed following the general procedure “Solid-phase peptide synthesis (SPPS)”. The resin was washed with DCM and dried under vacuum. The mass of the dried peptidyl resin was 725 mg.
  • the crude peptide was precipitated following the procedure “Resin cleavage and side-chain deprotection of the peptides” using a cocktail of 92.5:2.5:2.5:2.5 TFA/TIPS/DODT/H 2 O v/v/v/v (10 mL/g resin) at r.t. for 2 hours.
  • the segment 12A was obtained following the general procedure “Ligation of IL-2 segments 1 and 2 and photodeprotection” with 34 mg (6.56 ⁇ mol; 1.1 equiv.) of segment 1A and 19 mg (4.9 ⁇ mol; 1.0 equiv.) of segment 2A dissolved in 241 ⁇ L of 9.5:0.5 v/v DMSO/H 2 O solution containing 0.1 M oxalic acid.
  • segment 34A was obtained following the general procedure “Ligation of IL-2 segments 3 and 4 and Fmoc deprotection” with 69 mg (17.5 ⁇ mol; 1.1 equiv.) of segment 3A and 59 mg (16.6 ⁇ mol; 1.0 equiv.) of segment 4A dissolved in 1100 ⁇ L of 9.9:0.1 DMSO/H 2 O v/v containing 0.1 M oxalic acid.
  • the segment 1234A was obtained following the general procedure “Final ligation” with 45 mg (5.1 ⁇ mol; 1.2 equiv.) of segment 12A and 31 mg (16.6 ⁇ mol; 1.0 equiv.) of segment 34A dissolved in 220 ⁇ L of 9.5:0.5 DMSO/H 2 O v/v containing 0.1 M oxalic acid.
  • IL2-Seg1234-A linear protein (11.7 mg, 0.736 ⁇ mol) was dissolved in aqueous 6 M Gu ⁇ HCl containing 0.1 M Tris and 30 mM reduced glutathione (15 ⁇ M protein concentration) and the mixture was gently shaken at 50° C. for 2 hours.
  • Folding of the linear rearranged protein (method 1): After completion of rearrangement reaction, the sample was cooled to room temperature and diluted with 0.1 M Tris and 1.5 mM oxidized glutathione, pH 8.0 (5 ⁇ M protein concentration). The folding was allowed to proceed for 20 hours at room temperature. Then, the sample was acidified with TFA to pH 3 and purified by preparative HPLC using a C4 column (20 ⁇ 250 mm) kept at room temperature with a two-step gradient of 5 to 40 to 95% acetonitrile with 0.1% TFA in 60 min, at a flow rate of 10.0 mL/min, using CH 3 CN/H 2 O with 0.1% TFA (v/v) as mobile phase. The fractions containing the product were pooled and lyophilized to give pure folded protein Composition A as a white powder in 98% purity (2.2 mg, 19% yield for folding and purification steps).
  • IL-2 composition A folded protein (39.76 mg, 1 equiv.) were first dissolved in 8 mL of 10 mM sodium acetate buffer, 8.4% Sucrose, 0.02% polysorbate80 pH 5.0. The solution was supplemented with 22 mL 50 mM sodium acetate buffer pH 5.0 and 30 kDa DBCO-polyethylene glycol polymer (392.05 mg, 5 equiv) and the reaction was gently mixed at 25° C. for 17 hours.
  • the reaction mixture was loaded 10 mL at a time on a HiTrap Capto S ImpAct column (5 mL) and purified at a flow rate of 2.5 mL/min with a gradient of 20 CV from 50 mM sodium acetate buffer pH 5.0 to 50 mM sodium acetate buffer with 1 M NaCl.
  • the fractions containing the IL-2 composition A1 PEGylated protein were pooled together and dialyzed against 10 mM sodium acetate buffer, 8.4% Sucrose, 0.02% polysorbate80 pH 5.0 to obtain 12.26 mg of protein fraction IL-2 composition A1 PEGylated protein as determined by BCA (31% yield for PEGylation and purifications steps).
  • the purity and identity of the pure PEGylated protein was confirmed by analytical RP-HPLC, MALDI-TOF and analytical size exclusion.
  • Fmoc-Opr IL2 (72-102)-Phe- ⁇ -ketoacid segment 3B (See residues 71-103 of SEQ ID NO: 4) was synthesized by automated Fmoc-SPPS synthesis in analogy to the procedures described for the synthesis of segment 3A to yield segment 3B as a white solid in >98% purity. The isolated yield based on the resin loading was 18% (200 mg).
  • the segment 34B was obtained following the general procedure “Ligation of IL-2 segments 3 and 4 and Fmoc deprotection” with 37 mg (9.3 ⁇ mol; 1.2 equiv.) of segment 3B and 27 mg (7.8 ⁇ mol; 1.0 equiv.) of segment 4A dissolved in 517 ⁇ L of 9.8:0.2 DMSO/H 2 O v/v containing 0.1 M oxalic acid.
  • segment 1234B was obtained following the general procedure “Final ligation” with 34 mg (3.8 ⁇ mol; 1.2 equiv.) of segment 12A and 23 mg (3.2 ⁇ mol; 1.0 equiv.) of segment 34B dissolved in 214 ⁇ L of 9.5:0.5 DMSO/H 2 O v/v containing 0.1 M oxalic acid.
  • IL2-Seg1234-B linear protein (14.5 mg, 0.913 ⁇ mol) was dissolved in aqueous 6 M Gu-HCl containing 0.1 M Tris and 30 mM reduced glutathione (61 mL, 15 ⁇ M protein concentration) and the mixture was gently shaken at 50° C. for 2 hours.
  • the fractions containing the PEGylated IL2-Seg1234-B1 protein were pooled together and lyophilized to obtain 2.5 mg of IL2-Seg1234-B1 PEGylated protein as a white powder in 98% purity. (29% yield for PEGylation and purifications steps).
  • the purity and identity of the pure PEGylated protein was confirmed by analytical RP-HPLC, MALDI-TOF, SEC-HPLC and SDS-page.
  • Opr-IL2(42-69)-Leu-photoprotected-a-ketoacid segment 2C (See residues 41-70 of SEQ ID NO: 5) was synthesized by automated Fmoc-SPPS synthesis in analogy to the procedures described for the synthesis of segment 2A to yield segment 2C as a white solid in 94% purity.
  • the isolated yield based on the resin loading was 19.7% (153.7 mg).
  • MALDI-TOF was used to confirm the desired product mass was obtained.
  • the segment 12C was obtained following the general procedure “Ligation of IL-2 segments 1 and 2 and photodeprotection” with 90 mg of segment 1A (17.4 ⁇ mol; 1.1 equiv.) and 56 mg (14.5 ⁇ mol; 1.0 equiv.) of segment 2C dissolved in 1157 ⁇ L of 9.5:0.5 v/v DMSO/H 2 O solution containing 0.1 M oxalic acid.
  • segment 1234C was obtained following the general procedure “Final ligation” with 46.7 mg (5.24 ⁇ mol; 1.2 equiv.) of segment 12C and 32 mg (4.41 ⁇ mol; 1.0 equiv.) of segment 34A dissolved in 683 ⁇ L of 9.5:0.5 DMSO/H 2 O v/v containing 0.1 M oxalic acid.
  • IL2-Seg1234-C linear protein (16.6 mg, 1.04 ⁇ mol) was dissolved in aqueous 6 M Gu-HCl containing 0.1 M Tris and 30 mM reduced glutathione (69 mL, 15 ⁇ M protein concentration) and the mixture was gently shaken at 50° C. for 2 hours.
  • the fractions containing the PEGylated IL2-Seg1234-C1 protein were pooled together and lyophilized to obtain 1.4 mg of IL2-Seg1234-C1 PEGylated protein as a white powder in >99% purity. (25% yield for PEGylation and purifications steps). The purity and identity of the pure PEGylated protein was confirmed by analytical RP-HPLC and MALDI-TOF.
  • IL2 (1-39)-Leu- ⁇ -ketoacid segment 1D (See residues 1-40 of SEQ ID NO: 6) was synthesized by automated Fmoc-SPPS synthesis in analogy to the procedures described for the synthesis of segment 1A to yield segment 1D as a white solid in 98% purity.
  • the isolated yield based on the resin loading was 20% (209 mg).
  • MALDI-TOF was used to confirm the desired product mass was obtained.
  • the segment 12D was obtained following the general procedure “Ligation of IL-2 segments 1 and 2 and photodeprotection” with 60 mg (11.7 ⁇ mol; 1.1 equiv.) of segment 1D and 38 mg (9.7 ⁇ mol; 1.0 equiv.) of segment 2A dissolved in 780 ⁇ L of 9.5:0.5 v/v DMSO/H 2 O solution containing 0.1 M oxalic acid.
  • segment 1234D was obtained following the general procedure “Final ligation” with 35.2 mg (5.24 ⁇ mol; 1.2 equiv.) of segment 12D and 26 mg (3.62 ⁇ mol; 1.0 equiv.) of segment 34B dissolved in 270 ⁇ L of 9.5:0.5 DMSO/H 2 O v/v containing 0.1 M oxalic acid.
  • IL2-Seg1234-D linear protein (9 mg, 0.568 ⁇ mol) was dissolved in aqueous 6 M Gu-HCl containing 0.1 M Tris and 30 mM reduced glutathione (38 mL, 15 ⁇ M protein concentration) and the mixture was gently shaken at 50° C. for 2 hours.
  • fractions containing the PEGylated IL2-Seg1234-D1 protein were pooled together and lyophilized to obtain 0.1 mg of IL2-Seg1234-D1 PEGylated protein as a white powder in >98% purity. (11% yield for PEGylation and purifications steps).
  • IL2 (1-39)-Leu- ⁇ -ketoacid segment 1E (See residues 1-40 of SEQ ID NO: 7) was synthesized by automated Fmoc-SPPS synthesis in analogy to the procedures described for the synthesis of segment 1A to yield segment 1E as a white solid in 97% purity. The isolated yield based on the resin loading was 17% (180 mg).
  • the segment 12E was obtained following the general procedure “Ligation of IL-2 segments 1 and 2 and photodeprotection” with 60 mg (11.7 ⁇ mol; 1.1 equiv.) of segment 1E and 38 mg (9.7 ⁇ mol; 1.0 equiv.) of segment 2A dissolved in 780 ⁇ L of 9.5:0.5 v/v DMSO/H 2 O solution containing 0.1 M oxalic acid.
  • segment 1234E was obtained following the general procedure “Final ligation” with 17.5 mg (1.98 ⁇ mol; 1.2 equiv.) of segment 12E and 12 mg (1.66 ⁇ mol; 1.0 equiv.) of segment 34B dissolved in 256 ⁇ L of 9.5:0.5 DMSO/H 2 O v/v containing 0.1 M oxalic acid.
  • IL2-Seg1234-E linear protein (5.8 mg, 0.366 ⁇ mol) was dissolved in aqueous 6 M Gu-HCl containing 0.1 M Tris and 30 mM reduced glutathione (24 mL, 15 ⁇ M protein concentration) and the mixture was gently shaken at 50° C. for 2 hours.
  • the fractions containing the PEGylated IL2-Seg1234-E1 protein were pooled together and lyophilized to obtain 0.2 mg of IL2-Seg1234-E1 PEGylated protein as a white powder in >98% purity. (12% yield for PEGylation and purifications steps). The purity and identity of the pure PEGylated protein was confirmed by analytical RP-HPLC, MALDI-TOF.
  • Opr-IL2(42-69)-Leu-photoprotected- ⁇ -ketoacid segment 2F was synthesized by automated Fmoc-SPPS synthesis in analogy to the procedures described for the synthesis of segment 2A to yield segment 2F as a white solid in 99% purity.
  • the isolated yield based on the resin loading was 35% (269 mg).
  • the segment 12F was obtained following the general procedure “Ligation of IL-2 segments 1 and 2 and photodeprotection” with 34 mg (6.6 ⁇ mol; 1.1 equiv.) of segment 1A and 19 mg (4.9 ⁇ mol; 1.0 equiv.) of segment 2F dissolved in 385 ⁇ L of 9.5:0.5 v/v DMSO/H 2 O solution containing 0.1 M oxalic acid.
  • segment 1234F was obtained following the general procedure “Final ligation” with 25.5 mg (2.89 ⁇ mol; 1.2 equiv.) of segment 12F and 17.5 mg (2.42 ⁇ mol; 1.0 equiv.) of segment 34B dissolved in 373 ⁇ L of 9.5:0.5 DMSO/H 2 O v/v containing 0.1 M oxalic acid.
  • IL2-Seg1234-F linear protein 8.7 mg, 0.549 ⁇ mol was dissolved in aqueous 6 M Gu-HCl containing 0.1 M Tris and 30 mM reduced glutathione (37 mL, 15 ⁇ M protein concentration) and the mixture was gently shaken at 50° C. for 2 hours.
  • the fractions containing the product were pooled and lyophilized to give pure folded IL2-Seg1234-F (0.2 mg, 2% yield for folding and purification steps).
  • the purity and identity of the pure folded protein was confirmed by analytical RP-HPLC and MALDI-TOF.
  • T-cells were obtained from healthy donor buffy coat by peripheral blood mononuclear cells (PBMC) purification using ficoll gradient centrifugation followed by negative isolation with magnetic beads and then cryopreserved until further use. Pan T-cells were thawed and incubated overnight in T-cell medium (RPMI 10% FCS, 1% Glutamine, 1% NEAA, 25) ⁇ M ⁇ MeoH, 1% NaPyruvate) followed by two washing steps with PBS.
  • PBMC peripheral blood mononuclear cells
  • Cells were resuspended in PBS and distributed at 200,000 cells per well followed by incubation for 40 min at 37° C./5% CO 2 with either aldesleukin, unconjugated IL-2 polypeptide (Composition A), PEGylated IL-2 polypeptide (Composition A1), or another indicated variant provided herein. After incubation, cells were fixed and permeabilized using the Transcription Factor Phospho Buffer kit followed by a surface and intracellular immunostaining for CD4, CD8, CD25, FoxP3 and pSTAT5 to enable cell subset identification and measure of levels of STAT5 phosphorylation. The FACS measurement was done either with a NovoCyte or a Quanteon Flow Cytometer from Acea.
  • Tregs were classified as CD4+CD25+FoxP3+ cells and Teff as CD8+ T cells.
  • EC50 results of STAT5 phosphorylation assay from the indicated variants in various immune cell types is shown below in Table 6. More detailed information about the data found in Table 6 below can be found in FIG. 4 C and FIG. 4 D .
  • both the unconjugated and PEGylated IL-2 polypeptide activated STAT5 in Tregs from mouse and cynomolgus monkeys with comparable potency to that for human Tregs, thus demonstrating cross-reactivity to mouse and cynomolgus IL-2R (Table 7).
  • Example 9 Binding Affinity of PEGylated IL-2 Polypeptide (Composition A1) to ⁇ and ⁇ Subunits of the IL-2 Receptor
  • the binding affinities of PEGylated IL-2 polypeptide (Composition A1) and SEQ ID NO: 2 (aldesleukin) to the IL-2R alpha and beta subunits was measured using bio-layer interferometry (BLI) technology.
  • Biotinylated IL-R2 ⁇ and ⁇ were individually loaded onto Streptavidin Biosensors SAX2 and the sensor immersed into 1x Octet® kinetic buffer to set the baseline.
  • the sensors were incubated in the analyte solution for 300 s followed by an incubation in kinetics buffer for 600 s to measure the dissociation.
  • Octet® Analysis studio was used to calculate Kd values.
  • Composition A1 had a higher affinity for the alpha subunit than aldesleukin (2.37 versus 12.23 nM, respectively) whereas binding to the beta subunit was abolished ( FIG. 5 ). Composition A1 therefore represents an ⁇ -enhanced, ⁇ -dead IL-2 polypeptide.
  • PK/PD Single-dose pharmacokinetic/pharmacodynamic studies were performed in C57BL/6 mice receiving 5 daily subcutaneous (sc) injections of 0.3 mg/kg protein equivalents of aldesleukin or a single subcutaneous injection of 0.1 or 0.3 mg/kg of PEGylated IL-2 polypeptide Composition A1. Blood was sampled at various timepoints in K 2 EDTA, plasma was generated by centrifugation and stored at -80° C. until PK analysis and cell pellets were freshly subjected to staining for flow cytometry analysis.
  • Cell pellets were treated with 1 ⁇ Lyse/Fix buffer (BD Bioscience, 558050) during 10 min. After washing, cells were stained with anti-CD3, ant-CD335, and anti-CD25 antibodies for 30 min at 4° C. Cells were then permeabilized using cold BD Perm Buffer III and stained with antibodies against Ki67, Siglec-F, CD4, CD8, FoxP3, CD62L, CD44 or pSTAT5.
  • the FACS fluorescence activated cell sorting
  • Concentrations of Composition A1 in plasma were determined using a qualified human IL-2 LegendPlex bead assay (Biolegend, #740717, #740368, #740758).
  • PK data were subjected to a non-compartmental PK analysis by using the Phoenix WinNonlin software version 6.3.
  • the linear/log trapezoidal rule was applied in obtaining the PK parameters.
  • the PK profile of the modified IL-2 polypeptide ( FIG. 6 ) peaked at 6 hours and concentration declined with a long half-life between 26 and 30 hours by virtue of the PEGylation.
  • PK parameters (Table 8) showed a dose proportional increase in exposure. This PK profile is superior to the wild-type polypeptide whose half-life in mouse is reported to be within minutes.
  • the immuno PD profiles of aldesleukin and PEGylated IL-2 polypeptide Composition A1 were assessed concurrently in the same study and monitored for 14 days.
  • a single dose treatment with the PEGylated IL-2 polypeptide led to a strong and sustained STAT5 phosphorylation in the Treg population (CD3+, CD4+, CD25Hi, FoxP3+) with no or minimal effect on CD8+ Teff cells(CD3+, CD8+, CD4-) and NK cells (CD3-, CD49b+) ( FIG. 7 ).
  • a 5-dose treatment with aldesleukin led to a more modest STAT5 phosphorylation profile in Treg cells and also no effect on CD8+ T eff cells and NK cells ( FIG.
  • Example 11 Composition A1 Suppresses Keyhole Limpet Hemocyanin-Induced Delayed Type Hypersensitivity
  • Delayed type hypersensitivity represents a local T effector recall response to a previously encountered antigen.
  • mice are first sensitized to keyhole limpet hemocyanin (KLH) by immunization s.c. with KLH and then rechallenged several days later with an intradermal injection of the same antigen into the ear resulting in local tissue inflammation and swelling.
  • KLH keyhole limpet hemocyanin
  • Composition A1 was administered at 0.3 mg/kg s.c.
  • Composition A1 therefore potently suppresses antigen-driven tissue inflammation.

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WO2026083295A1 (en) 2024-10-16 2026-04-23 Bright Peak Therapeutics Ag Trispecific compositions comprising il-2, vegf binding domains, and pd-1 binding domains

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