Activatable il-21 polypeptides and methods of use thereof and single domain human serum albumin antibodies

Abstract

Provided herein are IL-21 polypeptide prodrugs comprising IL-21, a half-life extension element, an IL-21 blocking element and a protease cleavable linker. Also provided herein are pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, host cells for making such polypeptide prodrugs. Also disclosed are methods of using the polypeptide prodrugs in the treatment of diseases, conditions and disorders.

Description

Attorney Docket No.: 761146.332320 ACTIVATABLE IL-21 POLYPEPTIDES AND METHODS OF USE THEREOF AND SINGLE DOMAIN HUMAN SERUM ALBUMIN ANTIBODIES

[0001] The present application claims the benefit of U.S. Provisional Application No.63 / 585,550, filed on September 26, 2023, U.S. Provision Application No.63 / 594,006, filed on October 29, 2023, U.S. Provision Application No.63 / 563,713, filed on March 11, 2024, U.S. Provision Application No. 63 / 575,026, filed on April 5, 2024, the contents of each of which are incorporated herein by reference. 1. BACKGROUND

[0002] Interleukin-21 (IL-21) is a is a cytokine that is produced mainly by T cells and natural killer T cells that has pleiotropic actions on a wide range of immune and non-immune cell types. IL-21 has diverse effects on a broad range of cell types including, but not limited to, CD4+and CD8+T cells, B cells, macrophages, monocytes, and dendritic cells (DCs). The functional receptor for IL-21 is composed of the IL-21 receptor (IL-21R) and the common cytokine receptor 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 has been considered a promising therapeutic agent for oncology. Unfortunately, due to the biology of cytokines and the inability to effectively target and control their activity, cytokines have not achieved the hoped for clinical advantages in the treatment of tumors. This is exacerbated by the need to administer large quantities of cytokines (i.e., IL-21) in order to achieve the desired levels of cytokine at the intended site of cytokine action (e.g., a tumor microenvironment).

[0004] Inducible IL-12, IL-2, and interferon prodrugs have been described in International Publication Nos.: WO2019 / 222294, WO2019 / 222295, WO2019 / 222296, WO2021 / 097376.

[0005] There remains an unmet medical need for improved methods for treating cancer. 2. SUMMARY

[0006] The disclosure relates to inducible IL-21 prodrugs that contain at least one polypeptide chain, and can contain two or more polypeptides, if desired. The inducible IL-21 prodrug comprises an IL-21 polypeptide, an IL-21 blocking element, a half-life extension element, and a protease cleavable polypeptide linker.

[0007] Inducible IL-21 prodrugs of this disclosure have attenuated IL-21 receptor agonist activity and the circulating half-life is extended. The inducible IL-21 receptor agonist activity is attenuated through the blocking element. The half-life extension element can also contribute to attenuation, for example through steric effects. The blocking element is capable of blocking all or some of the receptor agonist activity of the IL-21 by noncovalently binding to the IL-21 and / or sterically blocking receptor binding. Upon cleavage of the protease cleavable linker a form of the IL-21 is released that is active (e.g., more 1 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 active than the IL-21 polypeptide prodrug). Typically, the released IL-21 is at least 10 x more active than the IL-21 polypeptide prodrug. Preferably, the released IL-21 is at least 20 x, at least 30 x, at least 50 x, at least 100 x, at least 200 x, at least 300 x, at least 500 x, at least 1000 x, at least about 10,000 x or more active than the inducible IL-21 prodrug.

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

[0009] The inducible IL-21 prodrug can comprise at least one of each of an IL-21 polypeptide [A], an IL-21 blocking element [D], a half-life extension element [H], and a protease-cleavable polypeptide linker [L]. The IL-21 polypeptide and the IL-21 blocking element or the half-life extension element can be operably linked by the protease-cleavable polypeptide linker and the inducible IL-21 prodrug has attenuated IL-21 receptor activating activity. The IL-21 receptor activating activity of the inducible IL-21 prodrug is at least about 10X less than the IL-21 receptor activating activity of the polypeptide that contains the IL-21 polypeptide that is produced by cleavage of the protease cleavable linker.

[0010] The inducible IL-21 prodrug can have the formula:

[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].

[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 polypeptide linker that is optionally protease-cleavable, and [L2’] is a protease-cleavable polypeptide linker. L2 can be a protease-cleavable polypeptide linker. L1 or L2 or both L1 and L2 are cleaved by two or more different proteases.

[0018] The inducible IL-21 prodrug disclosed herein can comprise an antigen binding fragment of an antibody as the IL-21 blocking element. The antigen binding fragment of an antibody can comprise as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain. The inducible IL-21 prodrug can comprise: a first polypeptide comprising the IL-21 polypeptide, at least an antigen binding portion of an 2 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 antibody light chain or an antigen binding portion of an antibody heavy chain, and the half-life-extension element; wherein the IL-21 polypeptide and the antigen binding portion of the antibody light chain or the antigen binding portion of the antibody heavy chain and / or half-life extension element are operably linked by the protease cleavable linker; and second polypeptide comprising at least an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the first polypeptide, or an antibody light chain that is complementary to the heavy chain in the first polypeptide and together with said light chain forms an IL-21 binding site.

[0019] The inducible IL-21 prodrug can contain two polypeptide chains. The first polypeptide chain can comprise an IL-21 polypeptide, a half-life extension element, a first IL-21 blocking element, a protease- cleavable polypeptide linker, and a portion of an antibody heavy chain constant region or a light chain; wherein the first blocking element is IL-21R. The IL-21 polypeptide and the first blocking element and / or the half-life extension element are operably linked by the protease cleavable linker. The second polypeptide can comprise a second IL-21 blocking element and a portion of an antibody heavy chain constant region or an antibody light chain. The second blocking element and the portion of the antibody heavy chain constant region or the antibody light chain are linked through a linker that is optionally protease cleavable. The second blocking element can be a common gamma chain receptor. When the antibody heavy chain constant region is on the first polypeptide, the antibody light chain is on the second polypeptide and when the antibody heavy chain constant region is on the second polypeptide, the antibody light chain is on the first polypeptide.

[0020] The inducible IL-21 prodrug can comprise a first polypeptide selected from the group consisting of SEQ ID NOs.18-23 and 26-31, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 18-23 and 26-31. The inducible IL-21 prodrug can comprise a second polypeptide selected from the group consisting of SEQ ID NO.17 or 25 or an amino acid sequence that has at least 80% identity to SEQ ID NO.17 or 25.

[0021] The inducible IL-21 prodrug can comprise a first polypeptide selected from the group consisting of SEQ ID NOs.18-23 and 26-31, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 18-23 and 26-31, and a second polypeptide selected from the group consisting of SEQ ID NO.17 or 25 or an amino acid sequence that has at least 80% identity to SEQ ID NO.17 or 25.

[0022] The inducible IL-21 prodrug can comprise a first polypeptide chain selected from the group consisting of SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65. The inducible IL-21 prodrug can further comprise 3 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 a second polypeptide chain selected from the group consisting of SEQ ID NOs.: 10, 40, 52, and 64 or an amino acid sequence that has at least 80% identity to SEQ ID NOs.: 10, 40, 52, and 64.

[0023] The inducible IL-21 prodrug can comprise a first polypeptide chain selected from the group consisting of SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 39, 41, 51, 58, 59, 63, and 65, and a second polypeptide chain selected from the group consisting of SEQ ID NOs.: 10, 40, 52, and 64 or an amino acid sequence that has at least 80% identity to SEQ ID NOs.: 10, 40, 52, and 64.

[0024] The inducible IL-21 prodrug can comprise the amino acid sequence selected from the group consisting of SEQ ID NOs.: 44-50 or an amino acid sequence that has at least 80% identity to SEQ ID NOs: and 44-50.

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

[0026] The disclosure relates to inducible IL-21 prodrug comprising a first polypeptide selected from the group consisting of SEQ ID NOs.72, 110-113, 117-126, 130-136, 138-144, 153-194, 221-235, 241-243, 245-247, 250, 251, 253-255, 257, 258, 260-264, 267-269, 271-290, 475, 476, and 449-463, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 72.110-113, 117-126, 130-136, 138-144, 153-208, 221-235, 241-243, 245-247, 250, 251, 253-255, 257, 258, 260-264, 267-269, 271-290, 475, and 476, and a second polypeptide selected from the group consisting of SEQ ID NO.71, 127-129, 137, or 300-306 or an amino acid sequence that has at least 80% identity to SEQ ID NO.71, 127-129, 137, or 300-306. The inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 130, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 129. The inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 131, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 129. The inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 140, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 137. The inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 143, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 137. The inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid sequence of SEQ ID NO: 169, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 301. The inducible IL-21 prodrug can comprise a first polypeptide comprising the amino acid 4 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 sequence of SEQ ID NO: 185, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 303.

[0027] The half-life extension element can comprise a serum albumin binding domain, a serum albumin, transferrin, or immunoglobulin Fc, or fragment thereof. The half-life extension element can also be a blocking element.

[0028] The IL-21 blocking element can comprise a ligand-binding domain or fragment of a cognate receptor for the IL-21 polypeptide, or an antibody or antigen-binding fragment of an antibody that binds to the IL-21 polypeptide. The IL-21 blocking element inhibits activation of the IL-21 receptor by the inducible IL-21 prodrug.

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

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

[0031] The cathepsin can be cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, or cathepsin G. The matrix metalloprotease (MMP) is MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP19, or MMP20.

[0032] The disclosure also relates to a nucleic acid encoding the inducible IL-21 prodrug. The nucleic acid can comprise a circular vector. The nucleic acid can comprise DNA. The nucleic acid can comprise RNA. Disclosed herein are methods of making the pharmaceutical composition comprising culturing the host cell under suitable conditions for expression and collection of the inducible IL-21 prodrug. Disclosed herein is a nucleic acid composition comprising one or more nucleic acid sequences encoding an inducible IL-21 prodrug. The inducible IL-21 prodrug comprises at least one of each of an IL-21 polypeptide [A]; an IL-21 blocking element [D]; a half-life extension element [H]; and a protease- cleavable polypeptide linker [L]. The IL-21 polypeptide and the IL-21 blocking element or the half-life extension element are operably linked by the protease-cleavable polypeptide linker and the inducible IL- 21 prodrug has attenuated IL-21 receptor activity.

[0033] The nucleic composition can comprise one or more nucleic acid sequences encoding an inducible IL-21 prodrug containing two polypeptide chains. The inducible IL-21 prodrug can comprise an antigen 5 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 binding fragment of an antibody as the IL-21 blocking element. The antigen binding fragment of an antibody can comprise as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain. The inducible IL- 21 prodrug can comprise: a first polypeptide comprising the IL-21 polypeptide, at least an antigen binding portion of an antibody light chain or an antigen binding portion of an antibody heavy chain, and the half- life-extension element; wherein the IL-21 polypeptide and the antigen binding portion of the antibody light chain or the antigen binding portion of the antibody heavy chain and / or half-life extension element are operably linked by the protease cleavable linker; and second polypeptide comprising at least an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the first polypeptide, or an antibody light chain that is complementary to the heavy chain in the first polypeptide and together with said light chain forms an IL-21 binding site.

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

[0035] The disclosure also relates to therapeutic methods that include administering to a subject in need thereof an effective amount of an inducible IL-21 prodrug, nucleic acid that encodes the inducible IL-21 prodrug, vector or host cells that contain such a nucleic acid, and pharmaceutical compositions of any of the foregoing. Typically, the subject has, or is at risk of developing cancer, a proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease, or a host-versus-graft disease. The methods disclosed herein are particularly suitable for treating cancer. The inducible IL-21 prodrug can be administered intravenously. 6 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0036] The disclosure relates to single domain antibody fragments (sdAbs) (such as VHH, and VH) that have binding specificity for human serum albumin, methods of making, and uses thereof. Such sdAbs can be used, for example, for extending the in vivo serum half-life of therapeutic agents (e.g., proteins, peptides and the like) through the engineering of fusion proteins and conjugates using well-known methods and as described herein. Preferred sdAbs compete with FcRn (human FcRn) for binding to human serum albumin and, preferably, bind to domain 1 (DI) of human serum albumin.

[0037] As described and exemplified herein, contrary to the well-accepted knowledge in the art that anti- albumin binding domains that interfere with albumin binding to FcRn are not suitable for half-life extension applications (e.g., by inclusion in fusion proteins or conjugates with short half-life therapeutic proteins), the inventors have discovered that sdAbs that compete with FcRn (human FcRn) for binding to human serum albumin can effectively extend the half-life (e.g., the in vivo serum half-life) of short half- life therapeutic agents (e.g., proteins, peptides).

[0038] The sdAbs disclosed herein comprise a CDR1, CDR2, and a CDR3. The CDR1 comprises any one of SEQ ID NO: 656, SEQ ID NO: 659, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 691, or a variant of any of the foregoing comprising up to about 2 amino acid substitutions. The CDR2 comprises any one of SEQ ID NO: 657, SEQ ID NO: 660; SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 692, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions. The CDR3 comprises any one of SEQ ID NO: 658, SEQ ID NO: 661, SEQ ID NO: 667 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions.

[0039] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 656, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 657, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 658, or a variant thereof comprising up to about 3 amino acid substitutions.

[0040] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 659, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO:660, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO:660, or a variant thereof comprising up to about 3 amino acid substitutions.

[0041] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 664, or a variant thereof comprising up to about 3 amino acid substitutions; and 7 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

[0042] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 665, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

[0043] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 666, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

[0044] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 692, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

[0045] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 664, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

[0046] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 665, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

[0047] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 666, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions. 8 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0048] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 692, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

[0049] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 664, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

[0050] The sdAb can comprise CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 665, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

[0051] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 666, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

[0052] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 692, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

[0053] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 662; a CDR2 that consists of SEQ ID NO: 692; and a CDR3 that consists of SEQ ID NO: 667.

[0054] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 691; a CDR2 that consists of SEQ ID NO: 664; and a CDR3 that consists of SEQ ID NO: 667.

[0055] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 663; a CDR2 that consists of SEQ ID NO: 665; and a CDR3 that consists of SEQ ID NO: 667. 9 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0056] The sdAb can comprise a CDR1 that consists of SEQ ID NO: 663; a CDR2 that consists of SEQ ID NO: 666; and a CDR3 that consists of SEQ ID NO: 667.

[0057] The sdAb can comprise CDR1 that consists of SEQ ID NO: 656; a CDR2 that consists of SEQ ID NO: 2; and a CDR3 that consists of SEQ ID NO: 658.

[0058] The sdAb can comprise CDR1 that consists of SEQ ID NO: 659; a CDR2 that consists of SEQ ID NO:660; and a CDR3 that consists of SEQ ID NO: 661.

[0059] The sdAbs disclosed herein can compete with neonatal Fc receptor (FcRn) for binding to HSA. The sdAbs can have binding specificity for domain one of human serum albumin.

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

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

[0062] The sdAb’s disclosed herein further comprise one or more framework regions. The framework region can be derived from a human germline.

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

[0064] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 656; a CDR2 comprising the amino acid sequence of SEQ ID NO: 657; a CDR3 comprising the amino acid sequence of SEQ ID NO: 658; and a FR1, a FR2, a FR3, and a FR4. The amino acid residue at position 24 of FR1 can be a serine or an alanine, the amino acid at position 44 of FR2 can be a aspartic acid or a glycine; the amino acid at position 45 of FR2 can be a arginine or a leucine; the amino acid at position 49 of FR2 can be a serine or alanine, the amino acid at position 78 of FR3 can be a leucine or a phenylalanine, and the amino acid at position 79 of F3 can be a asparagine or a lysine. The FR1, FR2, FR3, FR4 can be from human IGHV3-23 germ line.

[0065] The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 659; a CDR2 comprising the amino acid sequence of SEQ ID NO: 660; a CDR3 comprising the amino acid sequence of SEQ ID NO: 661; and a FR1, a FR2, a FR3, and a FR4. The amino acid residue at position 10 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 44 of F2 can be a glutamic acid or a glycine, the amino acid at position 45 of F2 can be a arginine or a leucine, the amino acid at position

[0078] of F3 can be a threonine or a serine, and the amino acid at position 79 can be F3 is a leucine or a valine. The FR1, FR2, FR3, FR4 can be from human IGHV3-23 germ line.

[0066] The sdAbs disclosed herein can further comprises a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 685, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 686, SEQ ID NO:33, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 34, SEQ ID NO: 689, or SEQ ID NO: 690, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0067] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 670, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0068] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 671, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0069] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 673, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions. 11 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0070] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 669, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 671, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0071] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 678, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0072] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 679, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0073] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0074] The sdAb can comprise FR1 comprising the amino acid sequence of SEQ ID NO: 669, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0075] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid 12 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 sequence of SEQ ID NO: 686, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0076] The sdAb can comprise FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0077] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0078] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 686, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 689, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0079] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 689, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0080] The sdAb can comprise a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 689, or a variant thereof comprising up to about 13 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

[0081] The sdAb can comprise the amino acid sequence of any one of SEQ ID NOs: 286-290 or 608- 648, or an amino acid sequence of at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 286-290 or 608-648.

[0082] The sdAb can comprise the amino acid sequence of SEQ ID NO: 608, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 608.

[0083] The sdAb can comprise the amino acid sequence of SEQ ID NO: 609, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 609.

[0084] The sdAb can comprise the amino acid sequence of SEQ ID NO: 611, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 611.

[0085] The sdAb can comprise the amino acid sequence of SEQ ID NO: 618, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 618.

[0086] The sdAb can comprise the amino acid sequence of SEQ ID NO: 622, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 622.

[0087] The sdAb can comprise the amino acid sequence of SEQ ID NO: 624, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 624.

[0088] The sdAb can comprise the amino acid sequence of SEQ ID NO: 625, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 625.

[0089] The sdAb can comprise the amino acid sequence of SEQ ID NO: 626, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 626.

[0090] The sdAb can comprise the amino acid sequence of SEQ ID NO: 628, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 628.

[0091] The sdAb can comprise the amino acid sequence of SEQ ID NO: 632, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 632.

[0092] The sdAb can comprise the amino acid sequence of SEQ ID NO: 635, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 635.

[0093] The sdAb can comprise the amino acid sequence of SEQ ID NO: 637, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 637.

[0094] The sdAb can comprise the amino acid sequence of SEQ ID NO: 642, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 642. 14 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0095] The sdAb can comprise the amino acid sequence of SEQ ID NO: 644, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 644.

[0096] The sdAb can comprise the amino acid sequence of SEQ ID NO: 645, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 645.

[0097] The sdAb can comprise the amino acid sequence of SEQ ID NO: 646, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO: 646.

[0098] The sdAbs disclosed herein can comprise a half-life of at least about 5 hours to about 40 hours.

[0099] The disclosure further relates to pharmaceutical compositions comprising the sdAbs disclosed herein or nucleic acids encoding the sdAbs.

[0100] The disclosure further relates to a nucleic acid encoding the sdAbs disclosed herein. The nucleic acid encoding sdAbs can be a circular vector, DNA, RNA, or an expression vector comprising a circular vector, DNA, or RNA. Additionally, the disclosure relates to an isolated host cell comprising an expression vector encoding for a nucleic acid of a sdAb.

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

[0102] The disclosure further relates to a nucleic acid encoding the fusion polypeptide. The nucleic acid composition encoding fusion polypeptide can be a circular vector, DNA, RNA, or an expression vector comprising a circular vector, DNA, or RNA. Additionally, the disclosure relates to an isolated host cell comprising an expression vector encoding for a nucleic acid composition of an fusion polypeptide.

[0103] The disclosure also relates to a pharmaceutical composition of the fusion polypeptide and methods of making pharmaceutical compositions of the fusion polypeptide. The disclosure further relates to methods for treating a disease, such as cancer, that comprise administering to a subject in need thereof an effective amount of the fusion polypeptide, a nucleic acid encoding the fusion polypeptide, an expression vector for the fusion polypeptide, or a pharmaceutical composition comprising an effective amount of any of these. 15 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] The drawings are not necessarily to scale or exhaustive. Instead, the emphasis is generally placed upon illustrating the principles of the inventions described herein. The accompanying drawings, which constitute a part of the specification, illustrate several embodiments consistent with the disclosure and, together with the description, serve to explain the principles of the disclosure. In the drawings:

[0105] FIGs.1A-1O are schematic illustrations depicting various inducible IL-21 prodrugs.

[0106] FIG.2 is a SDS-PAGE gel showing the results of protein cleavage assays. The IL-21 prodrugs were run in both cleaved and uncleaved form.

[0107] FIGs.3A is a graph showing results of analyzing WW50008 (half-life extended mouse IL-21) in a syngeneic MC38 mouse tumor model. It shows average tumor volume over time in mice treated with 1 μg, 10 μg, 100 μg and 300 μg of WW50008. WW50008 was dosed twice a week for 3 weeks (total of 6 doses). Data show 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 with the 100ug dose level. The 300ug dose level was not tolerated by the animals.

[0108] FIG.3B is a graph showing body weight averages for the animals treated in data shown in FIG. 3A. Each line in the plot represents the body weight average of the group over time. The 300 µg dose of WW50008 was not tolerated by the animals.

[0109] FIG.4 is a graph showing results of analyzing WW50008 in a syngeneic B16F10 mouse tumor model. It shows average tumor volume over time in mice treated with 10 μg, 33 μg, 100 μg and 200 μg WW50008 (square, triangle upside, triangle downside and diamond respectively). Vehicle alone is indicated by circle. The data show tumor volume was inhibited over time in a dose-dependent manner in mice treated with WW50008 at the higher concentrations.

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

[0111] FIG.6 is a graph showing results of analyzing WW50008 (half-life extended mouse IL-21) in a syngeneic EMT6 mouse tumor model. It shows average tumor volume over time in mice treated with 10 μg, 33 μg, 100 μg and 200 μg WW50008 (square, triangle upside, triangle downside and diamond respectively). Vehicle alone is indicated by circle. The data show tumor volume was inhibited over time in a dose-dependent manner in mice treated with WW50008 at the concentrations of 10 μg and 33μg. WW50008 was not tolerated at 200 μg dose in this model. 16 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

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

[0113] FIGs.8A-8D shows the results of the efficacy and TILs analysis of MC38 tumors treated with a half-life extended IL-21 (WW50008). FIG.8A is a graph showing tumor volume (mm3) in mice treated with vehicle or 100 µg WW50008 on days 1, 4, 8 and 11. FIG.8B are graphs showing an increase in lymphocyte infiltrates in WW50008 treated group compared to vehicle as measured by total cells per mg of tissue and frequency of CD45+ TILs. FIG.8C are graphs showing NK cell counts and NK IFN- gamma+ cell frequency in mice treated with vehicle and WW50008. FIG.8D are graphs showing frequency of CD8+ Tetramer+ T cells (Granzyme B+, INF-gamma+, and TNF+) in mice treated with vehicle and WW50008.

[0114] FIGs.9A-9E shows the results of the efficacy and TILs analysis of EMT-6 tumors upon in vivo dosing with half-life extended human IL-2 (WW0177) or half-life extended mouse IL-21 (WW50008). FIG.9A is a graph showing tumor volume in mice treated with vehicle, WW0177 (10 µg) or WW50008 (100 µg) on days 1, 4, 8, and 11. FIG.9B shows frequency of CD8 and CD8+ granzyme B+, INF- gamma+, and TNF+ infiltrates in vehicle, WW0177 and WW50008 treated groups. FIG.9C are graphs showing polyfunctionality of cytokine production in vehicle, WW0177 and WW50008 treated CD8+ T cells. FIG.9D are graphs showing the frequency of Treg cells and CD8 / Treg ratio in Vehicle, WW0177, and WW5008 treated groups. FIG.9E shows a heat map depicting the differential gene expression in tumors treated with WW0177 and WW50008 using NanoString analysis.

[0115] FIGs.10A and 10B show efficacy of half-life extended IL-21 and IL-21 prodrug at different dose levels (dose dependent effect) (FIG.10A) in the MC38 mouse model. No detrimental effect observed in body weight of these animals at any of the dose levels tested (FIG.10B). FIGs.11A-11D is a graph showing the results of EMT-6 mouse tumor re-challenge. FIG.11A is a graph showing tumor volume over time in mice treated with half-life extended human IL-2 (squares), half-life extended mouse IL-21 (triangles), and vehicle (circles). The majority of animals treated with half-life extended IL-21 survived the study and were used for a re-challenge study. FIG.11B is a graph showing average tumor volume over days post re-challenge in mice that survived EMT-6 challenge (see, FIG 11A triangles) and were confirmed to have complete regression of tumors. These animals were re-challenged with the EMT-6 cell line (circles) and naïve mice were used as controls (square). Only naïve animals developed tumors. The results show that treatment with half-life extended IL-21 induced immunological memory to EMT-6. FIG.11C are representative microscopic images from an IFN ^ ELISPOT assay. 17 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Splenocytes were isolated EMT-6 tumor bearing mice that had complete remission (CR) following treatment with half-life extended human IL-2 or half-life extended mouse IL-21, and splenocytes were incubated with EMT-6 cells for 24 hours. Splenocytes responded to EMT-6 cells and produced IFN ^. FIG.11D is a graph depicting the average relative spot volume on the IFNγ ELISPOT plate. The results show that treatment with IL-2 and IL-21 induced immunological memory to EMT-6.

[0116] FIGs.12A-12D are graphs showing that half-life extended IL-21 and inducible IL-21 prodrug have comparable anti-tumor activity in a syngeneic MC38 tumor model using mice that express human IL-21 receptor, but that inducible IL-21 prodrug (FIG.12C) has better tolerability and a broader therapeutic window. FIGs.12A and 12C show average tumor volume over days in mice treated with half- life extended IL-21 (FIG.12A) and inducible IL-21 prodrug (FIG.12C). FIGs. 12B and 12D show body weight averages for the animals treated in data shown in FIGs.12A and 12C. Each line in the plot represents the body weight average of the group over time. The therapeutic window is maximum tolerated dose (MTD) / minimum effective dose (MED).

[0117] FIGs.13A-13C are graphs showing results of analyzing WW50334 in a syngeneic MC38 tumor model using mice that express human IL-21 receptor. FIG.13A shows average tumor volume over time in mice treated with 3.3 μg, 10 μg, 33 μg, and 100 μg (dose per mouse per injection) of WW50334. FIGs. 13B is a graph showing body weight averages for the animals treated in FIG.13A. Each line in the plot represents the body weight average of the group over time. FIG.13C are graphs showing body weights for animals treated in FIG.13A. The total nanomoles of WW50334 delivered per mouse over the dosing period are shown. The top dose of 100 μg was not tolerated, inducing animal deaths.

[0118] FIGs.14A-14C are graphs showing results of analyzing WW50112 / WW50347 in a syngeneic MC38 tumor model in mice that express human IL-21 receptor. FIG.14A shows average tumor volume over time in mice treated with 200 μg, 600 μg, and 1800 μg (dose per mouse per injection) of WW50112 / WW50347. FIGs.14B is a graph showing body weight averages for the animals treated in FIG.14A. Each line in the plot represents the body weight average of the group over time. FIG.14C are graphs showing body weights for each animal treated in FIG.14A. The total nanomoles of WW50334 delivered per mouse over the dosing period are shown.

[0119] FIGs.15A-15C are graphs showing results of analyzing WW50112 / WW50348 in a syngeneic MC38 tumor model in mice that express human IL-21 receptor. FIG.15A shows average tumor volume over time in mice treated with 200 μg, 600 μg, and 1800 μg (dose per mouse per injection) of WW50112 / WW50348. FIGs.15B is a graph showing body weight averages for the animals treated in 18 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 FIG.15A. Each line in the plot represents the body weight average of the group over time. FIG.15C are graphs showing body weights for each animal treated in FIG.15A.

[0120] FIGs.16A and 16B are graphs showing results of analyzing WW50260 in a syngeneic MC38 tumor model in mice that express human IL-21 receptor. FIG.16A shows average tumor volume over time in mice treated with 142 μg, 425 μg, and 1,277 μg (dose per mouse per injection) of WW50260. FIGs.16B is a graph showing body weight averages for the animals treated in FIG.16A. Each line in the plot represents the body weight average of the group over time.

[0121] FIGs.17A-17D are graphs showing results of analyzing WW50334 (human IL-21 half-life extended), WW50260, WW50347 / WW50112, WW050348 / WW50112 in a syngeneic MC38 tumor model in mice that express human IL-21 receptor. FIG.17A shows the corresponding average serum levels of WW50334 over time. FIG.17B shows the corresponding average serum levels of WW50260 over time. FIG.17C shows the corresponding average serum levels of WW50112 / WW50347 over time. FIG.17D shows the corresponding average serum levels of WW50112 / WW50348 over time.

[0122] FIGs.18A-18G are graphs showing exposure of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysate. FIG.18A shows WW050334 (human IL-21 half-life extended) over time in serum (triangles) and in tumor lysate (squares). FIG.18B shows intact WW50260 over time in serum (triangles) and in tumor lysate (squares) and FIG.18C shows cleaved WW50260 over time in serum (triangles) and in tumor lysate (squares). FIG.18D shows intact WW50347 / WW50112 over time in serum (triangles) and in tumor lysate (squares) and FIG.18E shows cleaved WW50347 / WW50112 over time in serum (triangles) and in tumor lysate (squares). FIG.18F shows intact WW50348 / WW50112 over time in serum (triangles) and in tumor lysate (squares) and FIG.18G shows cleaved WW50348 / WW50112 over time in serum (triangles) and in tumor lysate (squares).

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

[0124] FIGs.20A and 20B show the results of the TILs analysis in EMT-6 tumors upon in vivo dosing with half-life extended mouse IL-21 and vehicle in the EMT-6 mouse model. FIG.20A shows a heat map depicting the differential gene expression in IL-21 and vehicle. FIG.20B shows a graph showing the fold change in genes expressed between IL-21 and vehicle.357 significant differentially expressed genes were identified.

[0125] FIGs.21A-21B shows the results of the TILs analysis in EMT-6 tumors upon in vivo dosing with half-life extended mouse IL-21 and half-life extended human IL-2 in the EMT-6 mouse model. FIG.21A shows a heat map depicting the differential gene expression in IL-21, IL-2, and vehicle. FIG.21B shows 19 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 a graph showing the fold change in genes expressed between IL-21 and IL-2.35 significant differentially expressed genes were identified.

[0126] FIGs.22A-22F are graphs showing results of analyzing inducible mouse IL-21 prodrugs in syngeneic MC38 mouse tumor model. They show average tumor volume over time in mice treated with 250 μg, 1,000 μg, and 1500 μg (dose per mouse per injection) of inducible IL-21 prodrug. Mice were dosed twice a week for two weeks (total of 4 doses). The following constructs were analyzed: IL-21 half- life extended (WW50008, FIG.22A), WW50076 (FIG.22B), WW50079 (FIG.22C), WW50080 (FIG. 22D), WW50256 (FIG.22E), WW50290 (FIG.22F). Data show that tumor volume was inhibited over time in a dose-dependent manner in mice treated with WW50076 (FIG.31B), WW50256 (FIG.22E), and WW50290 (FIG.22F). The 1,000 μg, and 1500 μg doses were not tolerated in mice treated with WW50079 and WW50080.

[0127] FIGs.23A-32F are graphs showing body weight averages for animals treated in data shown in FIGs.22A-22F. Each line in the plot represents the body weight average of the group over time.

[0128] FIGs.24A-24O are graphs showing body weight for each animal treated in data shown in FIGs. 22A-22F. Each line in the plot represents the body weight of the animal over time.

[0129] ).

[0130] FIGs.25A-25H are graphs showing results of analyzing inducible IL-21 prodrugs in a syngeneic MC38 mouse tumor model in human IL-21 receptor knock-in mice. It shows average tumor volume over time in mice treated with 67 μg, 200 μg, 600 μg and 1800 μg (dose per mouse per injection) of WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50388 / WW50394, WW50465 / WW50473, or WW50468 / WW50473. Data show the tumor volume was inhibited over time in a dose-dependent manner.

[0131] FIGs.26A-26H are graphs showing body weight averages for the animals treated in data shown in FIGs.25A-25H. Each line in the plot represents the body weight average of the group over time.

[0132] FIGs.27A-27H are graphs showing the corresponding average serum levels of WW50387 / WW50394, WW50454 / WW50464, WW50446 / WW50463, WW50467 / WW50473, WW50385 / WW50394, WW50465 / WW50473, WW50388 / WW50394 and WW50468 / WW50473, respectively, for the animals treated in data shown in FIGs.25A-25H and 26A-26H.

[0133] FIGs.28A-28P are graphs showing exposure of intact and cleaved inducible IL-21 prodrugs in serum and tumor lysate. 20 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0134] FIGs.29A-29C are images of capillary western blots showing the stability in murine serum (FIG. 29A), cynomolgus murine serum (FIG. 29B), and / or pooled human serum (FIG.29C) of inducible IL-21 prodrugs. All inducible IL-21 prodrugs were stable in serum, showing no cleavage species.

[0135] FIG.30 is a graph showing results of analyzing mean tumor volumes after dosing WW50387 / WW50394 administered at 47, 94, 188, 375, 750, 1200, and 1500 µg on Days 0, 3, 7, and 10 after treatment initiation. The study was carried out through Day 28 after treatment initiation, measuring tumor volumes 2x weekly during the course of the study. Data show the tumor volume was inhibited over time in a dose-dependent manner. The minimum efficacious dose (MED), defined as the dose at which 50% of the subjects showed tumor control during the dosing period was determined to be 94 µg, while the maximum tolerated dose (MTD), defined as the dose where no subjects were lost due to drug toxicity, was determined to be 1500 µg. The therapeutic window for WW50387 / WW50394 in this syngeneic tumor volume was determined to be 16.

[0136] FIGs.31A-31H are graphs showing the individual plots of treatment with WW50387 / WW50394 which reveals that tumor volume decreases with increasing dose level up to 1500 µg. FIGs.31I-31J show tumor volume for the 188ug and 375ug dose levels respectively for the non-cleavable control.

[0137] FIGs.32A and 32B are graphs showing body weight of the mice over time after the initiation of dosing with the inducible IL-21 prodrug. FIG.32A shows the plots of mean body weight of the mice expressed as grams across the dosing groups. The 750 µg and 1500 µg dose groups both showed a reduction in body weight at Day 14, 4 days after the last dose on Day 10. Both groups recovered their pretreatment body weights with the help of additional feeding with diet gels. FIG.32B shows the plots of mean body weight of the subjects expressed as percent body weight change from the initial measurement at Day -1 at the time of randomization across the dosing groups.

[0138] FIGs.33A-33J shows plots of the individual body weights for all subjects in all dosing groups. FIGs.33I-33J show tumor volume for the 188 µg and 375 µg dose levels respectively for the non- cleavable control.

[0139] FIG.34A and 34B show IL-21 prodrug treatment results in immunity to tumor rechallenge for animals deemed as complete responders from the initial study. FIG.34A shows the original group of tumor growth responses from where the CR animals were recruited. A total of 24 WW50387 / WW50394- treated mice with CR were rechallenged by inoculating with MC38-BC2 to the left flank on Day 88. FIG. 34B shows that all rechallenged mice rejected the second inoculation and did not show regrowth of their primary tumors, surviving until the last observation day (Day 116) while strain and age-matched naïve mice similarly inoculated showed a consistent tumor growth of their left flank tumors. 21 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0140] FIGs.35A-35H show the results of the efficacy and TILs analysis of MC38 tumors treated with WW50387 / WW50394. FIG.35is a graph showing tumor volume (mm3) in mice treated with WW50387 / WW50394. FIG.35B are graphs showing the percentage of M1 and M2 macrophages in mice treated with WW50387 / WW50394 and vehicle. FIGs.35C and 35D are graphs showing NK cell frequency and NK IFN-gamma+ cell frequency in mice treated with vehicle and WW50387 / WW50394. FIG.35E is a graph showing the frequency of CD4+ T conventional cells in mice treated with vehicle and WW50387 / WW50394. FIG.35F is a graph showing the percentage of CD8+ T cell infiltrates. FIG. 35G are graphs showing frequency of CD8+ Tetramer+ T cells (Granzyme B+, INF-gamma+, and TNF+) in mice treated with vehicle and WW50387 / WW50394. FIG.35H are pie graphs showing the polyfunctionality of cytokine CD8+ T cell production in mice treated with WW50387 / WW50394 and vehicle.

[0141] FIG.36 is a graph showing the percentage of total activity of WW50387 / WW50394 processing in primary human dissociated tumor samples (glioblastoma multiforme, colorectal cancer, cervical cancer, bladder cancer, and endometrial cancer).

[0142] FIGs.37A and 37B show B16F10-OVA tumor volume (FIG. 37A) and body weight (FIG.37B) for a range of doses with WW50387 / WW50394, including 125, 250, 500, and 1000 µg. Anti-tumor activity was observed across the dose range with complete control of tumor growth for the 250, 500, and 1000 µg dose levels through the dosing schedule and up to Day 21. All dosing regimens were well tolerated with no treatment-related deaths or considerable weight loss observed.

[0143] FIG.38A-38E show the individual tumor volumes for the treatment groups. These plots again show the superior control of tumors at the 250µg dose level of WW50387 / WW50394.

[0144] FIG.39 shows the complete abrogation of efficacy of WW50387 / WW50394 at the 500µg dose level when CD8 T cells were depleted using an αCD8 antibody, underscoring the effector T cell dependency for this B16F10-OVA model when treated with WW50387 / WW50394.

[0145] FIG.40A and 40B are plots of the exposure determined by the bioanalytical WW50387 / WW50394 assay in serum after dosing with WW50387 / WW50394. FIG.40A shows the dose proportional exposure that was detected in the serum ~24 hours after dosing of WW50387 / WW50394.  FIG.40B shows that this dose proportional exposure is also confirmed on Day 11, ~72 hours after the third dose of WW50387 / WW50394 administered on day 8.

[0146] FIGs.41A and 41B show the dose-normalized exposure data for the 125, 250, 500, and 1000 µg of WW50387 / WW50394 groups as well as the 500 µg dose of WW50387 / WW50394 / αCD8 depletion 22 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 group. Both Day 2 (FIG.41A) and Day 11 (FIG.41B) plots demonstrate dose-proportional exposure across the dose range.

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

[0148] FIGs.43A and 43B are graphs showing tumor volume after administration of WW50387 / WW50394 once weekly for a total of 2 doses (FIG.43A) and after a single dose (FIG.43B). FIG.43A is a graph showing results of mean tumor volumes after dosing WW50387 / WW50394 at 188, 375, and 750 µg on Days 0 and 7. FIG.43B is a graph showing results of analyzing mean tumor volumes after dosing WW50387 / WW50394 administered at 750, 1000, and 1500 µg on Days 0. Triangles below the X axis indicate dosing day.

[0149] FIGs.44A-44F are tables showing sequence alignments of humanized variants of llama anti- human serum albumin VHH antibodies. Alignments of each llama VHH, the chosen human germline Vh template protein(s), and humanized variants are shown in FIG.44A (1HUM19) FIG.44A discloses SEQ ID NOS 701-703 and 608-616, respectively, in order of appearance), FIG.44B (3HUM181) FIG.44B discloses SEQ ID NOS 704-706 and 625-634, respectively, in order of appearance), FIG.44C (2HUM45 on a IGHV3-23 backbone) ) FIG.44C discloses SEQ ID NOS 707-709 and 635-639, respectively, in order of appearance), FIG.44D, (2HUM45 on an IGHV3-11 backbone) FIG.44D discloses SEQ ID NOS 710-712 and 640-648, respectively, in order of appearance), FIG.44E (1HUM19 variants with modified CDR sequences) FIG.44E discloses SEQ ID NOS 713-715, 617-624 and 649-651, respectively, in order of appearance), and FIG.44F (3HUM181 variants with modified CDR sequences) FIG.44F discloses SEQ ID NOS 716-718, 652 and 653, respectively, in order of appearance). The CDRs are indicated.

[0150] FIGs.45A and 45B are graphs showing predicted pharmacokinetics (PK) (µg / mL) over time of anti-albumin VHHs with varying binding affinities to mouse (FIG.45A) or human serum albumin (FIG. 45B).

[0151] FIGs.46A and 46B are plots for efficacy (FIG.46A) and effect on body weight (FIG.46B) for the WW50387 / WW50394 + αPD-1 combination study. Generally, efficacy improves with the increased dose level of WW50387 / WW50394 and addition of αPD-1. All treatment regimens were well tolerated showing no decreases in body weight during the 28-day course of the study post treatment initiation.

[0152] FIGs.47A-47D show efficacy of WW50387 / WW50394 dosed at 94 µg (FIG.47A) or 375 µg (FIG.47B) with or without αPD-1. At the 94 µg dose were no differences between 23 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 WW50387 / WW50394, αPD-1, or the WW50387 / WW50394 + αPD-1 combination. At the 375 µg dose the WW50387 / WW50394 + αPD-1 combination was highly significantly (****p<0.0001) more efficacious than αPD-1 alone, but not WW50387 / WW50394 alone. (FIG.47C).

[0153] FIGs.48A and 48B are plots for efficacy (FIG.48A) and effect on body weight (FIG.48B) for the WW50387 / WW50394 + αPD-L1 combination study. Generally, efficacy improved with the increased dose level of WW50387 / WW50394 and addition of αPD-L1. All treatment regimens were well tolerated showing no decreases in body weight during the 28-day course of the study post treatment initiation.

[0154] FIGs.49A-4D show efficacy of WW50387 / WW50394 dosed at 94 µg (FIG.49A) or 375 µg (FIG.49B) with or without αPD-L1. At the 94 µg dose there was a trend for better efficacy when WW50387 / WW50394 was combined with αPD-L1, but this did not reach significance. At the 375 µg dose the WW50387 / WW50394+αPD-L1 combination was highly significantly (****p<0.0001) more efficacious than αPD-L1 alone, and significantly (**p<0.0063) more efficacious than WW50387 / WW50394 alone. Furthermore, the combinations showed increased CRs compared to 94 µg WW50387 / WW50394 alone (3 CRs vs 0 CRs) and 300 µg WW50387 / WW50394 alone (6 CRs vs 2 CRs) (FIG.49C, CR table summary in FIG.49D).

[0155] FIGs.50A and 50B are plots for efficacy (FIG.50A) and effect on body weight (FIG.50B) for the WW50387 / WW50394 + αCTLA-4 combination study. Generally, efficacy improved with the increased dose level of WW50387 / WW50394 and addition of αCTLA-4. All treatment regimens were well tolerated showing no decreases in body weight during the 28-day course of the study post treatment initiation.

[0156] FIGs.51A-51D show efficacy of WW50387 / WW50394 dosed at 94 µg (FIG.51A) or 375 µg (FIG.51B) with or without αCTLA-4. At the 94 µg dose there was highly significantly (****p<0.0001) better efficacy when WW50387 / WW50394 was combined with αCTLA-4 compared to either WW50387 / WW50394 or αCTLA-4 treatment alone. At the 375 µg dose the WW50387 / WW50394+ αCTLA-4 combination was highly significantly (****p<0.0001) more efficacious than αCTLA-4 alone, and there was a trend for better efficacy that did not reach significance when compared to WW50387 / WW50394 alone. Furthermore, the combinations showed increased CRs compared to 94 µg WW50387 / WW50394 alone (4 CRs vs 0 CRs) and 300 µg WW50387 / WW50394 alone (4 CRs vs 2 CRs) (FIG.51C, CR table summary in FIG.51D).

[0157] FIGs.52A and 52B are plots for efficacy (FIG.52A) and effect on body weight (FIG.52B) for the WW50387 / WW50394 + αLAG3 combination study. Generally, efficacy improved with the increased 24 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 dose level of WW50387 / WW50394 and addition of αLAG3. All treatment regimens were well tolerated showing no decreases in body weight during the 28-day course of the study post treatment initiation.

[0158] FIGs.53A-53D show efficacy of WW50387 / WW50394 dosed at 94 µg (FIG.53A) or 375 µg (FIG.53B) with or without αLAG3. At the 94 µg dose there was a trend for better efficacy when WW50387 / WW50394 was combined with αLAG3, but this did not reach significance. At the 375 µg dose the WW50387 / WW50394 + αLAG3 combination was highly significantly (****p<0.0001) more efficacious than αLAG3 alone but not WW50387 / WW50394 alone. Furthermore, this combination failed to produce more complete responses (CRs) than WW50387 / WW50394 (2 CR vs 2 CRs) but did show an increase of 1 CR for the 94 µg WW50387 / WW50394 + αLAG3 vs the 0 CRs observed with 94 µg WW50387 / WW50394 alone (FIG.53C, CR table summary in FIG.53D).

[0159] FIGs.54A-54N show the individual tumor volume plots for all 15 groups in the study.

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

[0161] FIGs.56A-56D are graphs showing exposure in the serum (FIGs.56A and 56B) and in the tumor (FIGs.56C and 56D) for total inducible IL-21 prodrug (FIGs.56A and 56C) and free human IL- 21 (FIGs.56B and 56D) after a single dose and after three doses of WW50387 / WW50394 over the 168- hour time course for each. While ADA response was evident from an accelerated clearance of IL-21 prodrug starting at 72, 96, 120 and 168 hours post the 3rddose in all tested animals, these ADAs did not reduce the exposure (AUCINF) of Total inducible IL-21 prodrug compared to the 1stdose (5.5x105vs 5.0x105hr*nM) nor did they reduce exposure of free IL-21 (1.0x102vs 1.1x102hr*nM). There was a reduction observed in the Cmaxof Total inducible IL-21 after the 3rddose compared to the 1stdose (2.0 x102vs 3.3x102nM) but no reduction observed in the Cmaxof free IL-21 (0.9 vs 0.9 nM). There was also a reduction observed for the exposure (AUC) of Total inducible IL-21 after the 3rddose compared to the 1stdose (7.9x103vs 2.0x104hr*nM) but no reduced exposure of free IL-21 (1.6x102vs 1.4x102hr*nM).

[0162] FIGs.57A and 57B are graphs showing the time course of IFNγ levels after a single dose of WW50387 / WW50394. IFNγ levels in the serum are near the lower limits of quantitation in the serum but increase over time in the tumor, peaking at 1028 pg / mL at 96 hours after treatment.

[0163] FIG.58 shows an increase of pSTAT3 expression over total STAT3 in tumors after a single dose (squares) or 3 doses (triangles) of WW50387 / WW50394 compared to PBS control (circles). pSTAT3 relative levels peak at 5.8 ECL units / g protein at 24 hours after a single dose and at 11.8 ECL units / g protein at 12 hours after the third dose of WW50387 / WW50394. 25 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0164] FIG.59 shows multiplex-immunofluorescence (M-IF) staining pictures of MC38 tumors resected from animals treated with vehicle or WW50387 / WW50394. Tumors were collected at day 10 after initial dosing. Tumors were fixed in formalin and paraffin embedded to obtain slices for staining. Staining was performed after antigen retrieval using the automated M-IF COMETTMsystem from Lunaphore. Different markers were used for the staining: DAPI, CD3, CD4, CD8, CD11b, CD11c, CD103, F4 / 80 and Granzyme B (GZMB). Combinations of these different markers were used to identify specific cell populations. DAPI stains nuclei, CD3+CD8+ (CTL), CD3+CD8+ GZMB+ (activated CTL), CD3+CD4+ (CD4+ T cells) F4 / 80 (macrophages) CD11b (myeloid cells) CD11c+CD103+ (cross presenting DCs). Data show an increase of immune infiltration in tumors from WW50387 / WW50394 treated animals including activated CTLs (enriched in highlighted white square). The presence of immune hubs or lymphocyte aggregates is only observed in the tumors from animals treated with WW50387 / WW50394.

[0165] FIGs.60A and 60B are graphs showing results of analyzing half-life extended IL-2 and half-life extended IL-21 in a Renca mouse tumor model. FIG.60A shows tumor volume over time in mice treated with half-life extended IL-2 (WW0177), half-life extended IL-21 (WW50008), or vehicle. FIG.60B is a graph showing body weight averages for the animals treated in data shown in FIG.60A. Each line in the plot represents the body weight average of the group over time.

[0166] FIGs.61A and 61B show the results of the TILs analysis of Renca tumors treated with half-life extended IL-2 (WW0177), half-life extended IL-21 (WW50008), or vehicle. FIG.61A is a graph showing CD8+ T cell abundance in tumor tissue over time in mice treated with WW0177, WW50008 or vehicle. FIG.61B are pie charts showing polyfunctionality of CD8+ T cell cytokine production (i.e. percent of cells expressing 0-5 of the effector molecules IFNγ, TNF, GzmA, GzmB, Prf1) in tumors of mice treated with half-life extended IL-2 (WW0177), half-life extended IL-12 (WW50008), or vehicle.

[0167] FIGs.62A-62I show that IL-21 activates Type I IFN signaling in the TME. FIG.62A is a graph depicting pathways identified which were significantly enriched with a p value <0.05 and enrichment greater than 3-fold. EMT-6 tumor bearing mice were randomized into treatment groups (Day 0), dosed twice a week with IL-21-HLE or IL-2-HLE for two weeks, and tumors were harvested for Nanostring analysis of bulk RNA. Pathway analysis was performed on the differentially expressed genes using Partek Software. FIGs.62B and 62C are graphs showing individual tumor volumes over time in EMT-6 tumor bearing mice that were randomized into treatment groups (Day 0). Mice were dosed every third day with anti-IFNAR1 or isotype control for a total of 8 doses. Mice were dosed twice a week with IL-21-HLE or vehicle for two weeks. Tumors and body weight were measured twice weekly. FIGs.62D-62I are graphs showing normalized counts for specific genes identified in the type-I IFN related pathways. p values 26 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 represent the results of a one-way ANOVA: * p<0.05, **p<0.005, *** p<0.001, ****p<0.0001. The data shows specific activation of type-I IFN genes triggered by IL-21 treatment and the inhibitory effect that blocking type-I IFN signaling has on IL-21 anti-tumor efficacy.

[0168] FIGs.63A-63C are representative immunofluorescence images showing staining for DAPI and CD45 at various timepoints (FIG.63A), DAPI, CD8, CD4, F4 / 80, CD11b and Granzyme B at day 12 (FIG.63B), and DAPI, CD8, CD4, TCF1, CD11c, CD103 and NCR1 at day 12 (FIG.63C) in EMT-6 tumor bearing mice treated with vehicle, IL-21-HLE or IL-2-HLE. The data shows a long duration increase of immune infiltration for those animals treated with IL-21-HLE. With special mention to CD8+ T cells (CD8) and macrophages (F4 / 80).

[0169] FIGs.64A-64E are graphs showing that IL-21 promotes increased presence of intratumoral CD8+ T cells that express effector cytokines. FIG.64A shows density of total CD8+ T cells in EMT-6 tumors from animals treated with vehicle, IL-21-HLE or IL-2-HLE. FIG.64B shows density of Granzyme B+ CD8+ T cells in EMT-6 tumors from animals treated with vehicle, IL-21-HLE or IL-2- HLE . FIG.64C shows density of Perforin+ CD8+ T cells in EMT-6 tumors from animals treated with vehicle, IL-21-HLE or IL-2-HLE . FIG.64D shows density of TCF1+ CD8+ T cells in EMT-6 tumors from animals treated with vehicle, IL-21-HLE or IL-2-HLE . FIG.64E is a spatial pseudo-color dot plot of CD8+ T cell infiltration, Granzyme B+ CD8+ T cells and stem-like (TCF1+) CD8+ T cells in EMT-6 tumors from animals treated with vehicle, IL-21-HLE or IL-2-HLE. The data shows that IL-21 treatment induces infiltration of activated and stem-like CD8+ T cells compared to vehicle or IL-2-HLE treatment.

[0170] FIGs.65A-65E shows treatment with IL-21 results in robust anti-tumor efficacy in an immune checkpoint inhibitor (CPI) resistant tumor model. Renca tumor bearing mice were randomized into treatment groups and dosed with vehicle or either IL-21-HLE or IL-2-HLE twice weekly for two weeks. FIG.65A is a graph showing average tumor volume over time. FIG.65B is a graph showing average body weight over time. FIGs.65C-65E are graphs showing individual spider plots of tumor burden from animals treated with vehicle, IL-21 HLE or IL-2-HLE. The data shows that IL-21treatment can induced efficacy in an animal tumor model resistant to CPI treatment and where IL-2-HLE treatment fails to produce significant efficacy.

[0171] FIGs.66A-66F show that IL-21 treatment leads to sustained intratumoral CD8+ T cell population that are polyfunctional (express multiple effector molecules). Renca tumor bearing mice were randomized into treatment groups and dosed with vehicle, IL-21-HLE, or IL-2-HLE twice weekly for two weeks. Tumors were collected at indicated timepoints and enzymatically and mechanically dissociated for single cell suspensions. Cells were stained for flow cytometry analysis. FIG.66A is a graph showing 27 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 CD8+ T cell counts per mg of tissue over time. FIG.66B is a graph showing the ratio of CD8+ T cells to Tregs (CD4+ Foxp3+ T cells) in tumors over time. FIGs.66C-66E are graphs showing cytokine production from CD8+ T cells on day 14. FIG.66F are pie charts showing the frequency of polyfunctional CD8+ T cells in the tumor based on co-expression of Granzyme A, Granzyme B, IFNg, TNF, and Perforin over time. p values represent the results of a one-way ANOVA: *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. The data shows that IL-21-HLE treatment can induce a sustained intratumoral infiltration of activated CD8+ T cells able to produce multiple effector cytokines over time.

[0172] FIGs.67A-67C are immunofluorescence images from Renca tumor bearing mice treated with vehicle, IL-21-HLE, or IL-2-HLE. FIG.67A is an immunofluorescence showing DAPI and CD45 in mice administered with vehicle, IL-21 HLE, or IL-2 HLE. FIG.67B is an immunofluorescence showing DAPI, CD4, CD8, F4 / 80, CD11c in mice administered with vehicle, IL-21 HLE, or IL-2 HLE. FIG.67C is an immunofluorescence image showing higher magnification of the white boxed area in corresponding images in (FIG.67B), DAPI, CD4, CD8, F4 / 80, CD11c, and Granzyme B in mice administered with vehicle, IL-21 HLE, or IL-2 HLE. Shading legends are indicated in figures. The data shows that IL-21- HLE treatment can induce a quantifiable intratumoral infiltration of different immune populations, specially CD8+ T cells. CD4+ T cells and macrophages.

[0173] FIGs.68A-68E are graphs showing results of analyzing WW50387 / WW50394 and WW51100 / WW50394 in a syngeneic MC38 mouse tumor model. FIG.68A is a graph showing results of analyzing mean tumor volumes after dosing WW50387 / WW50394 administered at 375 µg and WW51100 / WW50394 administered at 47, 94, 188, 375, 750, 1200, and 1500 µg on Days 0, 3, 7, and 10 after treatment initiation. The study was carried out through Day 28 after treatment initiation, measuring tumor volumes 2x weekly during the study. Data show the tumor volume was inhibited over time in a dose-dependent manner. The minimum efficacious dose (MED), defined as the dose at which the mean tumor growth inhibition (TGI) of the groups is greater than 50%, was determined to be 47 µg, while the maximum tolerated dose (MTD), defined as the dose where no subjects were lost due to drug toxicity, was determined to be 750 µg. The therapeutic window for WW51100 / WW50394 in the MC38 syngeneic tumor volume was determined to be 16. Arrows show the dosing days. FIG.68B are graphs showing the individual plots of the treatments which reveals that efficacy improves with dose level up to 1500 µg, with one outlier in the 1500 µg treatment group which initially lost control of tumor growth but recovered to shrink the tumor to less than baseline levels. FIG.68C shows the plots of mean body weight of the subjects expressed as grams across the dosing groups. FIG.68D shows the plots of mean body weight of the subjects expressed as percent body weight change from the initial measurement at Day -1 at the time 28 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 of randomization across the dosing groups. Red arrows show the dosing days. FIG.68E are plots of the percentage change from initial weight of individual subjects in all dosing groups.

[0174] FIG.69 is a graph showing plasma concentration profiles for VHH human IL-2 fusion proteins comprising an anti-human serum albumin VHH antibody derived from llama (WW10308, WW10311, WW10312, WW10313, WW10314, WW10315, and WW10316) over time. The graph shows that the concentration for each of the VHH-IL-2 fusion proteins followed a similar, non-linear pharmacokinetic (PK) profile, with a steep terminal elimination phase. 4. DETAILED DESCRIPTION A. IL-21 Polypeptide Prodrug

[0175] The disclosure relates to inducible IL-21 polypeptide prodrugs that contain at least one polypeptide chain, and can contain two or more polypeptide chains, if desired. The inducible IL-21 prodrugs comprise an IL-21 or a mutein thereof, a half-life extension element, an IL-21 blocking element, and a protease cleavable linker. The inducible IL-21 prodrugs can be encoded by the nucleic acids disclosed herein.

[0176] The inducible IL-21 prodrugs of this disclosure have attenuated IL-21 receptor agonist activity and the circulating half-life is extended. The IL-21 receptor agonist activity is attenuated through the blocking element, which is capable of blocking all or some of the receptor agonist activity of the IL-21 or mutein within the prodrug, typically by noncovalently binding to the IL-21 or mutein within the prodrug and / or sterically blocking their binding to the receptor. The half-life extension element can also contribute to attenuation, for example through steric effects. The half-life extension element can also act as a blocking element that is capable of blocking all or some of the receptor agonist activity of IL-21. For instance, the half-life extension element can contribute to blocking when the half-life extension element is adjacent to the IL-21 polypeptide.

[0177] Upon cleavage of the protease cleavable linker a form of IL-21 is released that is active (e.g., more active than the inducible IL-21 prodrug). Typically, the released IL-21 is at least 10 x more active than the inducible IL-21 prodrug. Preferably, the released Il-21 is at least 20 x, at least 30 x, at least 50 x, at least 100 x, at least 200 x, at least 300 x, at least 500 x, at least 1000 x, at least about 10,000X or more active than the inducible IL-21 prodrug. Typically, cleavage of the protease cleavable linker releases IL- 21 from both the IL-21 blocking element and the half-life extension element.

[0178] The form of IL-21 that is released upon cleavage of the inducible IL-21 prodrug typically has a short half-life, which is often substantially similar to the half-life of naturally occurring IL-21. Even though the half-life of the inducible IL-21 prodrug is extended, toxicity is reduced or eliminated because 29 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 the agonist activity of the circulating inducible IL-21 prodrug is attenuated and active IL-21 is targeted to the desired site of activity (e.g., tumor microenvironment).

[0179] It will be appreciated by those skilled in the art, that the number of polypeptide chains, and the location of the elements, the half-life extension element, the protease cleavable linker(s), and the blocking element (and components of such elements, such as a VH or VL domain) on the polypeptide chains can vary and is often a matter of design preference. All such variations are encompassed by this disclosure.

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

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

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

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

[0184] The IL-21 polypeptide and the blocking element and the half-life extension element are operably linked by the protease-cleavable polypeptide. For example, the polypeptide can be any of Formulas (I)- (VI). 30 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[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) – (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 is optionally protease-cleavable, and [L2’] is a protease-cleavable polypeptide linker. [L1] and [L2], or [L1] and [L2’], can have the same or different amino acid sequence and / or protease-cleavage site (when L2 is protease-cleavable) as desired. [H] can also optionally provide blocking. The protease cleavable linker can comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), particularly ALFKSSFP (SEQ ID NO: 198).

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

[0193] For example, the inducible IL-21 prodrug can have a first polypeptide of Formulas (X-XI) or (XXV-XXVIII). Formula X: [D]-[L1]-[A]-[L2]-[H], Formula XI: [H]-[L1]-[A]-[L2]-[D], Formula XXV: [A]-[L1]-[H]-[L3]-[D], Formula XXVI: [D]-[L3]-[H]-[L1]-[A], Formula XXVII: [A]-[L1]-[D]-[L3]-[H], or Formula XXVIII: [H]-[L3]-[D]-[L1]-[A]. In Formulas (X)-(XI) and (XXV)-(XXVIII), [A] is a IL-21 polypeptide, [D] is a IL-21 antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH), [H] is a half-life extension element, [L1] is a protease-cleavable polypeptide linker, and [L2] is a protease-cleavable linker, and [L3] is a polypeptide linker that is optionally protease-cleavable. [L1] and [L2] can have the same or different amino acid sequence and or protease-cleavage site, as desired. The inducible IL-21 prodrug can also have a second polypeptide comprising an antibody light 31 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 chain (VL+CL) or light chain variable domain (VL) that is complementary to the VH + CH1 or VH on the first polypeptide. The protease cleavable linker can comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), particularly ALFKSSFP (SEQ ID NO: 198).

[0194] The inducible IL-21 prodrug disclosed herein can comprise a first polypeptide chain that includes an IL-21 polypeptide and a IL-21 antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that are linked through a protease cleavable linker, and a second polypeptide chain that includes a half-life extension element and an IL-21 antibody light chain (VL+CL) or light chain variable domain (VL) that is complementary to the VH + CH1 or VH on the first polypeptide that are linked through an optionally protease cleavable linker.

[0195] The inducible IL-21 prodrug disclosed herein can comprise a first polypeptide chain that includes a IL-21 polypeptide and an antibody light chain (VL+CL) or light chain variable domain (VL) that are linked through a protease cleavable linker, and a second polypeptide chain that includes a half-life extension element and a IL-21 antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that is complementary to the VL+CL or VL of the first polypeptide chain and which are linked through an optional protease cleavable linker.

[0196] The inducible IL-21 prodrugs disclosed herein can comprise two blocking elements on two different polypeptide chains. The first polypeptide chain can comprise IL-21R and the second polypeptide chain can comprise the common gamma chain. In embodiments, a first polypeptide chain can comprise IL-21R and an antibody heavy chain constant region (i.e., CH1) operably linked through an optionally protease cleavable linker, and a second polypeptide chain can comprise the common gamma chain and an antibody light chain or fragment thereof operably linked through an optionally protease cleavable linker. The heavy chain constant region on the first polypeptide chain together with the light chain form a dimer (i.e., dimerize) such that the IL-21R and the common gamma chain form binding sites for IL-21. Typically, the first polypeptide chain also comprises IL-21 and a half-life extension element. For example, the inducible IL-21 prodrug can have a first polypeptide chain of Formulas (XII-XV). Formula 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)-(XV), [A] is an IL-21 polypeptide, [D] is an IL-21R, [X] is a heavy chain constant region (i.e., CH1), [H] is a half-life extension element, [L1] is a protease-cleavable polypeptide linker, and [L2] is optionally protease-cleavable. [L1] and [L2] can have the same or different amino acid sequences and / or a protease cleavage site (when L2 is protease cleavable), as desired. The inducible IL-21 prodrug can have a second 32 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 polypeptide chain comprising a common gamma chain and a light chain operably linked through an optionally protease cleavable linker.

[0197] In embodiments, a first polypeptide chain can comprise the common gamma chain and a heavy chain constant region (i.e., CH1) operably linked through an optionally protease cleavable linker, and a second polypeptide chain can comprise the IL-21R and a light chain operably linked through an optionally protease cleavable linker. The heavy chain constant region on the first polypeptide chain together with the light chain form a dimer (i.e., dimerize) such that the IL-21R and the common gamma chain form binding sites for IL-21.

[0198] The first polypeptide chain can also comprise IL-21 and optionally a half-life extension element. The second polypeptide chain can also comprise an optional half-life extension element. In some embodiments, only one of the first and the second polypeptide contains the half-life extension element. When the first polypeptide chain contains the half-life extension element, the second polypeptide may not contain a half-life extension element.

[0199] In embodiments, at least one of the half-life extension element, the blocking element, or a component of the half-life extension element or blocking element is on a separate polypeptide. For example, the first polypeptide can comprise IL-21 and either an antibody light chain (VL + CL) or light chain variable domain (VL), or an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that is operably linked to IL-21 through a protease cleavable linker. The second polypeptide can comprise the half-life extension element and either an antibody light chain (VL + CL) or light chain variable domain (VL), or an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that is operably linked to the half-life extension element through an optionally protease-cleavable linker. When the antibody heavy chain constant region is on the first polypeptide, the antibody light chain is on the second polypeptide and when the antibody heavy chain constant region is on the second polypeptide, the antibody light chain is on the first polypeptide. The portion of the antibody heavy chain together with the complementary light chain associate to form a binding site for IL-21. In an example, the first polypeptide can comprise IL-21 and a portion of an antibody light chain that are linked through a protease-cleavable linker. The second polypeptide comprises a half-life extension element and a portion of an antibody heavy chain that is complementary to the antibody light chain that are linked through an optionally protease-cleavable linker. The portion of the antibody heavy chain together with the complementary light chain associate to form a binding site for IL-21. In another example, the first polypeptide can comprise IL-21 and a portion of an antibody heavy chain that are linked through a protease-cleavable linker. The second polypeptide can comprise a half-life extension element and a 33 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 portion of an antibody light chain that are linked through an optionally protease-cleavable linker. The portion of the antibody heavy chain together with the complementary light chain associate to form a binding site for IL-21.

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

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

[0202] For example the inducible IL-21 prodrug can have a first polypeptide of Formula XVI: [D]-[L2]- [H], and a second polypeptide of Formula XVII: [A]-[L1]-[H]. For example the inducible IL-21 prodrug can have a first polypeptide of Formula XVIII: [H]-[L2]-[D], and a second polypeptide of Formula XIX: [H]-[L1]-[A]. In Formulas [XVI]-[XIX], [A] is a IL-21 polypeptide, [D] is a blocking element, [H] is a 34 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 half-life extension element, [L1] is a protease cleavable linker, and [L2] is an optionally protease cleavable linker.

[0203] In embodiments, each polypeptide chain can contain one IL-21 polypeptide, one half-life extension element, and one blocking element. For example, a first polypeptide chain can comprise an IL- 21 polypeptide, a half-life extension element, and a blocking element, and the second polypeptide chain can comprise an IL-21 polypeptide, a half-life extension element, and a blocking element. For example, the inducible IL-21 prodrug can have a first polypeptide chain of any one of 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); and a second polypeptide chain of any one of the Formulas 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);

[0216] In Formulas (I) – (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 is optionally protease-cleavable, and [L2’] is a protease-cleavable polypeptide linker. [L1] and [L2], or [L1] and [L2’], can have the same or different amino acid sequence and / or protease-cleavage site (when L2 is protease-cleavable) as desired. [H] can also optionally provide blocking.

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

[0218] For example, the inducible IL-21 prodrug can have a first polypeptide chain of any one of Formulas (XX)-(XXVI): 35 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 [H]-[L1]-[A]-[L1]-[D] (XX); [D]-[L1]-[A]-[L1]-[H] (XXI); [D]-[L2]-[H]-[L1]-[A] (XXII); [D]-[L2]-[H]-[L1]-[A] (XXIII); [H]-[L2]-[D]-[L1]-[A] (XXIV); [A]-[L1]-[D]-[L2]-[H] (XXV); [A]-[L1]-[H]-[L2]-[D] (XXVI);

[0219] and a second polypeptide chain of any one of Formulas: XXVII: [H’]-[L2]-[D’] or XXVIII: [D’]- [L2]-[H’].

[0220] In Formulas [XX-XXVIII] [A] is a IL-21 polypeptide, [D] is a first IL-21 blocking element, [H] is a first half-life extension element, [D’] is a second IL-21 blocking element, [H’] is a second half-life extension element, [L1] is a protease cleavable linker, and [L2] is an optional protease cleavable linker.

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

[0222] The half-life extension element increases the in vivo half-life and provides altered pharmacodynamics and pharmacokinetics of the inducible IL-21 prodrugs. Without being bound by theory, the half-life extension element alters pharmacodynamic properties including alteration of tissue distribution, penetration, and diffusion of the inducible IL-21 prodrug. In some embodiments, the half-life extension element can improve tissue targeting, tissue penetration, diffusion within the tissue, and enhanced efficacy as compared with a protein without a half-life extension element. Without being bound by theory, an exemplary way to improve the pharmacokinetics of a polypeptide is by expression of an element in the polypeptide chain that binds to receptors that are recycled to the plasma membrane of cells rather than degraded in the lysosomes, such as the FcRn receptor on endothelial cells and transferrin receptor. Three types of proteins, e.g., human IgGs, HSA (or fragments), and transferrin, persist for much 36 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 longer in human serum than would be predicted just by their size, which is a function of their ability to bind to receptors that are recycled rather than degraded in the lysosome. These proteins, or fragments that retain FcRn binding, are routinely linked to other polypeptides to extend their serum half-life. HSA may also be directly bound to the pharmaceutical compositions or bound via a short linker. Fragments of HSA may also be used. HSA and fragments thereof can function as both a blocking element and a half-life extension element. Human IgGs and Fc fragments can also carry out a similar function.

[0223] The serum half-life extension element can also be an antigen-binding polypeptide that binds to a protein with a long serum half-life such as serum albumin (e.g., HSA), transferrin and the like. Examples of such polypeptides include antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a single chain variable fragment (scFv), an antigen binding fragment (Fab), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic antibody binding and / or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include a ligand for a desired receptor, a ligand-binding portion of a receptor, a lectin, and peptides that bind to or associate with one or more target antigens. The antibodies and fragments thereof can function as both a blocking element and a half-life extension element.

[0224] The half-life extension element can also function as both a blocking element and a half-life extension element. For instance, the half-life extension element (e.g., HSA binding polypeptide) can function as a blocking element when adjacent to the IL-21 polypeptide.

[0225] The half-life extension element as provided herein is preferably a human serum albumin (HSA), an antigen binding polypeptide that binds human serum albumin, or an immunoglobulin Fc or fragment thereof. The half-life extension element can comprise an Fc domain or a fragment thereof. For example, the Fc domain can comprise a CH2 and CH3 domain or fragment thereof. For example, the Fc domain can comprise a constant domain of the heavy chain polypeptide. Typically, the half-life extension element is an antibody or fragment thereof (particularly a dAb), e.g., an antibody or fragment that binds serum albumin (particularly HSA), an immunoglobulin Fc or fragment thereof, or serum albumin (particularly HSA). 37 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0226] Preferably, the half-life extension element is a single domain antibody fragment (sdAb). The sdAb can comprise or consist of SEQ ID NO: 602-606, 608-653, or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 602 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 603 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 604 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 605 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 606 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 608 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 609 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 610 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 611 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 612 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 613 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 614 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 615 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 616 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 617 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 618 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 619 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 620 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 621 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 622 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 623 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 624 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 625 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 625 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 626 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 627 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 628 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 629 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 630 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 631 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 632 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 633 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 634 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 635 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 636 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 637 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 638 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 639 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 640 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 641 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 642 or a variant thereof. The sdAb can comprise or consist of 38 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 SEQ ID NO: 643 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 644 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 645 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 46 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 647 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 648 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 649 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 650 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 651 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 652 or a variant thereof. The sdAb can comprise or consist of SEQ ID NO: 653 or a variant thereof. The sdAb can comprise or consist of the amino acid sequence of any of SEQ ID NOs: 602-606, 608-653, or an amino acid sequence that has at least about 80% identity to any of SEQ ID NOs: 608-653. For example, the sdAbs can comprise or consist of an amino acid sequence that has at least about 81% identity, at least about 82% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to any of SEQ ID NOs: 602, 603, 604, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, or 653.

[0001] Compounds 210-221 are specific examples of inducible IL-21 prodrugs that comprise a half-life extension element that is a sdAb that comprises or consists of any one of SEQ ID NOs: 602-606 or 608- 653 or a variant thereof. Compound 210 comprises a sdAb that comprises SEQ ID NO: 608. Compound 211 comprises a sdAb that comprises SEQ ID NO: 609. Compound 212 comprises a sdAb that comprises SEQ ID NO: 611. Compound 213 comprises a sdAb that comprises SEQ ID NO: 612. Compound 214 comprises a sdAb that comprises SEQ ID NO: 625. Compound 215 comprises a sdAb that comprises SEQ ID NO: 626. Compound 216 comprises a sdAb that comprises SEQ ID NO: 628. Compound 217 comprises a sdAb that comprises SEQ ID NO: 632. Compound 218 comprises a sdAb that comprises SEQ ID NO: 635. Compound 219 comprises a sdAb that comprises SEQ ID NO: 637. Compound 220 comprises a sdAb that comprises SEQ ID NO: 642. Compound 221 comprises a sdAb that comprises SEQ ID NO: 644. Compound 222 comprises a sdAb that comprises SEQ ID NO: 645. Compound 223 comprises a sdAb that comprises SEQ ID NO: 646. Compound 224 comprises a sdAb that comprises 39 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 SEQ ID NO: 618. Compound 225 comprises a sdAb that comprises SEQ ID NO: 622. Compound 226 comprises a sdAb that comprises SEQ ID NO: 624.

[0227] The half-life extension element disclosed herein can comprise a first half-life extension element and a second half-life extension element. The first and second half-life extension element can heterodimerize. The first and second half-life extension elements can include one or more modifications that promote heterodimerization of the first and second half-life extension elements. For example, one or more amino acid modifications can be made to the first half-life extension element. For example, one or more amino acid modifications can be made to the second half-life extension element. The inducible IL- 21 prodrugs disclosed herein can comprise a first half-life extension element and a second half-life extension element, each of which comprises a CH3 domain. In embodiments, the half-life extension element comprising a CH3 domain is a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or fragment thereof). The CH3 domains of the first and second half-life extension elements can be altered by the “knobs-into-holes” technology. See, e.g., WO1996 / 027011; Ridgway et al., Protein Eng., 9(7):617- 621 (1996); Merchant et al., Nat. Biotechnol., 16(7):677-681 (1998). Using the knob-into-holes method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of the first half-life extension element and the second half-life extension element each containing an altered CH3 domain. This occurs by introducing a bulky residue into the CH3 domain of one of the half-life extension elements, which acts as the “knob.” Then, in order to accommodate the bulky residue, a “hole” is formed in the other half-life extension domain that can accommodate the knob. Either of the altered CH3 domains can be the “knob” while the other can be the “hole.” The introduction of a disulfide bridge can further stabilize the heterodimers.

[0228] In embodiments, the knobs-into-holes approach can be used to promote heterodimerization between two different half-life extension elements (e.g., between a first half-life extension element and a second half-life extension element).

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

[0230] The first half-life extension element can comprise a heavy chain polypeptide or portion thereof (e.g., the heavy chain constant regions of an Fc domain, or fragment thereof) that comprises one or more 40 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 amino acid mutations that creates a “knob,” and the second half-life extension element can comprise a heavy chain polypeptide or portion thereof (e.g., the heavy chain constant regions of an Fc domain, or fragment thereof) that comprises one or more amino acid mutations that create a “hole.” The first half-life extension element can comprise a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) that comprises one or more amino acid mutations that creates a “hole,” and the second half-life extension element can comprise a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) that comprises one or more amino acid mutations that create a “knob.” The techniques and procedures for introducing a hole or knob in a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) are known by those skilled in the art.

[0231] The half-life extension element of an inducible IL-21 prodrug extends the half-life of the inducible IL-21 prodrug by at least about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, about 10 days or more. C. Blocking Element

[0232] The blocking element can be any element that binds to IL-21 and inhibits the ability of the IL-21 polypeptide to bind and activate its receptor. The blocking element can inhibit the ability of IL-21 to bind and / or activate its receptor e.g., by sterically blocking and / or by noncovalently binding to IL-21 within the prodrug. The blocking element disclosed herein can bind to IL-21. Examples of suitable blocking elements include the full-length cognate receptor of IL-21 and an IL-21-binding fragment or mutein thereof. The cognate receptor for IL-21 can be the IL-21R receptor or a portion thereof and / or the common gamma chain or a portion thereof.

[0233] Antibodies and antigen-binding fragments thereof that bind IL-21, including an antigen-binding fragment (Fab), a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a dAb and the like, can also be used. Other suitable antigen-binding domains that bind IL-21 can also be used, including non-immunoglobulin proteins that mimic antibody binding and / or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block binding of IL-21 to its cognate receptor. Advantageously, such moieties can also function as half-life extending elements. For example, a peptide that is modified by conjugation to a water-soluble polymer, such as PEG, can sterically inhibit or prevent binding of the 41 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 cytokine to its receptor. Polypeptides, or fragments thereof, that have long serum half-lives can also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin and the like, as well as fragments and muteins of such polypeptides.

[0234] IL-21 blocking elements that are particularly suitable are single chain variable fragments (scFv), Fab fragments, or full length or an IL-21-binding fragment or mutein of the cognate receptor of an IL-21 (e.g., IL-21R receptor or the common gamma chain). Typically, the IL-21 blocking element will be an IL- 21-binding Fab, dAb, scFv, or cognate IL-21 receptor or fragment thereof.

[0235] Also disclosed herein is an inducible IL-21 polypeptide that contains a blocking element having specificity for IL-21 and further contains a half-life extension element.

[0236] The blocking element can contain two or more components that are present on the same polypeptide chain or on separate polypeptide chains. A first polypeptide chain can include an antibody light chain (VL+CL) or light chain variable domain (VL) and a second polypeptide can include an antibody heavy chain Fab fragment (VH + CH1) or heavy chain variable domain (VH) that is complementary to the VL+ CL or VL on the first polypeptide. In such situations, these components can associate in the polypeptide complex to form an antigen-binding site, such as an Fab that binds IL-21 and attenuates IL-21 activity. D. Protease Cleavable Linker

[0237] As disclosed herein, the inducible IL-21 prodrug comprises one or more linker sequences. A linker sequence serves to provide flexibility between the polypeptides, such that, for example, the blocking element is capable of inhibiting the activity of IL-21. The linker can be located between the IL- 21 subunit, the half-life extension element, and / or the blocking element. As described herein the inducible IL-21 prodrug comprises a protease cleavable linker. The protease cleavable linker can comprise one or more cleavage sites for one or more desired proteases. Preferably, the desired protease is enriched or selectively expressed at the desired target site of IL-21 activity (e.g., the tumor microenvironment). Thus, the inducible IL-21 prodrug is preferentially or selectively cleaved at the target site of desired IL-21 activity.

[0238] Suitable linkers are typically less than about 100 amino acids. Such linkers can be of different lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino acids, from 1 amino acid to 60 amino acids, from 1 to 70 amino acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from 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 42 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 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 from about 5 amino acids to about 30 amino acids.

[0239] Preferably the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked domain. In a preferred embodiment, the linker is cleavable by a cleaving agent, e.g., an enzyme. Preferably, the linker comprises a protease cleavage site. In some cases, the linker comprises one or more cleavage sites. The linker can comprise a single protease cleavage site. The linker can also comprise 2 or more protease cleavage sites. For example, 2 cleavage sites, 3 cleavage sites, 4, cleavage sites, 5 cleavage sites, or more. In cases the linker comprises 2 or more protease cleavage sites, the cleavage sites can be cleaved by the same protease or different proteases. A linker comprising two or more cleavage sites is referred to as a “tandem linker.” The two or more cleavage sites can be arranged in any desired orientation, including, but not limited to one cleavage site adjacent to another cleavage site, one cleavage site overlapping another cleavage site, or one cleavage site following by another cleavage site with intervening amino acids between the two cleavage sites.

[0240] Of particular interest in the present invention are disease specific protease-cleavable linkers. Also preferred are protease-cleavable linkers that are preferentially cleaved at a desired location in the body, such as the tumor microenvironment, relative to the peripheral circulation. For example, the rate at which the protease-cleavable linker is cleaved in 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 in the desired location in the body, e.g., the tumor microenvironment, in comparison to in the peripheral circulation (e.g., in plasma).

[0241] Proteases known to be associated with diseased cells or tissues include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin S, Cathepsin K, Cathepsin L, kallikreins, hKl, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, MMP19, MMP20, urokinase plasminogen activator (uPA), enterokinase, 43 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAPa), dipeptidyl peptidase, meprins, granzymes and dipeptidyl peptidase IV (DPPIV / CD26). Proteases capable of cleaving linker amino acid sequences (which can be encoded by the chimeric nucleic acid sequences provided herein) can, for example, be selected from the group consisting of a prostate specific antigen (PSA), a matrix metalloproteinase (MMP), an A Disintigrin and a Metalloproteinase (ADAM), a plasminogen activator, a cathepsin, a caspase, a tumor cell surface protease, and an elastase. The MMP can, for example, be 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 can be cleaved by a cathepsin, such as, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin S, Cathepsin E, Cathepsin G, Cathepsin K and / or Cathepsin L. Preferably, the linker can be cleaved by MMP14 or Cathepsin L.

[0242] Proteases useful for cleavage of linkers and for use in cleaving the inducible IL-21 polypeptide prodrug disclosed herein are presented in Table 1, and exemplary proteases and their cleavage site are presented in Table 2. Table 1. Proteases relevant to inflammation and cancer Protease Specificity Other aspects Secreted by killer T cells: nt l h e;\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Protease Specificity Other aspects narrow central pore; also associated with inflammation ; e e n nTable 2. Exemplary Proteases and Protease Recognition Sequences Protease Cleavage Domain Sequence SEQ ID NO:\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Protease Cleavage Domain Sequence SEQ ID NO: MMP9 LEATA 378 MMP11 GGAANLVRGG 37946 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0243] Exemplary protease cleavable linkers include, but are not limited to kallikrein cleavable linkers, thrombin cleavable linkers, chymase cleavable linkers, carboxypeptidase A cleavable linkers, cathepsin cleavable linkers, elastase cleavable linkers, FAP cleavable linkers, ADAM cleavable linkers, PR-3 cleavable linkers, granzyme M cleavable linkers, a calpain cleavable linkers, a matrix metalloproteinase (MMP) cleavable linkers, a plasminogen activator cleavable linkers, a caspase cleavable linkers, a tryptase cleavable linkers, or a tumor cell surface protease. Specifically, MMP9 cleavable linkers, ADAM cleavable linkers, CTSL1 cleavable linkers, FAPα cleavable linkers, and cathepsin cleavable linkers. Some preferred protease-cleavable linkers are cleaved by a MMP and / or a cathepsin. Also preferred are linkers cleaved by MMP14 or CTSL1.

[0244] The linker sequences disclosed herein are typically less than 100 amino acids. Such linker sequences can be of different lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino acids, from 1 amino acid to 60 amino acids, from 1 to 70 amino acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from 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 from about 5 amino acids to about 30 amino acids.

[0245] Preferably the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked domains.

[0246] In some embodiments, the linker comprises the sequence GPAGLYAQ (SEQ ID NO: 195); GPAGMKGL (SEQ ID NO: 196); PGGPAGIG (SEQ ID NO: 197); ALFKSSFP (SEQ ID NO: 198); ALFFSSPP (SEQ ID NO: 199); LAQRLRSS (SEQ ID NO: 200); LAQKLKSS (SEQ ID NO; 201); GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (SEQ ID NO: 202); RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (SEQ ID NO: 203); KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR (SEQ ID NO: 204); RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (SEQ ID NO: 205); KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (SEQ ID NO: 206); SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (SEQ ID NO: 207); RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (SEQ ID NO: 208); GGGALFKSSFPLAQKLKSSPGGPAGIGGGR (SEQ ID NO: 209); RGPGGPAGIGPLAQKLKSSALFKSSFPGGG (SEQ ID NO: 210); RGGPLAQKLKSSPGGPAGIGALFKSSFPGK (SEQ ID NO: 211); 47 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (SEQ ID NO: 212); GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (SEQ ID NO: 213); GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (SEQ ID NO: 214); RGGALFKSSFPLAQKLKSSGPAGLYAQGGK (SEQ ID NO: 215); RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (SEQ ID NO: 216); SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (SEQ ID NO: 217); KGGPGGPAGIGPLAQRLRSSALFKSSFPGR (SEQ ID NO: 218); KSGPGGPAGIGALFFSSPPLAQKLKSSGGR (SEQ ID NO: 219); or SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG (SEQ ID NO: 220)

[0247] Certain preferred linkers comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), particularly ALFKSSFP (SEQ ID NO: 198). The linkers disclosed herein can comprise one or more cleavage motifs or functional variants that are the same or different. The linkers can comprise 1, 2, 3, 4, 5, or more cleavage motifs or functional variants. Linkers comprising 30 amino acids can contain 2 cleavage motifs or functional variants, 3 cleavage motifs or functional variants or more. A “functional variant” of a linker retains the ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease) and are not cleaved or cleaved with low efficiency in the periphery (e.g., serum). For example, the functional variants retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a linker comprising any one of SEQ ID NOs: 195-220 or 447-448.

[0248] The linkers comprising more than one cleavage motif can be selected from SEQ ID NOs: 195- 201 or 447-448 and combinations thereof. Linkers comprising more than one cleavage motif can comprise the amino acids selected from SEQ ID NO: 202-220.

[0249] The linker can comprise both ALFKSSFP (SEQ ID NO: 198) and GPAGLYAQ (SEQ ID NO: 195). The linker can comprise two cleavage motifs that each have the sequence GPAGLYAQ (SEQ ID NO: 195). Alternatively or additionally, the linker can comprise two cleavage motifs that each have the sequence ALFKSSFP (SEQ ID NO: 198). The linker can comprise a third cleavage motif that is the same or different.

[0250] In some embodiments, the linker comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% identical to SEQ ID NOs: 195 to SEQ ID NO: 220 or 447-448 over the full length of SEQ ID NO: 195-220 or SEQ ID NOS 447-448. 48 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

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

[0252] The functional variants can contain at least one or more amino acid substitutions, deletions, or insertions relative to the linkers comprising SEQ ID NOs: 195-220 or 447-448. The functional variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations comparted to the linkers comprising SEQ ID NOs: 195-220 or 447-448. In some preferred embodiments, the functional variant differs from the linker comprising SEQ ID NOs: 195-220 by less than 10, less, than 8, less than 5, less than 4, less than 3, less than 2, or one amino acid alterations, e.g., amino acid substitutions or deletions. In other embodiments, the functional variant may comprise 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 substitution can be a conservative substitution or a non-conservative substitution, but preferably is a conservative substitution. In some embodiments, the functional variants comprise 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 variants of the linkers may comprise 1, 2, 3, 4, or 5 or more non-conservative amino acid substitutions compared to the linkers comprising SEQ ID NOs: 195-220 or 447-448. Non-conservative amino acid substitutions could be recognized by one of skill in the art. The functional variant of the linker preferably contains no more than 1, 2, 3, 4, or 5 amino acid deletions.

[0254] The amino acid sequences disclosed in the linkers can be described by the relative linear position in the linker with respect to the scissile bond. As will be well-understood by persons skilled in the art, linkers comprising 8 amino acid protease substrates (e.g., SEQ ID NOs: 195-201 or 447-448) contain amino acid at positions P4, P3, P2, P1, P1’, P2’, P3’, P4’, wherein the scissile bond is between P1 and P1’. For example, amino acid positions for the linker comprising the sequence GPAGLYAQ (SEQ ID NO: 195) can be described as follows (SEQ ID NO: 195): G P A G L Y A Q

[0255] Amino acids positions for the linker comprising the sequence ALFKSSFP (SEQ ID NO: 198) can be described as follows (SEQ ID NO: 198): 49 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 A L F K S S F P P4 P3 P2 P1 P1’ P2’ P3’ P4’

[0256] Preferabnd P1’for SEQ ID NOs: 195-201 or 447-448) are not substituted.

[0257] In 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 function variant of SEQ ID NO: 198. As described herein, a functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) can comprise one or more amino acid substitutions, and substantially retain their ability to be cleaved by a protease. Specifically, the functional variants of GPAGLYAQ (SEQ ID NO: 195) are cleaved by MMP14, and the functional variants of ALFKSSFP (SEQ ID NO: 198) are cleaved by Capthepsin L (CTSL1). The functional variants also retain their ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease). For example, the functional variants of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a linker comprising amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.

[0258] Preferably, the functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) comprise no more than 1, 2, 3, 4, or 5 conservative amino acid substitutions compared to GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). Preferably, the amino acids at position P1 and P1’ are not substituted. The amino acids at positions P1 and P1’ in SEQ ID NO: 195 are G and L, and the amino acids at positions P1 and P1’ in SEQ ID NO: 198 are K and S.

[0259] The functional variant of GPAGLYAQ (SEQ ID NO: 195) can preferably comprise one or more of the following: a) an arginine amino acid substitution at position P4, b) a leucine, valine, asparagine, or proline amino acid substitution at position P3, c) a asparagine amino acid substitution at position P2, d) a histidine, asparagine, or glycine amino acid substitution at position P1, e) a asparagine, isoleucine, or leucine amino acid substitution at position P1’, f) a tyrosine or arginine amino acid substitution at position P2’, g) a glycine, arginine, or alanine amino acid substitution at position P3’, h) or a serine, glutamine, or lysine amino acid substitution at position P4’. The following amino acid substitutions are disfavored in functional variants of GPAGLYAQ (SEQ ID NO: 195): 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 50 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 P4’. In some embodiments, the functional variant of GPAGLYAQ (SEQ ID NO: 195) does not comprise an amino acid substitution at position P1 and / or P1’.

[0260] The amino acid substitution of the functional variant of GPAGLYAQ (SEQ ID NO: 195) preferably comprises an amino acid substitution at position P4 and / or P4’. For example, the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprise a leucine at position P4, or serine, glutamine, lysine, or phenylalanine at position P4. Alternatively or additionally, the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprise a glycine, phenylalanine, or a proline at position P4’.

[0261] In some embodiments, the amino acid substitutions at position P2 or P2’ of GPAGLYAQ (SEQ ID NO: 195) are not preferred.

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

[0263] The functional variants of LFKSSFP (SEQ ID NO: 448) preferably comprises hydrophobic amino acid substitutions. The functional variant of LFKSSFP (SEQ ID NO: 448) can preferably comprise one or more of the following: (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at position P4; (b) lysine, histidine, glycine, proline, asparagine, phenylalanine at position P3; (c) arginine, leucine, alanine, glutamine, or histatine at position P2; (d) phenylalanine, histidine, threonine, alanine, or glutamine at position P1; (e) histidine, leucine, lysine, alanine, isoleucine, arginine, phenylalanine, asparagine, glutamic acid, or glycine at position P1’, (f) phenylalanine, leucine, isoleucine, lysine, alanine, glutamine, or proline at position P2’; (g) phenylalanine, leucine, glycine, serine, valine, histidine, alanine, or asparagine at position P3’; and phenylalanine, histidine, glycine, alanine, serine, valine, glutamine, lysine, or leucine.

[0264] The inclusion of aspartic acid and / or glutamic acid in functional variants of SEQ ID NO: 448 are generally disfavored and avoided. The following amino acid substitutions are also disfavored in functional variants of LFKSSFP (SEQ ID NO: 448): (a) alanine, serine, or glutamic acid at position P3; (b) proline, threonine, glycine, or aspartic acid at position P2; (c) proline at position P1; (d) proline at position P1’; (e) glycine at position P2’; (f) lysine or glutamic acid at position P3’; (g) aspartic acid at position P4’.

[0265] The amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 448) preferably comprises an amino acid substitution at position P4 and / or P1. In some embodiments, an amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 448) at position P4’ is not preferred. 51 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0266] In some embodiments, the functional variant of LFKSSFP (SEQ ID NO: 448) comprises the amino acid sequence selected from SEQ ID NOs: 296- 374. 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 stable prodrug under physiological conditions with the amino acid sequences (e.g. domains) that they link, while being capable of being cleaved by a protease. For example, the linker is stable (e.g., not cleaved or cleaved with low efficiency) in the circulation and cleaved with higher efficiency at a target site (i.e. a tumor microenvironment). Accordingly, inducible IL- 21 prodrugs that include the linkers disclosed herein can, if desired, have a prolonged circulation half-life and / or lower biological activity in the circulation in comparison to the components of the inducible IL-21 prodrugs as separate molecular entities. Yet, when in the desired location (e.g., tumor microenvironment) the linkers can be efficiently cleaved to release the components that are joined together by the linker and restoring or nearly restoring the half-life and biological activity of the components as separate molecular entities.

[0268] The linker desirably remains stable in the circulation for at least 2 hours, at least 5, hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or longer.

[0269] In some embodiments, the linker is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% in the circulation as compared to the target location. The linker is also stable in the absence of an enzyme capable of cleaving the linker. However, upon expose to a suitable enzyme (i.e., a protease), the linker is cleaved resulting in separation of the linked domain. E. Exemplary Inducible IL-21 Prodrugs

[0270] Compounds 1-52 are specific examples of inducible IL-21 prodrugs that comprise a single polypeptide chain for use according to this disclosure. Further details of exemplary IL-21 prodrugs are described in Table 38. Table 38. Inducible IL-21 prodrugs IL-21 Prodrug Polypeptide Chain\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Compound 2 SEQ ID NO: 3 Compound 3 SEQ ID NO: 453 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Compound 26 SEQ ID NO: 83 Compound 27 SEQ ID NO: 8454 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Compound 51 SEQ ID NO: 108 Compound 52 SEQ ID NO: 109ain attenuated IL-21 activity in the periphery and that release active IL-21 upon protease cleavage in the tumor microenvironment can also be used in accordance with this disclosure. For example, the inducible IL-21 prodrugs can include a polypeptide that comprises or consists of an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 2-8, 44- 50, and 72-109.

[0272] Compounds 53-222 are specific examples of inducible IL-21 prodrugs that comprise two polypeptide chains for use according to this disclosure. Further details of exemplary IL-21 prodrugs is described in Table 39. Table 39. Inducible IL-21 prodrugs IL-21 Prodrug First Polypeptide Second Polypeptide55 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Compound 64 SEQ ID NO: 29 SEQ ID NO: 25 Compound 65 SEQ ID NO: 30 SEQ ID NO: 2556 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Compound 88 SEQ ID NO: 135 SEQ ID NO: 129 Compound 89 SEQ ID NO: 136 SEQ ID NO: 13757 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Compound 112 SEQ ID NO: 168 SEQ ID NO: 300 Compound 113 SEQ ID NO: 169 SEQ ID NO: 30158 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Compound 126 SEQ ID NO: 192 SEQ ID NO: 303 Compound 127 SEQ ID NO: 193 SEQ ID NO: 30459 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Compound 150 SEQ ID NO: 228 SEQ ID NO: 137 Compound 151 SEQ ID NO: 229 SEQ ID NO: 13760 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Compound 174 SEQ ID NO: 261 SEQ ID NO: 306 Compound 175 SEQ ID NO: 262 SEQ ID NO: 30661 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Compound 198 SEQ ID NO: 288 SEQ ID NO: 304 Compound 199 SEQ ID NO: 289 SEQ ID NO: 30562 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

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

[0274] For example, the inducible IL-21 prodrugs can include 1) a first polypeptide that comprises or consists of an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any one of SEQ ID NOs: 9, 18-30, 41, 51, 58, 59, 63, 65, 69, 70, 72, 110-144, 153-194, 221-247, 250-255, 256, 258, 260-269, 281-290, 289, 449-462, 475,476, 479-491, and 655 and 2) a second polypeptide that comprises or consists of an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical 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. F. sdAbs that bind Serum Albumin

[0275] The disclosure also relates to single domain antibody fragments (sdAbs) (such as VHH, and VH) that have binding specificity for human serum albumin, methods of making, and uses thereof. Such sdAbs can be used, for example, for extending the in vivo serum half-life of therapeutic agents (e.g., proteins, peptides and the like) through the engineering of fusion proteins and conjugates using well-known methods and as described herein. Preferred sdAbs compete with FcRn (human FcRn) for binding to human serum albumin and, preferably, bind to domain 1 (DI) of human serum albumin.

[0276] As described and exemplified herein, contrary to the well-accepted knowledge in the art that anti- albumin binding domains that interfere with albumin binding to FcRn are not suitable for half-life extension applications (e.g., by inclusion in fusion proteins or conjugates with short half-life therapeutic proteins), the inventors have discovered that sdAbs that compete with FcRn (human FcRn) for binding to human serum albumin can effectively extend the half-life (e.g., the in vivo serum half-life) of short half- life therapeutic agents (e.g., proteins, peptides).

[0277] The sdAbs of this disclosure include, but are not limited to, heavy chain variable domain (VH), variable heavy domain of heavy chain (VHH), single domain shark variable domain of new antigen receptor (VNAR), or light chain variable (VL) domain (e.g., a kappa VL domain or a lambda VL domain). 63 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0278] The sdAbs comprise one or more complementary determining regions (CDRs), typically three CDRs, of an immunoglobulin single variable domain that has binding specificity for serum albumin. The CDRs of the sdAbs disclosed herein can be derived from a camelid. The sdAbs can be humanized and comprise one or more FRs, that can be derived from a human germline and typically include back mutations. The sdAbs disclosed herein can comprise a CDR1, a CDR2 and / or a CDR3. Preferably, the sdAbs disclosed herein comprise CDR1, CDR2, and CDR3.

[0279] The sdAbs can comprise a CDR1 comprising an amino acid sequence of Formula (I): X1X2X3X4X5X6AX7X8G (I).

[0280] In Formula (I), X1is glycine or aspartic acid, X2is glycine, arginine, phenylalanine, leucine, valine, serine, tyrosine, histidine, or threonine, X3is threonine, glycine, or serine, X4is phenylalanine, valine, tyrosine, leucine, isoleucine, or arginine, X5is aspartic acid, serine, threonine, or arginine, X6is aspartic acid, serine, glycine, or glutamic acid, X7is arginine, glycine, serine, threonine, methionine, valine, isoleucine, histidine, or asparagine and X8is arginine, glycine, leucine, methionine, isoleucine or valine.

[0281] The sdAbs can comprise a CDR2 comprising an amino acid of Formula (II) A I S X1S X2X3X4T X5Y X6X7X8V K G (II) (SEQ ID NO: 693)

[0282] In Formulas (II), X1is isoleucine or alanine, X2is glycine or threonine, X3is glutamic acid or glycine, X4is arginine, threonine, or serine, X5is asparagine, arginine, or tyrosine, X6is glycine, arginine, serine, or glutamic acid, X7is glutamic acid, lysine, aspartic acid, or glutamic acid, and X8is serine or glutamic acid.

[0283] The sdAbs can comprise a CDR3 comprising an amino acid sequence of Formula (III): A X1G D W Y H L X2Q X3X4X5X6X7X8I X9X10(III) (SEQ ID NO: 694).

[0284] In Formula (III), X1is serine or alanine, X2is valine, isoleucine, or threonine, X3is glycine or glutamic acid, X4is threonine, glutamic acid, or histidine, X5is glycine or asparagine, X6us methionine or asparagine, X7is serine, alanine, or methionine, X8is tyrosine or phenylalanine, X9is arginine or tryptophan, and X10is asparagine or tyrosine.

[0285] In embodiments, the sdAbs, comprises a CDR1 of Formula (I), a CDR2 of Formula (II), and a CDR3 of Formula (III).

[0286] The sdAbs disclosed herein can comprise a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 656, 659, 662, 663, 691 or a variant of any of the foregoing as described herein. The sdAbs disclosed herein can include a CDR1 that comprises or consists of SEQ ID NO: 656. The sdAbs disclosed herein can include a CDR1 that comprises or consists of SEQ ID NO: 659. The sdAbs disclosed 64 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 herein can include a CDR1 that comprises or consists of SEQ ID NO: 662. The sdAbs disclosed herein can comprise a CDR1 that comprises or consists of SEQ ID NO: 663. The sdAbs disclosed herein can comprise a CDR1 that comprises or consists of SEQ ID NO: 691.

[0287] The sdAb disclosed herein can comprise a CDR2 comprising the amino acid sequence of any one of SEQ ID NO: 657, 660, 664, 665, 666, 38 or a variant of any of the foregoing as described herein. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 657. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 660. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 664. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 665. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 666. The sdAbs disclosed herein can include a CDR2 that comprises or consists of SEQ ID NO: 38.

[0288] The sdAbs disclosed herein can comprise a CDR3 as defined by any one of SEQ ID NOs: 658, 661, or 667, or a variant of any of the foregoing as described herein. The sdAbs disclosed herein can include a CDR3 that comprises or consists of SEQ ID NO: 658. The sdAbs disclosed herein can include a CDR3 that comprises or consists of SEQ ID NO: 661. The sdAbs disclosed herein can include a CDR3 that comprises or consists of SEQ ID NO: 667.

[0289] The sdAbs disclosed herein can comprise a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 656, 659, 662, 663, 691, or a variant of any of the foregoing, a CDR2 comprising any one of SEQ ID NO: 657, 660, 664, 665, 666, 38, or a variant of any of the foregoing, and a CDR3, such that the sdAbs binds human serum albumin.

[0290] The sdAbs disclosed herein can comprise a CDR1 comprising the amino acid sequence of any one of SEQ ID NO: 656, 659, 662, 663, 691, or a variant of any of the foregoing, a CDR3 comprising any one of SEQ ID NO: 658, 661, or 667, or a variant of any of the foregoing, and a CDR2, such that the sdAbs binds human serum albumin.

[0291] The sdAbs disclosed herein can comprise a CDR2 comprising any one of SEQ ID NO: 657, 660, 664, 665, 666, 38, or a variant of any of the foregoing, a CDR3 comprising any one of SEQ ID NO: 658, 661, or 667, or a variant of any of the foregoing, and a CDR1, such that the sdAbs binds human serum albumin.

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

[0293] The sdAbs disclosed herein can comprise a CDR1 comprising any one of SEQ ID NO: 656, 659, 662, 663 and 691, or a variant of any of the foregoing; a CDR2 comprising any one of SEQ ID NO: 657, 65 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 660, 664, 665, 666 and 38 or a variant of any of the foregoing; and a CDR3 comprising any one of SEQ ID NO: 658, 661, and 667, or a variant of any of the foregoing.

[0294] The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 656 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 657 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 658 or variant thereof. The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 656, CDR2 that comprises or consists of SEQ ID NO: 657, and CDR3 that comprises or consists of SEQ ID NO: 658.

[0295] The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 659 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 660 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 661 or variant thereof. The sdAbs can comprise CDR1 that comprises or consists of SEQ ID NO: 659, CDR2 that comprises or consists of SEQ ID NO: 660, and CDR3 that comprises or consists of SEQ ID NO: 661.

[0296] The sdAbs can comprise CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof, CDR1 that comprises or consists of SEQ ID NO: 662, 691 or 663 or variant of any of the foregoing, and CDR2 that comprises or consists of SEQ ID NO: 38, 664, 665 or 666 or variant of any of the foregoing. The sdAbs can comprise CDR3 that comprises or consists of SEQ ID NO: 667, CDR1 that comprises or consists of SEQ ID NO: 662, 691 or 663, and CDR2 that comprises or consists of SEQ ID NO: 38, 664, 665 or 666.

[0297] For example, the sdAbs can comprise a) CDR1 that comprises or consists of SEQ ID NO: 662 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 664 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; b) CDR1 that comprises or consists of SEQ ID NO: 662, CDR2 that comprises or consists of SEQ ID NO: 664, and CDR3 that comprises or consists of SEQ ID NO: 667; c) CDR1 that comprises or consists of SEQ ID NO: 662 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 665 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; d) CDR1 that comprises or consists of SEQ ID NO: 662, CDR2 that comprises or consists of SEQ ID NO: 665, and CDR3 that comprises or consists of SEQ ID NO: 667; e) CDR1 that comprises or consists of SEQ ID NO: 662 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 666 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; f) CDR1 that comprises or consists of SEQ ID NO: 662, CDR2 that comprises or consists of SEQ ID NO: 666, and CDR3 that comprises or consists of SEQ ID NO: 667; g) CDR1 that comprises or consists of SEQ ID NO: 662 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; h) CDR1 that 66 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 comprises or consists of SEQ ID NO: 662, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 667; i) CDR1 that comprises or consists of SEQ ID NO: 691 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 664 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; j) CDR1 that comprises or consists of SEQ ID NO: 691, CDR2 that comprises or consists of SEQ ID NO: 664, and CDR3 that comprises or consists of SEQ ID NO: 667; k) CDR1 that comprises or consists of SEQ ID NO: 691 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 665 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; l) CDR1 that comprises or consists of SEQ ID NO: 691, CDR2 that comprises or consists of SEQ ID NO: 665, and CDR3 that comprises or consists of SEQ ID NO: 667; m) CDR1 that comprises or consists of SEQ ID NO: 691 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 666 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; n) CDR1 that comprises or consists of SEQ ID NO: 691, CDR2 that comprises or consists of SEQ ID NO: 666, and CDR3 that comprises or consists of SEQ ID NO: 667; o) CDR1 that comprises or consists of SEQ ID NO: 691 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; p) CDR1 that comprises or consists of SEQ ID NO: 691, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 667; q) CDR1 that comprises or consists of SEQ ID NO: 663 or variant thereof, a CDR2 that comprises or consists of SEQ ID NO: 664 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; r) CDR1 that comprises or consists of SEQ ID NO: 663, a CDR2 that comprises or consists of SEQ ID NO: 664, and CDR3 that comprises or consists of SEQ ID NO: 667; s) CDR1 that comprises or consists of SEQ ID NO: 663 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 665 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; t) CDR1 that comprises or consists of SEQ ID NO: 663, CDR2 that comprises or consists of SEQ ID NO: 665, and CDR3 that comprises or consists of SEQ ID NO: 667; u) CDR1 that comprises or consists of SEQ ID NO: 663 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 666 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; v) CDR1 that comprises or consists of SEQ ID NO: 663, CDR2 that comprises or consists of SEQ ID NO: 666, and CDR3 that comprises or consists of SEQ ID NO: 667; w) CDR1 that comprises or consists of SEQ ID NO: 663 or variant thereof, CDR2 that comprises or consists of SEQ ID NO: 38 or variant thereof, and CDR3 that comprises or consists of SEQ ID NO: 667 or variant thereof; x) CDR1 that comprises or consists of SEQ ID NO: 663, CDR2 that comprises or consists of SEQ ID NO: 38, and CDR3 that comprises or consists of SEQ ID NO: 667. 67 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0298] Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 662; a CDR2 that comprises or consists of SEQ ID NO: 38; and a CDR3 that comprises or consists of SEQ ID NO: 667. Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 691; a CDR2 that comprises or consists of SEQ ID NO: 664; and a CDR3 that comprises or consists of SEQ ID NO: 667. Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 663; a CDR2 that comprises or consists of SEQ ID NO: 665; and a CDR3 that comprises or consists of SEQ ID NO: 667. Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 663; a CDR2 that comprises or consists of SEQ ID NO: 666; and a CDR3 that comprises or consists of SEQ ID NO: 667. Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 656; a CDR2 that comprises or consists of SEQ ID NO: 657; and a CDR3 that comprises or consists of SEQ ID NO: 658. Certain preferred sdAbs comprise a CDR1 that comprises or consists of SEQ ID NO: 659; a CDR2 that comprises or consists of SEQ ID NO: 660; and a CDR3 that comprises or consists of SEQ ID NO: 661.

[0299] The CDRs of the sdAbs disclosed herein are interposed between stretches amino acids referred to as framework regions (FRs). The sdAbs disclosed herein can comprise FR1, FR2, FR3, and / or F4. In certain embodiments, the FRs regions of the sdAbs disclosed herein comprise 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 of amino acid sequence homology to human FRs. Typically, the FRs can be derived from a human germline gene. Exemplary human germline genes include, but are not limited to, 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, IGHV3-43, 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 sdAbs include FRs derived from human germline gene IGHV3-11 and / or IGHV3-23.

[0300] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 668, 669, 31, or a variant of any of the foregoing. In some instances, the sdAbs disclosed herein can comprise a FR1 comprising an amino acid sequence that has at least 80% identity to any one of SEQ ID NOs.: SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing. The sdAbs disclosed herein can include a FR1 that comprises or consists of SEQ ID NO: 668. The sdAbs disclosed herein can include a FR1 that comprises or consists of SEQ ID NO: 669. The sdAbs disclosed herein can include a FR1 that comprises or consists of SEQ ID NO: 31. 68 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0301] The sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a variant of any of the foregoing. In some instances, the sdAbs disclosed herein can comprise a FR2 comprising an amino acid sequence that has at least 80% identity to any one of SEQ ID NOs: 670-674, 678-682, 32, 33, or a variant of any of the foregoing. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 670. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 671. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 672. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 673. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 674. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 678. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 679. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 680. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 681. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 682. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 32. The sdAbs disclosed herein can include a FR2 that comprises or consists of SEQ ID NO: 33.

[0302] The sdAbs disclosed herein can comprise a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing. In some instances, the sdAbs disclosed herein can comprise a FR2 comprising an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 675. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 676. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 34. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 35. The sdAbs disclosed herein can include a FR3 that comprises or consists of SEQ ID NO: 36.

[0303] The sdAbs disclosed herein can comprise a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof. In some instances, the sdAbs disclosed herein can comprise a FR4 comprising an amino acid sequence that has at least 80% identity to SEQ ID NO: 677, or a variant thereof. The sdAbs disclosed herein can include a FR4 that comprises or consists of SEQ ID NO: 677.

[0304] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing; and a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a 69 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a variant of any of the foregoing.

[0305] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing; and a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing.

[0306] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 677, or a variant thereof.

[0307] The sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a variant of any of the foregoing; and a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing.

[0308] The sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 670-674, 24-682, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 670-674, 24-682, 32, 33, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 677, or a variant thereof.

[0309] The sdAbs disclosed herein can comprise a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 677, or a variant thereof.

[0310] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing; a FR2 70 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 comprising the amino acid sequence of any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a variant of any of the foregoing; and a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing.

[0311] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing; a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 677, or a variant thereof.

[0312] The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 677, or a variant thereof.

[0313] The sdAbs disclosed herein can comprise a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a variant of any of the foregoing; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 675, 676, 34, 35, or 36, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 677, or a variant thereof.

[0314] Typically, the sdAbs disclosed herein have all four FRs (e.g., a FR1, a FR2, a FR3, and a FR4). The sdAbs disclosed herein can comprise a FR1 comprising the amino acid sequence of any one of SEQ 71 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 ID NO: 668, 669, 31, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 668, 669, 31, or a variant of any of the foregoing; a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 670-674, 678-682, 32, 33, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 670-674, 678-682, 686, 687, or a variant of any of the foregoing; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, 676, 688, 689, or 690, or a variant of any of the foregoing, or an amino acid sequence that has at least 80% identity to any one of SEQ ID NO: 675, 676, 688, 689, or 690, or a variant of any of the foregoing; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 677, or a variant thereof.

[0315] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 670, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0316] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 671, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0317] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 672, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0318] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 673, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0319] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 669, a FR2 that comprises or consists of SEQ ID NO: 670, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0320] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 669, a FR2 that comprises or consists of SEQ ID NO: 671, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0321] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 669, a FR2 that comprises or consists of SEQ ID NO: 672, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677. 72 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0322] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 669, a FR2 that comprises or consists of SEQ ID NO: 673, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0323] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 674, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0324] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 678, a FR3 that comprises or consists of SEQ ID NO: 683; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0325] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 679, a FR3 that comprises or consists of SEQ ID NO: 683; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0326] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 680, a FR3 that comprises or consists of SEQ ID NO: 683; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0327] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 681, a FR3 that comprises or consists of SEQ ID NO: 683; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0328] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 669, a FR2 that comprises or consists of SEQ ID NO: 24, a FR3 that comprises or consists of SEQ ID NO: 683; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0329] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 669, a FR2 that comprises or consists of SEQ ID NO: 679, a FR3 that comprises or consists of SEQ ID NO: 29; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0330] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 669, a FR2 that comprises or consists of SEQ ID NO: 680, a FR3 that comprises or consists of SEQ ID NO: 683; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0331] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 669, a FR2 that comprises or consists of SEQ ID NO: 681, a FR3 that comprises or consists of SEQ ID NO: 683; and a FR4 that comprises or consists of SEQ ID NO: 677. 73 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0332] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 682, a FR3 that comprises or consists of SEQ ID NO: 676; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0333] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 669, a FR2 that comprises or consists of SEQ ID NO: 678, a FR3 that comprises or consists of SEQ ID NO: 684; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0334] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 686, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0335] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 687, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0336] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 681, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0337] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 668, a FR2 that comprises or consists of SEQ ID NO: 679, a FR3 that comprises or consists of SEQ ID NO: 675; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0338] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 686, a FR3 that comprises or consists of SEQ ID NO: 688; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0339] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 687, a FR3 that comprises or consists of SEQ ID NO: 688; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0340] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 681, a FR3 that comprises or consists of SEQ ID NO: 688; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0341] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 679, a FR3 that comprises or consists of SEQ ID NO: 688; and a FR4 that comprises or consists of SEQ ID NO: 677. 74 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0342] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 686, a FR3 that comprises or consists of SEQ ID NO: 689; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0343] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 687, a FR3 that comprises or consists of SEQ ID NO: 689; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0344] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 681, a FR3 that comprises or consists of SEQ ID NO: 689; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0345] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 679, a FR3 that comprises or consists of SEQ ID NO: 689; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0346] The sdAbs disclosed herein can comprise a FR1 that comprises or consists of SEQ ID NO: 685, a FR2 that comprises or consists of SEQ ID NO: 682, a FR3 that comprises or consists of SEQ ID NO: 690; and a FR4 that comprises or consists of SEQ ID NO: 677.

[0347] The sdAbs disclosed herein can comprise or consist of any one of the sdAbs disclosed in Table 51. Table 51. Exemplary sdAbs sdAb FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 7 7 775 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 1HUM19_D SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: 668 NO: 662 NO: 673 NO: 692 NO: 675 NO: 667 NO: 677 610 7 7 7 7 7 7 7 7 7 7 776 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 1HUM19_Q SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: 669 NO: 663 NO: 670 NO: 666 NO: 675 NO: 667 NO: 677 623 7 7 7 7 7 7 7 7 7 7 777 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 2HUM45_A SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: NO: 668 NO: NO: 686 NO: 660 NO: 675 NO: 661 NO: 677 635 7 7 7 7 7 7 7 7 7 7 778 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 2HUM45_M SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 685 NO: 659 NO: 679 NO: 660 NO: 689 NO: 661 NO: 677 NO:647 7 7 7 7 7 7 y of SEQID NOs: 608-648 or 649-653, or an amino acid sequence that has at least about 80% identity to any of SEQ ID NOs: 608-648 or 649-653. For example, the sdAbs can comprise or consist of an amino acid sequence that has at least about 81% identity, at least about 82% identity, at least about 83% identity, at least about 84% identity, at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to any of SEQ ID NOs: 608, 609, 41, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, or 653.

[0349] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 608, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 608. 79 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0350] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 609, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 609.

[0351] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 612, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 612.

[0352] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 610, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 610.

[0353] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 611, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 611.

[0354] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 613, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 613.

[0355] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 614, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 614.

[0356] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 615, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 615.

[0357] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 616, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 616.

[0358] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 617, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 617.

[0359] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 618, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 618.

[0360] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 619, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 619.

[0361] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 620, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 620.

[0362] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 621, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 621.

[0363] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 622, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 622.

[0364] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 623, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 623.

[0365] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 624, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 624. 80 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0366] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 625, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 625.

[0367] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 626, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 626.

[0368] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 627, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 627.

[0369] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 628, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 628.

[0370] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 629, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 629.

[0371] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 630, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 630.

[0372] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 631, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 631.

[0373] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 632, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 632.

[0374] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 633, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 633.

[0375] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 634, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 634.

[0376] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 635, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 635.

[0377] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 636, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 636.

[0378] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 637, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 637.

[0379] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 638, or an amino acid sequence that has at least about 80% identity to SEQ ID NOs: 638.

[0380] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 639, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 639.

[0381] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 640, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 640. 81 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0382] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 641, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 641.

[0383] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 642, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 642.

[0384] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 643, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 643.

[0385] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 644, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 644.

[0386] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 645, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 645.

[0387] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 646, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 646.

[0388] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 647, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 647.

[0389] The sdAbs can comprise or consist of the amino acid sequence of SEQ ID NO: 648, or an amino acid sequence that has at least about 80% identity to SEQ ID NO: 648.

[0390] The sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 649, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 649.

[0391] The sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 650, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 650.

[0392] The sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 651, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 651.

[0393] The sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 652, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 652.

[0394] The sdAb can comprise or consist of the amino acid sequence of SEQ ID NO: 653, or an amino acid sequence that has at least 80% identity to SEQ ID NO: 653.

[0395] The sdAbs disclosed herein include functional variants that differ in amino acid sequence in comparison, for example, to any one of SEQ ID NOs.: 609-648 or 649-653. A “functional variant” of a sdAb retains the ability to bind to serum albumin.

[0396] The sdAbs disclosed herein can comprise one or more amino acid substitutions. The amino acid substitution can be a conservative substitution or a non-conservative substitution, but preferably is a 82 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 conservative substitution. Conservative amino acid substitutions are generally made in accordance with the following Table 52. Table 52. Exemplary amino acid substitutions Amino Acid Exemplary Substitutions

[0397] As described herein, a sdAb of this disclosure can contain a CDR1 that comprises or consists of SEQ ID NO: 656, 659, 662, 663, or 691 or a variant of any of the forgoing. A variant of SEQ ID NO: 83 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 656, 659, 662, 663, or 691 has at least about 80% or at least about 90% amino acid sequence identity to SEQ ID NO: 656, 659, 663, 663, or 691; a variant of SEQ ID NO: 656, 659, 662, 663 or 691 can contain one or two amino acid substitutions. As amino acid substitutions in the CDRs (i.e., CDR1, CDR2 and / or CDR3) can affect binding to serum albumin, it is preferred that any such substitutions are conservative substitutions. One or more amino acids in a CDR can be replaced with a human germ line amino acid, for example, when it is desirable to decrease the binding affinity.

[0398] As described herein, a sdAb of this disclosure can contain a CDR2 that comprises or consists of SEQ ID NO: 657, 660, 664, 665, 666 or 692 or a variant of any of the forgoing. A variant of SEQ ID NO: 657, 660, 664, 665, 666 or 692 has at least about 80%, at least about 85%, or at least about 90% amino acid sequence identity to SEQ ID NO: 657, 660, 664, 665, 666 or 692; a variant of SEQ ID NO: 657, 660, 664, 665, 666 or 692 can contain one or two or three amino acid substitutions.

[0399] As described herein, a sdAb of this disclosure can contain a CDR3 that comprises or consists of SEQ ID NO: 658, 661 or 667 or a variant of any of the forgoing. A 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 to SEQ ID NO: 658, 661 or 667; a variant of SEQ ID NO: 658, 661 or 667 can contain one or two or three amino acid substitutions.

[0400] As described herein, a sdAb of this disclosure can contain FRs and these sequences can generally include amino acid substitutions without causing a loss of binding activity. As described herein, a sdAb of this disclosure can contain a FR1 that comprises or consists of SEQ ID NO: 13, 14, or 31 or a variant of any of the forgoing. A variant of SEQ ID NO: 658, 661 or 667 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 658, 661 or 667.

[0401] As described herein, a sdAb of this disclosure can contain a FR2 that comprises or consists of SEQ ID NO: 670-674, 678-682, 686, or 687 or a variant of any of the forgoing. A variant of SEQ ID NO: 670-674, 678-682, 686, or 687 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 670-674, 678-682, 686, or 687.

[0402] As described herein, a sdAb of this disclosure can contain a FR3 that comprises or consists of SEQ ID NO: 675, 676, 683, 684, 688, 689, or 690 or a variant of any of the forgoing. A variant of SEQ ID NO: 675, 676, 683, 684, 688, 689, or 690 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 675, 676, 683, 684, 688, 689, or 690. 84 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0403] As described herein, a sdAb of this disclosure can contain a FR4 that comprises or consists of SEQ ID NO: 677 or a variant thereof. A variant of SEQ ID NO: 677 has at least about 80%, at least about 85%, at least about 90% or at least about 95% amino acid sequence identity to SEQ ID NO: 677.

[0404] The sdAb disclosed herein comprising the amino acid sequence of any one of SEQ ID NO: 608- 648 or 649-653 can comprises any one of the following amino acids: 1) the amino acid at residue at position 678 can be selected from alanine or serine; 2) the amino acid at position 44 can be selected from glutamic acid, glycine, or aspartic acid; 3) the amino acid at position 45 can be selected from arginine, or leucine; 4) the amino acid at position 78 can be selected from asparagine, serine, threonine, or leucine; or 5) the amino acid at position 79 can be selected from valine, leucine, or phenylalanine.

[0405] The sdAbs disclosed herein are particularly suitable for use in preparing fusion polypeptides and conjugates, e.g., that have extended serum half-life. The fusion polypeptide can comprise a continuous polypeptide chain that comprises a sdAb as disclosed herein and an amino acid sequence of interest, typically a therapeutic protein or peptide. The sdAb can be fused to the amino acid sequence of interest directly, or indirectly, for example through an amino acid linker. If desired, the fusion polypeptide can associate with one or more other polypeptides to form a functional polypeptide complex. For example, when it is desired to provide an extended half-life form of a protein that is a heterodimer, a fusion polypeptide that contains a sdAb disclosed herein and one member of the heterodimer can be prepared, and it can associate with the other member of the heterodimer to form a functional polypeptide complex.

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

[0407] The amino acid sequence of interest can be any desired amino acid sequence. Exemplary amino acid sequences of interest 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] Exemplary fusion polypeptides and conjugates disclosed herein are half-life extended cytokines. The half-life extended cytokines can be a fusion polypeptide or a conjugate that comprises a sdAb or non- immunoglobulin format as disclosed herein and a cytokine polypeptide, and optionally a linker (e.g., cleavable or non-cleavable linker). Any cytokine of interest can be suitable for inclusion in a half- life extended cytokine of this disclosure. Exemplary cytokines include IL-21 and / or IL-2. Preferably, the cytokine is IL-21. Preferably, the cytokine is IL-2. 85 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

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

[0410] Exemplary half-life extended IL-2, as disclosed herein, can comprise the amino acid sequence of any one of SEQ ID NOs: 696-699. For example, the half-life extended IL-2 can comprise SEQ ID NO: 696. For example, the half-life extended IL-2 can comprise SEQ ID NO: 697. For example, the half-life extended IL-2 can comprise SEQ ID NO: 698. For example, the half-life extended IL-2 can comprise SEQ ID NO: 699.

[0411] This disclosure also relates to conjugates. Like the fusion polypeptides described herein, conjugates comprise a sdAb as described herein and an amino acid sequence of interest. In the conjugates of this disclosure, the sdAb is covalently bonded directly or indirectly to the amino acid sequence of interest. The sdAb can be conjugated to the amino acid sequence of interest at the C-terminus of the amino acid sequence of interest, at the N-terminus of the amino acid sequence of interest, or internally, for example through, an amino acid side chain of an amino acid in the amino acid sequence of interest. Many suitable linkers and functional groups that can be used to prepare conjugates as described herein are well-known in the art. For example, homo- or hetero-bifunctional cross-linkers can be used to link a sdAb disclosed herein to an amino acid sequence of interest, for example through an amine group on the sdAb and a carboxyl group on the amino acid sequence of interest. In some aspects, the sdAb is indirectly conjugated to the amino acid sequence of interest through a linker that includes a peptide or polypeptide amino acid sequence. Such linkers can be non-cleavable or cleavable, for example by protease (e.g., a protease with activity that is associated with diseased cells or tissues as disclosed herein).

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

[0413] The sdAb and the amino acid sequence of interest can be linked by a non-cleavable linker. The sdAb and the amino acid sequence of interest is preferably linked by a cleavable linker. The cleavable linker can comprise one or more cleavage sites for one or more desired protease. Preferably, the desired protease activity is enriched or selectively expressed at the desired target site of the amino acid sequence 86 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 of interest, such as a desired cite of cytokine activity (e.g., the tumor microenvironment). The linker is preferentially or selectively cleaved at the target site. Suitable protease-cleavable linkers are described herein.

[0414] The fusion polypeptide can comprise a sdAb comprising or consisting of the amino acid sequence of a) SEQ ID NO: 608 or variant thereof; b) SEQ ID NO: 609 or a variant thereof; c) SEQ ID NO: 610, or a variant thereof; d) SEQ ID NO: 611 or a variant thereof; e) SEQ ID NO: 612, or a variant thereof; f) SEQ ID NO: 613 or a variant thereof; g) SEQ ID NO: 614 or a variant thereof; h) SEQ ID NO: 615 or a variant thereof; i) SEQ ID NO: 616 or variant thereof; j) SEQ ID NO: 617, or a variant thereof; k) SEQ ID NO: 618 or a variant thereof; l) SEQ ID NO: 619 or a variant thereof; m) SEQ ID NO: 620 or a variant thereof; n) SEQ ID NO: 621 or a variant thereof; o) SEQ ID NO: 622 or a variant thereof; o) SEQ ID NO: 623 or a variant thereof; p) SEQ ID NO: 624 or a variant thereof; q) SEQ ID NO: 625 or a variant thereof; r) SEQ ID NO: 626 or a variant thereof; s) SEQ ID NO: 627 or a variant thereof; t) SEQ ID NO: 628 a variant thereof; u) SEQ ID NO: 629 or a variant thereof; v) SEQ ID NO: 630 or a variant thereof; w) SEQ ID NO: 631 or a variant thereof; x) SEQ ID NO: 632 or a variant thereof, y) SEQ ID NO: 633 or a variant thereof, z) SEQ ID NO: 634 or a variant thereof; za) SEQ ID NO: 635 or a variant thereof; zb) SEQ ID NO: 636 or a variant thereof; zc) SEQ ID NO: 637 or a variant thereof; zd) SEQ ID NO: 638 or a variant thereof; ze) SEQ ID NO: 639 or a variant thereof; zf) SEQ ID NO: 640 or a variant thereof; zg) SEQ ID NO: 641 or a variant thereof; zh) SEQ ID NO: 642 or a variant thereof; zi) SEQ ID NO: 643 or a variant thereof; zj) SEQ ID NO: 644 or a variant thereof; zk) SEQ ID NO: 645 or a variant thereof; zl) SEQ ID NO: 646 or a variant thereof; zm) SEQ ID NO: 647 or a variant thereof; or zn) SEQ ID NO: 648 or a variant thereof.

[0415] Additional moieties and / or linkers can be present, as appropriate. Additional specific applications of uses for the sdAbs are disclosed herein. F. Pharmaceutical Compositions

[0002] This disclosure provides pharmaceutical compositions comprising an inducible IL-21 prodrug described herein. The pharmaceutical compositions typically further comprise a pharmaceutically acceptable carrier. The disclosure further relates to pharmaceutical compositions comprising a sdAb and non-immunoglobulin formats disclosed herein or a fusion polypeptide or conjugate as disclosed. The pharmaceutical formulations or compositions comprise a sdAbs described herein, the fusion polypeptides or conjugates described herein as described herein and typically a pharmaceutically acceptable carrier. 87 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0416] The pharmaceutical compositions can take a variety of forms, e.g., liquid, lyophilized, and typically contain a suitable pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers (or excipients) are the non-active ingredient components of the pharmaceutical composition and are not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical formulation or composition in which it is contained. Carriers are frequently selected to minimize 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 the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptides. Matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. Certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are those suitable for administration of the chimeric polypeptides or nucleic acid sequences encoding the chimeric polypeptides to humans or other subjects.

[0418] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives are optionally present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic, although the formulation can be hypertonic or hypotonic if desired. The pH of the solution is generally about 5 to about 8 or from about 7 to 7.5.

[0419] Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners and the like are optionally necessary or desirable. 88 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0420] Compositions for oral administration include powders or granules, suspension or solutions in water or non-aqueous media, capsules, sachets, or tables. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders are optionally desirable.

[0421] The pharmaceutical compositions described herein are suitable for use as a medicament and can be administered by an appropriate route of administration e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In some embodiments, the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. Dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently. An "effective dose" refers to amounts of the active ingredient that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology and may be determined using known methods.

[0422] The pharmaceutical compositions (e.g., the inducible IL-21 prodrugs described herein, sdAbs described herein, the fusion polypeptides or conjugates described herein) described herein can be encapsulated in nanoparticles. The nanoparticles can be fullerenes, liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. The inducible IL-21 prodrugs described herein can be conjugated to the surface of liposomes. The inducible IL-21prodrugs described herein can be encapsulated within the shell of a liposome. The liposome can be a cationic liposome.

[0423] While parenteral administration is generally preferred, the inducible IL-21 prodrugs, sdAbs, fusion polypeptides, conjugates, or nucleic acid sequences encoding the inducible IL-21 prodrugs can be administered by a vector. There are a number of compositions and methods which can be used to deliver the nucleic acid molecules and / or polypeptides to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express and preferably secrete the encoded chimeric polypeptide or to therapeutically deliver nucleic acids to a subject.

[0424] As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the nucleic acid molecule and / or polypeptide in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, 89 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general and methods of making them are described by Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997). The construction of replication-defective adenoviruses has been described (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 benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.

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

[0426] The inducible IL-21 prodrugs described herein can be delivered by subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003). The provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006 / 110728.

[0427] Non-viral based delivery methods, can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding polypeptides, wherein the nucleic acids are operably linked to an expression control sequence. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.). Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to 90 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 make the inducible IL-21 prodrugs described herein by expression in a suitable host cell, such as CHO cells.

[0428] Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, e.g., β-actin promoter or EF1α promoter, or from hybrid or chimeric promoters (e.g., CMV promoter fused to the β-actin promoter). Of course, promoters from the host cell or related species are also useful herein.

[0429] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5′ or 3′ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 base pairs (bp) in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0430] The promoter and / or the enhancer can be inducible (e.g., chemically or physically regulated). A chemically regulated promoter and / or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal. A physically regulated promoter and / or enhancer can, for example, be regulated by environmental factors, such as temperature and light. Optionally, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize the expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region can be active in a cell type specific manner. Optionally, in certain vectors, the promoter and / or enhancer region can be active in all eukaryotic cells, independent of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the β-actin promoter, the EF1α promoter, and the retroviral long terminal repeat (LTR).

[0431] The vectors also can include, for example, origins of replication and / or markers. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, 91 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. G. Therapeutic Applications

[0432] Also provided herein, are methods and uses for the treatment of a disease, disorder or condition comprising administering to a subject in need thereof an inducible IL-21 prodrug as described herein. Also provided herein, are methods and uses for the treatment of a disease, disorder or condition comprising administering to a subject in need thereof a sdAbs as described herein, or a fusion polypeptide or conjugate as described herein. Diseases, disorders, or conditions include, but are not limited to, cancer, inflammatory disease, an immunological disorder, autoimmune disease, infectious disease (i.e., bacterial, viral, or parasitic disease). 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 sdAbs provided herein, or a fusion polypeptide provided herein or conjugates described herein. Illustrative suitable cancers, in particular solid tumors, such as sarcomas and carcinomas. For examples, the methods and compositions disclosed herein can be used to treat acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, 92 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T- cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and 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 can be used to treat adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, non-Hodgkin 93 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 lymphoma, squamous 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 cancer (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability high or mismatch repair deficient cancer, microsatellite instability high or mismatch repair deficient colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial carcinoma, tumor mutational burden high cancer, cutaneous squamous cell carcinoma (cSCC), triple negative breast cancer (TNBC), urothelial carcinoma, colorectal cancer or oesophageal carcinoma.

[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. As an example, the methods and compositions disclosed herein can be used to treat melanoma in subjects with unresectable or metastatic melanoma. As another example, the methods and compositions disclosed herein can be used for the adjuvant treatment of subjects with melanoma with involvement of lymph node(s) following complete resection.

[0439] In some embodiments, provided herein is a method of enhancing an immune response in a subject in need thereof by administering an effective amount of an inducible IL-21 prodrug described herein to the subject. The enhanced immune response may prevent, delay, or treat the onset of cancer, a tumor, or a viral disease. In some embodiments, the methods described herein increase the activity of Natural Killer Cells and T lymphocytes.

[0440] The method can further involve the administration of one or more additional agents to treat cancer, such as chemotherapeutic agents (e.g., Adriamycin, Cerubidine, Bleomycin, Alkeran, Velban, 94 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine), immuno-oncology agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-LAG3, anti- CD47, anti-GD2), cellular therapies (e.g., CAR-T, T-cell therapy), oncolytic viruses and the like. Non- limiting examples of anti-cancer agents that can be used include acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-nl interferon alpha-n3; interferon beta-I; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone 95 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinzolidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride.

[0441] In some embodiments of the methods described herein, the inducible IL-21 prodrugs described herein is administered in combination with an agent for the treatment of the particular disease, disorder, or condition. This disclosure relates to a therapeutic combination of any of the sdAbs as described herein, or a fusion polypeptide or conjugate described herein in combination with one or more additional agents to for the treatment of a particular disease or condition. Agents include, but are not limited to, therapies involving antibodies, small molecules (e.g., chemotherapeutics), hormones (steroidal, peptide, and the like), radiotherapies (γ-rays, C-rays, and / or the directed delivery of radioisotopes, microwaves, UV radiation and the like), gene therapies (e.g., antisense, retroviral therapy and the like) and other immunotherapies. In some embodiments, the inducible IL-21 prodrugs described herein or is administered in combination with anti-diarrheal agents, anti-emetic agents, analgesics and / or non-steroidal anti- inflammatory agents.

[0442] This disclosure relates to a therapeutic combination of any of the inducible IL-21 prodrugs as described herein in combination with one or more additional agents to treat cancer (such as lymphoma), such as chemotherapeutic agents (e.g., cyclophosphamide, mechlorethamine, melphalan, chlorambucil, ifosfamide, busulfan, N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mitomycin, diaziquone (AZQ), cisplatin, carboplatin, oxaliplatin, procarbazine, hexamethylmelamine, methotrexate, pemetrexed, fluorouracil (e.g.5-fluorouracil), capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, thioguanine, mercaptopurine, vincristine, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, etoposide, teniposide, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, mitoxantrone, actinomycin, bleomycin, bisantrene, gemcitabine, cytarabine, and the like), immuno- oncology agents and immune checkpoint inhibitors (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti- LAG3, anti-CD47, anti-GD2), oncolytic viruses and the like.

[0443] The inducible IL-21 prodrugs described herein can be combined with any desired additional anti- cancer agent. The s inducible cytokine prodrugs described herein can be combined with any desired anti- PD-1 antibody or any desired anti-PD-L1 antibody. 96 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0444] Exemplary anti-PD-1 antibodies that can be combined with the inducible IL-21 prodrugs as described herein, include, but are not limited to, AMP-224 (AstraZeneca), 609A (3SBio), 704 (3SBio), 705 (3SBio), ABBV-181 (AbbVie), ADU-1503 / bion-004 (Chinook Therapeutics), AGEN2034 / balstilimab (Agenus), AK103 (Akeso), AK104 (Akeso), AK112 (Akeso), AK123 (Akeso), AMG 256 (Amgen), AMG 404 (Amgen), ANB030 (AnaptysBio), ANKEBIO Anti-PD1 product (Anhui Anke Biotechnology), Anti PD-1 / Anti-CD47 (DiNonA), ASKG915 (Ask Gene Pharmaceuticals), AV-MEL-1 (Aivita Biomedical), BCD-100 (Biocad CJSC), BI 754091 (Boehringer Ingelheim), BiCKI-IL-7 (OSE Immunotherapeutics), Boehringer-PD-1-unknown (Boehringer Ingelheim), BSK-050K01 (Biosion), Camrelizumab (Jiangsu Hengrui Medicine), CB201 (Crescendo Biologics), CB213 (Crescendo Biologics), CC-90006 (AnaptsBio), cetrelimab (J&J), chPD1 (Kiromic Biopharma), CMAB819 (Mabpharm), CS1003 (CStone Pharmaceuticals), CS17938 (Shenzhen Chipscreen Biosciences), CTX- 8371 (Compass Therapeutics), CX-072 (CytomX Therapeutics), CX-188 (CytomX Therapeutics), cypalizumab (Harbin Gloria Pharmaceuticals), DB004 (DotBio), EMB02 (EpimAb Biotherapeutics), Geptanblimab / genolimzumab (Apollomics), GS19 (Suzhou Zelgen Biopharmaceuticals), HLX10 (Shanghai Henlius Biotech), HX008 (Taizhou HanZhong Pharmaceuticals), HY003 (Juventas Cell Therapy), IBI315 / BH2950 (Innovent Biologics), IBI318 (Innovent Biologics), IBI319 (Innovent Biologics), IMM1802 (ImmuneOnco Biopharma), IMT200 (TrueBinding), Jemperli / dostarlimab (AnaptysBio), JTX-4014 (Jounce Therapeutics), Keytruda / pembrolizumab (Merck), LBL-006 (Nanjing Leads Biolabs), Libtayo / cemiplimab-rwlc (Regeneron Pharmaceuticals), LVGN3616 (Lyvgen Biopharma), LXF821 (Novartis), LY01015 (Luye Pharma Group), LY3462817 (Eli Lilly), MCLA-134 (Merus N.V.), MEDI5752 (AstraZeneca), NIR178 (Novartis), ONCR-177 (Oncorus), ONO-4685 (Ono Pharmaceutical), Opdivo / nivolumab (Ono Pharmaceutical), MGD019 (MacroGenius), PD1-GDT CAR- T (Kiromic Biopharma), penpulimab (Akeso), PSB205 (Qilu Puget Sound Biotherapeutics), PT-001 (Merck), PT627 (Merck), RB-M1 (Refuge Biotechnologies), Retifanlimab (MacroGenics), 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), Tebotelimab (MacroGenics), tislelizumab (BeiGene), TSR-075 (AnaptsBio), Tuhura-DO / PD-1-unknown (Tuhura Biopharma), toripalimab (Shanghai Junshi Biosciences), sintilimab (Innovent Biologics), Unicar-CAR-T&PD-1- unknown (Shanghai Unicar-Therapy Bio-Medicine Technology), Xdivane (Xbrane Biopharma), XmAb20717 (Xencor), XmAb23104 (Xencor), YBL-006 (Y-Biologics), and zimberelimab (Arcus Biosciences). 97 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0445] The anti-PD-1 antibody that can be combined with the inducible IL-21 prodrugs described herein is typically an approved anti-PD-1 antibody. Approved anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA), dostarlimab (JEMPERLI), cemiplimab-rwlc (LIBATYO), nivolumab (OPDIVO), camrelizumab, tislelizumab, toripalimab, and sintilimab (TYVYT).

[0446] Exemplary anti-PD-L1 antibodies that can be combined with the inducible IL-21 prodrugs described herein include, but are not limited to, 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), 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 alfa / GSK4045154 (Merck), CA-170 / aupm-170 (Dr. Reddy’s Laboratories), CCX559 (ChemoCentryx), CDR101 (CDR-Life), cosibelimab (Checkpoint Therapeutics), CTX-8371 (Compass Therapeutics), DiNonA-Solid Tumors-unknown (DiNonA), DR30207 (Zhejiang Doer Biologics), DuoBody-PD-L1x4-1BB (Ligand Pharmaceuticals), envafolimab (Alphamab Oncology), EPIM-001 (Elpis Biopharmaceuticals), ES101 (Elpiscience Biopharma), INBRX-105 (Inhibrx), FAZ053 (Novartis), FS118 (F-star Therapeutics), GB262 (Genor Biopharma), GS-4224 (Gilead), GT900008 (Kintor Pharmaceuticals), GX-P2 (Genexine), Hamni-PS-L1 / CD47-Unknown (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 / durvalumab (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-unknown (Jubilant Therapeutics), KD033 (Kadmon Holdings), KN046 (Alphamab Oncology), KY1003 (Sanofi), KY1043 (Sanofi), LY3300054 (Eli Lilly), LY3415244 (Eli Lilly), MRNA-6981 (Moderna), MSB2311 (Transcenta Holding), MT-6035 (Molecular Templates), ND021 / NM21-1480 (Numab Therapeutics), OX001R (Oxford BioTherapeutics), PD-L1 based BsAbs (I-Mab), PD-L1 Boltbody ISAC (Bolt Biotherapeutics), PDL-GEX (Glycotope GmbH), 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 98 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 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 monoclonals), Vaximm (VXM10), and YBL- 013 (Y-Biologics).

[0447] The anti-PD-L1 antibody that can be combined with the inducible IL-21 prodrugs described herein is typically an approved anti-PD-L1 antibody. Approved anti-PD-1 antibodies include, but are not limited to, avelumab (BAVENCIO), durvalumab (IMFINZI), and atezolizumab (TECENTRIQ).

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

[0449] All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present disclosure. When a range of values is expressed, it includes embodiments using any particular value within the range. Further, reference to values stated in ranges includes each and every value within that range. All ranges are inclusive of their endpoints and combinable. When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The use of “or” will mean “and / or” unless the specific context of its use dictates otherwise.

[0450] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted. 99 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0451] As used herein, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly indicates otherwise. The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.

[0452] Unless otherwise indicated, the terms "at least," "less than," and "about," or similar terms preceding a series of elements or a range are to be understood to refer to every element in the series or range. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0453] As used herein, the terms “activatable,” “activate,” “induce,” and “inducible” refers to a polypeptide complex that has an attenuated activity form (e.g., attenuated receptor binding and / or agonist activity) and an activated form. The polypeptide complex is activated by protease cleavage of the linker that causes the blocking element and half-life extension element to dissociate from the polypeptide complex. The induced / activated polypeptide complex can bind with increased affinity / avidity to the IL-21 receptor.

[0454] The terms “antibody” and “immunoglobulin” are used interchangeably herein. An antibody or immunoglobulin, as used herein, is intended to refer to immunoglobulin molecules comprised of two heavy (H) chains. Typically, antibodies in mammals (e.g., humans, rodents, and monkeys) comprise four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multi specific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, or tetrameric antibodies comprising two heavy chain and two light chain molecules. One of skill in the art would recognize that other forms of antibodies exist (e.g. camelid and shark antibodies). 100 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0455] The term “attenuated” as used herein refers to an IL-21 receptor agonist that has decreased receptor agonist activity as compared to the IL-21 receptor’s. An attenuated IL-21 agonist can have at least about 10X, at least about 50X, at least about 100X, at least about 250X, at least about 500X, at least about 1000X or less agonist activity as compared to the receptor’s naturally occurring agonist. When an IL-21 polypeptide complex that contains IL-21 as described herein is described as “attenuated” or having “attenuated activity”, it is meant that the IL-12 polypeptide complex is an attenuated IL-12 receptor agonist.

[0456] The term "cancer" refers to the physiological condition in mammals in which a population of cells is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate and / or certain morphological features. Often cancers can be in the form of a tumor or mass, but may exist alone within the subject, or may circulate in the blood stream as independent cells, such a leukemic or lymphoma cells. The term cancer includes all types of cancers and metastases, including hematological malignancy, solid tumors, sarcomas, carcinomas and other solid and non-solid tumors. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., triple negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancers. Triple negative breast cancer refers to breast cancer that is negative for expression of the genes for estrogen receptor (ER), progesterone receptor (PR), and Her2 / neu.

[0457] A "conservative" amino acid substitution, as used herein, generally refers to substitution of one amino acid residue with another amino acid residue from within a recognized group which can change the structure of the peptide but biological activity of the peptide is substantially retained. Conservative substitutions of amino acids are known to those skilled in the art. Conservative substitutions of amino acids can include, but not limited to, substitutions made amongst 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 instance, a person of ordinary skill in the art reasonably expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of 101 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 an amino acid with a structurally related amino acid will not have a major effect on the biological activity of the resulting molecule.

[0458] As used herein, the term “half-life extension element” in the context of the polypeptide complex disclosed herein, refers to a chemical element, preferable a polypeptide that increases the serum half-life and improve pK, for example, by altering its size (e.g., to be above the kidney filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination.

[0459] As used herein, the term “operably linked” in the context of a polypeptide complex refers to the orientation of the components of a polypeptide complex that permits the components to function in their intended manner. For example, a polypeptide comprising an IL-21 subunit and an IL-21 blocking element are operably linked by a protease cleavable linker in a polypeptide complex when the IL-21 blocking element is capable of inhibiting the IL-21 receptor-activating activity of the IL-21 polypeptide, but upon cleavage of the protease cleavable linker the inhibition of the IL-21 receptor-activating activity of the IL- 21 polypeptide by the IL-21 blocking element is decreased or eliminated, for example because the IL-21 blocking element can diffuse away from the IL-21.

[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 herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more.

[0461] The term “subject” herein to refers to any animal, such as any mammal, including but not limited to, humans, non-human primates, rodents, and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0462] As used herein, the term “therapeutically effective amount” refers to an amount of a compound described herein (i.e., a IL-21 polypeptide complex) that is sufficient to achieve a desired pharmacological or physiological effect under the conditions of administration. For example, a “therapeutically effective amount” can be an amount that is sufficient to reduce the signs or symptoms of a disease or condition (e.g., a tumor). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. A therapeutically effective amount of a pharmaceutical composition can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmaceutical composition to elicit a desired response in the individual. An ordinarily skilled clinician can determine appropriate amounts to administer to achieve the desired therapeutic benefit based on these and other considerations. 102 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 5. EQUIVALENTS

[0463] It will be readily apparent to those skilled in the art that other suitable modifications and adaptions of the methods of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments. Having now described certain compounds and methods in detail, the same will be more clearly understood by reference to the following examples, which are introduced for illustration only and not intended to be limiting. 6. EXAMPLES

[0464] The present invention is further described by the following examples, which are not intended to be limiting in any way. Example 1: HuT78 pSTAT3 AlphaLISA Assay HuT78 cells (ATCC, TIB-161) were plated in suspension at a concentration of 100,000 cells / well in HBSS media and serum starved for 2 hours at 37oC and 5% CO2. After serum starvation, the cells were stimulated with a dilution series of recombinant human or mouse IL-21 or activatable human or mouse IL-21 for 20 minutes at 37oC and 5% CO2. Activity of uncleaved and cleaved activatable IL-21 was tested. Cleaved inducible IL-21 was generated by incubation with active CTSL or ELA. Post stimulation, the cells were lysed and pSTAT3 Tyr705 levels were measured using AlphaLISA Surefire Ultra pSTAT3 (Tyr705) Assay Kit (Perkin Elmer, #ALSU-PST3-A10K). See, summarized data in Table 45. Table 45. Activity of control IL-21 and IL-21 prodrug on the HuT78 pSTAT3 AlphaLISA asssay Heavy Light Intact Cleaved EC50 Fold Change Ch i Ch i EC50 ( M) ( M) (I t t C t)Example 2: Protease cleavage of IL-21 prodrugs

[0465] One of skill in the art would be familiar with methods of setting up protein cleavage assay.50 µg of protein in 1xPBS pH 7.4 were cleaved with 1 µg active CTSL (R&D Systems catalog # 952-CY-010) 103 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 or ELA (Sigma Aldrich catalog # 324682, Elastase, Porcine Pancreas, High Purity, Crystallized) in a total volume of 100 µL and incubated at room temperature for up to 16 hours. Digested protein was subsequently used in functional assays or stored at -80oC prior to testing. Extent of cleavage was monitored by SDS PAGE using methods well known in the art (see example cleavage gel FIG.2). Full cleavage of the IL-21 prodrugs by CTSL or elastase protease was seen. Full cleavage is not seen in controls. Example 3. IL-21 Dimerization β-galactosidase Reporter Assay

[0466] IL-21 β-galactosidase reporter cells (DiscoverX, Eurofins), purchased from the manufacturer in a “Thaw and Use” format, were plated for 24 hours at 37oC and 5% CO2 according to the manufacturer’s directions. The next day, the plated cells were stimulated with a dilution series of recombinant hIL-21 or activatable hIL-21 for 6 hours at 37oC and 5% CO2. In some experiments, 1.5% human albumin was included. Activity of uncleaved and cleaved activatable IL-21 was tested. Cleaved inducible IL-21 was generated by incubation with active CTSL (R&D Systems) or ELA-1 (Sigma Aldrich catalog # 324682, Elastase, Porcine Pancreas, High Purity, Crystallized). The PathHunter® eXpress IL-21 assay (DiscoverX, Eurofins) uses Enzyme Fragment Complementation (EFC) technology, where the β - galactosidase (β -gal) enzyme contains two fragments; the ProLink (PK) and Enzyme Acceptor (EA). When forced to complement through the IL-21-IL-21R interaction, they form an active β -gal enzyme that hydrolyzes a substrate to form a chemiluminescent signal, which is used to quantify IL-21 activity. The results are summarized in Table 46. Table 46. Activity of inducible IL-21 prodrugs in a IL-21 β-galactosidase reporter assay Heavy Light Intact Cleaved EC50 Fold Change Ch i Ch i EC50 ( M) ( M) (I t t C t)\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 WW50108 WW50102 not active 4.87 N / A WW50118 WW50112 not active 3.25 N / A\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 WW50385 WW50394 565 5.08 111.2 WW50387 WW50394 368 8.08 45.5106 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Example 4: Mouse T-Blast Assay

[0467] Splenocytes from C57BL / 6 mice were stimulated with 2µg / mL Concanavalin A (ConA) to generate Tblasts. Cells were incubated in RMPI supplemented with 10% FBS, penicillin / streptomycin and 2-mercaptoethanol at 37°C with 5% CO2 for 72 hours. Tblasts harvested, washed with HBSS, plated at 100,000 cells per well and serum starved for 2 hours at 37°C. Tblasts were stimulated with titrated amounts of mouse IL-21 or activatable mouse IL-21 for 20 minutes at 37°C. Activity of uncleaved and cleaved activatable mouse IL-21 was tested. Cleaved activatable IL-21 was generated by incubation with active CTSL or ELA. Cells were lysed and pStat3 measured with an AlphaLISA SureFire Ultra pStat3 (Tyr705) assay kit from PerkinElmer. As sumatized in Table 47 cleaved IL-21 prodrugs induced phosphorylation of Stat3 in murine Tblasts. Table 47. Activity of inducible IL-21 prodrugs in Mouse T-Blast assay. Heavy Light AssIntact Cleaved EC50 Fold Change ChainChainayEC50 (nM) (nM) (Intact / Cut)xamp e : - ur ne r mary e ssay

[0468] B cells were isolated from C57BL / 6 splenocytes using CD19 positive selection beads from Miltenyi. Cells plated at 100,000 cells per well in round bottom 96 well plates in RMPI supplemented with 10% FBS, penicillin / streptomycin and 2-mercaptoethanol. Cells stimulated with 1µg / mL soluble anti-IgM and titrated amounts of mouse IL-21 or activatable mouse IL-21. Activity of uncleaved and cleaved activatable mouse IL-21 was tested. Cells were incubated for 48 hours at 37°C with 5% CO2. IL- 21 activity was assessed by measuring proliferation with Cell Titer Glo assay (Promega). Results are summarized in Table 48. Table 48. Activity of inducible IL-21 prodrugs in murine primary B-cell assay Heavy Light AssayIntact Cleaved EC50 Fold Change )\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 WW50081Murine B cell5.84 0.0389150WW50084 WW50085Murine B cell1.87 0.443 4.22

[0469] Previously frozen human PBMCs were thawed, washed and counted in pre-warmed X-Vivo15 media. Cells were washed with HBSS, plated at 100,000 cells per well and serum starved for 2 hours at 37°C. PBMCs were stimulated with titrated amounts of uncleaved and cleaved activatable IL-21 for 20 minutes at 37°C. Cells were lysed and pStat3 measured with an AlphaLISA SureFire Ultra pStat3 (Tyr705) assay kit from PerkinElmer. As summarized in Table 49 cleaved inducible IL-21 prodrugs induce phosphorylation of Stat3 in human PBMCs. Table 49. Activity of inducible IL-21 prodrugs in human primary Heavy Light nAssIntact Cleaved EC50 Fold Change ChainChaiayEC50 (nM) (nM) (Intact / Cut)\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 WW50385 WW50393 Human PBMC 1.35 0.0218 61.9 WW50387 WW50393 Human PBMC 2.65 0.0367 72.2Example 7: MC38 Experiments (Study MC38-e52)

[0470] The MC38 cell line, a rapidly growing colon adenocarcinoma cell line, was used. Using this tumor model, the ability of IL-21 prodrugs to affect tumor growth and body weight was examined. Table 3. Agents and treatment regimen Group N Agent Dose Route Schedule09 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 4 8 WW50008 100 µg / animal ip biwk x 3 5 8 WW50008 300 µg / animal ip biwk x 3Female C57BL / 6 mice were set up with 5x105MC38 tumor cells in 0% Matrigel sc in flank. Cell injection volume was 0.1 mL / mouse. Mouse age at start date was 8 to 12 weeks. Pair matches were performed when tumors reach an average size of 100 - 150 mm3and began treatment. This was Day 1 of the study. Body weights were taken at initiation and then biweekly to the end. Caliper measurements were taken biweekly to the end. Any adverse reactions were reported immediately. Any individual animal with a single observation of > than 25% body weight loss or three consecutive measurements of >20% body weight loss was euthanized. Any group with a mean body weight loss of >20 % or >10% mortality stopped dosing; the group was not euthanized, and recovery was allowed. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint were euthanized. If the group treatment related body weight loss was recovered to within 10% of the original weights, dosing resumed at a lower dose or less frequent dosing schedule. Exceptions to non-treatment body weight % recovery were allowed on a case-by-case basis. Endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1500 mm3or 40 days, whichever came first. When the endpoint was reached, the animals were euthanized. Results are shown in FIGs.3A- 3B. Dose dependent efficacy observed (Ave. tumor volume vs. time) FIG.3A and tolerability FIG.3B. Example 8: B16F10 experiments (study B16F10-E4832-U2104)

[0472] The B16F10 cell line, a rapidly growing murine melanoma cell line, was used. Using this tumor model, the ability of IL-21 prodrugs to affect tumor growth was examined. Table 4. Agents and treatment regimen Group N Agent Dose Route Schedule 1 8 Vehicle - i biwk x 3110 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Procedures:

[0473] 40 JAX female C57BL / 6 mice were set up with 2x105B16F10 tumor cells in 0% Matrigel sc in right flank. Cell Injection Volume was 0.1 mL / mouse. Mouse age at start date was 6 to 8 weeks. Animals were shaved and ear tagged prior to injection. One mouse at a time was immobilized and the site of injection was disinfected with an alcohol swab. During implantation, a new syringe and needle was used for every mouse to minimize tumor ulcerations. Animals were monitored weekly for palpable tumors, or any changes in appearance or behavior. Once tumors were palpable, tumors were measured 2 times a week using calipers. Tumor volume will were calculated using the following equation: (longest diameter * shortest diameter2) / 2. Once tumors reached an average size of 50-90 mm3 size, tumors and body weights were measured 2 times per week for the duration of the study. This was Day 1 of the study. One individual was responsible for tumor measurements for the duration of the study. Body weight loss was calculated based on the BW of the mouse on the first day of treatment. If body weight loss of >20% was observed, the animal was monitored daily for signs of recovery for up to 72 hours. If there were no signs of recovery, the animal was sacrificed for humane reasons as per IACUC protocol regulations. The endpoint of the experiment was a tumor volume of 3000 mm3or 25 days, whichever came first. When the endpoint was reached, the animals were euthanized. Results are shown in FIGs.4 and 5A-5E. Example 9: EMT6 experiments (study EMT6-E4832-U2105)

[0474] The EMT6 cell line, a rapidly growing breast cancer cell line, was used. Using this tumor model, the ability of IL-21 prodrugs to affect tumor growth was examined. Table 5. Agents and Treatment: Group N Agent Dose Route Schedule 1 8 Vehicle - ip biwk x 3Procedures:

[0475] 40 JAX female BALB / c mice were set up with 2.5x105EMT6 tumor cells in 0% Matrigel sc in flank. Cell injection volume was 0.1 mL / mouse. Mouse age at start date was 6 to 8 weeks. Mouse age at start date was 6 to 8 weeks. Animals were shaved and ear tagged prior to injection. One mouse at a time 111 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 was immobilized and the site of injection was disinfected with an alcohol swab. During implantation, a new syringe and needle was used for every mouse to minimize tumor ulcerations. Animals were monitored weekly for palpable tumors, or any changes in appearance or behavior. Once tumors were palpable, tumors were measured 2 times a week using calipers. Tumor volume was calculated using the following equation: (longest diameter * shortest diameter2) / 2. Once tumors reached an average size of 50- 90 mm3 size, tumors and body weights were measured 2 times per week for the duration of the study. This was Day 1 of study. One individual was responsible for tumor measurements for the duration of the study. Body weight loss was calculated based on the BW of the mouse on the first day of treatment. If body weight loss of >20% was observed, the animal was monitored daily for signs of recovery for up to 72 hours. If there were no signs of recovery, the animal was sacrificed for humane reasons as per IACUC protocol regulations. The endpoint of the experiment was a tumor volume of 2000 mm3or 30 days, whichever came first. When the endpoint was reached, the animals were euthanized. Results are shown in FIGs.6 and 10A-10E. Example 10. Efficacy and TILs Analysis of MC38 Tumors Treated with Half-Life Extended IL-21

[0476] Female C57BL / 6 mice were inoculated with 1x105MC38 cells in 50% Matrigel on the flank. Tumors were allowed to grow for 13 days and were randomized for dosing (average tumor volume 120 mm3). Mice were treated with vehicle or 100µg WW50008 by intraperitoneal injection (IP) on days 1, 4, 8 and 11 after randomization. Body weight and tumor sizes were measured three times per week. Groups consisted of 10 animals, 5 remained on study to measure efficacy and 5 mice were sacrificed on day 5 for analysis. Animals remaining on study were monitored for 25 days or euthanized when tumor burden reached 2000 mm3volume. For day 5 analysis, tumors were collected, weighed, and digested using enzymatic and manual dissociation to obtain a single cell suspension of tumor infiltrating lymphocytes (TILs). Single cell suspensions were stimulated with PMA / ionomycin / BFA for 4 hours and then stained with fluorescently labeled antibodies for analysis by flow cytometry. Samples were run on a Cytek Aurora flow cytometer and data analyzed using Flow Jo and GraphPad Prism software. Measurements from the efficacy study showed a reduction in tumor burden with WW50008 treatment. Results are shown in FIG.8A. All mice from WW50008 group had complete regressions (CR) of tumors with regressions starting at day 5. Meanwhile all mice from vehicle group were euthanized due to tumor burden.

[0477] Analysis of TILs showed an increase in lymphocyte infiltrates in WW50008 treated group compared to vehicle as measured by total cells per mg of tissue and frequency of CD45+ TILs (FIG.8B). WW50008 treatment led to an increase in total counts of NK cells within the tumor and IFNɣ production 112 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 (FIG.8C). Along with increased NK cells, WW50008 treated animals had increased CD8+ Tetramer+ T cells compared to vehicle (FIG.8D). This tetramer staining peptide is derived from the p15e envelope protein from endogenous (AKV / MCF) Murine Leukemia Virus which is overexpressed by MC38 tumor cells. These CD8+ Tetramer+ T cells made more pro-inflammatory cytokines such as Granzyme B, IFNɣ and TNF than vehicle treated mice. Together these data demonstrate WW50008 treatment leads to tumor regression mediated by increased immune infiltrates consisting of activated NK and tumor-specific effector CD8+ T cells. Example 11. Efficacy and TILs analysis of EMT-6 tumors treated with half-life extended IL-2 or IL-21.

[0478] Female BALB / c mice were inoculated with 1x105EMT-6 cells in 50% Matrigel on the flank. Tumors were allowed to grow for 8 days, and then mice were randomized for dosing (average tumor volume 100 mm3). Mice were treated with vehicle, 10µg WW0177 (half-life extended human IL-2) or 100µg WW50008 (half-life extended mouse IL-21) by intraperitoneal injection (IP) on days 1, 4, 8 and 11 after randomization. Body weight and tumor sizes were measured three times per week. Groups consisted of 15 animals, 8 remained on study to measure efficacy, and 5 mice were sacrificed on day 12 for analysis. Animals remaining on study were monitored for 30 days or euthanized when tumor burden reached 2000 mm3volume. For day 12 analysis tumors were collected, weighed, and digested using enzymatic and manual dissociation to obtain a single cell suspension of tumor infiltrating lymphocytes (TILs). Single cell suspensions were stimulated with PMA / ionomycin / BFA for 4 hours and then stained with fluorescently labeled antibodies for analysis by flow cytometry. Samples were run on a Cytek Aurora flow cytometer and data analyzed using Flow Jo and GraphPad Prism software. Efficacy study results showed a reduced tumor burden with both WW0177 and WW50008 treatment (FIG.9A). WW0177 treated animals had reduced tumor burden compared to vehicle but tumors persisted and continued to grow, albeit at a slower rate. At the end of study, two mice from WW0177 had tumors <100 mm3, but all remaining mice had reached the tumor burden endpoint. Conversely, all mice treated with WW50008 had complete regressions (CRs) of tumors. WW50008 treatment led to reduced tumor sizes starting around day 15 and tumors continued to decrease until end of study or CR reached. All mice from the vehicle group reached tumor burden endpoint by day 25 and were sacrificed.

[0479] Analysis of TILs showed an increase in CD8+ T cell infiltrates in both WW0177 and WW50008 treated animals compared to vehicle (FIG.9B). Additionally, CD8+ T cells from both WW0177 and WW50008 treated mice produced effector cytokines such as Granzyme B, IFNɣ and TNF at higher 113 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 frequencies than vehicle mice (FIG.9B). Of note, WW50008 treatment led to higher frequencies of these pro-inflammatory cytokines than treatment with WW0177. Within the CD8+ T cell compartment, cells were able to make multiple cytokines at the same time, defined as polyfunctionality (FIG.9C). Examining this polyfunctionality of cytokine production, both WW0177 and WW50008 treated CD8+ T cells were more polyfunctional than vehicle treated T cells. WW50008 treated CD8+ T cells were more polyfunctional than WW0177 treated T cells, with 11% of WW50008 treated CD8+ T cells producing all three effector cytokines compared to 4% for WW0177 treated. WW50008 T cells also had a higher frequency of double positive producers than WW0177 T cells, about 30% vs 26% (FIG.9C).

[0480] Within the CD4+ T cells population of TILs, WW0177 led to an increase in CD4+ Foxp3+ regulatory T cells (Tregs) compared to vehicle. Treatment with WW50008 did not expand Tregs compared to vehicle. Calculating the ratio of CD8+ T cells to Tregs, WW50008 treated mice had a more favorable CD8 / Treg ratio than vehicle or WW0177 treated animals (FIG.9D).

[0481] 500,000 cells from tumor single cell suspensions (day 12) were pelleted and cells lysed using RLT+ Lysis Buffer (Qiagen) and snap frozen. RNA samples isolated from tumors treated with vehicle control, WW0177 or WW50008 were shipped to Canopy Biosciences and analyzed using the NanoString technology pan-cancer cassette. Data analysis was preformed using the Counter software and Prism GraphPad. WW50008 treatment of tumors induces differential upregulation or downregulation of 35 exclusive genes compared to WW0177 (FIG.9E and FIG.21B). Comparison genes significantly differentially expressed between vehicle and IL-21 (WW50008) are shown in FIGs.20A and 20B. Differential transcriptional regulation is driven by WW50008 when compared to WW0177. Comparison genes significantly differentially expressed between IL-2 and IL-21 are shown in FIGs.21A and 21B.

[0482] Together these data demonstrate that treatment with WW50008 (half-life extended IL-21) leads to complete regression of tumors mediated by immune activation. Treatment with WW50008 is superior to WW0177 (half-life extended IL-2) treatment in the EMT-6 model (poorly immunogenic) with greater efficacy and activation of pro-inflammatory immune cells. Example 12. Efficacy in MC38 Tumors Treated with Half-Life Extended IL-21 and IL-21 Prodrug

[0483] The MC38 cell line, a rapidly growing colon adenocarcinoma cell line, was used. Using this tumor model, the ability of IL-21 prodrug to affect tumor growth and body weight was examined. Table 6. Agents and Treatment Regiment Group N Agent Dose Route Schedule\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 2 8 WW50008 10 µg / animal ip biwk x 2 3 8 WW50076 100 µg / animal ip biwk x 3 i l i i kce the ulcerations. Female C57BL / 6 mice were set up with 5x105MC38 tumor cells in 0% Matrigel sc in flank. Cell injection volume was 0.1 mL / mouse. Mouse age at start date was 8 to 12 weeks. Pair matches were performed when tumors reach an average size of 100 - 150 mm3 and began treatment. This was Day 1 of the study. Body weights were taken at initiation and then biweekly to the end. Caliper measurements were taken biweekly to the end. Any adverse reactions were reported immediately. Any individual animal with a single observation of > than 25% body weight loss or three consecutive measurements of >20% body weight loss was euthanized. Any group with a mean body weight loss of >20 % or >10% mortality stopped dosing; the group was not euthanized, and recovery was allowed. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint were euthanized. If the group treatment related body weight loss was recovered to within 10% of the original weights, dosing resumed at a lower dose or less frequent dosing schedule. Exceptions to non-treatment body weight % recovery were allowed on a case-by-case basis. Endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1500 mm3 or 40 days, whichever came first. When the endpoint was reached, the animals were euthanized. Results are shown in FIGs.10A (Ave. Tumor volume vs time) and 10B (Ave. body weigh vs time). Example 13. EMT-6 Tumor Re-Challenged

[0485] As a demonstration that treatment with IL-21 half-life extended (HLE) is able to induce immunological memory, animals previously treated with mouse IL-21-HLE (WW50008) which survived initial EMT-6 inoculation were re-challenged with the same cell line. Specifically, 113 days post initial inoculation and 45 days after confirmation of complete regressions, those animals were inoculated with 100,000 EMT-6 cells + 50% matrigel on the flank opposite to the initial inoculation site. As a control, naïve animals of matched age were inoculated with the same number of cells and followed to assess tumor growth. As shown in FIGs.11A-11B, only the naïve animals developed tumors, while the animals which survived the first tumor inoculation due to treatment with IL-21-HLE were protected against a second inoculation of EMT-6 cells. 115 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Example 14. Biocytogen hIL21R MC38 study with inducible IL-21 prodrugs

[0486] A validated slow growing clone (BC2) of the cell line MC38, derived from C57BL / 6 murine colon adenocarcinoma cells was used for this study. These cells were implanted into B-hIL21R humanized knock-in mice where exons 3-6 of mouse Il21r gene that encode the extracellular domain were replaced by human IL21R exons 3-6 (Biocytogen).

[0487] Table 7. Agents and Treatment Regimen (Efficacy Arm) Group N Agent Dose (µg / mouse) Route Schedule 1 8 Vehicle - ip biwk x 2Tab e 8. Agents and Dos ng Reg men (PK arm) Group N Agent DoseTerminal Sample (µg / mouse)Route ScheduleCollection (N) 8h 8h 8h 8h 8h116 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320

[0488] Cells in an exponential growth phase were harvested, prepared, and suspended in serum-free medium to obtain 10.0x106cells per mL, and matrigel was added 1:1, so each 0.1 mL delivered the number of cells needed per inoculation. B-hIL21R mice (Jiangsu Biocytogen Co., Ltd., China Production- Stock# 110766F), with an age range of 10 weeks and a weight range of 1725 g, were lightly anesthetized before implantation, and each mouse was subcutaneously injected with 0.5x106MC38-BC2 tumor cells in 0.1 mL serum-free medium containing 50% Matrigel in the right flank for tumor development. Tumor- bearing animals were enrolled in 18 study groups when mean tumor volume (TV) reached approximately ~150 mm3(125-175 mm3). Each group consisted of 8 mice (Table 7: Groups 1-18) in the efficacy arm. Each single-dose group consisted of 8 mice (Table 8: Groups 2-6) in the PK arm, with an additional vehicle group (Group 1, n=4) and a no treatment group (n=3). The physical grouping based on baseline measurements was done the day before treatment initiation (Day -1). The day treatment was initiated is represented as Day 0. Blood samples for pharmacokinetic analysis were collected from each group at 24 hours and 48 hours after the 1stdose from 4 animals per time-point. Animals were monitored until the defined study endpoints so that each individual mouse was terminated when TV >2000 mm3or any other humane endpoint such as 20% body weight loss without recovery to 15% within 2 consecutive readings was reached. The study continued even after the termination of the control group until day 30 post study initiation. The minimum efficacious dose (MED) was then calculated on a per molar basis as shown by significant tumor growth inhibition for any group, while the maximum tolerated dose (MTD) was calculated on a per molar basis as no deaths from treatments, and no unrecoverable weight loss of over 20%. Therapeutic window (TW) is calculated and defined as MTD / MED.

[0489] Results are shown in FIGs.13A-13C (average volume vs time, body weight vs time for hIL-21 HLE-WW50324), FIGs.14A-14C (average volume vs time, body weight vs time for WW50112 / WW50347), FIGs.15A-15C (average volume vs time, body weight vs time for WW50112 / WW50348), and FIGs.16A-16B (average volume vs time, body weight vs time for WW50260). After 24 hours 4 mice from the vehicle and each treatment group (groups 1-6) were euthanized, and serum and tumor samples were collected. After 48 hours the remaining 4 mice from each of the treatment groups (Groups 2-6) and the no treatment control mice (group 7) were euthanized for tumor and serum collections. Serum samples 4 and 24 hour from the first and last dose in the efficacy arm, and terminal 24 and 48 hours after a single dose in the PK arm and tumor lysates (24 and 48 hours) after a single dose in the PK arm were analyzed using electrochemiluminescence immunoassays (Meso Scale Discovery MESOTMQuickPlex SQ 120 reader) designed to detect total and free human IL-21 in mouse serum and tumor lysates. Results are shown in FIGs.17A-17D and 18A-18G. 117 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Example 15. IL-21 ELISPOT

[0490] Spleens from EMT-6 IL-2-half-life extended (HLE) (WW0177) or IL-21-HLE (WW50008) treated complete remission (CR) mice were collected 30 days post CR. Spleens from naïve BALB / c mice were used as negative controls. Spleens were dissociated into single cell suspension, and EMT-6 cells were harvested from cell culture flasks. Cells were counted, and splenocytes were plated in duplicate alone or with EMT-6 cells at a 5:1 ratio. Cells were cultured on a pre-coated mouse IFNγ ELISPOT plates (Mabtech) and incubated for 24 hours at 37°C with 5% CO2. Plates were developed following the manufacturer’s protocol. The developed plates were shipped to ZellNet Consulting Inc, where plates were imaged and read for spot counts and volume. Results are show in FIG.11C and 11D. Example 16. Serum Stability

[0491] Stability of inducible IL-21 prodrugs was assessed by incubation in pooled healthy donor serum from healthy humans, cynomolgus monkeys, or mice. Pooled human and cynomolgus monkey serum were purchased from BioIVT. Pooled murine serum was generated in house from C57BL / 6 mice purchased from Jackson Laboratories. Intact inducible IL-21 prodrugs were incubated in serum at 37°C for 0 hours (immediately frozen), 24 hours, or 72 hours. After incubation, samples were frozen at -80°C until all samples were collected. Samples were thawed on ice, ran under reduced conditions and cleavage was detected using JESS Capillary Western Blot (Protein Simple) for human IL-21 using an antibody purchased from LSBio (#C170343). As show in FIGs.29A-29C all inducible IL-21 prodrugs were stable in serum, showing no cleavage species. Example 17. Primary Cell Assay for Screening Anti-IL-21 Antibodies

[0492] Previously frozen human PBMCs were thawed, washed and counted in pre-warmed X-Vivo15 media. Cells were washed with HBSS, plated at 100,000 cells per well and serum starved for 2 hours at 37°C. Titrated amounts of anti-IL-21 antibodies were pre-incubated with 0.5nM of recombinant human IL-21 (WW50289) for 30 minutes at room temperature. After serum starvation, PBMCs were stimulated with either 0.5nM recombinant IL-21 or 0.5nM recombinant IL-21 with titrated amounts of antibodies for 20 minutes at 37°C. Cells were lysed and pStat3 measured with an AlphaLISA SureFire Ultra pStat3 (Tyr705) assay kit from PerkinElmer. As summarized in Table 50, most anti-IL-21 antibodies inhibit IL- 21 induced phosphorylation of Stat3 in human PBMCs to varying degrees. 118 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Table 50. Activity of anti-IL-21 antibodies in a primary cell assay Heavy Chain / Ab #LightIntact IC50 Chain(nM)Example 18. CT26 Efficacy Tumor Mouse Model

[0493] Female BALB / c mice were inoculated with 1x105CT26 cells in 50% Matrigel on the flank. Tumors were allowed to grow for 7-8 days until mice were randomized for dosing (average tumor volume 100 mm3). Mice were treated with vehicle, 9µg WW0177 (half-life extended IL-2) or 10µg and 75 µg of WW50008 (half-life extended IL-21) by intraperitoneal injection (IP) on days 1, 4, 8 and 11 after randomization. Body weight and tumor sizes were measured three times per week. Animals were monitored for 30 days or euthanized when tumor burden reached 2000 mm3volume. Tumor size was plotted against time using GraphPad Prism software to assess tumor growth inhibition. Results are shown in FIG.19B. 119 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Example 19. Renca Efficacy Tumor Mouse Model

[0494] Female BALB / c mice were inoculated with 1x105Renca cells in 50% Matrigel on the flank. Tumors were allowed to grow for 7-8 days until mice were randomized for dosing (average tumor volume 100 mm3). Mice were treated with vehicle, 9µg WW0177 (half-life extended IL-2) or 10µg and 75 µg of WW50008 (half-life extended IL-21) by intraperitoneal injection (IP) on days 1, 4, 8 and 11 after randomization. Body weight and tumor sizes were measured three times per week. Animals were monitored for 56 days or euthanized when tumor burden reached 2000 mm3volume. Tumor size was plotted against time using GraphPad Prism software to assess tumor growth inhibition. Results are shown in FIG.19A. Example 20. IL-21 Luciferase Reporter Assay

[0495] IL-21 luciferase reporter cells (Promega), purchased from the manufacturer in a “Thaw and Use” format, were plated according to the manufacturer’s directions and stimulated with a dilution series of recombinant hIL-21 or activatable hIL-21 for 6 hours at 37oC and 5% CO2. Activity of uncleaved and cleaved activatable IL-21 was tested. Cleaved inducible IL-21 was generated by incubation with active protease (e.g. elastase). IL-21 activity was assessed by quantification of luciferase activity using Bio- Glo™ Reagent (Promega), which allows for the measurement of luciferase activity by luminescence readout. Results are summarized in Table 53. Table 53: Activity of inducible IL-21 Promega assay Intact Cleaved Light Fold Change Heav EC50 EC50 )\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 WW50444 WW50463 0.235 0.0174 13.5 WW50446 WW50463 0308 00063 492Example 21. MC38-e662 Study. Efficacy and Tolerability of Mouse IL-21 Prodrugs in MC38 Mice

[0496] The MC38 cell line, a rapidly growing colon adenocarcinoma cell line, was used. Using this tumor model, the ability of IL-21 prodrugs to affect tumor growth and body weight was examined. Table 9. Agents and Treatment Regiment Group N Agent Dose Route Schedule 1 8 Vehicle - i biwk x 2\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 14 8 WW50256 1,000 µg / animal ip biwk x 2 15 8 WW50256 1,500 µg / animal ip biwk x 2 i l i i kof cells to reduce ulcerations. MC38 cells used for implantation were harvested during log phase growth and resuspended in cold RPMI serum-free medium (in the absence of matrigel). Tumors were initiated by anesthetizing each animal with isoflurane and then subcutaneously implanting 5 x 105MC38 cells (in a 0.1 mL suspension) into the right flank of each test animal. Mouse ages at the start date were 8 to 12 weeks and body weights (BW) ranged from 16.7 to 24.3g. When tumors reached an average size of 100 - 150 mm3, pair matches were performed and treatment was initiated, representing Day 1 of the study. Body weights were measured at initiation and then biweekly to the end of the study. Tumor size was measured by digital calipers biweekly to the end of the study. Any adverse reactions were reported immediately. Any individual animal with a single observation of > than 25% body weight loss or three consecutive measurements of >20% body weight loss was euthanized. Endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1500 mm3or 40 days, whichever came first. When the endpoint was reached, the animals were euthanized. Results are shown in FIGs.22A-22F (average tumor volume vs. time) and FIGs.23A-23F and 24A-24O (average body weight vs. time). Example 22. Albumin binding to Inducible IL-21 prodrugs

[0498] To assess the binding affinity of inducible IL-21 prodrugs to albumin, a biolayer interferometry assay was conducted with an Octet Qke instrument (ForteBio / Sartorius). A biotinylated His-tagged and Avi-tagged human albumin (Acro Biosystems) was captured onto streptavidin biosensors (Sartorius) at 1 µg / mL in 1X kinetics buffer (Sartorius) for 300 seconds. Next, a titration of the inducible IL-21 prodrugs, 100 nM to 1.56 nM (2-fold dilutions) in 1X kinetics buffer for 300 seconds was performed. A blank buffer well was used for reference subtraction. Then, sensors were dipped into 1X kinetics buffer for 600 seconds. Sensors were initially dipped into 1x kinetics buffer for 60 seconds and subsequently between each step for 120 seconds to provide a baseline prior to the step. Agitation at all steps was 1000 rpm. Data were analyzed by background subtracting a reference well (no albumin) and then a global group fit of the binding curves using a 1:1 binding model to generate KDs (ForteBio Data Analysis Software v8.2). Results are shown below in Table 10. 122 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Table 10. Inducible IL-21 prodrugs binding to human serum albumin by biolayer interferometry Ab Ab KD* Heavy Light Orientation KD (M) kon(1 / Ms) kdis(1 / s) WW50 HSA-X-hIL21-LXL- 4 3 3 4 3 3 4 3 3 4 4 4Example 23. IL-21 Binding to Antibodies

[0499] To assess the binding affinity of IL-21 antibodies to human IL-21, a biolayer interferometry assay was conducted with an Octet Qke instrument (ForteBio / Sartorius). Full length human IgG1 antibodies were captured onto anti-human IgG-Fc (AHC) biosensors (Sartorius) at 5 µg / mL in 1X kinetics buffer (Sartorius) for 300 seconds. Next, a titration of human IL-21, 100 nM to 1.56 nM (2-fold dilutions) in 1x 123 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 kinetics buffer was added for 300 seconds. A blank buffer well was used for reference subtraction. Then, sensors were dipped into 1X kinetics buffer for 600 seconds. Sensors were initially dipped into 1x kinetics buffer for 60 seconds and subsequently between each step for 120 seconds to provide a baseline prior to the step. Data were analyzed by background subtracting a reference well (no Ab) and then a global group fit of the binding curves using a 1:1 binding model to generate KDs (ForteBio Data Analysis Software v8.2). Results are shown below in Table 11. Table 11. Anti-IL-21 IgGs binding to human IL-21 by biolayer interferometry Sample ID KD (M) kon(1 / Ms) kdis(1 / s) ADI 76957 299E 11 125E+06 373E 05. IL-21 Prodrugs in Biocytogen human IL-21 Receptor Mice Bearing Syngeneic MC38 (Slow-Growing) Murine Colon Carcinoma

[0500] A validated slow growing clone (BC2) of the cell line MC38, derived from C57BL / 6 murine colon adenocarcinoma cells was used for this study. These cells were implanted into B-hIL21R humanized knock-in mice where exons 3-6 of mouse Il21r gene that encode the extracellular domain were replaced by human IL21R exons 3-6 (Biocytogen). Table 12. Agents and Treatment Regimen (Efficacy Arm) Group N Agent Dose (µg / mouse) Route Schedule 1 8 V hi l i biwk x 2\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 12 8 WW50454 / WW50464 600 ip biwk x 2 13 8 WW50454 / WW50464 1800 ip biwk x 2 i i kGroup N Agent Dose Route SchedulTerminal Sample (µg / mouse)eCollection (N) ) ) ) ) ) ) ) ) )p g p , p p , p medium to obtain 10.0x106cells per mL, and matrigel was added 1:1, so each 0.1 mL delivered the number of cells needed per inoculation. B-hIL21R mice (Jiangsu Biocytogen Co., Ltd., China Production- Stock# 110766F), with an age range of 15-17 weeks and a weight range of 19-36 g, were lightly anesthetized before implantation, and each mouse was subcutaneously injected with 0.5x106MC38-BC2 tumor cells in 0.1 mL serum-free medium containing 50% Matrigel in the right flank for tumor development. Tumor-bearing animals were enrolled in 18 study groups when mean tumor volume (TV) reached approximately ~150 mm3(125-175 mm3). Each group consisted of 8 mice (Table 12: Groups 1- 18). Each single-dose group consisted of 8 mice (Table 13: Groups 2-5) in the PK arm, with an additional vehicle group (Group 1, n=4). The physical grouping based on baseline measurements was done the day before treatment initiation (Day -1). The day treatment was initiated is represented as Day 0. Blood samples for pharmacokinetic analysis from the efficacy arm were collected at 4 hours after the 1st(Day 0) and 4th(Day 10) treatments from 4 animals in each group, and at 24 hours after the 1stand 4thtreatments from the remaining 4 animals in each group. Animals were monitored until the defined study endpoints so that each individual mouse was terminated when TV >2000 mm3or any other humane endpoint such as 20% body weight loss without recovery to 15% within 2 consecutive readings was reached. The study 125 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 continued even after the termination of the control group until day 28 post study initiation. The minimum efficacious dose (MED) was then calculated on a per molar basis as shown by significant tumor growth inhibition for any group, while the maximum tolerated dose (MTD) was calculated on a per molar basis as no deaths from treatments, and no unrecoverable weight loss of over 20%. Therapeutic window (TW) is calculated and defined as MTD / MED. Results are shown in FIGs.12C-12D.

[0502] After 24 hours 4 mice from the vehicle and each treatment group (PK Arm groups 1-5) were euthanized, and serum and tumor samples were collected. After 48 hours the remaining 4 mice from each of the treatment groups (PK Arm groups 2-5) were euthanized for tumor and serum collections. Serum samples (4 and 24 hour from the first and last dose in the efficacy arm, and terminal 24 and 48 hours after a single dose in the PK arm) and tumor lysates (24 and 48 hours after a single dose in the PK arm) were analyzed using electrochemiluminescence immunoassays (Meso Scale Discovery MESOTMQuickPlex SQ 120 reader) designed to detect total and free human IL-21 in mouse serum and tumor lysates. Results are shown in FIGs.28A-28P. Example 25. In vivo Efficacy and PK Evaluation of Inducible IL-21 Prodrugs in Biocytogen human IL-21 Receptor Mice Bearing Syngeneic MC38 (Slow-Growing) Murine Colon Carcinoma

[0503] A validated slow growing clone (BC2) of the cell line MC38, derived from C57BL / 6 murine colon adenocarcinoma cells was used for this study. These cells were implanted into B-hIL21R humanized knock-in mice where exons 3-6 of mouse Il21r gene that encode the extracellular domain were replaced by human IL21R exons 3-6 (Biocytogen). Table 14. Agents and Treatment Regimen (Efficacy Arm) Group N Agent Dose (µg / mouse) Route Schedule 1 8 Vehicle - i biwk x 2\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 14 8 WW50468 / WW50473 67 ip biwk x 2 15 8 WW50468 / WW50473 200 ip biwk x 2 i i kGroup N Agent Dose RoutTerminal Sample (µg / mouse)e ScheduleCollection (N) ) ) ) ) ) ) ) ) ), , medium to obtain 10.0x106cells per mL, and matrigel was added 1:1, so each 0.1 mL delivered the number of cells needed per inoculation. B-hIL21R mice (Jiangsu Biocytogen Co., Ltd., China Production- Stock# 110766F), with an age range of 7-12 weeks and a weight range of 17-25 g, were lightly anesthetized before implantation and each mouse was subcutaneously injected with 0.5x106MC38-BC2 tumor cells in 0.1 mL serum-free medium containing 50% Matrigel in the right flank for tumor development. Tumor-bearing animals were enrolled in 18 study groups when mean tumor volume (TV) reached approximately ~150 mm3(125-175 mm3). Each group consisted of 8 mice (Table 14 Groups 1- 18). Each single-dose group consisted of 8 mice (Table 15: Groups 2-5) in the PK arm, with an additional vehicle group (Group 1, n=4). The physical grouping based on baseline measurements was done the day before treatment initiation (Day -1). The day treatment was initiated is represented as Day 0. Blood samples for pharmacokinetic analysis from the efficacy arm were collected at 4 hours after the 1st(Day 0) and 4th(Day 10) treatments from 4 animals in each group, and at 24 hours after the 1stand 4thtreatments from the remaining 4 animals in each group. Animals were monitored until the defined study endpoints where each individual mouse was terminated when TV >2000 mm3or any other humane endpoint such as 20% body weight loss without recovery to 15% within 2 consecutive readings was reached. The study continued even after the termination of the control group until day 28 post study initiation. The minimum efficacious dose (MED) was then calculated on a per molar basis as shown by significant tumor growth 127 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 inhibition for any group, while the maximum tolerated dose (MTD) was calculated on a per molar basis as no deaths from treatments, and no unrecoverable weight loss of over 20%. Therapeutic window (TW) is calculated and defined as MTD / MED. Results are shown in FIGs.25A-25H and FIGs.26A-26H.

[0505] After 24 hours 4 mice from the vehicle and each treatment group (PK arm groups 1-5) were euthanized, and serum and tumor samples were collected. After 48 hours the remaining 4 mice from each of the treatment groups (PK Arm groups 2-5) were euthanized for tumor and serum collections. Serum samples (4 and 24 hour from the first and last dose in the efficacy arm, and terminal 24 and 48 hours after a single dose in the PK arm) and tumor lysates (24 and 48 hours after a single dose in the PK arm) were analyzed using electrochemiluminescence immunoassays (Meso Scale Discovery MESOTMQuickPlex SQ 120 reader) designed to detect total and free human IL-21 in mouse serum and tumor lysates. Results are shown in FIGs.27A-27H and 28A-28P. Example 26. In vivo Efficacy and PK Evaluation of Inducible IL-21 Prodrugs in Biocytogen human IL-21 Receptor Mice Bearing Syngeneic MC38 (Slow-Growing) Murine Colon Carcinoma

[0506] A validated slow growing clone (BC2) of the cell line MC38, derived from C57BL / 6 murine colon adenocarcinoma cells was used for this study. These cells were implanted into B-hIL21R humanized knock-in mice where exons 3-6 of mouse Il21r gene that encode the extracellular domain were replaced by human IL21R exons 3-6 (Biocytogen). Table 16. Agents and Treatment Regimen (Efficacy Arm) Group N Agent Dose (µg / mouse) Route Schedule 1 8 Vehicle - ip biwk x 2\\4144-9392-5971 v1Attorney Docket No.: 761146.332320 Table 17. Agents and Dosing Regimen (PK arm) Group N Agent DoseTerminal Sample (µg / mouse)Route ScheduleCollection (N) 1 4 V hi l i 24h t d 4) 8h 8h 8h 8h 8hmedium to obtain 10.0x106cells per mL, and matrigel was added 1:1, so each 0.1 mL delivered the number of cells needed per inoculation. B-hIL21R mice (Jiangsu Biocytogen Co., Ltd., China Production- Stock# 110766F), with an age range of 10 weeks and a weight range of 1725 g, were lightly anesthetized before implantation, and each mouse was subcutaneously injected with 0.5x106MC38-BC2 tumor cells in 0.1 mL serum-free medium containing 50% Matrigel in the right flank for tumor development. Tumor- bearing animals were enrolled in 18 study groups when mean tumor volume (TV) reached approximately ~150 mm3(125-175 mm3). Each group consisted of 8 mice (Table 16: Groups 1-18) in the efficacy arm. Each single-dose group consisted of 8 mice (Table 17: Groups 2-6) in the PK arm, with an additional vehicle group (Group 1, n=4) and a no treatment group (n=3). The physical grouping based on baseline measurements was done the day before treatment initiation (Day -1). The day treatment was initiated is represented as Day 0. Blood samples for pharmacokinetic analysis from the efficacy arm were collected at 4 hours after the 1st(Day 0) and 4th(Day 10) treatments from 4 animals in each group, and at 24 hours after the 1stand 4thtreatments from the remaining 4 animals in each group. Animals were monitored until the defined study endpoints so that each individual mouse was terminated when TV >2000 mm3or any other humane endpoint such as 20% body weight loss without recovery to 15% within 2 consecutive readings was reached. The study continued even after the termination of the control group until day 30 post study initiation. The minimum efficacious dose (MED) was then calculated on a per molar basis as shown by significant tumor growth inhibition for any group, while the maximum tolerated dose (MTD) was calculated on a per molar basis as no deaths from treatments, and no unrecoverable weight loss of 129 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 over 20%. Therapeutic window (TW) is calculated and defined as MTD / MED. Results are shown in FIGs.12A-12B, 13A-13C, 14A-14C, 15A-15C, and 16A-16B.

[0508] After 24 hours 4 mice from the vehicle and each treatment group (PK arm groups 1-6) were euthanized, and serum and tumor samples were collected. After 48 hours the remaining 4 mice from each of the treatment groups (PK arm groups 2-6) and the no treatment control mice (PK arm group 7) were euthanized for tumor and serum collections. Serum samples 4 and 24 hour from the first and last dose in the efficacy arm, and terminal 24 and 48 hours after a single dose in the PK arm (and tumor lysates (24 and 48 hours after a single dose in the PK arm were analyzed using electrochemiluminescence immunoassays (Meso Scale Discovery MESOTMQuickPlex SQ 120 reader) designed to detect total and free human IL-21 in mouse serum and tumor lysates. Results are shown in FIGs.17A-17D and 18A-18G. Example 27. IL-21 Antibody Discovery Materials and methods Antigen preparation

[0509] Antigens were biotinylated using the EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Scientific, Cat #21425). The antigens were concentrated to ~1mg / mL and buffer exchanged into PBS before addition of 1:7.5 molar ratio biotinylation reagent. The mixture was held at 4oC overnight prior to another buffer exchange to remove free biotin in the solution. Biotinylation was confirmed through streptavidin sensor binding of the labeled proteins on a ForteBio Octet. Naïve Library Selections

[0510] Eight naïve human synthetic yeast libraries each of ~109diversity were propagated as previously described (see, e.g., Y. Xu et al, PEDS 26(10), 663-70 (2013); WO2009036379; WO2010105256; and WO2012009568.) For the first two rounds of selection, a magnetic bead sorting technique utilizing the Miltenyi MACS system was performed, as previously described (see, e.g., Siegel et al, J Immunol Methods 286(1-2), 141-153 (2004).) Briefly, yeast cells (~1010cells / library) were incubated with 10 nM biotinylated human IL-21 Fc fusion for 30 min at 30°C in wash buffer (phosphate-buffered saline (PBS) / 0.1% bovine serum albumin (BSA)). After washing once with 40 mL ice-cold wash buffer, the cell pellet was resuspended in 20 mL wash buffer, and Streptavidin MicroBeads (500 μl) were added to the yeast and incubated for 15 min at 4°C. Next the yeast were pelleted, resuspended in 5 mL wash buffer, and loaded onto a Miltenyi LS column. After the 5 mL were loaded, the column was washed 3 times with 130 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 3 mL wash buffer. The column was then removed from the magnetic field, and the yeast were eluted with 5 mL of growth media and then grown overnight.

[0511] The following rounds of selection were performed using flow cytometry (FACS). Yeast were pelleted, washed three times with wash buffer, and incubated at 3...

Claims

Attorney Docket No.: 761146.332320 CLAIMS 1. An inducible IL-21 prodrug comprising at least one of each of: a) an IL-21 polypeptide [A]; b) an IL-21 blocking element [D]; c) a half-life extension element [H]; and c) a protease-cleavable polypeptide linker [L]; wherein the IL-21 polypeptide and the IL-21 blocking element or the half-life extension element are operably linked by the protease-cleavable polypeptide linker and the inducible IL-21 prodrug has attenuated IL-21 receptor activating activity, wherein the IL-21 receptor activating activity of the inducible IL-21 prodrug is at least about 10X less than the IL-21 receptor activating activity of the polypeptide that contains the IL-21 polypeptide that is produced by cleavage of the protease cleavable linker.

2. The inducible IL-21 prodrug of claim 1, wherein the inducible IL-21 prodrug has the 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] wherein [A] is an IL-21 polypeptide, [D] is an IL-21 blocking element, [H] is a half-life extension element, [L1] is a protease-cleavable polypeptide linker, [L2] is a polypeptide linker that is optionally protease-cleavable, and [L2’] is a protease-cleavable polypeptide linker.

3. The inducible IL-21 prodrug of any one of the preceding claims, wherein the half-life extension element comprises a serum albumin binding domain, a serum albumin, transferrin, or immunoglobulin Fc, or fragment thereof.

4. The inducible IL-21 prodrug of any one of the preceding claims, wherein the IL-21 blocking element comprises a ligand-binding domain or fragment of a cognate receptor for the IL-21 polypeptide, or an antibody or antigen-binding fragment of an antibody that binds to the IL-21 polypeptide. 281 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 5. The inducible IL-21 prodrug of claim 4, wherein the antibody or antigen-binding fragment is a single domain antibody, a Fab, or a scFv that binds the IL-21 polypeptide.

6. The inducible IL-21 prodrug of claim 4, wherein the cognate receptor for the IL-21 is the IL-21 receptor or the IL-21 common gamma chain.

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

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

9. The inducible IL-21 prodrug of any one of the preceding claims, wherein each protease-cleavable polypeptide linker independently comprises a sequence that is capable of being cleaved by a protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, cathepsin L, an elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), an ADAM, a FAP, a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease.

10. The inducible IL-21 prodrug of any one of claims 2-9, wherein L2 is a protease-cleavable polypeptide linker.

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

12. The inducible IL-21 prodrug of 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 of claim 9, wherein the matrix metalloprotease (MMP) is MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP19, or MMP20. 282 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 14. The inducible IL-21 prodrug of any one of the proceeding claims comprising the amino acid sequence selected from the group consisting of SEQ ID NOs.: 44-50 or an amino acid sequence that has at least 80% identity to SEQ ID NOs: and 44-50.

15. A nucleic acid encoding the inducible IL-21 prodrug of any one of the preceding claims.

16. A vector comprising the nucleic acid of claim 15.

17. A host cell comprising the vector of claim 16.

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

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

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

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

22. A nucleic acid encoding the inducible IL-21 prodrug as defined in claim 21.

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

24. The nucleic acid of claim 22, wherein the nucleic acid comprises DNA.

25. The nucleic acid of claim 22, wherein the nucleic acid comprises RNA. 283 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 26. An expression vector comprising the nucleic acid of any once of claims 22-25.

27. An isolated host cell comprising the vector of claim 26.

28. A method of making a pharmaceutical composition, comprising culturing the isolated host cell of claim 27 under suitable conditions for expression of the polypeptide complex.

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

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

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

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

33. The nucleic acid composition of claim 32, wherein the nucleic acid comprises a circular vector.

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

35. The nucleic acid composition of claim 33, wherein the nucleic acid comprises RNA. 284 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 36. An expression vector comprising the nucleic acid composition of any once of claims 32-35.

37. An isolated host cell comprising the vector of claim 36.

38. An inducible IL-21 prodrug comprising an IL-21 polypeptide, a half-life extension element, an IL-21 blocking element and a protease cleavable linker, wherein the IL-21 blocking element is an antigen binding fragment of an antibody, wherein 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 comprises: 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 half-life extension element are operably linked by the protease cleavable linker; and ii. a second polypeptide comprising at least an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the first polypeptide, or an antibody light chain that is complementary to the heavy chain in the first polypeptide and together with said light chain forms an IL-21 binding site.

39. An inducible IL-21 prodrug comprising: i. 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 an antibody heavy chain constant region or a light chain; wherein the first blocking element is IL-21R; and wherein the IL-21 polypeptide and the first blocking element and / or the half-life extension element are operably linked by the protease cleavable linker; and ii. a second polypeptide that comprises a second IL-21 blocking element and a portion of an antibody heavy chain constant region or an antibody light chain; wherein the second blocking element and the portion of the antibody heavy chain constant region or the antibody light chain are linked through a linker that is optionally protease cleavable; wherein the second blocking element is a common gamma chain receptor; and 285 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 wherein when the antibody heavy chain constant region is on the first polypeptide, the antibody light chain is on the second polypeptide and when the antibody heavy chain constant region is on the second polypeptide, the antibody light chain is on the first polypeptide.

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

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

42. The inducible IL-21 prodrug of any one of claims 39-40, 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 of any one of claims 39-40, wherein protease is selected from matrix metalloprotease (MMP) is MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, or MMP14.

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

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

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

47. The inducible IL-21 prodrug of claim 38, wherein the blocking element binds the IL-21. 286 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 48. The inducible IL-21 prodrug of claim 39, wherein the first blocking element and the second blocking element binds the IL-21.

49. A nucleic acid encoding a polypeptide as defined in any one of claims 38-48.

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

51. The nucleic acid of claim 49, comprising DNA.

52. The nucleic acid of claims 49, comprising RNA.

53. An expression vector comprising the nucleic acid of any one of claims 49-52.

54. An isolated host cell comprising the vector of claim 53.

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

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

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

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

59. The inducible IL-21 prodrug of any one of claims 1-1,421 or 38-48 or the nucleic acid composition of any one of claims 15, 22-25, 32-35, or the nucleic acid of any one of claims 49-53, wherein the IL-21 is a mutein. 287 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 60. An inducible IL-21 prodrug comprising a first polypeptide selected from the group consisting of SEQ ID NOs.18-23 and 26-31, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 18-23 and 26-31.

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

62. An inducible IL-21 prodrug comprising a first polypeptide selected from the group consisting of SEQ ID NOs.18-23 and 26-31, or an amino acid sequence that has at least 80% identity to SEQ ID NOs: 18-23 and 26-31, and a second polypeptide selected from the group consisting of SEQ ID NO. 17 or 25 or an amino acid sequence that has at least 80% identity to SEQ ID NO.17 or 25.

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

64. An inducible IL-21 prodrug further comprising a second polypeptide chain selected from the group consisting of SEQ ID NOs.: 10, 40, 52, and 64 or an amino acid sequence that has at least 80% identity to SEQ ID NOs.: 10, 40, 52, and 64.

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

66. A nucleic acid encoding a polypeptide as defined in any one of claims 60-65.

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

68. The nucleic acid composition of claim 66, comprising DNA. 288 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 69. The nucleic acid composition of claim 66, comprising RNA.

70. An expression vector comprising the nucleic acid of any one of claims 66-69.

71. An isolated host cell comprising the vector of claim 70.

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

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

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

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

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

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 extension element, a blocking element and a protease cleavable linker, wherein the IL-21 blocking element is an antigen binding fragment of an antibody, wherein 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 comprises: 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 289 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 light chain or the antigen binding portion of an antibody heavy chain and / or half-life extension element are operably linked by the protease cleavable linker; and ii. a second polypeptide comprising at least an antigen binding portion of an antibody heavy chain that is complementary to the light chain in the first polypeptide, or an antibody light chain that is complementary to the heavy chain in the first polypeptide and together with said light chain forms and IL-21 binding site.

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

79. The nucleic acid composition of claims 77 or 78, comprising a circular vector.

80. The nucleic acid composition of claims 77 or 78, comprising DNA.

81. The nucleic acid composition of claims 77 or 78, comprising RNA.

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

83. An isolated host cell comprising the vector of claim 82. 290 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 84. An inducible IL-21 prodrug comprising the amino acid sequence selected from the group consisting of SEQ ID NOs.: 72-109 or an amino acid sequence that has at least about 80% identity to SEQ ID NOs.: 72-109.

85. A nucleic acid encoding the inducible IL-21 prodrug as defined in claim 84.

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

87. The nucleic acid of claim 85, wherein the nucleic acid comprises DNA.

88. The nucleic acid of claim 85, wherein the nucleic acid comprises RNA.

89. An expression vector comprising the nucleic acid of any once of claims 85-88.

90. An isolated host cell comprising the vector of claim 89.

91. A method of making a pharmaceutical composition, comprising culturing the isolated host cell of claim 90 under suitable conditions for expression of the polypeptide complex.

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

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

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

95. An inducible IL-21 prodrug comprising a first polypeptide selected from the group consisting of SEQ ID NOs.72, 110-113, 117-126, 130-136, 138-144, 153-194, 221-235, 241-243, 245-247, 250, 251, 253-255, 257, 258, 260-264, 267-269, 271-290, 475, 476, 449-463, and 655, or an amino acid sequence 291 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 that has at least 80% identity to SEQ ID NOs: 72.110-113, 117-126, 130-136, 138-144, 153-208, 221- 235, 241-243, 245-247, 250, 251, 253-255, 257, 258, 260-264, 267-269, 271-290, 475, 476, 449-463, and 655, and a second polypeptide selected from the group consisting of SEQ ID NO.71, 127-129, 137, or 300-306 or an amino acid sequence that has at least 80% identity to SEQ ID NO.71, 127-129, 137, or 300-306.

96. The inducible IL-21 prodrug of 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 of 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 of 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 of 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 of 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 of 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 the inducible IL-21 prodrug as defined in claim 95-101. 292 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 103. The nucleic acid of any one of claims 95-102, wherein the nucleic acid comprises a circular vector.

104. The nucleic acid of any one of claims 95-102, wherein the nucleic acid comprises DNA.

105. The nucleic acid of any one of claims 102-104, wherein the nucleic acid comprises RNA.

106. An expression vector comprising the nucleic acid of any once of claims 102-105.

107. An isolated host cell comprising the vector of claim 106.

108. A method of making a pharmaceutical composition, comprising culturing the isolated host cell of claim 107 under suitable conditions for expression of the polypeptide complex.

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

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

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

112. An inducible IL-21 prodrug comprising: 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 cleavable polypeptide linker comprising the amino acid sequence of SEQ ID NO: 198 or a variant thereof, [H] is a half-life extension element comprising the amino acid sequence of any one of SEQ ID NOs: 602-606, 608-653, or a variant of any of the foregoing, [L2] is a protease cleavable 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 the amino acid sequence of any one of SEQ ID NOs: 291-299 or a variant thereof, and 293 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 2) a second polypeptide chain comprising an antibody light chain of SEQ ID NO: 129 or a variant thereof that is complementary to the heavy chain in the first polypeptide and together with the light chain forms an IL-2 binding site.

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

114. The inducible IL-21 prodrug of any one of claims 112 or 113, wherein the half-life extension element in the inducible IL-21 prodrug is selected from the group consisting of SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, SEQ ID NO: 606, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 619, SEQ ID NO: 620, SEQ ID NO: 621, SEQ ID NO: 622, SEQ ID NO: 623, SEQ ID NO: 624, SEQ ID NO: 625, SEQ ID NO: 626, SEQ ID NO: 627, SEQ ID NO: 628, SEQ ID NO: 629, SEQ ID NO: 630, SEQ ID NO: 631, SEQ ID NO: 632, SEQ ID NO: 633, SEQ ID NO: 634, SEQ ID NO: 635, SEQ ID NO: 636, SEQ ID NO: 637, SEQ ID NO: 638, SEQ ID NO: 639, SEQ ID NO: 640, SEQ ID NO: 641, SEQ ID NO: 642, SEQ ID NO: 643, SEQ ID NO: 644, SEQ ID NO: 645, SEQ ID NO: 646, SEQ ID NO: 647, SEQ ID NO: 648, SEQ ID NO: 649, SEQ ID NO: 650, SEQ ID NO: 651, SEQ ID NO: 652, and SEQ ID NO:

653.

115. The inducible IL-21 prodrug of any one of claims 112-114, wherein the inducible IL-21 prodrug comprises 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 as defined in any one of claims 112-114.

117. The nucleic acid of claim 116, comprising a circular vector. 294 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 118. The nucleic acid of claim 116, comprising DNA.

119. The nucleic acid of claim 116, comprising RNA.

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

121. An isolated host cell comprising the vector of claim 120.

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

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

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

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

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

127. The inducible IL-21 prodrug of any one of claims 112-114, wherein the inducible IL-21 prodrug comprises 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), 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), 295 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 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 (SEQ ID NO: 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 86 (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: 163 / SEQ ID NO: 300), Compound 108 (SEQ ID NO: 164 / SEQ ID NO: 300), Compound 109 (SEQ ID NO: 165 / SEQ ID NO: 300), Compound 110 (SEQ ID NO: 166 / SEQ ID NO: 300), Compound 111 (SEQ ID NO: 167 / SEQ ID NO: 300), Compound 112 (SEQ ID NO: 168 / SEQ ID NO: 300), Compound 113 (SEQ ID NO: 169 / SEQ ID NO: 301), Compound 114 (SEQ ID NO: 170 / SEQ ID NO: 301), Compound 115 (SEQ ID NO: 171 / SEQ ID NO: 301), Compound 116 (SEQ ID NO: 172 / SEQ ID NO: 301), Compound 117 (SEQ ID NO: 173 / SEQ ID NO: 301), Compound 118 (SEQ ID NO: 174 / SEQ ID NO: 301), Compound 119 (SEQ ID NO: 175 / SEQ ID NO: 301), Compound 110 (SEQ ID NO: 176 / SEQ ID NO: 301), Compound 111 (SEQ ID NO: 177 / SEQ ID NO: 302), Compound 112 (SEQ ID NO: 178 / SEQ ID NO: 302), Compound 113 (SEQ ID NO: 179 / SEQ ID NO: 302), Compound 114 (SEQ ID NO: 180 / SEQ ID NO: 302), Compound 115 (SEQ ID NO: 181 / SEQ ID NO: 302), Compound 116 (SEQ ID NO: 182 / SEQ ID NO: 302), Compound 117 (SEQ ID NO: 183 / SEQ ID NO: 302), Compound 118 (SEQ ID NO: 184 / SEQ ID NO: 302), Compound 296 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 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: 226 / SEQ ID NO: 129), Compound 149 (SEQ ID NO: 227 / SEQ ID NO: 137), Compound 150 (SEQ ID NO: 228 / SEQ ID NO: 137), Compound 151 (SEQ ID NO: 229 / SEQ ID NO: 137), Compound 152 (SEQ ID NO: 230 / SEQ ID NO: 304), Compound 153 (SEQ ID NO: 231 / SEQ ID NO: 304), Compound 154 (SEQ ID NO: 232 / SEQ ID NO: 304), Compound 155 (SEQ ID NO: 233 / SEQ ID NO: 305), Compound 156 (SEQ ID NO: 234 / SEQ ID NO: 305), Compound 157 (SEQ ID NO: 235 / SEQ ID NO: 305), Compound 158 (SEQ ID NO: 241 / SEQ ID NO: 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 (SEQ ID NO: 297 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 268 / SEQ ID NO: 306), Compound 180 (SEQ ID NO: 269 / 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 / 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), 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 (sdAbs) that has binding specificity for human serum albumin comprising a CDR1, CDR2, and a CDR3; wherein CDR1 comprises any one of SEQ ID NO: 656, SEQ ID NO: 659, SEQ ID NO: 662, SEQ ID NO: 663, SEQ ID NO: 691, or a variant of any of the foregoing comprising up to about 2 amino acid substitutions; CDR2 comprises any one of SEQ ID NO: 657, SEQ ID NO: 660; SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 692, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; and 298 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 CDR3 comprises any one of SEQ ID NO: 658, SEQ ID NO: 661, SEQ ID NO: 667 or a variant of any of the foregoing comprising up to about 3 amino acid substitutions.

129. The sdAb of claim 128, wherein the sdAb comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 656, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 657, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 658, or a variant thereof comprising up to about 3 amino acid substitutions; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 659, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO:660, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO:660, or a variant thereof comprising up to about 3 amino acid substitutions; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 664, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 665, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 666, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 692, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 299 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 664, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 665, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions; (9) CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 666, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions; (10) CDR1 comprising the amino acid sequence of SEQ ID NO: 691, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 692, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions; (11) CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 664, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions; (12) CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 665, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions; 300 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 (13) a CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 666, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions; or (14) The sdAb can comprise a CDR1 comprising the amino acid sequence of SEQ ID NO: 663, or a variant thereof comprising up to about 2 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 692, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 667, or a variant thereof comprising up to about 3 amino acid substitutions.

130. The sdAb of any one of the preceding claims, wherein the sdAb comprises: (1) a CDR1 that consists of SEQ ID NO: 662; a CDR2 that consists of SEQ ID NO: 692; and a CDR3 that consists of SEQ ID NO: 667; (2) a CDR1 that consists of SEQ ID NO: 691; a CDR2 that consists of SEQ ID NO: 664; and a CDR3 that consists of SEQ ID NO: 667; (3) a CDR1 that consists of SEQ ID NO: 663; a CDR2 that consists of SEQ ID NO: 665; and a CDR3 that consists of SEQ ID NO: 667; (4) a CDR1 that consists of SEQ ID NO: 663; a CDR2 that consists of SEQ ID NO: 666; and a CDR3 that consists of SEQ ID NO: 667; (5) a CDR1 that consists of SEQ ID NO: 656; a CDR2 that consists of SEQ ID NO: 2; and a CDR3 that consists of SEQ ID NO: 658; or (6) a CDR1 that consists of SEQ ID NO: 659; a CDR2 that consists of SEQ ID NO:660; and a CDR3 that consists of SEQ ID NO:

661.

131. The sdAb of any one of claims 128-130, sdAb competes with neonatal Fc receptor (FcRn) for binding to HSA. The sdAbs can have binding specificity for domain one of human serum albumin.

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

133. The sdAb of claim 132, wherein the sdAb is a heavy chain variable domain (VH). 301 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 134. The sdAb of any one of claims 131 or 132, wherein the sdAb is a variable heavy domain of heavy chain (VHH).

135. The sdAb of any one of claims 128-135, wherein the sdAb is camelid, human, or humanized.

136. The sdAb of claim 135, wherein the sdAb is humanized.

137. The sdAb of any one of claims 128-136, further comprises one or more framework regions.

138. The sdAb of claim 138, wherein the sdAb comprises a framework region derived from a human germline.

139. The sdAb of any one of claims 128-138, wherein the sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 662, SEQ ID NO: 663, or SEQ ID NO: 691; a CDR2 comprising the amino acid sequence of SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, or SEQ ID NO: 692; a CDR3 comprising the amino acid sequence of SEQ ID NO: 667; and a FR1, a FR2, a FR3, and a FR4; wherein the amino acid residue at position 24 of FR1 can be a serine or an alanine, the amino acid at position 44 of F2 can be a glutamic acid or a glycine, the amino acid at position 45 of F2 can be a arginine or a leucine, and the amino acid at position 79 of F3 is a leucine or a valine; and wherein FR1, FR2, FR3, FR4 can be from human IGHV3-23 germ line.

140. The sdAb of any one of claims 128-138, wherein the sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 656; a CDR2 comprising the amino acid sequence of SEQ ID NO: 657; a CDR3 comprising the amino acid sequence of SEQ ID NO: 658; and a FR1, a FR2, a FR3, and a FR4; wherein the amino acid residue at position 24 of FR1 can be a serine or an alanine, the amino acid at position 44 of FR2 can be a aspartic acid or a glycine; the amino acid at position 45 of FR2 can be a arginine or a leucine; the amino acid at position 49 of FR2 can be a serine or alanine, the amino acid at position 78 of FR3 can be a leucine or a phenylalanine, and the amino acid at position 79 of F3 can be a asparagine or a lysine; and wherein the FR1, FR2, FR3, FR4 can be from human IGHV3-23 germ line. 302 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 141. The sdAb of any one of claims 128-138, wherein the sdAb comprises: a CDR1 comprising the amino acid sequence of SEQ ID NO: 659; a CDR2 comprising the amino acid sequence of SEQ ID NO: 660; a CDR3 comprising the amino acid sequence of SEQ ID NO: 661; and a FR1, a FR2, a FR3, and a FR4; wherein the amino acid residue at position 44 of F2 can be a glutamic acid or a glycine, the amino acid at position 45 of F2 can be a arginine or a leucine, the amino acid at position [78] of F3 can be a threonine or a serine, and the amino acid at position 79 can be F3 is a leucine or a valine; and wherein FR1, FR2, FR3, FR4 can be from human IGHV3-23 germ line.

142. The sdAb of any one of claims 128-141, wherein the sdAb further comprises a FR1 comprising the amino acid sequence of any one of SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 685, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of any one of SEQ ID NO: 670, SEQ ID NO: 671, SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680, SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 686, SEQ ID NO:33, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of any one of SEQ ID NO: 675, SEQ ID NO: 676, SEQ ID NO: 34, SEQ ID NO: 689, or SEQ ID NO: 690, or a variant of any of the foregoing comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; 143. The sdAb of claim 143, wherein the sdAb comprises: (1) a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 670, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; (2) a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 671, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the 303 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; (3) a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 673, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; (4) a FR1 comprising the amino acid sequence of SEQ ID NO: 669, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 671, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; (5) a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 678, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; (6) a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 679, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; (7) a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid 304 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; (8) a FR1 comprising the amino acid sequence of SEQ ID NO: 669, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 683, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; (9) a FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 686, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; (10) FR1 comprising the amino acid sequence of SEQ ID NO: 668, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 675, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; (11) a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 34, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; (12) a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 686, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 689, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; 305 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 (13) a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 689, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions; or (14) a FR1 comprising the amino acid sequence of SEQ ID NO: 685, or a variant thereof comprising up to about 3 amino acid substitutions; a FR2 comprising the amino acid sequence of SEQ ID NO: 681, or a variant thereof comprising up to about 3 amino acid substitutions; a FR3 comprising the amino acid sequence of SEQ ID NO: 689, or a variant thereof comprising up to about 3 amino acid substitutions; and a FR4 comprising the amino acid sequence of SEQ ID NO: 677, or a variant thereof comprising up to about 3 amino acid substitutions.

144. The sdAb of any one of claims 128-143, wherein the sdAb comprises the amino acid sequence of any one of SEQ ID NOs: 286-290 or 608-648, or an amino acid sequence of at least about 85% identical to the amino acid sequence of any one of SEQ ID NOs: 286-290 or 608-648.

145. The sdAb of any one of claims 128-144, wherein the sdAb can comprise the amino acid sequence of SEQ ID NO: 608, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO:

608.

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

609.

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

611.

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

618. 306 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 149. The sdAb of any one of claims 128-144, comprises the amino acid sequence of SEQ ID NO: 622, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO:

622.

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

624.

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

625.

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

626.

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

628.

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

632.

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

635.

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

637. 307 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 157. The sdAb of any one of claims 128-144, comprises the amino acid sequence of SEQ ID NO: 642, or an amino acid sequence of at least about 85% identical to the amino acid sequence of SEQ ID NO:

642.

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

644.

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

645.

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

646.

161. A nucleic acid encoding the sdAb of any one of claims 128-160.

162. A vector comprising the nucleic acid of claim 161.

163. A host cell comprising the vector of claim 162.

164. A fusion polypeptide comprising: a) the sdAb of any one of claims 128-160; and b) a domain of interest.

165. The fusion polypeptide of claim 164, wherein the domain sequence of interest is a polypeptide with biological activity.

166. The fusion polypeptide of any one of claims 164 or 165, further comprising an optionally cleavable linker, wherein the optionally cleavable linker is operably linked to the sdAb and the domain acid sequence of interest. 308 \\4144-9392-5971 v1Attorney Docket No.: 761146.332320 167. The fusion polypeptide of claim 166, wherein the polypeptide with biological activity comprises a cytokine.

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

169. The fusion polypeptide of 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 of claim 168, wherein protease is selected from matrix metalloprotease (MMP) is MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, or MMP14. 309 \\4144-9392-5971 v1