WO2025072443A2 - Polypeptides il-21 activables et leurs procédés d'utilisation et anticorps d'albumine sérique humaine à domaine unique - Google Patents

Polypeptides il-21 activables et leurs procédés d'utilisation et anticorps d'albumine sérique humaine à domaine unique Download PDF

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WO2025072443A2
WO2025072443A2 PCT/US2024/048555 US2024048555W WO2025072443A2 WO 2025072443 A2 WO2025072443 A2 WO 2025072443A2 US 2024048555 W US2024048555 W US 2024048555W WO 2025072443 A2 WO2025072443 A2 WO 2025072443A2
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amino acid
acid sequence
compound
variant
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WO2025072443A3 (fr
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William Winston
Heather R. BRODKIN
Jose Andres SALMERON-GARCIA
Philipp W. STEINER
Cynthia Seidel-Dugan
Jenna M. SULLIVAN
Amanda T. VO
Nels Peter NIELSON
Courtney E. PERCY
Daniel Hicklin
Tiziana CAFARELLI
Celesztina D. NAGY-DOMONKOS
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Werewolf Therapeutics Inc
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Werewolf Therapeutics Inc
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    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • Interleukin-21 is a is a cytokine that is produced mainly by T cells and natural killer T cells that has pleiotropic actions on a wide range of immune and non-immune cell types. IL-21 has diverse effects on a broad range of cell types including, but not limited to, CD4 + and CD8 + T cells, B cells, macrophages, monocytes, and dendritic cells (DCs).
  • the functional receptor for IL-21 is composed of the IL-21 receptor (IL-21R) and the common cytokine receptor ⁇ chain, which is also a subunit of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15.
  • IL-21 is a potent immune agonist and has been considered a promising therapeutic agent for oncology.
  • cytokines due to the biology of cytokines and the inability to effectively target and control their activity, cytokines have not achieved the hoped for clinical advantages in the treatment of tumors. This is exacerbated by the need to administer large quantities of cytokines (i.e., IL-21) in order to achieve the desired levels of cytokine at the intended site of cytokine action (e.g., a tumor microenvironment).
  • inducible IL-12, IL-2, and interferon prodrugs have been described in International Publication Nos.: WO2019/222294, WO2019/222295, WO2019/222296, WO2021/097376. [05] There remains an unmet medical need for improved methods for treating cancer. 2. SUMMARY [06] The disclosure relates to inducible IL-21 prodrugs that contain at least one polypeptide chain, and can contain two or more polypeptides, if desired.
  • the inducible IL-21 prodrug comprises an IL-21 polypeptide, an IL-21 blocking element, a half-life extension element, and a protease cleavable polypeptide linker.
  • Inducible IL-21 prodrugs of this disclosure have attenuated IL-21 receptor agonist activity and the circulating half-life is extended.
  • the inducible IL-21 receptor agonist activity is attenuated through the blocking element.
  • the half-life extension element can also contribute to attenuation, for example through steric effects.
  • the blocking element is capable of blocking all or some of the receptor agonist activity of the IL-21 by noncovalently binding to the IL-21 and/or sterically blocking receptor binding.
  • the released IL-21 Upon cleavage of the protease cleavable linker a form of the IL-21 is released that is active (e.g., more 1 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 active than the IL-21 polypeptide prodrug).
  • the released IL-21 is at least 10 x more active than the IL-21 polypeptide prodrug.
  • the released IL-21 is at least 20 x, at least 30 x, at least 50 x, at least 100 x, at least 200 x, at least 300 x, at least 500 x, at least 1000 x, at least about 10,000 x or more active than the inducible IL-21 prodrug.
  • the form of IL-21 that is released upon cleavage of the inducible IL-21 prodrug typically has a short half-life, which is often substantially similar to the half-life of naturally occurring cytokine. Even though the half-life of the inducible IL-21 prodrug is extended, toxicity is reduced or eliminated because the agonist activity of the circulating inducible cytokine prodrug is attenuated and active IL-21 is targeted to the desired site of activity (e.g., tumor microenvironment).
  • the inducible IL-21 prodrug can comprise at least one of each of an IL-21 polypeptide [A], an IL-21 blocking element [D], a half-life extension element [H], and a protease-cleavable polypeptide linker [L].
  • the IL-21 polypeptide and the IL-21 blocking element or the half-life extension element can be operably linked by the protease-cleavable polypeptide linker and the inducible IL-21 prodrug has attenuated IL-21 receptor activating activity.
  • the IL-21 receptor activating activity of the inducible IL-21 prodrug is at least about 10X less than the IL-21 receptor activating activity of the polypeptide that contains the IL-21 polypeptide that is produced by cleavage of the protease cleavable linker.
  • the inducible IL-21 prodrug can have the formula: [011] [A]-[L1]-[H]-[L2]-[D]; [012] [D]-[L2]-[H]-[L1]-[A]; [013] [A]-[L1]-[D]-[L2]-[H]; [014] [H]-[L2]-[D]-[L1]-[A]; [015] [H]-[L1]-[A]-[L2’]-[D]; or [016] [D]-[L1]-[A]-[L2’]-[H].
  • [A] is an IL-21 polypeptide
  • [D] is an IL-21 blocking element
  • [H] is a half-life extension element
  • [L1] is a protease-cleavable polypeptide linker
  • [L2] is a polypeptide linker that is optionally protease-cleavable
  • [L2’] is a protease-cleavable polypeptide linker.
  • L2 can be a protease-cleavable polypeptide linker.
  • L1 or L2 or both L1 and L2 are cleaved by two or more different proteases.
  • the inducible IL-21 prodrug disclosed herein can comprise an antigen binding fragment of an antibody as the IL-21 blocking element.
  • the antigen binding fragment of an antibody can comprise as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain.
  • the inducible IL-21 prodrug can comprise: a first polypeptide comprising the IL-21 polypeptide, at least an antigen binding portion of an 2 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 antibody light chain or an antigen binding portion of an antibody heavy chain, and the half-life-extension element; wherein the IL-21 polypeptide and the antigen binding portion of the antibody light chain or the antigen binding portion of the antibody heavy chain and/or half-life extension element are operably linked by the protease cleavable linker; and second polypeptide comprising at least an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the first polypeptide, or an antibody light chain that is complementary to the heavy chain in the first polypeptide and together with said light chain forms an IL-21 binding site.
  • the inducible IL-21 prodrug can contain two polypeptide chains.
  • the first polypeptide chain can comprise an IL-21 polypeptide, a half-life extension element, a first IL-21 blocking element, a protease- cleavable polypeptide linker, and a portion of an antibody heavy chain constant region or a light chain; wherein the first blocking element is IL-21R.
  • the IL-21 polypeptide and the first blocking element and/or the half-life extension element are operably linked by the protease cleavable linker.
  • the second polypeptide can comprise a second IL-21 blocking element and a portion of an antibody heavy chain constant region or an antibody light chain.
  • the second blocking element and the portion of the antibody heavy chain constant region or the antibody light chain are linked through a linker that is optionally protease cleavable.
  • the second blocking element can be a common gamma chain receptor.
  • the antibody heavy chain constant region is on the first polypeptide
  • the antibody light chain is on the second polypeptide
  • the antibody heavy chain constant region is on the second polypeptide
  • the antibody light chain is on the first polypeptide.
  • the inducible IL-21 prodrug can comprise a first polypeptide selected from the group consisting of SEQ ID NOs.18-23 and 26-31, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 18-23 and 26-31.
  • the inducible IL-21 prodrug can comprise a second polypeptide selected from the group consisting of SEQ ID NO.17 or 25 or an amino acid sequence that has at least 80% identity to SEQ ID NO.17 or 25.
  • the inducible IL-21 prodrug can comprise a first polypeptide selected from the group consisting of SEQ ID NOs.18-23 and 26-31, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 18-23 and 26-31, and a second polypeptide selected from the group consisting of SEQ ID NO.17 or 25 or an amino acid sequence that has at least 80% identity to SEQ ID NO.17 or 25.
  • the inducible IL-21 prodrug can comprise a first polypeptide chain selected from the group consisting of SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65.
  • the inducible IL-21 prodrug can further comprise 3 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 a second polypeptide chain selected from the group consisting of SEQ ID NOs.: 10, 40, 52, and 64 or an amino acid sequence that has at least 80% identity to SEQ ID NOs.: 10, 40, 52, and 64.
  • the inducible IL-21 prodrug can comprise a first polypeptide chain selected from the group consisting of SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65, and a second polypeptide chain selected from the group consisting of SEQ ID NOs.: 10, 40, 52, and 64 or an amino acid sequence that has at least 80% identity to SEQ ID NOs.: 10, 40, 52, and 64.
  • the inducible IL-21 prodrug can comprise the amino acid sequence selected from the group consisting of SEQ ID NOs.: 44-50 or an amino acid sequence that has at least 80% identity to SEQ ID NOs: and 44-50.
  • the disclosure relates to an inducible IL-21 prodrug comprising the amino acid sequence selected from the group consisting of SEQ ID NOs.: 72-109 or an amino acid sequence that has at least about 80% identity to SEQ ID NOs.: 72-109.
  • the disclosure relates to inducible IL-21 prodrug comprising a first polypeptide selected from the group consisting of SEQ ID NOs.72, 110-113, 117-126, 130-136, 138-144, 153-194, 221-235, 241-243, 245-247, 250, 251, 253-255, 257, 258, 260-264, 267-269, 271-290, 475, 476, and 449-463, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 72.110-113, 117-126, 130-136, 138-144, 153-208, 221-235, 241-243, 245-247, 250, 251, 253-255, 257, 258, 260-264, 267-269, 271-290, 475, and 476, and a second polypeptide selected from the group consisting of SEQ ID NO.71, 127-129, 137, or 300-306 or an amino acid sequence that has at least 80% identity to SEQ ID NO.71
  • the inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 130, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 129.
  • the inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 129.
  • the inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 140, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 137.
  • the inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 143, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 137.
  • the inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 169, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 301.
  • the inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid 4 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 sequence of SEQ ID NO: 185, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 303.
  • the half-life extension element can comprise a serum albumin binding domain, a serum albumin, transferrin, or immunoglobulin Fc, or fragment thereof.
  • the half-life extension element can also be a blocking element.
  • the IL-21 blocking element can comprise a ligand-binding domain or fragment of a cognate receptor for the IL-21 polypeptide, or an antibody or antigen-binding fragment of an antibody that binds to the IL-21 polypeptide.
  • the IL-21 blocking element inhibits activation of the IL-21 receptor by the inducible IL-21 prodrug.
  • the antibody or antigen-binding fragment can be a single domain antibody, a Fab, or a scFv that binds the IL-21 polypeptide.
  • the cognate receptor for the IL-21 can be the IL-21 receptor or the IL-21 common gamma chain.
  • Each protease-cleavable polypeptide linker independently comprises a sequence that is capable of being cleaved by a protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, cathepsin L, an elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), an ADAM, a FAP, a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease.
  • a protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, cathepsin L, an elastase, PR-3, granzyme M, a calpain,
  • the cathepsin can be cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, or cathepsin G.
  • the matrix metalloprotease (MMP) is MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP19, or MMP20.
  • MMP matrix metalloprotease
  • the disclosure also relates to a nucleic acid encoding the inducible IL-21 prodrug.
  • the nucleic acid can comprise a circular vector.
  • the nucleic acid can comprise DNA.
  • the nucleic acid can comprise RNA.
  • Disclosed herein are methods of making the pharmaceutical composition comprising culturing the host cell under suitable conditions for expression and collection of the inducible IL-21 prodrug.
  • a nucleic acid composition comprising one or more nucleic acid sequences encoding an inducible IL-21 prodrug.
  • the inducible IL-21 prodrug comprises at least one of each of an IL-21 polypeptide [A]; an IL-21 blocking element [D]; a half-life extension element [H]; and a protease- cleavable polypeptide linker [L].
  • the IL-21 polypeptide and the IL-21 blocking element or the half-life extension element are operably linked by the protease-cleavable polypeptide linker and the inducible IL- 21 prodrug has attenuated IL-21 receptor activity.
  • the nucleic composition can comprise one or more nucleic acid sequences encoding an inducible IL-21 prodrug containing two polypeptide chains.
  • the inducible IL-21 prodrug can comprise an antigen 5 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 binding fragment of an antibody as the IL-21 blocking element.
  • the antigen binding fragment of an antibody can comprise as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain.
  • the inducible IL- 21 prodrug can comprise: a first polypeptide comprising the IL-21 polypeptide, at least an antigen binding portion of an antibody light chain or an antigen binding portion of an antibody heavy chain, and the half- life-extension element; wherein the IL-21 polypeptide and the antigen binding portion of the antibody light chain or the antigen binding portion of the antibody heavy chain and/or half-life extension element are operably linked by the protease cleavable linker; and second polypeptide comprising at least an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the first polypeptide, or an antibody light chain that is complementary to the heavy chain in the first polypeptide and together with said light chain forms an IL-21 binding site.
  • the nucleic acid composition can comprise one or more nucleic acid sequences encoding an inducible IL-21 prodrug comprising an inducible IL-21 prodrug comprising: a first polypeptide comprising a IL-21 polypeptide, a half-life extension element, a first IL-21 blocking element, a protease- cleavable polypeptide linker, and a portion of an antibody heavy chain constant region or a light chain; wherein the first blocking element is IL-21R; and wherein the IL-21 polypeptide and the first blocking element or the half-life extension element are operably linked by the protease cleavable linker; and a second polypeptide that comprises a second blocking element and a portion of an antibody heavy chain constant region or an antibody light chain; wherein the second blocking element and the portion of the antibody heavy chain constant region or the antibody light chain are linked through a linker that is optionally protease cleavable; wherein the second blocking element is a common gam
  • the disclosure also relates to therapeutic methods that include administering to a subject in need thereof an effective amount of an inducible IL-21 prodrug, nucleic acid that encodes the inducible IL-21 prodrug, vector or host cells that contain such a nucleic acid, and pharmaceutical compositions of any of the foregoing.
  • the subject has, or is at risk of developing cancer, a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease, or a host-versus-graft disease.
  • the methods disclosed herein are particularly suitable for treating cancer.
  • the inducible IL-21 prodrug can be administered intravenously. 6 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 [036]
  • the disclosure relates to single domain antibody fragments (sdAbs) (such as VHH, and VH) that have binding specificity for human serum albumin, methods of making, and uses thereof.
  • sdAbs single domain antibody fragments
  • Such sdAbs can be used, for example, for extending the in vivo serum half-life of therapeutic agents (e.g., proteins, peptides and the like) through the engineering of fusion proteins and conjugates using well-known methods and as described herein.
  • therapeutic agents e.g., proteins, peptides and the like
  • Preferred sdAbs compete with FcRn (human FcRn) for binding to human serum albumin and, preferably, bind to domain 1 (DI) of human serum albumin.
  • the sdAbs disclosed herein comprise a CDR1, CDR2, and a CDR3.
  • the CDR1 comprises any one of SEQ ID NO: 656, SEQ ID NO: 659, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 691, or a variant of any of the foregoing comprising up to about 2 amino acid substitutions.
  • the CDR2 comprises any one of SEQ ID NO: 657, SEQ ID NO: 660; SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 692, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions.
  • the CDR3 comprises any one of SEQ ID NO: 658, SEQ ID NO: 661, SEQ ID NO: 667 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 656, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 657, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 658, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 659, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO:660, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO:660, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 664, or a variant thereof comprising up to about 3 amino acid substitutions; and 7 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 665, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 666, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 692, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 664, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 665, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 666, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 692, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 664, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 665, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 666, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 692, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a CDR1 that consists of SEQ ID NO: 662; a CDR2 that consists of SEQ ID NO: 692; and a CDR3 that consists of SEQ ID NO: 667.
  • the sdAb can comprise a CDR1 that consists of SEQ ID NO: 691; a CDR2 that consists of SEQ ID NO: 664; and a CDR3 that consists of SEQ ID NO: 667.
  • the sdAb can comprise a CDR1 that consists of SEQ ID NO: 663; a CDR2 that consists of SEQ ID NO: 665; and a CDR3 that consists of SEQ ID NO: 667.
  • the sdAb can comprise a CDR1 that consists of SEQ ID NO: 663; a CDR2 that consists of SEQ ID NO: 666; and a CDR3 that consists of SEQ ID NO: 667. [057]
  • the sdAb can comprise CDR1 that consists of SEQ ID NO: 656; a CDR2 that consists of SEQ ID NO: 2; and a CDR3 that consists of SEQ ID NO: 658.
  • the sdAb can comprise CDR1 that consists of SEQ ID NO: 659; a CDR2 that consists of SEQ ID NO:660; and a CDR3 that consists of SEQ ID NO: 661.
  • the sdAbs disclosed herein can compete with neonatal Fc receptor (FcRn) for binding to HSA.
  • the sdAbs can have binding specificity for domain one of human serum albumin.
  • the sdAb can be a heavy chain variable domain (VH), a variable heavy domain of heavy chain (VHH), a single domain shark variable domain of new antigen receptor (VNAR), or a light chain variable (VL) domain.
  • VH and VHH are preferred sdAbs.
  • the sdAb is camelid, human, or humanized.
  • the sdAb is humanized.
  • the sdAb’s disclosed herein further comprise one or more framework regions.
  • the framework region can be derived from a human germline.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, SEQ ID NO: 663, or SEQ ID NO: 691; a CDR2 comprising the amino acid sequence of SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, or SEQ ID NO: 692; a CDR3 comprising the amino acid sequence of SEQ ID NO: 667; and a FR1, a FR2, a FR3, and a FR4.
  • the amino acid residue at position 24 of FR1 can be a serine or an alanine
  • the amino acid at position 44 of F2 can be a glutamic acid or a glycine
  • the amino acid at position 45 of F2 can be a arginine or a leucine
  • the amino acid at position 79 of F3 is a leucine or a valine.
  • FR1, FR2, FR3, FR4 can be from human IGHV3-23 germ line.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 656; a CDR2 comprising the amino acid sequence of SEQ ID NO: 657; a CDR3 comprising the amino acid sequence of SEQ ID NO: 658; and a FR1, a FR2, a FR3, and a FR4.
  • the amino acid residue at position 24 of FR1 can be a serine or an alanine
  • the amino acid at position 44 of FR2 can be a aspartic acid or a glycine
  • the amino acid at position 45 of FR2 can be a arginine or a leucine
  • the amino acid at position 49 of FR2 can be a serine or alanine
  • the amino acid at position 78 of FR3 can be a leucine or a phenylalanine
  • the amino acid at position 79 of F3 can be a asparagine or a lysine.
  • the FR1, FR2, FR3, FR4 can be from human IGHV3-23 germ line.
  • the sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 659; a CDR2 comprising the amino acid sequence of SEQ ID NO: 660; a CDR3 comprising the amino acid sequence of SEQ ID NO: 661; and a FR1, a FR2, a FR3, and a FR4.
  • the amino acid residue at position 10 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 44 of F2 can be a glutamic acid or a glycine
  • the amino acid at position 45 of F2 can be a arginine or a leucine
  • the amino acid at position [78] of F3 can be a threonine or a serine
  • the amino acid at position 79 can be F3 is a leucine or a valine.
  • the FR1, FR2, FR3, FR4 can be from human IGHV3-23 germ line.
  • the sdAbs disclosed herein can further comprises a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 685, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 686, SEQ ID NO:33, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 34, SEQ ID NO: 689,
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 670, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 671, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 673, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 669, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 671, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 678, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 679, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise FR1 comprising the amino acid sequence of SEQ ID NO: 669, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid 12 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 sequence of SEQ ID NO: 686, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 686, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 689, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 689, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 689, or a variant thereof comprising up to about 13 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.
  • the sdAb can comprise the amino acid sequence of any one of SEQ ID NOs: 286-290 or 608- 648, or an amino acid sequence of at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 286-290 or 608-648.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 608, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 608.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 609, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 609.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 611, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 611. [085] The sdAb can comprise the amino acid sequence of SEQ ID NO: 618, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 618. [086] The sdAb can comprise the amino acid sequence of SEQ ID NO: 622, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 622.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 624, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 624.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 625, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 625.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 626, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 626.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 628, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 628.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 632, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 632.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 635, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 635.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 637, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 637.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 642, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 642.
  • 14 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 644, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 644.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 645, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 645.
  • the sdAb can comprise the amino acid sequence of SEQ ID NO: 646, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 646.
  • the sdAbs disclosed herein can comprise a half-life of at least about 5 hours to about 40 hours.
  • the disclosure further relates to pharmaceutical compositions comprising the sdAbs disclosed herein or nucleic acids encoding the sdAbs.
  • the disclosure further relates to a nucleic acid encoding the sdAbs disclosed herein.
  • the nucleic acid encoding sdAbs can be a circular vector, DNA, RNA, or an expression vector comprising a circular vector, DNA, or RNA. Additionally, the disclosure relates to an isolated host cell comprising an expression vector encoding for a nucleic acid of a sdAb.
  • the disclosure further relates to a fusion polypeptide comprising a sdAb as disclosed herein and an amino acid sequence of interest.
  • the fusion polypeptide can further comprise an optionally cleavable linker.
  • the optionally cleavable linker can be operably linked to the sdAb and the amino acid sequence of interest.
  • the optionally cleavable linker can be operably linked to the N-terminus of the sdAb.
  • the optionally cleavable linker can be operably linked to the C-terminus of the sdAb.
  • the optionally cleavable linker is preferably cleavable.
  • the optionally cleavable linker can comprise a cleavable moiety that is a substrate for one or more proteases. In some instances, the optionally cleavable linker is not cleavable.
  • the disclosure further relates to a nucleic acid encoding the fusion polypeptide.
  • the disclosure further relates to methods for treating a disease, such as cancer, that comprise administering to a subject in need thereof an effective amount of the fusion polypeptide, a nucleic acid encoding the fusion polypeptide, an expression vector for the fusion polypeptide, or a pharmaceutical composition comprising an effective amount of any of these.
  • a disease such as cancer
  • FIGs.1A-1O are schematic illustrations depicting various inducible IL-21 prodrugs.
  • FIG.2 is a SDS-PAGE gel showing the results of protein cleavage assays. The IL-21 prodrugs were run in both cleaved and uncleaved form.
  • FIGs.3A is a graph showing results of analyzing WW50008 (half-life extended mouse IL-21) in a syngeneic MC38 mouse tumor model.
  • FIG.3B is a graph showing body weight averages for the animals treated in data shown in FIG. 3A. Each line in the plot represents the body weight average of the group over time. The 300 ⁇ g dose of WW50008 was not tolerated by the animals.
  • FIG.4 is a graph showing results of analyzing WW50008 in a syngeneic B16F10 mouse tumor model. It shows average tumor volume over time in mice treated with 10 ⁇ g, 33 ⁇ g, 100 ⁇ g and 200 ⁇ g WW50008 (square, triangle upside, triangle downside and diamond respectively). Vehicle alone is indicated by circle. The data show tumor volume was inhibited over time in a dose-dependent manner in mice treated with WW50008 at the higher concentrations.
  • FIGs.5A-5E are spider plots showing activity of WW50008 (half-life extended mouse IL-21) in a B16F10 mouse model corresponding to the data shown in FIG.4. Each line in the plots is the tumor volume over time for a single mouse.
  • FIG.6 is a graph showing results of analyzing WW50008 (half-life extended mouse IL-21) in a syngeneic EMT6 mouse tumor model. It shows average tumor volume over time in mice treated with 10 ⁇ g, 33 ⁇ g, 100 ⁇ g and 200 ⁇ g WW50008 (square, triangle upside, triangle downside and diamond respectively). Vehicle alone is indicated by circle. The data show tumor volume was inhibited over time in a dose-dependent manner in mice treated with WW50008 at the concentrations of 10 ⁇ g and 33 ⁇ g. WW50008 was not tolerated at 200 ⁇ g dose in this model.
  • FIGs.7A-7E are spider plots showing activity of WW50008 (half-life extended IL-21) in a EMT6 mouse model corresponding to the data shown in FIG.6. Each line in the plots is the tumor volume over time for a single mouse.
  • FIGs.8A-8D shows the results of the efficacy and TILs analysis of MC38 tumors treated with a half-life extended IL-21 (WW50008).
  • FIG.8A is a graph showing tumor volume (mm 3 ) in mice treated with vehicle or 100 ⁇ g WW50008 on days 1, 4, 8 and 11.
  • FIG.8B are graphs showing an increase in lymphocyte infiltrates in WW50008 treated group compared to vehicle as measured by total cells per mg of tissue and frequency of CD45+ TILs.
  • FIG.8C are graphs showing NK cell counts and NK IFN- gamma+ cell frequency in mice treated with vehicle and WW50008.
  • FIG.8D are graphs showing frequency of CD8+ Tetramer+ T cells (Granzyme B+, INF-gamma+, and TNF+) in mice treated with vehicle and WW50008.
  • FIGs.9A-9E shows the results of the efficacy and TILs analysis of EMT-6 tumors upon in vivo dosing with half-life extended human IL-2 (WW0177) or half-life extended mouse IL-21 (WW50008).
  • FIG.9A is a graph showing tumor volume in mice treated with vehicle, WW0177 (10 ⁇ g) or WW50008 (100 ⁇ g) on days 1, 4, 8, and 11.
  • FIG.9B shows frequency of CD8 and CD8+ granzyme B+, INF- gamma+, and TNF+ infiltrates in vehicle, WW0177 and WW50008 treated groups.
  • FIG.9C are graphs showing polyfunctionality of cytokine production in vehicle, WW0177 and WW50008 treated CD8+ T cells.
  • FIG.9D are graphs showing the frequency of Treg cells and CD8/Treg ratio in Vehicle, WW0177, and WW5008 treated groups.
  • FIG.9E shows a heat map depicting the differential gene expression in tumors treated with WW0177 and WW50008 using NanoString analysis.
  • FIGs.10A and 10B show efficacy of half-life extended IL-21 and IL-21 prodrug at different dose levels (dose dependent effect) (FIG.10A) in the MC38 mouse model. No detrimental effect observed in body weight of these animals at any of the dose levels tested (FIG.10B).
  • FIGs.11A-11D is a graph showing the results of EMT-6 mouse tumor re-challenge.
  • FIG.11A is a graph showing tumor volume over time in mice treated with half-life extended human IL-2 (squares), half-life extended mouse IL-21 (triangles), and vehicle (circles). The majority of animals treated with half-life extended IL-21 survived the study and were used for a re-challenge study.
  • FIG.11B is a graph showing average tumor volume over days post re-challenge in mice that survived EMT-6 challenge (see, FIG 11A triangles) and were confirmed to have complete regression of tumors. These animals were re-challenged with the EMT-6 cell line (circles) and na ⁇ ve mice were used as controls (square).
  • FIG.11C are representative microscopic images from an IFN ⁇ ELISPOT assay. 17 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 Splenocytes were isolated EMT-6 tumor bearing mice that had complete remission (CR) following treatment with half-life extended human IL-2 or half-life extended mouse IL-21, and splenocytes were incubated with EMT-6 cells for 24 hours. Splenocytes responded to EMT-6 cells and produced IFN ⁇ .
  • FIG.11D is a graph depicting the average relative spot volume on the IFN ⁇ ELISPOT plate.
  • FIGs.12A-12D are graphs showing that half-life extended IL-21 and inducible IL-21 prodrug have comparable anti-tumor activity in a syngeneic MC38 tumor model using mice that express human IL-21 receptor, but that inducible IL-21 prodrug (FIG.12C) has better tolerability and a broader therapeutic window.
  • FIGs.12A and 12C show average tumor volume over days in mice treated with half- life extended IL-21 (FIG.12A) and inducible IL-21 prodrug (FIG.12C).
  • FIGs.13A-13C are graphs showing results of analyzing WW50334 in a syngeneic MC38 tumor model using mice that express human IL-21 receptor.
  • FIG.13A shows average tumor volume over time in mice treated with 3.3 ⁇ g, 10 ⁇ g, 33 ⁇ g, and 100 ⁇ g (dose per mouse per injection) of WW50334.
  • FIGs. 13B is a graph showing body weight averages for the animals treated in FIG.13A.
  • FIG.13C are graphs showing body weights for animals treated in FIG.13A. The total nanomoles of WW50334 delivered per mouse over the dosing period are shown. The top dose of 100 ⁇ g was not tolerated, inducing animal deaths.
  • FIGs.14A-14C are graphs showing results of analyzing WW50112/WW50347 in a syngeneic MC38 tumor model in mice that express human IL-21 receptor.
  • FIG.14A shows average tumor volume over time in mice treated with 200 ⁇ g, 600 ⁇ g, and 1800 ⁇ g (dose per mouse per injection) of WW50112/WW50347.
  • FIGs.14B is a graph showing body weight averages for the animals treated in FIG.14A. Each line in the plot represents the body weight average of the group over time.
  • FIG.14C are graphs showing body weights for each animal treated in FIG.14A. The total nanomoles of WW50334 delivered per mouse over the dosing period are shown.
  • FIGs.15A-15C are graphs showing results of analyzing WW50112/WW50348 in a syngeneic MC38 tumor model in mice that express human IL-21 receptor.
  • FIG.15A shows average tumor volume over time in mice treated with 200 ⁇ g, 600 ⁇ g, and 1800 ⁇ g (dose per mouse per injection) of WW50112/WW50348.
  • FIGs.15B is a graph showing body weight averages for the animals treated in 18 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 FIG.15A. Each line in the plot represents the body weight average of the group over time.
  • FIG.15C are graphs showing body weights for each animal treated in FIG.15A.
  • FIGs.16A and 16B are graphs showing results of analyzing WW50260 in a syngeneic MC38 tumor model in mice that express human IL-21 receptor.
  • FIG.16A shows average tumor volume over time in mice treated with 142 ⁇ g, 425 ⁇ g, and 1,277 ⁇ g (dose per mouse per injection) of WW50260.
  • FIGs.16B is a graph showing body weight averages for the animals treated in FIG.16A. Each line in the plot represents the body weight average of the group over time.
  • FIGs.17A-17D are graphs showing results of analyzing WW50334 (human IL-21 half-life extended), WW50260, WW50347/WW50112, WW050348/WW50112 in a syngeneic MC38 tumor model in mice that express human IL-21 receptor.
  • FIG.17A shows the corresponding average serum levels of WW50334 over time.
  • FIG.17B shows the corresponding average serum levels of WW50260 over time.
  • FIG.17C shows the corresponding average serum levels of WW50112/WW50347 over time.
  • FIG.17D shows the corresponding average serum levels of WW50112/WW50348 over time.
  • FIGs.18A-18G are graphs showing exposure of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysate.
  • FIG.18A shows WW050334 (human IL-21 half-life extended) over time in serum (triangles) and in tumor lysate (squares).
  • FIG.18B shows intact WW50260 over time in serum (triangles) and in tumor lysate (squares) and
  • FIG.18C shows cleaved WW50260 over time in serum (triangles) and in tumor lysate (squares).
  • FIG.18D shows intact WW50347/WW50112 over time in serum (triangles) and in tumor lysate (squares) and
  • FIG.18E shows cleaved WW50347/WW50112 over time in serum (triangles) and in tumor lysate (squares).
  • FIG.18F shows intact WW50348/WW50112 over time in serum (triangles) and in tumor lysate (squares) and
  • FIG.18G shows cleaved WW50348/WW50112 over time in serum (triangles) and in tumor lysate (squares).
  • FIGs.19A and 19B show efficacy of half-life extended mouse IL-21 at different dose levels in the Renca mouse model (FIG.19A) and in the CT26 mouse model (FIG.19B).
  • FIGs.20A and 20B show the results of the TILs analysis in EMT-6 tumors upon in vivo dosing with half-life extended mouse IL-21 and vehicle in the EMT-6 mouse model.
  • FIG.20A shows a heat map depicting the differential gene expression in IL-21 and vehicle.
  • FIG.20B shows a graph showing the fold change in genes expressed between IL-21 and vehicle.357 significant differentially expressed genes were identified.
  • FIGs.21A-21B shows the results of the TILs analysis in EMT-6 tumors upon in vivo dosing with half-life extended mouse IL-21 and half-life extended human IL-2 in the EMT-6 mouse model.
  • FIG.21A shows a heat map depicting the differential gene expression in IL-21, IL-2, and vehicle.
  • FIG.21B shows 19 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 a graph showing the fold change in genes expressed between IL-21 and IL-2.35 significant differentially expressed genes were identified.
  • FIGs.22A-22F are graphs showing results of analyzing inducible mouse IL-21 prodrugs in syngeneic MC38 mouse tumor model.
  • mice show average tumor volume over time in mice treated with 250 ⁇ g, 1,000 ⁇ g, and 1500 ⁇ g (dose per mouse per injection) of inducible IL-21 prodrug. Mice were dosed twice a week for two weeks (total of 4 doses). The following constructs were analyzed: IL-21 half- life extended (WW50008, FIG.22A), WW50076 (FIG.22B), WW50079 (FIG.22C), WW50080 (FIG. 22D), WW50256 (FIG.22E), WW50290 (FIG.22F). Data show that tumor volume was inhibited over time in a dose-dependent manner in mice treated with WW50076 (FIG.31B), WW50256 (FIG.22E), and WW50290 (FIG.22F).
  • FIG.34A and 34B show IL-21 prodrug treatment results in immunity to tumor rechallenge for animals deemed as complete responders from the initial study.
  • FIG.34A shows the original group of tumor growth responses from where the CR animals were recruited. A total of 24 WW50387/WW50394- treated mice with CR were rechallenged by inoculating with MC38-BC2 to the left flank on Day 88.
  • FIG. 34B shows that all rechallenged mice rejected the second inoculation and did not show regrowth of their primary tumors, surviving until the last observation day (Day 116) while strain and age-matched na ⁇ ve mice similarly inoculated showed a consistent tumor growth of their left flank tumors.
  • FIG.44A discloses SEQ ID NOS 701-703 and 608-616, respectively, in order of appearance
  • FIG.44B (3HUM181)
  • FIG.44B discloses SEQ ID NOS 704-706 and 625-634, respectively, in order of appearance
  • FIG.44C (2HUM45 on a IGHV3-23 backbone)
  • FIG.44C discloses SEQ ID NOS 707-709 and 635-639, respectively, in order of appearance
  • FIG.44D discloses SEQ ID NOS 710-712 and 640-648, respectively, in order of appearance
  • FIG.44E (1HUM19 variants with modified CDR sequences
  • FIG.44E discloses SEQ ID NOS 713-715, 617-624 and 649-651, respectively, in order of appearance)
  • FIG.44E discloses SEQ ID NOS 713-715, 617-624 and 649-651, respectively, in order of appearance
  • FIG.44E discloses SEQ ID NOS 713-715, 617-6
  • FIGs.53A-53D show efficacy of WW50387/WW50394 dosed at 94 ⁇ g (FIG.53A) or 375 ⁇ g (FIG.53B) with or without ⁇ LAG3.
  • 94 ⁇ g dose there was a trend for better efficacy when WW50387/WW50394 was combined with ⁇ LAG3, but this did not reach significance.
  • 375 ⁇ g dose the WW50387/WW50394 + ⁇ LAG3 combination was highly significantly (****p ⁇ 0.0001) more efficacious than ⁇ LAG3 alone but not WW50387/WW50394 alone.
  • FIGs.56A-56D are graphs showing exposure in the serum (FIGs.56A and 56B) and in the tumor (FIGs.56C and 56D) for total inducible IL-21 prodrug (FIGs.56A and 56C) and free human IL- 21 (FIGs.56B and 56D) after a single dose and after three doses of WW50387/WW50394 over the 168- hour time course for each.
  • FIGs.57A and 57B are graphs showing the time course of IFN ⁇ levels after a single dose of WW50387/WW50394. IFN ⁇ levels in the serum are near the lower limits of quantitation in the serum but increase over time in the tumor, peaking at 1028 pg/mL at 96 hours after treatment.
  • FIG.58 shows an increase of pSTAT3 expression over total STAT3 in tumors after a single dose (squares) or 3 doses (triangles) of WW50387/WW50394 compared to PBS control (circles).
  • FIG.59 shows multiplex-immunofluorescence (M-IF) staining pictures of MC38 tumors resected from animals treated with vehicle or WW50387/WW50394. Tumors were collected at day 10 after initial dosing. Tumors were fixed in formalin and paraffin embedded to obtain slices for staining.
  • M-IF multiplex-immunofluorescence
  • DAPI DAPI stains nuclei, CD3+CD8+ (CTL), CD3+CD8+ GZMB+ (activated CTL), CD3+CD4+ (CD4+ T cells) F4/80 (macrophages) CD11b (myeloid cells) CD11c+CD103+ (cross presenting DCs).
  • FIGs.60A and 60B are graphs showing results of analyzing half-life extended IL-2 and half-life extended IL-21 in a Renca mouse tumor model.
  • FIG.60A shows tumor volume over time in mice treated with half-life extended IL-2 (WW0177), half-life extended IL-21 (WW50008), or vehicle.
  • FIG.60B is a graph showing body weight averages for the animals treated in data shown in FIG.60A.
  • FIGs.61A and 61B show the results of the TILs analysis of Renca tumors treated with half-life extended IL-2 (WW0177), half-life extended IL-21 (WW50008), or vehicle.
  • FIG.61A is a graph showing CD8+ T cell abundance in tumor tissue over time in mice treated with WW0177, WW50008 or vehicle.
  • FIG.61B are pie charts showing polyfunctionality of CD8+ T cell cytokine production (i.e.
  • p values 26 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 represent the results of a one-way ANOVA: * p ⁇ 0.05, **p ⁇ 0.005, *** p ⁇ 0.001, ****p ⁇ 0.0001.
  • the data shows specific activation of type-I IFN genes triggered by IL-21 treatment and the inhibitory effect that blocking type-I IFN signaling has on IL-21 anti-tumor efficacy.
  • FIGs.63A-63C are representative immunofluorescence images showing staining for DAPI and CD45 at various timepoints (FIG.63A), DAPI, CD8, CD4, F4/80, CD11b and Granzyme B at day 12 (FIG.63B), and DAPI, CD8, CD4, TCF1, CD11c, CD103 and NCR1 at day 12 (FIG.63C) in EMT-6 tumor bearing mice treated with vehicle, IL-21-HLE or IL-2-HLE.
  • the data shows a long duration increase of immune infiltration for those animals treated with IL-21-HLE.
  • CD8+ T cells CD8+ T cells (CD8) and macrophages (F4/80).
  • FIGs.64A-64E are graphs showing that IL-21 promotes increased presence of intratumoral CD8+ T cells that express effector cytokines.
  • FIG.64A shows density of total CD8+ T cells in EMT-6 tumors from animals treated with vehicle, IL-21-HLE or IL-2-HLE.
  • FIG.64B shows density of Granzyme B+ CD8+ T cells in EMT-6 tumors from animals treated with vehicle, IL-21-HLE or IL-2- HLE .
  • FIG.64C shows density of Perforin+ CD8+ T cells in EMT-6 tumors from animals treated with vehicle, IL-21-HLE or IL-2-HLE .
  • FIG.64D shows density of TCF1+ CD8+ T cells in EMT-6 tumors from animals treated with vehicle, IL-21-HLE or IL-2-HLE .
  • FIG.64E is a spatial pseudo-color dot plot of CD8+ T cell infiltration, Granzyme B+ CD8+ T cells and stem-like (TCF1+) CD8+ T cells in EMT-6 tumors from animals treated with vehicle, IL-21-HLE or IL-2-HLE.
  • TCF1+ stem-like CD8+
  • FIGs.65A-65E shows treatment with IL-21 results in robust anti-tumor efficacy in an immune checkpoint inhibitor (CPI) resistant tumor model.
  • Renca tumor bearing mice were randomized into treatment groups and dosed with vehicle or either IL-21-HLE or IL-2-HLE twice weekly for two weeks.
  • FIG.65A is a graph showing average tumor volume over time.
  • FIG.65B is a graph showing average body weight over time.
  • FIGs.65C-65E are graphs showing individual spider plots of tumor burden from animals treated with vehicle, IL-21 HLE or IL-2-HLE. The data shows that IL-21treatment can induced efficacy in an animal tumor model resistant to CPI treatment and where IL-2-HLE treatment fails to produce significant efficacy.
  • FIGs.66A-66F show that IL-21 treatment leads to sustained intratumoral CD8+ T cell population that are polyfunctional (express multiple effector molecules). Renca tumor bearing mice were randomized into treatment groups and dosed with vehicle, IL-21-HLE, or IL-2-HLE twice weekly for two weeks. Tumors were collected at indicated timepoints and enzymatically and mechanically dissociated for single cell suspensions. Cells were stained for flow cytometry analysis.
  • FIG.66A is a graph showing 27 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 CD8+ T cell counts per mg of tissue over time.
  • FIG.66B is a graph showing the ratio of CD8+ T cells to Tregs (CD4+ Foxp3+ T cells) in tumors over time.
  • FIGs.66C-66E are graphs showing cytokine production from CD8+ T cells on day 14.
  • FIG.66F are pie charts showing the frequency of polyfunctional CD8+ T cells in the tumor based on co-expression of Granzyme A, Granzyme B, IFNg, TNF, and Perforin over time. p values represent the results of a one-way ANOVA: *p ⁇ 0.05, **p ⁇ 0.005, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • FIGs.67A-67C are immunofluorescence images from Renca tumor bearing mice treated with vehicle, IL-21-HLE, or IL-2-HLE.
  • FIG.67A is an immunofluorescence showing DAPI and CD45 in mice administered with vehicle, IL-21 HLE, or IL-2 HLE.
  • FIG.67B is an immunofluorescence showing DAPI, CD4, CD8, F4/80, CD11c in mice administered with vehicle, IL-21 HLE, or IL-2 HLE.
  • FIG.67C is an immunofluorescence image showing higher magnification of the white boxed area in corresponding images in (FIG.67B), DAPI, CD4, CD8, F4/80, CD11c, and Granzyme B in mice administered with vehicle, IL-21 HLE, or IL-2 HLE. Shading legends are indicated in figures. The data shows that IL-21- HLE treatment can induce a quantifiable intratumoral infiltration of different immune populations, specially CD8+ T cells. CD4+ T cells and macrophages.
  • FIGs.68A-68E are graphs showing results of analyzing WW50387/WW50394 and WW51100/WW50394 in a syngeneic MC38 mouse tumor model.
  • FIG.68A is a graph showing results of analyzing mean tumor volumes after dosing WW50387/WW50394 administered at 375 ⁇ g and WW51100/WW50394 administered at 47, 94, 188, 375, 750, 1200, and 1500 ⁇ g on Days 0, 3, 7, and 10 after treatment initiation. The study was carried out through Day 28 after treatment initiation, measuring tumor volumes 2x weekly during the study. Data show the tumor volume was inhibited over time in a dose-dependent manner.
  • FIG.69 is a graph showing plasma concentration profiles for VHH human IL-2 fusion proteins comprising an anti-human serum albumin VHH antibody derived from llama (WW10308, WW10311, WW10312, WW10313, WW10314, WW10315, and WW10316) over time.
  • the graph shows that the concentration for each of the VHH-IL-2 fusion proteins followed a similar, non-linear pharmacokinetic (PK) profile, with a steep terminal elimination phase.
  • PK non-linear pharmacokinetic
  • the disclosure relates to inducible IL-21 polypeptide prodrugs that contain at least one polypeptide chain, and can contain two or more polypeptide chains, if desired.
  • the inducible IL-21 prodrugs comprise an IL-21 or a mutein thereof, a half-life extension element, an IL-21 blocking element, and a protease cleavable linker.
  • the inducible IL-21 prodrugs can be encoded by the nucleic acids disclosed herein. [0176]
  • the inducible IL-21 prodrugs of this disclosure have attenuated IL-21 receptor agonist activity and the circulating half-life is extended.
  • the IL-21 receptor agonist activity is attenuated through the blocking element, which is capable of blocking all or some of the receptor agonist activity of the IL-21 or mutein within the prodrug, typically by noncovalently binding to the IL-21 or mutein within the prodrug and/or sterically blocking their binding to the receptor.
  • the half-life extension element can also contribute to attenuation, for example through steric effects.
  • the half-life extension element can also act as a blocking element that is capable of blocking all or some of the receptor agonist activity of IL-21. For instance, the half-life extension element can contribute to blocking when the half-life extension element is adjacent to the IL-21 polypeptide.
  • IL-21 Upon cleavage of the protease cleavable linker a form of IL-21 is released that is active (e.g., more active than the inducible IL-21 prodrug).
  • the released IL-21 is at least 10 x more active than the inducible IL-21 prodrug.
  • the released Il-21 is at least 20 x, at least 30 x, at least 50 x, at least 100 x, at least 200 x, at least 300 x, at least 500 x, at least 1000 x, at least about 10,000X or more active than the inducible IL-21 prodrug.
  • cleavage of the protease cleavable linker releases IL- 21 from both the IL-21 blocking element and the half-life extension element.
  • the form of IL-21 that is released upon cleavage of the inducible IL-21 prodrug typically has a short half-life, which is often substantially similar to the half-life of naturally occurring IL-21.
  • the inducible IL-21 prodrug can comprise a single polypeptide chain.
  • the single polypeptide complex comprises an IL-21 polypeptide or a mutein thereof [A], a blocking element [D], a half-life extension element [H], and a protease cleavable linker [L].
  • the IL-21 [A] polypeptide can be operably linked to the blocking element, the half-life extension element, or both the blocking element and the half-life extension element by a protease cleavable linker.
  • the protease cleavable linker can comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), particularly ALFKSSFP (SEQ ID NO: 198).
  • the single polypeptide complex can comprise an IL-21 polypeptide [A], a blocking element [D], a half-life extension element [H], and a protease cleavable linker having the amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) [L], particularly ALFKSSFP (SEQ ID NO: 198).
  • the IL-21 [A] polypeptide can be operably linked to the blocking element, the half-life extension element, or both the blocking element and the half-life extension element by a protease cleavable linker.
  • the single polypeptide complex can comprise a IL-21 polypeptide [A], a blocking element [D], a half-life extension element [H], and a protease cleavable linker having the amino acid sequence GPAGLYAQ (SEQ ID NO: 195) [L].
  • the IL-21 [A] polypeptide can be operably linked to the blocking element, the half-life extension element, or both the blocking element and the half-life extension element by a protease cleavable linker.
  • the single polypeptide complex can comprise a IL-21 polypeptide [A], a blocking element [D], a half-life extension element [H], and a protease cleavable linker having the amino acid sequence ALFKSSFP (SEQ ID NO: 198) [L].
  • the IL-21 [A] polypeptide can be operably linked to the blocking element, the half-life extension element, or both the blocking element and the half-life extension element by a protease cleavable linker.
  • the IL-21 polypeptide and the blocking element and the half-life extension element are operably linked by the protease-cleavable polypeptide.
  • the polypeptide can be any of Formulas (I)- (VI).
  • [L1] and [L2], or [L1] and [L2’] can have the same or different amino acid sequence and/or protease-cleavage site (when L2 is protease-cleavable) as desired.
  • [H] can also optionally provide blocking.
  • the protease cleavable linker can comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), particularly ALFKSSFP (SEQ ID NO: 198).
  • the inducible IL-21 prodrugs disclosed herein may contain one half-life extension element and one blocking element, such elements can contain two or more components that are present on the same polypeptide chain or on different polypeptide chains.
  • components of the blocking element can be present on separate polypeptide chains.
  • a first polypeptide chain can include an antibody light chain (VL+CL) or light chain variable domain (VL) and a second polypeptide can include an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that is complementary to the VL+ CL or VL on the first polypeptide.
  • these components can associate in the peptide complex to form an antigen-binding site, such as a Fab that binds IL-21 and attenuates IL-21 activity.
  • the inducible IL-21 prodrug can have a first polypeptide of Formulas (X-XI) or (XXV-XXVIII).
  • Formula X [D]-[L1]-[A]-[L2]-[H]
  • Formula XI [H]-[L1]-[A]-[L2]-[D]
  • Formula XXV [A]-[L1]-[H]-[L3]-[D]
  • Formula XXVI [D]-[L3]-[H]-[L1]-[A]
  • Formula XXVII [A]-[L1]-[D]-[L3]-[H]]
  • Formula XXVIII [H]-[L3]-[D]-[L1]-[A].
  • [A] is a IL-21 polypeptide
  • [D] is a IL-21 antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH)
  • [H] is a half-life extension element
  • [L1] is a protease-cleavable polypeptide linker
  • [L2] is a protease-cleavable linker
  • [L3] is a polypeptide linker that is optionally protease-cleavable.
  • [L1] and [L2] can have the same or different amino acid sequence and or protease-cleavage site, as desired.
  • the inducible IL-21 prodrug can also have a second polypeptide comprising an antibody light 31 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 chain (VL+CL) or light chain variable domain (VL) that is complementary to the VH + CH1 or VH on the first polypeptide.
  • the protease cleavable linker can comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), particularly ALFKSSFP (SEQ ID NO: 198).
  • the inducible IL-21 prodrug disclosed herein can comprise a first polypeptide chain that includes an IL-21 polypeptide and a IL-21 antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that are linked through a protease cleavable linker, and a second polypeptide chain that includes a half-life extension element and an IL-21 antibody light chain (VL+CL) or light chain variable domain (VL) that is complementary to the VH + CH1 or VH on the first polypeptide that are linked through an optionally protease cleavable linker.
  • [A] is an IL-21 polypeptide
  • [D] is an IL-21R
  • [X] is a heavy chain constant region (i.e., CH1)
  • [H] is a half-life extension element
  • [L1] is a protease-cleavable polypeptide linker
  • [L2] is optionally protease-cleavable.
  • [L1] and [L2] can have the same or different amino acid sequences and/or a protease cleavage site (when L2 is protease cleavable), as desired.
  • the inducible IL-21 prodrug can have a second 32 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 polypeptide chain comprising a common gamma chain and a light chain operably linked through an optionally protease cleavable linker.
  • a first polypeptide chain can comprise the common gamma chain and a heavy chain constant region (i.e., CH1) operably linked through an optionally protease cleavable linker
  • a second polypeptide chain can comprise the IL-21R and a light chain operably linked through an optionally protease cleavable linker.
  • the heavy chain constant region on the first polypeptide chain together with the light chain form a dimer (i.e., dimerize) such that the IL-21R and the common gamma chain form binding sites for IL-21.
  • the first polypeptide chain can also comprise IL-21 and optionally a half-life extension element.
  • the second polypeptide chain can also comprise an optional half-life extension element. In some embodiments, only one of the first and the second polypeptide contains the half-life extension element. When the first polypeptide chain contains the half-life extension element, the second polypeptide may not contain a half-life extension element.
  • the first polypeptide can comprise IL-21 and either an antibody light chain (VL + CL) or light chain variable domain (VL), or an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that is operably linked to IL-21 through a protease cleavable linker.
  • VL + CL antibody light chain
  • VL light chain variable domain
  • VH + CH1 antibody heavy chain Fab fragment
  • VH heavy chain variable domain
  • the second polypeptide can comprise the half-life extension element and either an antibody light chain (VL + CL) or light chain variable domain (VL), or an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that is operably linked to the half-life extension element through an optionally protease-cleavable linker.
  • VL + CL antibody light chain
  • VH + CH1 antibody heavy chain Fab fragment
  • VH heavy chain variable domain
  • the first polypeptide can comprise IL-21 and a portion of an antibody light chain that are linked through a protease-cleavable linker.
  • the second polypeptide comprises a half-life extension element and a portion of an antibody heavy chain that is complementary to the antibody light chain that are linked through an optionally protease-cleavable linker.
  • the portion of the antibody heavy chain together with the complementary light chain associate to form a binding site for IL-21.
  • the first polypeptide can comprise IL-21 and a portion of an antibody heavy chain that are linked through a protease-cleavable linker.
  • the second polypeptide can comprise a half-life extension element and a 33 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 portion of an antibody light chain that are linked through an optionally protease-cleavable linker.
  • the portion of the antibody heavy chain together with the complementary light chain associate to form a binding site for IL-21.
  • the inducible IL-21 prodrugs disclosed herein may contain at least one half- life extension element and at least one blocking element, and such elements can contain two or more components that are present on the same polypeptide chain or on different polypeptide chains.
  • the first polypeptide chain can comprise a first half-life extension element, and a second polypeptide chain can comprise a second half-life extension element.
  • the first half-life extension element can comprise a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) that optionally comprises one or more amino acid mutations that creates a “knob,” and the second half-life extension element can comprise a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) that optionally comprises one or more amino acid mutations that create a “hole.”
  • the first half-life extension element can comprise a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) that optionally comprises one or more amino acid mutations that creates a “hole,” and the second half-life extension element can comprise a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) that optionally comprises one or more amino acid mutations that create a “knob.”
  • the first half-life extension element and the second half-life extension element can form a heterodimer through disulfide bonds or a optionally protease cleavable linker, for example.
  • the first polypeptide chain and second polypeptide chain can each comprise a half-life extension element.
  • the first polypeptide chain can comprise the first half-life extension element, an IL- 21 polypeptide, and a blocking element
  • the second polypeptide chain can comprise the second half- life extension element.
  • the first polypeptide chain can comprise the first half-life extension element and an IL-21 polypeptide
  • the second polypeptide chain can comprise the second half-life extension element and a blocking element.
  • the first polypeptide chain can comprise the first half-life extension element and a blocking element
  • the second polypeptide chain can comprise the second half-life extension element and a IL-21 polypeptide.
  • the inducible IL-21 prodrug can have a first polypeptide of Formula XVI: [D]-[L2]- [H], and a second polypeptide of Formula XVII: [A]-[L1]-[H].
  • the inducible IL-21 prodrug can have a first polypeptide of Formula XVIII: [H]-[L2]-[D], and a second polypeptide of Formula XIX: [H]-[L1]-[A].
  • each polypeptide chain can contain one IL-21 polypeptide, one half-life extension element, and one blocking element.
  • [L1] and [L2], or [L1] and [L2’] can have the same or different amino acid sequence and/or protease-cleavage site (when L2 is protease-cleavable) as desired.
  • [H] can also optionally provide blocking.
  • the inducible IL-21 prodrugs disclosed herein can also comprise one or more additional blocking elements.
  • the first polypeptide chain can comprise one or more additional blocking elements.
  • the second polypeptide chain can comprise one or more additional blocking elements.
  • the inducible IL-21 prodrug can have a first polypeptide chain of any one of Formulas (XX)-(XXVI): 35 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 [H]-[L1]-[A]-[L1]-[D] (XX); [D]-[L1]-[A]-[L1]-[H] (XXI); [D]-[L2]-[H]-[L1]-[A] (XXII); [D]-[L2]-[H]-[L1]-[A] (XXIII); [H]-[L2]-[D]-[L1]-[A] (XXIV); [A]-[L1]-[D]-[L2]-[H] (XXV); [A]-[L1]-[H]-[L2]-[D] (XXVI); [0219
  • [A] is a IL-21 polypeptide
  • [D] is a first IL-21 blocking element
  • [H] is a first half-life extension element
  • [D’] is a second IL-21 blocking element
  • [H’] is a second half-life extension element
  • [L1] is a protease cleavable linker
  • [L2] is an optional protease cleavable linker.
  • the inducible IL-21 prodrug comprises: (i) IL-21; (ii) a half-life extension element selected from an antibody or fragment thereof (particularly a dAb), e.g., an antibody or fragment that binds serum albumin (particularly HSA), an immunoglobulin Fc or fragment thereof, or serum albumin (particularly HSA); (iii) a blocking element selected from an IL-21 binding Fab, dAB, scFV, or cognate IL-21 receptor or fragment thereof; and a protease cleavable linker, e.g., a linker cleavable by MMP14 or CTSL1 such as the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), particularly ALFKSSFP (SEQ ID NO: 198).
  • a dAb an antibody or fragment that binds serum albumin
  • a blocking element selected from an IL-21 binding Fab, dAB, scF
  • the half-life extension element increases the in vivo half-life and provides altered pharmacodynamics and pharmacokinetics of the inducible IL-21 prodrugs. Without being bound by theory, the half-life extension element alters pharmacodynamic properties including alteration of tissue distribution, penetration, and diffusion of the inducible IL-21 prodrug. In some embodiments, the half-life extension element can improve tissue targeting, tissue penetration, diffusion within the tissue, and enhanced efficacy as compared with a protein without a half-life extension element.
  • an exemplary way to improve the pharmacokinetics of a polypeptide is by expression of an element in the polypeptide chain that binds to receptors that are recycled to the plasma membrane of cells rather than degraded in the lysosomes, such as the FcRn receptor on endothelial cells and transferrin receptor.
  • an element in the polypeptide chain that binds to receptors that are recycled to the plasma membrane of cells rather than degraded in the lysosomes, such as the FcRn receptor on endothelial cells and transferrin receptor.
  • Three types of proteins, e.g., human IgGs, HSA (or fragments), and transferrin persist for much 36 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 longer in human serum than would be predicted just by their size, which is a function of their ability to bind to receptors that are recycled rather than degraded in the lysosome.
  • HSA may also be directly bound to the pharmaceutical compositions or bound via a short linker. Fragments of HSA may also be used. HSA and fragments thereof can function as both a blocking element and a half-life extension element. Human IgGs and Fc fragments can also carry out a similar function.
  • the serum half-life extension element can also be an antigen-binding polypeptide that binds to a protein with a long serum half-life such as serum albumin (e.g., HSA), transferrin and the like.
  • polypeptides include antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a single chain variable fragment (scFv), an antigen binding fragment (Fab), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like.
  • scFv single chain variable fragment
  • Fab antigen binding fragment
  • single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like.
  • antigen-binding domains include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
  • non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
  • antigen-binding polypeptides include a ligand for a desired receptor, a ligand-binding portion of a receptor, a lectin, and peptides that bind to or associate with one or more target antigens.
  • the antibodies and fragments thereof can function as both a blocking element and a half-life extension element.
  • the half-life extension element can also function as both a blocking element and a half-life extension element.
  • the half-life extension element e.g., HSA binding polypeptide
  • the half-life extension element can function as a blocking element when adjacent to the IL-21 polypeptide.
  • the half-life extension element is a single domain antibody fragment (sdAb).
  • the sdAb can comprise or consist of SEQ ID NO: 602-606, 608-653, or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 602 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 603 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 604 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 605 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 630 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 631 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 632 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 633 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 634 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 635 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 636 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 637 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 638 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 639 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 640 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 641 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 642 or a variant thereof.
  • the sdAb can comprise or consist of 38 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 SEQ ID NO: 643 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 644 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 645 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 46 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 647 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 648 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 649 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 650 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 651 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 652 or a variant thereof.
  • the sdAb can comprise or consist of SEQ ID NO: 653 or a variant thereof.
  • the sdAb can comprise or consist of the amino acid sequence of any of SEQ ID NOs: 602-606, 608-653, or an amino acid sequence that has at least about 80% identity to any of SEQ ID NOs: 608-653.
  • the sdAbs can comprise or consist of an amino acid sequence that has at least about 81% identity, at least about 82% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to any of SEQ ID NOs: 602, 603, 604, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 6
  • Compounds 210-221 are specific examples of inducible IL-21 prodrugs that comprise a half-life extension element that is a sdAb that comprises or consists of any one of SEQ ID NOs: 602-606 or 608- 653 or a variant thereof.
  • Compound 210 comprises a sdAb that comprises SEQ ID NO: 608.
  • Compound 211 comprises a sdAb that comprises SEQ ID NO: 609.
  • Compound 212 comprises a sdAb that comprises SEQ ID NO: 611.
  • Compound 213 comprises a sdAb that comprises SEQ ID NO: 612.
  • Compound 214 comprises a sdAb that comprises SEQ ID NO: 625.
  • Compound 215 comprises a sdAb that comprises SEQ ID NO: 626.
  • Compound 216 comprises a sdAb that comprises SEQ ID NO: 628.
  • Compound 217 comprises a sdAb that comprises SEQ ID NO: 632.
  • Compound 218 comprises a sdAb that comprises SEQ ID NO: 635.
  • Compound 219 comprises a sdAb that comprises SEQ ID NO: 637.
  • Compound 220 comprises a sdAb that comprises SEQ ID NO: 642.
  • Compound 221 comprises a sdAb that comprises SEQ ID NO: 644.
  • Compound 222 comprises a sdAb that comprises SEQ ID NO: 645.
  • Compound 223 comprises a sdAb that comprises SEQ ID NO: 646.
  • Compound 224 comprises a sdAb that comprises 39 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 SEQ ID NO: 618.
  • Compound 225 comprises a sdAb that comprises SEQ ID NO: 622.
  • Compound 226 comprises a sdAb that comprises SEQ ID NO: 624.
  • the half-life extension element disclosed herein can comprise a first half-life extension element and a second half-life extension element.
  • the first and second half-life extension element can heterodimerize.
  • the first and second half-life extension elements can include one or more modifications that promote heterodimerization of the first and second half-life extension elements.
  • one or more amino acid modifications can be made to the first half-life extension element.
  • one or more amino acid modifications can be made to the second half-life extension element.
  • the inducible IL- 21 prodrugs disclosed herein can comprise a first half-life extension element and a second half-life extension element, each of which comprises a CH3 domain.
  • the half-life extension element comprising a CH3 domain is a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or fragment thereof).
  • the CH3 domains of the first and second half-life extension elements can be altered by the “knobs-into-holes” technology.
  • knob-into-holes method the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of the first half-life extension element and the second half-life extension element each containing an altered CH3 domain. This occurs by introducing a bulky residue into the CH3 domain of one of the half-life extension elements, which acts as the “knob.” Then, in order to accommodate the bulky residue, a “hole” is formed in the other half-life extension domain that can accommodate the knob.
  • Either of the altered CH3 domains can be the “knob” while the other can be the “hole.”
  • the introduction of a disulfide bridge can further stabilize the heterodimers.
  • the knobs-into-holes approach can be used to promote heterodimerization between two different half-life extension elements (e.g., between a first half-life extension element and a second half-life extension element).
  • the IL-21 prodrugs described herein can comprise two half-life extension elements (i.e., a first half-life element and a second-half-life extension element).
  • the first half-life extension element and the second half-life extension element can be linked via a linker.
  • the first half-life extension element and the second half-life extension element can be an Fc domain or fragment thereof.
  • the first half-life extension element and the second half-life extension element can be an antibody, or a fragment, variant, or derivative thereof.
  • the first half-life extension element can comprise a heavy chain polypeptide or portion thereof (e.g., the heavy chain constant regions of an Fc domain, or fragment thereof) that comprises one or more 40 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 amino acid mutations that creates a “knob”
  • the second half-life extension element can comprise a heavy chain polypeptide or portion thereof (e.g., the heavy chain constant regions of an Fc domain, or fragment thereof) that comprises one or more amino acid mutations that create a “hole.”
  • the first half-life extension element can comprise a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) that comprises one or more amino acid mutations that creates a “hole
  • the half-life extension element of an inducible IL-21 prodrug extends the half-life of the inducible IL-21 prodrug by at least about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, about 10 days or more.
  • the blocking element can be any element that binds to IL-21 and inhibits the ability of the IL-21 polypeptide to bind and activate its receptor.
  • the blocking element can inhibit the ability of IL-21 to bind and/or activate its receptor e.g., by sterically blocking and/or by noncovalently binding to IL-21 within the prodrug.
  • the blocking element disclosed herein can bind to IL-21.
  • Suitable blocking elements include the full-length cognate receptor of IL-21 and an IL-21-binding fragment or mutein thereof.
  • the cognate receptor for IL-21 can be the IL-21R receptor or a portion thereof and/or the common gamma chain or a portion thereof.
  • Antibodies and antigen-binding fragments thereof that bind IL-21 including an antigen-binding fragment (Fab), a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like, can also be used.
  • Fab antigen-binding fragment
  • scFv single chain variable fragment
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VHH camelid-type nanobody
  • Suitable antigen-binding domains that bind IL-21 can also be used, including non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
  • suitable blocking polypeptides include polypeptides that sterically inhibit or block binding of IL-21 to its cognate receptor.
  • moieties can also function as half-life extending elements.
  • a peptide that is modified by conjugation to a water-soluble polymer can sterically inhibit or prevent binding of the 41 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 cytokine to its receptor.
  • Polypeptides, or fragments thereof, that have long serum half-lives can also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin and the like, as well as fragments and muteins of such polypeptides.
  • the linker is cleavable by a cleaving agent, e.g., an enzyme.
  • the linker comprises a protease cleavage site.
  • the linker comprises one or more cleavage sites.
  • the linker can comprise a single protease cleavage site.
  • the linker can also comprise 2 or more protease cleavage sites. For example, 2 cleavage sites, 3 cleavage sites, 4, cleavage sites, 5 cleavage sites, or more.
  • the linker comprises 2 or more protease cleavage sites, the cleavage sites can be cleaved by the same protease or different proteases.
  • the linkers disclosed herein can comprise one or more cleavage motifs or functional variants that are the same or different.
  • the linkers can comprise 1, 2, 3, 4, 5, or more cleavage motifs or functional variants.
  • Linkers comprising 30 amino acids can contain 2 cleavage motifs or functional variants, 3 cleavage motifs or functional variants or more.
  • a “functional variant” of a linker retains the ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease) and are not cleaved or cleaved with low efficiency in the periphery (e.g., serum).
  • the functional variant of the linker preferably contains no more than 1, 2, 3, 4, or 5 amino acid deletions.
  • the amino acid sequences disclosed in the linkers can be described by the relative linear position in the linker with respect to the scissile bond.
  • linkers comprising 8 amino acid protease substrates e.g., SEQ ID NOs: 195-201 or 447-448 contain amino acid at positions P4, P3, P2, P1, P1’, P2’, P3’, P4’, wherein the scissile bond is between P1 and P1’.
  • amino acid positions for the linker comprising the sequence GPAGLYAQ can be described as follows (SEQ ID NO: 195): G P A G L Y A Q [0255]
  • Amino acids positions for the linker comprising the sequence ALFKSSFP can be described as follows (SEQ ID NO: 198): 49 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 A L F K S S F P P4 P3 P2 P1 P1’ P2’ P3’ P4’ [0256]
  • Preferab nd P1’for SEQ ID NOs: 195-201 or 447-448) are not substituted.
  • the linker comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) or a functional variant of SEQ ID NO: 195 or a function variant of SEQ ID NO: 198.
  • a functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) can comprise one or more amino acid substitutions, and substantially retain their ability to be cleaved by a protease.
  • the functional variants of GPAGLYAQ (SEQ ID NO: 195) are cleaved by MMP14, and the functional variants of ALFKSSFP (SEQ ID NO: 198) are cleaved by Capthepsin L (CTSL1).
  • the functional variants also retain their ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease).
  • the functional variants of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a linker comprising amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.
  • the functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) comprise no more than 1, 2, 3, 4, or 5 conservative amino acid substitutions compared to GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198).
  • the amino acids at position P1 and P1’ are not substituted.
  • the amino acids at positions P1 and P1’ in SEQ ID NO: 195 are G and L
  • the amino acids at positions P1 and P1’ in SEQ ID NO: 198 are K and S.
  • the functional variant of GPAGLYAQ can preferably comprise one or more of the following: a) an arginine amino acid substitution at position P4, b) a leucine, valine, asparagine, or proline amino acid substitution at position P3, c) a asparagine amino acid substitution at position P2, d) a histidine, asparagine, or glycine amino acid substitution at position P1, e) a asparagine, isoleucine, or leucine amino acid substitution at position P1’, f) a tyrosine or arginine amino acid substitution at position P2’, g) a glycine, arginine, or alanine amino acid substitution at position P3’, h) or a serine, glutamine, or lysine amino acid substitution at position P4’.
  • GPAGLYAQ SEQ ID NO: 195
  • the functional variant of GPAGLYAQ does not comprise an amino acid substitution at position P1 and/or P1’.
  • the amino acid substitution of the functional variant of GPAGLYAQ (SEQ ID NO: 195) preferably comprises an amino acid substitution at position P4 and/or P4’.
  • the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprise a leucine at position P4, or serine, glutamine, lysine, or phenylalanine at position P4.
  • the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprise a glycine, phenylalanine, or a proline at position P4’.
  • the amino acid substitutions at position P2 or P2’ of GPAGLYAQ are not preferred.
  • the functional variant of GPAGLYAQ comprises the amino acid sequence selected from SEQ ID NOs: 221- 295.
  • Specific functional variants of GPAGLYAQ include GPLGLYAQ (SEQ ID NO: 259), and GPAGLKGA (SEQ ID NO: 249).
  • the functional variants of LFKSSFP (SEQ ID NO: 448) preferably comprises hydrophobic amino acid substitutions.
  • the functional variant of LFKSSFP can preferably comprise one or more of the following: (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at position P4; (b) lysine, histidine, glycine, proline, asparagine, phenylalanine at position P3; (c) arginine, leucine, alanine, glutamine, or histatine at position P2; (d) phenylalanine, histidine, threonine, alanine, or glutamine at position P1; (e) histidine, leucine, lysine, alanine, isoleucine, arginine, phenylalanine, asparagine, glutamic acid, or glycine at position P1’, (f) phenylalanine, leucine, isoleucine, lysine, alanine, glut
  • aspartic acid and/or glutamic acid are generally disfavored and avoided.
  • the following amino acid substitutions are also disfavored in functional variants of LFKSSFP (SEQ ID NO: 448): (a) alanine, serine, or glutamic acid at position P3; (b) proline, threonine, glycine, or aspartic acid at position P2; (c) proline at position P1; (d) proline at position P1’; (e) glycine at position P2’; (f) lysine or glutamic acid at position P3’; (g) aspartic acid at position P4’.
  • the amino acid substitution of the functional variant of LFKSSFP preferably comprises an amino acid substitution at position P4 and/or P1. In some embodiments, an amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 448) at position P4’ is not preferred. 51 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 [0266] In some embodiments, the functional variant of LFKSSFP (SEQ ID NO: 448) comprises the amino acid sequence selected from SEQ ID NOs: 296- 374.
  • LFKSSFP Specific functional variants of LFKSSFP (SEQ ID NO: 448) include ALFFSSPP (SEQ ID NO: 199), ALFKSFPP (SEQ ID NO: 346), ALFKSLPP (SEQ ID NO: 347); ALFKHSPP (SEQ ID NO: 335); ALFKSIPP (SEQ ID NO: 348); ALFKSSLP (SEQ ID NO: 356); or SPFRSSRQ (SEQ ID NO: 297).
  • the linkers disclosed herein can form a stable prodrug under physiological conditions with the amino acid sequences (e.g. domains) that they link, while being capable of being cleaved by a protease.
  • the linker desirably remains stable in the circulation for at least 2 hours, at least 5, hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or longer.
  • the linker is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% in the circulation as compared to the target location.
  • the linker is also stable in the absence of an enzyme capable of cleaving the linker.
  • the inducible IL-21 prodrugs can include a polypeptide that comprises or consists of an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 2-8, 44- 50, and 72-109.
  • Compounds 53-222 are specific examples of inducible IL-21 prodrugs that comprise two polypeptide chains for use according to this disclosure. Further details of exemplary IL-21 prodrugs is described in Table 39. Table 39.
  • F. sdAbs that bind Serum Albumin The disclosure also relates to single domain antibody fragments (sdAbs) (such as VHH, and VH) that have binding specificity for human serum albumin, methods of making, and uses thereof.
  • sdAbs single domain antibody fragments
  • Such sdAbs can be used, for example, for extending the in vivo serum half-life of therapeutic agents (e.g., proteins, peptides and the like) through the engineering of fusion proteins and conjugates using well-known methods and as described herein.
  • Preferred sdAbs compete with FcRn (human FcRn) for binding to human serum albumin and, preferably, bind to domain 1 (DI) of human serum albumin.
  • the sdAbs of this disclosure include, but are not limited to, heavy chain variable domain (VH), variable heavy domain of heavy chain (VHH), single domain shark variable domain of new antigen receptor (VNAR), or light chain variable (VL) domain (e.g., a kappa VL domain or a lambda VL domain).
  • VH heavy chain variable domain
  • VHH variable heavy domain of heavy chain
  • VNAR single domain shark variable domain of new antigen receptor
  • VL light chain variable domain
  • the sdAbs comprise one or more complementary determining regions (CDRs), typically three CDRs, of an immunoglobulin single variable domain that has binding specificity for serum albumin.
  • CDRs of the sdAbs disclosed herein can be derived from a camelid.
  • the sdAbs can be humanized and comprise one or more FRs, that can be derived from a human germline and typically include back mutations.
  • the sdAbs disclosed herein can comprise a CDR1, a CDR2 and/or a CDR3.
  • the sdAbs disclosed herein comprise CDR1, CDR2, and CDR3.
  • the sdAbs can comprise a CDR1 comprising an amino acid sequence of Formula (I): X 1 X 2 X 3 X 4 X 5 X 6 AX 7 X 8 G (I).
  • X 1 is glycine or aspartic acid
  • X 2 is glycine, arginine, phenylalanine, leucine, valine, serine, tyrosine, histidine, or threonine
  • X 3 is threonine, glycine, or serine
  • X 4 is phenylalanine, valine, tyrosine, leucine, isoleucine, or arginine
  • X 5 is aspartic acid, serine, threonine, or arginine
  • X 6 is aspartic acid, serine, glycine, or glutamic acid
  • X 7 is arginine, glycine, serine, threonine, methionine, valine, isoleucine, histidine, or asparagine
  • X 8 is arginine, glycine, leucine, methionine, isoleucine or valine.
  • the sdAbs can comprise a CDR2 comprising an amino acid of Formula (II) A I S X 1 S X 2 X 3 X 4 T X 5 Y X 6 X 7 X 8 V K G (II) (SEQ ID NO: 693) [0282]
  • X 1 is isoleucine or alanine
  • X 2 is glycine or threonine
  • X 3 is glutamic acid or glycine
  • X 4 is arginine, threonine, or serine
  • X 5 is asparagine, arginine, or tyrosine
  • X 6 is glycine, arginine, serine, or glutamic acid
  • X 7 is glutamic acid, lysine, aspartic acid, or glutamic acid
  • X 8 is serine or glutamic acid.
  • the sdAbs can comprise a CDR3 comprising an amino acid sequence of Formula (III): A X 1 G D W Y H L X 2 Q X 3 X 4 X 5 X 6 X 7 X 8 I X 9 X 10 (III) (SEQ ID NO: 694).
  • X 1 is serine or alanine
  • X 2 is valine, isoleucine, or threonine
  • X 3 is glycine or glutamic acid
  • X 4 is threonine, glutamic acid, or histidine
  • X 5 is glycine or asparagine
  • X 6 us methionine or asparagine
  • X 7 is serine, alanine, or methionine
  • X 8 is tyrosine or phenylalanine
  • X 9 is arginine or tryptophan
  • X 10 is asparagine or tyrosine.
  • the sdAbs comprises a CDR1 of Formula (I), a CDR2 of Formula (II), and a CDR3 of Formula (III).
  • the sdAbs disclosed herein can comprise a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 656, 659, 662, 663, 691 or a variant of any of the foregoing as described herein.
  • the sdAbs disclosed herein can include a CDR1 that comprises or consists of SEQ ID NO: 656.
  • the sdAbs disclosed herein can include a CDR1 that comprises or consists of SEQ ID NO: 659.
  • the sdAbs disclosed 64 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 herein can include a CDR1 that comprises or consists of SEQ ID NO: 662.
  • the sdAbs disclosed herein can comprise a CDR1 that comprises or consists of SEQ ID NO: 663.
  • the sdAbs disclosed herein can comprise a CDR1 that comprises or consists of SEQ ID NO: 691.
  • the sdAb disclosed herein can comprise a CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 657, 660, 664, 665, 666, 38 or a variant of any of the foregoing as described herein.
  • the sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 657.
  • the sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 660.
  • the sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 664.
  • the sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 665.
  • the sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 666.
  • the sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 38.
  • the sdAbs disclosed herein can comprise a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 656, 659, 662, 663, 691, or a variant of any of the foregoing, a CDR2 comprising any one of SEQ ID NO: 657, 660, 664, 665, 666, 38, or a variant of any of the foregoing, and a CDR3, such that the sdAbs binds human serum albumin.
  • the sdAbs disclosed herein can comprise a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 656, 659, 662, 663, 691, or a variant of any of the foregoing, a CDR3 comprising any one of SEQ ID NO: 658, 661, or 667, or a variant of any of the foregoing, and a CDR2, such that the sdAbs binds human serum albumin.
  • the sdAbs disclosed herein can comprise a CDR2 comprising any one of SEQ ID NO: 657, 660, 664, 665, 666, 38, or a variant of any of the foregoing, a CDR3 comprising any one of SEQ ID NO: 658, 661, or 667, or a variant of any of the foregoing, and a CDR1, such that the sdAbs binds human serum albumin.
  • the sdAbs disclosed herein have all three CDRs (e.g., a CDR1, a CDR2, and a CDR3).
  • the sdAbs disclosed herein can comprise a CDR1 comprising any one of SEQ ID NO: 656, 659, 662, 663 and 691, or a variant of any of the foregoing; a CDR2 comprising any one of SEQ ID NO: 657, 65 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 660, 664, 665, 666 and 38 or a variant of any of the foregoing; and a CDR3 comprising any one of SEQ ID NO: 658, 661, and 667, or a variant of any of the foregoing.
  • the sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 656 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 657 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 658 or variant thereof.
  • the sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 656, CDR2 that comprises or consists of SEQ ID NO: 657, and CDR3 that comprises or consists of SEQ ID NO: 658.
  • Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 663; a CDR2 that comprises or consists of SEQ ID NO: 665; and a CDR3 that comprises or consists of SEQ ID NO: 667. Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 663; a CDR2 that comprises or consists of SEQ ID NO: 666; and a CDR3 that comprises or consists of SEQ ID NO: 667.
  • Certain preferred sdAbs include FRs derived from human germline gene IGHV3-11 and/or IGHV3-23.
  • the sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 668, 669, 31, or a variant of any of the foregoing.
  • the sdAbs disclosed herein can comprise a FR1 comprising an amino acid sequence that has at least 80% identity to any one of SEQ ID NOs.: SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing.
  • the sdAbs disclosed herein can include a FR1 that comprises or consists of SEQ ID NO: 668.
  • the sdAbs disclosed herein can comprise a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 677, or a variant thereof.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 669, a FR2 that comprises or consists of SEQ ID NO: 672, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 674, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 678, a FR3 that comprises or consists of SEQ ID NO: 683; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 682, a FR3 that comprises or consists of SEQ ID NO: 676; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 669, a FR2 that comprises or consists of SEQ ID NO: 678, a FR3 that comprises or consists of SEQ ID NO: 684; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 686, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 687, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 681, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 687, a FR3 that comprises or consists of SEQ ID NO: 688; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 681, a FR3 that comprises or consists of SEQ ID NO: 688; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 679, a FR3 that comprises or consists of SEQ ID NO: 688; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 686, a FR3 that comprises or consists of SEQ ID NO: 689; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 687, a FR3 that comprises or consists of SEQ ID NO: 689; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 681, a FR3 that comprises or consists of SEQ ID NO: 689; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 679, a FR3 that comprises or consists of SEQ ID NO: 689; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 682, a FR3 that comprises or consists of SEQ ID NO: 690; and a FR4 that comprises or consists of SEQ ID NO: 677.
  • the sdAbs disclosed herein can comprise or consist of any one of the sdAbs disclosed in Table 51.
  • Exemplary sdAbs sdAb FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 7 7 7 75 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 1HUM19_D SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: 668 NO: 662 NO: 673 NO: 692 NO: 675 NO: 667 NO: 677 610 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 76 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 1HUM19_Q SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: 669 NO: 663 NO: 670 NO: 666 NO: 675 NO: 667
  • the sdAbs can comprise or consist of an amino acid sequence that has at least about 81% identity, at least about 82% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to any of SEQ ID NOs: 608, 609, 41, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639,
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 608, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 608. 79 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 [0350]
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 609, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 609.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 612, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 612.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 610, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 610.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 611, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 611.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 613, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 613.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 614, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 614.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 615, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 615.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 616, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 616.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 617, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 617.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 618, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 618.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 619, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 619.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 620, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 620.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 621, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 621.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 622, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 622.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 623, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 623.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 624, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 624. 80 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 [0366]
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 625, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 625.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 626, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 626.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 627, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 627.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 628, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 628.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 629, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 629.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 630, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 630.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 631, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 631.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 632, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 632.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 633, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 633.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 634, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 634.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 635, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 635.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 636, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 636.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 637, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 637.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 638, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 638.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 639, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 639.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 640, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 640.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 641, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 641.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 642, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 642.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 643, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 643.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 644, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 644.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 645, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 645.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 646, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 646.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 647, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 647.
  • the sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 648, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 648.
  • the sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 649, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 649.
  • the sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 650, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 650.
  • the sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 651, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 651.
  • the sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 652, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 652.
  • the sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 653, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 653.
  • the sdAbs disclosed herein include functional variants that differ in amino acid sequence in comparison, for example, to any one of SEQ ID NOs.: 609-648 or 649-653.
  • a “functional variant” of a sdAb retains the ability to bind to serum albumin.
  • the sdAbs disclosed herein can comprise one or more amino acid substitutions.
  • the amino acid substitution can be a conservative substitution or a non-conservative substitution, but preferably is a 82 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 conservative substitution.
  • Conservative amino acid substitutions are generally made in accordance with the following Table 52. Table 52.
  • a sdAb of this disclosure can contain a CDR1 that comprises or consists of SEQ ID NO: 656, 659, 662, 663, or 691 or a variant of any of the forgoing.
  • a sdAb of this disclosure can contain a CDR2 that comprises or consists of SEQ ID NO: 657, 660, 664, 665, 666 or 692 or a variant of any of the forgoing.
  • Phage generated from the libraries was panned on solid-phase coated recombinant HSA (100 ⁇ g/mL in 100 mM NaHCO 3 pH 8.2) for 3 rounds. 380 phagemid colonies were randomly selected and analyzed by ELISA for the presence of antigen specific VHHs in their periplasmic extracts (ELISA using crude periplasmic extracts including soluble VHHs). The antigen used for panning and ELISA screening was the same one as used for immunization, using uncoated blocked wells as negative controls (blank). Additionally, wells were coated with rhesus/cynomolgus monkey serum albumin (CSA) or mouse serum albumin (MSA) to check for cross- reactivity.
  • CSA rhesus/cynomolgus monkey serum albumin
  • MSA mouse serum albumin
  • VHHs belonging to the same CDR3 group are very similar and their amino acid sequences suggest that they are from clonally-related B-cells resulting from somatic hypermutation or from the same B cell but diversified due to cloning induced errors during library construction.
  • Nanobodies belonging to the same CDR3 group are expected to recognize the same epitope but their other characteristics (e.g. affinity, potency, stability, expression yield, etc.) can be different.
  • VHHs were expressed using the Expi293 Expression System (ThermoFisher). Gene synthesis was conducted to assemble VHHs with a C terminal 6x His tag (SEQ ID NO: 695) and C-tag or with a C- terminal human IgG1 Fc tag. Sequences were cloned into pcDNA3.4 (ThermoFisher), including an amino terminal signal sequence to enable secretion.
  • VHH plasmids were transiently transfected according to the Expi293 Expression System protocol. VHH-containing supernatants were harvested, and VHHs were purified using either Ni-NTA or MabSelect SuRe LX resin. [0542] Purified VHHs were characterized by analytical SEC and SDS-PAGE. For SDS-PAGE, three ⁇ g of protein were assessed as non-reduced and reduced samples. Samples were run on a 4-12% Bis-tris gel run in 1X MOPS SDS Running buffer according to the manufacturer's instructions. Summaries of final yield and percent monomer (by SEC) are shown in Table 27 and Table 28. All VHH’s were >95% purity by SDS-PAGE. Table 27.
  • VHH CDR3 ID Clone Group Yield (mg/L) Percent monomer 149 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 Construct VHH CDR3 ID Clone Group Yield (mg/L) Percent monomer .
  • VHHs containing a human IgG1 Fc tag at the C-terminus were then captured onto anti-human IgG Fc capture biosensors (AHC) at 5 ⁇ g/mL in 1X PBS with 0.05% Tween 20 for 300 seconds.
  • a second baseline step was performed by dipping sensors in 1X PBS with 0.05% Tween 20 for 120 seconds.
  • a titration of albumin was loaded onto sensors for 300 seconds.
  • VHHs containing a human IgG1 Fc tag at the C-terminus were then captured onto anti-human IgG Fc capture biosensors (AHC) at 5 ⁇ g/mL in 1X PBS with 0.05% Tween 20 for 300 seconds (the NOC17 and 10GE control VHHs contained a mouse IgG1 Fc tag and were captured onto anti-mouse IgG Fc biosensors [AMC]).
  • a second baseline step was performed by dipping sensors in 1X PBS with 0.05% Tween 20 for 120 seconds.
  • individual domains of human albumin Domain I, Domain II, or Domain III [Albumin Bioscience]
  • VHHs containing a human IgG1 Fc tag at the C-terminus were then captured onto anti-human IgG Fc capture biosensors (AHC) at 5 ⁇ g/mL in 1X PBS with 0.05% Tween 20 for 300 seconds.
  • a second baseline step was 153 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 performed by dipping sensors in 1X PBS with 0.05% Tween 20 for 120 seconds. Sensors were quenched by binding human IgG Fc fragment at 200 ⁇ g/mL for 600 seconds, and the baseline step was repeated (120 seconds).
  • Human serum albumin (HSA) was then loaded onto sensors at a concentration of 100 nM (300 seconds).
  • VHHs (1HUM19, 3HUM181, and 2HUM45) were found to be competitive with one another for binding to HSA, and therefore belong to the same epitope bin.1HUM19, 3HUM181, and 2HUM45 did not compete with 10GE or NOC17 for binding to albumin, and therefore 1HUM19, 3HUM181, and 2HUM45 belong to different epitope bins from 10GE and NOC17. [0547] When establishing criteria to identify VHHs of interest, it was anticipated that any lead VHH options should not compete with FcRn for binding to albumin. FcRn (Neonatal Fc receptor) is responsible for the pH-dependent recycling and long half-life of albumin.
  • Biotinylated VHHs containing an Avi-tag at the C-terminus were then captured onto streptavidin biosensors (SA) at 2 ⁇ g/mL in 0.5X PBS with 0.05% Tween 20 for 300 seconds.
  • SA streptavidin biosensors
  • a second baseline step was performed by dipping sensors in 0.5X PBS with 0.05% Tween 20 for 120 seconds.
  • human serum albumin at 2 uM was loaded onto sensors for 300 seconds. Sensors were dipped into 100 mM phosphate buffer, pH 6, 150 mM NaCl, 0.05% Tween 20 as a baseline for the next step (120 seconds).
  • each humanized variant was to maintain albumin binding affinity equivalent to that of the corresponding llama VHH, increase the humanness of each VHH, and ensure Protein A binding.
  • FIG.44A (1HUM19), FIG.44B (3HUM181), FIG.44C (2HUM45 on a IGHV3-23 backbone), FIG.44D, (2HUM45 on an IGHV3-11 backbone), FIG.44E (1HUM19 variants with modified CDR sequences), and FIG.44F (3HUM181 variants with modified CDR sequences).
  • FIG.44A (1HUM19)
  • FIG.44B (3HUM181)
  • FIG.44C (2HUM45 on a IGHV3-23 backbone
  • FIG.44D (2HUM45 on an IGHV3-11 backbone
  • FIG.44E (1HUM19 variants with modified CDR sequences
  • FIG.44F 3HUM181 variants with modified CDR sequences.
  • VHHs were transiently transfected according to the 155 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 Expi293 Expression System protocol. VHH-containing supernatants were harvested, and VHHs were purified using MabSelect Protein A resin (Cytiva). VHHs did not contain any tags for purification, as all humanized variants were capable of binding to ProA. [0552] Purified VHHs were characterized by analytical SEC and SDS-PAGE.
  • Sensors were dipped in 1X PBS with 0.05% Tween 20 for 60 seconds to provide an initial baseline. Biotinylated HSA or MSA was then captured onto streptavidin sensors (SA) at 2 ⁇ g/mL in 1X PBS with 0.05% Tween 20 for 300 seconds. A second baseline step was performed by dipping sensors in 1X PBS with 0.05% Tween 20 for 120 seconds. Next, a single concentration of each VHH (500 nM) was loaded onto sensors for 300 seconds to measure association. Sensors were dipped into 1X PBS with 0.05% Tween 20 for 600 seconds to allow for dissociation of albumin from the VHH.
  • SA streptavidin sensors
  • Non-specific binding of a control VHH to human and mouse albumin was assessed by running the experiment as described, but using an anti-IL-2 VHH as the analyte. Lack of non-specific binding to sensors was confirmed by associating three of the anti-albumin VHHs, but including no albumin as the load protein. All kinetic runs were performed at 30°C. [0554] Data were analyzed by fitting binding curves using a 1:1 binding model to generate approximate KD (equilibrium constant, Kdis/Kon), kon (association rate constant), and kdis (dissociation rate constant) values (ForteBio Data Analysis Software v8.2). Results are shown in Table 33. Table 33.
  • Sensors were dipped in 1X PBS with 0.05% Tween 20 for 60 seconds to provide an initial baseline.
  • Biotinylated HAS, MSA, or CSA was then captured onto streptavidin sensors (SA) at 2 ⁇ g/mL in 1X PBS with 0.05% Tween 20 for 600 seconds.
  • SA streptavidin sensors
  • a second baseline step was performed by dipping sensors in 1X PBS with 0.05% Tween 20 for 120 seconds.
  • a titration of each VHH was loaded onto sensors for 300 seconds to measure association (7 point, serial 1:2 dilution, with 200, 400, or 2000 nM as the highest concentration).
  • Each VHH was diluted to 10 ⁇ M or 4 ⁇ M in PBS with the addition of 2X final concentration Protein Thermal Shift Dye Kit (ThermoFisher). Next, samples were subjected to a temperature ramp (25°C to 95°C, with 0.3°C increase every 15 seconds) and fluorescence based melting curve was measured using an Applied Biosystems StepOnePlus qPCR machine. For a number of humanized VHHs, the melting temperature could not be determined; although a number of conditions were assessed by varying both protein and dye concentrations, no single clear profile was obtained. The melting temperatures of humanized VHH variants are listed in Table 35. Table 35.
  • the amount of aggregate present was initially assessed for protein at 1 mg/mL to establish a baseline, and again after three freeze/thaw cycles. Protein aliquots were frozen at –80°C and thawed at room temperature. Aggregation state was also determined for protein aliquots that were incubated for 2 and 4 weeks at 4°C or 37°C. For all samples, fifty ⁇ L at a final concentration of 0.5 mg/mL were injected and run for 20 minutes. Results are shown in Table 36. Table 36.
  • PK was simulated for anti-albumin VHHs with the following KDs for mouse-serum albumin (MSA): No-binding, 10, 50, 100, 250, 500, 1000 and 5000 nM. PK simulations shown for a 1 mg/kg dose, suggested that at least 1000 nM KD was essential to decrease exposure of the anti-VHHs.
  • MSA mouse-serum albumin
  • PK was simulated for anti-albumin VHHs with the following KDs for human-serum albumin (HSA): No-binding, 10, 50, 100, 250, 500, 1000 and 5000 nM. PK simulations shown for a 1 mg/kg dose, suggested that at least a 5000 nM KD was essential to substantially decrease exposure of the anti-VHHs.
  • HSA human-serum albumin
  • Control and cleavage reactions were incubated at 22°C for 2 hours in a thermocycler, and then cleavage was assessed by SDS-PAGE. Three ⁇ g of protein from each sample was removed. All samples were assessed under non-reducing conditions only; 4X Laemmli sample buffer (Bio-Rad 1610747) was added, and samples were heated to 70°C for 10 minutes.
  • mice inoculated with MC38-BC2 were used to assess efficacy and preliminary PK assessments for inducible IL-21 prodrugs.
  • WW50387/WW50394 was included for comparison.
  • Table 37 provides a summary of the agents and treatment. Table 37.
  • tumors reached an average size of 150 mm 3 (calculated as longest diameter * shortest diameter 2 )/2), treatment was initiated (Day 0 of study start), and tumors and body weights were measured 2 times per week for the duration of the study.
  • Example 40 Example 40.
  • the MC38 BC-2 Cell line was injected as 0.5x10 6 cells into the flank of B-hIL21R mice at 1:1 with Matrigel. Dosing was initiated when tumor volumes reached ⁇ 150 mm 3 (range 93.35 – 254.85 mm 3 ).
  • Table 40 summarizes the treatment groups and dose schedules. [0567]
  • %TGI % Tumor Growth inhibition
  • B-hIL21R mice (Jiangsu Biocytogen Co., Ltd., China Production- Stock# 110766F), with an age range of 7-12 weeks and a weight range of 17-25 g, were lightly anesthetized before implantation and each mouse was subcutaneously injected with 0.5x10 6 MC38-BC2 tumor cells in 0.1 mL serum-free medium containing 50% Matrigel in the right flank for tumor development.
  • Tumor-bearing animals were enrolled in 2 study groups when mean tumor volume (TV) reached approximately ⁇ 150 mm 3 (125-175 mm 3 ). Each group consisted of 10 mice (Table 42 Groups 1- 2). Two animals from each group were sacrificed and tumors collected on day 10.
  • Tumors were fixed in formalin and paraffin embedded for generation of slices to stain. After antigen retrieval, tumor slices staining was performed with the COMET-Lunaphore automated M-IF equipment using a series of stains and markers including DAPI, CD3, CD4, CD8, CD11b, CD11c, CD103, F4/80 and Granzyme B (GZMB). Images were generated, and combination of these markers was used to identify different immune populations in the following way: CD3+CD8+ (CTLs), CD3+CD8+GZMB+(activated CTLs), CD3+CD4+ (CD4 T cells), CD11b (myeloid cells), F4/80 (macrophages), CD11c+CD103+ (cross- presenting DCs).
  • CTLs CD3+CD8+
  • CD3+CD8+GZMB+(activated CTLs) CD3+CD4+
  • CD4 T cells CD11b (myeloid cells)
  • F4/80 macrophages
  • Example 42. Renca Tumor Mouse Model Female BALB/c mice were inoculated on the flank with 2x10 6 Renca cells in 50% Matrigel. Tumors were allowed to grow for 7-8 days until mice were randomized for dosing (average tumor volume 100 mm 3 ). Mice were treated with vehicle, 10 ⁇ g WW0177 (IL-2-HLE), or 75 ⁇ g of WW50008 (IL-21- HLE) by intraperitoneal injection (IP) on days 1, 4, 8 and 11 after randomization. Body weight and tumor sizes were measured three times per week and plotted versus time using GraphPad Prism software to assess tumor growth inhibition.
  • IL-2-HLE IL-2-HLE
  • IP intraperitoneal injection
  • mice were sacrificed for analysis; tumors were collected, weighed, and digested using enzymatic and manual dissociation to obtain a single cell suspension of tumor infiltrating lymphocytes (TILs). A portion of the single cell suspensions were left unstimulated and the rest were stimulated with PMA/ionomycin/BFA for 4 hours and then stained with fluorescently labeled antibodies for analysis by flow cytometry. Samples were run on a Cytek Aurora flow cytometer and data analyzed using Flow Jo and GraphPad Prism software.
  • TILs tumor infiltrating lymphocytes
  • mice treated with WW50008 showed reduced tumor burden and reduced weight loss when compared to vehicle and mice treated with WW0177 (IL-2 HLE). Results are shown in FIGs. 60A and 60B. The numbers of CD8+ T cells did not change in vehicle mice over time. Mice treated with WW0177 demonstrated an increase of CD8+ T cells in tumors, peaking at day 10 which decreased levels similar to vehicle by day 20. Mice treated with WW50008 had increased CD8+ T cells in the tumor at day 10 which were sustained at later timepoints.
  • CD8+ T cells within tumors from mice treated with WW50008 were more polyfunctional, producing multiple cytokines (IFN-g, TNF, GzmA, GzmB, and Prf1 were measured) compared to CD8+ T cells in mice treated with vehicle and/or WW0177. Results are shown in FIGs.61A and 61B.
  • mice were treated with vehicle, 10 ⁇ g WW0177 (IL-2-HLE), or 100 ⁇ g of WW50008 (IL-21- HLE) by intraperitoneal injection (IP) on days 1, 4, 8 and 11 after randomization. Tumors were harvested on day 10, dissociated and bulk RNA isolated. RNA was examined using Nanostring mouse PanCancer IO panel and analyzed using Nanostring nCounter and Partek software. Pathway analysis comparing differentially expressed genes highlights several type I IFN related pathways which were upregulated with WW50008 treatment compared to WW0177. Results are shown in FIG.62A. Dot plots show IFN responsive genes differentially expressed between vehicle, WW50008 and WW0177 treatment. Results are shown in FIGs.62D-62I.
  • mice Female BALB/c mice were inoculated on the flank with 1x10 5 EMT-6 cells in 50% Matrigel. Tumors were allowed to grow for 7-8 days until mice were randomized for dosing (average tumor volume 100 mm 3 ). Mice were treated with vehicle, 10 ⁇ g WW0177 (IL-2-HLE), or 50 ⁇ g of WW50008 (IL-21- HLE) by intraperitoneal injection (IP) on days 1, 4 and 8 after randomization. Mice were dosed with isotype control (BioXCell, clone MOPC-21) or anti-IFNAR1 (BioXCell, clone MAR1-5A3) every 3 days starting on day 0 through day 22 for a total of 8 doses.
  • isotype control BioXCell, clone MOPC-21
  • anti-IFNAR1 BioXCell, clone MAR1-5A3
  • mice were treated with vehicle, 10 ⁇ g WW0177 (IL-2-HLE), or 50 ⁇ g of WW50008 (IL-21- HLE) by intraperitoneal injection (IP) on days 1, 4 and 8 after randomization. Tumors were harvested on Day 5, 9, 12 and 16, and stored in 10% neutral buffered formalin for at least 72 hours prior to being embedded into FFPE blocks. These blocks were used to generate slides for Multiplex Immunofluorescence (M-IF) staining. [0576] Staining was performed after antigen retrieval using the automated M-IF COMET TM system from Lunaphore.
  • M-IF Multiplex Immunofluorescence
  • Various markers were used to identify several different cell populations: DAPI, CD3, CD8, CD4, CD11c, F4/80, CD103, CD45, TCF1, Nkp46, Perforin (Prf1), and Granzyme B (GZMB). Combinations of these different markers were used to identify and quantify specific cell populations using Halo software.
  • Representative images demonstrate robust and sustained infiltration of CD45+ cells in WW50008 treated tumors. Vehicle and WW0177 treated tumors show CD45+ cell infiltration at early timepoints (day 5-9) which retract overtime (day 12-16). Results are shown in FIG.63A. Images from day 12 tumors reveal increased diversity of immune cells in WW50008 treated tumors compared to vehicle and WW0177 treated tumors.
  • Immune infiltrates in vehicle and WW0177 tumors mainly consist of F4/80+ and CD11b+ cells.
  • WW50008 treated tumors have CD8+ and CD4+ T cells and NK cells Results are shown in FIG.63B. Additionally, WW50008 treated tumors have increased CD103+ CD11c+ DCs in close proximity to CD8+ T cells. Results are shown in FIG.63C.
  • Quantification of M-IF show increased stem-like TCF1+ CD8+ T cells in WW50008 treated tumors compared to vehicle and WW0177.
  • WW50008 treated CD8+ T cells had higher expression of Perforin (Prf1) and Granzyme B (GZMB).
  • Tumors were harvested on 173 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 Day 5, 9, 12 and 16, and stored in 10% neutral buffered formalin for at least 72 hours prior to being embedded into FFPE blocks. These blocks were used to generate slides for Multiplex Immunofluorescence (M-IF) staining.
  • M-IF Multiplex Immunofluorescence
  • Staining was performed after antigen retrieval using the automated M-IF COMET TM system from Lunaphore.
  • Various markers were used to identify several different cell populations: DAPI, CD3, CD8, CD4, CD11c, F4/80, CD103, CD45, TCF1, NCR1, Perforin (Prf1), and Granzyme B (GZMB).
  • mice Female BALB/c mice were inoculated on the flank with 2x10 6 Renca cells in 50% Matrigel. Tumors were allowed to grow for 7-8 days until mice were randomized for dosing (average tumor volume 100 mm 3 ). Mice were treated with vehicle, 10 ⁇ g WW0177 (IL-2-HLE), or 75 ⁇ g of WW50008 (IL-21- HLE) by intraperitoneal injection (IP) on days 1, 4, 8 and 11 after randomization. Body weight and tumor sizes were measured three times per week and plotted versus time using GraphPad Prism software to assess tumor growth inhibition. Animals were monitored for 56 days or euthanized when tumor burden reached 2000 mm 3 volume.
  • IP intraperitoneal injection
  • WW50008 treated mice showed reduced tumor burden (FIG.65A) and reduced weight loss (FIG. 65B) when compared to Vehicle and WW0177 groups. Individual spider plots showed that tumors regressed in the WW50008 (IL-21-HLE) treated group even after reaching high volume (FIGs.65C-65E).
  • Example 47. Renca Study, TILs Analysis Female BALB/c mice were inoculated on the flank with 2x10 6 Renca cells in 50% Matrigel. Tumors were allowed to grow for 7-8 days until mice were randomized for dosing (average tumor volume 100 mm 3 ).
  • mice were treated with vehicle, 10 ⁇ g WW0177 (IL-2-HLE), or 75 ⁇ g of WW50008 (IL-21- HLE) by intraperitoneal injection (IP) on days 1, 4, 8 and 11 after randomization.
  • Body weight and tumor sizes were measured three times per week and plotted versus time using GraphPad Prism software to 174 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 assess tumor growth inhibition. Animals were monitored for up to 56 days, cohorts of animals were sacrificed at specific time-points for tumor analysis or euthanized when tumor burden reached 2000 mm 3 volume.
  • mice were sacrificed for analysis; tumors were collected, weighed, and digested using enzymatic and manual dissociation to obtain a single cell suspension of tumor infiltrating lymphocytes (TILs). A portion of the single cell suspensions were left unstimulated, and the rest were stimulated with PMA/ionomycin/BFA for 4 hours and then stained with fluorescently labeled antibodies for analysis by flow cytometry. Samples were run on a Cytek Aurora flow cytometer and data analyzed using Flow Jo and GraphPad Prism software. [0586] Examining the T cell compartment, numbers of CD8+ T cells did not change in Vehicle mice over time.
  • TILs tumor infiltrating lymphocytes
  • WW0177 mice demonstrated an increase of CD8+ T cells, peaking at day 10 which dramatically decreased back to similar levels as Vehicle by day 20.
  • WW50008 treated animals had increased CD8+ T cells in the tumor at day 10 which were sustained at later timepoints (FIG.66A).
  • Analysis of the CD8+ T cell ratio to Tregs also demonstrated that treatment with IL-21-HLE (WW50008) resulted in a positive ratio picking at day 10 and maintained during the study duration (FIG.66B).
  • CD8+ T cells from WW50008 mice were able to produced higher levels of effector molecules, GzmA, GzmB, and Prf1(FIGs.66C-66E) and were more polyfunctional, producing multiple cytokines simultaneously (IFN-g, TNF, GzmA, GzmB, and Prf1 were measured) compared to Vehicle and WW0177 CD8+ T cells (FIG.66F).
  • Example 48. Renca Study, Multi-IF Analysis [0587] Female BALB/c mice were inoculated on the flank with 2x10 6 Renca cells in 50% Matrigel. Tumors were allowed to grow for 7-8 days until mice were randomized for dosing (average tumor volume 100 mm 3 ).
  • mice were treated with vehicle, 10 ⁇ g WW0177 (IL-2-HLE), or 75 ⁇ g of WW50008 (IL-21- HLE) by intraperitoneal injection (IP) on days 1, 4, 8 and 11 after randomization.
  • Body weight and tumor sizes were measured three times per week and plotted versus time using GraphPad Prism software to assess tumor growth inhibition. Animals were monitored for up to 56 days, cohorts of animals were sacrificed at specific time-points for tumor analysis or euthanized when tumor burden reached 2000 mm 3 volume.
  • tumors were harvested on Day 14 and stored in 10% neutral buffered formalin for at least 72 hours prior to being embedded into FFPE blocks. These blocks were used to generate slides for M-IF staining.
  • WW5008 treated tumors had increased immune clusters containing Granzyme B producing CD8+ T cells, CD4+ T cells, F4/80+ cells and CD11c+ cells (FIGs. 67B and 67C).
  • Example 49. MC38 Mouse Study of WW51100/WW50394 [0589] A study was conducted to determine the dose range of WW51100/WW50394 that is efficacious and tolerated in MC38 tumor bearing mice. Table 43 summarizes the treatment groups and dosage of WW51100/WW50394. D-PBS was used as a vehicle control, WW51100/WW50394 was dosed at 47, 94, 188, 375, 750, 1200, and 1500 ⁇ g/mouse per dose.
  • VHHs were first fused to the amino terminus of human IL-2 and joined by a non-cleavable glycine-serine linker. Gene synthesis was conducted to assemble VHHs with a C terminal 6x His tag and sequences were cloned into pcDNA3.4 (ThermoFisher), including an amino terminal signal sequence to enable secretion. Plasmids were transiently transfected according to the Expi293 Expression System protocol.
  • VHH-IL-2 fusion proteins were harvested and proteins were purified by ion metal affinity chromatography using Ni Sepharose® 6Fast Flow resin (Cytiva).
  • Purified VHH-IL-2 fusion proteins were characterized by analytical SEC and SDS-PAGE. For SDS-PAGE, three ⁇ g of protein were assessed as non-reduced and reduced samples. Samples were run on a 4-12% Bis-tris gel run in 1X MOPS SDS Running buffer according to the manufacturer’s instructions. A summary of final yield and percent monomer (by SEC) is shown in Table 58. Table 58.
  • VHH Clone CDR3 Yield (mg/L) Percent monomer ID Group . . y g
  • Pharmacokinetics of the VHH-IL-2 constructs were performed in C57BL/6j mice (Jackson Labs - Bar Harbor, Maine).126 female C57BL/6j mice at age of 7-8 weeks were given an intraperitoneal (IP) dose of 1 mg/kg VHH-IL-2 on Day 0 in the study carried out by NeoSome Life Sciences (NeoSome Life Sciences LLC, 5 Fortune Dr., Billerica, MA).
  • submandibular puncture survival bleeds using 4-5 mm lancets to draw blood at 4 hr, 8 hr, 12 hr, 24 hr, 48 hr, 72hr timepoints
  • terminal cardiac puncture terminal blood collection on days 4, 5 and 7 following euthanasia
  • VHH-IL-2 constructs were used to generate an 8-point calibration standard curve.
  • MSD plate Prior to analysis, the MSD plate was washed with 150 ⁇ l/well of Wash Buffer, then standard, controls, blank and samples diluted with minimum required dilution (MRD) in Diluent 2 were loaded to the plate in duplicate and incubated at room temperature with shaking for 2 hours. Following incubation, the plate was washed, and 25 ⁇ l of Detection antibody solution was added to each well and incubated for 2 more hours. The MSD plate was washed a final time and 150 ⁇ l of MSD Gold Read Buffer was added to the wells and subsequent excitation of the Sulfo-tagged streptavidin via an electrochemical reaction of Ru(bpy)3 to generate luminescence was read on MSD Sector Plate reader.
  • MSD Gold Read Buffer 150 ⁇ l of MSD Gold Read Buffer was added to the wells and subsequent excitation of the Sulfo-tagged streptavidin via an electrochemical reaction of Ru(bpy)3 to generate luminescence was read on MSD Sector Plate reader.
  • VHH-IL-2 constructs The plasma concentration-time profiles for VHH-IL-2 constructs are illustrated graphically in FIG.69, and the derived PK parameters are summarized in Table 55.
  • Concentration vs time profiles for VHH-IL-2 fusions followed a similar, non-linear PK profile, with a steep terminal elimination phase; suggesting saturation of any target-mediated clearance pathways.
  • Estimated plasma half-life of the VHHs was around 7.7 to 29.8 hr which was shorter than the presumed half-life of albumin in wild type mice and shorter than observed for 10GE control. However, it is important to note that the terminal half-life could not be accurately calculated for any of the constructs during the 7-day observation period due to the multiphasic profile of the concentration-time course curve.
  • VHH-IL-2 Plasma Half-Life (T 1/2 ) and pharmacokinetic values of the VHH-IL-2 constructs C max T last C AUC AUC Construct ID VHH Clone T last last INF 1/2 (hr) T max (hr) (pg/mL) (hr) (pg/mL) (hr*pg/mL) (hr*pg/mL) 8 9 8 8 8 8 8 .
  • VHHs were first fused to the amino terminus of human IL-2 and joined by a non-cleavable glycine-serine linker. Gene synthesis was conducted to assemble VHHs with a C terminal 6x His tag and sequences were cloned into pcDNA3.4 (ThermoFisher), including an amino terminal signal sequence to enable secretion. Plasmids were transiently transfected according to the Expi293 Expression System protocol.
  • VHH-IL-2 fusion proteins were harvested and proteins were purified by ion metal affinity chromatography using Ni Sepharose® 6Fast Flow resin (Cytiva). Purified VHH-IL-2 fusion proteins were characterized by analytical SEC and SDS-PAGE. For SDS- PAGE, three ⁇ g of protein were assessed as non-reduced and reduced samples. Samples were run on a 4- 12% Bis-tris gel run in 1X MOPS SDS Running buffer according to the manufacturer’s instructions. A summary of final yield and percent monomer (by SEC) is shown in Table 56. 180 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 Table 56.
  • VHH Clone CDR3 Yield (mg/L) Percent monomer ID Group
  • Pharmacokinetics of the VHH-IL-2 constructs were performed in C57BL/6j mice (Jackson Labs - Bar Harbor, Maine).126 female C57BL/6j mice at age of 7-8 weeks were given an intraperitoneal (IP) dose of 1 mg/kg VHH-IL-2 on Day 0 in the study carried out by NeoSome Life Sciences (NeoSome Life Sciences LLC, 5 Fortune Dr., Billerica, MA).
  • submandibular puncture survival bleeds using 4-5 mm lancets to draw blood at 4hr, 8hr, 12hr, 24 hr, 48hr, 72hr timepoints
  • terminal cardiac puncture terminal blood collection on days 4, 5 and 7 following euthanasia
  • VHH-IL-2 constructs were used to generate an 8-point calibration standard curve.
  • MSD MSD plate was washed with 150 ⁇ l/well of Wash Buffer, then standard, controls, blank and samples diluted with minimum required dilution (MRD) in Diluent 2 were loaded to the plate in duplicate 181 ⁇ 4144-9392-5971 v1 Attorney Docket No.: 761146.332320 and incubated at room temperature with shaking for 2 hours. Following incubation, the plate was washed, and 25 ⁇ l of Detection antibody solution was added to each well and incubated for 2 more hours.
  • MRD minimum required dilution
  • MSD plate was washed a final time and 150 ⁇ l of MSD Gold Read Buffer was added to the wells and subsequent excitation of the Sulfo-tagged streptavidin via an electrochemical reaction of Ru(bpy)3 to generate luminescence was read on MSD Sector Plate reader. 51.1.4 Results [0601] The linear portions of standard curves generated in Prism (GraphPad Software) were then used to quantify VHH-IL-2 concentration in the plasma samples. Pharmacokinetic parameters were estimated by noncompartmental analysis based on the mean plasma concentration vs time data using Phoenix WinNonlin Version 8.1. All calculations used the nominal doses and nominal sampling times.
  • AUC last (Area under the curve to the last measurable concentration) and AUC Inf (Area under the curve to infinity) were calculated using the log-linear trapezoidal method. Terminal half-life (T 1 ⁇ 2 ) was calculated using log- linear regression of the concentration data including at least the last three sampling time-points with measurable concentrations. Descriptive statistics for major PK parameters were then calculated.
  • the plasma concentration-time profiles for VHH-IL-2 constructs are illustrated graphically in FIG.69, and the derived PK parameters are summarized in Table 57. Concentration vs time profiles for VHH-IL-2 fusions followed a similar, non-linear PK profile, with a steep terminal elimination phase; suggesting saturation of any target-mediated clearance pathways.
  • Plasma Half-Life (T 1/2 ) and pharmacokinetic values of the VHH-IL-2 constructs C max T la C AUC AUC Construct ID VHH Clone T (hr) T (hr st last last INF 1/2 max ) (pg/mL) (hr) (pg/mL) (hr*pg/mL) (hr*pg/mL) 8 9 8 8 8 8 8 .

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Abstract

L'invention concerne des promédicaments polypeptidiques d'IL-21 comprenant l'IL-21, un élément d'extension de demi-vie, un élément de blocage d'IL-21 et un lieur clivable par protéase. L'invention concerne également des compositions pharmaceutiques associées, ainsi que des acides nucléiques, des vecteurs d'expression recombinants, des cellules hôtes pour fabriquer de tels promédicaments polypeptidiques. L'invention concerne en outre des procédés d'utilisation des promédicaments polypeptidiques dans le traitement de maladies, d'états et de troubles.
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WO2026072455A1 (fr) * 2024-09-26 2026-04-02 Werewolf Therapeutics, Inc. Anticorps à domaine unique ciblant l'albumine sérique humaine

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996027011A1 (fr) 1995-03-01 1996-09-06 Genentech, Inc. Procede d'obtention de polypeptides heteromultimeriques
WO2006110728A2 (fr) 2005-04-12 2006-10-19 The Uab Research Foundation Agregats de tegument immunogenes
WO2009036379A2 (fr) 2007-09-14 2009-03-19 Adimab, Inc. Bibliothèques d'anticorps synthétiques rationnelles et leurs utilisations
WO2010105256A1 (fr) 2009-03-13 2010-09-16 Adimab, Inc. Banques d'anticorps synthétiques, conçues de façon rationnelle, et leurs utilisations
WO2012009568A2 (fr) 2010-07-16 2012-01-19 Adimab, Llc Banques d'anticorps
WO2019222296A1 (fr) 2018-05-14 2019-11-21 Werewolf Therapeutics, Inc. Polypeptides d'interleukine 12 activables et procédés d'utilisation associés
WO2019222295A1 (fr) 2018-05-14 2019-11-21 Werewolf Therapeutics, Inc. Polypeptides d'interleukine 2 activables et procédés d'utilisation associés
WO2021097376A1 (fr) 2019-11-14 2021-05-20 Werewolf Therapeutics, Inc. Polypeptides de cytokine activables et leurs méthodes d'utilisation

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021142471A1 (fr) * 2020-01-11 2021-07-15 AskGene Pharma, Inc. Nouvelles cytokines masquées et leurs procédés d'utilisation
MX2024003889A (es) * 2021-09-30 2024-04-22 Yunovia Co Ltd Inmunocitocina que contiene la muteina de il-21r.
CN116496408A (zh) * 2022-01-21 2023-07-28 广东菲鹏制药股份有限公司 白细胞介素21及其受体复合物
CA3249997A1 (fr) * 2022-06-29 2024-01-04 Beijing Neox Biotech Limited Polypeptides d’il-21 et méthodes d’utilisation

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996027011A1 (fr) 1995-03-01 1996-09-06 Genentech, Inc. Procede d'obtention de polypeptides heteromultimeriques
WO2006110728A2 (fr) 2005-04-12 2006-10-19 The Uab Research Foundation Agregats de tegument immunogenes
WO2009036379A2 (fr) 2007-09-14 2009-03-19 Adimab, Inc. Bibliothèques d'anticorps synthétiques rationnelles et leurs utilisations
WO2010105256A1 (fr) 2009-03-13 2010-09-16 Adimab, Inc. Banques d'anticorps synthétiques, conçues de façon rationnelle, et leurs utilisations
WO2012009568A2 (fr) 2010-07-16 2012-01-19 Adimab, Llc Banques d'anticorps
WO2019222296A1 (fr) 2018-05-14 2019-11-21 Werewolf Therapeutics, Inc. Polypeptides d'interleukine 12 activables et procédés d'utilisation associés
WO2019222294A1 (fr) 2018-05-14 2019-11-21 Werewolf Therapeutics, Inc. Polypeptides de cytokine activables et leurs procédés d'utilisation
WO2019222295A1 (fr) 2018-05-14 2019-11-21 Werewolf Therapeutics, Inc. Polypeptides d'interleukine 2 activables et procédés d'utilisation associés
WO2021097376A1 (fr) 2019-11-14 2021-05-20 Werewolf Therapeutics, Inc. Polypeptides de cytokine activables et leurs méthodes d'utilisation

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
"Remington: The Science and Practice of Pharmacy", 2005, LIPPICOTT WILLIAMS & WILKINS
COFFIN ET AL.: "Retroviruses", 1997, COLD SPRING HARBOR LABORATORY PRESS
DAVIDSON ET AL., J. VIROL., vol. 61, 1987, pages 1226 - 39
ESTEP ET AL., MABS, vol. 5, no. 2, 2013, pages 270 - 278
GARCEAGISSMANN, CURRENT OPINION IN BIOTECHNOLOGY, vol. 15, 2004, pages 513 - 7
HAJ-AHMAD ET AL., J. VIROL., vol. 57, 1986, pages 267 - 74
HOEFMAN ET AL., ANTIBODIES, vol. 4, no. 3, 2015, pages 141 - 156
MADAN ET AL., BLOOD, vol. 138, no. 1, 2021, pages 1654
MASSIE ET AL., MOL. CELL. BIOL., vol. 6, 1986, pages 2872 - 83
MERCHANT ET AL., NAT. BIOTECHNOL., vol. 16, no. 7, 1998, pages 677 - 681
PEPPERL-KLINDWORTH ET AL., GENE THERAPY, vol. 10, 2003, pages 278 - 84
RIDGWAY ET AL., PROTEIN ENG., vol. 9, no. 7, 1996, pages 617 - 621
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2012, COLD SPRING HARBOR LABORATORY PRESS
SIEGEL ET AL., J IMMUNOL METHODS, vol. 286, no. 1-2, 2004, pages 141 - 153
Y. XU ET AL., PEDS, vol. 26, no. 10, 2013, pages 663 - 70
ZHANG ET AL., BIOTECHNIQUES, vol. 15, 1993, pages 868 - 72

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2026072455A1 (fr) * 2024-09-26 2026-04-02 Werewolf Therapeutics, Inc. Anticorps à domaine unique ciblant l'albumine sérique humaine

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