Activatable IL-21 polypeptide and method of use thereof, and single-domain human serum albumin antibody

Abstract

This specification provides IL-21 polypeptide prodrugs comprising IL-21, a half-life extender, an IL-21 blocking element, and a protease-cleaving linker. This specification also provides pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for constructing such polypeptide prodrugs. Methods for using polypeptide prodrugs in the treatment of diseases, conditions, and disorders are also disclosed. This disclosure relates to inducible IL-21 prodrugs containing at least one polypeptide chain, and optionally two or more polypeptides.

Description

[Technical Field]

[0001] This application claims the interests of U.S. Provisional Application No. 63 / 585,550 filed on 26 September 2023, U.S. Provisional Application No. 63 / 594,006 filed on 29 October 2023, U.S. Application No. 63 / 563,713 filed on 11 March 2024, and U.S. Provisional Application No. 63 / 575,026 filed on 5 April 2024, the contents of each of these are incorporated herein by reference. [Background technology]

[0002] 1. Background technology Interleukin-21 (IL-21) is a cytokine primarily produced by T cells and natural killer T cells, and acts multifaceted on a wide range of immune and non-immune cell types. IL-21 is CD4 + and CD8 + It exerts diverse effects on a wide range of cell types, including but not limited to T cells, B cells, macrophages, monocytes, and dendritic cells (DCs). The functional receptor for IL-21 consists of the IL-21 receptor (IL-21R) and a common cytokine receptor γ chain, which is also a subunit of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15.

[0003] IL-21 is a potent immune agonist and is considered a promising therapeutic agent for oncology. Unfortunately, due to the biological properties of cytokines and the inability to effectively target and control their activity, cytokines have not achieved the expected clinical benefits in tumor treatment. This is exacerbated by the need to administer large doses of cytokines (e.g., IL-21) to achieve desired cytokine levels at the intended site of action (e.g., the tumor microenvironment).

[0004] Inducible IL-12, IL-2, and interferon prodrugs are described in International Publications WO2019 / 222294, WO2019 / 222295, WO2019 / 222296, and WO2021 / 097376. The medical need for improved methods to treat cancer remains unmet. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2019 / 222294 [Patent Document 2] International Publication No. 2019 / 222295 [Patent Document 3] International Publication No. 2019 / 222296 [Patent Document 4] International Publication No. 2021 / 097376 [Overview of the Initiative]

[0006] 2. Outline of the Invention This disclosure relates to an inducible IL-21 prodrug comprising at least one polypeptide chain, and optionally two or more polypeptides. The inducible IL-21 prodrug comprises an IL-21 polypeptide, an IL-21 blocking element, a half-life extending element, and a protease-cleaving polypeptide linker.

[0007] The inducible IL-21 prodrugs of this disclosure have attenuated IL-21 receptor agonist activity and prolonged circulating half-life. The inducible IL-21 receptor agonist activity is attenuated via blocking elements. Half-life prolonging elements may also contribute to the attenuation, for example, through steric effects. Blocking elements can block all or part of the receptor agonist activity of IL-21 by non-covalent binding to IL-21 and / or by sterically blocking receptor binding. Cleavage of the protease-cleaving linker releases an active form of IL-21 (e.g., more active than the IL-21 polypeptide prodrug). Typically, the released IL-21 is at least 10-fold more active than the IL-21 polypeptide prodrug. Preferably, the released IL-21 is at least 20 times, at least 30 times, at least 50 times, at least 1000 times, at least 200 times, at least 300 times, at least 500 times, at least 1000 times, at least about 10,000 times or more active than the inducible IL-21 prodrug.

[0008] The form of IL-21 released by cleavage of an inducible IL-21 prodrug typically has a short half-life, which is often substantially similar to that of naturally occurring cytokines. Even if the half-life of the inducible IL-21 prodrug is extended, the toxicity is reduced or eliminated because the agonist activity of the circulating inducible cytokine prodrug is attenuated and the active IL-21 is targeted to the desired site of activity (e.g., the tumor microenvironment).

[0009] An inducible IL-21 prodrug may contain at least one each of an IL-21 polypeptide [A], an IL-21 blocking element [D], a half-life extension element [H], and a protease-cleaving polypeptide linker [L]. The IL-21 polypeptide and the IL-21 blocking element or half-life extension element may be operably linked by the protease-cleaving polypeptide linker, and the inducible IL-21 prodrug has attenuated IL-21 receptor activating activity. The IL-21 receptor activating activity of an inducible IL-21 prodrug is at least about 10 times lower than the IL-21 receptor activating activity of a polypeptide containing the IL-21 polypeptide produced by cleavage of the protease-cleaving linker.

[0010] The inducible IL-21 prodrug is formulated as follows:

[0011] [A]-[L1]-[H]-[L2]-[D];

[0012] [D]-[L2]-[H]-[L1]-[A];

[0013] [A]-[L1]-[D]-[L2]-[H];

[0014] [H]-[L2]-[D]-[L1]-[A];

[0015] [H]-[L1]-[A]-[L2']-[D]; or

[0016] [D]-[L1]-[A]-[L2']-[H] may be present.

[0017] [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 potentially protease-cleavable polypeptide linker, and [L2'] is a protease-cleavable polypeptide linker. L2 may be a protease-cleavable polypeptide linker. L1 or L2 or both L1 and L2 are cleaved by two or more different proteases.

[0018] The inducible IL-21 prodrugs disclosed herein may include an antigen-binding fragment of an antibody as an IL-21 blocking element. The antigen-binding fragment of an antibody may include, as a separate component, 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 may include a first polypeptide comprising an 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 a half-life extender, 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 the half-life extender are operably linked by a protease-cleaving linker; and a second polypeptide comprising at least an antigen-binding portion of an antibody heavy chain complementary to the light chain of the first polypeptide, or an antibody light chain complementary to the heavy chain of the first polypeptide and forming an IL-21 binding site together with the light chain.

[0019] An inducible IL-21 prodrug may contain two polypeptide chains. The first polypeptide chain may include an IL-21 polypeptide, a half-life extender, a first IL-21 blocking element, a protease-cleaving polypeptide linker, and a portion of the constant region or light chain of an antibody heavy chain, the first blocking element being IL-21R. The IL-21 polypeptide and the first blocking element and / or half-life extender are operably linked by a protease-cleaving linker. The second polypeptide may include a second IL-21 blocking element and a portion of the constant region or light chain of an antibody heavy chain. The second blocking element and the portion of the constant region or light chain of an antibody heavy chain are linked via a linker that may be protease-cleaving. The second blocking element may be a common gamma chain receptor. If the constant region of the antibody heavy chain is on the first polypeptide, the antibody light chain is on the second polypeptide; if the constant region of the antibody heavy chain is on the second polypeptide, the antibody light chain is on the first polypeptide.

[0020] The inducible IL-21 prodrug may contain a first polypeptide selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs. 18-23 and 26-31, or SEQ ID NOs. 18-23 and 26-31. The inducible IL-21 prodrug may contain a second polypeptide selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs. 17 or 25, or SEQ ID NOs. 17 or 25.

[0021] An inducible IL-21 prodrug may comprise a first polypeptide selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs. 18-23 and 26-31, or SEQ ID NOs. 18-23 and 26-31, and a second polypeptide selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs. 17 or 25, or SEQ ID NOs. 17 or 25.

[0022] An inducible IL-21 prodrug may comprise a first polypeptide chain selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs. 39, 41, 51, 58, 59, 63, and 65, or SEQ ID NOs. 39, 41, 51, 58, 59, 63, and 65. The inducible IL-21 prodrug may further comprise a second polypeptide chain selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs. 10, 40, 52, and 64, or SEQ ID NOs. 10, 40, 52, and 64.

[0023] An inducible IL-21 prodrug may comprise a first polypeptide chain selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65, or SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65, and a second polypeptide chain selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs: 10, 40, 52, and 64, or SEQ ID NOs: 10, 40, 52, and 64.

[0024] The inducible IL-21 prodrug may contain an amino acid sequence selected from the group consisting of SEQ ID NOs. 44-50 or an amino acid sequence having at least 80% identity with SEQ ID NOs. 44-50.

[0025] This disclosure relates to an inducible IL-21 prodrug comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 72 to 109, or an amino acid sequence having at least about 80% identity with SEQ ID NOs. 72 to 109.

[0026] This disclosure relates to Sequence IDs 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 Sequence IDs 72, 110-113, 117-126, 130-136, 138-144, 153-208, 221-235, 241-243, 245-247, 250, 251, 253 The present invention relates to an inducible IL-21 prodrug comprising: a first polypeptide selected from the group consisting of amino acid sequences having at least 80% identity with sequence numbers ~255, 257, 258, 260~264, 267~269, 271~290, 475, and 476; and a second polypeptide selected from the group consisting of amino acid sequences having at least 80% identity with sequence numbers 71, 127~129, 137, or 300~306, or sequence numbers 71, 127~129, 137, or 300~306. The inducible IL-21 prodrug may comprise a first polypeptide containing the amino acid sequence of sequence number 130, and a second polypeptide containing the amino acid sequence of sequence number 129. The inducible IL-21 prodrug may comprise a first polypeptide containing the amino acid sequence of sequence number 131, and a second polypeptide containing the amino acid sequence of sequence number 129. An inducible IL-21 prodrug may contain a first polypeptide containing the amino acid sequence of SEQ ID NO: 140 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 137. An inducible IL-21 prodrug may contain a first polypeptide containing the amino acid sequence of SEQ ID NO: 143 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 137. An inducible IL-21 prodrug may contain a first polypeptide containing the amino acid sequence of SEQ ID NO: 169 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 301. An inducible IL-21 prodrug may contain a first polypeptide containing the amino acid sequence of SEQ ID NO: 185 and a second polypeptide containing the amino acid sequence of SEQ ID NO: 303.

[0027] Half-life extending elements may include serum albumin-binding domains, serum albumin, transferrin, or immunoglobulin Fc or its fragments. Half-life extending elements may also be blocking elements.

[0028] The IL-21 blocking element may include a ligand-binding domain or fragment of a congener 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 the activation of the IL-21 receptor by an inducible IL-21 prodrug.

[0029] The antibody or antigen-binding fragment may be a single-domain antibody, Fab, or scFv that binds to the IL-21 polypeptide. The homologous receptor for IL-21 may be the IL-21 receptor or the IL-21 common gamma chain.

[0030] Each protease-cleavable polypeptide linker independently contains a sequence that can be cleaved by a protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, cathepsin L, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), ADAM, FAP, plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface proteases.

[0031] Cathepsins may be cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, or cathepsin G. Matrix metalloproteinases (MMPs) are MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP19, or MMP20.

[0032] This disclosure also relates to nucleic acids encoding inducible IL-21 prodrugs. The nucleic acids may include circular vectors. The nucleic acids may include DNA. The nucleic acids may include RNA. This specification discloses a method for preparing a pharmaceutical composition, comprising culturing host cells under conditions suitable for the expression and collection of an inducible IL-21 prodrug. This specification discloses 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 each of an IL-21 polypeptide [A], an IL-21 blocking element [D], a half-life extension element [H], and a protease-cleaving polypeptide linker [L]. The IL-21 polypeptide and the IL-21 blocking element or half-life extension element are operably linked by the protease-cleaving polypeptide linker, and the inducible IL-21 prodrug has attenuated IL-21 receptor activity.

[0033] The nucleic acid composition may comprise one or more nucleic acid sequences encoding an inducible IL-21 prodrug containing two polypeptide chains. The inducible IL-21 prodrug may comprise an antigen-binding fragment of an antibody as an IL-21 blocking element. The antigen-binding fragment of the antibody may comprise, as separate components, at least an antigen-binding portion of the antibody light chain and at least an antigen-binding portion of the complementary antibody heavy chain. An inducible IL-21 prodrug may comprise a first polypeptide comprising an 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 a half-life extender, 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 the half-life extender are operably linked by a protease-cleaving linker; and a second polypeptide comprising at least an antigen-binding portion of an antibody heavy chain complementary to the light chain of the first polypeptide, or an antibody light chain complementary to the heavy chain of the first polypeptide and forming an IL-21 binding site together with the light chain.

[0034] The nucleic acid composition may contain one or more nucleic acid sequences encoding an inducible IL-21 prodrug, comprising: a first polypeptide comprising an IL-21 polypeptide, a half-life extension element, a first IL-21 blocking element, a protease-cleavable polypeptide linker, and a portion of the constant region or light chain of an antibody heavy chain, wherein the first blocking element is IL-21R, and the IL-21 polypeptide and the first blocking element or half-life extension element are operably linked by a protease-cleavable linker; and a second polypeptide comprising a second blocking element and a portion of the constant region or light chain of an antibody heavy chain, wherein the second blocking element and the portion of the constant region or light chain of an antibody heavy chain are linked via a linker that may be protease-cleavable, and the second blocking element is a common gamma chain receptor; and If the constant region of the antibody heavy chain is on the first polypeptide, the antibody light chain is on the second polypeptide; if the constant region of the antibody heavy chain is on the second polypeptide, the antibody light chain is on the first polypeptide.

[0035] This disclosure also relates to therapeutic methods comprising administering an effective amount of an inducible IL-21 prodrug, a nucleic acid encoding an inducible IL-21 prodrug, a vector or host cell containing such nucleic acid, and any of the aforementioned pharmaceutical compositions to a subject in need. Typically, the subject has or is at risk of developing cancer, proliferative disorders, neoplastic diseases, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases. The methods disclosed herein are particularly suitable for treating cancer. The inducible IL-21 prodrug may be administered intravenously.

[0036] This disclosure relates to single-domain antibody fragments (sdAbs) (such as VHH and VH) having binding specificity to human serum albumin, methods for producing the same, and uses thereof. Such sdAbs may be used to extend the in vivo serum half-life of therapeutic agents (e.g., proteins, peptides, etc.) by engineering fusion proteins and conjugates, for example, using well-known methods, 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.

[0037] Contrary to the widely accepted knowledge in the art that anti-albumin-binding domains that interfere with albumin binding to FcRn are unsuitable for half-life extension applications (e.g., by inclusion in fusion proteins or conjugates with short-lived therapeutic proteins), as described and illustrated herein, we have discovered that sdAbs competing with FcRn (human FcRn) for binding to human serum albumin can effectively extend the half-life (e.g., in vivo serum half-life) of short-lived therapeutic agents (e.g., proteins, peptides).

[0038] The sdAb disclosed herein includes CDR1, CDR2, and CDR3. CDR1 includes a variant of any of the aforementioned sequences containing one of SEQ ID NOs. 656, 659, 662, 663, or 691, or up to approximately two amino acid substitutions. CDR2 includes a variant of any of the aforementioned sequences containing one of SEQ ID NOs. 657, 660, 664, 665, 666, or 692, or up to approximately three amino acid substitutions. CDR3 includes a variant of any of the aforementioned sequences containing one of SEQ ID NOs. 658, 661, or 667, or up to approximately three amino acid substitutions.

[0039] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 656 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 657 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 658 or a variant thereof containing up to approximately three amino acid substitutions.

[0040] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 659 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 660 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 660 or a variant thereof containing up to approximately three amino acid substitutions.

[0041] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 662 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 664 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0042] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 662 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 665 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0043] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 662 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 666 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0044] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 662 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 692 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0045] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 691 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 664 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0046] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 691 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 665 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0047] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 691 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 666 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0048] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 691 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 692 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0049] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 663 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 664 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0050] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 663 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 665 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0051] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 663 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 666 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0052] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 663 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 692 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions.

[0053] sdAb may include CDR1 consisting of sequence number 662, CDR2 consisting of sequence number 692, and CDR3 consisting of sequence number 667.

[0054] sdAb may include CDR1 consisting of sequence number 691, CDR2 consisting of sequence number 664, and CDR3 consisting of sequence number 667.

[0055] sdAb may include CDR1 consisting of sequence number 663, CDR2 consisting of sequence number 665, and CDR3 consisting of sequence number 667.

[0056] sdAb may include CDR1 consisting of sequence number 663, CDR2 consisting of sequence number 666, and CDR3 consisting of sequence number 667.

[0057] sdAb may include CDR1 consisting of sequence number 656, CDR2 consisting of sequence number 2, and CDR3 consisting of sequence number 658.

[0058] sdAb may include CDR1 consisting of sequence number 659, CDR2 consisting of sequence number 660, and CDR3 consisting of sequence number 661.

[0059] The sdAbs disclosed herein may compete with the embryonic Fc receptor (FcRn) for binding to HSA. The sdAbs may have binding specificity to domain 1 of human serum albumin.

[0060] The sdAb may be a heavy chain variable domain (VH), a heavy chain variable heavy chain domain (VHH), a novel antigen receptor single-domain shark variable domain (VNAR), or a light chain variable domain (VL). VH and VHH are preferred sdAbs.

[0061] Typically, the sdAb is camelid, humanoid, or humanized. Preferably, the sdAb is humanized.

[0062] The sdAb disclosed herein further comprises one or more framework regions. The framework regions may be derived from the human germline.

[0063] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 662, SEQ ID NO: 663, or SEQ ID NO: 691; CDR2 containing the amino acid sequence of SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, or SEQ ID NO: 692; CDR3 containing the amino acid sequence of SEQ ID NO: 667; and FR1, FR2, FR3, and FR4. The amino acid residue at position 24 of FR1 may be serine or alanine; the amino acid at position 44 of F2 may be glutamic acid or glycine; the amino acid at position 45 of F2 may be arginine or leucine; and the amino acid at position 79 of F3 may be leucine or valine. FR1, FR2, FR3, and FR4 may be derived from the human IGHV3-23 germline.

[0064] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 656, CDR2 containing the amino acid sequence of SEQ ID NO: 657, CDR3 containing the amino acid sequence of SEQ ID NO: 658, and FR1, FR2, FR3, and FR4. The amino acid residue at position 24 of FR1 may be serine or alanine, the amino acid at position 44 of FR2 may be aspartic acid or glycine, the amino acid at position 45 of FR2 may be arginine or leucine, the amino acid at position 49 of FR2 may be serine or alanine, the amino acid at position 78 of FR3 may be leucine or phenylalanine, and the amino acid at position 79 of FR3 may be asparagine or lysine. FR1, FR2, FR3, and FR4 may be derived from the human IGHV3-23 germline.

[0065] sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 659, CDR2 containing the amino acid sequence of SEQ ID NO: 660, CDR3 containing the amino acid sequence of SEQ ID NO: 661, and FR1, FR2, FR3, and FR4. The amino acid residue at position 44 of F2 may be glutamic acid or glycine, the amino acid at position 45 of F2 may be arginine or leucine, the amino acid at position

[78] of F3 may be threonine or serine, and the amino acid at position 79 of F3 may be leucine or valine. FR1, FR2, FR3, and FR4 may be derived from the human IGHV3-23 germline.

[0066] The sdAb disclosed herein may further include FR1 comprising any one amino acid sequence from SEQ ID NO: 668, SEQ ID NO: 669, or SEQ ID NO: 685, or a variant of any of the aforementioned sequences including up to approximately three amino acid substitutions; FR2 comprising any one amino acid sequence from 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, or SEQ ID NO: 33, or a variant of any of the aforementioned sequences including up to approximately three amino acid substitutions; FR3 comprising any one amino acid sequence from any one of SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 34, SEQ ID NO: 689, or SEQ ID NO: 690, or a variant of any of the aforementioned sequences including up to approximately three amino acid substitutions; and FR4 comprising the amino acid sequence of SEQ ID NO: 677 or a variant thereof including up to approximately three amino acid substitutions.

[0067] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 670 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0068] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 671 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0069] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 673 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0070] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 669 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 671 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0071] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 678 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 683 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0072] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 679 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 683 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0073] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 681 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 683 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0074] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 669 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 681 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 683 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0075] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 686 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0076] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 681 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0077] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 685 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 681 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 34 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0078] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 685 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 686 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 689 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0079] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 685 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 33 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 689 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0080] sdAb may include FR1 containing the amino acid sequence of SEQ ID NO: 685 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 681 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 689 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions.

[0081] sdAb may contain one of the amino acid sequences of sequence numbers 286-290 or 608-648, or an amino acid sequence that is at least approximately 85% identical to one of the amino acid sequences of sequence numbers 286-290 or 608-648.

[0082] sdAb may contain the amino acid sequence of SEQ ID NO: 608, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 608.

[0083] sdAb may contain the amino acid sequence of SEQ ID NO: 609, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 609.

[0084] sdAb may contain the amino acid sequence of SEQ ID NO: 611, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 611.

[0085] sdAb may contain the amino acid sequence of SEQ ID NO: 618, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 618.

[0086] sdAb may contain the amino acid sequence of SEQ ID NO: 622, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 622.

[0087] sdAb may contain the amino acid sequence of SEQ ID NO: 624, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 624.

[0088] sdAb may contain the amino acid sequence of SEQ ID NO: 625, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 625.

[0089] sdAb may contain the amino acid sequence of SEQ ID NO: 626, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 626.

[0090] sdAb may contain the amino acid sequence of SEQ ID NO: 628, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 628.

[0091] sdAb may contain the amino acid sequence of SEQ ID NO: 632, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 632.

[0092] sdAb may contain the amino acid sequence of SEQ ID NO: 635, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 635.

[0093] sdAb may contain the amino acid sequence of SEQ ID NO: 637, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 637.

[0094] sdAb may contain the amino acid sequence of SEQ ID NO: 642, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 642.

[0095] sdAb may contain the amino acid sequence of SEQ ID NO: 644, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 644.

[0096] sdAb may contain the amino acid sequence of SEQ ID NO: 645, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 645.

[0097] sdAb may contain the amino acid sequence of SEQ ID NO: 646, or an amino acid sequence that is at least approximately 85% identical to the amino acid sequence of SEQ ID NO: 646.

[0098] The sdAb disclosed herein may have a half-life of at least about 5 hours to about 40 hours.

[0099] This disclosure further relates to a pharmaceutical composition comprising an sdAb disclosed herein or a nucleic acid encoding such sdAb.

[0100] This disclosure further relates to the nucleic acid encoding sdAb disclosed herein. The nucleic acid encoding sdAb may be a circular vector, DNA, RNA, or an expression vector comprising a circular vector, DNA, or RNA. In addition, this disclosure relates to isolated host cells containing an expression vector encoding the nucleic acid of sdAb.

[0101] This disclosure further relates to a fusion polypeptide comprising the sdAb disclosed herein and the amino acid sequence of interest. The fusion polypeptide may further comprise a linker that may be cleavable. The linker that may be cleavable may be operably ligated to the sdAb and the amino acid sequence of interest. The linker that may be cleavable may be operably ligated to the N-terminus of the sdAb. The linker that may be cleavable may be operably ligated to the C-terminus of the sdAb. The linker that may be cleavable is preferably cleavable. The linker that may be cleavable may comprise a cleavable moiety that is a substrate of one or more proteases. In some cases, the linker that may be cleavable is not cleavable.

[0102] This disclosure further relates to nucleic acids encoding fusion polypeptides. The nucleic acid composition encoding a fusion polypeptide may be a circular vector, DNA, RNA, or an expression vector comprising a circular vector, DNA, or RNA. In addition, this disclosure relates to isolated host cells containing an expression vector encoding a nucleic acid composition of a fusion polypeptide.

[0103] This disclosure also relates to pharmaceutical compositions of fusion polypeptides and methods for preparing pharmaceutical compositions of fusion polypeptides. This disclosure further relates to a method for treating a disease such as cancer, which includes administering to a subject in need a pharmaceutical composition containing an effective amount of a fusion polypeptide, a nucleic acid encoding a fusion polypeptide, an expression vector for a fusion polypeptide, or any of these in an effective amount.

[0104] 3. Brief explanation of the drawing The drawings do not necessarily need to be to scale or exhaustive. Rather, the emphasis is generally on illustrating the principles of the invention described herein. The accompanying drawings, which constitute part of this specification, illustrate several embodiments according to this disclosure and, together with this specification, serve to illustrate the principles of this disclosure. [Brief explanation of the drawing]

[0105] [Figure 1A] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1B] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1C] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1D] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1E] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1F] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1G] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1H] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1I] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1J] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1K] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1L] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1M] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 1N] This is a schematic diagram showing an inducible IL-21 prodrug. [Figure 10] This is a schematic diagram showing an inducible IL-21 prodrug.

[0106] [Figure 2] This is an SDS-PAGE gel showing the results of a protein cleavage assay. IL-21 prodrug was electrophoresed in both cleavage and non-cleavage forms.

[0107] [Figure 3A] This graph shows the analysis results of WW50008 (extended half-life mouse IL-21) in a syngeneic MC38 mouse tumor model. It shows the mean tumor volume over time in mice treated with 1 μg, 10 μg, 100 μg, and 300 μg of WW50008. WW50008 was administered twice a week for 3 weeks (total of 6 doses). The data indicate that tumor growth was inhibited over time in a dose-dependent manner in mice treated with WW50008. The highest degree of tumor growth inhibition was achieved at a dose level of 100 μg. The 300 μg dose level was not tolerated by the animals.

[0108] [Figure 3B] Figure 3A shows a graph illustrating the average body weight of the treated animals in the data presented. Each line in the plot represents the average body weight of the group over time. The 300 μg dose of WW50008 was not tolerated by the animals.

[0109] [Figure 4] This graph shows the analysis results of WW50008 in a syngeneic B16F10 mouse tumor model. It shows the average tumor volume over time in mice treated with 10 μg, 33 μg, 100 μg, and 200 μg of WW50008 (represented by squares, upward-pointing triangles, downward-pointing triangles, and rhombuses, respectively). Vehicle treatment alone is indicated by circles. These data demonstrate that tumor volume was suppressed over time in a dose-dependent manner in mice treated with higher concentrations of WW50008.

[0110] [Figure 5] A-E are spider plots showing the activity of WW50008 (extended half-life mouse IL-21) in the B16F10 mouse model, corresponding to the data shown in Figure 4. Each line in the plot represents the tumor volume over time for a single mouse.

[0111] [Figure 6] This graph shows the analysis results of WW50008 (extended half-life mouse IL-21) in a syngeneic EMT6 mouse tumor model. It shows the mean tumor volume over time in mice treated with 10 μg, 33 μg, 100 μg, and 200 μg of WW50008 (represented by squares, upward-pointing triangles, downward-pointing triangles, and rhombuses, respectively). Vehicles alone are indicated by circles. These data demonstrate that tumor volume was suppressed over time in a dose-dependent manner in mice treated with 10 μg and 33 μg of WW50008. WW50008 was not tolerated at a dose of 200 μg in this model.

[0112] [Figure 7] A-E are spider plots showing the activity of WW50008 (extended half-life IL-21) in the EMT6 mouse model corresponding to the data shown in Figure 6. Each line in the plot represents the tumor volume over time for a single mouse.

[0113] [Figure 8A] This shows the efficacy and TIL analysis results for MC38 tumors treated with extended half-life IL-21 (WW50008). The graph shows tumor volume (mm3) in mice treated with vehicle or 100 μg of WW50008 on days 1, 4, 8, and 11. [Figure 8B] This shows the efficacy and TIL analysis results for MC38 tumors treated with extended half-life IL-21 (WW50008). The graph shows the increase in lymphocyte infiltration in the WW50008-treated group compared to the vehicle, measured by the ratio of total cells and CD45+ TILs per 1 mg of tissue. [Figure 8C] This shows the efficacy and TIL analysis results for MC38 tumors treated with extended half-life IL-21 (WW50008). The graphs show the NK cell count and the relative abundance of NK IFN-γ+ cells in mice treated with the vehicle and WW50008. [Figure 8D]This shows the efficacy and TIL analysis results for MC38 tumors treated with extended half-life IL-21 (WW50008). The graph shows the relative abundance of CD8+ tetramer+ T cells (granzyme B+, INF-γ+, and TNF+) in mice treated with the vehicle and WW50008.

[0114] [Figure 9A] This document presents the efficacy of in vivo administration of extended-half-life human IL-2 (WW0177) or extended-half-life mouse IL-21 (WW50008) on EMT-6 tumors and the results of TIL analysis. The graphs show tumor volume in mice treated with vehicle, WW0177 (10 μg), or WW50008 (100 μg) on ​​days 1, 4, 8, and 11. [Figure 9B] This report presents the efficacy of in vivo administration of extended-half-life human IL-2 (WW0177) or extended-half-life mouse IL-21 (WW50008) on EMT-6 tumors and the results of TIL analysis. The proportions of CD8 and CD8+ granzyme B+, INF-γ+, and TNF+ infiltrates in the vehicle, WW0177, and WW50008 treatment groups are also shown. [Figure 9C] This shows the efficacy of in vivo administration of extended-half-life human IL-2 (WW0177) or extended-half-life mouse IL-21 (WW50008) on EMT-6 tumors and the results of TIL analysis. The graph shows the multifunctionality of cytokine production in CD8+ T cells treated with the vehicle, WW0177, and WW50008. [Figure 9D] This report presents the efficacy of in vivo administration of extended-half-life human IL-2 (WW0177) or extended-half-life mouse IL-21 (WW50008) on EMT-6 tumors and the results of TIL analysis. The graphs show the proportion of Treg cells and the CD8 / Treg ratio in the vehicle, WW0177, and WW5008 treatment groups. [Figure 9E]This report presents the efficacy of in vivo administration of extended-half-life human IL-2 (WW0177) or extended-half-life mouse IL-21 (WW50008) to EMT-6 tumors and the results of TIL analysis. A heatmap showing differential gene expression in tumors treated with WW0177 and WW50008, using NanoString analysis, is also presented.

[0115] [Figure 10A] Figures A and B show the efficacy (dose-dependent effect) of different dose levels of extended-half-life IL-21 and IL-21 prodrugs in the MC38 mouse model (A). No adverse effects on the body weight of these animals were observed at any of the dose levels tested (B). [Figure 10B] Figures A and B show the efficacy (dose-dependent effect) of different dose levels of extended-half-life IL-21 and IL-21 prodrugs in the MC38 mouse model (A). No adverse effects on the body weight of these animals were observed at any of the dose levels tested (B). [Figure 11A] This graph shows the results of the EMT-6 mouse tumor rechallenge study. It shows the tumor volume over time in mice treated with extended-half-life human IL-2 (square), extended-half-life mouse IL-21 (triangle), and vehicle (circle). The majority of animals treated with extended-half-life IL-21 survived this study and were used in the rechallenge trial. [Figure 11B] This graph shows the results of EMT-6 mouse tumor rechallenge. It shows the average tumor volume as a percentage of days after rechallenge in mice that survived the EMT-6 challenge (see triangle A) and showed complete tumor regression. These animals were then rechallenged with the EMT-6 cell line (circles), and naive mice were used as controls (squares). Only the naive animals developed tumors. These results indicate that treatment with extended half-life IL-21 induced immunological memory against EMT-6. [Figure 11C]This graph shows the results of tumor rechallenge in EMT-6 mice. The images are representative microscopic images from the IFNγ ELISPOT assay. Splenocytes were isolated from EMT-6 tumor-carrying mice that achieved complete remission (CR) after treatment with extended-half-life human IL-2 or extended-half-life mouse IL-21, and the splenocytes were incubated with EMT-6 cells for 24 hours. The splenocytes responded to the EMT-6 cells by producing IFNγ. [Figure 11D] This graph shows the results of EMT-6 mouse tumor rechallenge. The graph also shows the average relative spot volume on the IFNγ ELISPOT plate. These results indicate that treatment with IL-2 and IL-21 induced immunological memory against EMT-6.

[0116] [Figure 12] Graphs A-D show that in a syngeneic MC38 tumor model using mice expressing the human IL-21 receptor, the extended half-life IL-21 and the inducible IL-21 prodrug have comparable antitumor activity, but the inducible IL-21 prodrug (C) is better tolerated and has a wider therapeutic range. A and C show the average tumor volume over days in mice treated with the extended half-life IL-21 (A) and the inducible IL-21 prodrug (C). B and D show the average body weight of the treated animals in the data shown in A and C. Each line in the plot represents the average body weight of the group over time. The therapeutic range is the maximum tolerable dose (MTD) / minimum effective dose (MED).

[0117] [Figure 13A] This graph shows the analysis results of WW50334 in a syngeneic MC38 tumor model using mice expressing the human IL-21 receptor. It shows the mean tumor volume over time in mice treated with WW50334 at doses of 3.3 μg, 10 μg, 33 μg, and 100 μg (per single injection per mouse). [Figure 13B]This graph shows the analysis results of WW50334 in a syngeneic MC38 tumor model using mice expressing the human IL-21 receptor. The graph for group A shows the average body weight of the treated animals. Each line in the plot represents the average body weight of the group over time. [Figure 13C] This graph shows the analysis results of WW50334 in a syngeneic MC38 tumor model using mice expressing the human IL-21 receptor. Graph A shows the body weight of the treated animals. It shows the total nanomoles of WW50334 delivered per mouse during the administration period. The highest dose of 100 μg was not tolerated and induced animal death.

[0118] [Figure 14A] This graph shows the analysis results of WW50112 / WW50347 in a syngeneic MC38 tumor model of mice expressing the human IL-21 receptor. It shows the mean tumor volume over time in mice treated with WW50112 / WW50347 at doses of 200 μg, 600 μg, and 1800 μg (per single injection per mouse). [Figure 14B] This graph shows the analysis results for WW50112 / WW50347 in a syngeneic MC38 tumor model of mice expressing the human IL-21 receptor. The graph for group A shows the average body weight of the treated animals. Each line in the plot represents the average body weight of the group over time. [Figure 14C] This graph shows the analysis results of WW50112 / WW50347 in a syngeneic MC38 tumor model of mice expressing the human IL-21 receptor. Graph A shows the body weight of each treated animal. The graph shows the total nanomoles of WW50334 delivered per mouse during the administration period.

[0119] [Figure 15A] This graph shows the analysis results of WW50112 / WW50348 in a syngeneic MC38 tumor model of mice expressing the human IL-21 receptor. It shows the mean tumor volume over time in mice treated with 200 μg, 600 μg, and 1800 μg (dose per single injection per mouse) of WW50112 / WW50348. [Figure 15B] This graph shows the analysis results for WW50112 / WW50348 in a syngeneic MC38 tumor model of mice expressing the human IL-21 receptor. The graph for group A shows the average body weight of the treated animals. Each line in the plot represents the average body weight of the group over time. [Figure 15C] This graph shows the analysis results for WW50112 / WW50348 in a syngeneic MC38 tumor model of mice expressing the human IL-21 receptor. Graph A shows the body weight of each treated animal.

[0120] [Figure 16] Graphs A and B show the results of the analysis of WW50260 in a syngeneic MC38 tumor model of mice expressing the human IL-21 receptor. Graph A shows the mean tumor volume over time in mice treated with WW50260 at doses of 142 μg, 425 μg, and 1,277 μg (per single injection per mouse). Graph B shows the mean body weight of the treated animals in A. Each line in the plot represents the mean body weight of the group over time.

[0121] [Figure 17A] This graph shows the analysis results for WW50334 (human IL-21 half-life extended variant), WW50260, WW50347 / WW50112, and WW050348 / WW50112 in a syngeneic MC38 tumor model of mice expressing the human IL-21 receptor. The corresponding mean serum levels for WW50334 are shown over time. [Figure 17B] This graph shows the analysis results for WW50334 (human IL-21 half-life extended variant), WW50260, WW50347 / WW50112, and WW050348 / WW50112 in a syngeneic MC38 tumor model of mice expressing the human IL-21 receptor. The corresponding mean serum levels for WW50260 are shown over time. [Figure 17C]This graph shows the analysis results for WW50334 (human IL-21 half-life extended variant), WW50260, WW50347 / WW50112, and WW050348 / WW50112 in a syngeneic MC38 tumor model of mice expressing the human IL-21 receptor. The corresponding mean serum levels for WW50112 / WW50347 are shown over time. [Figure 17D] This graph shows the analysis results for WW50334 (human IL-21 half-life extended variant), WW50260, WW50347 / WW50112, and WW050348 / WW50112 in a syngeneic MC38 tumor model of mice expressing the human IL-21 receptor. The corresponding mean serum levels for WW50112 / WW50348 are shown over time.

[0122] [Figure 18-1] This graph shows exposure levels to intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates. Column A shows the levels of WW050334 (human IL-21 with extended half-life) in serum (triangle) and tumor lysates (square) over time. [Figure 18-2] This graph shows exposure levels of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates. Graph B shows intact WW50260 in serum (triangle) and tumor lysates (square) over time, while graph C shows cleaved WW50260 in serum (triangle) and tumor lysates (square) over time. [Figure 18-3] This graph shows exposure levels of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates. Column D shows the time course of intact WW50347 / WW50112 in serum (triangle) and tumor lysates (square), while Column E shows the time course of cleaved WW50347 / WW50112 in serum (triangle) and tumor lysates (square). [Figure 18-4]This graph shows exposure levels of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates. F shows the time course of intact WW50348 / WW50112 in serum (triangle) and tumor lysates (square), while G shows the time course of cleaved WW50348 / WW50112 in serum (triangle) and tumor lysates (square).

[0123] [Figure 19] Figures A and B demonstrate the efficacy of extended half-life mouse IL-21 at different dose levels in the Renca mouse model (A) and the CT26 mouse model (B).

[0124] [Figure 20A] This shows the results of TIL analysis in EMT-6 tumors after in vivo administration of extended-half-life mouse IL-21 and vehicle in an EMT-6 mouse model. A shows a heatmap representing differential gene expression in IL-21 and vehicle. B shows a graph illustrating the multiplicative change in gene expression between IL-21 and vehicle. 357 genes with significantly differential expression were identified. [Figure 20B] This shows the results of TIL analysis in EMT-6 tumors after in vivo administration of extended-half-life mouse IL-21 and vehicle in the MT-6 mouse model. A shows a heatmap representing differential gene expression in IL-21 and vehicle. B shows a graph illustrating the multiplicative change in gene expression between IL-21 and vehicle. 357 genes with significantly differential expression were identified.

[0125] [Figure 21A] This shows the results of TIL analysis in EMT-6 tumors after in vivo administration of extended-half-life mouse IL-21 and extended-half-life human IL-2 in an EMT-6 mouse model. A shows a heatmap representing differential gene expression in IL-21, IL-2, and the vehicle. B shows a graph illustrating the multiplicative changes in gene expression between IL-21 and IL-2. 35 genes with significantly differential expression were identified. [Figure 21B] This shows the results of TIL analysis in EMT-6 tumors after in vivo administration of extended-half-life mouse IL-21 and extended-half-life human IL-2 in an EMT-6 mouse model. A shows a heatmap representing differential gene expression in IL-21, IL-2, and the vehicle. B shows a graph illustrating the multiplicative changes in gene expression between IL-21 and IL-2. 35 genes with significantly differential expression were identified.

[0126] [Figure 22A] Graphs A-F show the results of the analysis of inducible mouse IL-21 prodrugs in a syngeneic MC38 mouse tumor model. These show the mean tumor volume over time in mice treated with 250 μg, 1,000 μg, and 1,500 μg (dose per single injection per mouse) of inducible IL-21 prodrugs. Mice were administered twice a week for two weeks (a total of four doses). The following constructs were analyzed: IL-21 half-life extended (WW50008, Figure 22A), WW50076 (Figure 22B), WW50079 (Figure 22C), WW50080 (Figure 22D), WW50256 (Figure 22E), and WW50290 (Figure 22F). The data show that tumor volume was suppressed over time in a dose-dependent manner in mice treated with WW50076 (Figure 31B), WW50256 (Figure 22E), and WW50290 (Figure 22F). The doses of 1,000 μg and 1,500 μg were not tolerated in mice treated with WW50079 and WW50080. [Figure 22B]Graphs A-F show the results of the analysis of inducible mouse IL-21 prodrugs in a syngeneic MC38 mouse tumor model. These show the mean tumor volume over time in mice treated with 250 μg, 1,000 μg, and 1,500 μg (dose per single injection per mouse) of inducible IL-21 prodrugs. Mice were administered twice a week for two weeks (a total of four doses). The following constructs were analyzed: IL-21 half-life extended (WW50008, Figure 22A), WW50076 (Figure 22B), WW50079 (Figure 22C), WW50080 (Figure 22D), WW50256 (Figure 22E), and WW50290 (Figure 22F). The data show that tumor volume was suppressed over time in a dose-dependent manner in mice treated with WW50076 (Figure 31B), WW50256 (Figure 22E), and WW50290 (Figure 22F). The doses of 1,000 μg and 1,500 μg were not tolerated in mice treated with WW50079 and WW50080. [Figure 22C] Graphs A-F show the results of the analysis of inducible mouse IL-21 prodrugs in a syngeneic MC38 mouse tumor model. These show the mean tumor volume over time in mice treated with 250 μg, 1,000 μg, and 1,500 μg (dose per single injection per mouse) of inducible IL-21 prodrugs. Mice were administered twice a week for two weeks (a total of four doses). The following constructs were analyzed: IL-21 half-life extended (WW50008, Figure 22A), WW50076 (Figure 22B), WW50079 (Figure 22C), WW50080 (Figure 22D), WW50256 (Figure 22E), and WW50290 (Figure 22F). The data show that tumor volume was suppressed over time in a dose-dependent manner in mice treated with WW50076 (Figure 31B), WW50256 (Figure 22E), and WW50290 (Figure 22F). The doses of 1,000 μg and 1,500 μg were not tolerated in mice treated with WW50079 and WW50080. [Figure 22D]Graphs A-F show the results of the analysis of inducible mouse IL-21 prodrugs in a syngeneic MC38 mouse tumor model. These show the mean tumor volume over time in mice treated with 250 μg, 1,000 μg, and 1,500 μg (dose per single injection per mouse) of inducible IL-21 prodrugs. Mice were administered twice a week for two weeks (a total of four doses). The following constructs were analyzed: IL-21 half-life extended (WW50008, Figure 22A), WW50076 (Figure 22B), WW50079 (Figure 22C), WW50080 (Figure 22D), WW50256 (Figure 22E), and WW50290 (Figure 22F). The data show that tumor volume was suppressed over time in a dose-dependent manner in mice treated with WW50076 (Figure 31B), WW50256 (Figure 22E), and WW50290 (Figure 22F). The doses of 1,000 μg and 1,500 μg were not tolerated in mice treated with WW50079 and WW50080. [Figure 22E] Graphs A-F show the results of the analysis of inducible mouse IL-21 prodrugs in a syngeneic MC38 mouse tumor model. These show the mean tumor volume over time in mice treated with 250 μg, 1,000 μg, and 1,500 μg (dose per single injection per mouse) of inducible IL-21 prodrugs. Mice were administered twice a week for two weeks (a total of four doses). The following constructs were analyzed: IL-21 half-life extended (WW50008, Figure 22A), WW50076 (Figure 22B), WW50079 (Figure 22C), WW50080 (Figure 22D), WW50256 (Figure 22E), and WW50290 (Figure 22F). The data show that tumor volume was suppressed over time in a dose-dependent manner in mice treated with WW50076 (Figure 31B), WW50256 (Figure 22E), and WW50290 (Figure 22F). The doses of 1,000 μg and 1,500 μg were not tolerated in mice treated with WW50079 and WW50080. [Figure 22F]Graphs A-F show the results of the analysis of inducible mouse IL-21 prodrugs in a syngeneic MC38 mouse tumor model. These show the mean tumor volume over time in mice treated with 250 μg, 1,000 μg, and 1,500 μg (dose per single injection per mouse) of inducible IL-21 prodrugs. Mice were administered twice a week for two weeks (a total of four doses). The following constructs were analyzed: IL-21 half-life extended (WW50008, Figure 22A), WW50076 (Figure 22B), WW50079 (Figure 22C), WW50080 (Figure 22D), WW50256 (Figure 22E), and WW50290 (Figure 22F). The data show that tumor volume was suppressed over time in a dose-dependent manner in mice treated with WW50076 (Figure 31B), WW50256 (Figure 22E), and WW50290 (Figure 22F). The doses of 1,000 μg and 1,500 μg were not tolerated in mice treated with WW50079 and WW50080.

[0127] [Figure 23A] Figures 22A to 22F show graphs illustrating the average body weight of treated animals based on the data presented. Each line in the plot represents the average body weight of the group over time. [Figure 23B] Figures 22A to 22F show graphs illustrating the average body weight of treated animals based on the data presented. Each line in the plot represents the average body weight of the group over time. [Figure 23C] Figures 22A to 22F show graphs illustrating the average body weight of treated animals based on the data presented. Each line in the plot represents the average body weight of the group over time. [Figure 23D] Figures 22A to 22F show graphs illustrating the average body weight of treated animals based on the data presented. Each line in the plot represents the average body weight of the group over time. [Figure 23E] Figures 22A to 22F show graphs illustrating the average body weight of treated animals based on the data presented. Each line in the plot represents the average body weight of the group over time. [Figure 23F] Figures 22A to 22F show graphs illustrating the average body weight of treated animals based on the data presented. Each line in the plot represents the average body weight of the group over time.

[0128] [Figure 24A] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24B] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24C] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24D] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24E] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24F] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24G] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24H] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24I] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24J] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24K] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24L]Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24M] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24N] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time. [Figure 24O] Figures 22A to 22F are graphs showing the weight of each treated animal in the data presented. Each line in the plot represents the animal's weight over time.

[0129] ).

[0130] [Figure 25A] Graphs A-H show the results of the analysis of inducible IL-21 prodrugs in syngeneic MC38 mouse tumor models of human IL-21 receptor knock-in mice. They show the mean tumor volume over time in mice treated with WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50388 / WW50394, WW50465 / WW50473, or WW50468 / WW50473 at doses of 67 μg, 200 μg, 600 μg, and 1800 μg (per single injection per mouse). The data indicate that tumor volume was suppressed over time in a dose-dependent manner. [Figure 25B]Graphs A-H show the results of the analysis of inducible IL-21 prodrugs in syngeneic MC38 mouse tumor models of human IL-21 receptor knock-in mice. They show the mean tumor volume over time in mice treated with WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50388 / WW50394, WW50465 / WW50473, or WW50468 / WW50473 at doses of 67 μg, 200 μg, 600 μg, and 1800 μg (per single injection per mouse). The data indicate that tumor volume was suppressed over time in a dose-dependent manner. [Figure 25C] Graphs A-H show the results of the analysis of inducible IL-21 prodrugs in syngeneic MC38 mouse tumor models of human IL-21 receptor knock-in mice. They show the mean tumor volume over time in mice treated with WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50388 / WW50394, WW50465 / WW50473, or WW50468 / WW50473 at doses of 67 μg, 200 μg, 600 μg, and 1800 μg (per single injection per mouse). The data indicate that tumor volume was suppressed over time in a dose-dependent manner. [Figure 25D] Graphs A-H show the results of the analysis of inducible IL-21 prodrugs in syngeneic MC38 mouse tumor models of human IL-21 receptor knock-in mice. They show the mean tumor volume over time in mice treated with WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50388 / WW50394, WW50465 / WW50473, or WW50468 / WW50473 at doses of 67 μg, 200 μg, 600 μg, and 1800 μg (per single injection per mouse). The data indicate that tumor volume was suppressed over time in a dose-dependent manner. [Figure 25E]Graphs A-H show the results of the analysis of inducible IL-21 prodrugs in syngeneic MC38 mouse tumor models of human IL-21 receptor knock-in mice. They show the mean tumor volume over time in mice treated with WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50388 / WW50394, WW50465 / WW50473, or WW50468 / WW50473 at doses of 67 μg, 200 μg, 600 μg, and 1800 μg (per single injection per mouse). The data indicate that tumor volume was suppressed over time in a dose-dependent manner. [Figure 25F] Graphs A-H show the results of the analysis of inducible IL-21 prodrugs in syngeneic MC38 mouse tumor models of human IL-21 receptor knock-in mice. They show the mean tumor volume over time in mice treated with WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50388 / WW50394, WW50465 / WW50473, or WW50468 / WW50473 at doses of 67 μg, 200 μg, 600 μg, and 1800 μg (per single injection per mouse). The data indicate that tumor volume was suppressed over time in a dose-dependent manner. [Figure 25G] Graphs A-H show the results of the analysis of inducible IL-21 prodrugs in syngeneic MC38 mouse tumor models of human IL-21 receptor knock-in mice. They show the mean tumor volume over time in mice treated with WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50388 / WW50394, WW50465 / WW50473, or WW50468 / WW50473 at doses of 67 μg, 200 μg, 600 μg, and 1800 μg (per single injection per mouse). The data indicate that tumor volume was suppressed over time in a dose-dependent manner. [Figure 25H]Graphs A-H show the results of the analysis of inducible IL-21 prodrugs in syngeneic MC38 mouse tumor models of human IL-21 receptor knock-in mice. They show the mean tumor volume over time in mice treated with WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50388 / WW50394, WW50465 / WW50473, or WW50468 / WW50473 at doses of 67 μg, 200 μg, 600 μg, and 1800 μg (per single injection per mouse). The data indicate that tumor volume was suppressed over time in a dose-dependent manner.

[0131] [Figure 26A] Figures A through H are graphs showing the average body weight of treated animals in the data presented in Figures 25A through 25H. Each line in the plot represents the average body weight of the group over time. [Figure 26B] Figures A through H are graphs showing the average body weight of treated animals in the data presented in Figures 25A through 25H. Each line in the plot represents the average body weight of the group over time. [Figure 26C] Figures A through H are graphs showing the average body weight of treated animals in the data presented in Figures 25A through 25H. Each line in the plot represents the average body weight of the group over time. [Figure 26D] Figures A through H are graphs showing the average body weight of treated animals in the data presented in Figures 25A through 25H. Each line in the plot represents the average body weight of the group over time. [Figure 26E] Figures A through H are graphs showing the average body weight of treated animals in the data presented in Figures 25A through 25H. Each line in the plot represents the average body weight of the group over time. [Figure 26F] Figures A through H are graphs showing the average body weight of treated animals in the data presented in Figures 25A through 25H. Each line in the plot represents the average body weight of the group over time. [Figure 26G] Figures A through H are graphs showing the average body weight of treated animals in the data presented in Figures 25A through 25H. Each line in the plot represents the average body weight of the group over time. [Figure 26H] Figures A through H are graphs showing the average body weight of treated animals in the data presented in Figures 25A through 25H. Each line in the plot represents the average body weight of the group over time.

[0132] [Figure 27A] Figures A to H are graphs showing the corresponding mean serum levels for WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50465 / WW50473, WW50388 / WW50394, and WW50468 / WW50473 for the treated animals in the data shown in Figures 25A to 25H and Figures 26A to 26H. [Figure 27B] Figures A to H are graphs showing the corresponding mean serum levels for WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50465 / WW50473, WW50388 / WW50394, and WW50468 / WW50473 for the treated animals in the data shown in Figures 25A to 25H and Figures 26A to 26H. [Figure 27C] Figures A to H are graphs showing the corresponding mean serum levels for WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50465 / WW50473, WW50388 / WW50394, and WW50468 / WW50473 for the treated animals in the data shown in Figures 25A to 25H and Figures 26A to 26H. [Figure 27D]Figures A to H are graphs showing the corresponding mean serum levels for WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50465 / WW50473, WW50388 / WW50394, and WW50468 / WW50473 for the treated animals in the data shown in Figures 25A to 25H and Figures 26A to 26H. [Figure 27E] Figures A to H are graphs showing the corresponding mean serum levels for WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50465 / WW50473, WW50388 / WW50394, and WW50468 / WW50473 for the treated animals in the data shown in Figures 25A to 25H and Figures 26A to 26H. [Figure 27F] Figures A to H are graphs showing the corresponding mean serum levels for WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50465 / WW50473, WW50388 / WW50394, and WW50468 / WW50473 for the treated animals in the data shown in Figures 25A to 25H and Figures 26A to 26H. [Figure 27G] Figures A to H are graphs showing the corresponding mean serum levels for WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50465 / WW50473, WW50388 / WW50394, and WW50468 / WW50473 for the treated animals in the data shown in Figures 25A to 25H and Figures 26A to 26H. [Figure 27H]Figures A to H are graphs showing the corresponding mean serum levels for WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50465 / WW50473, WW50388 / WW50394, and WW50468 / WW50473 for the treated animals in the data shown in Figures 25A to 25H and Figures 26A to 26H.

[0133] [Figure 28-1] Graphs A through P show the exposure levels of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates. [Figure 28-2] Graphs A through P show the exposure levels of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates. [Figure 28-3] Graphs A through P show the exposure levels of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates. [Figure 28-4] Graphs A through P show the exposure levels of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates. [Figure 28-5] Graphs A through P show the exposure levels of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates. [Figure 28-6] Graphs A through P show the exposure levels of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates. [Figure 28-7] Graphs A through P show the exposure levels of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates. [Figure 28-8] Graphs A through P show the exposure levels of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysates.

[0134] [Figure 29A]Images A–C are capillary Western blot images showing the stability of inducible IL-21 prodrugs in mouse serum (A), cynomolgus monkey mouse serum (B), and / or pooled human serum (C). All inducible IL-21 prodrugs were stable in serum, and no cleavage species were observed. [Figure 29B] Images A–C are capillary Western blot images showing the stability of inducible IL-21 prodrugs in mouse serum (A), cynomolgus monkey mouse serum (B), and / or pooled human serum (C). All inducible IL-21 prodrugs were stable in serum, and no cleavage species were observed. [Figure 29C] Images A–C are capillary Western blot images showing the stability of inducible IL-21 prodrugs in mouse serum (A), cynomolgus monkey mouse serum (B), and / or pooled human serum (C). All inducible IL-21 prodrugs were stable in serum, and no cleavage species were observed.

[0135] [Figure 30] This graph shows the results of an analysis of the mean tumor volume after administration of WW50387 / WW50394 at doses of 47, 94, 188, 375, 750, 1200, and 1500 μg on days 0, 3, 7, and 10 after the start of treatment. This study was conducted until day 28 after the start of treatment, and tumor volume was measured twice a week during the study period. The data show that tumor volume was suppressed over time in a dose-dependent manner. The minimum effective dose (MED), defined as the dose at which 50% of subjects showed tumor control during the treatment period, was determined to be 94 μg, and the maximum tolerable dose (MTD), defined as the dose at which no subjects were lost due to drug toxicity, was determined to be 1500 μg. The therapeutic range of WW50387 / WW50394 in this syngeneic tumor volume was determined to be 16.

[0136] [Figure 31A]Graphs A-H show individual plots of treatment with WW50387 / WW50394, clearly demonstrating a decrease in tumor volume as the dose level increases up to 1500 μg. Graphs I and J show tumor volumes for unresectable controls at dose levels of 188 ug and 375 ug, respectively. [Figure 31B] Same as above. [Figure 31C] Same as above. [Figure 31D] Same as above. [Figure 31E] Same as above. [Figure 31F] Same as above. [Figure 31G] Same as above. [Figure 31H] Same as above. [Figure 31I] Same as above. [Figure 31J] Same as above.

[0137] [Figure 32] Graphs A and B show the time course of mouse body weight after the start of administration of the inducible IL-21 prodrug. Graph A shows the mean body weight of mice, expressed in grams, across all treatment groups. Both the 750 μg and 1500 μg dose groups showed a decrease in body weight on day 14 (four days after the final dose on day 10). Both groups recovered to pre-treatment body weight with additional feeding of edible gel. Graph B shows the mean body weight of subjects, expressed as the percentage change in body weight from the initial measurement at the randomization point on day -1, across all treatment groups.

[0138] [Figure 33A] This shows a plot of individual body weight for all subjects in all treatment groups. [Figure 33B] This shows a plot of individual body weight for all subjects in all treatment groups. [Figure 33C] This shows a plot of individual body weight for all subjects in all treatment groups. [Figure 33D] This shows a plot of individual body weight for all subjects in all treatment groups. [Figure 33E]This shows a plot of individual body weight for all subjects in all treatment groups. [Figure 33F] This shows a plot of individual body weight for all subjects in all treatment groups. [Figure 33G] This shows a plot of individual body weight for all subjects in all treatment groups. [Figure 33H] This shows a plot of individual body weight for all subjects in all treatment groups. [Figure 33I] The plot shows individual body weight for all subjects in all treatment groups. I and J represent tumor volume for un-amputated controls at dose levels of 188 μg and 375 μg, respectively. [Figure 33J] The plot shows individual body weight for all subjects in all treatment groups. I and J represent tumor volume for un-amputated controls at dose levels of 188 μg and 375 μg, respectively.

[0139] [Figure 34] Figures A and B show the results of IL-21 prodrug treatment for immunization against tumor rechallenge in animals considered to have achieved a complete response in the initial study. Figure A shows the primary group of tumor growth response, from which CR animals were selected. A total of 24 WW50387 / WW50394 treated mice that showed CR were rechallenged by inoculation of MC38-BC2 into the left flank on day 88. Figure B shows that all rechallenged mice refused the second inoculation, showed no regrowth of the primary tumor, and survived until the final observation day (day 116), whereas similarly inoculated naive mice of the same strain and age showed consistent tumor growth in the left flank.

[0140] [Figure 35A] The efficacy and TIL analysis results for MC38 tumors treated with WW50387 / WW50394 are shown. Figure 35 is a graph showing tumor volume (mm3) in mice treated with WW50387 / WW50394. [Figure 35B]This shows the efficacy and TIL analysis results for MC38 tumors treated with WW50387 / WW50394. The graph shows the percentage of M1 and M2 macrophages in mice treated with WW50387 / WW50394 and the vehicle. [Figure 35C] The efficacy and TIL analysis results for MC38 tumors treated with WW50387 / WW50394 are shown. C and D are graphs showing the relative abundance of NK cells and NK IFN-γ+ cells in mice treated with the vehicle and WW50387 / WW50394. [Figure 35D] The efficacy and TIL analysis results for MC38 tumors treated with WW50387 / WW50394 are shown. C and D are graphs showing the relative abundance of NK cells and NK IFN-γ+ cells in mice treated with the vehicle and WW50387 / WW50394. [Figure 35E] This shows the efficacy and TIL analysis results for MC38 tumors treated with WW50387 / WW50394. The graph shows the relative abundance of CD4+ conventional T cells in mice treated with the vehicle and WW50387 / WW50394. [Figure 35F] This shows the efficacy and TIL analysis results for MC38 tumors treated with WW50387 / WW50394. The graph shows the percentage of CD8+ T cell infiltration. [Figure 35G] This shows the efficacy and TIL analysis results for MC38 tumors treated with WW50387 / WW50394. The graph shows the relative abundance of CD8+ tetramer+ T cells (granzyme B+, INF-γ+, and TNF+) in mice treated with the vehicle and WW50387 / WW50394. [Figure 35H] This shows the efficacy and TIL analysis results for MC38 tumors treated with WW50387 / WW50394. The pie chart shows the multifunctionality of cytokine CD8+ T cell production in mice treated with WW50387 / WW50394 and the vehicle.

[0141] [Figure 36]This graph shows the percentage of total activity of WW50387 / WW50394 processing in primary human dissociative tumor samples (glioblastoma multiforme, colorectal cancer, cervical cancer, bladder cancer, and endometrial cancer).

[0142] [Figure 37] A and B show the B16F10-OVA tumor volume (A) and body weight (B) for WW50387 / WW50394 in a dose range including 125, 250, 500, and 1000 μg. Throughout this dose range, antitumor activity was observed at dose levels of 250, 500, and 1000 μg, completely controlling tumor growth up to day 21 of the administration schedule. All administration regimens were well tolerated, and no treatment-related deaths or significant weight loss were observed.

[0143] [Figure 38] A-E show the individual tumor volume in the treatment group. These plots also demonstrate excellent tumor control with WW50387 / WW50394 at a dose level of 250 μg.

[0144] [Figure 39] This study demonstrates that the efficacy of WW50387 / WW50394 at a dose level of 500 μg was completely rendered ineffective when CD8 T cells were depleted using αCD8 antibody, highlighting the effector T cell dependence of this B16F10-OVA model when treated with WW50387 / WW50394.

[0145] [Figure 40] Plots A and B show exposure levels determined by bioanalytical assays of WW50387 / WW50394 in serum after administration of WW50387 / WW50394. Plot A shows the dose-proportional exposure level detected in serum approximately 24 hours after administration of WW50387 / WW50394. Plot B shows that this dose-proportional exposure level was also confirmed on day 11 (approximately 72 hours after the third administration of WW50387 / WW50394 on day 8).

[0146] [Figure 41] A and B show dose-normalized exposure data for the 125, 250, 500, and 1000 μg WW50387 / WW50394 groups, as well as the 500 μg dose WW50387 / WW50394 / αCD8 depletion group. Both plots on Day 2 (A) and Day 11 (B) demonstrate dose-proportional exposure across the entire dose range.

[0147] [Figure 42] A and B are graphs showing the proportion of CD8 T cells in the CD45+ cell population of blood, demonstrating that CD8 T cells were effectively depleted. Vehicle + IgG is represented by circles, Vehicle + αCD8 by squares, WW50387 / WW50394 + IgG by diamonds, and WW50387 / WW50394 + αCD8 by triangles.

[0148] [Figure 43] A and B are graphs showing tumor volumes after a total of two once-weekly administrations of WW50387 / WW50394 (A), and after a single administration (B). A is a graph showing the results of mean tumor volumes after WW50387 / WW50394 was administered at 188, 375, and 750 μg on Day 0 and Day 7. B is a graph showing the results of analysis of mean tumor volumes after WW50387 / WW50394 was administered at 750, 1000, and 1500 μg on Day 0. The triangles below the X-axis indicate the administration days.

[0149] [Figure 44A]Tables A-F show the sequence alignments of humanized variants of the llama anti-human serum albumin VHH antibody. The alignments of each llama VHH, selected human germline Vh template protein(s), and humanized variant are shown as follows: A(1HUM19) (A discloses sequence numbers 701-703 and 608-616 in the order listed), B(3HUM181) (B discloses sequence numbers 704-706 and 625-634 in the order listed), and C(2HUM45 on the IGHV3-23 backbone) (C discloses sequence numbers 707-709 and 635-639 in the order listed). The CDRs are shown in D (2HUM45 on the IGHV3-11 backbone) (D discloses sequence numbers 710-712 and 640-648 in the order they are listed), E (1HUM19 variant with a modified CDR sequence) (E discloses sequence numbers 713-715, 617-624 and 649-651 in the order they are listed), and F (3HUM181 variant with a modified CDR sequence) (F discloses sequence numbers 716-718, 652 and 653 in the order they are listed). [Figure 44B]Tables A-F show the sequence alignments of humanized variants of the llama anti-human serum albumin VHH antibody. The alignments of each llama VHH, selected human germline Vh template protein(s), and humanized variant are shown as follows: A(1HUM19) (A discloses sequence numbers 701-703 and 608-616 in the order listed), B(3HUM181) (B discloses sequence numbers 704-706 and 625-634 in the order listed), and C(2HUM45 on the IGHV3-23 backbone) (C discloses sequence numbers 707-709 and 635-639 in the order listed). The CDRs are shown in D (2HUM45 on the IGHV3-11 backbone) (D discloses sequence numbers 710-712 and 640-648 in the order they are listed), E (1HUM19 variant with a modified CDR sequence) (E discloses sequence numbers 713-715, 617-624 and 649-651 in the order they are listed), and F (3HUM181 variant with a modified CDR sequence) (F discloses sequence numbers 716-718, 652 and 653 in the order they are listed). [Figure 44C]Tables A-F show the sequence alignments of humanized variants of the llama anti-human serum albumin VHH antibody. The alignments of each llama VHH, selected human germline Vh template protein(s), and humanized variant are shown as follows: A(1HUM19) (A discloses sequence numbers 701-703 and 608-616 in the order listed), B(3HUM181) (B discloses sequence numbers 704-706 and 625-634 in the order listed), and C(2HUM45 on the IGHV3-23 backbone) (C discloses sequence numbers 707-709 and 635-639 in the order listed). The CDRs are shown in D (2HUM45 on the IGHV3-11 backbone) (D discloses sequence numbers 710-712 and 640-648 in the order they are listed), E (1HUM19 variant with a modified CDR sequence) (E discloses sequence numbers 713-715, 617-624 and 649-651 in the order they are listed), and F (3HUM181 variant with a modified CDR sequence) (F discloses sequence numbers 716-718, 652 and 653 in the order they are listed). [Figure 44D]Tables A-F show the sequence alignments of humanized variants of the llama anti-human serum albumin VHH antibody. The alignments of each llama VHH, selected human germline Vh template protein(s), and humanized variant are shown as follows: A(1HUM19) (A discloses sequence numbers 701-703 and 608-616 in the order listed), B(3HUM181) (B discloses sequence numbers 704-706 and 625-634 in the order listed), and C(2HUM45 on the IGHV3-23 backbone) (C discloses sequence numbers 707-709 and 635-639 in the order listed). The CDRs are shown in D (2HUM45 on the IGHV3-11 backbone) (D discloses sequence numbers 710-712 and 640-648 in the order they are listed), E (1HUM19 variant with a modified CDR sequence) (E discloses sequence numbers 713-715, 617-624 and 649-651 in the order they are listed), and F (3HUM181 variant with a modified CDR sequence) (F discloses sequence numbers 716-718, 652 and 653 in the order they are listed). [Figure 44E]Tables A-F show the sequence alignments of humanized variants of the llama anti-human serum albumin VHH antibody. The alignments of each llama VHH, selected human germline Vh template protein(s), and humanized variant are shown as follows: A(1HUM19) (A discloses sequence numbers 701-703 and 608-616 in the order listed), B(3HUM181) (B discloses sequence numbers 704-706 and 625-634 in the order listed), and C(2HUM45 on the IGHV3-23 backbone) (C discloses sequence numbers 707-709 and 635-639 in the order listed). The CDRs are shown in D (2HUM45 on the IGHV3-11 backbone) (D discloses sequence numbers 710-712 and 640-648 in the order they are listed), E (1HUM19 variant with a modified CDR sequence) (E discloses sequence numbers 713-715, 617-624 and 649-651 in the order they are listed), and F (3HUM181 variant with a modified CDR sequence) (F discloses sequence numbers 716-718, 652 and 653 in the order they are listed). [Figure 44F]Tables A-F show the sequence alignments of humanized variants of the llama anti-human serum albumin VHH antibody. The alignments of each llama VHH, selected human germline Vh template protein(s), and humanized variant are shown as follows: A(1HUM19) (A discloses sequence numbers 701-703 and 608-616 in the order listed), B(3HUM181) (B discloses sequence numbers 704-706 and 625-634 in the order listed), and C(2HUM45 on the IGHV3-23 backbone) (C discloses sequence numbers 707-709 and 635-639 in the order listed). The CDRs are shown in D (2HUM45 on the IGHV3-11 backbone) (D discloses sequence numbers 710-712 and 640-648 in the order they are listed), E (1HUM19 variant with a modified CDR sequence) (E discloses sequence numbers 713-715, 617-624 and 649-651 in the order they are listed), and F (3HUM181 variant with a modified CDR sequence) (F discloses sequence numbers 716-718, 652 and 653 in the order they are listed).

[0150] [Figure 45] Graphs A and B show the predicted pharmacokinetics (PK) (μg / mL) over time of anti-albumin VHH with different binding affinities to mouse (A) or human serum albumin (B).

[0151] [Figure 46] Plots A and B show the efficacy (A) and effect on body weight (B) of the WW50387 / WW50394 + αPD-1 combination study. In general, efficacy improved with increasing dose levels of WW50387 / WW50394 and the addition of αPD-1. All treatment regimens were well tolerated, and no weight loss was observed after the start of treatment during the 28-day study.

[0152] [Figure 47A]A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αPD-1. At the 94 μg dose, there was no difference between WW50387 / WW50394, αPD-1, or the combination of WW50387 / WW50394 + αPD-1. At the 375 μg dose, the combination of WW50387 / WW50394 + αPD-1 was highly significantly (****p<0.0001) more effective than αPD-1 alone, but not highly significantly more effective than WW50387 / WW50394 alone (C). [Figure 47B] A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αPD-1. At the 94 μg dose, there was no difference between WW50387 / WW50394, αPD-1, or the combination of WW50387 / WW50394 + αPD-1. At the 375 μg dose, the combination of WW50387 / WW50394 + αPD-1 was highly significantly (****p<0.0001) more effective than αPD-1 alone, but not highly significantly more effective than WW50387 / WW50394 alone (C). [Figure 47C] A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αPD-1. At the 94 μg dose, there was no difference between WW50387 / WW50394, αPD-1, or the combination of WW50387 / WW50394 + αPD-1. At the 375 μg dose, the combination of WW50387 / WW50394 + αPD-1 was highly significantly (****p<0.0001) more effective than αPD-1 alone, but not highly significantly more effective than WW50387 / WW50394 alone (C). [Figure 47D]A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αPD-1. At the 94 μg dose, there was no difference between WW50387 / WW50394, αPD-1, or the combination of WW50387 / WW50394 + αPD-1. At the 375 μg dose, the combination of WW50387 / WW50394 + αPD-1 was highly significantly (****p<0.0001) more effective than αPD-1 alone, but not highly significantly more effective than WW50387 / WW50394 alone (C).

[0153] [Figure 48] Plots A and B show the efficacy (A) and effect on body weight (B) of the WW50387 / WW50394 + αPD-L1 combination study. In general, efficacy improved with increasing dose levels of WW50387 / WW50394 and the addition of αPD-L1. All treatment regimens were well tolerated, and no weight loss was observed after the start of treatment during the 28-day study.

[0154] [Figure 49A] A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αPD-L1. At the 94 μg dose, there was a tendency for efficacy to improve when WW50387 / WW50394 was combined with αPD-L1, but this did not reach statistical significance. At the 375 μg dose, the combination of WW50387 / WW50394 + αPD-L1 was highly significantly (****p<0.0001) more effective than αPD-L1 alone, and significantly (**p<0.0063) more effective than WW50387 / WW50394 alone. Furthermore, these combinations showed an increase in complete response (CR) compared to 94 μg of WW50387 / WW50394 alone (3 CRs vs. 0 CRs), and an increase in CR compared to 300 μg of WW50387 / WW50394 alone (6 CRs vs. 2 CRs) (Summary table of CRs in C and D). [Figure 49B]A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αPD-L1. At the 94 μg dose, there was a tendency for efficacy to improve when WW50387 / WW50394 was combined with αPD-L1, but this did not reach statistical significance. At the 375 μg dose, the combination of WW50387 / WW50394 + αPD-L1 was highly significantly (****p<0.0001) more effective than αPD-L1 alone, and significantly (**p<0.0063) more effective than WW50387 / WW50394 alone. Furthermore, these combinations showed an increase in complete response (CR) compared to 94 μg of WW50387 / WW50394 alone (3 CRs vs. 0 CRs), and an increase in CR compared to 300 μg of WW50387 / WW50394 alone (6 CRs vs. 2 CRs) (Summary table of CRs in C and D). [Figure 49C] A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αPD-L1. At the 94 μg dose, there was a tendency for efficacy to improve when WW50387 / WW50394 was combined with αPD-L1, but this did not reach statistical significance. At the 375 μg dose, the combination of WW50387 / WW50394 + αPD-L1 was highly significantly (****p<0.0001) more effective than αPD-L1 alone, and significantly (**p<0.0063) more effective than WW50387 / WW50394 alone. Furthermore, these combinations showed an increase in complete response (CR) compared to 94 μg of WW50387 / WW50394 alone (3 CRs vs. 0 CRs), and an increase in CR compared to 300 μg of WW50387 / WW50394 alone (6 CRs vs. 2 CRs) (Summary table of CRs in C and D). [Figure 49D]A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αPD-L1. At the 94 μg dose, there was a tendency for efficacy to improve when WW50387 / WW50394 was combined with αPD-L1, but this did not reach statistical significance. At the 375 μg dose, the combination of WW50387 / WW50394 + αPD-L1 was highly significantly (****p<0.0001) more effective than αPD-L1 alone, and significantly (**p<0.0063) more effective than WW50387 / WW50394 alone. Furthermore, these combinations showed an increase in complete response (CR) compared to 94 μg of WW50387 / WW50394 alone (3 CRs vs. 0 CRs), and an increase in CR compared to 300 μg of WW50387 / WW50394 alone (6 CRs vs. 2 CRs) (Summary table of CRs in C and D).

[0155] [Figure 50] Plots A and B show the efficacy (A) and effect on body weight (B) of the WW50387 / WW50394 + αCTLA-4 combination study. In general, efficacy improved with increasing dose levels of WW50387 / WW50394 and the addition of αCTLA-4. All treatment regimens were well tolerated, and no weight loss was observed after the start of treatment during the 28-day study.

[0156] [Figure 51A]A to D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αCTLA-4. At the 94 μg dose, when WW50387 / WW50394 was combined with αCTLA-4, highly significantly (****p<0.0001) higher efficacy was observed compared to either WW50387 / WW50394 or αCTLA-4 treatment alone. At the 375 μg dose, the combination of WW50387 / WW50394 + αCTLA-4 was highly significantly (****p<0.0001) more effective than αCTLA-4 alone, and showed a trend of improved efficacy that did not reach statistical significance when compared to WW50387 / WW50394 alone. Furthermore, these combinations showed an increase in CR compared to 94 μg of WW50387 / WW50394 alone (4 CR cases vs. 0 CR cases), and showed an increase in CR compared to 300 μg of WW50387 / WW50394 alone (4 CR cases vs. 2 CR cases) (summary table of CR for C and D). [Figure 51B] A to D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αCTLA-4. At the 94 μg dose, when WW50387 / WW50394 was combined with αCTLA-4, highly significantly (****p<0.0001) higher efficacy was observed compared to either WW50387 / WW50394 or αCTLA-4 treatment alone. At the 375 μg dose, the combination of WW50387 / WW50394 + αCTLA-4 was highly significantly (****p<0.0001) more effective than αCTLA-4 alone, and showed a trend of improved efficacy that did not reach statistical significance when compared to WW50387 / WW50394 alone. Furthermore, these combinations showed an increase in CR compared to 94 μg of WW50387 / WW50394 alone (4 CR cases vs. 0 CR cases), and showed an increase in CR compared to 300 μg of WW50387 / WW50394 alone (4 CR cases vs. 2 CR cases) (summary table of CR for C and D). [Figure 51C]A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αCTLA-4. At the 94 μg dose, the combination of WW50387 / WW50394 with αCTLA-4 showed significantly higher efficacy (****p<0.0001) compared to either WW50387 / WW50394 or αCTLA-4 treatment alone. At the 375 μg dose, the combination of WW50387 / WW50394 + αCTLA-4 was significantly more effective (****p<0.0001) than αCTLA-4 alone, and compared to WW50387 / WW50394 alone, there was a tendency for improved efficacy, although it did not reach statistical significance. Furthermore, these combinations showed an increase in complete response (CR) compared to 94 μg of WW50387 / WW50394 alone (4 CRs vs. 0 CRs), and an increase in CR compared to 300 μg of WW50387 / WW50394 alone (4 CRs vs. 2 CRs) (Summary table of CRs in C and D). [Figure 51D] A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αCTLA-4. At the 94 μg dose, the combination of WW50387 / WW50394 with αCTLA-4 showed significantly higher efficacy (****p<0.0001) compared to either WW50387 / WW50394 or αCTLA-4 treatment alone. At the 375 μg dose, the combination of WW50387 / WW50394 + αCTLA-4 was significantly more effective (****p<0.0001) than αCTLA-4 alone, and compared to WW50387 / WW50394 alone, there was a tendency for improved efficacy, although it did not reach statistical significance. Furthermore, these combinations showed an increase in complete response (CR) compared to 94 μg of WW50387 / WW50394 alone (4 CRs vs. 0 CRs), and an increase in CR compared to 300 μg of WW50387 / WW50394 alone (4 CRs vs. 2 CRs) (Summary table of CRs in C and D).

[0157] [Figure 52]Plots A and B show the efficacy (A) and effect on body weight (B) of the WW50387 / WW50394 + αLAG3 combination study. In general, efficacy improved with increasing dose levels of WW50387 / WW50394 and the addition of αLAG3. All treatment regimens were well tolerated, and no weight loss was observed after the start of treatment during the 28-day study.

[0158] [Figure 53A] A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αLAG3. At the 94 μg dose, there was a tendency for efficacy to improve when WW50387 / WW50394 was combined with αLAG3, but this did not reach statistical significance. At the 375 μg dose, the combination of WW50387 / WW50394 + αLAG3 was highly statistically (****p<0.0001) more effective than αLAG3 alone, but not highly statistically more effective than WW50387 / WW50394 alone. Furthermore, although this combination did not produce more complete responses (CRs) than WW50387 / WW50394 (2 CRs vs. 2 CRs), it showed an increase in CRs compared to 1 CR observed with 94 μg WW50387 / WW50394 + αLAG3, compared to 0 CRs observed with 94 μg WW50387 / WW50394 alone (Summary table of CRs in C and D). [Figure 53B]A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αLAG3. At the 94 μg dose, there was a tendency for efficacy to improve when WW50387 / WW50394 was combined with αLAG3, but this did not reach statistical significance. At the 375 μg dose, the combination of WW50387 / WW50394 + αLAG3 was highly statistically (****p<0.0001) more effective than αLAG3 alone, but not highly statistically more effective than WW50387 / WW50394 alone. Furthermore, although this combination did not produce more complete responses (CRs) than WW50387 / WW50394 (2 CRs vs. 2 CRs), it showed an increase in CRs compared to 1 CR observed with 94 μg WW50387 / WW50394 + αLAG3, compared to 0 CRs observed with 94 μg WW50387 / WW50394 alone (Summary table of CRs in C and D). [Figure 53C] A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αLAG3. At the 94 μg dose, there was a tendency for efficacy to improve when WW50387 / WW50394 was combined with αLAG3, but this did not reach statistical significance. At the 375 μg dose, the combination of WW50387 / WW50394 + αLAG3 was highly statistically (****p<0.0001) more effective than αLAG3 alone, but not highly statistically more effective than WW50387 / WW50394 alone. Furthermore, although this combination did not produce more complete responses (CRs) than WW50387 / WW50394 (2 CRs vs. 2 CRs), it showed an increase in CRs compared to 1 CR observed with 94 μg WW50387 / WW50394 + αLAG3, compared to 0 CRs observed with 94 μg WW50387 / WW50394 alone (Summary table of CRs in C and D). [Figure 53D]A-D show the efficacy of WW50387 / WW50394 administered at 94 μg (A) or 375 μg (B) in the presence or absence of αLAG3. At the 94 μg dose, there was a tendency for efficacy to improve when WW50387 / WW50394 was combined with αLAG3, but this did not reach statistical significance. At the 375 μg dose, the combination of WW50387 / WW50394 + αLAG3 was highly statistically (****p<0.0001) more effective than αLAG3 alone, but not highly statistically more effective than WW50387 / WW50394 alone. Furthermore, although this combination did not produce more complete responses (CRs) than WW50387 / WW50394 (2 CRs vs. 2 CRs), it showed an increase in CRs compared to 1 CR observed with 94 μg WW50387 / WW50394 + αLAG3, compared to 0 CRs observed with 94 μg WW50387 / WW50394 alone (Summary table of CRs in C and D).

[0159] [Figure 54A] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54B] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54C] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54D] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54E] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54F] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54G] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54H] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54I] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54J]The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54K] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54L] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54M] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54N] The plots of individual tumor volumes for all 15 groups in the study are shown. [Figure 54O] The plots of individual tumor volumes for all 15 groups in the study are shown.

[0160] [Figure 55] A and B are graphs showing the total inducible IL-21 prodrug and free human IL-21 levels in serum (A) and tumor (B) over 168 hours after a single dose of WW50387 / WW50394.

[0161] [Figure 56-1]Graphs A-D show the time-course exposure to total inducible IL-21 prodrugs (A and C) and free human IL-21 (B and D) in serum (A and B) and tumors (C and D) over 168 hours after a single dose and three doses of WW50387 / WW50394, respectively. In all test animals, an ADA response was evident from the acceleration of IL-21 prodrug clearance at 72, 96, 120, and 168 hours after the third dose. However, these ADAs did not reduce the total inducible IL-21 prodrug exposure (AUCINF) compared to the first dose (5.5 × 10⁵ vs. 5.0 × 10⁵ hr*nM), nor did they reduce the exposure to free IL-21 (1.0 × 10² vs. 1.1 × 10² hr*nM). After the third dose, the Cmax of total inducible IL-21 decreased compared to the first dose (2.0 × 10² vs. 3.3 × 10² nM), but there was no decrease in the Cmax of free IL-21 (0.9 vs. 0.9 nM). Similarly, after the third dose, the exposure (AUC) of total inducible IL-21 decreased compared to the first dose (7.9 × 10³ vs. 2.0 × 10⁴ hr*nM), but there was no decrease in the exposure of free IL-21 (1.6 × 10² vs. 1.4 × 10² hr*nM). [Figure 56-2]Graphs A-D show the time-course exposure to total inducible IL-21 prodrugs (A and C) and free human IL-21 (B and D) in serum (A and B) and tumors (C and D) over 168 hours after a single dose and three doses of WW50387 / WW50394, respectively. In all test animals, an ADA response was evident from the acceleration of IL-21 prodrug clearance at 72, 96, 120, and 168 hours after the third dose. However, these ADAs did not reduce the total inducible IL-21 prodrug exposure (AUCINF) compared to the first dose (5.5 × 10⁵ vs. 5.0 × 10⁵ hr*nM), nor did they reduce the exposure to free IL-21 (1.0 × 10² vs. 1.1 × 10² hr*nM). After the third dose, the Cmax of total inducible IL-21 decreased compared to the first dose (2.0 × 10² vs. 3.3 × 10² nM), but there was no decrease in the Cmax of free IL-21 (0.9 vs. 0.9 nM). Similarly, after the third dose, the exposure (AUC) of total inducible IL-21 decreased compared to the first dose (7.9 × 10³ vs. 2.0 × 10⁴ hr*nM), but there was no decrease in the exposure of free IL-21 (1.6 × 10² vs. 1.4 × 10² hr*nM).

[0162] [Figure 57] Graphs A and B show the time course of IFNγ levels after a single administration of WW50387 / WW50394. Serum IFNγ levels are near the lower limit of quantification in serum, but they increase over time in tumors, peaking at 1028 pg / mL 96 hours after treatment.

[0163] [Figure 58] The increase in pSTAT3 expression relative to total STAT3 in tumors after a single dose (square) or three doses (triangle) of WW50387 / WW50394 is shown compared to the PBS control (circle). The relative level of pSTAT3 peaked at 5.8 ECL units / protein (g) 24 hours after a single dose of WW50387 / WW50394 and at 11.8 ECL units / protein (g) 12 hours after the third dose.

[0164] [Figure 59] The images show multiplex immunofluorescence (M-IF) stained MC38 tumors excised from animals treated with a vehicle or WW50387 / WW50394. Tumors were collected 10 days after the initial dose. Tumors were fixed in formalin, embedded in paraffin, and sections for staining were obtained. After antigen recovery, staining was performed using the Lunaphore automated M-IF COMET® system. Different markers, namely DAPI, CD3, CD4, CD8, CD11b, CD11c, CD103, F4 / 80, and granzyme B (GZMB), were used for staining. Specific cell populations were identified using combinations of these different markers. DAPI stains the nucleus, CD3+CD8+ (CTL), CD3+CD8+GZMB+ (activated CTL), CD3+CD4+ (CD4+ T cells), F4 / 80 (macrophages), CD11b (myeloid cells), and CD11c+CD103+ (cross-presented DCs). The data show increased immune infiltration in tumors derived from WW50387 / WW50394 treated animals, including activated CTLs (enriched within the highlighted white box). The presence of immune hubs or lymphocyte aggregates is observed only in tumors derived from animals treated with WW50387 / WW50394.

[0165] [Figure 60] Graphs A and B show the results of analyzing extended half-life IL-2 and extended half-life IL-21 in the Renca mouse tumor model. Graph A shows the tumor volume over time in mice treated with extended half-life IL-2 (WW0177), extended half-life IL-21 (WW50008), or a vehicle. Graph B shows the average body weight of the treated animals in the data shown in A. Each line in the plot represents the average body weight of the group over time.

[0166] [Figure 61]A and B show the results of TIL analysis of Renca tumors treated with extended half-life IL-2 (WW0177), extended half-life IL-21 (WW50008), or a vehicle. A is a graph showing the abundance of CD8+ T cells in tumor tissue over time from mice treated with WW0177, WW50008, or a vehicle. B is a pie chart showing the pluripotency of cytokine production by CD8+ T cells in tumors from mice treated with extended half-life IL-2 (WW0177), extended half-life IL-12 (WW50008), or a vehicle (i.e., the percentage of cells expressing 0 to 5 effector molecules: IFNγ, TNF, GzmA, GzmB, and Prf1).

[0167] [Figure 62A] A-I show that IL-21 activates type I IFN signaling in the TME. A is a graph representing identified pathways that were significantly enriched with p-values ​​< 0.05 and enrichment levels greater than 3-fold. EMT-6 tumor-bearing mice were randomly assigned to treatment groups (day 0), administered IL-21-HLE or IL-2-HLE twice weekly for 2 weeks, and tumors were collected for bulk RNA nanostring analysis. Pathway analysis was performed for differentially expressed genes using Partek software. B and C are graphs showing individual tumor volume over time in EMT-6 tumor-bearing mice randomly assigned to treatment groups (day 0). Mice were administered anti-IFNAR1 or isotype control a total of 8 times every 3 days. Mice were administered IL-21-HLE or vehicle twice weekly for 2 weeks. Tumors and body weight were measured twice weekly. D-I are graphs showing normalized counts of specific genes identified in type I IFN-related pathways. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data demonstrate the inhibitory effect of IL-21-induced specific activation of type I IFN genes and blocking of type I IFN signaling on the antitumor efficacy of IL-21. [Figure 62B]A-I show that IL-21 activates type I IFN signaling in the TME. A is a graph representing identified pathways that were significantly enriched with p-values ​​< 0.05 and enrichment levels greater than 3-fold. EMT-6 tumor-bearing mice were randomly assigned to treatment groups (day 0), administered IL-21-HLE or IL-2-HLE twice weekly for 2 weeks, and tumors were collected for bulk RNA nanostring analysis. Pathway analysis was performed for differentially expressed genes using Partek software. B and C are graphs showing individual tumor volume over time in EMT-6 tumor-bearing mice randomly assigned to treatment groups (day 0). Mice were administered anti-IFNAR1 or isotype control a total of 8 times every 3 days. Mice were administered IL-21-HLE or vehicle twice weekly for 2 weeks. Tumors and body weight were measured twice weekly. D-I are graphs showing normalized counts of specific genes identified in type I IFN-related pathways. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data demonstrate the inhibitory effect of IL-21-induced specific activation of type I IFN genes and blocking of type I IFN signaling on the antitumor efficacy of IL-21. [Figure 62C]A-I show that IL-21 activates type I IFN signaling in the TME. A is a graph representing identified pathways that were significantly enriched with p-values ​​< 0.05 and enrichment levels greater than 3-fold. EMT-6 tumor-bearing mice were randomly assigned to treatment groups (day 0), administered IL-21-HLE or IL-2-HLE twice weekly for 2 weeks, and tumors were collected for bulk RNA nanostring analysis. Pathway analysis was performed for differentially expressed genes using Partek software. B and C are graphs showing individual tumor volume over time in EMT-6 tumor-bearing mice randomly assigned to treatment groups (day 0). Mice were administered anti-IFNAR1 or isotype control a total of 8 times every 3 days. Mice were administered IL-21-HLE or vehicle twice weekly for 2 weeks. Tumors and body weight were measured twice weekly. D-I are graphs showing normalized counts of specific genes identified in type I IFN-related pathways. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data demonstrate the inhibitory effect of IL-21-induced specific activation of type I IFN genes and blocking of type I IFN signaling on the antitumor efficacy of IL-21. [Figure 62D]A-I show that IL-21 activates type I IFN signaling in the TME. A is a graph representing identified pathways that were significantly enriched with p-values ​​< 0.05 and enrichment levels greater than 3-fold. EMT-6 tumor-bearing mice were randomly assigned to treatment groups (day 0), administered IL-21-HLE or IL-2-HLE twice weekly for 2 weeks, and tumors were collected for bulk RNA nanostring analysis. Pathway analysis was performed for differentially expressed genes using Partek software. B and C are graphs showing individual tumor volume over time in EMT-6 tumor-bearing mice randomly assigned to treatment groups (day 0). Mice were administered anti-IFNAR1 or isotype control a total of 8 times every 3 days. Mice were administered IL-21-HLE or vehicle twice weekly for 2 weeks. Tumors and body weight were measured twice weekly. D-I are graphs showing normalized counts of specific genes identified in type I IFN-related pathways. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data demonstrate the inhibitory effect of IL-21-induced specific activation of type I IFN genes and blocking of type I IFN signaling on the antitumor efficacy of IL-21. [Figure 62E]A-I show that IL-21 activates type I IFN signaling in the TME. A is a graph representing identified pathways that were significantly enriched with p-values ​​< 0.05 and enrichment levels greater than 3-fold. EMT-6 tumor-bearing mice were randomly assigned to treatment groups (day 0), administered IL-21-HLE or IL-2-HLE twice weekly for 2 weeks, and tumors were collected for bulk RNA nanostring analysis. Pathway analysis was performed for differentially expressed genes using Partek software. B and C are graphs showing individual tumor volume over time in EMT-6 tumor-bearing mice randomly assigned to treatment groups (day 0). Mice were administered anti-IFNAR1 or isotype control a total of 8 times every 3 days. Mice were administered IL-21-HLE or vehicle twice weekly for 2 weeks. Tumors and body weight were measured twice weekly. D-I are graphs showing normalized counts of specific genes identified in type I IFN-related pathways. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data demonstrate the inhibitory effect of IL-21-induced specific activation of type I IFN genes and blocking of type I IFN signaling on the antitumor efficacy of IL-21. [Figure 62F]A-I show that IL-21 activates type I IFN signaling in the TME. A is a graph representing identified pathways that were significantly enriched with p-values ​​< 0.05 and enrichment levels greater than 3-fold. EMT-6 tumor-bearing mice were randomly assigned to treatment groups (day 0), administered IL-21-HLE or IL-2-HLE twice weekly for 2 weeks, and tumors were collected for bulk RNA nanostring analysis. Pathway analysis was performed for differentially expressed genes using Partek software. B and C are graphs showing individual tumor volume over time in EMT-6 tumor-bearing mice randomly assigned to treatment groups (day 0). Mice were administered anti-IFNAR1 or isotype control a total of 8 times every 3 days. Mice were administered IL-21-HLE or vehicle twice weekly for 2 weeks. Tumors and body weight were measured twice weekly. D-I are graphs showing normalized counts of specific genes identified in type I IFN-related pathways. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data demonstrate the inhibitory effect of IL-21-induced specific activation of type I IFN genes and blocking of type I IFN signaling on the antitumor efficacy of IL-21. [Figure 62G]A-I show that IL-21 activates type I IFN signaling in the TME. A is a graph representing identified pathways that were significantly enriched with p-values ​​< 0.05 and enrichment levels greater than 3-fold. EMT-6 tumor-bearing mice were randomly assigned to treatment groups (day 0), administered IL-21-HLE or IL-2-HLE twice weekly for 2 weeks, and tumors were collected for bulk RNA nanostring analysis. Pathway analysis was performed for differentially expressed genes using Partek software. B and C are graphs showing individual tumor volume over time in EMT-6 tumor-bearing mice randomly assigned to treatment groups (day 0). Mice were administered anti-IFNAR1 or isotype control a total of 8 times every 3 days. Mice were administered IL-21-HLE or vehicle twice weekly for 2 weeks. Tumors and body weight were measured twice weekly. D-I are graphs showing normalized counts of specific genes identified in type I IFN-related pathways. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data demonstrate the inhibitory effect of IL-21-induced specific activation of type I IFN genes and blocking of type I IFN signaling on the antitumor efficacy of IL-21. [Figure 62H]A-I show that IL-21 activates type I IFN signaling in the TME. A is a graph representing identified pathways that were significantly enriched with p-values ​​< 0.05 and enrichment levels greater than 3-fold. EMT-6 tumor-bearing mice were randomly assigned to treatment groups (day 0), administered IL-21-HLE or IL-2-HLE twice weekly for 2 weeks, and tumors were collected for bulk RNA nanostring analysis. Pathway analysis was performed for differentially expressed genes using Partek software. B and C are graphs showing individual tumor volume over time in EMT-6 tumor-bearing mice randomly assigned to treatment groups (day 0). Mice were administered anti-IFNAR1 or isotype control a total of 8 times every 3 days. Mice were administered IL-21-HLE or vehicle twice weekly for 2 weeks. Tumors and body weight were measured twice weekly. D-I are graphs showing normalized counts of specific genes identified in type I IFN-related pathways. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data demonstrate the inhibitory effect of IL-21-induced specific activation of type I IFN genes and blocking of type I IFN signaling on the antitumor efficacy of IL-21. [Figure 62I]A-I show that IL-21 activates type I IFN signaling in the TME. A is a graph representing identified pathways that were significantly enriched with p-values ​​< 0.05 and enrichment levels greater than 3-fold. EMT-6 tumor-bearing mice were randomly assigned to treatment groups (day 0), administered IL-21-HLE or IL-2-HLE twice weekly for 2 weeks, and tumors were collected for bulk RNA nanostring analysis. Pathway analysis was performed for differentially expressed genes using Partek software. B and C are graphs showing individual tumor volume over time in EMT-6 tumor-bearing mice randomly assigned to treatment groups (day 0). Mice were administered anti-IFNAR1 or isotype control a total of 8 times every 3 days. Mice were administered IL-21-HLE or vehicle twice weekly for 2 weeks. Tumors and body weight were measured twice weekly. D-I are graphs showing normalized counts of specific genes identified in type I IFN-related pathways. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data demonstrate the inhibitory effect of IL-21-induced specific activation of type I IFN genes and blocking of type I IFN signaling on the antitumor efficacy of IL-21.

[0168] [Figure 63A] Images A-C are representative immunofluorescence images showing staining of DAPI and CD45 (A) at various time points in EMT-6 tumor-bearing mice treated with vehicle, IL-21-HLE, or IL-2-HLE; DAPI, CD8, CD4, F4 / 80, CD11b, and granzyme B (B) at day 12; and DAPI, CD8, CD4, TCF1, CD11c, CD103, and NCR1 (C) at day 12. These data demonstrate a long-term increase in immune infiltration in animals treated with IL-21-HLE. CD8+ T cells (CD8) and macrophages (F4 / 80) are particularly noteworthy. [Figure 63B]Images A-C are representative immunofluorescence images showing staining of DAPI and CD45 (A) at various time points in EMT-6 tumor-bearing mice treated with vehicle, IL-21-HLE, or IL-2-HLE; DAPI, CD8, CD4, F4 / 80, CD11b, and granzyme B (B) at day 12; and DAPI, CD8, CD4, TCF1, CD11c, CD103, and NCR1 (C) at day 12. These data demonstrate a long-term increase in immune infiltration in animals treated with IL-21-HLE. CD8+ T cells (CD8) and macrophages (F4 / 80) are particularly noteworthy. [Figure 63C] Images A-C are representative immunofluorescence images showing staining of DAPI and CD45 (A) at various time points in EMT-6 tumor-bearing mice treated with vehicle, IL-21-HLE, or IL-2-HLE; DAPI, CD8, CD4, F4 / 80, CD11b, and granzyme B (B) at day 12; and DAPI, CD8, CD4, TCF1, CD11c, CD103, and NCR1 (C) at day 12. These data demonstrate a long-term increase in immune infiltration in animals treated with IL-21-HLE. CD8+ T cells (CD8) and macrophages (F4 / 80) are particularly noteworthy.

[0169] [Figure 64]Graphs A-E show that IL-21 promotes the increased presence of intratumoral CD8+ T cells expressing effector cytokines. A shows the density of total CD8+ T cells in animal-derived EMT-6 tumors treated with vehicle, IL-21-HLE, or IL-2-HLE. B shows the density of granzyme B+CD8+ T cells in animal-derived EMT-6 tumors treated with vehicle, IL-21-HLE, or IL-2-HLE. C shows the density of perforin+CD8+ T cells in animal-derived EMT-6 tumors treated with vehicle, IL-21-HLE, or IL-2-HLE. D shows the density of TCF1+CD8+ T cells in animal-derived EMT-6 tumors treated with vehicle, IL-21-HLE, or IL-2-HLE. E is a spatial pseudo-color dot plot of CD8+ T cell infiltration, granzyme B+ CD8+ T cells, and stem cell-like (TCF1+) CD8+ T cells in animal-derived EMT-6 tumors treated with vehicle, IL-21-HLE, or IL-2-HLE. These data indicate that IL-21 treatment induces activated and stem cell-like CD8+ T cell infiltration compared to vehicle or IL-2-HLE treatment.

[0170] [Figure 65] Figures A-E demonstrate that IL-21 treatment provides robust antitumor efficacy in immune checkpoint inhibitor (CPI) resistant tumor models. Renca tumor-bearing mice were randomly assigned to treatment groups and administered either a vehicle, IL-21-HLE, or IL-2-HLE twice weekly for two weeks. Figure A shows the mean tumor volume over time. Figure B shows the mean body weight over time. Figures C-E show individual spider plots of tumor volume from animals treated with vehicle, IL-21 HLE, or IL-2-HLE. These data suggest that IL-21 treatment may induce efficacy in animal tumor models that are resistant to CPI treatment and do not show significant efficacy with IL-2-HLE treatment.

[0171] [Figure 66-1]Figures A-F demonstrate that IL-21 treatment results in a persistent intratumoral population of multifunctional (expressing multiple effector molecules) CD8+ T cells. Renca tumor-bearing mice were randomly assigned to treatment groups and administered either vehicle, IL-21-HLE, or IL-2-HLE twice weekly for two weeks. Tumors were collected at the time shown and enzymatically and mechanically dissociated for single-cell suspension. Cells were stained for flow cytometry analysis. Figure A is a graph showing the number of CD8+ T cells per 1 mg of tissue over time. Figure B is a graph showing the ratio of CD8+ T cells to Treg (CD4+Foxp3+ T cells) in the tumor over time. Figures C-E show cytokine production from CD8+ T cells on day 14. Figure F is a pie chart showing the proportion of multifunctional CD8+ T cells in the tumor based on the co-expression of granzyme A, granzyme B, IFNg, TNF, and perforin over time. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data suggest that IL-21-HLE treatment can induce persistent intratumoral invasion of activated CD8+ T cells capable of producing multiple effector cytokines over time. [Figure 66-2]Figures A-F demonstrate that IL-21 treatment results in a persistent intratumoral population of multifunctional (expressing multiple effector molecules) CD8+ T cells. Renca tumor-bearing mice were randomly assigned to treatment groups and administered either vehicle, IL-21-HLE, or IL-2-HLE twice weekly for two weeks. Tumors were collected at the time shown and enzymatically and mechanically dissociated for single-cell suspension. Cells were stained for flow cytometry analysis. Figure A is a graph showing the number of CD8+ T cells per 1 mg of tissue over time. Figure B is a graph showing the ratio of CD8+ T cells to Treg (CD4+Foxp3+ T cells) in the tumor over time. Figures C-E show cytokine production from CD8+ T cells on day 14. Figure F is a pie chart showing the proportion of multifunctional CD8+ T cells in the tumor based on the co-expression of granzyme A, granzyme B, IFNg, TNF, and perforin over time. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data suggest that IL-21-HLE treatment can induce persistent intratumoral invasion of activated CD8+ T cells capable of producing multiple effector cytokines over time. [Figure 66-3]Figures A-F demonstrate that IL-21 treatment results in a persistent intratumoral population of multifunctional (expressing multiple effector molecules) CD8+ T cells. Renca tumor-bearing mice were randomly assigned to treatment groups and administered either vehicle, IL-21-HLE, or IL-2-HLE twice weekly for two weeks. Tumors were collected at the time shown and enzymatically and mechanically dissociated for single-cell suspension. Cells were stained for flow cytometry analysis. Figure A is a graph showing the number of CD8+ T cells per 1 mg of tissue over time. Figure B is a graph showing the ratio of CD8+ T cells to Treg (CD4+Foxp3+ T cells) in the tumor over time. Figures C-E show cytokine production from CD8+ T cells on day 14. Figure F is a pie chart showing the proportion of multifunctional CD8+ T cells in the tumor based on the co-expression of granzyme A, granzyme B, IFNg, TNF, and perforin over time. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data suggest that IL-21-HLE treatment can induce persistent intratumoral invasion of activated CD8+ T cells capable of producing multiple effector cytokines over time. [Figure 66-4]Figures A-F demonstrate that IL-21 treatment results in a persistent intratumoral population of multifunctional (expressing multiple effector molecules) CD8+ T cells. Renca tumor-bearing mice were randomly assigned to treatment groups and administered either vehicle, IL-21-HLE, or IL-2-HLE twice weekly for two weeks. Tumors were collected at the time shown and enzymatically and mechanically dissociated for single-cell suspension. Cells were stained for flow cytometry analysis. Figure A is a graph showing the number of CD8+ T cells per 1 mg of tissue over time. Figure B is a graph showing the ratio of CD8+ T cells to Treg (CD4+Foxp3+ T cells) in the tumor over time. Figures C-E show cytokine production from CD8+ T cells on day 14. Figure F is a pie chart showing the proportion of multifunctional CD8+ T cells in the tumor based on the co-expression of granzyme A, granzyme B, IFNg, TNF, and perforin over time. The p-values ​​represent the results of one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. These data suggest that IL-21-HLE treatment can induce persistent intratumoral invasion of activated CD8+ T cells capable of producing multiple effector cytokines over time.

[0172] [Figure 67]Images A-C are immunofluorescence images of Renca tumor-bearing mice treated with vehicle, IL-21-HLE, or IL-2-HLE. Image A shows immunofluorescence of DAPI and CD45 in mice administered with vehicle, IL-21-HLE, or IL-2-HLE. Image B shows immunofluorescence of DAPI, CD4, CD8, F4 / 80, and CD11c in mice administered with vehicle, IL-21-HLE, or IL-2-HLE. Image C shows immunofluorescence of DAPI, CD4, CD8, F4 / 80, CD11c, and granzyme B in mice administered with vehicle, IL-21-HLE, or IL-2-HLE, with the area enclosed in the white frame in the corresponding image of (B) shown at high magnification. The legend for shading is shown in the figure. These data demonstrate that IL-21-HLE treatment can induce quantifiable intratumoral invasion of different immune populations, particularly CD8+ T cells, CD4+ T cells, and macrophages.

[0173] [Figure 68A] This graph shows the results of analyzing WW50387 / WW50394 and WW51100 / WW50394 in syngeneic MC38 mouse tumor models. The graph shows the results of analyzing the average tumor volume after administration of WW50387 / WW50394 at a dose of 375 μg and WW51100 / WW50394 at doses of 47, 94, 188, 375, 750, 1200, and 1500 μg on days 0, 3, 7, and 10 after the start of treatment. This study was conducted until day 28 after the start of treatment, and tumor volume was measured twice a week during the study. The data indicate that tumor volume was suppressed over time in a dose-dependent manner. The minimum effective dose (MED), defined as the dose at which the group mean tumor growth inhibition (TGI) exceeds 50%, was determined to be 47 μg. The maximum tolerable dose (MTD), defined as the dose at which no subjects are lost due to drug toxicity, was determined to be 750 μg. The therapeutic range for WW51100 / WW50394 in MC38 syngeneic tumor volume was determined to be 16. Arrows indicate administration days. [Figure 68B]This graph shows the results of an analysis of WW50387 / WW50394 and WW51100 / WW50394 in syngeneic MC38 mouse tumor models. The graph shows individual plots for each treatment, clearly demonstrating improved efficacy up to a dose level of 1500 μg. However, there was one outlier in the 1500 μg treatment group, which initially lost control of tumor growth but subsequently recovered, shrinking the tumor to below baseline levels. [Figure 68C] This graph shows the results of the analysis of WW50387 / WW50394 and WW51100 / WW50394 in the syngeneic MC38 mouse tumor model. It also shows a plot of the average body weight of the subjects, expressed in grams, across all treatment groups. [Figure 68D] This graph shows the results of the analysis of WW50387 / WW50394 and WW51100 / WW50394 in the syngeneic MC38 mouse tumor model. It plots the average body weight of the subjects as a percentage change from the initial measurement at the randomization point (day -1) across all treatment groups. Red arrows indicate the day of administration. [Figure 68E] This graph shows the results of the analysis of WW50387 / WW50394 and WW51100 / WW50394 in the syngeneic MC38 mouse tumor model. It plots the percentage change from the initial body weight of each individual subject in all treatment groups.

[0174] [Figure 69] This graph shows the time-course plasma concentration profiles of VHH-human IL-2 fusion proteins (WW10308, WW10311, WW10312, WW10313, WW10314, WW10315, and WW10316) containing llama-derived anti-human serum albumin VHH antibodies. The graph shows that the concentrations of each VHH-IL-2 fusion protein followed similar nonlinear pharmacokinetic (PK) profiles, with a steep slope in the terminal elimination phase. [Modes for carrying out the invention]

[0175] 4. Detailed explanation A. IL-21 polypeptide prodrug This disclosure relates to an inducible IL-21 polypeptide prodrug containing at least one polypeptide chain, and optionally two or more polypeptide chains. The inducible IL-21 prodrug comprises IL-21 or its mutaine, a half-life extender, an IL-21 blocking element, and a protease-cleaving linker. The inducible IL-21 prodrug may be encoded by nucleic acids disclosed herein.

[0176] The inducible IL-21 prodrugs of this disclosure have attenuated IL-21 receptor agonist activity and prolonged circulating half-life. The IL-21 receptor agonist activity is attenuated via blocking elements, which can block all or part of the receptor agonist activity of IL-21 or mutein in the prodrug by non-covalently binding to and / or sterically blocking their binding to the receptor. Half-life prolonging elements may also contribute to attenuation, for example, through steric effects. Half-life prolonging elements can also act as blocking elements that can block all or part of the receptor agonist activity of IL-21. For example, half-life prolonging elements can contribute to blocking when they are adjacent to the IL-21 polypeptide.

[0177] Cleavage of the protease-cleaving linker releases an active form of IL-21 (e.g., more active than the inducible IL-21 prodrug). Typically, the released IL-21 is at least 10 times more active than the inducible IL-21 prodrug. Preferably, the released IL-21 is at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 500 times, at least 1000 times, at least about 10,000 times or more more active than the inducible IL-21 prodrug. Typically, cleavage of the protease-cleaving linker releases IL-21 from both the IL-21 blocking element and the half-life extending element.

[0178] The form of IL-21 released by cleavage of an inducible IL-21 prodrug typically has a short half-life, which is often substantially similar to that of naturally occurring IL-21. Even if the half-life of the inducible IL-21 prodrug is extended, the toxicity is reduced or eliminated because the agonist activity of the circulating inducible IL-21 prodrug is attenuated and the active IL-21 is targeted to the desired site of activity (e.g., the tumor microenvironment).

[0179] Those skilled in the art will understand that the number of polypeptide chains, as well as the positions of elements on the polypeptide chains, half-life extenders, protease-cleaving linkers(s), and blocking elements (and components of such elements, such as VH or VL domains), can vary and are often a matter of design preference. All such variations are encompassed in this disclosure.

[0180] Inducible IL-21 prodrugs may comprise a single polypeptide chain. Typically, a single polypeptide complex comprises an IL-21 polypeptide or its mutain [A], a blocking element [D], a half-life extension element [H], and a protease-cleaving linker [L]. The IL-21[A] polypeptide may be operably linked by the protease-cleaving linker to the blocking element, the half-life extension element, or both. The protease-cleaving linker may comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), particularly ALFKSSFP (SEQ ID NO: 198).

[0181] A single polypeptide complex may include an IL-21 polypeptide [A], a blocking element [D], a half-life extension element [H], and a protease-cleaving linker [L] having the amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), particularly ALFKSSFP (SEQ ID NO: 198). The IL-21[A] polypeptide may be operably linked by the protease-cleaving linker to the blocking element, the half-life extension element, or both the blocking element and the half-life extension element.

[0182] A single polypeptide complex may include an IL-21 polypeptide [A], a blocking element [D], a half-life extension element [H], and a protease-cleaving linker [L] having the amino acid sequence GPAGLYAQ (SEQ ID NO: 195). The IL-21[A] polypeptide may be operably linked by the protease-cleaving linker to the blocking element, the half-life extension element, or both the blocking element and the half-life extension element.

[0183] A single polypeptide complex may include an IL-21 polypeptide [A], a blocking element [D], a half-life extension element [H], and a protease-cleaving linker [L] having the amino acid sequence ALFKSSFP (SEQ ID NO: 198). The IL-21[A] polypeptide may be operably linked by the protease-cleaving linker to the blocking element, the half-life extension element, or both the blocking element and the half-life extension element.

[0184] The IL-21 polypeptide, blocking element, and half-life extender element are operably linked by a protease-cleaving polypeptide. For example, the polypeptide may be any of formulas (I) to (VI).

[0185] [A]-[L1]-[H]-[L2]-[D] (I);

[0186] [D]-[L2]-[H]-[L1]-[A] (II);

[0187] [A]-[L1]-[D]-[L2]-[H] (III);

[0188] [H]-[L2]-[D]-[L1]-[A] (IV);

[0189] [H]-[L1]-[A]-[L2']-[D] (V);

[0190] [D]-[L1]-[A]-[L2']-[H] (VI).

[0191] In formulas (I) to (VI), [A] is an IL-21 polypeptide, [D] is an IL-21 blocking element (e.g., IL-21R or an antibody or antigen-binding fragment), [H] is a half-life extension element, [L1] is a protease-cleavable polypeptide linker, [L2] is a polypeptide linker that may be protease-cleavable, and [L2'] is a protease-cleavable polypeptide linker. [L1] and [L2], or [L1] and [L2'], may optionally have the same or different amino acid sequences and / or protease-cleavage sites (if L2 is protease-cleavable). [H] may also optionally perform blocking. The protease-cleavable linker may include the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), in particular ALFKSSFP (SEQ ID NO: 198).

[0192] The inducible IL-21 prodrugs disclosed herein may contain one half-life extension element and one blocking element, such elements may contain two or more components present on the same polypeptide chain or on different polypeptide chains. As an example, and as disclosed and illustrated herein, the components of the blocking element may be present on separate polypeptide chains. For example, the first polypeptide chain may contain an antibody light chain (VL+CL) or a light chain variable domain (VL), and the second polypeptide may contain an antibody heavy chain Fab fragment (VH+CH1) or a heavy chain variable domain (VH) complementary to the VL+CL or VL on the first polypeptide. In such a scenario, these components may associate in the peptide complex to form an antigen-binding site such as Fab that binds to IL-21 and attenuates IL-21 activity.

[0193] For example, an inducible IL-21 prodrug may have a first polypeptide of formula (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], or Formula XXVIII:[H]-[L3]-[D]-[L1]-[A]. In formulas (X) to (XI) and (XXV) to (XXVIII), [A] is an IL-21 polypeptide, [D] is an 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, and [L3] is a polypeptide linker that may be protease-cleavable. [L1] and [L2] may optionally have the same or different amino acid sequences and / or protease cleavage sites. The inducible IL-21 prodrug may have a second polypeptide comprising an antibody light chain (VL+CL) or light chain variable domain (VL) complementary to VH+CH1 or VH on the first polypeptide. The protease-cleaving linker may include the sequence GPAGLYAQ (sequence number 195) or ALFKSSFP (sequence number 198), in particular ALFKSSFP (sequence number 198).

[0194] The inducible IL-21 prodrugs disclosed herein may include a first polypeptide chain comprising an IL-21 polypeptide linked via a protease-cleavable linker and an IL-21 antibody heavy chain Fab fragment (VH+CH1) or heavy chain variable domain (VH), and a second polypeptide chain linked via a potentially protease-cleavable linker and comprising a half-life extension element and an IL-21 antibody light chain (VL+CL) or light chain variable domain (VL) complementary to VH+CH1 or VH on the first polypeptide.

[0195] The inducible IL-21 prodrugs disclosed herein may include a first polypeptide chain comprising an IL-21 polypeptide linked via a protease-cleaving linker and an antibody light chain (VL+CL) or a light chain variable domain (VL), and a second polypeptide chain comprising a half-life extension element linked via an optional protease-cleaving linker and an IL-21 antibody heavy chain Fab fragment (VH+CH1) or a heavy chain variable domain (VH) complementary to the VL+CL or VL of the first polypeptide chain.

[0196] The inducible IL-21 prodrugs disclosed herein may comprise two blocking elements on two different polypeptide chains. The first polypeptide chain may comprise IL-21R, and the second polypeptide chain may comprise a common gamma chain. In various embodiments, the first polypeptide chain may comprise IL-21R and an antibody heavy chain constant region (i.e., CH1) operably linked via a linker that may be protease-cleavable, and the second polypeptide chain may comprise a common gamma chain and an antibody light chain or fragment thereof operably linked via a linker that may be protease-cleavable. The heavy chain constant region on the first polypeptide chain dimerizes with the light chain (i.e., dimerizes), thereby forming the IL-21R and common gamma chain with the IL-21 binding site. Typically, the first polypeptide chain also comprises IL-21 and a half-life extension element. For example, an inducible IL-21 prodrug may have a first polypeptide chain of formula (XII-XV). Formulas XII:[A]-[L1]-[D]-[L2]-[X]-[L2]-[H], Formula XIII:[H]-[L2]-[X]-[L2]-[D]-[L1]-[A], Formula XIV:[H]-[L1]-[A]-[L1]-[D]-[L2]-[X], and Formula XV:[X]-[L2]-[D]-[L1]-[A]-[L1]-[H]. In formulas (XII) to (XV), [A] is the IL-21 polypeptide, [D] is IL-21R, [X] is the heavy chain constant region (i.e., CH1), [H] is the half-life extension element, [L1] is the protease-cleavable polypeptide linker, and [L2] may be protease-cleavable. [L1] and [L2] may optionally have the same or different amino acid sequences and / or protease cleavage sites (if L2 is protease-cleavable). The inducible IL-21 prodrug may have a second polypeptide chain comprising a common gamma chain and a light chain operably linked via a linker that may be protease-cleavable.

[0197] In various embodiments, the first polypeptide chain may include a common gamma chain and a heavy chain constant region (i.e., CH1) operably linked via a linker that may be protease-cleavable, and the second polypeptide chain may include IL-21R and a light chain operably linked via a linker that may be protease-cleavable. The heavy chain constant region on the first polypeptide chain dimerizes with the light chain (i.e., dimerizes), thereby forming the IL-21R and common gamma chain with the IL-21 binding site.

[0198] The first polypeptide chain may contain IL-21 and optionally contain a half-life extender. The second polypeptide chain may also optionally contain a half-life extender. In some embodiments, only one of the first or second polypeptides contains a half-life extender. If the first polypeptide chain contains a half-life extender, the second polypeptide does not need to contain a half-life extender.

[0199] In various embodiments, the half-life extension element, the blocking element, or at least one component of the half-life extension element or the blocking element lies on a separate polypeptide. For example, the first polypeptide may include 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) operably linked to IL-21 via a protease-cleaving linker. The second polypeptide may include 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) operably linked to the half-life extension element via a linker that may be protease-cleaving. If the antibody heavy chain constant region is on the first polypeptide, the antibody light chain lies on the second polypeptide, and if the antibody heavy chain constant region is on the second polypeptide, the antibody light chain lies on the first polypeptide. A portion of the antibody heavy chain associates with a complementary light chain to form an IL-21 binding site. In one example, the first polypeptide may include a portion of the antibody light chain and IL-21 linked via a protease-cleaving linker. The second polypeptide includes a half-life extension element linked via a potentially protease-cleaving linker and a portion of the antibody heavy chain complementary to the antibody light chain. A portion of the antibody heavy chain associates with a complementary light chain to form an IL-21 binding site. In another example, the first polypeptide may include a portion of the antibody heavy chain and IL-21 linked via a protease-cleaving linker. The second polypeptide may include a half-life extension element linked via a potentially protease-cleaving linker and a portion of the antibody light chain. A portion of the antibody heavy chain associates with a complementary light chain to form an IL-21 binding site.

[0200] As disclosed herein, the inducible IL-21 prodrugs disclosed herein may contain at least one half-life extender and at least one blocking element, such elements may contain two or more components present on the same polypeptide chain or on different polypeptide chains. The first polypeptide chain may contain the first half-life extender, and the second polypeptide chain may contain the second half-life extender. The first half-life extender may contain a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) which may contain one or more amino acid mutations that produce a “knob,” and the second half-life extender may contain a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) which may contain one or more amino acid mutations that produce a “hole.” The first half-life extension element may include a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that may contain one or more amino acid mutations that produce a “hole,” and the second half-life extension element may include a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) that may contain one or more amino acid mutations that produce a “knob.” The first and second half-life extension elements may form a heterodimer (i.e., heterodimerize). The first and second half-life extension elements may form a heterodimer via a linker that may, for example, be a disulfide bond or protease-cleavable.

[0201] The first polypeptide chain and the second polypeptide chain can each contain half-life extenders. For example, the first polypeptide chain may contain a first half-life extender, an IL-21 polypeptide, and a blocking element, and the second polypeptide chain may contain a second half-life extender. For example, the first polypeptide chain may contain a first half-life extender and an IL-21 polypeptide, and the second polypeptide chain may contain a second half-life extender and a blocking element. For example, the first polypeptide chain may contain a first half-life extender and a blocking element, and the second polypeptide chain may contain a second half-life extender and an IL-21 polypeptide.

[0202] For example, an inducible IL-21 prodrug may have a first polypeptide of formula XVI:[D]-[L2]-[H] and a second polypeptide of formula XVII:[A]-[L1]-[H]. For example, an inducible IL-21 prodrug may have a first polypeptide of formula XVIII:[H]-[L2]-[D] and a second polypeptide of formula XIX:[H]-[L1]-[A]. In formulas [XVI] to [XIX], [A] is the IL-21 polypeptide, [D] is a blocking element, [H] is a half-life extension element, [L1] is a protease-cleaving linker, and [L2] is a linker that may be protease-cleaving.

[0203] In various embodiments, each polypeptide chain may comprise one IL-21 polypeptide, one half-life extender, and one blocking element. For example, a first polypeptide chain may comprise an IL-21 polypeptide, a half-life extender, and a blocking element, and a second polypeptide chain may comprise an IL-21 polypeptide, a half-life extender, and a blocking element. For example, an inducible IL-21 prodrug may have formulas [I~VI]:

[0204] [A]-[L1]-[H]-[L2]-[D] (I);

[0205] [D]-[L2]-[H]-[L1]-[A] (II);

[0206] [A]-[L1]-[D]-[L2]-[H] (III);

[0207] [H]-[L2]-[D]-[L1]-[A] (IV);

[0208] [H]-[L1]-[A]-[L2']-[D] (V);

[0209] [D]-[L1]-[A]-[L2']-[H] (VI) One of the following first polypeptide chains, Equations I-VI:

[0210] [A]-[L1]-[H]-[L2]-[D] (I);

[0211] [D]-[L2]-[H]-[L1]-[A] (II);

[0212] [A]-[L1]-[D]-[L2]-[H] (III);

[0213] [H]-[L2]-[D]-[L1]-[A] (IV);

[0214] [H]-[L1]-[A]-[L2']-[D] (V);

[0215] [D]-[L1]-[A]-[L2']-[H] (VI); It may have any one of the following as a second polypeptide chain.

[0216] In formulas (I) to (VI), [A] is an IL-21 polypeptide, [D] is an IL-21 blocking element (e.g., IL-21R or an antibody or antigen-binding fragment), [H] is a half-life extension element, [L1] is a protease-cleavable polypeptide linker, [L2] is a polypeptide linker that may be protease-cleavable, and [L2'] is a protease-cleavable polypeptide linker. [L1] and [L2], or [L1] and [L2'], may optionally have the same or different amino acid sequences and / or protease-cleavage sites (if L2 is protease-cleavable). [H] may also optionally perform blocking.

[0217] The inducible IL-21 prodrugs disclosed herein may also include one or more additional blocking elements. For example, the first polypeptide chain may include one or more additional blocking elements. For example, the second polypeptide chain may include one or more additional blocking elements.

[0218] For example, the inducible IL-21 prodrug is given by formulas (XX)~(XXVI): [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) One of the following first polypeptide chains,

[0219] Formula: XXVII:[H']-[L2]-[D'] or XXVIII:[D']-[L2]-[H'], a second polypeptide chain, It can have.

[0220] In formulas [XX~XXVIII], [A] is an IL-21 polypeptide, [D] is the first IL-21 blocking element, [H] is the first half-life extension element, [D'] is the second IL-21 blocking element, [H'] is the second half-life extension element, [L1] is a protease-cleaving linker, and [L2] is an optional protease-cleaving linker.

[0221] In some embodiments, the inducible IL-21 prodrug comprises (i) IL-21, (ii) an antibody or fragment thereof (especially dAb), e.g., an antibody or fragment that binds to serum albumin (especially HSA), immunoglobulin Fc or fragment thereof, or a half-life extension element selected from serum albumin (especially HSA), (iii) a blocking element selected from IL-21-binding Fab, dAB, scFV, or a congeneral IL-21 receptor or fragment thereof, and a protease-cleavable linker, e.g., a linker cleavable by MMP14 or CTSL1, such as sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), especially ALFKSSFP (SEQ ID NO: 198).

[0222] B. Half-life extension factors Half-life extenders prolong the in vivo half-life of inducible IL-21 prodrugs, resulting in alterations to pharmacokinetics and efficacy. While not theoretically bound, half-life extenders alter the pharmacokinetic properties of inducible IL-21 prodrugs, including changes in tissue distribution, penetration, and diffusion. In some embodiments, half-life extenders may improve tissue targeting, tissue penetration, intra-tissue diffusion, and efficacy compared to proteins without them. While not theoretically bound, an exemplary method of improving polypeptide pharmacokinetics involves expressing elements within the polypeptide chain that bind to receptors that are recycled to the cell membrane rather than being degraded in lysosomes (e.g., FcRn receptors and transferrin receptors on endothelial cells). Three proteins, e.g., human IgG, HSA (or fragments), and transferrin, persist in human serum much longer than their size would suggest, which is related to their ability to bind to receptors that are recycled rather than degraded in lysosomes. These proteins, or fragments that retain FcRn binding ability, are conventionally linked with other polypeptides to extend their serum half-life. HSAs may be directly bound to the pharmaceutical composition or via a short linker. Fragments of HSAs can also be used. HSAs and their fragments can function as both blocking and half-life extending elements. Human IgG and Fc fragments can perform similar functions.

[0223] Serum half-life extending elements may also be antigen-binding polypeptides that bind to proteins with long serum half-lives, such as serum albumin (e.g., HSA) and transferrin. Examples of such polypeptides include antibodies and their fragments, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), antigen-binding fragments (Fab), single-domain antibodies (e.g., heavy-chain variable domain (VH), light-chain variable domain (VL), and camel-type nanobody variable domain (VHH)), and dAbs. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as antikalin, affilin, affibody molecules, affimers, affitins, alpha bodies, avimers, DARPin, finomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin, and the CTLA4 scaffold. Further examples of antigen-binding polypeptides include ligands for a desired receptor, ligand-binding moieties of receptors, lectins, and peptides that bind to or associate with one or more target antigens. Antibodies and their fragments can function as both blocking and half-life-extending elements.

[0224] Half-life extenders can function as both blocking and half-life extenders. For example, a half-life extender (e.g., an HSA-binding polypeptide) can function as a blocking element when adjacent to an IL-21 polypeptide.

[0225] The half-life extenders provided herein are preferably human serum albumin (HSA), antigen-binding polypeptides that bind to human serum albumin, or immunoglobulin Fc or a fragment thereof. The half-life extenders may include an Fc domain or a fragment thereof. For example, the Fc domain may include CH2 and CH3 domains or fragments thereof. For example, the Fc domain may include the constant domain of a heavy chain polypeptide. Typically, the half-life extenders are antibodies or fragments thereof (particularly dAb), for example, antibodies or fragments that bind to serum albumin (particularly HSA), immunoglobulin Fc or a fragment thereof, or serum albumin (particularly HSA).

[0226] Preferably, the half-life extension element is a single-domain antibody fragment (sdAb). The sdAb may include or be derived from SEQ ID NOs. 602-606, 608-653, or their variants. The sdAb may include or be derived from SEQ ID NOs. 602 or its variants. The sdAb may include or be derived from SEQ ID NOs. 603 or its variants. The sdAb may include or be derived from SEQ ID NOs. 604 or its variants. The sdAb may include or be derived from SEQ ID NOs. 605 or its variants. The sdAb may include or be derived from SEQ ID NOs. 606 or its variants. The sdAb may include or be derived from SEQ ID NOs. 608 or its variants. The sdAb may include or be derived from SEQ ID NOs. 609 or its variants. The sdAb may include or be derived from SEQ ID NOs. 610 or its variants. sdAb may contain or be derived from sequence number 611 or its variants. sdAb may contain or be derived from sequence number 612 or its variants. sdAb may contain or be derived from sequence number 613 or its variants. sdAb may contain or be derived from sequence number 614 or its variants. sdAb may contain or be derived from sequence number 615 or its variants. sdAb may contain or be derived from sequence number 616 or its variants. sdAb may contain or be derived from sequence number 617 or its variants. sdAb may contain or be derived from sequence number 618 or its variants. sdAb may contain or be derived from sequence number 619 or its variants. sdAb may contain or be derived from sequence number 620 or its variants. sdAb may contain or be derived from sequence number 621 or its variants. sdAb may contain or consist of sequence number 622 or a variant thereof.sdAb may contain or be derived from sequence number 623 or its variants. sdAb may contain or be derived from sequence number 624 or its variants. sdAb may contain or be derived from sequence number 625 or its variants. sdAb may contain or be derived from sequence number 625 or its variants. sdAb may contain or be derived from sequence number 626 or its variants. sdAb may contain or be derived from sequence number 627 or its variants. sdAb may contain or be derived from sequence number 628 or its variants. sdAb may contain or be derived from sequence number 629 or its variants. sdAb may contain or be derived from sequence number 630 or its variants. sdAb may contain or be derived from sequence number 631 or its variants. sdAb may contain or be derived from sequence number 632 or its variants. sdAb may contain or be derived from sequence number 633 or its variants. sdAb may contain or be derived from sequence number 634 or its variants. sdAb may contain or be derived from sequence number 635 or its variants. sdAb may contain or be derived from sequence number 636 or its variants. sdAb may contain or be derived from sequence number 637 or its variants. sdAb may contain or be derived from sequence number 638 or its variants. sdAb may contain or be derived from sequence number 639 or its variants. sdAb may contain or be derived from sequence number 640 or its variants. sdAb may contain or be derived from sequence number 641 or its variants. sdAb may contain or be derived from sequence number 642 or its variants. sdAb may contain or be derived from sequence number 643 or its variants.sdAb may contain or be derived from sequence number 644 or its variants. sdAb may contain or be derived from sequence number 645 or its variants. sdAb may contain or be derived from sequence number 46 or its variants. sdAb may contain or be derived from sequence number 647 or its variants. sdAb may contain or be derived from sequence number 648 or its variants. sdAb may contain or be derived from sequence number 649 or its variants. sdAb may contain or be derived from sequence number 650 or its variants. sdAb may contain or be derived from sequence number 651 or its variants. sdAb may contain or be derived from sequence number 652 or its variants. sdAb may contain or be derived from sequence number 653 or its variants. sdAb contains, or may contain, an amino acid sequence that is at least approximately 80% identical to any of the amino acid sequences of sequence numbers 602-606 or 608-653, or any of the amino acid sequences of sequence numbers 608-653.For example, sdAb has at least approximately 81% identity, at least approximately 82% identity, and at least approximately 8 It contains, or may consist of, an amino acid sequence having 3% 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. (0001) Compounds 210-221 are specific examples of inducible IL-21 prodrugs containing a half-life extension element which is an sdAb containing or consisting of one of the sequence numbers 602-606 or 608-653 or a variant thereof. Compound 210 contains an sdAb containing sequence number 608. Compound 211 contains an sdAb containing sequence number 609. Compound 212 contains an sdAb containing sequence number 611. Compound 213 contains an sdAb containing sequence number 612. Compound 214 contains an sdAb containing sequence number 625. Compound 215 contains an sdAb containing sequence number 626. Compound 216 contains an sdAb containing sequence number 628. Compound 217 contains an sdAb containing sequence number 632. Compound 218 contains an sdAb containing sequence number 635. Compound 219 contains an sdAb containing sequence number 637. Compound 220 contains an sdAb including SEQ ID NO: 642. Compound 221 contains an sdAb including SEQ ID NO: 644. Compound 222 contains an sdAb including SEQ ID NO: 645. Compound 223 contains an sdAb including SEQ ID NO: 646. Compound 224 contains an sdAb including SEQ ID NO: 618. Compound 225 contains an sdAb including SEQ ID NO: 622. Compound 226 contains an sdAb including SEQ ID NO: 624.

[0227] The half-life extenders disclosed herein may comprise a first half-life extender and a second half-life extender. The first and second half-life extenders can be heterodimerized. The first and second half-life extenders may comprise one or more modifications that facilitate heterodimerization of the first and second half-life extenders. For example, one or more amino acid modifications may be made to the first half-life extender. For example, one or more amino acid modifications may be made to the second half-life extender. The inducible IL-21 prodrugs disclosed herein may comprise a first half-life extender and a second half-life extender, each comprising a CH3 domain. In various embodiments, the half-life extender comprising a CH3 domain is a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or a fragment thereof). The CH3 domains of the first and second half-life extenders may be modified by the "knob-into-hole" technique. See, for example, WO1996 / 027011, Ridgway et al., Protein Eng., 9(7):617-621 (1996), and Merchant et al., Nat. Biotechnol., 16(7):677-681 (1998). By using the knob-into-hole method, the interaction surface of the two CH3 domains is modified, enhancing the heterodimerization of the first and second half-life extension elements, each containing the modified CH3 domain. This occurs by introducing a bulky residue into one of the CH3 domains of the half-life extension element to act as a "knob." Then, a "hole" capable of accepting the knob is formed in the other half-life extension domain to accommodate the bulky residue. Either of the modified CH3 domains may be the "knob," or the other may be the "hole." The heterodimer can be further stabilized by introducing disulfide bridges.

[0228] In some embodiments, the knob-into-hole technique may be used to facilitate heterodimerization between two different half-life extension elements (e.g., between a first half-life extension element and a second half-life extension element).

[0229] In some embodiments, the IL-21 prodrug described herein may comprise two half-life extension elements (i.e., a first half-life extension element and a second half-life extension element). In some embodiments, the first half-life extension element and the second half-life extension element may be linked via a linker. The first half-life extension element and the second half-life extension element may be an Fc domain or a fragment thereof. The first half-life extension element and the second half-life extension element may be an antibody, or a fragment, variant, or derivative thereof.

[0230] The first half-life extension element may include a heavy chain polypeptide or a portion thereof (e.g., the heavy chain constant region of the Fc domain or a fragment thereof) containing one or more amino acid mutations that produce a “knob,” and the second half-life extension element may include a heavy chain polypeptide or a portion thereof (e.g., the heavy chain constant region of the Fc domain or a fragment thereof) containing one or more amino acid mutations that produce a “hole.” Techniques and procedures for introducing holes or knobs into a heavy chain polypeptide or a portion thereof (e.g., the Fc domain or a fragment thereof) are known to those skilled in the art.

[0231] The half-life-extending elements of the inducible IL-21 prodrug extend the half-life of the inducible IL-21 prodrug by at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days or more.

[0232] C. Blocking elements The blocking element may be any element that binds to IL-21 and inhibits the ability of the IL-21 polypeptide to bind to its receptor and / or activate the receptor. The blocking element can inhibit the ability of IL-21 to bind to its receptor and / or activate the receptor, for example, by sterically blocking IL-21 in the prodrug and / or by non-covalently binding to IL-21 in the prodrug. The blocking elements disclosed herein can bind to IL-21. Examples of preferred blocking elements include the full-length congener receptor of IL-21 and its IL-21 binding fragment or mutaine. The congener receptor for IL-21 may be the IL-21R receptor or a portion thereof and / or the common gamma chain or a portion thereof.

[0233] Furthermore, antibodies and their antigen-binding fragments that bind to IL-21 can be used, including antigen-binding fragments (Fab), polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies (e.g., heavy-chain variable domain (VH), light-chain variable domain (VL), and camel-type nanobody variable domain (VHH)), dAbs, etc. Other suitable antigen-binding domains that bind to IL-21 can also be used, which include non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as antikalin, affilin, affibody molecules, affimers, affitins, alpha bodies, avimers, DARPin, finomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block IL-21 from binding to its homologous receptors. Advantageously, such parts can also function as half-life extenders. For example, peptides modified by conjugation to water-soluble polymers such as PEG can sterically inhibit or prevent the binding of cytokines to their receptors. Polypeptides or fragments with long serum half-lives, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin, as well as fragments of such polypeptides and mutains, can also be used.

[0234] Particularly suitable IL-21 blocking elements are single-chain variable fragments (scFv), Fab fragments, or the full-length or IL-21-binding fragments or mutaines (e.g., IL-21R receptor or common gamma chain) of IL-21-related receptors. Typically, IL-21 blocking elements are IL-21-binding Fab, dAb, scFv, or IL-21-related receptors or their fragments.

[0235] This specification also discloses inducible IL-21 polypeptides containing a blocking element having specificity for IL-21 and further containing a half-life extending element.

[0236] A blocking element can contain two or more components present on the same polypeptide chain or on separate polypeptide chains. The first polypeptide chain may contain an antibody light chain (VL+CL) or a light chain variable domain (VL), and the second polypeptide may contain an antibody heavy chain Fab fragment (VH+CH1) or a heavy chain variable domain (VH) complementary to VL+CL or VL on the first polypeptide. In such a scenario, these components can associate within the polypeptide complex to form antigen-binding sites such as Fab that bind to IL-21 and attenuate IL-21 activity.

[0237] D. Protease-cleaving linker As disclosed herein, an inducible IL-21 prodrug comprises one or more linker sequences. The linker sequences help to provide flexibility between polypeptides, for example, blocking elements, so that the activity of IL-21 can be inhibited. The linker may be located between IL-21 subunits, half-life extension elements, and / or blocking elements. As described herein, an inducible IL-21 prodrug comprises a protease-cleaving linker. The protease-cleaving linker may comprise one or more cleavage sites for one or more desired proteases. Preferably, the desired protease is enriched or selectively expressed at a desired target site for IL-21 activity (e.g., the tumor microenvironment). Thus, the inducible IL-21 prodrug is preferentially or selectively cleaved at a target site for desired IL-21 activity.

[0238] Preferred linkers are typically less than about 100 amino acids. Such linkers can be of different lengths, such as 1 amino acid (e.g., Gly) to 30 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 50 amino acids, 1 amino acid to 60 amino acids, 1 to 70 amino acids, 1 to 80 amino acids, 1 to 90 amino acids, and 1 to 100 amino acids. In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acids in length. Preferred linkers are typically about 5 amino acids to about 30 amino acids.

[0239] Preferably, the linker length varies by 2 to 30 amino acids and is optimized for each condition so that the linker does not impose constraints on the conformation or interaction of the linking domain. In preferred embodiments, the linker can be cleaved by a cleaving agent, such as an enzyme. Preferably, the linker contains a protease cleavage site. In some cases, the linker contains one or more cleavage sites. The linker may contain a single protease cleavage site. The linker may also contain two or more protease cleavage sites. For example, two cleavage sites, three cleavage sites, four cleavage sites, five cleavage sites, or more. If the linker contains two or more protease cleavage sites, the cleavage sites may be cleaved by the same protease or different proteases. A linker containing two or more cleavage sites is called a “tandem linker”. The two or more cleavage sites may be arranged in any desired orientation, for example, one cleavage site may be adjacent to another, one cleavage site may overlap with another, or one cleavage site may be followed by another, with an amino acid interposed between the two cleavage sites, but this is not limited to these arrangements.

[0240] Of particular interest in the present invention are disease-specific protease-cleaving linkers. Protease-cleaving linkers that are preferentially cleaved at desired locations within the body, such as the tumor microenvironment, compared to peripheral circulation are also preferred. For example, the rate at which a protease-cleaving linker is cleaved within the tumor microenvironment can be at least about 10 times, at least about 100 times, at least about 1000 times, or at least about 10,000 times faster at a desired location within the body (e.g., within the tumor microenvironment) compared to peripheral circulation (e.g., in plasma).

[0241] Proteases known to be associated with diseased cells or tissues include serine proteases, cysteine ​​proteases, aspartate proteases, threonine proteases, glutamate proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, cathepsins B, C, D, E, G, S, K, L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromericin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase, elastase-like proteases, subtilisin-like proteases, actinidine, bromelain, calpain, caspases, and caspase- 3. Includes, but is not limited to, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, plasmmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, MMP19, MMP20, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAPa), dipeptidyl peptidase, meprin, granzyme, and dipeptidyl peptidase IV (DPPIV / CD26). Proteases capable of cleaving linker amino acid sequences (which may be encoded by chimeric nucleic acid sequences provided herein) may be selected from, for example, the group consisting of prostate-specific antigen (PSA), matrix metalloproteinase (MMP), A Disintigrin and a Metalloproteinase (ADAM), plasminogen activator, cathepsin, caspase, tumor cell surface protease, and elastase.MMPs may be, for example, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 14 (MMP14), matrix metalloproteinase 19 (MMP19), or matrix metalloproteinase 20 (MMP20). In addition, or alternatively, the linker may be cleaved by cathepsins such as cathepsin B, cathepsin C, cathepsin D, cathepsin S, cathepsin E, cathepsin G, cathepsin K, and / or cathepsin L. Preferably, the linker may be cleaved by MMP14 or cathepsin L.

[0242] Table 1 shows proteases useful for linker cleavage and cleavage of the inducible IL-21 polypeptide prodrugs disclosed herein, and Table 2 shows exemplary proteases and their cleavage sites. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0243] Examples of protease-cleaving linkers include, but are not limited to, kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin, elastase, FAP, ADAM, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP) cleaving linkers, plasminogen activator, caspase, tryptase, or tumor cell surface proteases. Specifically, these include MMP9, ADAM, CTSL1, FAPα, and cathepsin. Some preferred protease-cleaving linkers are cleaved by MMPs and / or cathepsins. Linkers cut by MMP14 or CTSL1 are also preferred.

[0244] The linker sequences disclosed herein are typically less than 100 amino acids. Such linker sequences may be of varying lengths, such as 1 amino acid (e.g., Gly) to 30 amino acids, 1 amino acid to 40 amino acids, 1 amino acid to 50 amino acids, 1 amino acid to 60 amino acids, 1 to 70 amino acids, 1 to 80 amino acids, 1 to 90 amino acids, and 1 to 100 amino acids. In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acids in length. Preferred linkers are typically about 5 amino acids to about 30 amino acids.

[0245] Preferably, the linker length varies by 2 to 30 amino acids and is optimized for each condition so that the linker does not impose constraints on the conformation or interaction of the linking domain.

[0246] In some embodiments, the linker is the sequence GPAGLYAQ (sequence number 195);GPAGMKGL (sequence number 196);PGGPAGIG (sequence number 197);ALFKSSFP (sequence number 198);ALFFSSPP (sequence number 199);LAQRLRSS (sequence number 200);LAQKLKSS (sequence number 201);GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (sequence number 202);RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (sequence number 203);KG GGPAGLYAQGPAGLYAQGPAGLYAQGSR (Sequence No. 204);RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (Sequence No. 205);KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (Sequence No. 206);SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (Sequence No. 207);RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (Sequence No. 208);GGGALFKSSFPLAQKLKSSPGGPAGIGGGR (Sequence code 209);RGPGGPAGIGPLAQKLKSSALFKSSFPGGG(Sequence code 210);RGGPLAQKLKSSPGGPAGIGALFKSSFPGK(Sequence code 211);RSGGPAGLYAQALFKSSFPLAQKLKSSGGG(Sequence code 212);GGPLAQKLKSSALFKSSFPGPAGLYAQGGR(Sequence code 213);GGALFKSSFPGPAGLYAQPLAQKLKSSGGK(Sequence code 214);RGGALFKSSFPLAQKLKS Includes SGPAGLYAQGGK (sequence number 215);RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (sequence number 216);SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (sequence number 217);KGGPGGPAGIGPLAQRLRSSALFKSSFPGR (sequence number 218);KSGPGGPAGIGALFFSSPPLAQKLKSSGGR (sequence number 219); or SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG (sequence number 220).

[0247] Certain preferred linkers include the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), particularly ALFKSSFP (SEQ ID NO: 198). Linkers disclosed herein may contain one or more identical or different cleavage motifs or functional variants. A linker may contain one, two, three, four, five, or more cleavage motifs or functional variants. A linker containing 30 amino acids may contain two cleavage motifs or functional variants, three 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 expressing high levels of protease) and is not cleaved or is cleaved with low efficiency in the periphery (e.g., serum). For example, a functional variant retains at least approximately 50%, 55%, 60%, 70%, 80%, 85%, 95%, or more of the cleavage efficiency of a linker containing any one of sequence numbers 195-220 or 447-448.

[0248] Linkers containing more than one cleavage motif may be selected from SEQ ID NOs: 195-201 or 447-448 and combinations thereof. Linkers containing more than one cleavage motif may contain amino acids selected from SEQ ID NOs: 202-220.

[0249] A linker may contain both ALFKSSFP (sequence number 198) and GPAGLYAQ (sequence number 195). A linker may contain two cleavage motifs, each having the sequence GPAGLYAQ (sequence number 195). Alternatively, a linker may contain two more cleavage motifs, each having the sequence ALFKSSFP (sequence number 198). A linker may contain a third cleavage motif, either the same or different.

[0250] In some embodiments, the linker includes an amino acid sequence that is identical to SEQ ID NOs. 195-220 or 447-448 by 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% over the entire length of SEQ ID NOs. 195-220 or 447-448.

[0251] This disclosure also relates to functional variants of linkers including SEQ ID NOs. 195-220 or 447-448. Functional variants of linkers including SEQ ID NOs. 195-220 or 447-448 generally differ from SEQ ID NOs. 195-220 or 447-448 by one or more amino acids (including substitutions, deletions, insertions, or any combination thereof) and substantially retain their ability to be cleaved by proteases.

[0252] A functional variant may contain at least one amino acid substitution, deletion, or insertion compared to a linker containing SEQ ID NOs. 195-220 or 447-448. A functional variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to a linker containing SEQ ID NOs. 195-220 or 447-448. In some preferred embodiments, a functional variant differs from a linker containing SEQ ID NOs. 195-220 by having less than 10, less than 8, less than 5, less than 4, less than 3, less than 2, or 1 amino acid change, e.g., an amino acid substitution or deletion. In other embodiments, a functional variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NOs. 195-220 or 447-448. The amino acid substitutions may be conservative or non-conservative, but are preferably conservative. In some embodiments, the functional variant includes an amino acid sequence that is at least about 90% identical to SEQ ID NOs. 195-220 or 447-448.

[0253] In other embodiments, the functional variant of the linker may include one, two, three, four, or five or more non-conservative amino acid substitutions compared to the linker containing SEQ ID NOs. 195-220 or 447-448. Non-conservative amino acid substitutions may be recognizable to those skilled in the art. The functional variant of the linker preferably includes one or fewer, two or fewer, three or fewer, four or fewer, or five or fewer amino acid deletions.

[0254] The amino acid sequences disclosed in a linker can be described by their relative linear positions within the linker with respect to the cleavage bond. As those skilled in the art will understand, a linker containing an 8-amino acid protease substrate (e.g., SEQ ID NOs: 195-201 or 447-448) contains amino acids at positions P4, P3, P2, P1, P1', P2', P3', and P4', with the cleavage bond located between P1 and P1'. For example, the amino acid positions of a linker containing the sequence GPAGLYAQ (SEQ ID NOs: 195) can be described as follows (SEQ ID NOs: 195): [Table 59]

[0255] The amino acid positions of the linker containing the sequence ALFKSSFP (sequence number 198) can be described as follows (sequence number 198): [Table 60]

[0256] Preferably, the amino acids surrounding the cleavage site (for example, positions P1 and P1' of sequence numbers 195-201 or 447-448) are not substituted.

[0257] In various embodiments, 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 functional variant of SEQ ID NO: 198. As described herein, functional variants of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) contain one or more amino acid substitutions and can substantially retain the ability to be cleaved by proteases. Specifically, the functional variant of GPAGLYAQ (SEQ ID NO: 195) is cleaved by MMP14, and the functional variant of ALFKSSFP (SEQ ID NO: 198) is cleaved by captepsin L (CTSL1). The functional variants also retain the ability to be cleaved efficiently at target sites (e.g., tumor microenvironments expressing high levels of proteases). For example, functional variants of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retain at least approximately 50%, 55%, 60%, 70%, 80%, 85%, 95%, or more of the cleavage efficiency of linkers containing the amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.

[0258] Preferably, the functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) contains one, two, three, four, or five or fewer conservative amino acid substitutions compared to GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). Preferably, the amino acids at positions P1 and P1' are not substituted. The amino acids at positions P1 and P1' in SEQ ID NO: 195 are G and L, and the amino acids at positions P1 and P1' in SEQ ID NO: 198 are K and S.

[0259] A functional variant of GPAGLYAQ (SEQ ID NO: 195) may preferably include one or more of the following: a) arginine amino acid substitution at position P4; b) leucine, valine, asparagine, or proline amino acid substitution at position P3; c) asparagine amino acid substitution at position P2; d) histidine, asparagine, or glycine amino acid substitution at position P1; e) asparagine, isoleucine, or leucine amino acid substitution at position P1'; f) tyrosine or arginine amino acid substitution at position P2'; g) glycine, arginine, or alanine amino acid substitution at position P3'; h) serine, glutamine, or lysine amino acid substitution at position P4'. In functional variants of GPAGLYAQ (SEQ ID NO: 195), the following amino acid substitutions are unfavorable: a) arginine or isoleucine at position P3; b) alanine at position P2; c) valine at position P1; d) arginine, glycine, asparagine, or threonine at position P1'; e) aspartic acid or glutamic acid at position P2'; f) isoleucine at position P3'; g) valine at position P4'. In some embodiments, functional variants of GPAGLYAQ (SEQ ID NO: 195) do not contain amino acid substitutions at position P1 and / or P1'.

[0260] The amino acid substitutions of the functional variant of GPAGLYAQ (SEQ ID NO: 195) preferably include an amino acid substitution at position P4 and / or P4'. For example, the functional variant of GPAGLYAQ (SEQ ID NO: 195) may include leucine at position P4, or serine, glutamine, lysine, or phenylalanine at position P4. Alternatively, the functional variant of GPAGLYAQ (SEQ ID NO: 195) may also include glycine, phenylalanine, or proline at position P4'.

[0261] In some embodiments, amino acid substitutions at position P2 or P2' of GPAGLYAQ (SEQ ID NO: 195) are undesirable.

[0262] In some embodiments, functional variants of GPAGLYAQ (SEQ ID NO: 195) include amino acid sequences selected from SEQ ID NOs: 221-295. Specific functional variants of GPAGLYAQ (SEQ ID NO: 195) include GPGLYAQ (SEQ ID NO: 259) and GPAGLKGA (SEQ ID NO: 249).

[0263] Functional variants of LFKSSFP (SEQ ID NO: 448) preferably include hydrophobic amino acid substitutions. Functional variants of LFKSSFP (SEQ ID NO: 448) preferably include one or more of the following: (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at position P4; (b) lysine, histidine, glycine, proline, asparagine, or 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) position P (f) Histidine, leucine, lysine, alanine, isoleucine, arginine, phenylalanine, asparagine, glutamic acid, or glycine at position P2'; (g) Phenylalanine, leucine, isoleucine, lysine, alanine, glutamine, or proline at position P3'; and phenylalanine, histidine, glycine, serine, valine, histidine, alanine, or asparagine at position P3'.

[0264] The inclusion of aspartic acid and / or glutamic acid in the functional variant of SEQ ID NO: 448 is generally undesirable and should be avoided. In the functional variant of LFKSSFP (SEQ ID NO: 448), the following amino acid substitutions are also undesirable: (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'.

[0265] The amino acid substitutions of the functional variant of LFKSSFP (SEQ ID NO: 448) preferably include amino acid substitutions at positions P4 and / or P1. In some embodiments, amino acid substitutions of the functional variant of LFKSSFP (SEQ ID NO: 448) at position P4' are undesirable.

[0266] In some embodiments, functional variants of LFKSSFP (SEQ ID NO: 448) include amino acid sequences selected from SEQ ID NOs: 296-374. 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).

[0267] The linkers disclosed herein can form a physiologically stable prodrug together with the amino acid sequence (e.g., domain) they link, while also being cleaved by proteases. For example, the linkers are stable in circulation (e.g., not cleaved or cleaved with low efficiency) and cleaved with higher efficiency at a target site (i.e., the tumor microenvironment). Thus, an inducible IL-21 prodrug containing the linkers disclosed herein may, if desired, have a longer circulating half-life and / or reduced circulating biological activity compared to the components of the inducible IL-21 prodrug as separate molecular entities. However, at a desired site (e.g., the tumor microenvironment), the linkers can be efficiently cleaved to release the components linked together by the linkers, restoring or nearly restoring the half-life and biological activity of the components as separate molecular entities.

[0268] The linker preferably remains stable in 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.

[0269] In some embodiments, the linker is cleaved by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or less than 1% in circulation compared to the target site. The linker is also stable in the absence of an enzyme capable of cleaving it. However, upon exposure to a suitable enzyme (i.e., a protease), the linker is cleaved, resulting in the separation of the linked domains.

[0270] E. Examples of inducible IL-21 prodrugs Compounds 1-52 are specific examples of inducible IL-21 prodrugs comprising a single polypeptide chain for use in accordance with this disclosure. Further details of exemplary IL-21 prodrugs are provided in Table 38. [Table 38-1] [Table 38-2]

[0271] Amino acid sequence variants of compounds 1 to 52 that retain attenuated IL-21 activity in the periphery and release active IL-21 by protease cleavage in the tumor microenvironment can also be used in accordance with this disclosure. For example, an inducible IL-21 prodrug may include a polypeptide containing or comprising 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 to 8, 44 to 50, and 72 to 109.

[0272] Compounds 53-222 are specific examples of inducible IL-21 prodrugs comprising two polypeptide chains for use in accordance with this disclosure. Further details of exemplary IL-21 prodrugs are provided in Table 39. [Table 39-1] [Table 39-2] [Table 39-3] [Table 39-4] [Table 39-5] [Table 39-6] [Table 39-7]

[0273] Amino acid sequence variants of compounds 53-222 that retain attenuated IL-21 activity in the periphery and release active IL-21 by protease cleavage in the tumor microenvironment can also be used in accordance with this disclosure.

[0274] For example, an inducible IL-21 prodrug is 1) 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% 1) A first polypeptide containing or comprising an amino acid sequence that is 10% identical to or comprising a second polypeptide containing or comprising 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: 10, 17, 25, 40, 52, 64, 71, 128, 129, 137, 127, 300, 301, 302, 303, 304, 305, and 306.

[0275] F. sdAb that binds to serum albumin This disclosure also relates to single-domain antibody fragments (sdAbs) (such as VHH and VH) having binding specificity to human serum albumin, methods for producing the same, and uses thereof. Such sdAbs may be used to extend the in vivo serum half-life of therapeutic agents (e.g., proteins, peptides, etc.) by engineering fusion proteins and conjugates, for example, using well-known methods, 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.

[0276] Contrary to the widely accepted knowledge in the art that anti-albumin-binding domains that interfere with albumin binding to FcRn are unsuitable for half-life extension applications (e.g., by inclusion in fusion proteins or conjugates with short-lived therapeutic proteins), as described and illustrated herein, we have discovered that sdAbs competing with FcRn (human FcRn) for binding to human serum albumin can effectively extend the half-life (e.g., in vivo serum half-life) of short-lived therapeutic agents (e.g., proteins, peptides).

[0277] The sdAbs of this disclosure include, but are not limited to, heavy chain variable domains (VH), heavy chain variable heavy chain domains (VHH), novel antigen receptor single-domain shark variable domains (VNAR), or light chain variable (VL) domains (e.g., kappa VL domain or lambda VL domain).

[0278] An sdAb comprises one or more complementarity-determining regions (CDRs), typically three CDRs, of an immunoglobulin monovariate domain having binding specificity to serum albumin. The CDRs of the sdAb disclosed herein may be derived from camelid animals. The sdAb may also contain one or more FRs in its humanized form, the FRs may be derived from the human germline and may typically include revertant mutations. The sdAb disclosed herein may contain CDR1, CDR2, and / or CDR3. Preferably, the sdAb disclosed herein comprises CDR1, CDR2, and CDR3.

[0279] sdAb may contain CDR1 with the amino acid sequence of formula (I): X1X2X3X4X5X6AX7X8G (I).

[0280] In formula (I), X1 is glycine or aspartic acid, X2 is glycine, arginine, phenylalanine, leucine, valine, serine, tyrosine, histidine, or threonine, X3 is threonine, glycine, or serine, X4 is phenylalanine, valine, tyrosine, leucine, isoleucine, or arginine, X5 is aspartic acid, serine, threonine, or arginine, X6 is aspartic acid, serine, glycine, or glutamic acid, X7 is arginine, glycine, serine, threonine, methionine, valine, isoleucine, histidine, or asparagine, and X8 is arginine, glycine, leucine, methionine, isoleucine, or valine.

[0281] sdAb may contain CDR2 containing the amino acid of formula (II). AIS X1S X2X3X4T X5Y X6X7X8V KG (II) (Sequence ID 693)

[0282] In formula (II), X1 is isoleucine or alanine, X2 is glycine or threonine, X3 is glutamic acid or glycine, X4 is arginine, threonine, or serine, X5 is asparagine, arginine, or tyrosine, X6 is glycine, arginine, serine, or glutamic acid, X7 is glutamic acid, lysine, aspartic acid, or glutamic acid, and X8 is serine or glutamic acid.

[0283] sdAb may contain a CDR3 having the amino acid sequence of formula (III): A X1G DWYHL X2Q X3X4X5X6X7X8I X9X 10 (III) (Sequence ID 694).

[0284] In formula (III), X1 is serine or alanine, X2 is valine, isoleucine, or threonine, X3 is glycine or glutamic acid, X4 is threonine, glutamic acid, or histidine, X5 is glycine or asparagine, X6 is methionine or asparagine, X7 is serine, alanine, or methionine, X8 is tyrosine or phenylalanine, X9 is arginine or tryptophan, and X 10 It is either asparagine or tyrosine.

[0285] In various embodiments, sdAb includes CDR1 of formula (I), CDR2 of formula (II), and CDR3 of formula (III).

[0286] The sdAb disclosed herein may comprise a CDR1 comprising any one of the amino acid sequences of SEQ ID NOs. 656, 659, 662, 663, and 691 described herein, or a variant of any of the aforementioned sequences. The sdAb disclosed herein may comprise a CDR1 comprising or consisting of SEQ ID NOs. 656 may comprise a CDR1 comprising or consisting of SEQ ID NOs. 662 may comprise a CDR1 comprising or consisting of SEQ ID NOs. 663 may comprise a CDR1 comprising or consisting of SEQ ID NOs. 691 may comprise a CDR1 comprising or consisting of SEQ ID NOs.

[0287] The sdAb disclosed herein may include a CDR2 comprising any one of the amino acid sequences of SEQ ID NOs. 657, 660, 664, 665, 666, and 38 described herein, or a variant of any of the aforementioned sequences. The sdAb disclosed herein may include a CDR2 comprising or consisting of SEQ ID NOs. 657. The sdAb disclosed herein may include a CDR2 comprising or consisting of SEQ ID NOs. 660. The sdAb disclosed herein may include a CDR2 comprising or consisting of SEQ ID NOs. 664. The sdAb disclosed herein may include a CDR2 comprising or consisting of SEQ ID NOs. 665. The sdAb disclosed herein may include a CDR2 comprising or consisting of SEQ ID NOs. 666. The sdAb disclosed herein may include a CDR2 comprising or consisting of SEQ ID NOs. 38.

[0288] The sdAb disclosed herein may include a CDR3 defined by any one of sequence numbers 658, 661, or 667 described herein, or a variant of any of the aforementioned sequences. The sdAb disclosed herein may include a CDR3 containing or consisting of sequence number 658. The sdAb disclosed herein may include a CDR3 containing or consisting of sequence number 661. The sdAb disclosed herein may include a CDR3 containing or consisting of sequence number 667.

[0289] The sdAb disclosed herein may include CDR1 containing any one amino acid sequence of SEQ ID NOs. 656, 659, 662, 663, 691, or a variant of any of the aforementioned sequences, CDR2 containing any one of SEQ ID NOs. 657, 660, 664, 665, 666, 38, or a variant of any of the aforementioned sequences, and CDR3.

[0290] The sdAb disclosed herein may include CDR1 containing one of the amino acid sequences of SEQ ID NOs. 656, 659, 662, 663, and 691, or a variant of any of the aforementioned sequences, CDR3 containing one of SEQ ID NOs. 658, 661, or 667, or a variant of any of the aforementioned sequences, and CDR2.

[0291] The sdAb disclosed herein may include CDR2 containing any one of SEQ ID NOs. 657, 660, 664, 665, 666, 38, or a variant of any of the aforementioned sequences, CDR3 containing any one of SEQ ID NOs. 658, 661, or 667, or a variant of any of the aforementioned sequences, and CDR1.

[0292] Typically, the sdAb disclosed herein has all three CDRs (e.g., CDR1, CDR2, and CDR3).

[0293] The sdAb disclosed herein may include CDR1 containing any one of sequence numbers 656, 659, 662, 663, and 691, or a variant of any of the aforementioned sequences; CDR2 containing any one of sequence numbers 657, 660, 664, 665, 666, and 38, or a variant of any of the aforementioned sequences; and CDR3 containing any one of sequence numbers 658, 661, and 667, or a variant of any of the aforementioned sequences.

[0294] sdAb may include CDR1 containing or consisting of SEQ ID NO: 656 or a variant thereof, CDR2 containing or consisting of SEQ ID NO: 657 or a variant thereof, and CDR3 containing or consisting of SEQ ID NO: 658 or a variant thereof.

[0295] sdAb may include CDR1 containing or consisting of SEQ ID NO: 659 or a variant thereof, CDR2 containing or consisting of SEQ ID NO: 660 or a variant thereof, and CDR3 containing or consisting of SEQ ID NO: 661 or a variant thereof.

[0296] sdAb may include CDR3 containing or consisting of sequence number 667 or a variant thereof, CDR1 containing or consisting of sequence number 662, 691, or 663 or a variant of any of the aforementioned sequences, and CDR2 containing or consisting of sequence number 38, 664, 665, or 666 or a variant of any of the aforementioned sequences.

[0297] For example, sdAb is a) CDR1 containing or consisting of SEQ ID NO: 662 or its variant, CDR2 containing or consisting of SEQ ID NO: 664 or its variant, and CDR3 containing or consisting of SEQ ID NO: 667 or its variant; b) CDR1 containing or consisting of SEQ ID NO: 662, CDR2 containing or consisting of SEQ ID NO: 664, and CDR3 containing or consisting of SEQ ID NO: 667; c) CDR1 containing or consisting of SEQ ID NO: 662 or its variant, CDR2 containing or consisting of SEQ ID NO: 665, and CDR3 containing or consisting of SEQ ID NO: 667; e) CDR1 containing or consisting of SEQ ID NO: 662 or its variant, CDR2 containing or consisting of SEQ ID NO: 666, and CDR3 containing or consisting of SEQ ID NO: 667 or its variant. R3, f) CDR1 containing or consisting of sequence number 662, CDR2 containing or consisting of sequence number 666, and CDR3 containing or consisting of sequence number 667, g) CDR1 containing or consisting of sequence number 662 or its variant, CDR2 containing or consisting of sequence number 38 or its variant, and CDR3 containing or consisting of sequence number 667 or its variant, h) CDR1 containing or consisting of sequence number 662, CDR2 containing or consisting of sequence number 38, and sequence number i) CDR3 containing or consisting of sequence number 667, j) CDR1 containing or consisting of sequence number 691 or its variant, CDR2 containing or consisting of sequence number 664 or its variant, and CDR3 containing or consisting of sequence number 667 or its variant, k) CDR1 containing or consisting of sequence number 691, CDR2 containing or consisting of sequence number 664, and CDR3 containing or consisting of sequence number 667,l) CDR2 containing or consisting of SEQ ID NO: 665 or its variant, and CDR3 containing or consisting of SEQ ID NO: 667 or its variant, m) CDR1 containing or consisting of SEQ ID NO: 691, CDR2 containing or consisting of SEQ ID NO: 665, and CDR3 containing or consisting of SEQ ID NO: 667, n) CDR1 containing or consisting of SEQ ID NO: 691, CDR2 containing or consisting of SEQ ID NO: 666, and CDR3 containing or consisting of SEQ ID NO: 667, o) CDR1 containing or consisting of SEQ ID NO: 691, CDR2 containing or consisting of SEQ ID NO: 666, and CDR3 containing or consisting of SEQ ID NO: 667, p) CDR1 containing or consisting of SEQ ID NO: 691, CDR1 containing or consisting of SEQ ID NO: 38, and CDR2 containing or consisting of SEQ ID NO: 667 or its variant, ) CDR2 consisting of the same, and CDR3 containing or consisting of SEQ ID NO: 667, q) CDR1 containing or consisting of SEQ ID NO: 663 or a variant thereof, CDR2 containing or consisting of SEQ ID NO: 664 or a variant thereof, and CDR3 containing or consisting of SEQ ID NO: 667 or a variant thereof, r) CDR1 containing or consisting of SEQ ID NO: 663, CDR2 containing or consisting of SEQ ID NO: 664, and CDR3 containing or consisting of SEQ ID NO: 667, s) CDR1 containing or consisting of SEQ ID NO: 663 or a variant thereof, CDR2 containing or consisting of SEQ ID NO: 665 or a variant thereof, and CDR3 containing or consisting of SEQ ID NO: 667, t) CDR1 containing or consisting of SEQ ID NO: 663, CDR2 containing or consisting of SEQ ID NO: 665, and CDR3 containing or consisting of SEQ ID NO: 667, u) CDR1 containing or consisting of SEQ ID NO: 663 or a variant thereof, CDR2 containing or consisting of SEQ ID NO: 666 or a variant thereof,and CDR3 containing or consisting of SEQ ID NO: 667 or a variant thereof, v) CDR1 containing or consisting of SEQ ID NO: 663, CDR2 containing or consisting of SEQ ID NO: 666, and CDR3 containing or consisting of SEQ ID NO: 667, w) CDR1 containing or consisting of SEQ ID NO: 663 or a variant thereof, CDR2 containing or consisting of SEQ ID NO: 38 or a variant thereof, and CDR3 containing or consisting of SEQ ID NO: 667, x) CDR1 containing or consisting of SEQ ID NO: 663, CDR2 containing or consisting of SEQ ID NO: 38, and CDR3 containing or consisting of SEQ ID NO: 667.

[0298] Certain preferred sdAb include CDR1 containing or consisting of SEQ ID NO: 662, CDR2 containing or consisting of SEQ ID NO: 38, and CDR3 containing or consisting of SEQ ID NO: 667. Certain preferred sdAb includes CDR1 containing or consisting of SEQ ID NO: 691, CDR2 containing or consisting of SEQ ID NO: 664, and CDR3 containing or consisting of SEQ ID NO: 667. Certain preferred sdAb includes CDR1 containing or consisting of SEQ ID NO: 663, CDR2 containing or consisting of SEQ ID NO: 665, and CDR3 containing or consisting of SEQ ID NO: 667. Certain preferred sdAb includes CDR1 containing or consisting of SEQ ID NO: 663, CDR2 containing or consisting of SEQ ID NO: 666, and CDR3 containing or consisting of SEQ ID NO: 667. Certain preferred sdAb include CDR1 containing or consisting of SEQ ID NO: 656, CDR2 containing or consisting of SEQ ID NO: 657, and CDR3 containing or consisting of SEQ ID NO: 658. Certain preferred sdAb also includes CDR1 containing or consisting of SEQ ID NO: 659, CDR2 containing or consisting of SEQ ID NO: 660, and CDR3 containing or consisting of SEQ ID NO: 661.

[0299] The CDRs of the sdAbs disclosed herein are inserted between amino acid intervals called framework regions (FRs). The sdAbs disclosed herein may include FR1, FR2, FR3, and / or F4. In certain embodiments, the FR regions of the sdAbs disclosed herein contain at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more amino acid sequence homology to human FRs. Typically, FRs may be derived from human germline genes. Exemplary human germline genes include IGHV3-7, IGHV3-9, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16, IGHV3-19, IGHV3-20, IGHV3-21, IGHV3-22, IGHV3-23, IGHV3-25, IGHV3-29, IGHV3-30, IGHV3-32, IGHV3-33, IGHV3-35, IGHV3-36, IGHV3-37, IGHV3-38, IGHV3-41, IGHV3-42, IGHV3-41, IGHV3-42, IGHV3-36, IGHV3-37, IGHV3-38, IGHV3-41, IGHV3-42, IGHV3-41 This includes, but is not limited to, HV3-43, IGHV3-47, IGHV3-38, IGHV3-49, IGHV3-50, IGHV3-52, IGHV3-53, IGHV3-54, IGHV3-57, IGHV3-60, IGHV3-62, IGHV3-63, IGHV3-64, IGHV3-66, IGHV3-69, IGHV3-71, IGHV3-72, IGHV3-73, IGHV3-74, IGHV3-75, IGHV3-76, or IGHV3-79. Certain preferred sdAb contain FRs derived from the human germline genes IGHV3-11 and / or IGHV3-23.

[0300] The sdAb disclosed herein may comprise an FR1 containing any one of the amino acid sequences of SEQ ID NOs. 668, 669, or 31, or a variant of any of the aforementioned sequences. In some cases, the sdAb disclosed herein may comprise an FR1 containing an amino acid sequence having at least 80% identity with any one of the SEQ ID NOs. 668, 669, or 31, or a variant of any of the aforementioned sequences. The sdAb disclosed herein may comprise an FR1 containing or consisting of SEQ ID NOs. 669 may comprise an FR1 containing or consisting of SEQ ID NOs. 31 may comprise an FR1 containing or consisting of SEQ ID NOs.

[0301] The sdAbs disclosed herein may include an FR2 comprising any one amino acid sequence from SEQ ID NOs. 670-674, 678-682, 32, or 33, or a variant of any of the aforementioned sequences. In some cases, the sdAbs disclosed herein may include an FR2 comprising an amino acid sequence having at least 80% identity with any one of SEQ ID NOs. 670-674, 678-682, 32, or 33, or a variant of any of the aforementioned sequences. The sdAbs disclosed herein may include an FR2 comprising or consisting of SEQ ID NOs. 670. 671. 672. 673. 674. The sdAb disclosed herein may include FR2 containing or consisting of SEQ ID NO: 678. The sdAb disclosed herein may include FR2 containing or consisting of SEQ ID NO: 679. The sdAb disclosed herein may include FR2 containing or consisting of SEQ ID NO: 680. The sdAb disclosed herein may include FR2 containing or consisting of SEQ ID NO: 681. The sdAb disclosed herein may include FR2 containing or consisting of SEQ ID NO: 682. The sdAb disclosed herein may include FR2 containing or consisting of SEQ ID NO: 32. The sdAb disclosed herein may include FR2 containing or consisting of SEQ ID NO: 33.

[0302] The sdAb disclosed herein may include an FR3 containing any one of the amino acid sequences of SEQ ID NOs. 675, 676, 34, 35, or 36, or a variant of any of the aforementioned sequences. In some cases, the sdAb disclosed herein may include an FR2 containing an amino acid sequence having at least 80% identity with any one of SEQ ID NOs. 675, 676, 34, 35, or 36, or a variant of any of the aforementioned sequences. The sdAb disclosed herein may include an FR3 containing or consisting of SEQ ID NOs. 675. 676. 34. 35. 36.

[0303] The sdAb disclosed herein may include an FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof. In some cases, the sdAb disclosed herein may include an FR4 containing an amino acid sequence having at least 80% identity with SEQ ID NO: 677 or a variant thereof. The sdAb disclosed herein may include an FR4 containing or consisting of SEQ ID NO: 677.

[0304] The sdAb disclosed herein may include FR1 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, and 31, or a variant of any of the aforementioned sequences, or an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, and 31, or a variant of any of the aforementioned sequences; and FR2 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 670-674, 678-682, 32, and 33, or a variant of any of the aforementioned sequences.

[0305] The sdAb disclosed herein may include FR1 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, or 31, or a variant of any of the aforementioned sequences, or an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, or 31, or a variant of any of the aforementioned sequences; and FR3 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 675, 676, 34, 35, or 36, or a variant of any of the aforementioned sequences.

[0306] The sdAb disclosed herein may include FR1 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, or 31, or a variant of any of the aforementioned sequences, or an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, or 31, or a variant of any of the aforementioned sequences, and FR4 comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NOs. 677 or a variant thereof, or an amino acid sequence having at least 80% identity with SEQ ID NOs. 677 or a variant thereof.

[0307] The sdAb disclosed herein may include FR2 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 670-674, 678-682, 32, or 33, or a variant of any of the aforementioned sequences, or an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 670-674, 678-682, 32, or 33, or a variant of any of the aforementioned sequences; and FR3 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 675, 676, 34, 35, or 36, or a variant of any of the aforementioned sequences.

[0308] The sdAb disclosed herein may include FR2, which comprises an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 670-674, 24-682, 32, or 33, or a variant of any of the aforementioned sequences, or an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 677 or a variant thereof, or an amino acid sequence having at least 80% identity with SEQ ID NOs. 677 or a variant thereof.

[0309] The sdAb disclosed herein may include FR3 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 675, 676, 34, 35, or 36, or a variant of any of the aforementioned sequences, or an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 675, 676, 34, 35, or 36, or a variant of any of the aforementioned sequences; and FR4 comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NOs. 677 or a variant thereof, or an amino acid sequence having at least 80% identity with SEQ ID NOs. 677 or a variant thereof.

[0310] The sdAb disclosed herein comprises FR1 having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, and 31, or a variant of any of the aforementioned sequences, or one of the amino acid sequences of SEQ ID NOs. 668, 669, and 31, or a variant of any of the aforementioned sequences, and one of the amino acid sequences of SEQ ID NOs. 670-674, 678-682, 32, and 33, or a variant of any of the aforementioned sequences, or SEQ ID NOs. 670-674, 678- FR2 may include an amino acid sequence having at least 80% identity with one of 682, 32, or 33, or a variant of any of the aforementioned sequences, and FR3 may include an amino acid sequence having at least 80% identity with one of sequence numbers 675, 676, 34, 35, or 36, or a variant of any of the aforementioned sequences, or one of sequence numbers 675, 676, 34, 35, or 36, or a variant of any of the aforementioned sequences.

[0311] The sdAb disclosed herein may include FR1 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, or 31, or a variant of any of the aforementioned sequences, or an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, or 31, or a variant of any of the aforementioned sequences; FR2 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 670-674, 678-682, 32, or 33, or a variant of any of the aforementioned sequences; and FR4 comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NOs. 677 or a variant thereof, or an amino acid sequence having at least 80% identity with SEQ ID NOs. 677 or a variant thereof.

[0312] The sdAb disclosed herein may include FR1 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, or 31, or a variant of any of the aforementioned sequences, or an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, or 31, or a variant of any of the aforementioned sequences; FR3 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 675, 676, 34, 35, or 36, or a variant of any of the aforementioned sequences; and FR4 comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NOs. 677 or a variant thereof, or an amino acid sequence having at least 80% identity with SEQ ID NOs. 677 or a variant thereof.

[0313] The sdAb disclosed herein may include FR2 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 670-674, 678-682, 32, or 33, or a variant of any of the aforementioned sequences, or an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 670-674, 678-682, 32, or 33, or a variant of any of the aforementioned sequences; FR3 comprising an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 675, 676, 34, 35, or 36, or a variant of any of the aforementioned sequences; and FR4 comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO. 677 or a variant thereof, or an amino acid sequence having at least 80% identity with SEQ ID NO. 677 or a variant thereof.

[0314] Typically, the sdAb disclosed herein has all four FRs (e.g., FR1, FR2, FR3, and FR4). The sdAb disclosed herein has FR1 which includes an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 668, 669, 31, or a variant of any of the aforementioned sequences, or one of the amino acid sequences of SEQ ID NOs. 668, 669, 31, or a variant of any of the aforementioned sequences, and one of the amino acid sequences of SEQ ID NOs. 670-674, 678-682, 32, 33, or a variant of any of the aforementioned sequences, or one of the SEQ ID NOs. 670-674, 678-682, 686, 687, or a variant of any of the aforementioned sequences. It may include FR2 containing an amino acid sequence having at least 80% identity with the above sequence; FR3 containing an amino acid sequence having at least 80% identity with one of the amino acid sequences of SEQ ID NOs. 675, 676, 688, 689, or 690, or a variant of any of the above sequences, or one of the amino acid sequences of SEQ ID NOs. 675, 676, 688, 689, or 690, or a variant of any of the above sequences; and FR4 containing an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NOs. 677 or a variant thereof, or an amino acid sequence having at least 80% identity with SEQ ID NOs. 677 or a variant thereof.

[0315] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 670, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0316] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 671, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0317] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 672, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0318] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 673, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0319] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 669, FR2 containing or comprising SEQ ID NO: 670, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0320] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 669, FR2 containing or comprising SEQ ID NO: 671, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0321] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 669, FR2 containing or comprising SEQ ID NO: 672, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0322] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 669, FR2 containing or comprising SEQ ID NO: 673, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0323] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 674, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0324] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 678, FR3 containing or comprising SEQ ID NO: 683, and FR4 containing or comprising SEQ ID NO: 677.

[0325] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 679, FR3 containing or comprising SEQ ID NO: 683, and FR4 containing or comprising SEQ ID NO: 677.

[0326] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 680, FR3 containing or comprising SEQ ID NO: 683, and FR4 containing or comprising SEQ ID NO: 677.

[0327] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 681, FR3 containing or comprising SEQ ID NO: 683, and FR4 containing or comprising SEQ ID NO: 677.

[0328] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 669, FR2 containing or comprising SEQ ID NO: 24, FR3 containing or comprising SEQ ID NO: 683, and FR4 containing or comprising SEQ ID NO: 677.

[0329] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 669, FR2 containing or comprising SEQ ID NO: 679, FR3 containing or comprising SEQ ID NO: 29, and FR4 containing or comprising SEQ ID NO: 677.

[0330] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 669, FR2 containing or comprising SEQ ID NO: 680, FR3 containing or comprising SEQ ID NO: 683, and FR4 containing or comprising SEQ ID NO: 677.

[0331] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 669, FR2 containing or comprising SEQ ID NO: 681, FR3 containing or comprising SEQ ID NO: 683, and FR4 containing or comprising SEQ ID NO: 677.

[0332] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 682, FR3 containing or comprising SEQ ID NO: 676, and FR4 containing or comprising SEQ ID NO: 677.

[0333] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 669, FR2 containing or comprising SEQ ID NO: 678, FR3 containing or comprising SEQ ID NO: 684, and FR4 containing or comprising SEQ ID NO: 677.

[0334] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 686, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0335] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 687, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0336] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 681, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0337] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 668, FR2 containing or comprising SEQ ID NO: 679, FR3 containing or comprising SEQ ID NO: 675, and FR4 containing or comprising SEQ ID NO: 677.

[0338] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 685, FR2 containing or comprising SEQ ID NO: 686, FR3 containing or comprising SEQ ID NO: 688, and FR4 containing or comprising SEQ ID NO: 677.

[0339] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 685, FR2 containing or comprising SEQ ID NO: 687, FR3 containing or comprising SEQ ID NO: 688, and FR4 containing or comprising SEQ ID NO: 677.

[0340] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 685, FR2 containing or comprising SEQ ID NO: 681, FR3 containing or comprising SEQ ID NO: 688, and FR4 containing or comprising SEQ ID NO: 677.

[0341] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 685, FR2 containing or comprising SEQ ID NO: 679, FR3 containing or comprising SEQ ID NO: 688, and FR4 containing or comprising SEQ ID NO: 677.

[0342] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 685, FR2 containing or comprising SEQ ID NO: 686, FR3 containing or comprising SEQ ID NO: 689, and FR4 containing or comprising SEQ ID NO: 677.

[0343] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 685, FR2 containing or comprising SEQ ID NO: 687, FR3 containing or comprising SEQ ID NO: 689, and FR4 containing or comprising SEQ ID NO: 677.

[0344] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 685, FR2 containing or comprising SEQ ID NO: 681, FR3 containing or comprising SEQ ID NO: 689, and FR4 containing or comprising SEQ ID NO: 677.

[0345] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 685, FR2 containing or comprising SEQ ID NO: 679, FR3 containing or comprising SEQ ID NO: 689, and FR4 containing or comprising SEQ ID NO: 677.

[0346] The sdAb disclosed herein may include FR1 containing or comprising SEQ ID NO: 685, FR2 containing or comprising SEQ ID NO: 682, FR3 containing or comprising SEQ ID NO: 690, and FR4 containing or comprising SEQ ID NO: 677.

[0347] The sdAb disclosed herein may include or consist of any one of the sdAb disclosed in Table 51. [Table 51-1] [Table 51-2] [Table 51-3] [Table 51-4]

[0348] The sdAb disclosed herein comprises, or may comprise, any amino acid sequence of any of SEQ ID NOs. 608-648 or 649-653, or an amino acid sequence having at least about 80% identity with any of SEQ ID NOs. 608-648 or 649-653. For example, sdAb has at least approximately 81% identity, at least approximately 82% identity, at least approximately 83% identity, and at least approximately 83% identity with any of sequence numbers 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, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, or 653. It may contain or consist of an amino acid sequence having approximately 84% identity, at least approximately 85% identity, at least approximately 86% identity, at least approximately 87% identity, at least approximately 88% identity, at least approximately 89% identity, at least approximately 90% identity, at least approximately 91% identity, at least approximately 92% identity, at least approximately 93% identity, at least approximately 94% identity, at least approximately 95% identity, at least approximately 96% identity, at least approximately 97% identity, at least approximately 98% identity, or at least approximately 99% identity.

[0349] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 608, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 608.

[0350] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 609, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 609.

[0351] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 612, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 612.

[0352] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 610, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 610.

[0353] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 611, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 611.

[0354] sdAb contains, or may contain, the amino acid sequence of SEQ ID NO: 613, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 613.

[0355] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 614, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 614.

[0356] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 615, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 615.

[0357] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 616, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 616.

[0358] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 617, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 617.

[0359] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 618, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 618.

[0360] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 619, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 619.

[0361] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 620, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 620.

[0362] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 621, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 621.

[0363] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 622, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 622.

[0364] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 623, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 623.

[0365] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 624, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 624.

[0366] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 625, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 625.

[0367] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 626, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 626.

[0368] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 627, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 627.

[0369] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 628, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 628.

[0370] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 629, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 629.

[0371] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 630, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 630.

[0372] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 631, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 631.

[0373] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 632, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 632.

[0374] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 633, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 633.

[0375] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 634, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 634.

[0376] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 635, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 635.

[0377] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 636, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 636.

[0378] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 637, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 637.

[0379] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 638, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 638.

[0380] sdAb contains, or may contain, the amino acid sequence of SEQ ID NO: 639, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 639.

[0381] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 640, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 640.

[0382] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 641, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 641.

[0383] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 642, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 642.

[0384] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 643, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 643.

[0385] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 644, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 644.

[0386] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 645, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 645.

[0387] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 646, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 646.

[0388] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 647, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 647.

[0389] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 648, or an amino acid sequence having at least approximately 80% identity with SEQ ID NO: 648.

[0390] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 649, or an amino acid sequence having at least 80% identity with SEQ ID NO: 649.

[0391] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 650, or an amino acid sequence having at least 80% identity with SEQ ID NO: 650.

[0392] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 651, or an amino acid sequence having at least 80% identity with SEQ ID NO: 651.

[0393] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 652, or an amino acid sequence having at least 80% identity with SEQ ID NO: 652.

[0394] sdAb contains, or may consist of, the amino acid sequence of SEQ ID NO: 653, or an amino acid sequence having at least 80% identity with SEQ ID NO: 653.

[0395] The sdAbs disclosed herein include functional variants with different amino acid sequences compared to, for example, any one of SEQ ID NOs. 609-648 or 649-653. The “functional variants” of the sdAb retain the ability to bind to serum albumin.

[0396] The sdAb disclosed herein may include one or more amino acid substitutions. The amino acid substitutions may be conservative or non-conservative, but are preferably conservative. Conservative amino acid substitutions are generally carried out according to Table 52 below. [Table 52]

[0397] As described herein, the sdAb of this disclosure may include a CDR1 comprising or consisting of a variant of SEQ ID NOs. 656, 659, 662, 663, or 691 or any of the aforementioned sequences. Variants of SEQ ID NOs. 656, 659, 662, 663, or 691 have at least about 80% or at least about 90% amino acid sequence identity with SEQ ID NOs. 656, 659, 662, 663, or 691, and variants of SEQ ID NOs. 656, 659, 662, 663, or 691 may contain one or two amino acid substitutions. Since amino acid substitutions in the CDR (i.e., CDR1, CDR2, and / or CDR3) can affect binding to serum albumin, it is preferable that any such substitutions are conservative substitutions. One or more amino acids in the CDR may be replaced, for example, with human germline amino acids if it is desirable to reduce binding affinity.

[0398] As described herein, the sdAb of this disclosure may include a CDR2 comprising or consisting of a variant of SEQ ID NO: 657, 660, 664, 665, 666, or 692 or any of the aforementioned sequences. The variants of SEQ ID NO: 657, 660, 664, 665, 666, or 692 have at least about 80%, at least about 85%, or at least about 90% amino acid sequence identity with SEQ ID NO: 657, 660, 664, 665, 666, or 692, and the variants of SEQ ID NO: 657, 660, 664, 665, 666, or 692 may comprise one, two, or three amino acid substitutions.

[0399] As described herein, the sdAb of this disclosure may include a CDR3 comprising or consisting of a variant of SEQ ID NO: 658, 661, or 667 or any of the aforementioned sequences. The variant of SEQ ID NO: 658, 661, or 667 has at least about 80%, at least about 85%, or at least about 90% amino acid sequence identity with SEQ ID NO: 658, 661, or 667, and the variant of SEQ ID NO: 658, 661, or 667 may contain one, two, or three amino acid substitutions.

[0400] As described herein, the sdAbs of this disclosure may contain FRs, and these sequences may generally contain amino acid substitutions without causing loss of binding activity. As described herein, the sdAbs of this disclosure may contain or consist of a variant of SEQ ID NOs. 13, 14, or 31 or any of the aforementioned sequences. Variants of SEQ ID NOs. 658, 661, or 667 have at least about 80%, at least about 85%, at least about 90%, or at least about 95% amino acid sequence identity with SEQ ID NOs. 658, 661, or 667.

[0401] As described herein, the sdAb of this disclosure may include or comprise FR2 comprising a variant of SEQ ID NOs. 670-674, 678-682, 686, or 687 or any of the aforementioned sequences. The variants of SEQ ID NOs. 670-674, 678-682, 686, or 687 have at least about 80%, at least about 85%, at least about 90%, or at least about 95% amino acid sequence identity with SEQ ID NOs. 670-674, 678-682, 686, or 687.

[0402] As described herein, the sdAb of this disclosure may include or comprise an FR3 comprising a variant of SEQ ID NO: 675, 676, 683, 684, 688, 689, or 690 or any of the aforementioned sequences. Variants of SEQ ID NO: 675, 676, 683, 684, 688, 689, or 690 have at least about 80%, at least about 85%, at least about 90%, or at least about 95% amino acid sequence identity with SEQ ID NO: 675, 676, 683, 684, 688, 689, or 690.

[0403] As described herein, the sdAb of this disclosure may include or comprise FR4 comprising SEQ ID NO: 677 or a variant thereof. Variants of SEQ ID NO: 677 have at least about 80%, at least about 85%, at least about 90%, or at least about 95% amino acid sequence identity with SEQ ID NO: 677.

[0404] An sdAb disclosed herein, comprising any one amino acid sequence of SEQ ID NOs. 608-648 or 649-653, may contain any one of the following amino acids: 1) the amino acid at position 678 may be selected from alanine or serine; 2) the amino acid at position 44 may be selected from glutamic acid, glycine, or aspartic acid; 3) the amino acid at position 45 may be selected from arginine or leucine; 4) the amino acid at position 78 may be selected from asparagine, serine, threonine, or leucine; or 5) the amino acid at position 79 may be selected from valine, leucine, or phenylalanine.

[0405] The sdAbs disclosed herein are particularly suitable for use in the preparation of fusion polypeptides and conjugates having an extended serum half-life, for example. The fusion polypeptide may comprise a continuous polypeptide chain containing the sdAb disclosed herein and a target amino acid sequence, typically a therapeutic protein or peptide. The sdAb can be fused to the target amino acid sequence directly or indirectly, for example, via an amino acid linker. Optionally, the fusion polypeptide can associate with one or more other polypeptides to form a functional polypeptide complex. For example, if it is desired to obtain a half-life extended protein that is a heterodimer, a fusion polypeptide containing the sdAb disclosed herein and one member of the heterodimer may be prepared and associated with the other member of the heterodimer to form a functional polypeptide complex.

[0406] In non-limiting examples, the fusion polypeptide is of formula V or VI: [A1]-[L1]-[D1] (V) [D1]-[L1]-[A1] (VI) It can be represented by, In the formula, A1 is an sdAb disclosed herein, L1 is a linker connecting or ligating A1 to D1, or is absent, and D1 is the amino acid sequence of interest.

[0407] The target amino acid sequence may be any desired amino acid sequence. Examples of the target amino acid sequence include, but are not limited to, cytokines. Preferably, the cytokine is IL-21 and / or IL-2. Preferably, the cytokine is IL-21. Preferably, the cytokine is IL-2.

[0408] The exemplary fusion polypeptides and conjugates disclosed herein are half-life extended cytokines. A half-life extended cytokine may be a fusion polypeptide or complex comprising an sdAb or non-immunoglobulin format disclosed herein, a cytokine polypeptide, and optionally a linker (e.g., a cleavable or non-cleavable linker). Cytokines of any purpose may be suitable for inclusion in the half-life extended cytokines of this disclosure. Exemplary cytokines include IL-21 and / or IL-2. Preferably, the cytokine is IL-21. Preferably, the cytokine is IL-2.

[0409] The half-life extended cytokine may further comprise the linker described herein. The linker may be a cleavage linker as disclosed herein. Preferably, the cleavage linker is a protease-cleavage linker. The desired protease activity is concentrated or selectively present at the desired target site of cytokine activity (e.g., the tumor microenvironment). The linker is preferentially or selectively cleaved at the target site.

[0410] The exemplary half-life extended IL-2 sequences disclosed herein may comprise any one of the amino acid sequences of SEQ ID NOs. 696 to 699. For example, the half-life extended IL-2 sequence may comprise SEQ ID NOs. 697. 698. 699.

[0411] This disclosure also relates to conjugates. Similar to the fusion polypeptides described herein, the conjugates comprise the sdAb and the target amino acid sequence described herein. In the conjugates of this disclosure, the sdAb is covalently bonded to the target amino acid sequence directly or indirectly. The sdAb may be conjugated to the target amino acid sequence at its C-terminus, N-terminus, or internally, for example, via the amino acid side chains of the amino acids in the target amino acid sequence. Many suitable linkers and functional groups are known in the art and can be used to prepare the conjugates described herein. For example, homobifunctional or heterobifunctional crosslinkers can be used to link the sdAb disclosed herein to the target amino acid sequence, for example, via the amine group of the sdAb and the carboxyl group of the target amino acid sequence. In some embodiments, the sdAb is indirectly conjugated to the target amino acid sequence via a linker comprising a peptide or polypeptide amino acid sequence. Such linkers may be incapable of cleavage, or they may be cleavable by, for example, a protease (e.g., a protease having activity related to disease cells or tissues as disclosed herein).

[0412] The sdAb and the target amino acid sequence in the fusion polypeptide and conjugate of this disclosure may be linked to each other directly or indirectly. The sdAb and the target amino acid sequence may be linked via a suitable amino acid, peptide, or polypeptide linker. Such linker sequences may be naturally occurring or non-natural sequences. Preferably, the linker sequence is a non-natural sequence.

[0413] The sdAb and the target amino acid sequence may be linked by a non-cleaving linker. Preferably, the sdAb and the target amino acid sequence are linked by a cleaving linker. The cleaving linker may include one or more cleavage sites for one or more desired proteases. Preferably, the desired protease activity is concentrated or selectively expressed at a desired target site of the target amino acid sequence, such as a desired site of cytokine activity (e.g., tumor microenvironment). The linker is preferentially or selectively cleaved at the target site. Suitable protease-cleaving linkers are described herein.

[0414] The fusion polypeptide is a) SEQ ID NO: 608 or its variant, b) SEQ ID NO: 609 or its variant, c) SEQ ID NO: 610 or its variant, d) SEQ ID NO: 611 or its variant, e) SEQ ID NO: 612 or its variant, f) SEQ ID NO: 613 or its variant, g) SEQ ID NO: 614 or its variant, h) SEQ ID NO: 615 or its variant, i) SEQ ID NO: 616 or its variant, j) SEQ ID NO: 617 or its variant, k) SEQ ID NO: 618 or its variant, l) SEQ ID NO: 619 or its variant, m) SEQ ID NO: 620 or its variant, n) SEQ ID NO: 621 or its variant, o) SEQ ID NO: 622 or its variant, o) SEQ ID NO: 623 or its variant, p) SEQ ID NO: 624 or its variant, q) SEQ ID NO: 625 or its variant, r) SEQ ID NO: 626 or its variant, s) SEQ ID NO: 627 or its variant, t) SEQ ID NO: 628 or its variant, u) SEQ ID NO: 62 It may contain or consist of the amino acid sequence of 9 or its variant, v) SEQ ID NO: 630 or its variant, w) SEQ ID NO: 631 or its variant, x) SEQ ID NO: 632 or its variant, y) SEQ ID NO: 633 or its variant, z) SEQ ID NO: 634 or its variant, za) SEQ ID NO: 635 or its variant, zb) SEQ ID NO: 636 or its variant, zc) SEQ ID NO: 637 or its variant, zd) SEQ ID NO: 638 or its variant, ze) SEQ ID NO: 639 or its variant, zf) SEQ ID NO: 640 or its variant, zg) SEQ ID NO: 641 or its variant, zh) SEQ ID NO: 642 or its variant, zi) SEQ ID NO: 643 or its variant, zj) SEQ ID NO: 644 or its variant, zk) SEQ ID NO: 645 or its variant, zl) SEQ ID NO: 646 or its variant, zm) SEQ ID NO: 647 or its variant, or zn) SEQ ID NO: 648 or its variant.

[0415] Additional parts and / or linkers may be present as needed. Further specific application examples of the use of sdAb are disclosed herein. (0002) F. Pharmaceutical Compositions This disclosure provides pharmaceutical compositions comprising the inducible IL-21 prodrug described herein. The pharmaceutical compositions typically further comprise a pharmaceutically acceptable carrier. This disclosure further relates to pharmaceutical compositions comprising the sdAb and non-immunoglobulin format, or the disclosed fusion polypeptide or conjugate. The pharmaceutical formulation or pharmaceutical composition comprises the sdAb described herein, the fusion polypeptide or conjugate described herein as described herein, and typically a pharmaceutically acceptable carrier.

[0416] Pharmaceutical compositions can take various forms, such as liquids and lyophilized materials, and typically contain a suitable pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier (or excipient) is a non-active component of the pharmaceutical composition that is not biologically or otherwise undesirable; that is, it is administered to the subject without causing undesirable biological effects or interacting in a harmful manner with other components of the pharmaceutical formulation or composition in which it is contained. The carrier is often selected to minimize the degradation of the active ingredient and to minimize adverse side effects in the subject.

[0417] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffer solutions such as Ringer's solution, and glucose solutions. Other carriers include sustained-release formulations such as semipermeable matrices of solid hydrophobic polymers containing immunogenic polypeptides. The matrix may be in the form of molded articles such as films, liposomes, or microparticles. Certain carriers may be more preferred depending on the route of administration and the concentration of the composition to be administered. Carriers are suitable for administering chimeric polypeptides or nucleic acid sequences encoding chimeric polypeptides to humans or other subjects.

[0418] Parenteral formulations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including physiological saline and buffer media. Parenteral vehicles include sodium chloride solution, glucose Ringer's solution, glucose and sodium chloride, lactated Ringer's solution, or fixative oils. Intravenous vehicles include liquids and nutritional supplements, electrolyte supplements (such as those based on glucose Ringer's solution), etc. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may be optionally present. Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make it isotonic, but the formulation may be hypertonic or hypotonic as needed. The pH of the solution is generally about 5 to about 8 or about 7 to 7.5.

[0419] Topical formulations include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powder, or oily bases, thickeners, etc., are necessary or desirable as needed.

[0420] Compositions for oral administration include powders or granules, suspensions or solutions in water or a non-aqueous medium, capsules, pouches, or tablets. Depending on the circumstances, thickeners, flavoring agents, diluents, emulsifiers, dispersion aids, or binders may be present.

[0421] The pharmaceutical compositions described herein are suitable for use as pharmaceuticals and may be administered by an appropriate route of administration, for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the route of administration depends on the type of treatment and the type of compound contained in the pharmaceutical composition. The drug regimen is determined by the attending physician and other clinical factors. The dosage for a given patient depends on many factors, including the patient's size, body surface area, age, sex, the specific compound administered, the time and route of administration, the type of therapy, the patient's overall health, and other drugs administered concurrently. "Effective dose" refers to the amount of active ingredient that is sufficient to affect the course and severity of the disease and result in reduction or remission of such condition, and which can be determined using known methods.

[0422] The pharmaceutical compositions described herein (e.g., the inducible IL-21 prodrug, the sdAb, the fusion polypeptide, or conjugate described herein) may be encapsulated in nanoparticles. The nanoparticles may be fullerenes, liquid crystals, liposomes, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. The inducible IL-21 prodrug described herein may be conjugated on the surface of a liposome. The inducible IL-21 prodrug described herein may be encapsulated within the shell of a liposome. The liposome may be a cationic liposome.

[0423] Parenteral administration is generally preferred, but inducible IL-21 prodrugs, sdAbs, fusion polypeptides, conjugates, or nucleic acid sequences encoding inducible IL-21 prodrugs can be administered by vector. There are numerous compositions and methods that can be used to deliver nucleic acid molecules and / or polypeptides to cells, either in vitro or in vivo, for example, via expression vectors. These methods and compositions can be mainly classified into two classes: virus-based delivery systems and non-virus-based delivery systems. Such methods are well known in the art and are readily adaptable for use in the compositions and methods described herein. Using such compositions and methods, cells can be transfected or transduced in vitro or in vivo to generate, for example, cell lines that express (and preferably secrete) encoded chimeric polypeptides, or nucleic acids can be delivered therapeutically.

[0424] As used herein, a plasmid or viral vector is an activator that transports the disclosed nucleic acid into a cell without degradation and contains a promoter that results in the expression of nucleic acid molecules and / or polypeptides in the target cell. Viral vectors include, for example, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, polioviruses, Sindbis, and other RNA viruses (including these viruses with an HIV backbone). Any viral family that shares properties of these viruses that make them suitable for use as vectors is also preferred. Retroviral vectors and methods for their preparation are generally described in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997). The construction of adenoviruses lacking replication ability is described below (Berkner et al., J.Virol.61:1213-20 (1987); Massie et al., Mol.Cell. Biol.6:2872-83 (1986); Haj-Ahmad et al., J.Virol.57:267-74 (1986); Davidson et al., J.Virol.61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72 (1993)). The use and advantages of these viruses as vectors are that while the viruses can replicate within the initial infected cells, they cannot form new initial infectious viral particles, thus limiting the range to which the viruses can spread to other cell types. Recombinant adenoviruses have been shown to achieve high efficiency after being delivered directly in vivo to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and many other tissue sites. Other useful systems include, for example, replicated and host-limited non-replicating vaccinia virus vectors.

[0425] Inducible IL-21 prodrugs and / or nucleic acid molecules can be delivered via virus-like particles. Virus-like particles (VLPs) consist of viral proteins (or more) derived from the structural proteins of a virus. Methods for preparing and using virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0426] The inducible IL-21 prodrugs described herein may be delivered by concentrated viral components (DBs). DBs transport proteins to target cells by membrane fusion. Methods for preparing and using DBs are described, for example, in Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003). The polypeptides provided may be delivered by enucleated aggregates. Methods for preparing and using enucleated aggregates are described in International Publication No. WO2006 / 110728.

[0427] Non-viral-based delivery methods may include expression vectors containing nucleic acid sequences encoding nucleic acid molecules and polypeptides, with the nucleic acids operably linked to expression regulatory sequences. Suitable vector backbones include those routinely used in the art (e.g., plasmids, artificial chromosomes, BACs, YACs, or PACs). Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, Wisconsin), Clonetech (Palo Alto, California), Stratagene (La Jolla, California), and Invitrogen / Life Technologies (Carlsbad, California). Vectors typically contain one or more regulatory regions. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription start sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to produce the inducible IL-21 prodrugs described herein by expression in suitable host cells, such as CHO cells.

[0428] Preferred promoters for controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, such as the genomes of viruses (e.g., polyomas, Simian virus 40 (SV40), adenoviruses, retroviruses, hepatitis B viruses, most preferably cytomegalovirus (CMV)), or heterologous mammalian promoters (e.g., β-actin promoters or EF1α promoters), or hybrid or chimeric promoters (e.g., CMV promoters fused to β-actin promoters). Naturally, promoters derived from host cells or related species are also useful herein.

[0429] An enhancer generally refers to a DNA sequence that functions without being located at a fixed distance from the transcription start site and can be located either at the 5' or 3' end of the transcription unit. Furthermore, enhancers can be located within introns and within the coding sequence itself. They are typically 10–300 base pairs (bp) in length and function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers may also contain response elements that mediate the regulation of transcription. Many enhancer sequences from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin) are known, and eukaryotic virus enhancers are usually used for general expression. Preferred examples include the SV40 enhancer located late at the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer located later at the origin of replication, and adenovirus enhancers.

[0430] Promoter and / or enhancer regions may be inducible (e.g., chemically or physically modulated). Chemically modulated promoters and / or enhancers may be modulated, for example, by the presence of alcohol, tetracycline, steroids, or metals. Physically modulated promoters and / or enhancers may be controlled, for example, by environmental factors (e.g., temperature and light). Optionally, promoter and / or enhancer regions may function as constitutive promoters and / or enhancers to maximize the expression of the region of the transcription unit to be transcribed. In certain vectors, promoter and / or enhancer regions may be cell-type specific in their activity. Optionally, in certain vectors, promoter and / or enhancer regions may be active in all eukaryotic cells, regardless of cell type. Preferred promoters of this type include the CMV promoter, SV40 promoter, β-actin promoter, EF1α promoter, and retroviral long-term repeats (LTRs).

[0431] Vectors may include, for example, origins of replication and / or markers. Marker genes can confer selectable phenotypes (e.g., antibiotic resistance) to cells. Marker products are used to determine whether a vector has been delivered to cells and, once delivered, whether it is being expressed. Examples of selective markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blastosidine. If such selective markers are successfully introduced into mammalian host cells, the transformed mammalian host cells may survive under selective pressure. Other examples of markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. Furthermore, expression vectors may include tag sequences designed to facilitate the manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences (e.g., sequences of GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG® tag (Kodak, New Haven, Connecticut)) are typically expressed as fusions with the encoded polypeptide. Such tags can be inserted at any location within the polypeptide, including carboxyl or amino termini.

[0432] G. Application to treatment This specification also provides methods and uses for treating diseases, disorders, or conditions, comprising administering the inducible IL-21 prodrug described herein to a subject requiring its use. This specification also provides methods and uses for treating diseases, disorders, or conditions, comprising administering the sdAb, or fusion polypeptide or conjugate described herein, to a subject requiring its use. Diseases, disorders, or conditions include, but are not limited to, cancer, inflammatory diseases, immunological disorders, autoimmune diseases, and infectious diseases (i.e., diseases caused by bacteria, viruses, or parasites). Preferably, the disease, disorder, or condition is cancer.

[0433] Any suitable cancer may be treated with the inducible IL-21 prodrugs described herein. Any suitable cancer may be treated with the sdAb provided herein, or the fusion polypeptide provided herein, or the conjugates described herein. Examples of suitable cancers are solid tumors in particular, such as sarcomas and carcinomas. For example, the methods and compositions disclosed herein may be used for acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumor, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, adenoid carcinoma, embryonic tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, ocular cancer, germ cell tumor, gallbladder cancer, gastric cancer. Cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, gliomas, head and neck cancers, hepatocellular carcinomas, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancers, intraocular melanomas, pancreatic islet cell tumors, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancers, oral cavity cancers, liver cancers, lobular carcinomas in situ, lung cancers, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary origin, midline cancer associated with the NUT gene, oral cancers (mouth It can be used to treat cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureteral cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma / thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0434] In certain embodiments, the methods and compositions disclosed herein are used to treat adrenocortical carcinoma, anal carcinoma, appendiceal carcinoma, astrocytoma, basal cell carcinoma, brain tumor, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, bronchial tumor, unknown primary carcinoma, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, adenoma, embryonic tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, ocular cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal carcinoma, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer. Cancer, liver cancer, non-invasive lobular carcinoma, lung cancer, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary origin, midline cancer associated with the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureteral cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer It can be used to treat cancer, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma / thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, non-Hodgkin lymphoma, squamous cell carcinoma of the head and neck, malignant pleural mesothelioma, and Wilms' tumor.

[0435] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, high microsatellite instability or mismatch repair deficiency cancer, high microsatellite instability or mismatch repair deficiency colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial cancer, high tumor mutational load cancer, cutaneous squamous cell carcinoma (cSCC), triple-negative breast cancer (TNBC), urothelial carcinoma, colorectal cancer, or esophageal cancer.

[0436] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat Merkel cell carcinoma (MCC), urothelial carcinoma (UC), renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), triple-negative breast cancer (TNBC), endometrial cancer, cutaneous squamous cell carcinoma (CSCC), basal cell carcinoma (BCC), melanoma, malignant pleural mesothelioma, classical Hodgkin lymphoma (cHL), squamous cell carcinoma of the head and neck (SCCHN), hepatocellular carcinoma (HCC), esophageal squamous cell carcinoma (ESCC), non-squamous non-small cell lung cancer, or nasopharyngeal carcinoma (NPC).

[0437] Preferably, the methods and compositions disclosed herein are used to treat colon cancer, lung cancer, melanoma, renal cell carcinoma, or breast cancer.

[0438] In certain preferred embodiments, the methods and compositions disclosed herein are used to treat melanoma. For example, the methods and compositions disclosed herein can be used to treat melanoma in subjects with unresectable or metastatic melanoma. For another example, the methods and compositions disclosed herein can be used in adjuvant therapy for subjects with melanoma involving lymph node involvement after complete excision.

[0439] In some embodiments, methods are provided herein for enhancing the immune response in subjects requiring enhancement of the immune response by administering an effective amount of the inducible IL-21 prodrug described herein to the subject. Enhancement of the immune response may prevent, delay, or treat the development of cancer, tumors, or viral diseases. In some embodiments, the methods described herein enhance the activity of natural killer cells and T lymphocytes.

[0440] This method may further include the administration of one or more additional agents for treating cancer, such as chemotherapeutic agents (e.g., Adriamycin, Seruvidine, Bleomycin, Alkeran, Verban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisanthren, Noantrone, Tiguanine, Cytaribine, Procalabidine), oncological immunotherapy agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-LAG3, anti-CD47, anti-GD2), cell therapies (e.g., CAR-T, T-cell therapy), and oncolytic viruses. Non-limiting examples of anticancer drugs that may be used include: asbicin; acralbicin; acodazole hydrochloride; acronin; adzeresin; aldesleukin; altretamine; ambomycin; amethantrone acetate; aminoglutethimide; amsacrin; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisanthren hydrochloride; bisnafido dimesylate; bizeresin; bleomycin sulfate; brakinal sodium; bropyrimin; busulfan; kakutinomycin; carsterone; calasemide; carvethymer; carboplatin; carmustine; carbicin hydrochloride; carzeresin; sedefingol; chlorambucil; sirolemycin; cisplatin; cladribine; cristol mesylate; cyclophosphamide; cytarabine; dacarb Zin; Dactinomycin; Daunorubicin hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine mesylate; Diadicone; Docetaxel; Doxorubicin; Dosorbinc hydrochloride; Doroxifene; Doroxifene citrate; Dromostanolone propionate; Duazomycin; Edatrexate; Eflornithine hydrochloride; Ersamitrusine; Enloplatin; Empromate; Epipropidine; Epir hydrochloride Bicin; Elbrozol; Esolubicin hydrochloride; Estramustine; Estramustine sodium phosphate; Etanidazole; Etoposide; Etoposide phosphate; Etoprine; Fadrozol hydrochloride; Fazarabine; Fenretinide; Furoxuridine; Fludarabine phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostoliesin sodium; Gemcitabine; Gemcitabine hydrochloride; Hydroxyurea; Idarubicin hydrochloride;Ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II or rIL2); interferon α-2a; interferon α-2b; interferon α-nl; interferon α-n3; interferon β-I; interferon γ-Ib; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; rialozol hydrochloride; lometrexol sodium; lomustine; loxoxantrone hydrochloride; masopropyl; mytansine; mechloretamine hydrochloride; Megestrol acetate; Melengestrol acetate; Melphalan; Menogalyl; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprin; Metsuredepa; Mitindomide; Mitocalcin; Mitochromin; Mitogiline; Mitomarcin; Mitomycin; Mitospel; Mitotan; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole; Nogaramycin; Ormaplatin; Oxythran; Paclitaxel; Pegaspargase; Periomycin; Pentamustine; Peplomycin sulfate; Perphosphamide; Pipobroman; Piposulfan ; Pyroxantrone hydrochloride; Plicamycin; Promethane; Porfimer sodium; Porphyromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofulin; Ribopurine; Logretimide; Safingol; Safingol hydrochloride; Semustine; Simtrazene; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Slofenucle; Talisomycin; Tecogalan sodium; Tegafur; Teloxant hydrochloride Ron; Temoporfin; Teniposide; Teroxylone; Testolactone; Thiamipurine; Thioguanine; Thiotepa; Thiazofulin; Tirapazamine; Toremifene citrate; Trestron acetate; Trisilibine phosphate; Trimethrexate; Trimethrexate glucuronide; Triptorelin; Tubrozol hydrochloride; Uracil mustard; Uredepa; Bubbreotide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Vinepidine sulfate; Bingricinate sulfate; Vinoleulosine sulfate; Vinorelbine tartrate;Examples include binzolidine sulfate; binzolidine sulfate; borozol; zeniplatin; dinostatin; and zolubicin hydrochloride.

[0441] In some embodiments of the methods described herein, the inducible IL-21 prodrugs described herein are administered in combination with an activating factor for the treatment of a particular disease, disorder, or condition. This disclosure relates to therapeutic combinations of any of the sdAbs described herein, or the fusion polypeptides or conjugates described herein, in combination with one or more additional activating factors for the treatment of a particular disease or condition. Activating factors include, but are not limited to, antibody-based therapies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiotherapy (gamma rays, C rays, and / or radioisotopes, microwaves, UV radiation, etc., with directional delivery), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapies. In some embodiments, the inducible IL-21 prodrugs described herein are administered in combination with antidiarrheal agents, antiemetic agents, analgesics, and / or nonsteroidal anti-inflammatory drugs.

[0442] This disclosure relates to the use of any of the inducible IL-21 prodrugs described herein as one or more additional agents for treating cancer (e.g., lymphoma), such as chemotherapeutic agents (e.g., cyclophosphamide, mechloretamine, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolomid, temozolomid, thiotepa, mitomycin, diaziquan (AZQ), cisplatin, carboplatin, oxaliplatin, procarbazine, hexamethylmelamine, methotrexate, pemetrexed, fluorouracil (e.g., 5-fluorouracil)). This relates to therapeutic combinations of drugs (such as capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nerarabine, cladribine, clofarabine, pentostatin, thioguanine, mercaptopurine, vincristine, vinblastine, vinorelbine, vindesine, vinflunin, paclitaxel, docetaxel, etoposide, teniposide, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, akurarubicin, mitoxantrone, actinomycin, bleomycin, bisanthren, gemcitabine, cytarabine, etc.), tumor immunotherapy agents and immune checkpoint inhibitors (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-LAG3, anti-CD47, anti-GD2), oncolytic viruses, etc.

[0443] The inducible IL-21 prodrugs described herein may be combined with any additional desired anticancer agent. The inducible cytokine prodrugs described herein may be combined with any desired anti-PD-1 antibody or any desired anti-PD-L1 antibody.

[0444] Exemplary anti-PD-1 antibodies that can be combined with the inducible IL-21 prodrugs described herein include AMP-224 (AstraZeneca), 609A (3SBio), 704 (3SBio), 705 (3SBio), ABBV-181 (AbbVie), ADU-1503 / bion-004 (Chinook Therapeutics), AGEN2034 / valstilimab (Agenus), AK103 (Akeso), AK104 (Akeso), AK112 (Akeso), AK123 (Akeso), AMG256 (Amgen), AMG404 (Amgen), ANB030 (AnaptysBio), ANKEBIO anti-PD1 product (Anhui Anke Biotechnology), anti-PD-1 / anti-CD47 (DiNonA), and ASKG915 (Ask Gene Pharmaceuticals), AV-MEL-1 (Aivita Biomedical), BCD-100 (Biocad CJSC), BI754091 (Boehringer Ingelheim), BiCKI-IL-7 (OSE Immunotherapeutics), Boehringer-PD-1-Unknown (Boehringer Ingelheim), BSK-050K01 (Biosion), Canrelizumab (Jiangsu Hengrui) Medicine), CB201 (Crescendo Biologics), CB213 (Crescendo Biologics), CC-90006 (AnaptsBio), Setrelimab (J&J), chPD1 (Kiromic Biopharma), CMAB819 (Mabpharm), CS1003 (CStone Pharmaceuticals), CS17938 (Shenzhen Chipscreen Biosciences), CTX-8371 (Compass CX-072 (CytomX Therapeutics), CX-188 (CytomX Therapeutics), Cipalizumab (Harbin Gloria Pharmaceuticals), DB004 (DotBio), EMB02 (EpimAbBiotherapeutics) GS19 (Suzhou Zelgen Biopharmaceuticals) HLX10 (Shanghai Henlius). Biotech, HX008 (Taizhou HanZhong Pharmaceuticals), HY003 (Juventus Cell Therapy), IBI315 / BH2950 (Innovent Biologics), IBI318 (Innovent Biologics), IBI319 (Innovent Biologics), IMM1802 (ImmuneOnco). Biopharma), IMT200 (TrueBinding), Jemperli / Synthetic (AnaptysBio), JTX-4014 (Jounce Therapeutics) , Keytruda / Backpack (Merck), LBL-006 (Nanjing Leads Biolabs), Libtayo / Backpack-rwlc (Regeneron Pharmaceuticals), LVGN3616 (Lyvgen Biopharma, LXF821 (Novartis), LY01015 (Luye Pharma Group), LY3462817 (Eli Lilly), MCLA-134 (Merus). NV, MEDI5752 (AstraZeneca), NIR178 (Novartis), ONCR-177 (Oncorus), ONO-4685 (Ono Pharmaceutical), Opdivo / Dilute (Ono). Pharmaceutical, MGD019 (MacroGenius), PD1-GDT CAR-T (Kiromic Biopharma). Biotherapeutics) PT-001 (Merck) PT627 (Merck) RB-M1 (Refuge). Biotechnologies) RG6139 (Roche), RG6279 (Roche), RTX-002 (RubrYc).(Therapeutics), Sasanlimab (Pfizer), Servier-PD1xLAG3-Unknown (Servier), SL-279252 / TAK-252 (Shattuck Labs), Sofusa anti-PD1 (Sorrento Therapeutics), Spartalizumab (Novartis), SSI-361 (Lyvgen Biopharma), Sym021 (Servier), Teboterimab (MacroGenics), Tithrelizumab (BeiGene), TSR-075 (AnaptsBio), Tuhura-DO / PD-1-Unknown (Tuhura Biopharma), Tripalimab (Shanghai Junshi Biosciences), Syntilimab (Innovent Biologics), Unicar-CAR-T&PD-1-Unknown (Shanghai Unicar-Therapy Bio-Medicine Technology), Xdivane (Xbrane) This includes, but is not limited to, Biopharma's XmAb20717 (Xencor), XmAb23104 (Xencor), YBL-006 (Y-Biologics), and zimbererimab (Arcus Biosciences).

[0445] Anti-PD-1 antibodies that can be combined with the inducible IL-21 prodrugs described herein are typically approved anti-PD-1 antibodies. Approved anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA), dostallimab (JEMPERLI), semiprimab-rwlc (LIBATYO), nivolumab (OPDIVO), camrelizumab, tisrelizumab, tripalimab, and cintilimab (TYVYT).

[0446] Exemplary anti-PD-L1 antibodies that can be combined with the inducible IL-21 prodrugs described herein include A167 (Sichuan Kelun), ABL501 (ABL Bio), ABL503 (ABL Bio), ABSK041 (Abbisko Therapeutics), ACE1708 (Acepodia), ACE-NK-PDL1 (Acepodia), ADG104 (Adagene), AK106 (Akeso), ALPN-202 (Alpine Immune Sciences), AN4005 (Adlai Nortye Biopharma), BMS-936559 / MDX-1105 (BMS), APL-502 / TQB2450 (Apollomics), Arbutus-PD-L1-Unknown (Arbutus Biopharma), and ASC22 (Ascletis Pharma), ATG-101(Antengene), AVA-004(Avacta Group), AVA021(Avacta Group), AVA027(Avacta Group), AVA-040-100(Avacta Group), AVA04-Vbp(Avacta Group), Bavencio / Avelumab(Merck), BCD-135(Biocad CJSC), BGB-A333 (BeiGene), Bintrafusp alpha / GSK4045154 (Merck), CA-170 / aupm-170 (Dr.Reddy's Laboratories), CCX559 (ChemoCentryx), CDR101 (CDR-Life), cosibelimab (Checkpoint Therapeutics), CTX-8371 (Compass Therapeutics), DiNonA-Solid Tumor-Unknown (DiNonA), DR30207 (Zhejiang Doer Biologics), DuoBody-PD-L1x4-1BB (Ligand Pharmaceuticals), Embafolimab (Alphamab Oncology), EPIM-001 (Elpis Biopharmaceuticals), ES101 (Elpiscience) Biopharma), INBRX-105 (Inhibrx), FAZ053 (Novartis), FS118 (F-starTherapeutics), GB262 (Genor Biopharma), GS-4224 (Gilead), GT900008 (Kintor Pharmaceuticals), GX-P2 (Genexine), Hamni-PS-L1 / CD47 Antibody (Hanmi Pharmaceutical), HBM7015 (HBM). Holdings, HBM9167 (HBM Holdings), HLX20 (Shanghai Henlius Biotech), HTI-1088 (Jiangsu Hengrui Medicine), IBI318 (Innovent Biologics), IBI322 (Innovent Biologics), IBI323 (Innovent Biologics, IGM-7354 (IGM Biosciences), IMC-001 (Sorrento Therapeutics), Imfinzi / Marine (AstraZeneca), IMM25 (ImmuneOnco Biopharma), IMM2502 (ImmuneOnco). Biopharma, IMM2503 (ImmuneOnco Biopharma), IMM2504 (ImmuneOnco Biopharma), INCB86550 (Incyte), IO103 (IO Biotech), JS003 (Shanghai Junshi Biosciences), Jubilant-PD-L1 Antibody (Jubilant Therapeutics, KD033 (Kadmon Holdings), KN046 (Alphamab Oncology), KY1003 (Sanofi), KY1043 (Sanofi), LY3300054 (Eli Lilly), LY3415244 (Eli Lilly)、MRNA-6981(Modern)、MSB2311(Transcenta Holding)、MT-6035(Molecular Templates)、ND021 / NM21-1480(Numab Therapeutics)、OX001R(Oxford). BioTherapeutics) PD-L1ベやBsAb(I-Mab), PD-L1 Boltbody ISAC(Bolt Biotherapeutics), PDL-GEX(GlycotopeGmbH), PMC-122 (PharmAbcine), PMI06 (D&D Pharmatech), Protheragen-RV-scFv-PDL1-unknown (Protheragen), PRS-344 (Pieris Pharmaceuticals), Q-1802 (Merck), RC98 (Yantai Rongchang Pharmaceutical), RV-scFv-PDL1 (Protheragen), SenI_TAAx22P (Hebei Senlang Biotechnology), SHC020 (Nanjing Sanhome Pharmaceutical), Sugemalimab (Ligand Pharmaceuticals), Atezolizumab (Roche), TST005 (Transcenta Holding), TT-01 (Topmunnity Therapeutics), TTX-siPDL1 (TransCode Therapeutics), UniCAR-T-PD-L1(GEMoaB This includes, but is not limited to, monoclonals, Vaximm (VXM10), and YBL-013 (Y-Biologics).

[0447] Anti-PD-L1 antibodies that can be combined with the inducible IL-21 prodrugs described herein are typically approved anti-PD-L1 antibodies. Approved anti-PD-L1 antibodies include, but are not limited to, avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ).

[0448] Anti-CTLA4 antibodies that can be combined with the inducible IL-21 prodrugs described herein are typically approved anti-CTLA4 antibodies. Approved anti-CTLA4 antibodies include, but are not limited to, ipilimumab (YERVOY) and tremelimumab (IMJUDO). Anti-LAG3 antibodies that can be combined with the inducible IL-21 prodrugs are typically approved anti-LAG3 antibodies. An example of an approved anti-LAG3 antibody is relatrimab (OPDUALAG).

[0449] I. Definition All published documents and patents cited in this disclosure are incorporated herein by reference in their entirety. To the extent that any material incorporated by reference conflicts with or is inconsistent with this disclosure, this disclosure shall prevail over any such material. No reference to any document herein constitutes prior art of this disclosure. Ranges of values, where expressed, include embodiments that use any specific value within the range. Furthermore, references to values ​​presented within a range include all values ​​within that range. All ranges include their endpoints and are combinable. Where values ​​are expressed as approximate by the use of the antecedent “about,” it should be understood that a specific value forms another embodiment. References to a specific numerical value include at least that specific value unless the context clearly indicates otherwise. The use of “or” shall mean “and / or” unless the specific context of its use indicates otherwise.

[0450] Various terms relating to aspects of this specification are used throughout this specification and the claims. Unless otherwise stated, such terms shall be given their ordinary meanings in the art. Other terms specifically defined shall be construed in accordance with the definitions presented herein. The techniques and practices described or referenced herein are generally well understood and commonly used by those skilled in the art, such as the widely used molecular cloning methodology described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Unless otherwise specified, practices involving the use of commercially available kits and reagents, where necessary, are generally carried out in accordance with the protocols and conditions defined by the manufacturers.

[0451] In this specification, the singular forms "a," "an," and "the" include the plural form unless the context clearly indicates otherwise. Terms such as "include" and "such as" are intended to convey unrestricted inclusion unless otherwise specified.

[0452] Unless otherwise specified, the terms “at least,” “less than,” and “about,” or similar terms preceding a series or range of elements should be understood to refer to all elements within the series or range. Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein, or can verify them by standard experimentation alone. Such equivalents are intended to be covered by the following claims.

[0453] As used herein, the terms “activatable,” “activate,” “inducible,” and “inducible” refer to polypeptide complexes having both a weakening activity form (e.g., weakening receptor binding and / or agonist activity) and an active form. These polypeptide complexes are activated by protease cleavage of the linker, which dissociates blocking and half-life-extending elements from the polypeptide complex. The induced / activated polypeptide complexes can bind to the IL-21 receptor with high affinity / binding affinity.

[0454] The terms “antibody” and “immunoglobulin” are used interchangeably herein. As used herein, antibody or immunoglobulin refers to an immunoglobulin molecule consisting of two heavy (H) chains. Typically, antibodies of mammals (e.g., humans, rodents, and monkeys) contain four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (CHI, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) sandwiched between more conserved regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs in the following order, from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Antibodies may include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, or tetrameric antibodies containing two heavy chain molecules and two light chain molecules. Those skilled in the art will recognize the existence of other forms of antibodies (e.g., camelid and shark antibodies).

[0455] As used herein, the term “attenuated” refers to an IL-21 receptor agonist whose receptor agonist activity is reduced compared to that of the IL-21 receptor. Attenuated IL-21 agonists may have agonist activity at least about 10 times, at least about 50 times, at least about 100 times, at least about 250 times, at least about 500 times, at least about 1000 times, or less, compared to the receptor’s naturally occurring agonist. When an IL-21 polypeptide complex containing IL-21 described herein is described as having “attenuated” or “attenuated activity”, it means that the IL-12 polypeptide complex is an attenuated IL-12 receptor agonist.

[0456] The term "cancer" refers to a physiological condition in mammals in which a population of cells is characterized by uncontrolled growth, immortality, metastatic ability, rapid growth and proliferation rates, and / or certain morphological features. Often, cancer can take the form of a tumor or mass, but it may also exist alone within a subject or circulate in the bloodstream as independent cells, such as leukemia cells or lymphoma cells. The term cancer encompasses any type of cancer and metastasis (including hematological malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors). Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include: squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer (e.g., triple-negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancers. Triple-negative breast cancer refers to breast cancer that is negative for the expression of the estrogen receptor (ER), progesterone receptor (PR), and Her2 / neu genes.

[0457] As used herein, a “conservative” amino acid substitution generally refers to a substitution in which one amino acid residue is replaced with another amino acid residue within a recognized group, thereby altering the structure of the peptide but substantially preserving its biological activity. Conservative amino acid substitutions are known to those skilled in the art. Conservative amino acid substitutions may include, but are not limited to, substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. For example, it can be reasonably expected by those skilled in the art that a single substitution of leucine to isoleucine or valine, a single substitution of aspartate to glutamate, a single substitution of threonine to serine, or similar substitutions from one amino acid to a structurally related amino acid will not have a significant effect on the biological activity of the resulting molecule.

[0458] As used herein, the term “half-life extension element” in the context of polypeptide complexes disclosed herein means a chemical element, preferably a polypeptide, that extends the serum half-life and improves pK by altering, for example, its size (e.g., to exceed the renal filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and excretion.

[0459] As used herein, the term “operatably linked” in the context of polypeptide complexes refers to the orientation of the components of a polypeptide complex to enable them to function as intended. For example, a polypeptide containing an IL-21 subunit and an IL-21 blocking element is operatably linked within the polypeptide complex by a protease-cleaving linker if the IL-21 blocking element can inhibit the IL-21 receptor activating activity of the IL-21 polypeptide, but if the protease-cleaving linker is cleaved, for example, the IL-21 blocking element can dissociate from IL-21, thereby reducing or eliminating the inhibition of the IL-21 receptor activating activity of the IL-21 polypeptide by the IL-21 blocking element.

[0460] As used herein, the terms “peptide,” “polypeptide,” or “protein” are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used interchangeably herein to refer to an amino acid sequence. It should be understood that the term polypeptide is not used herein to suggest a specific size or number of amino acids in a molecule, and that the peptides of the present invention may contain several or more amino acid residues.

[0461] In this specification, the term “subject” refers to any animal, for example, any mammal (including, but not limited to, humans, non-human primates, rodents, etc.). In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0462] As used herein, the term “therapeutic effective dose” refers to the amount of the compound described herein (i.e., the IL-21 polypeptide complex) that is sufficient to achieve the desired pharmacological or physiological effect under the conditions of administration. For example, a “therapeutic effective dose” may be an amount sufficient to reduce the signs or symptoms of a disease or condition (e.g., a tumor). Those skilled in the art will understand that the therapeutic effect does not need to be complete or curative, as long as it provides some benefit to the subject. The therapeutic effective dose of a pharmaceutical composition may vary depending on factors such as the individual’s medical condition, age, sex, and weight, as well as the ability of the pharmaceutical composition to induce the desired response in the individual. A clinician of ordinary skill can determine, based on these and other considerations, the appropriate dose to be administered to achieve the desired therapeutic benefit.

[0463] 5. Equivalents It will be readily apparent to those skilled in the art that other suitable modifications and optimizations of the methods of the present invention described herein can be made using suitable equivalents without departing from the scope of the disclosure or embodiments. While specific compounds and methods have been described in detail, these will be more clearly understood by referring to the following examples, which are introduced for illustrative purposes only and are not intended to limit the scope of the invention. [Examples]

[0464] 6. Examples The present invention will be further illustrated by the following examples, but these examples are not intended to be limiting. Example 1: HuT78 pSTAT3 AlphaLISA assay HuT78 cells (ATCC, TIB-161) were suspended in HBSS medium at a concentration of 100,000 cells / well and plated, then serum-starved at 37°C and 5% CO2 for 2 hours. After serum starvation, cells were stimulated at 37°C and 5% CO2 for 20 minutes with a dilution series of recombinant human or mouse IL-21, or activatable human or mouse IL-21. The activity of uncleaved and cleaved activatable IL-21 was tested. Cleaved inducible IL-21 was generated by incubation with activated CTSL or ELA. After stimulation, cells were lysed, and pSTAT3 Tyr705 levels were measured using the AlphaLISA Surefire Ultra pSTAT3 (Tyr705) assay kit (Perkin Elmer, #ALSU-PST3-A10K). See summary data in Table 45. [Table 45]

[0465] Example 2: Protease cleavage of IL-21 prodrug Those skilled in the art will be familiar with the method for preparing a protein cleavage assay. 50 μg of protein in 1×PBS (pH 7.4) was cleaved in 100 μL total volume with 1 μg ...

Claims

1. It is an inducible IL-21 prodrug, a) IL-21 polypeptide [A] and b) IL-21 blocking element [D], c) Half-life extension element [H], c) Protease-cleaving polypeptide linker [L], It includes at least one of each, The IL-21 polypeptide and the IL-21 blocking element or the half-life extension element are operably linked by the protease-cleaving polypeptide linker, the inducible IL-21 prodrug having attenuated IL-21 receptor activating activity, wherein the IL-21 receptor activating activity of the inducible IL-21 prodrug is at least about 10 times lower than the IL-21 receptor activating activity of a polypeptide containing the IL-21 polypeptide produced by cleavage of the protease-cleaving linker.

2. The inducible IL-21 prodrug is given by formula: [A]-[L1]-[H]-[L2]-[D] [D]-[L2]-[H]-[L1]-[A] [A]-[L1]-[D]-[L2]-[H] [H]-[L2]-[D]-[L1]-[A] [H]-[L1]-[A]-[L2']-[D] [D]-[L1]-[A]-[L2']-[H] The inducible IL-21 prodrug according to claim 1, wherein [A] is an IL-21 polypeptide, [D] is an IL-21 blocking element, [H] is a half-life extending element, [L1] is a protease-cleaving polypeptide linker, [L2] is a polypeptide linker that may be protease-cleaving, and [L2'] is a protease-cleaving polypeptide linker.

3. The half-life-extending element comprises a serum albumin-binding domain, serum albumin, transferrin, or immunoglobulin Fc or a fragment thereof, as described in any one of the prior claims, for the inducible IL-21 prodrug.

4. The inducible IL-21 prodrug according to any one of the prior claims, wherein the blocking element comprises a ligand-binding domain or fragment of a congener receptor for the IL-21 polypeptide, or an antibody or antigen-binding fragment of an antibody that binds to the IL-21 polypeptide.

5. The inducible IL-21 prodrug according to claim 4, wherein the antibody or antigen-binding fragment is a single-domain antibody, Fab, or scFv that binds to the IL-21 polypeptide.

6. The inducible IL-21 prodrug according to claim 4, wherein the congener receptor for IL-21 is an IL-21 receptor or a common IL-21 gamma chain.

7. The inducible IL-21 prodrug according to claim 3, wherein the half-life extension element is also a blocking element.

8. The inducible IL-21 prodrug according to any one of the prior claims, wherein the IL-21 blocking element inhibits the activation of the IL-21 receptor by the inducible IL-21 prodrug.

9. Each protease-cleavable polypeptide linker comprises a sequence that can be independently cleaved by a protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, cathepsin L, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), ADAM, FAP, plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface proteases, as described in any one of the prior claims.

10. L2 is a protease-cleaving polypeptide linker, as described in any one of claims 2 to 9, an inducible IL-21 prodrug.

11. An inducible IL-21 prodrug according to any one of the prior claims, wherein L1 or L2 or both L1 and L2 are cleaved by two or more different proteases.

12. The inducible IL-21 prodrug according to claim 9, wherein the cathepsin is cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, or cathepsin G.

13. The inducible IL-21 prodrug according to claim 9, wherein the matrix metalloproteinase (MMP) is MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP19, or MMP20.

14. An inducible IL-21 prodrug according to any one of the prior claims, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 44 to 50 or an amino acid sequence having at least 80% identity with SEQ ID NOs. 44 to 50.

15. A nucleic acid encoding an inducible IL-21 prodrug according to any one of the prior claims.

16. A vector comprising the nucleic acid described in claim 15.

17. A host cell comprising the vector according to claim 16.

18. A pharmaceutical composition comprising an inducible IL-21 prodrug according to any one of claims 1 to 14 or a nucleic acid according to claim 15.

19. A method for preparing a pharmaceutical composition, comprising culturing the host cells described in claim 17 under conditions suitable for the expression and collection of the inducible IL-21 prodrug.

20. A method for treating cancer or a cancer-related viral infection, comprising administering to a subject in need thereof the pharmaceutical composition according to claim 18 or one or more inducible IL-21 prodrugs according to any one of claims 1 to 14.

21. An inducible IL-21 prodrug comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-8 and 44-50, or an amino acid sequence having at least approximately 80% identity with SEQ ID NOs: 2-8 and 44-50.

22. A nucleic acid encoding an inducible IL-21 prodrug according to claim 21.

23. The nucleic acid according to claim 22, wherein the nucleic acid comprises a circular vector.

24. The nucleic acid according to claim 22, wherein the nucleic acid includes DNA.

25. The nucleic acid according to claim 22, wherein the nucleic acid includes RNA.

26. An expression vector comprising the nucleic acid described in any one of claims 22 to 25.

27. An isolated host cell containing the vector according to claim 26.

28. A method for preparing a pharmaceutical composition, comprising culturing the isolated host cells described in claim 27 under conditions suitable for the expression of the polypeptide complex.

29. The method according to claim 28, further comprising isolating the polypeptide complex.

30. A pharmaceutical composition comprising the inducible IL-21 prodrug according to claim 21 or the nucleic acid according to any one of claims 22 to 25.

31. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of the inducible IL-21 prodrug according to claim 21, the nucleic acid according to any one of claims 22 to 25, the expression vector according to claim 26, or the pharmaceutical composition according to claim 30.

32. A nucleic acid composition comprising one or more nucleic acid sequences encoding an inducible IL-21 prodrug, wherein the inducible IL-21 prodrug is a) IL-21 polypeptide [A] and b) IL-21 blocking element [D], c) Half-life extension element [H], c) Protease-cleaving polypeptide linker [L], It includes at least one of each, The IL-21 polypeptide and the IL-21 blocking element or the half-life extension element are operably linked by the protease-cleaving polypeptide linker, and the inducible IL-21 prodrug has attenuated IL-21 receptor activating activity. The nucleic acid composition.

33. The nucleic acid composition according to claim 32, wherein the nucleic acid comprises a cyclic vector.

34. The nucleic acid composition according to claim 32, wherein the nucleic acid comprises DNA.

35. The nucleic acid composition according to claim 33, wherein the nucleic acid comprises RNA.

36. An expression vector comprising the nucleic acid composition according to any one of claims 32 to 35.

37. An isolated host cell containing the vector according to claim 36.

38. An inducible IL-21 prodrug comprising an IL-21 polypeptide, a half-life extender, an IL-21 blocking element, and a protease-cleaving linker, wherein the IL-21 blocking element is an antigen-binding fragment of an antibody, and the antigen-binding fragment comprises, 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, and the inducible IL-21 prodrug is i. 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 the half-life extension element are operably linked by the protease-cleaving linker, ii. A second polypeptide comprising at least an antigen-binding portion of an antibody heavy chain complementary to the light chain of the first polypeptide, or an antibody light chain complementary to the heavy chain of the first polypeptide and forming an IL-21 binding site together with the light chain, The inducible IL-21 prodrug, which includes the above.

39. It is an inducible IL-21 prodrug, i. A first polypeptide comprising an IL-21 polypeptide, a half-life extension element, a first IL-21 blocking element, a protease-cleaving polypeptide linker, and a portion of the constant region or light chain of an antibody heavy chain, wherein the first blocking element is IL-21R, and the IL-21 polypeptide and the first blocking element and / or the half-life extension element are operably linked by the protease-cleaving linker, ii. A second polypeptide comprising a second IL-21 blocking element and a portion of the constant region of an antibody heavy chain or an antibody light chain, wherein the second blocking element and the portion of the constant region of the antibody heavy chain or the antibody light chain are linked via a linker that may be protease-cleavable, and the second blocking element is a common gamma chain receptor, comprising the second polypeptide, If the constant region of the antibody heavy chain is on the first polypeptide, the antibody light chain is on the second polypeptide, and if the constant region of the antibody heavy chain is on the second polypeptide, the antibody light chain is on the first polypeptide. The aforementioned inducible IL-21 prodrug.

40. The inducible IL-21 prodrug according to claim 38 or 39, wherein the half-life extending element is human serum albumin, an antigen-binding polypeptide that binds to human serum albumin, or immunoglobulin Fc or a fragment thereof.

41. The inducible IL-21 prodrug according to any one of claims 38 to 40, wherein the protease-cleavable linker comprises a sequence that can be cleaved by a protease selected from kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), ADAM, FAP, plasminogen activator, caspase, tryptase, or tumor protease.

42. The inducible IL-21 prodrug according to any one of claims 39 to 40, wherein the protease is selected from cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, or cathepsin G.

43. The inducible IL-21 prodrug according to any one of claims 39 to 40, wherein the protease is selected from matrix metalloproteases (MMPs) that are MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, or MMP14.

44. The inducible IL-21 prodrug according to any one of claims 39 to 40, wherein the protease-cleavable linker comprises at least two sequences that can be independently cleaved by a protease.

45. The inducible IL-21 prodrug according to any one of claims 39 to 40, wherein the protease-cleaving linker comprises a synthetic sequence.

46. The inducible IL-21 prodrug according to any one of claims 39 to 45, wherein each of the protease-cleaving linkers is cleaved by two or more different proteases.

47. The inducible IL-21 prodrug according to claim 38, wherein the blocking element is bound to the IL-21.

48. The inducible IL-21 prodrug according to claim 39, wherein the first blocking element and the second blocking element are bound to the IL-21.

49. A nucleic acid encoding a polypeptide according to any one of claims 38 to 48.

50. The nucleic acid according to claim 49, comprising a circular vector.

51. The nucleic acid according to claim 49, comprising DNA.

52. The nucleic acid according to claim 49, comprising RNA.

53. An expression vector comprising the nucleic acid described in any one of claims 49 to 52.

54. An isolated host cell comprising the vector according to claim 53.

55. A method for preparing a pharmaceutical composition, comprising culturing the isolated host cells described in claim 54 under conditions suitable for the expression of the inducible IL-21 prodrug.

56. The method according to claim 55, further comprising isolating the inducible IL-21 prodrug.

57. A pharmaceutical composition comprising an inducible IL-21 prodrug according to any one of claims 38 to 48 or a nucleic acid according to any one of claims 49 to 52.

58. A method for treating a tumor, comprising administering to a subject in need thereof an effective amount of an inducible IL-21 prodrug according to any one of claims 38 to 48, a nucleic acid according to any one of claims 49 to 52, an expression vector according to claim 53, or a pharmaceutical composition according to claim 57.

59. The inducible IL-21 prodrug according to any one of claims 1 to 14, 21 or 38 to 48, wherein the IL-21 is mutaine, or the nucleic acid composition according to any one of claims 15, 22 to 25 or 32 to 35, or the nucleic acid according to any one of claims 49 to 53.

60. An inducible IL-21 prodrug comprising a first polypeptide selected from the group consisting of amino acid sequences of SEQ ID NOs. 18-23 and 26-31, or sequences having at least 80% identity with SEQ ID NOs. 18-23 and 26-31.

61. The inducible IL-21 prodrug according to claim 60, further comprising a second polypeptide selected from the group consisting of SEQ ID NO: 17 or 25 or an amino acid sequence having at least 80% identity with SEQ ID NO: 17 or 25.

62. An inducible IL-21 prodrug comprising: a first polypeptide selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs. 18-23 and 26-31, or SEQ ID NOs. 18-23 and 26-31; and a second polypeptide selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs. 17 or 25, or SEQ ID NOs. 17 or 25.

63. An inducible IL-21 prodrug comprising a first polypeptide chain selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65, or SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65.

64. An inducible IL-21 prodrug further comprising a second polypeptide chain selected from the group consisting of amino acid sequences having at least 80% identity with sequence numbers 10, 40, 52, and 64, or sequence numbers 10, 40, 52, and 64.

65. An inducible IL-21 prodrug comprising: a first polypeptide chain selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65, or SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65; and a second polypeptide chain selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs: 10, 40, 52, and 64, or SEQ ID NOs: 10, 40, 52, and 64.

66. A nucleic acid encoding a polypeptide according to any one of claims 60 to 65.

67. The nucleic acid according to claim 66, comprising a circular vector.

68. The nucleic acid composition according to claim 66, comprising DNA.

69. The nucleic acid composition according to claim 66, comprising RNA.

70. An expression vector comprising the nucleic acid described in any one of claims 66 to 69.

71. Isolated host cells comprising the vector according to claim 70.

72. A method for preparing a pharmaceutical composition, comprising culturing the isolated host cells described in claim 71 under conditions suitable for the expression of the inducible IL-21 prodrug.

73. The method according to claim 72, further comprising isolating the inducible IL-21 prodrug.

74. A pharmaceutical composition comprising an inducible IL-21 prodrug according to any one of claims 60 to 65, or a nucleic acid according to any one of claims 66 to 69.

75. A method for treating a tumor, comprising administering to a subject in need thereof an effective amount of an inducible IL-21 prodrug according to any one of claims 60 to 65, a nucleic acid according to any one of claims 66 to 69, an expression vector according to claim 70, or a pharmaceutical composition according to claim 74.

76. The inducible IL-21 prodrug according to any one of claims 60 to 65 or the nucleic acid composition according to any one of claims 66 to 69, wherein the IL-21 is mutaine.

77. A nucleic acid composition comprising one or more nucleic acid sequences encoding an inducible IL-21 prodrug comprising an IL-21 polypeptide, a half-life extender, a blocking element, and a protease-cleaving linker, wherein the IL-21 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, 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, and the inducible IL-21 prodrug is i. 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 the half-life extension element are operably linked by the protease-cleaving linker, ii. A second polypeptide comprising at least an antigen-binding portion of an antibody heavy chain complementary to the light chain of the first polypeptide, or an antibody light chain complementary to the heavy chain of the first polypeptide and forming an IL-21 binding site together with the light chain, The nucleic acid composition comprising the above.

78. A nucleic acid composition comprising one or more nucleic acid sequences encoding an inducible IL-21 prodrug, wherein the inducible IL-21 prodrug is i. A first polypeptide comprising an IL-21 polypeptide, a half-life extension element, a first IL-21 blocking element, a protease-cleaving polypeptide linker, and a portion of the constant region or light chain of an antibody heavy chain, wherein the first blocking element is IL-21R, and the IL-21 polypeptide and the first blocking element or the half-life extension element are operably linked by the protease-cleaving linker, ii. A second polypeptide comprising a second blocking element and a portion of the constant region of an antibody heavy chain or an antibody light chain, wherein the second blocking element and the portion of the constant region of the antibody heavy chain or the antibody light chain are linked via a linker that may be protease-cleavable, and the second blocking element is a common gamma chain receptor, comprising the second polypeptide, If the constant region of the antibody heavy chain is on the first polypeptide, the antibody light chain is on the second polypeptide, and if the constant region of the antibody heavy chain is on the second polypeptide, the antibody light chain is on the first polypeptide. The nucleic acid composition.

79. A nucleic acid composition according to claim 77 or 78, comprising a cyclic vector.

80. A nucleic acid composition according to claim 77 or 78, comprising DNA.

81. A nucleic acid composition according to claim 77 or 78, comprising RNA.

82. An expression vector comprising the nucleic acid according to any one of claims 77 to 78.

83. An isolated host cell comprising the vector according to claim 82.

84. An inducible IL-21 prodrug comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 72 to 109, or an amino acid sequence having at least approximately 80% identity with SEQ ID NOs. 72 to 109.

85. A nucleic acid encoding an inducible IL-21 prodrug according to claim 84.

86. The nucleic acid according to claim 85, wherein the nucleic acid comprises a circular vector.

87. The nucleic acid according to claim 85, wherein the nucleic acid includes DNA.

88. The nucleic acid according to claim 85, wherein the nucleic acid includes RNA.

89. An expression vector comprising the nucleic acid according to any one of claims 85 to 88.

90. Isolated host cells comprising the vector according to claim 89.

91. A method for preparing a pharmaceutical composition, comprising culturing the isolated host cells described in claim 90 under conditions suitable for the expression of the polypeptide complex.

92. The method according to claim 91, further comprising isolating the polypeptide complex.

93. A pharmaceutical composition comprising the inducible IL-21 prodrug according to claim 84 or the nucleic acid according to any one of claims 85 to 88.

94. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of the inducible IL-21 prodrug according to claim 84, the nucleic acid according to any one of claims 85 to 88, the expression vector according to claim 89, or the pharmaceutical composition according to claim 93.

95. Sequence numbers 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, 449-463, and 655, or sequence numbers 72, 110-113, 117-126, 130-136, 138-144, 153-208, 221-235, 241-243, 245-247, 250, 251, 253-255 An inducible IL-21 prodrug comprising: a first polypeptide selected from the group consisting of amino acid sequences having at least 80% identity with 257, 258, 260-264, 267-269, 271-290, 475, 476, 449-463, and 655; and a second polypeptide selected from the group consisting of amino acid sequences having at least 80% identity with SEQ ID NOs. 71, 127-129, 137, or 300-306, or SEQ ID NOs. 71, 127-129, 137, or 300-306.

96. The inducible IL-21 prodrug according to claim 95, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 655, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

129.

97. The inducible IL-21 prodrug according to claim 95, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 131, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

129.

98. The inducible IL-21 prodrug according to claim 95, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 119, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

129.

99. The inducible IL-21 prodrug according to claim 95, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 143, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

137.

100. The inducible IL-21 prodrug according to claim 95, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 169, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

301.

101. The inducible IL-21 prodrug according to claim 95, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 185, and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

303.

102. A nucleic acid encoding an inducible IL-21 prodrug according to claims 95 to 101.

103. The nucleic acid according to any one of claims 95 to 102, wherein the nucleic acid comprises a circular vector.

104. The nucleic acid according to any one of claims 95 to 102, wherein the nucleic acid includes DNA.

105. The nucleic acid according to any one of claims 102 to 104, wherein the nucleic acid includes RNA.

106. An expression vector comprising the nucleic acid according to any one of claims 102 to 105.

107. An isolated host cell containing the vector according to claim 106.

108. A method for preparing a pharmaceutical composition, comprising culturing the isolated host cells described in claim 107 under conditions suitable for the expression of the polypeptide complex.

109. The method according to claim 108, further comprising isolating the polypeptide complex.

110. A pharmaceutical composition comprising an inducible IL-21 prodrug according to any one of claims 95 to 101, or a nucleic acid according to any one of claims 102 to 106.

111. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of an inducible IL-21 prodrug according to claims 95 to 101, a nucleic acid according to any one of claims 102 to 105, an expression vector according to claim 106, or a pharmaceutical composition according to claim 110.

112. It is an inducible IL-21 prodrug, 1) A first polypeptide chain having the formula: [A]-[L1]-[H]-[L2]-[D], wherein [A] is an IL-21 polypeptide comprising the amino acid sequence of SEQ ID NO: 654 or a variant thereof, [L1] is a protease-cleaving polypeptide linker comprising the amino acid sequence of SEQ ID NO: 198 or a variant thereof, [H] is a half-life extension element comprising any one amino acid sequence from SEQ ID NOs: 602-606, 608-653, or a variant thereof, [L2] is a protease-cleaving polypeptide linker comprising the amino acid sequence of SEQ ID NO: 198 or a variant thereof, and [D] is an antigen-binding portion of a heavy chain comprising any one amino acid sequence from SEQ ID NOs: 291-299 or a variant thereof, 2) A second polypeptide chain comprising an antibody light chain of SEQ ID NO: 129 or a variant thereof, which is complementary to the heavy chain of the first polypeptide and forms an IL-2 binding site together with the light chain, The inducible IL-21 prodrug, which includes the above.

113. The inducible IL-21 prodrug according to claim 112, wherein the half-life extending element in the inducible IL-2 prodrug comprises an amino acid sequence that is at least about 80% identical to any one of sequence numbers 602-606 or 608-653.

114. The half-life extension elements in the inducible IL-21 prodrug are sequence numbers 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, and 629. An inducible IL-21 prodrug according to any one of claims 112 or 113, selected from the group consisting of SEQ ID NOs: 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, and 653.

115. The inducible IL-21 prodrug is the inducible IL-21 prodrug according to any one of claims 112 to 114, comprising compound 210 (SEQ ID NO: 480 / SEQ ID NO: 129), compound 211 (SEQ ID NO: 481 / SEQ ID NO: 129), compound 212 (SEQ ID NO: 482 / SEQ ID NO: 129), compound 213 (SEQ ID NO: 483 / SEQ ID NO: 129), compound 214 (SEQ ID NO: 484 / SEQ ID NO: 129), compound 215 (SEQ ID NO: 485 / SEQ ID NO: 129), compound 216 (SEQ ID NO: 486 / SEQ ID NO: 129), compound 217 (SEQ ID NO: 487 / SEQ ID NO: 129), compound 218 (SEQ ID NO: 488 / SEQ ID NO: 129), compound 219 (SEQ ID NO: 489 / SEQ ID NO: 129), compound 220 (SEQ ID NO: 490 / SEQ ID NO: 129), compound 221 (SEQ ID NO: 491 / SEQ ID NO: 129), or compound 222 (SEQ ID NO: 655 / SEQ ID NO: 129).

116. A nucleic acid encoding a polypeptide according to any one of claims 112 to 114.

117. The nucleic acid according to claim 116, comprising a circular vector.

118. The nucleic acid according to claim 116, comprising DNA.

119. The nucleic acid according to claim 116, comprising RNA.

120. An expression vector comprising the nucleic acid according to any one of claims 116 to 119.

121. An isolated host cell containing the vector according to claim 120.

122. A method for preparing a pharmaceutical composition, comprising culturing the isolated host cells described in claim 120 under conditions suitable for the expression of the inducible IL-21 prodrug.

123. The method according to claim 122, further comprising isolating the inducible IL-21 prodrug.

124. A pharmaceutical composition comprising an inducible IL-21 prodrug according to any one of claims 112 to 115 or a nucleic acid according to any one of claims 116 to 119.

125. A method for treating a tumor, comprising administering to a subject in need thereof an effective amount of an inducible IL-21 prodrug according to any one of claims 112 to 115, a nucleic acid according to any one of claims 116 to 119, an expression vector according to claim 120, or a pharmaceutical composition according to claim 122.

126. The inducible IL-21 prodrug according to claim 85, wherein the inducible prodrug comprises any one of the inducible IL-21 prodrugs listed in Table 39.

127. The inducible IL-21 prodrugs are compound 53 (SEQ ID NO: 18 / SEQ ID NO: 17), compound 54 (SEQ ID NO: 19 / SEQ ID NO: 17), compound 55 (SEQ ID NO: 20 / SEQ ID NO: 17), compound 56 (SEQ ID NO: 21 / SEQ ID NO: 17), compound 57 (SEQ ID NO: 22 / SEQ ID NO: 17), compound 58 (SEQ ID NO: 23 / SEQ ID NO: 17), compound 59 (SEQ ID NO: 24 / SEQ ID NO: 17), compound 60 (SEQ ID NO: 25 / SEQ ID NO: 17), compound 61 (SEQ ID NO: 26 / SEQ ID NO: 25), compound 62 (SEQ ID NO: 27 / SEQ ID NO: 25), and compound 63 (SEQ ID NO: 28 / SEQ ID NO: 25). Compound 64 (SEQ ID NO: 29 / SEQ ID NO: 25), Compound 65 (SEQ ID NO: 30 / SEQ ID NO: 25), Compound 66 (SEQ ID NO: 69 / SEQ ID NO: 71), Compound 67 (SEQ ID NO: 70 / SEQ ID NO: 71), Compound 68 (SEQ ID NO: 72 / SEQ ID NO: 71), Compound 69 (SEQ ID NO: 110 / SEQ ID NO: 71), Compound 70 (SEQ ID NO: 111 / SEQ ID NO: 71), Compound 71 (SEQ ID NO: 112 / SEQ ID NO: 71), Compound 72 (SEQ ID NO: 113 / SEQ ID NO: 71), Compound 73 (SEQ ID NO: 117 / SEQ ID NO: 128), Compound 74 (SEQ ID NO: 118 / SEQ ID NO: 128), Compound 75 (Sequence No. Compound 119 / SEQ ID NO: 128), Compound 76 (SEQ ID NO: 120 / SEQ ID NO: 128), Compound 77 (SEQ ID NO: 121 / SEQ ID NO: 128), Compound 78 (SEQ ID NO: 122 / SEQ ID NO: 128), Compound 79 (SEQ ID NO: 123 / SEQ ID NO: 128), Compound 80 (SEQ ID NO: 124 / SEQ ID NO: 128), Compound 81 (SEQ ID NO: 125 / SEQ ID NO: 129), Compound 82 (SEQ ID NO: 126 / SEQ ID NO: 129), Compound 83 (SEQ ID NO: 130 / SEQ ID NO: 129), Compound 84 (SEQ ID NO: 131 / SEQ ID NO: 129), Compound 85 (SEQ ID NO: 132 / SEQ ID NO: 129), Compound 8 6 (SEQ ID NO: 133 / SEQ ID NO: 129), Compound 87 (SEQ ID NO: 134 / SEQ ID NO: 129), Compound 88 (SEQ ID NO: 135 / SEQ ID NO: 129), Compound 89 (SEQ ID NO: 136 / SEQ ID NO: 137), Compound 90 (SEQ ID NO: 138 / SEQ ID NO: 137), Compound 91 (SEQ ID NO: 139 / SEQ ID NO: 137), Compound 92 (SEQ ID NO: 140 / SEQ ID NO: 137), Compound 93 (SEQ ID NO: 141 / SEQ ID NO: 137), Compound 94 (SEQ ID NO: 142 / SEQ ID NO: 137), Compound 95 (SEQ ID NO: 143 / SEQ ID NO: 137), Compound 96 (SEQ ID NO: 144 / SEQ ID NO: 137),Compound 97 (SEQ ID NO: 153 / SEQ ID NO: 127), Compound 98 (SEQ ID NO: 154 / SEQ ID NO: 127), Compound 99 (SEQ ID NO: 155 / SEQ ID NO: 127), Compound 100 (SEQ ID NO: 156 / SEQ ID NO: 127), Compound 101 (SEQ ID NO: 157 / SEQ ID NO: 127), Compound 102 (SEQ ID NO: 158 / SEQ ID NO: 127), Compound 103 (SEQ ID NO: 159 / SEQ ID NO: 127), Compound 104 (SEQ ID NO: 160 / SEQ ID NO: 127), Compound 105 (SEQ ID NO: 161 / SEQ ID NO: 300), Compound 106 (SEQ ID NO: 162 / SEQ ID NO: 300), Compound 107 (SEQ ID NO: Compound 108 (Sequence No. 163 / Sequence No. 300), Compound 109 (Sequence No. 165 / Sequence No. 300), Compound 110 (Sequence No. 166 / Sequence No. 300), Compound 111 (Sequence No. 167 / Sequence No. 300), Compound 112 (Sequence No. 168 / Sequence No. 300), Compound 113 (Sequence No. 169 / Sequence No. 301), Compound 114 (Sequence No. 170 / Sequence No. 301), Compound 115 (Sequence No. 171 / Sequence No. 301), Compound 116 (Sequence No. 172 / Sequence No. 301), Compound 117 (Sequence No. 17 Compound 118 (Sequence No. 174 / Sequence No. 301), Compound 119 (Sequence No. 175 / Sequence No. 301), Compound 110 (Sequence No. 176 / Sequence No. 301), Compound 111 (Sequence No. 177 / Sequence No. 302), Compound 112 (Sequence No. 178 / Sequence No. 302), Compound 113 (Sequence No. 179 / Sequence No. 302), Compound 114 (Sequence No. 180 / Sequence No. 302), Compound 115 (Sequence No. 181 / Sequence No. 302), Compound 116 (Sequence No. 182 / Sequence No. 302), Compound 117 (Sequence No. 183 / Sequence No. Compound 302), Compound 118 (SEQ ID NO: 184 / SEQ ID NO: 302), Compound 119 (SEQ ID NO: 185 / SEQ ID NO: 303), Compound 120 (SEQ ID NO: 186 / SEQ ID NO: 303), Compound 121 (SEQ ID NO: 187 / SEQ ID NO: 303), Compound 122 (SEQ ID NO: 188 / SEQ ID NO: 303), Compound 123 (SEQ ID NO: 189 / SEQ ID NO: 303), Compound 124 (SEQ ID NO: 190 / SEQ ID NO: 303), Compound 125 (SEQ ID NO: 191 / SEQ ID NO: 303), Compound 126 (SEQ ID NO: 192 / SEQ ID NO: 303), Compound 127 (SEQ ID NO: 193 / SEQ ID NO: 304),Compound 128 (SEQ ID NO: 194 / SEQ ID NO: 304), Compound 129 (SEQ ID NO: 449 / SEQ ID NO: 304), Compound 130 (SEQ ID NO: 450 / SEQ ID NO: 304), Compound 131 (SEQ ID NO: 451 / SEQ ID NO: 304), Compound 132 (SEQ ID NO: 452 / SEQ ID NO: 304), Compound 133 (SEQ ID NO: 453 / SEQ ID NO: 304), Compound 134 (SEQ ID NO: 454 / SEQ ID NO: 304), Compound 135 (SEQ ID NO: 455 / SEQ ID NO: 305), Compound 136 (SEQ ID NO: 456 / SEQ ID NO: 305), Compound 137 (SEQ ID NO: 457 / SEQ ID NO: 305), Compound 138 (SEQ ID NO: 458 / SEQ ID NO: 305), Compound 139 (SEQ ID NO: 459 / SEQ ID NO: 305), Compound 140 (SEQ ID NO: 460 / SEQ ID NO: 305), Compound 141 (SEQ ID NO: 461 / SEQ ID NO: 305), Compound 142 (SEQ ID NO: 462 / SEQ ID NO: 305), Compound 143 (SEQ ID NO: 221 / SEQ ID NO: 128), Compound 144 (SEQ ID NO: 222 / SEQ ID NO: 128), Compound 145 (SEQ ID NO: 223 / SEQ ID NO: 128), Compound 146 (SEQ ID NO: 224 / SEQ ID NO: 129), Compound 147 (SEQ ID NO: 225 / SEQ ID NO: 129), Compound 148 (SEQ ID NO: 2 Compound 26 (Sequence No. 129), Compound 149 (Sequence No. 227 / Sequence No. 137), Compound 150 (Sequence No. 228 / Sequence No. 137), Compound 151 (Sequence No. 229 / Sequence No. 137), Compound 152 (Sequence No. 230 / Sequence No. 304), Compound 153 (Sequence No. 231 / Sequence No. 304), Compound 154 (Sequence No. 232 / Sequence No. 304), Compound 155 (Sequence No. 233 / Sequence No. 305), Compound 156 (Sequence No. 234 / Sequence No. 305), Compound 157 (Sequence No. 235 / Sequence No. 305), Compound 158 (Sequence No. 241 / Sequence No. Compound 300), Compound 159 (SEQ ID NO: 242 / SEQ ID NO: 300), Compound 160 (SEQ ID NO: 243 / SEQ ID NO: 300), Compound 161 (SEQ ID NO: 245 / SEQ ID NO: 301), Compound 162 (SEQ ID NO: 246 / SEQ ID NO: 301), Compound 163 (SEQ ID NO: 247 / SEQ ID NO: 301), Compound 164 (SEQ ID NO: 475 / SEQ ID NO: 302), Compound 165 (SEQ ID NO: 250 / SEQ ID NO: 302), Compound 166 (SEQ ID NO: 251 / SEQ ID NO: 302), Compound 167 (SEQ ID NO: 253 / SEQ ID NO: 303), Compound 168 (SEQ ID NO: 254 / SEQ ID NO: 303),Compound 169 (SEQ ID NO: 255 / SEQ ID NO: 303), Compound 170 (SEQ ID NO: 257 / SEQ ID NO: 306), Compound 171 (SEQ ID NO: 258 / SEQ ID NO: 306), Compound 172 (SEQ ID NO: 476 / SEQ ID NO: 306), Compound 173 (SEQ ID NO: 260 / SEQ ID NO: 306), Compound 174 (SEQ ID NO: 261 / SEQ ID NO: 306), Compound 175 (SEQ ID NO: 262 / SEQ ID NO: 306), Compound 176 (SEQ ID NO: 263 / SEQ ID NO: 306), Compound 177 (SEQ ID NO: 264 / SEQ ID NO: 306), Compound 178 (SEQ ID NO: 267 / SEQ ID NO: 306), Compound 179 ( Compound 180 (SEQ ID NO: 268 / SEQ ID NO: 306), Compound 181 (SEQ ID NO: 271 / SEQ ID NO: 128), Compound 182 (SEQ ID NO: 272 / SEQ ID NO: 128), Compound 183 (SEQ ID NO: 273 / SEQ ID NO: 129), Compound 184 (SEQ ID NO: 274 / SEQ ID NO: 129), Compound 185 (SEQ ID NO: 275 / SEQ ID NO: 137), Compound 186 (SEQ ID NO: 276 / SEQ ID NO: 137), Compound 187 (SEQ ID NO: 277 / SEQ ID NO: 306), Compound 188 (SEQ ID NO: 278 / SEQ ID NO: 306), Compound 189 (SEQ ID NO: 279 / Compound 190 (SEQ ID NO: 300), Compound 190 (SEQ ID NO: 289 / SEQ ID NO: 300), Compound 191 (SEQ ID NO: 281 / SEQ ID NO: 301), Compound 192 (SEQ ID NO: 282 / SEQ ID NO: 301), Compound 193 (SEQ ID NO: 283 / SEQ ID NO: 302), Compound 194 (SEQ ID NO: 284 / SEQ ID NO: 302), Compound 195 (SEQ ID NO: 285 / SEQ ID NO: 303), Compound 196 (SEQ ID NO: 286 / SEQ ID NO: 303), Compound 197 (SEQ ID NO: 287 / SEQ ID NO: 304), Compound 198 (SEQ ID NO: 288 / SEQ ID NO: 304), Compound 199 (SEQ ID NO: 289 / SEQ ID NO: 305) Compound 200 (SEQ ID NO: 290 / SEQ ID NO: 305), Compound 201 (SEQ ID NO: 39 / SEQ ID NO: 40), Compound 202 (SEQ ID NO: 41 / SEQ ID NO: 40), Compound 203 (SEQ ID NO: 51 / SEQ ID NO: 52), Compound 204 (SEQ ID NO: 58 / SEQ ID NO: 10), Compound 205 (SEQ ID NO: 59 / SEQ ID NO: 10), Compound 206 (SEQ ID NO: 63 / SEQ ID NO: 64), Compound 207 (SEQ ID NO: 65 / SEQ ID NO: 65), Compound 208 (SEQ ID NO: 9 / SEQ ID NO: 10), Compound 210 (SEQ ID NO: 480 / SEQ ID NO: 129), Compound 211 (SEQ ID NO: 481 / SEQ ID NO: 129),An inducible IL-21 prodrug according to any one of claims 112 to 114, comprising compound 212 (SEQ ID NO: 482 / SEQ ID NO: 129), compound 213 (SEQ ID NO: 483 / SEQ ID NO: 129), compound 214 (SEQ ID NO: 484 / SEQ ID NO: 129), compound 215 (SEQ ID NO: 485 / SEQ ID NO: 129), compound 216 (SEQ ID NO: 486 / SEQ ID NO: 129), compound 217 (SEQ ID NO: 487 / SEQ ID NO: 129), compound 218 (SEQ ID NO: 488 / SEQ ID NO: 129), compound 219 (SEQ ID NO: 489 / SEQ ID NO: 129), compound 220 (SEQ ID NO: 490 / SEQ ID NO: 129), compound 221 (SEQ ID NO: 491 / SEQ ID NO: 129), or compound 222 (SEQ ID NO: 655 / SEQ ID NO: 129).

128. A single-domain antibody fragment (sdAb) comprising CDR1, CDR2, and CDR3, having binding specificity to human serum albumin, CDR1 includes any one of the sequence substitutions 656, 659, 662, 663, and 691, or any variant of the sequence containing up to approximately two amino acid substitutions. CDR2 includes any one of SEQ ID NOs: 657, 660, 664, 665, 666, and 692, or any variant of the above sequence containing up to approximately three amino acid substitutions. CDR3 contains one of sequence numbers 658, 661, or 667, or any variant of the sequence containing up to approximately three amino acid substitutions. The aforementioned single-domain antibody fragment (sdAb).

129. The sdAb according to claim 128, wherein the sdAb is (1) CDR1 containing the amino acid sequence of SEQ ID NO: 656 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 657 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 658 or a variant thereof containing up to approximately three amino acid substitutions, (2) CDR1 containing the amino acid sequence of SEQ ID NO: 659 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 660 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 660 or a variant thereof containing up to approximately three amino acid substitutions, (3) CDR1 containing the amino acid sequence of SEQ ID NO: 662 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 664 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions, (4) CDR1 containing the amino acid sequence of SEQ ID NO: 662 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 665 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions, (5) CDR1 containing the amino acid sequence of SEQ ID NO: 662 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 666 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions, (6) CDR1 containing the amino acid sequence of SEQ ID NO: 662 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 692 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions, (7) CDR1 containing the amino acid sequence of SEQ ID NO: 691 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 664 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions, (8) CDR1 containing the amino acid sequence of SEQ ID NO: 691 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 665 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions, (9) CDR1 containing the amino acid sequence of SEQ ID NO: 691 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 666 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions, (10) CDR1 containing the amino acid sequence of SEQ ID NO: 691 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 692 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions, (11) CDR1 containing the amino acid sequence of SEQ ID NO: 663 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 664 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions, (12) CDR1 containing the amino acid sequence of SEQ ID NO: 663 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 665 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions, (13) CDR1 containing the amino acid sequence of SEQ ID NO: 663 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 666 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions, (14) The sdAb may include CDR1 containing the amino acid sequence of SEQ ID NO: 663 or a variant thereof containing up to approximately two amino acid substitutions, CDR2 containing the amino acid sequence of SEQ ID NO: 692 or a variant thereof containing up to approximately three amino acid substitutions, and CDR3 containing the amino acid sequence of SEQ ID NO: 667 or a variant thereof containing up to approximately three amino acid substitutions. The sdAb includes the aforementioned sdAb.

130. sdAb according to any one of the prior claims, wherein sdAb is (1) CDR1 consisting of sequence number 662, CDR2 consisting of sequence number 692, and CDR3 consisting of sequence number 667, (2) CDR1 consisting of sequence number 691, CDR2 consisting of sequence number 664, and CDR3 consisting of sequence number 667, (3) CDR1 consisting of sequence number 663, CDR2 consisting of sequence number 665, and CDR3 consisting of sequence number 667, (4) CDR1 consisting of sequence number 663, CDR2 consisting of sequence number 666, and CDR3 consisting of sequence number 667, (5) CDR1 consisting of sequence number 656, CDR2 consisting of sequence number 2, and CDR3 consisting of sequence number 658, (6) CDR1 consisting of sequence number 659, CDR2 consisting of sequence number 660, and CDR3 consisting of sequence number 661 The sdAb includes the aforementioned sdAb.

131. The sdAb according to any one of claims 128 to 130, wherein the sdAb competes with the fetal Fc receptor (FcRn) for binding to HSA. sdAb may have binding specificity to domain 1 of human serum albumin.

132. The sdAb according to any one of claims 128 to 131, wherein the sdAb is a heavy chain variable domain (VH), a heavy chain variable heavy chain domain (VHH), a novel antigen receptor single-domain shark variable domain (VNAR), or a light chain variable (VL) domain.

133. The sdAb according to claim 132, wherein the sdAb is a heavy chain variable domain (VH).

134. The sdAb according to claim 131 or 132, wherein the sdAb is a variable heavy chain domain (VHH) of the heavy chain.

135. The sdAb according to any one of claims 128 to 135, wherein the sdAb is a camelid type, a human type, or a humanized type.

136. The sdAb according to claim 135, wherein the sdAb is a humanized form.

137. The sdAb according to any one of claims 128 to 136, further comprising one or more framework regions.

138. The sdAb according to claim 138, wherein the sdAb includes a framework region derived from the human germline.

139. The sdAb according to any one of claims 128 to 138, wherein the sdAb is CDR1 containing the amino acid sequence of SEQ ID NO: 662, SEQ ID NO: 663, or SEQ ID NO: 691, Compilation R2 containing the amino acid sequence of SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, or SEQ ID NO: 692, CDR3 containing the amino acid sequence of SEQ ID NO: 667, and Including FR1, FR2, FR3, and FR4, The amino acid residue at position 24 of FR1 may be serine or alanine, the amino acid at position 44 of F2 may be glutamic acid or glycine, the amino acid at position 45 of F2 may be arginine or leucine, and the amino acid at position 79 of F3 may be leucine or valine, and FR1, FR2, FR3, and FR4 may be derived from the human IGHV3-23 germline, as described above.

140. The sdAb according to any one of claims 128 to 138, wherein the sdAb is CDR1 containing the amino acid sequence of SEQ ID NO: 656, CDR2 containing the amino acid sequence of SEQ ID NO: 657, CDR3 containing the amino acid sequence of SEQ ID NO: 658, and also includes FR1, FR2, FR3, and FR4, The amino acid residue at position 24 of FR1 may be serine or alanine, the amino acid at position 44 of FR2 may be aspartic acid or glycine, the amino acid at position 45 of FR2 may be arginine or leucine, the amino acid at position 49 of FR2 may be serine or alanine, the amino acid at position 78 of FR3 may be leucine or phenylalanine, the amino acid at position 79 of F3 may be asparagine or lysine, and The aforementioned FR1, FR2, FR3, and FR4 may be derived from the human IGHV3-23 germline, as described above.

141. The sdAb according to any one of claims 128 to 138, wherein the sdAb is CDR1 containing the amino acid sequence of SEQ ID NO: 659, CDR2 containing the amino acid sequence of SEQ ID NO: 660, CDR3 containing the amino acid sequence of SEQ ID NO: 661, and also includes FR1, FR2, FR3, and FR4, The amino acid residue at position 44 of F2 may be glutamic acid or glycine, the amino acid at position 45 of F2 may be arginine or leucine, the amino acid at position [78] of F3 may be threonine or serine, the amino acid at position 79 of F3 may be leucine or valine, and FR1, FR2, FR3, and FR4 may be derived from the human IGHV3-23 germline, as described above.

142. The sdAb according to any one of claims 128 to 141 further comprises: FR1 comprising any one amino acid sequence from SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 685, or any variant of the sequence comprising up to approximately three amino acid substitutions; FR2 comprising any one amino acid sequence from 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 any variant of the sequence comprising up to approximately three amino acid substitutions; FR3 comprising any one amino acid sequence from any one of SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 34, SEQ ID NO: 689, or SEQ ID NO: 690, or any variant of the sequence comprising up to approximately three amino acid substitutions; and FR4 comprising the amino acid sequence of SEQ ID NO: 677 or a variant thereof comprising up to approximately three amino acid substitutions.

143. The sdAb according to claim 143, wherein the sdAb is (1) FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 670 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (2) FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 671 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (3) FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 673 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (4) FR1 containing the amino acid sequence of SEQ ID NO: 669 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 671 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (5) FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 678 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 683 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (6) FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 679 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 683 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (7) FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 681 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 683 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (8) FR1 containing the amino acid sequence of SEQ ID NO: 669 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 681 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 683 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (9) FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 686 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (10) FR1 containing the amino acid sequence of SEQ ID NO: 668 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 681 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 675 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (11) FR1 containing the amino acid sequence of SEQ ID NO: 685 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 681 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 34 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (12) FR1 containing the amino acid sequence of SEQ ID NO: 685 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 686 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 689 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, (13) FR1 containing the amino acid sequence of SEQ ID NO: 685 or a variant thereof containing up to approximately 3 amino acid substitutions, FR2 containing the amino acid sequence of SEQ ID NO: 33 or a variant thereof containing up to approximately 3 amino acid substitutions, FR3 containing the amino acid sequence of SEQ ID NO: 689 or a variant thereof containing up to approximately 3 amino acid substitutions, and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions, or (14) FR1 containing the amino acid sequence of SEQ ID NO: 685 or a variant thereof containing up to approximately 3 amino acid substitutions; FR2 containing the amino acid sequence of SEQ ID NO: 681 or a variant thereof containing up to approximately 3 amino acid substitutions; FR3 containing the amino acid sequence of SEQ ID NO: 689 or a variant thereof containing up to approximately 3 amino acid substitutions; and FR4 containing the amino acid sequence of SEQ ID NO: 677 or a variant thereof containing up to approximately 3 amino acid substitutions. The sdAb includes the aforementioned sdAb.

144. The sdAb according to any one of claims 128 to 143, wherein the sdAb comprises one amino acid sequence of sequence numbers 286 to 290 or 608 to 648, or an amino acid sequence that is at least about 85% identical to one amino acid sequence of sequence numbers 286 to 290 or 608 to 648.

145. The sdAb according to any one of claims 128 to 144, wherein the sdAb may include the amino acid sequence of SEQ ID NO: 608, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

608.

146. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 609, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

609.

147. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 611, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

611.

148. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 618, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

618.

149. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 622, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

622.

150. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 624, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

624.

151. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 625, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

625.

152. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 626, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

626.

153. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 628, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

628.

154. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 632, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

632.

155. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 635, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

635.

156. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 637, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

637.

157. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 642, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

642.

158. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 644, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

644.

159. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 645, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

645.

160. The sdAb according to any one of claims 128 to 144, comprising the amino acid sequence of SEQ ID NO: 646, or an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO:

646.

161. A nucleic acid encoding sdAb according to any one of claims 128 to 160.

162. A vector comprising the nucleic acid according to claim 161.

163. A host cell comprising the vector according to claim 162.

164. a) sdAb according to any one of claims 128 to 160, and b) Target domain A fusion polypeptide containing [the specified component].

165. The fusion polypeptide according to claim 164, wherein the domain sequence for the purpose is a polypeptide having biological activity.

166. A fusion polypeptide according to any one of claims 164 or 165, further comprising a potentially cleavable linker, wherein the potentially cleavable linker is operably linked to the sdAb and the desired domain acid sequence.

167. The fusion polypeptide according to claim 166, wherein the polypeptide having biological activity comprises a cytokine.

168. The fusion polypeptide according to any one of claims 164 to 167, wherein the protease-cleavable linker comprises a sequence that can be cleaved by a protease selected from kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), ADAM, FAP, plasminogen activator, caspase, tryptase, or tumor protease.

169. The fusion polypeptide according to claim 168, wherein the protease is selected from cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, or cathepsin G.

170. The fusion polypeptide according to claim 168, wherein the protease is selected from matrix metalloproteases (MMPs) that are MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, or MMP14.

Images