Activatable il-12 polypeptides and methods of use thereof

Abstract

To provide activatable IL-12 polypeptides and methods of using the same.SOLUTION: Provided herein are IL-12 polypeptide complexes and / or IL23 polypeptide complexes comprising IL-12 or IL-23, a half-life-extending element, an IL-12 or IL-23 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 complexes. Also disclosed are methods of using the polypeptide complexes in the treatment of diseases, conditions and disorders.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 1. Sequence Listing This application claims the benefit of U.S. Provisional Patent Application No. 63 / 027,276, filed May 19, 2020, which is incorporated herein by reference in its entirety.

[0002] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on May 18, 2021, is named 761146_02320_SL.txt and is 1,294,403 bytes in size. [Background technology]

[0003] Interleukin-12 (IL-12) is a 70 kDa heterodimeric cytokine composed of two covalently linked glycosylated subunits (p35 and p40) (Lieschke et al., 1997; Jana et al., 2014). IL-12 is a potent immune antagonist and has been considered a promising therapeutic agent for oncology. However, IL-12 is highly potent and has a short serum half-life, resulting in a narrow therapeutic window. As a result, therapeutic administration of IL-12 results in undesirable systemic effects and toxicity. This is exacerbated by the need to administer large amounts of cytokines (e.g., IL-12) to achieve desired cytokine levels at the intended site of action (e.g., the tumor microenvironment). Unfortunately, due to the biology of cytokines and the inability to effectively target and control their activity, cytokines have not achieved the expected clinical benefits in tumor treatment.

[0004] To overcome the toxicity and short half-life issues that limit the clinical use of IL-12 in oncology, inducible IL-12 protein constructs are described in International Application Nos. PCT / US2019 / 032320 and PCT / US2019 / 032322. The inducible IL-12 polypeptide constructs comprise a single polypeptide containing IL-12, a blocking element, and a half-life extending element.

[0005] The inventors of the present invention have surprisingly found that IL-12 polypeptide complexes comprising two or more polypeptides have certain advantages, such as reduced aggregation and improved expression, leading to higher yields. Summary of the Invention

[0006] The present disclosure relates to an inducible IL-12 polypeptide complex that contains attenuated IL-12 and has a longer half-life compared to native IL-12. Optionally, the IL-12 can be a mutein. The IL-12 mutein can be aglycosylated or partially aglycosylated. The polypeptide complex disclosed herein comprises two or more polypeptide chains, and the complex includes the IL-12 subunits p35 and p40, a half-life-extending element, an IL-12 blocking element, and a protease-cleavable linker.

[0007] The inducible IL-12 polypeptide complex may comprise two different polypeptides. The first polypeptide may comprise an IL-12 subunit and, optionally, an IL-12 blocking element. The IL-12 blocking element, if present, is operably linked to the IL-12 subunit via a first protease-cleavable linker. The second polypeptide chain may comprise an IL-12 subunit operably linked to a half-life extending element via a second protease-cleavable linker and, optionally, an IL-12 blocking element. If present, the IL-12 blocking element may be operably linked to the IL-12 subunit via a protease-cleavable linker or may be operably linked to the half-life extending element via an optional protease-cleavable linker. Only one of the first and second polypeptides comprises an IL-12 blocking element. When the IL-12 subunit of the first polypeptide is p35, the IL-12 subunit of the second polypeptide is p40, and when the IL-12 subunit of the first polypeptide is p40, the IL-12 subunit of the second polypeptide is p35. A preferred blocking element of this complex is a single-chain antibody that binds to IL-12 or an antigen-binding fragment thereof. The cleavable linkers of this complex can be the same or different.

[0008] An inducible IL-12 polypeptide complex can comprise three distinct polypeptides. Typically, one polypeptide chain comprises either the p35 or p40 IL-12 subunit, but not both, a second polypeptide comprises the other IL-12 subunit, and a third polypeptide comprises at least a portion (component) of a blocking element. The first polypeptide can comprise an IL-12 subunit and, optionally, a half-life extending element. If present, the half-life extending element is operably linked to the IL-12 subunit via a protease-cleavable linker.

[0009] The second polypeptide can comprise an IL-12 subunit, at least an antigen-binding portion of an antibody light chain or an antigen-binding portion of an antibody heavy chain, and optionally a half-life extending element, where the half-life extending element, if present, is operably linked to the IL-12 subunit via a protease-cleavable linker, and the antibody heavy or light chain is a) operably linked to the IL-12 subunit via a second protease-cleavable linker, or b) optionally operably linked to the half-life extending element via a cleavable linker.

[0010] The third polypeptide may comprise an antigen-binding portion of an antibody heavy chain that is complementary to the light chain of the second polypeptide, or an antibody light chain that is complementary to the heavy chain of the second polypeptide and forms an IL-12 binding site together with the light chain. If the IL-12 subunit of the first polypeptide is p35, the IL-12 subunit of the second polypeptide is p40; if the IL-12 subunit of the first polypeptide is p40, the IL-12 subunit of the second polypeptide is p35. In this complex, the IL-12 blocking element is preferably an antigen-binding fragment of an antibody. The antigen-binding fragment comprises at least the antigen-binding portion of the antibody light chain and at least the antigen-binding portion of the complementary antibody heavy chain as separate components. The protease-cleavable linkers in this inducible IL-12 polypeptide complex may be the same or different.

[0011] The inducible polypeptide complex may comprise two different polypeptides in which p35 and p40 are located on the same polypeptide chain. The first polypeptide chain may comprise p35, p40, a half-life extending element, and at least an antigen-binding portion of an antibody light chain. p35 and p40 may be operably linked, the half-life extending element may be operably linked to p40 via a first protease-cleavable linker, and the antigen-binding portion of the antibody light chain may be operably linked to p35 via a protease-cleavable linker. Alternatively, the half-life extending element may be operably linked to p35 via a protease-cleavable linker, and the antigen-binding portion of the antibody light chain may be operably linked to p40 via a protease-cleavable linker. The second polypeptide comprises at least an antigen-binding portion of an antibody heavy chain that is complementary to the light chain of the second polypeptide and forms an IL-12 binding site together with the light chain. The protease-cleavable linkers in this complex may be the same or different.

[0012] In another embodiment, the first polypeptide chain can comprise p35, p40, a half-life extending element, and at least the antigen-binding portion of an antibody light chain. p35 and p40 can be operably linked, the half-life extending element can be operably linked to p40 via a first protease-cleavable linker, and the antigen-binding portion of the antibody light chain can be operably linked to p35 via a protease-cleavable linker. Alternatively, the half-life extending element can be operably linked to p35 via a protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain can be operably linked to p40 via a second protease-cleavable linker. The second polypeptide comprises at least the antigen-binding portion of an antibody light chain that is complementary to the heavy chain of the second polypeptide and forms an IL-12 binding site together with the light chain. The protease-cleavable linkers in this complex can be the same or different.

[0013] In one example, an IL-12 polypeptide complex includes a first polypeptide that does not include a blocking element, and a second polypeptide that includes the formula: [A]-[L1]-[B]-[L3]-[D] or [D]-[L3]-[B]-[L1]-[A] or [B]-[L1]-[A]-[L2]-[D] or [D]-[L1]-[A]-[L2]-[B], where A is an IL-12 subunit; L1 is a first protease-cleavable linker; L2 is a second protease-cleavable linker; L3 is an optional cleavable linker; B is a half-life extending element; and D is a blocking element.

[0014] In another example, the first polypeptide has the formula: [A]-[L1]-[D] or [D]-[L1]-[A]; the second polypeptide has the formula: [A']-[L2]-[B] or [B]-[L2]-[A'], where A is either p35 or p40, and if A is p35, A' is p40, and if A is p40, A' is p35; A' is either p35 or p40; L1 is a first protease-cleavable linker; L2 is a second protease-cleavable linker; B is a half-life extending element; and D is a blocking element.

[0015] In embodiments, the IL-12 polypeptide complex comprises a first polypeptide selected from the group consisting of SEQ ID NOs: 95-110, 119-126, and 135-143, or an amino acid sequence having at least 80% identity to SEQ ID NOs: 95-110, 119-126, and 135-143. A preferred IL-12 polypeptide complex comprises a first polypeptide comprising SEQ ID NO: 104 or SEQ ID NO: 136. A preferred IL-12 polypeptide complex comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 104 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18. Another preferred polypeptide complex comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 136 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18.

[0016] As described above, IL-12 can be a mutein, if desired. The IL-12 mutein retains IL-12 activity, e.g., intrinsic IL-12 receptor agonist activity. The IL-12 subunits, p35 and / or p40, can be muteins. Preferably, the IL-12 mutein has an altered glycosylation pattern. For example, the IL-12 mutein can be partially aglycosylated or fully aglycosylated.

[0017] The p35 and / or p40 subunits can contain one or more amino acid modifications, e.g., substitutions. For example, the p35 and / or p40 subunits can contain about one, about two, about three, about four, about five, about six, about seven, or more amino acid substitutions. Typically, the p35 and / or p40 subunits contain about one to about seven amino acid substitutions. The substitutions can be conservative or non-conservative, but are preferably conservative. A common modification alters the glycosylation pattern of the p35 and / or p40 subunits, thereby rendering the p35 and / or p40 subunits partially or fully aglycosylated. Preferably, the amino acid modification includes the substitution of an asparagine amino acid, e.g., asparagine to glutamine. In a particular example, the asparagine at amino acid position 16, 75, 85, 133, 151, 158, 201, 206, 221, 250, 267, 280, 282, 326, 400, 404, 425, 555, 572, 575, 582, or 602 of IL-12 p35 of SEQ ID NO: 434 can be mutated. In a particular example, the asparagine at amino acid position 103, 114, 163, 219, 227, or 282 of IL-12 p40 of SEQ ID NO: 18 can be mutated.

[0018] For example, a partially or fully aglycosylated IL-12 polypeptide can comprise a polypeptide selected from the group consisting of SEQ ID NOs: 104, 434, or 442-445, or an amino acid sequence having at least 80% identity to SEQ ID NOs: 104, 434, or 442-445.

[0019] The present disclosure also relates to single-chain IL-12-inducing polypeptides, which preferably comprise amino acids selected from the group consisting of SEQ ID NOs: 7, 9, 10, 18, 24-94, 110-118, and 127-134, or comprise an amino acid sequence having at least about 80% identity to SEQ ID NOs: 7, 9, 10, 18, 24-94, 110-118, and 127-134.

[0020] The present disclosure also relates to inducible IL-23 polypeptide complexes that contain attenuated IL-23 and have a longer half-life compared to native IL-23. Optionally, the IL-23 can be a mutein. The IL-23 mutein can be aglycosylated or partially aglycosylated. The polypeptide complexes disclosed herein comprise one or more polypeptide chains, and the complexes include the IL-23 subunits p19 and p40, a half-life-extending element, an IL-23 blocking element, and a protease-cleavable linker.

[0021] The inducible IL-23 polypeptide complex may comprise two different polypeptides. The first polypeptide may comprise an IL-23 subunit and, optionally, an IL-23 blocking element. The IL-23 blocking element, if present, is operably linked to the IL-23 subunit via a first protease-cleavable linker. The second polypeptide chain may comprise an IL-23 subunit operably linked to a half-life extending element via a second protease-cleavable linker and, optionally, an IL-23 blocking element. If present, the IL-23 blocking element may be operably linked to the IL-23 subunit via a protease-cleavable linker or may be operably linked to the half-life extending element via an optional protease-cleavable linker. Only one of the first and second polypeptides comprises an IL-23 blocking element. When the IL-23 subunit of the first polypeptide is p19, the IL-23 subunit of the second polypeptide is p40, and when the IL-23 subunit of the first polypeptide is p40, the IL-23 subunit of the second polypeptide is p40. A preferred blocking element of this complex is a single-chain antibody that binds to IL-23 or an antigen-binding fragment thereof. The cleavable linkers of this complex can be the same or different.

[0022] An inducible IL-23 polypeptide complex can comprise three distinct polypeptides. Typically, one polypeptide chain comprises either the p19 or p40 IL-23 subunit, but not both, a second polypeptide comprises the other IL-23 subunit, and a third polypeptide comprises at least a portion (component) of a blocking element. The first polypeptide can comprise an IL-23 subunit and, optionally, a half-life extending element. If present, the half-life extending element is operably linked to the IL-23 subunit via a protease-cleavable linker.

[0023] The second polypeptide can comprise an IL-23 subunit, at least an antigen-binding portion of an antibody light chain or an antigen-binding portion of an antibody heavy chain, and optionally a half-life extending element, where the half-life extending element, if present, is operably linked to the IL-23 subunit via a protease-cleavable linker, and the antibody heavy or light chain is a) operably linked to the IL-23 subunit via a second protease-cleavable linker, or b) optionally operably linked to the half-life extending element via a cleavable linker.

[0024] The third polypeptide may comprise an antigen-binding portion of an antibody heavy chain complementary to the light chain of the second polypeptide, or an antibody light chain complementary to the heavy chain of the second polypeptide and forming an IL-23-binding site together with the light chain. If the IL-23 subunit of the first polypeptide is p19, the IL-23 subunit of the second polypeptide is p40; if the IL-23 subunit of the first polypeptide is p40, the IL-23 subunit of the second polypeptide is p19. In this complex, the IL-23 blocking element is preferably an antigen-binding fragment of an antibody. The antigen-binding fragment comprises at least the antigen-binding portion of the antibody light chain and at least the antigen-binding portion of the complementary antibody heavy chain as separate components. The protease-cleavable linkers in this inducible IL-23 polypeptide complex may be the same or different.

[0025] The inducible polypeptide complex may comprise two different polypeptides in which p19 and p40 are located on the same polypeptide chain. The first polypeptide chain may comprise p19, p40, a half-life extending element, and at least an antigen-binding portion of an antibody light chain. p19 and p40 may be operably linked, the half-life extending element may be operably linked to p40 via a first protease-cleavable linker, and the antigen-binding portion of the antibody light chain may be operably linked to p19 via a protease-cleavable linker. Alternatively, the half-life extending element may be operably linked to p19 via a protease-cleavable linker, and the antigen-binding portion of the antibody light chain may be operably linked to p40 via a protease-cleavable linker. The second polypeptide comprises at least an antigen-binding portion of an antibody heavy chain that is complementary to the light chain of the second polypeptide and forms an IL-23 binding site together with the light chain. The protease-cleavable linkers of this complex may be the same or different.

[0026] In another format, the first polypeptide chain can comprise p19, p40, a half-life extending element, and at least the antigen-binding portion of an antibody heavy chain. p19 and p40 can be operably linked, and the half-life extending element can be operably linked to p40 via a protease-cleavable linker, or the antigen-binding portion of the antibody heavy chain can be operably linked to p19 via a protease-cleavable linker. Alternatively, the half-life extending element can be operably linked to p19 via a protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain can be operably linked to p40 via a second protease-cleavable linker. The second polypeptide comprises at least the antigen-binding portion of an antibody light chain that is complementary to the heavy chain of the second polypeptide and forms an IL-23 binding site together with the light chain. The protease-cleavable linkers in this complex can be the same or different.

[0027] In one example, an IL-23 polypeptide complex includes a first polypeptide that does not include a blocking element, and a second polypeptide has the formula: [A]-[L1]-[B]-[L3]-[D] or [D]-[L3]-[B]-[L1]-[A] or [B]-[L1]-[A]-[L2]-[D] or [D]-[L1]-[A]-[L2]-[B], where A is an IL-23 subunit; L1 is a first protease-cleavable linker; L2 is a second protease-cleavable linker; L3 is an optional cleavable linker; B is a half-life extending element; and D is a blocking element.

[0028] In another example, the first polypeptide has the formula: [A]-[L1]-[D] or [D]-[L1]-[A]; the second polypeptide has the formula: [A']-[L2]-[B] or [B]-[L2]-[A'], where A is either p19 or p40, and when A is p19, A' is p40, and when A is p40, A' is p19; A' is either p19 or p40; L1 is a first protease-cleavable linker; L2 is a second protease-cleavable linker; B is a half-life extending element; and D is a blocking element.

[0029] In embodiments, the IL-23 polypeptide complex comprises a first polypeptide selected from the group consisting of SEQ ID NOs: 423-428, or an amino acid sequence having at least 80% identity to SEQ ID NOs: 423-428. In embodiments, the IL-23 polypeptide complex comprises a second polypeptide selected from the group consisting of SEQ ID NOs: 18 or 433.

[0030] As described above, IL-23 can be a mutein, if desired. IL-23 muteins retain IL-23 activity, e.g., intrinsic IL-23 receptor agonist activity. IL-23 subunits, p19 and / or p40, can be muteins. Preferably, IL-23 muteins have an altered glycosylation pattern. For example, IL-23 muteins can be partially aglycosylated or fully aglycosylated.

[0031] The p19 and / or p40 subunits may contain one or more amino acid modifications, e.g., substitutions. For example, the p19 and / or p40 subunits may contain about one, about two, about three, about four, about five, or more amino acid substitutions. Typically, the p19 and / or p40 subunits contain one or two amino acid substitutions. The substitutions may be conservative or non-conservative, but are preferably conservative. A common modification alters the glycosylation pattern of the p19 and / or p40 subunits, thereby rendering the p19 and / or p40 subunits partially or fully aglycosylated. Preferably, the amino acid modification includes the substitution of an asparagine amino acid, e.g., asparagine to glutamine.

[0032] The present disclosure also relates to single-chain IL-23-inducing polypeptides, which preferably comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 422 or 429-432, or an amino acid sequence having at least about 80% identity to SEQ ID NO: 422 or SEQ ID NOs: 429-432.

[0033] The half-life extending elements disclosed herein are preferably human serum albumin, an antigen-binding polypeptide that binds to human serum albumin, or immunoglobulin Fc or a fragment thereof.

[0034] The protease-cleavable linker comprises a sequence that can be cleaved by a protease selected from kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), ADAM, FAP, plasminogen activator, caspase, tryptase, or tumor protease. The protease is preferably selected from cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, or cathepsin G. Alternatively, the protease is preferably selected from matrix metalloproteinases (MMPs), such as MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, or MMP14.

[0035] In embodiments, the protease-cleavable linker comprises at least two sequences that can be independently cleaved by a protease. The protease-cleavable linker may comprise a synthetic sequence. In embodiments, each of the protease-cleavable linkers is cleaved by two or more different proteases.

[0036] The blocking elements described herein can be any element that binds to IL-12 or IL-23. The blocking elements disclosed herein can bind to p35, p40, or the p35p40 heterodimer complex. The blocking elements disclosed herein can bind to p19, p40, or the p19p40 heterodimer complex. The blocking elements are preferably single-chain variable fragments (scFv) or Fab.

[0037] The present disclosure also relates to nucleic acids encoding the IL-12 polypeptide complexes described herein. The present disclosure also relates to nucleic acids encoding the IL-23 polypeptide complexes described herein. Nucleic acid compositions encoding the IL-12 polypeptide complexes or IL-23 polypeptide complexes described herein can comprise circular vectors, DNA, or RNA. Also provided herein are expression vectors comprising nucleic acids encoding the IL-12 polypeptide complexes or IL-23 polypeptide complexes described herein. In embodiments, provided herein are host cells comprising the vectors. The present disclosure also relates to methods for making pharmaceutical compositions, comprising culturing isolated host cells under conditions suitable for expression of the polypeptide complex.

[0038] Also provided herein are pharmaceutical compositions comprising the IL-12 polypeptide complexes disclosed herein. Also provided herein are pharmaceutical compositions comprising the IL-23 polypeptide complexes.

[0039] The present disclosure also relates to a method for treating a tumor, comprising administering to a subject in need thereof an effective amount of an IL-12 polypeptide complex, a nucleic acid encoding an IL-12 polypeptide complex, or a pharmaceutical composition thereof disclosed herein. The present disclosure also relates to a method for treating a tumor, comprising administering to a subject in need thereof an effective amount of an IL-23 polypeptide complex, a nucleic acid encoding an IL-23 polypeptide complex, or a pharmaceutical composition thereof disclosed herein. Any suitable tumor, such as melanoma or breast cancer, can be treated according to the methods disclosed herein. The present invention provides, for example, the following items. (Item 1) 1. A polypeptide complex comprising IL-12, a half-life extending element, an IL-12 blocking element, and a protease-cleavable linker, wherein the IL-12 blocking element is a single chain antibody that binds to IL-12 or an antigen-binding fragment thereof, the complex comprising: i. a first polypeptide comprising an IL-12 subunit and optionally said IL-12 blocking element, wherein said IL-12 blocking element, if present, is operably linked to said IL-12 subunit via a first protease-cleavable linker; ii. a second polypeptide chain comprising an IL-12 subunit operably linked to a half-life extending element via a second protease-cleavable linker, and optionally said IL-12 blocking element, wherein said IL-12 blocking element, if present, is operably linked to said IL-12 subunit via a first protease-cleavable linker. or optionally via a protease-cleavable linker, operably linked to said half-life extending element; only one of the first polypeptide and the second polypeptide comprises the IL-12 blocking element; The polypeptide complex, wherein when the IL-12 subunit of the first polypeptide is p35, the IL-12 subunit of the second polypeptide is p40, and when the IL-12 subunit of the first polypeptide is p40, the IL-12 subunit of the second polypeptide is p35. (Item 2) 2. The polypeptide of item 1, wherein the first protease-cleavable linker and the second protease-cleavable linker are the same. (Item 3) 1. A polypeptide complex comprising IL-12, a half-life extending element, an IL-12 blocking element, and a protease-cleavable linker, wherein the IL-12 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, the complex comprising: i. a first polypeptide comprising an IL-12 subunit and, optionally, a half-life extending element, wherein the half-life extending element, if present, is operably linked to the IL-12 subunit via a first protease-cleavable linker; ii. a second polypeptide comprising an IL-12 subunit, at least an antigen-binding portion of an antibody light chain or an antigen-binding portion of an antibody heavy chain, and optionally a half-life extending element; wherein the half-life extending element, if present, is operably linked to the IL-12 subunit via a first protease-cleavable linker, and the antibody heavy chain or the antibody light chain is a) operably linked to the IL-12 subunit via a second protease-cleavable linker, or b) optionally operably linked to the half-life extending element via a cleavable linker; and iii. a third polypeptide comprising at least an antigen-binding portion of an antibody heavy chain that is complementary to the light chain of the second polypeptide, or an antibody light chain that is complementary to the heavy chain of the second polypeptide and that, together with the light chain, forms an IL-12 binding site; The polypeptide complex, wherein when the IL-12 subunit of the first polypeptide is p35, the IL-12 subunit of the second polypeptide is p40, and when the IL-12 subunit of the first polypeptide is p40, the IL-12 subunit of the second polypeptide is p35. (Item 4) 4. The polypeptide of item 3, wherein the first protease-cleavable linker and the second protease-cleavable linker are the same. (Item 5) 1. A polypeptide complex comprising IL-12, a half-life extending element, an IL-12 blocking element, and a protease-cleavable linker, wherein the IL-12 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, the complex comprising: i. a first polypeptide chain comprising p35, p40, a half-life extending element, and at least an antigen-binding portion of an antibody light chain, wherein p35 and p40 are operably linked, the half-life extending element is operably linked to p40 via a first protease-cleavable linker, and the antigen-binding portion of the antibody light chain is operably linked to p35 via a second protease-cleavable linker; or the half-life extending element is operably linked to p35 via a first protease-cleavable linker, and the antigen-binding portion of the antibody light chain is operably linked to a second protease-cleavable linker. the first polypeptide chain operably linked to p40 via two protease-cleavable linkers; and ii. the polypeptide complex comprising a second polypeptide, wherein the second polypeptide comprises at least an antigen-binding portion of an antibody heavy chain that is complementary to a light chain of the second polypeptide and that, together with the light chain, forms an IL-12 binding site. (Item 6) 1. A polypeptide complex comprising IL-12, a half-life extending element, an IL-12 blocking element, and a protease-cleavable linker, wherein the IL-12 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, the complex comprising: iii. a first polypeptide chain comprising p35, p40, a half-life extending element, and at least an antigen-binding portion of an antibody heavy chain, wherein p35 and p40 are operably linked, the half-life extending element is operably linked to p40 via a first protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain is operably linked to p35 via a second protease-cleavable linker; or the half-life extending element is operably linked to p35 via a first protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain is operably linked to p40 via a second protease-cleavable linker; and iv. The polypeptide complex comprising a second polypeptide that is complementary to a heavy chain of the second polypeptide and comprises at least an antigen-binding portion of an antibody light chain that forms, in combination with the light chain, an IL-12 binding site. (Item 7) 7. The polypeptide according to item 5 or 6, wherein the first protease-cleavable linker and the second protease-cleavable linker are the same. (Item 8) wherein the first polypeptide does not contain a blocking element and the second polypeptide has the formula: [A]-[L1]-[B]-[L3]-[D] or [D]-[L3]-[B]-[L1]-[A] or [B]-[L1]-[A]-[L2]-[D] or [D]-[L1]-[A]-[L2]-[B], wherein A is the IL-12 subunit; L1 is the first protease-cleavable linker; L2 is the second protease-cleavable linker; L3 is the optionally cleavable linker; B is the half-life extending element; 4. The polypeptide complex of item 1 or 3, wherein D is the blocking element. (Item 9) the first polypeptide having the formula: [A]-[L1]-[D] or [D]-[L1]-[A]; and said second polypeptide comprises the formula: [A']-[L2]-[B] or [B]-[L2]-[A'], wherein A is either p35 or p40, and if A is p35, A' is p40, and if A is p40, A' is p35; A' is either p35 or p40; L1 is the first protease-cleavable linker; L2 is the second protease-cleavable linker; B is the half-life extending element; 2. The polypeptide complex of item 1, wherein D is the blocking element. (Item 10) the first polypeptide comprises the formula: [A]-[L1]-[B] or [B]-[L1]-[A]; and the second polypeptide has the formula: [A']-[L2]-[D] or [D]-[L2]-[A'], wherein: A is either p35 or p40; A' is either p35 or p40, and if A is p35, then A' is p40, and if A is p40, then A' is p35; L1 is the first protease-cleavable linker; L2 is the second protease-cleavable linker; B is the half-life extending element; 4. The polypeptide complex of item 3, wherein D is the blocking element. (Item 11) 11. The polypeptide conjugate of any one of items 1 to 10, wherein the half-life extending element is human serum albumin, an antigen-binding polypeptide that binds to human serum albumin, or immunoglobulin Fc or a fragment thereof. (Item 12) 11. The polypeptide conjugate according to any one of items 1 to 10, wherein the protease-cleavable linker comprises a sequence that can be cleaved by a protease selected from kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), ADAM, FAP, plasminogen activator, caspase, tryptase, or tumor protease. (Item 13) 11. The polypeptide complex according to any one of items 1 to 10, wherein the protease is selected from cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, or cathepsin G. (Item 14) 11. The polypeptide complex according to any one of items 1 to 10, wherein the protease is selected from matrix metalloproteinase (MMP) 1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, or MMP14. (Item 15) 11. The polypeptide complex according to any one of items 1 to 10, wherein the protease-cleavable linker comprises at least two sequences that can be independently cleaved by a protease. (Item 16) 11. The polypeptide complex of any one of items 1 to 10, wherein the protease-cleavable linker comprises a synthetic sequence. (Item 17) Item 11. The polypeptide complex of any one of the preceding items, wherein each of the protease-cleavable linkers is cleaved by two or more different proteases. (Item 18) 2. The polypeptide complex of item 1, wherein the single-chain antibody is a single-chain variable fragment (scFv). (Item 19) 7. The polypeptide complex of item 3, 5 or 6, wherein the antigen-binding fragment of the antibody is a Fab. (Item 20) 7. The polypeptide complex of any one of items 1, 3, 5, or 6, wherein the blocking element binds to the IL-12. (Item 21) the blocking element binds to p35, p40, or the p35p40 complex. 19. The polypeptide complex according to 17 or 18. (Item 22) 22. A nucleic acid encoding a polypeptide as defined in any one of items 1 to 21. (Item 23) 23. The nucleic acid of item 22, wherein the nucleic acid does not encode only p35 or p40. (Item 24) 1. A nucleic acid composition comprising one or more nucleic acid sequences encoding a polypeptide complex comprising IL-12, a half-life extending element, an IL-12 blocking element, and a protease-cleavable linker, wherein the IL-12 blocking element is a single chain antibody that binds to IL-12 or an antigen-binding fragment thereof, the complex comprising: iii. a first polypeptide comprising an IL-12 subunit and optionally said IL-12 blocking element, wherein said IL-12 blocking element, if present, is operably linked to said IL-12 subunit via a first protease-cleavable linker; iv. a second polypeptide chain comprising an IL-12 subunit operably linked to a half-life extending element via a second protease-cleavable linker, and optionally said IL-12 blocking element, wherein when said IL-12 blocking element is present, said second polypeptide chain is operably linked to said IL-12 subunit via a first protease-cleavable linker or is operably linked to said half-life extending element via a linker that is optionally protease-cleavable; only one of the first polypeptide and the second polypeptide comprises the IL-12 blocking element; The nucleic acid composition, wherein when the IL-12 subunit of the first polypeptide is p35, the IL-12 subunit of the second polypeptide is p40, and when the IL-12 subunit of the first polypeptide is p40, the IL-12 subunit of the second polypeptide is p35. (Item 25) 1. A nucleic acid composition comprising one or more nucleic acid sequences encoding a polypeptide complex comprising IL-12, a half-life extending element, an IL-12 blocking element, and a protease-cleavable linker, wherein the IL-12 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, wherein the complex comprises: i. a first polypeptide comprising an IL-12 subunit and, optionally, a half-life extending element, wherein the half-life extending element, if present, is operably linked to the IL-12 subunit via a first protease-cleavable linker; ii. a second polypeptide comprising an IL-12 subunit, at least an antigen-binding portion of an antibody light chain, and optionally a half-life extending element; wherein the half-life extending element, if present, is operably linked to the IL-12 subunit via a first protease-cleavable linker, and wherein the antibody heavy chain or the antibody light chain is a) operably linked to the IL-12 subunit via a second protease-cleavable linker, or b) optionally operably linked to the half-life extending element via a cleavable linker; and iii. a third polypeptide comprising at least an antigen-binding portion of an antibody heavy chain that is complementary to the light chain of the second polypeptide and that, together with the light chain, forms an IL-12 binding site; The nucleic acid composition, wherein when the IL-12 subunit of the first polypeptide is p35, the IL-12 subunit of the second polypeptide is p40, and when the IL-12 subunit of the first polypeptide is p40, the IL-12 subunit of the second polypeptide is p35. (Item 26) 1. A nucleic acid composition comprising one or more nucleic acid sequences encoding a polypeptide complex comprising IL-12, a half-life extending element, an IL-12 blocking element, and a protease-cleavable linker, wherein the IL-12 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, wherein the complex comprises: i. a first polypeptide chain comprising p35, p40, a half-life extending element, and at least an antigen-binding portion of an antibody light chain, wherein p35 and p40 are operably linked, the half-life extending element is operably linked to p40 via a first protease-cleavable linker, and the antigen-binding portion of the antibody light chain is operably linked to p35 via a second protease-cleavable linker; or the half-life extending element is operably linked to p35 via a first protease-cleavable linker, and the antigen-binding portion of the antibody light chain is operably linked to p40 via a second protease-cleavable linker; and ii. the nucleic acid composition comprising a second polypeptide, the second polypeptide comprising at least an antigen-binding portion of an antibody heavy chain that is complementary to a light chain of the second polypeptide and that, together with the light chain, forms an IL-12 binding site. (Item 27) 1. A nucleic acid composition comprising one or more nucleic acid sequences encoding a polypeptide complex comprising IL-12, a half-life extending element, an IL-12 blocking element, and a protease-cleavable linker, wherein the IL-12 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, wherein the complex comprises: iii. a first polypeptide chain comprising p35, p40, a half-life extending element, and at least an antigen-binding portion of an antibody heavy chain, wherein p35 and p40 are operably linked, the half-life extending element is operably linked to p40 via a first protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain is operably linked to p35 via a second protease-cleavable linker; or the half-life extending element is operably linked to p35 via a first protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain is operably linked to p40 via a second protease-cleavable linker; and iv. The nucleic acid composition comprising a second polypeptide, wherein the second polypeptide comprises at least an antigen-binding portion of an antibody light chain that is complementary to a heavy chain of the second polypeptide and that, together with the heavy chain, forms an IL-12 binding site. (Item 28) 28. The nucleic acid composition of any one of items 22 to 27, comprising a circular vector. (Item 29) 28. The nucleic acid composition according to any one of items 22 to 27, comprising DNA. (Item 30) 28. The nucleic acid composition of any one of items 22 to 27, comprising RNA. (Item 31) 28. An expression vector comprising the nucleic acid according to any one of Items 22 to 27. (Item 32) 32. An isolated host cell comprising the vector of item 31. (Item 33) 33. A method for producing a pharmaceutical composition, comprising culturing the isolated host cell of item 32 under conditions suitable for expression of the polypeptide complex. (Item 34) 34. The method of claim 33, further comprising isolating the polypeptide complex. (Item 35) A pharmaceutical composition comprising the protein complex according to any one of Items 1 to 21 or the nucleic acid according to any one of Items 22 to 31. (Item 36) A method for treating a tumor, comprising administering an effective amount of the polypeptide complex according to any one of Items 1 to 21, the nucleic acid according to any one of Items 22 to 30, the expression vector according to Item 31, or the pharmaceutical composition according to Item 34 to a subject in need of tumor treatment. (Item 37) An IL-12 polypeptide complex comprising a first polypeptide selected from the group consisting of SEQ ID NOs: 95 to 110, 119 to 126, and 135 to 143, or an amino acid sequence having at least 80% identity to SEQ ID NOs: 95 to 110, 119 to 126, and 135 to 143. (Item 38) An IL-12 polypeptide complex comprising a first polypeptide comprising SEQ ID NO: 104 or SEQ ID NO: 136. (Item 39) 1. An IL-12 polypeptide complex comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 104 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18. (Item 40) A polypeptide complex comprising a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 136 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18. (Item 41) A single-chain IL-12-inducing polypeptide comprising amino acids selected from the group consisting of SEQ ID NOs: 7, 9, 10, 18, 24 to 94, 110 to 118, and 127 to 134, or comprising an amino acid sequence having at least about 80% identity to SEQ ID NOs: 7, 9, 10, 18, 24 to 94, 110 to 118, and 127 to 134. (Item 42) 1. An inducible IL-12 polypeptide comprising p35, p40, a blocking element, and a half-life extending element, wherein the blocking element is an antibody or antigen-binding fragment having binding specificity for an epitope on IL-12 defined by the amino acids of Table 1. (Item 43) 43. A nucleic acid encoding a polypeptide as defined in any one of items 37 to 42. (Item 44) 43. The nucleic acid composition of any one of items 37 to 42, comprising a circular vector. (Item 45) 43. The nucleic acid composition of any one of items 37 to 42, comprising DNA. (Item 46) 43. The nucleic acid composition of any one of items 37 to 42, comprising RNA. (Item 47) 43. An expression vector comprising the nucleic acid according to any one of Items 37 to 42. (Item 48) 48. An isolated host cell comprising the vector of item 47. (Item 49) 49. A method for producing a pharmaceutical composition, comprising culturing the isolated host cell of item 48 under conditions suitable for expression of the polypeptide complex. (Item 50) 50. The method of claim 49, further comprising isolating the polypeptide complex. (Item 51) The protein complex according to any one of Items 37 to 40 or 42, or Item 4 48. A pharmaceutical composition comprising the polypeptide according to item 1 or the nucleic acid according to any one of items 43 to 47. (Item 52) A method for treating a tumor, comprising administering an effective amount of the polypeptide complex according to any one of Items 37 to 40 or 42, the nucleic acid according to any one of Items 43 to 47, the expression vector according to Item 48, or the pharmaceutical composition according to Item 51 to a subject in need of tumor treatment. (Item 53) 28. The polypeptide of any one of items 1 to 10 or the nucleic acid composition of any one of items 24 to 27, wherein the IL-12 is a mutein. (Item 54) 54. The polypeptide of item 53 or the nucleic acid composition of item 53, wherein the IL-12 is partially or fully aglycosylated. (Item 55) 55. The polypeptide of item 54 or the nucleic acid composition of item 54, wherein the p35 and / or the p40 is partially or fully aglycosylated. (Item 56) 56. The polypeptide of item 55 or the nucleic acid composition of item 55, wherein the p35 and / or the p40 is fully aglycosylated. (Item 57) 1. A polypeptide complex comprising IL-23, a half-life extending element, an IL-23 blocking element, and a protease-cleavable linker, wherein the IL-23 blocking element is a single chain antibody that binds to IL-23 or an antigen-binding fragment thereof, the complex comprising: v. a first polypeptide comprising an IL-23 subunit and optionally said IL-23 blocking element, wherein said IL-23 blocking element, if present, is operably linked to said IL-23 subunit via a first protease-cleavable linker; vi. a second polypeptide chain comprising an IL-23 subunit operably linked to a half-life extending element via a second protease-cleavable linker, and optionally said IL-23 blocking element, wherein said second polypeptide chain, when said IL-23 blocking element is present, is operably linked to said IL-23 subunit via a first protease-cleavable linker or is operably linked to said half-life extending element via a linker that is optionally protease-cleavable; only one of the first polypeptide and the second polypeptide comprises the IL-23 blocking element; The polypeptide complex, wherein when the IL-23 subunit of the first polypeptide is p19, the IL-23 subunit of the second polypeptide is p40, and when the IL-23 subunit of the first polypeptide is p40, the IL-23 subunit of the second polypeptide is p19. (Item 58) 58. The polypeptide of item 57, wherein the first protease-cleavable linker and the second protease-cleavable linker are the same. (Item 59) 1. A polypeptide complex comprising IL-23, a half-life extending element, an IL-23 blocking element, and a protease-cleavable linker, wherein the IL-23 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, wherein the complex comprises: iv. a first polypeptide comprising an IL-23 subunit and, optionally, a half-life extending element, wherein said half-life extending element, when present, inhibits a first protease cleavage. the first polypeptide operably linked to the IL-23 subunit via a reactive linker; v. a second polypeptide comprising an IL-12 subunit, at least an antigen-binding portion of an antibody light chain or an antigen-binding portion of an antibody heavy chain, and optionally a half-life extending element; wherein the half-life extending element, if present, is operably linked to the IL-23 subunit via a first protease-cleavable linker, and the antibody heavy chain or the antibody light chain is a) operably linked to the IL-23 subunit via a second protease-cleavable linker or b) optionally operably linked to the half-life extending element via a cleavable linker; and vi. a third polypeptide comprising at least an antigen-binding portion of an antibody heavy chain that is complementary to the light chain of the second polypeptide, or an antibody light chain that is complementary to the heavy chain of the second polypeptide and that, together with the light chain, forms an IL-23 binding site; The polypeptide complex, wherein when the IL-23 subunit of the first polypeptide is p19, the IL-23 subunit of the second polypeptide is p40, and when the IL-23 subunit of the first polypeptide is p40, the IL-23 subunit of the second polypeptide is p19. (Item 60) 60. The polypeptide of item 59, wherein the first protease-cleavable linker and the second protease-cleavable linker are the same. (Item 61) 1. A polypeptide complex comprising IL-23, a half-life extending element, an IL-23 blocking element, and a protease-cleavable linker, wherein the IL-23 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, wherein the complex comprises: v. a first polypeptide chain comprising p19, p40, a half-life extending element, and at least an antigen-binding portion of an antibody light chain, wherein p19 and p40 are operably linked, the half-life extending element is operably linked to p40 via a first protease-cleavable linker, and the antigen-binding portion of the antibody light chain is operably linked to p19 via a second protease-cleavable linker; or the half-life extending element is operably linked to p19 via a first protease-cleavable linker, and the antigen-binding portion of the antibody light chain is operably linked to p40 via a second protease-cleavable linker; and vi. the polypeptide complex comprising a second polypeptide, wherein the second polypeptide comprises at least an antigen-binding portion of an antibody heavy chain that is complementary to a light chain of the second polypeptide and that, together with the light chain, forms an IL-23 binding site. (Item 62) 1. A polypeptide complex comprising IL-23, a half-life extending element, an IL-23 blocking element, and a protease-cleavable linker, wherein the IL-23 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, wherein the complex comprises: vii. a first polypeptide chain comprising p19, p40, a half-life extending element, and at least an antigen-binding portion of an antibody heavy chain, wherein p19 and p40 are operably linked, the half-life extending element is operably linked to p40 via a first protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain is operably linked to p19 via a second protease-cleavable linker; or the half-life extending element is operably linked to p19 via a first protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain is operably linked to p40 via a second protease-cleavable linker; and viii. The polypeptide complex comprising a second polypeptide that is complementary to a heavy chain of said second polypeptide and comprises at least an antigen-binding portion of an antibody light chain that forms, in combination with the light chain, an IL-23 binding site. (Item 63) 63. The polypeptide according to item 61 or 62, wherein the first protease-cleavable linker and the second protease-cleavable linker are the same. (Item 64) wherein the first polypeptide does not contain a blocking element and the second polypeptide has the formula: [A]-[L1]-[B]-[L3]-[D] or [D]-[L3]-[B]-[L1]-[A] or [B]-[L1]-[A]-[L2]-[D] or [D]-[L1]-[A]-[L2]-[B], wherein A is the IL-23 subunit; L1 is the first protease-cleavable linker; L2 is the second protease-cleavable linker; L3 is the optionally cleavable linker; B is the half-life extending element; 60. The polypeptide complex of item 57 or 59, wherein D is the blocking element. (Item 65) the first polypeptide having the formula: [A]-[L1]-[D] or [D]-[L1]-[A]; and said second polypeptide comprises the formula: [A']-[L2]-[B] or [B]-[L2]-[A'], wherein A is either p19 or p40, and if A is p19, A' is p40, and if A is p40, A' is p19; A' is either p19 or p40; L1 is the first protease-cleavable linker; L2 is the second protease-cleavable linker; B is the half-life extending element; 58. The polypeptide complex of item 57, wherein D is the blocking element. (Item 66) the first polypeptide comprises the formula: [A]-[L1]-[B] or [B]-[L1]-[A]; and the second polypeptide has the formula: [A']-[L2]-[D] or [D]-[L2]-[A'], wherein: A is either p19 or p40; A' is either p19 or p40, and if A is p19, then A' is p40, and if A is p40, then A' is p19; L1 is the first protease-cleavable linker; L2 is the second protease-cleavable linker; B is the half-life extending element; 60. The polypeptide complex of item 59, wherein D is the blocking element. (Item 67) 67. The polypeptide conjugate of any one of items 57 to 66, wherein the half-life extending element is human serum albumin, an antigen-binding polypeptide that binds to human serum albumin, or immunoglobulin Fc or a fragment thereof. (Item 68) The protease-cleavable linker is a protease selected from kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), ADAM, FAP, plasminogen activator, caspase, tryptase, or tumor protease. 67. The polypeptide complex according to any one of Items 57 to 66, comprising a sequence that can be cleaved by an enzyme. (Item 69) 67. The polypeptide complex of any one of items 57 to 66, wherein the protease is selected from cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, or cathepsin G. (Item 70) 67. The polypeptide complex of any one of items 57 to 66, wherein the protease is selected from a matrix metalloproteinase (MMP), which is MMP 1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, or MMP14. (Item 71) 67. The polypeptide complex according to any one of items 57 to 66, wherein the protease-cleavable linker comprises at least two sequences that can be independently cleaved by a protease. (Item 72) 67. The polypeptide complex of any one of items 57 to 66, wherein the protease-cleavable linker comprises a synthetic sequence. (Item 73) Item 11. The polypeptide complex of any one of the preceding items, wherein each of the protease-cleavable linkers is cleaved by two or more different proteases. (Item 74) 58. The polypeptide conjugate of item 57, wherein the single-chain antibody is a single-chain variable fragment (scFv). (Item 75) 63. The polypeptide conjugate of items 59, 61 or 62, wherein the antigen-binding fragment of the antibody is a Fab. (Item 76) 63. The polypeptide complex of any one of paragraphs 57, 59, 61, or 62, wherein the blocking element binds to the IL-23. (Item 77) 75. The polypeptide complex of item 73 or 74, wherein the blocking element binds to p19, p40, or the p19p40 complex. (Item 78) A nucleic acid encoding a polypeptide as defined in any one of items 57 to 77. (Item 79) 79. The nucleic acid of item 78, wherein the nucleic acid does not encode only p19 or p40. (Item 80) 1. A nucleic acid composition comprising one or more nucleic acid sequences encoding a polypeptide complex comprising IL-23, a half-life extending element, an IL-23 blocking element, and a protease-cleavable linker, wherein the IL-23 blocking element is a single chain antibody that binds to IL-23 or an antigen-binding fragment thereof, and wherein the complex comprises: vii. a first polypeptide comprising an IL-23 subunit and optionally said IL-23 blocking element, wherein said IL-23 blocking element, if present, is operably linked to said IL-23 subunit via a first protease-cleavable linker; viii. A second polypeptide chain comprising an IL-23 subunit operably linked to a half-life extending element via a second protease-cleavable linker, and optionally said IL-23 blocking element, wherein said IL-23 blocking element, when present, is operably linked to said IL-23 subunit via a first protease-cleavable linker or optionally linked to said half-life extending element via a linker that is protease-cleavable. the second polypeptide chain operably linked to an extension element; only one of the first polypeptide and the second polypeptide comprises the IL-23 blocking element; The nucleic acid composition, wherein when the IL-23 subunit of the first polypeptide is p19, the IL-23 subunit of the second polypeptide is p40, and when the IL-23 subunit of the first polypeptide is p40, the IL-23 subunit of the second polypeptide is p19. (Item 81) 1. A nucleic acid composition comprising one or more nucleic acid sequences encoding a polypeptide complex comprising IL-23, a half-life extending element, an IL-23 blocking element, and a protease-cleavable linker, wherein the IL-23 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, and wherein the complex comprises: iv. a first polypeptide comprising an IL-23 subunit and, optionally, a half-life extending element, wherein the half-life extending element, if present, is operably linked to the IL-23 subunit via a first protease-cleavable linker; v. a second polypeptide comprising an IL-12 subunit, at least an antigen-binding portion of an antibody light chain, and optionally a half-life extending element; wherein the half-life extending element, if present, is operably linked to the IL-23 subunit via a first protease-cleavable linker, and wherein the antibody heavy chain or the antibody light chain is a) operably linked to the IL-23 subunit via a second protease-cleavable linker, or b) optionally operably linked to the half-life extending element via a cleavable linker; and vi. a third polypeptide comprising at least an antigen-binding portion of an antibody heavy chain that is complementary to the light chain of the second polypeptide and that, together with the light chain, forms an IL-23 binding site; The nucleic acid composition, wherein when the IL-23 subunit of the first polypeptide is p19, the IL-23 subunit of the second polypeptide is p40, and when the IL-23 subunit of the first polypeptide is p40, the IL-23 subunit of the second polypeptide is p19. (Item 82) 1. A nucleic acid composition comprising one or more nucleic acid sequences encoding a polypeptide complex comprising IL-23, a half-life extending element, an IL-23 blocking element, and a protease-cleavable linker, wherein the IL-23 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, and wherein the complex comprises: v. a first polypeptide chain comprising p19, p40, a half-life extending element, and at least an antigen-binding portion of an antibody light chain, wherein p19 and p40 are operably linked, the half-life extending element is operably linked to p40 via a first protease-cleavable linker, and the antigen-binding portion of the antibody light chain is operably linked to p19 via a second protease-cleavable linker; or the half-life extending element is operably linked to p19 via a first protease-cleavable linker, and the antigen-binding portion of the antibody light chain is operably linked to p40 via a second protease-cleavable linker; and vi. The nucleic acid composition comprising a second polypeptide, wherein the second polypeptide comprises at least an antigen-binding portion of an antibody heavy chain that is complementary to a light chain of the second polypeptide and that, together with the light chain, forms an IL-23 binding site. (Item 83) IL-23, half-life extension element, IL-23 blocking element, and protease cleavage element 1. A nucleic acid composition comprising one or more nucleic acid sequences encoding a polypeptide complex comprising an intercalable linker, wherein the IL-23 blocking element is an antigen-binding fragment of an antibody, the antigen-binding fragment comprising, as separate components, at least an antigen-binding portion of an antibody light chain and at least an antigen-binding portion of a complementary antibody heavy chain, the complex comprising: vii. a first polypeptide chain comprising p19, p40, a half-life extending element, and at least an antigen-binding portion of an antibody heavy chain, wherein p19 and p40 are operably linked, the half-life extending element is operably linked to p40 via a first protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain is operably linked to p19 via a second protease-cleavable linker; or the half-life extending element is operably linked to p19 via a first protease-cleavable linker, and the antigen-binding portion of the antibody heavy chain is operably linked to p40 via a second protease-cleavable linker; and viii. The nucleic acid composition comprising a second polypeptide, wherein the second polypeptide comprises at least an antigen-binding portion of an antibody light chain that is complementary to a heavy chain of the second polypeptide and that, together with the heavy chain, forms an IL-23 binding site. (Item 84) 84. The nucleic acid composition of any one of items 78 to 83, comprising a circular vector. (Item 85) 84. The nucleic acid composition of any one of items 78 to 83, comprising DNA. (Item 86) 84. The nucleic acid composition of any one of items 78 to 83, comprising RNA. (Item 87) 84. An expression vector comprising the nucleic acid according to any one of Items 78 to 83. (Item 88) 88. An isolated host cell comprising the vector of item 87. (Item 89) 89. A method for producing a pharmaceutical composition, comprising culturing the isolated host cell of item 88 under conditions suitable for expression of the polypeptide complex. (Item 90) 90. The method of claim 89, further comprising isolating the polypeptide complex. (Item 91) A pharmaceutical composition comprising the protein complex according to any one of Items 57 to 77 or the nucleic acid according to any one of Items 78 to 86. (Item 92) A method for treating a tumor, comprising administering an effective amount of the polypeptide complex according to any one of Items 57 to 77, the nucleic acid according to any one of Items 78 to 86, the expression vector according to Item 87, or the pharmaceutical composition according to Item 91 to a subject in need of tumor treatment. (Item 93) An IL-23 polypeptide complex comprising a first polypeptide selected from the group consisting of SEQ ID NOs: 423-428, or an amino acid sequence having at least 80% identity to SEQ ID NOs: 423-428. (Item 94) 94. The IL-23 polypeptide complex of Item 93, further comprising a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 104, 434, or 442-445, or an amino acid sequence having at least 80% identity to SEQ ID NO: 104, 434, or 442-445. (Item 95) A single-chain IL-23-inducing polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 422 or 429-432, or an amino acid sequence having at least about 80% identity to SEQ ID NO: 422 or SEQ ID NOs: 429-432. (Item 96) An inducible IL-23 polypeptide comprising p19, p40, a blocking element, and a half-life extending element, wherein the blocking element is an antibody or antigen-binding fragment having binding specificity for an epitope on IL-23. (Item 97) 97. A nucleic acid encoding a polypeptide as defined in any one of items 93 to 96. (Item 98) 98. The nucleic acid composition of any one of items 93 to 97, comprising a circular vector. (Item 99) 99. The nucleic acid composition of any one of items 93 to 98, comprising DNA. (Item 100) 99. The nucleic acid composition of any one of items 93 to 98, comprising RNA. (Item 101) An expression vector comprising the nucleic acid according to any one of Items 97 to 100. (Item 102) 102. An isolated host cell comprising the vector of item 101. (Item 103) 103. A method for producing a pharmaceutical composition, comprising culturing the isolated host cell of item 102 under conditions suitable for expression of the polypeptide complex. (Item 104) 104. The method of claim 103, further comprising isolating the polypeptide complex. (Item 105) A pharmaceutical composition comprising the polypeptide complex according to any one of items 93 to 96 or the nucleic acid according to any one of items 97 to 100. (Item 106) A method for treating a tumor, comprising administering an effective amount of the polypeptide complex according to any one of Items 93 to 96, the nucleic acid according to any one of Items 97 to 100, the expression vector according to Item 101, or the pharmaceutical composition according to Item 105 to a subject in need of tumor treatment. (Item 107) The polypeptide of any one of items 57 to 66 or the nucleic acid composition of any one of items 80 to 86, wherein the IL-23 is a mutein. (Item 108) Item 53. The polypeptide of item 53 or the nucleic acid composition of item 107, wherein the IL-23 is partially or fully aglycosylated. (Item 109) 109. The polypeptide of item 108 or the nucleic acid composition of item 108, wherein the p19 and / or the p40 is partially or fully aglycosylated. (Item 110) 110. The polypeptide of item 109 or the nucleic acid composition of item 109, wherein the p19 and / or the p40 is fully aglycosylated.

[0040] The drawings are not necessarily to scale. Instead, emphasis has generally been placed on illustrating the principles of the inventions described herein. The accompanying drawings, which form a part of this specification, illustrate several embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the disclosure. In the following drawings: [Brief explanation of the drawings]

[0041] [Figure 1A] FIG. 1 is a schematic diagram showing various inducible IL-12 complexes containing two or three polypeptide chains. [Figure 1B] FIG. 1 is a schematic diagram showing various inducible IL-12 complexes containing two or three polypeptide chains. [Figure 1C] FIG. 1 is a schematic diagram showing various inducible IL-12 complexes containing two or three polypeptide chains. [Figure 1D] FIG. 1 is a schematic diagram showing various inducible IL-12 complexes containing two or three polypeptide chains. [Figure 1E] FIG. 1 is a schematic diagram showing various inducible IL-12 complexes containing two or three polypeptide chains. [Figure 1F] FIG. 1 is a schematic diagram showing various inducible IL-12 complexes containing two or three polypeptide chains. [Figure 1G] FIG. 1 is a schematic diagram showing various inducible IL-12 complexes containing two or three polypeptide chains. [Figure 1H] FIG. 1 is a schematic diagram showing various inducible IL-12 complexes containing two or three polypeptide chains. [Figure 1I] FIG. 1 is a schematic diagram showing various inducible IL-12 complexes containing two or three polypeptide chains. [Figure 1J] FIG. 1 is a schematic diagram showing various inducible IL-12 complexes containing two or three polypeptide chains. [Figure 2A]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2B]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2C]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2D]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2E]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2F]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2G]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2H]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2I]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2J]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2K]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2L]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2M]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2N]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2O]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2P]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2Q]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2R]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 2S]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in a HEKBlue IL-12 reporter assay. IL-12 / STAT4 activation by heterodimeric IL-12 polypeptides compared to chimeric IL-12 (mouse p35 / human p40) or recombinant IL-12 (control). Squares represent the IL-12 activity of the uncleaved inducible heterodimer, and triangles represent the IL-12 activity of the cleaved heterodimer. Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 3A]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 luciferase reporter assay. The heterodimeric IL-12 polypeptides show activation of IL-12 signaling compared to recombinant human IL-12 (control). The black squares represent the activity of the uncleaved inducible heterodimeric IL-12 polypeptide (intact), and the white squares represent the activity of the cleaved inducible heterodimer (cleaved). The circles represent the activity of the control recombinant human IL-12. The respective EC50 values ​​are shown in the table. [Figure 3B]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 luciferase reporter assay. The heterodimeric IL-12 polypeptides show activation of IL-12 signaling compared to recombinant human IL-12 (control). The black squares represent the activity of the uncleaved inducible heterodimeric IL-12 polypeptide (intact), and the white squares represent the activity of the cleaved inducible heterodimer (cleaved). The circles represent the activity of the control recombinant human IL-12. The respective EC50 values ​​are shown in the table. [Figure 3C]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 luciferase reporter assay. The heterodimeric IL-12 polypeptides show activation of IL-12 signaling compared to recombinant human IL-12 (control). The black squares represent the activity of the uncleaved inducible heterodimeric IL-12 polypeptide (intact), and the white squares represent the activity of the cleaved inducible heterodimer (cleaved). The circles represent the activity of the control recombinant human IL-12. The respective EC50 values ​​are shown in the table. [Figure 3D]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 luciferase reporter assay. The heterodimeric IL-12 polypeptides show activation of IL-12 signaling compared to recombinant human IL-12 (control). The black squares represent the activity of the uncleaved inducible heterodimeric IL-12 polypeptide (intact), and the white squares represent the activity of the cleaved inducible heterodimer (cleaved). The circles represent the activity of the control recombinant human IL-12. The respective EC50 values ​​are shown in the table. [Figure 3E]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 luciferase reporter assay. The heterodimeric IL-12 polypeptides show activation of IL-12 signaling compared to recombinant human IL-12 (control). The black squares represent the activity of the uncleaved inducible heterodimeric IL-12 polypeptide (intact), and the white squares represent the activity of the cleaved inducible heterodimer (cleaved). The circles represent the activity of the control recombinant human IL-12. The respective EC50 values ​​are shown in the table. [Figure 3F]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 luciferase reporter assay. The heterodimeric IL-12 polypeptides show activation of IL-12 signaling compared to recombinant human IL-12 (control). The black squares represent the activity of the uncleaved inducible heterodimeric IL-12 polypeptide (intact), and the white squares represent the activity of the cleaved inducible heterodimer (cleaved). The circles represent the activity of the control recombinant human IL-12. The respective EC50 values ​​are shown in the table. [Figure 4A]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 T-Blast assay. Activation of IL-12 signaling by heterodimeric IL-12 polypeptides compared to IL-12 (control) is shown. Squares represent the activity of uncleaved inducible heterodimeric IL-12 polypeptides (intact), and triangles represent the activity of cleaved inducible heterodimeric IL-12 polypeptides. Circles represent the activity of the control (IL-12). EC50 values ​​are shown in the table. [Figure 4B]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 T-Blast assay. Activation of IL-12 signaling by heterodimeric IL-12 polypeptides compared to IL-12 (control) is shown. Squares represent the activity of uncleaved inducible heterodimeric IL-12 polypeptides (intact), and triangles represent the activity of cleaved inducible heterodimeric IL-12 polypeptides. Circles represent the activity of the control (IL-12). EC50 values ​​are shown in the table. [Figure 4C]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 T-Blast assay. Activation of IL-12 signaling by heterodimeric IL-12 polypeptides compared to IL-12 (control) is shown. Squares represent the activity of uncleaved inducible heterodimeric IL-12 polypeptides (intact), and triangles represent the activity of cleaved inducible heterodimeric IL-12 polypeptides. Circles represent the activity of the control (IL-12). EC50 values ​​are shown in the table. [Figure 4D]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 T-Blast assay. Activation of IL-12 signaling by heterodimeric IL-12 polypeptides compared to IL-12 (control) is shown. Squares represent the activity of uncleaved inducible heterodimeric IL-12 polypeptides (intact), and triangles represent the activity of cleaved inducible heterodimeric IL-12 polypeptides. Circles represent the activity of the control (IL-12). EC50 values ​​are shown in the table. [Figure 4E]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 T-Blast assay. Activation of IL-12 signaling by heterodimeric IL-12 polypeptides compared to IL-12 (control) is shown. Squares represent the activity of uncleaved inducible heterodimeric IL-12 polypeptides (intact), and triangles represent the activity of cleaved inducible heterodimeric IL-12 polypeptides. Circles represent the activity of the control (IL-12). EC50 values ​​are shown in the table. [Figure 4F]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 T-Blast assay. Activation of IL-12 signaling by heterodimeric IL-12 polypeptides compared to IL-12 (control) is shown. Squares represent the activity of uncleaved inducible heterodimeric IL-12 polypeptides (intact), and triangles represent the activity of cleaved inducible heterodimeric IL-12 polypeptides. Circles represent the activity of the control (IL-12). EC50 values ​​are shown in the table. [Figure 4G]

[0023] Figure 1 is a series of graphs showing the activity of fusion protein heterodimers in an IL-12 T-Blast assay. Activation of IL-12 signaling by heterodimeric IL-12 polypeptides compared to IL-12 (control) is shown. Squares represent the activity of uncleaved inducible heterodimeric IL-12 polypeptides (intact), and triangles represent the activity of cleaved inducible heterodimeric IL-12 polypeptides. Circles represent the activity of the control (IL-12). EC50 values ​​are shown in the table. [Figure 5]A series of SDS-PAGE gels comparing WW0663 (SEQ ID NO: 18) (a single polypeptide chain in which IL-12 subunits are joined using a linker designed to be non-cleavable) produced in a mammalian host cell line and purified by Protein A chromatography. Reducing and non-reducing conditions are compared. Analysis showed unintended cleavage of WW0663 at or near the linker connecting p35 and p40. In contrast, the heterodimer WW0750 / WW0636 showed only the desired product when produced in the same mammalian host cell line. [Figure 6] 1 is a graph showing the analysis of WW0749 / 636 in a syngeneic MC38 mouse tumor model. Mean tumor volume over time is shown for mice treated with 43 μg WW0749 / 636 (triangles), 170 μg WW0749 / 636 (inverted triangles), 340 μg WW0749 / 636 (diamonds), and 510 μg WW0749 / 636 (squares). Vehicle alone is indicated by open circles. [Figure 7] 7 shows a series of spider plots showing the activity of inducible IL-12 fusion proteins in an MC38 mouse xenograft model corresponding to the data shown in Figure 6. Each line in the plot is the tumor volume over time for one mouse. [Figure 8] 1 is a graph showing the analysis of WW0749 / 636 in a syngeneic MC38 mouse tumor model. Mean body weight (%) over time is shown for mice treated with 43 μg WW0749 / 636 (triangles), 170 μg WW0749 / 636 (inverted triangles), 340 μg WW0749 / 636 (diamonds), and 510 μg WW0749 / 636 (squares). Vehicle alone is indicated by open circles. [Figure 9]

[0023] Figure 9 shows a series of spider plots showing the effect of an inducible IL-12 fusion protein (WW0749 / 636) on body weight in an MC38 mouse xenograft model corresponding to the data shown in Figure 8. Each row of the plot is the body weight of one mouse over time. [Figure 10]1 is a graph showing the analysis of WW0751 / 636 in a syngeneic MC38 mouse tumor model. Mean tumor volume over time is shown for mice treated with 43 μg WW0751 / 636 (triangles), 170 μg WW0751 / 636 (inverted triangles), 340 μg WW0751 / 636 (diamonds), and 510 μg WW0751 / 636 (squares). Vehicle alone is indicated by open circles. The data show that tumor volume decreases over time in mice treated with WW0751 / 636 at all concentrations. [Figure 11] 1 shows a series of spider plots showing the activity of the fusion protein (WW0751 / 636) in an MC38 mouse xenograft model corresponding to the data shown in Figure 10. Each line in the plot is the tumor volume over time for one mouse. [Figure 12] 1 is a graph showing the analysis of WW0751 / 636 in a syngeneic MC38 mouse tumor model. Mean body weight (%) over time is shown for mice treated with 43 μg WW0751 / 636 (triangles), 170 μg WW0751 / 636 (inverted triangles), 340 μg WW0751 / 636 (diamonds), and 510 μg WW0751 / 636 (squares). Vehicle alone is indicated by open circles. [Figure 13] 13 shows a series of spider plots showing the effect of fusion proteins on body weight in the MC38 mouse xenograft model corresponding to the data shown in Figure 12. Each line in the plot is the body weight of one mouse over time. [Figure 14] 1 is a graph showing the analysis of WW0753 / 636 / 727 in a syngeneic MC38 mouse tumor model. Mean tumor volume over time is shown for mice treated with 52 μg of WW0753 / 636 / 727 (triangles), 207 μg of WW0753 / 636 / 727 (inverted triangles), 414 μg of WW0753 / 636 / 727 (diamonds), and 621 μg of WW0753 / 636 / 727 (squares). Vehicle alone is indicated by open circles. The data show that tumor volume decreased dose-dependently over time in mice treated with the higher concentrations of WW0753 / 636 / 727. [Figure 15]15 shows a series of spider plots showing the activity of the fusion protein (WW0753 / 636 / 727) in an MC38 mouse xenograft model corresponding to the data shown in Figure 14. Each line in the plot is the tumor volume over time for one mouse. [Figure 16] 1 is a graph showing the analysis of WW0753 / 636 / 727 in a syngeneic MC38 mouse tumor model. Mean body weight (%) over time is shown for mice treated with 52 μg WW0753 / 636 / 727 (triangles), 207 μg WW0753 / 636 / 727 (inverted triangles), 414 μg WW0753 / 636 / 727 (diamonds), and 621 μg WW0753 / 636 / 727 (squares). Vehicle alone is indicated by open circles. [Figure 17] 17 shows a series of spider plots showing the effect of the fusion protein (WW0753 / 636 / 727) on body weight in the MC38 mouse xenograft model corresponding to the data shown in Figure 16. Each line in the plot is the body weight of one mouse over time. [Figure 18] 1 is a graph showing the analysis of WW0755 / 636 / 727 in a syngeneic MC38 mouse tumor model. Mean tumor volume over time is shown for mice treated with 52 μg of WW0755 / 636 / 727 (triangles), 207 μg of WW0755 / 636 / 727 (inverted triangles), 414 μg of WW0755 / 636 / 727 (diamonds), and 621 μg of WW0755 / 636 / 727 (squares). Vehicle alone is indicated by open circles. [Figure 19] 19 shows a series of spider plots showing the activity of the fusion protein (WW0755 / 636 / 727) in the MC38 mouse xenograft model corresponding to the data shown in Figure 18. Each line in the plot is the tumor volume over time for one mouse. [Figure 20]1 is a graph showing the analysis of WW0755 / 636 / 727 in a syngeneic MC38 mouse tumor model. Mean body weight (%) over time is shown for mice treated with 52 μg WW0755 / 636 / 727 (triangles), 207 μg WW0755 / 636 / 727 (inverted triangles), 414 μg WW0755 / 636 / 727 (diamonds), and 621 μg WW0753 / 636 / 727 (squares). Vehicle alone is indicated by open circles. [Figure 21] 21 shows a series of spider plots showing the effect of the fusion protein (WW0755 / 636 / 727) on body weight in the MC38 mouse xenograft model corresponding to the data shown in Figure 20. Each line in the plot is the body weight of one mouse over time. [Figure 22] 1 is a graph showing the analysis of WW0749 / 636 in a syngeneic MC38 mouse tumor model. Mean tumor volume over time is shown for mice treated with 3.5 μg of WW0749 / 636 (diamonds), 14 μg of WW0749 / 636 (squares), and 43 μg of WW0749 / 636 (blue circles). Vehicle alone is indicated by black circles. [Figure 23] 23 shows a series of spider plots showing the activity of the fusion protein (WW0749 / 636) in an MC38 mouse xenograft model corresponding to the data shown in Figure 22. Each line in the plot is the tumor volume over time for one mouse. [Figure 24] 1 is a graph showing the analysis of WW0749 / 636 in a syngeneic MC38 mouse tumor model. Mean body weight (%) over time is shown for mice treated with 3.5 μg WW0749 / 636 (diamonds), 14 μg WW0749 / 636 (squares), and 43 μg WW0749 / 636 (blue circles). Vehicle alone is indicated by black circles. [Figure 25]

[0023] Figure 25 shows a series of spider plots showing the effect of the fusion protein (WW0749 / 636) on body weight in the MC38 mouse xenograft model corresponding to the data shown in Figure 24. Each line in the plot is the body weight of one mouse over time. [Figure 26]

[0023] Figure 1 shows the analysis of WW0753 / 636 / 727 in a syngeneic MC38 mouse tumor model. Mean tumor volume over time is shown for mice treated with 4.3 μg of WW0753 / 636 / 727 (diamonds), 17 μg of WW0753 / 636 / 727 (squares), and 52 μg of WW0753 / 636 / 727 (blue circles). Vehicle alone is indicated by black circles. [Figure 27] 27 shows a series of spider plots showing the activity of the fusion protein (WW0753 / 636 / 727) in the MC38 mouse xenograft model corresponding to the data shown in Figure 26. Each line in the plot is the tumor volume over time for one mouse. [Figure 28] 1 is a graph showing the analysis of WW0753 / 636 / 727 in a syngeneic MC38 mouse tumor model. Mean body weight (%) over time is shown for mice treated with 4.3 μg WW0753 / 636 / 727 (diamonds), 17 μg WW0753 / 636 / 727 (squares), and 52 μg WW0753 / 636 / 727 (blue circles). Vehicle alone is indicated by black circles. [Figure 29]

[0023] Figure 29 shows a series of spider plots showing the effect of the fusion protein (WW0753 / 636 / 727) on body weight in the MC38 mouse xenograft model corresponding to the data shown in Figure 28. Each line in the plot is the body weight of one mouse over time. [Figure 30] 13 is a graph showing the analysis of WW0757 / 636 in a syngeneic MC38 mouse tumor model, showing the mean tumor volume over time for mice treated with 14 μg WW0757 / 636 (diamonds), 43 μg WW0757 / 636 (squares), 86 μg WW0757 / 636 (circles), 170 μg WW0757 / 636 (upper triangles), 510 μg WW0757 / 636 (lower triangles), 765 μg WW0757 / 636 (stars), and 1,020 μg WW0757 / 636 (asterisks). Vehicle alone is indicated by open circles. [Figure 31]Figure 3 shows a series of spider plots depicting the activity of the fusion protein (WW0757 / 636) in an MC38 mouse xenograft model, corresponding to the data shown in Figure 30. Each line in the plot represents the tumor volume over time for one mouse. At 1,020 μg of WW0757 / 636, there were two treatment breaks on days 7 and 11 due to poor tolerability. [Figure 32] 13 is a graph showing the analysis of WW0757 / 636 in a syngeneic MC38 mouse tumor model, showing the mean body weight (%) over time for mice treated with 14 μg WW0757 / 636 (diamonds), 43 μg WW0757 / 636 (squares), 86 μg WW0757 / 636 (circles), 170 μg WW0757 / 636 (upper triangles), 510 μg WW0757 / 636 (lower triangles), 765 μg WW0757 / 636 (stars), and 1,020 μg WW0757 / 636 (asterisks). Vehicle alone is indicated by closed circles. [Figure 33] 3 shows a series of spider plots showing the effect of the fusion protein (WW0757 / 636) on body weight in the MC38 mouse xenograft model corresponding to the data shown in Figure 31. Each line in the plot is the body weight of one mouse over time. [Figure 34] 1 is a graph showing the analysis of WW0804 / 636 in a syngeneic MC38 mouse tumor model, showing the mean tumor volume over time for mice treated with 42 μg WW0804 / 636 (diamonds), 168 μg WW0804 / 636 (squares), 505 μg WW0804 / 636 (circles), 757 μg WW0804 / 636 (upper triangles), and 1,010 μg WW0804 / 636 (lower triangles). Vehicle alone is indicated by open circles. [Figure 35] Figure 34 shows a series of spider plots depicting the activity of the fusion protein (WW0804 / 636) in an MC38 mouse xenograft model, corresponding to the data shown in Figure 33. Each line in the plot represents the tumor volume over time for one mouse. WW0804 / 636 at 767 μg and 1,020 μg was withdrawn on day 11 due to poor tolerability. [Figure 36]1 is a graph showing the analysis of WW0804 / 636 in a syngeneic MC38 mouse tumor model, showing the mean body weight (%) over time for mice treated with 42 μg WW0804 / 636 (diamonds), 168 μg WW0804 / 636 (squares), 505 μg WW0804 / 636 (circles), 757 μg WW0804 / 636 (upper triangles), and 1,010 μg WW0804 / 636 (lower triangles). Vehicle alone is indicated by closed circles. [Figure 37]

[0033] Figure 36 shows a series of spider plots illustrating the effect of fusion protein (WW0804 / 636) on body weight in an MC38 mouse xenograft model, corresponding to the data shown in Figure 35. Each line in the plot represents the body weight of one mouse over time. WW0804 / 636 at 757 μg and 1,010 μg, respectively, had a washout day on day 11. [Figure 38] 1 is an image of an SDS-PAGE gel of aglycosylated IL-12 polypeptide constructs. The gel shows WW0924 (SEQ ID NO: 442) / WW0925 (SEQ ID NO: 443) in the first column. The gel shows WW0935 (SEQ ID NO: 444) / WW0936 (SEQ ID NO: 445) in the second column. The gel shows WW0924 (SEQ ID NO: 442) / WW0636 (SEQ ID NO: 18) in the third column. The gel shows WW0758 (SEQ ID NO: 104) / WW0925 (SEQ ID NO: 443) in the fourth column. [Figure 39A] 1 shows a series of graphs of SEC analysis of aglycosylated IL-12 polypeptide constructs derived from CHO cells, including fully aglycosylated WW0924 (SEQ ID NO: 442) / WW0925 (SEQ ID NO: 443). [Figure 39B] 1 shows a series of graphs of SEC analysis of aglycosylated IL-12 polypeptide constructs derived from CHO cells, including partially aglycosylated WW0935 (SEQ ID NO: 444) / WW0936 (SEQ ID NO: 445). [Figure 39C] 1 shows a series of graphs of SEC analysis of aglycosylated IL-12 polypeptide constructs derived from CHO cells, including fully aglycosylated WW0924 (SEQ ID NO: 442) / WW0925 (SEQ ID NO: 443). [Figure 39D]1 shows a series of graphs of SEC analysis of CHO cell-derived aglycosylated IL-12 polypeptide constructs, including the complete WW0758 (SEQ ID NO: 104) / WW0925 (SEQ ID NO: 443). [Figure 40] Figure 1 is a series of graphs showing the activity of fusion proteins in the HEKBlue IL23 reporter assay. (A) IL-23 / STAT3 activation in the absence of albumin is shown, comparing WW50009 (a half-life extended murine IL23 fusion protein (squares)) with murine IL23 (control (circles)). (B) IL-23 / STAT3 activation in the presence of albumin is shown, comparing WW50009 (a half-life extended murine IL23 fusion protein (squares)) with murine IL23 (control (circles)). The respective EC50 values ​​are shown in the table. The assay was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results confirm that the half-life extended murine IL23 fusion protein is active, regardless of the presence of albumin. [Figure 41] 1 is a graph showing the analysis of WW0757 / 636 in a syngeneic CT26 mouse tumor model. It shows the mean tumor volume over time in mice treated with 50 μg of WW0757 / 636 (diamonds) and 100 μg of WW0757 / 636 (squares). Vehicle alone is indicated by open circles. The data show that mice treated with high concentrations of WW0757 / 636 inhibited tumor volume growth over time in a dose-dependent manner. [Figure 42] 41 shows a series of spider plots showing the activity of fusion proteins in a CT26 mouse xenograft model corresponding to the data shown in Figure 41. Each line in the plot is the tumor volume over time for one mouse. [Figure 43]1 is a graph showing the analysis of WW0757 / 636 in a syngeneic B16F10 mouse tumor model. It shows the mean tumor volume over time in mice treated with 50 μg of WW0757 / 636 (diamonds) and 100 μg of WW0757 / 636 (squares). Vehicle alone is indicated by open circles. The data show that mice treated with high concentrations of WW0757 / 636 inhibited tumor volume growth over time in a dose-dependent manner. [Figure 44] 43 shows a series of spider plots depicting the activity of fusion proteins in a B16F10 mouse xenograft model corresponding to the data shown in Figure 43. Each line in the plot is the tumor volume over time for one mouse. [Figure 45] 1 is a graph showing the analysis of WW0757 / 636 in a syngeneic EMT6 mouse tumor model. It shows the mean tumor volume over time in mice treated with 50 μg of WW0757 / 636 (diamonds) and 100 μg of WW0757 / WW0636 (squares). Vehicle alone is indicated by large open circles. The data show that mice treated with high concentrations of WW0757 / WW0636 showed a dose-dependent inhibition of tumor volume growth over time. [Figure 46]

[0033] Figure 46 shows a series of spider plots showing the activity of fusion proteins in an EMT6 mouse xenograft model corresponding to the data shown in Figure 45. Each line in the plot is the tumor volume over time for one mouse. [Figure 47A]

[0023] Figure 1 is a series of graphs showing immune profiling and nanostring analysis of WW0757 / WW0636-treated MC38 mouse tumor extracts, demonstrating increased IFNg production by total CD8+ T cells, tetramer+ CD8+ T cells, and NK cells. [Figure 47B]

[0023] Figure 1 is a series of graphs showing immune profiling and nanostring analysis of WW0757 / WW0636-treated MC38 mouse tumor extracts, demonstrating increased IFNg production by total CD8+ T cells, tetramer+ CD8+ T cells, and NK cells. [Figure 47C]

[0023] Figure 1 is a series of graphs showing immune profiling and nanostring analysis of WW0757 / WW0636-treated MC38 mouse tumor extracts, demonstrating increased IFNg production by total CD8+ T cells, tetramer+ CD8+ T cells, and NK cells. [Figure 47D] 1 shows that CD25 and Tbet expression by tetramer+CD8+ T cells was activated. [Figure 47E] 1 shows that CD25 and Tbet expression by tetramer+CD8+ T cells was activated. [Figure 47F] Figure 1 shows CD25 production by CD4+ non-Tregs. P values ​​represent unpaired Student's T-test. *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001. [Figure 47G] Figure 1 shows Tbet production by CD4+ non-Tregs. P values ​​represent unpaired Student's T-test. *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001. [Figure 47H] IFNg production by CD4+ Non-Tregs is shown. P values ​​represent unpaired Student's T-test. *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001. [Figure 47I] Figure 1 shows TNF production by CD4+ Non-Tregs. P values ​​represent unpaired Student's T-test. *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001. [Figure 48A]

[0023] Figure 1 is a series of graphs showing that the IL-12 polypeptide complex WW0757 / WW0636 promotes a transcriptional shift toward immune activation.

[0024] Figure 1 shows a heat map analysis of statistically significant changes in transcriptional expression between vehicle and WW0757 / WW000636 treated animals. [Figure 48B] 1 is a series of graphs showing that the IL-12 polypeptide complex WW0757 / WW0636 promotes a transcriptional shift toward immune activation. 1 shows interferon signaling between vehicle and WW0757 / 0636 treated tumors. [Figure 48C]1 is a series of graphs showing that the IL-12 polypeptide complex WW0757 / WW0636 promotes a transcriptional shift toward immune activation. 2 shows a pathway scoring analysis of differences in immune cell function. [Figure 48D] 1 is a series of graphs showing that the IL-12 polypeptide complex WW0757 / WW0636 promotes a transcriptional shift toward immune activation. 2 shows a pathway scoring analysis of differences in immune cell function. [Figure 48E] 1 is a series of graphs showing that the IL-12 polypeptide complex WW0757 / WW0636 promotes a transcriptional shift toward immune activation. 2 shows a pathway scoring analysis of differences in immune cell function. [Figure 48F] 1 is a series of graphs showing that the IL-12 polypeptide complex WW0757 / WW0636 promotes a transcriptional shift toward immune activation. 2 shows pathway scoring analysis of differences in dendritic cell function between vehicle and WW0757 / 0636 treated tumors. [Figure 48G] 1 is a series of graphs showing that the IL-12 polypeptide complex WW0757 / WW0636 promotes a transcriptional shift toward immune activation. 2 shows pathway scoring analysis of differences in dendritic cell function between vehicle and WW0757 / 0636 treated tumors. [Figure 48H] 1 is a series of graphs showing that the IL-12 polypeptide complex WW0757 / WW0636 promotes a transcriptional shift toward immune activation. 2 shows pathway scoring analysis of differences in dendritic cell function between vehicle and WW0757 / 0636 treated tumors. [Figure 49] 1A-1C are graphs showing the analysis of WW5009 in a syngeneic MC38 mouse tumor model. (A) shows the mean tumor volume over time in mice treated with 1 μg WW5009 (filled circles), 10 μg WW5009 (squares), and 100 μg WW5009 (stars). Vehicle alone is shown as open circles. The data show that tumor volume decreased over time in the two highest dose groups, 10 and 100 μg. (B) shows the effect of WW5009 dosing on the mean body weight of the animals. [Figure 50]49A-49B are a series of spider plots showing the activity of WW5009 in an MC38 mouse xenograft model, corresponding to the data shown in Figures 49A-49B. Each line in the plot is the tumor volume over time for one mouse. DETAILED DESCRIPTION OF THE INVENTION

[0042] The present disclosure relates to inducible IL-12 polypeptide complexes that contain attenuated IL-12 and have a longer half-life compared to native IL-12. The IL-12 polypeptide complexes disclosed herein comprise two or more polypeptide chains, and the complexes include the IL-12 subunits p35 and p40, a half-life-extending element, an IL-12 blocking element, and a protease-cleavable linker. The activity of IL-12 in the complex (e.g., receptor-binding activity and / or receptor agonist activity) is attenuated by the action of the blocking element, which is connected to the complex by the protease-cleavable linker. Upon cleavage of the protease-cleavable linker(s), the blocking element and half-life-extending element separate from the IL-12 and can diffuse away from the IL-12 to generate active IL-12. The active IL-12 typically has substantially similar biological activity and half-life to native IL-12. 1A-1J show non-limiting examples of IL-12 polypeptide complexes disclosed herein. The present disclosure further relates to inducible IL-12 polypeptide complexes, as well as pharmaceutical compositions comprising nucleic acids encoding the polypeptides, and recombinant expression vectors and host cells for producing such polypeptides and complexes. Also provided herein are methods of using the disclosed IL-12 polypeptide complexes in the treatment of diseases, conditions, and disorders.

[0043] The IL-12 polypeptide complexes disclosed herein overcome the problems of toxicity and short half-life that have significantly limited the clinical use of IL-12, particularly in the field of oncology. The IL-12 polypeptide complexes comprise an IL-12 polypeptide with receptor agonist activity. However, with respect to the IL-12 polypeptide complexes, the IL-12 receptor agonist activity is attenuated and the circulating half-life is extended.

[0044] The IL-12 polypeptide complexes disclosed herein comprise at least two polypeptide chains, and can optionally comprise three or more polypeptide chains.

[0045] The present disclosure also relates to inducible IL-23 polypeptide complexes that contain attenuated IL-23 and have a longer half-life compared to native IL-23. The IL-23 polypeptide complexes disclosed herein comprise one or more polypeptide chains, where the complexes comprise the IL-23 subunits p19 and p40, a half-life-extending element, an IL-23 blocking element, and a protease-cleavable linker. The activity of IL-23 in the complex (e.g., receptor-binding activity and / or receptor agonist activity) is attenuated by the action of the blocking element, which is connected to the complex by the protease-cleavable linker. Upon cleavage of the protease-cleavable linker(s), the blocking element and half-life-extending element separate from IL-23 and can diffuse away from IL-23 to generate active IL-23. The active IL-23 typically has substantially similar biological activity and half-life to native IL-23. The present disclosure further relates to inducible IL-23 polypeptide complexes, as well as pharmaceutical compositions comprising nucleic acids encoding the polypeptides, and recombinant expression vectors and host cells for producing such polypeptides and complexes. Also provided herein are methods of using the disclosed IL-23 polypeptide complexes in the treatment of diseases, conditions, and disorders.

[0046] The IL-23 polypeptide conjugates disclosed herein overcome the problems of toxicity and short half-life that have significantly limited the clinical use of IL-23, particularly in the field of oncology. The IL-23 polypeptide conjugates comprise an IL-23 polypeptide with receptor agonist activity, but with the IL-23 polypeptide conjugates, the IL-23 receptor agonist activity is attenuated and the circulating half-life is extended.

[0047] The IL-23 polypeptide complexes disclosed herein comprise at least one polypeptide chain, and can optionally comprise two or more polypeptide chains.

[0048] Certain exemplary and preferred embodiments are described in detail herein, and the embodiments within the specification should not be construed as limiting the scope of the disclosure.

[0049] All publications and patents cited herein are incorporated by reference in their entirety. In the event that a document incorporated by reference contradicts or is inconsistent with the present specification, the present specification takes precedence over such document. The citation of any reference herein is not an admission that such reference is prior art to the present disclosure. When a range of values ​​is expressed, it includes embodiments using any specific value within the range. Furthermore, reference to values ​​stated in a range includes all values ​​within that range. All ranges are inclusive of their endpoints and are combinable. When values ​​are expressed as approximations, by use of the antecedent "about," it is 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" means "and / or" unless the specific context of its use dictates otherwise.

[0050] Throughout the specification and claims, various terms are used in connection with the described embodiments. Such terms shall be given their ordinary meaning in the art unless otherwise indicated. Other terms that are specifically defined shall be interpreted in a manner consistent with the definitions set forth herein. The techniques and procedures described or referenced herein are generally well understood by those of skill in the art and commonly employed using conventional methodologies, such as the widely used 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. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and conditions unless otherwise specified.

[0051] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. The terms "including," "e.g.," and the like are intended to convey inclusion without limitation, unless otherwise stated.

[0052] Unless otherwise indicated, the terms "at least," "less than," and "about," or similar terms preceding a series or range of elements, should 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.

[0053] As used herein, the terms "activatable," "activate," "induce," and "inducible" refer to a polypeptide complex having an attenuated active form (e.g., attenuated receptor binding and / or agonist activity) and an active form. The polypeptide complex is activated by protease cleavage of the linker, which releases the blocking element and half-life extending element from the polypeptide complex. The induced / activated polypeptide complex can bind to the IL-12 receptor with increased affinity / activity. The induced / activated polypeptide complex can bind to the IL-23 receptor with increased affinity / activity.

[0054] The terms "antibody" and "immunoglobulin" are used interchangeably herein. As used herein, antibody or immunoglobulin is intended to refer to an immunoglobulin molecule consisting of two heavy (H) chains. Generally, mammalian (e.g., human, rodent, and monkey) antibodies comprise four polypeptide chains: two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CHI, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, single-specific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, or tetrameric antibodies comprising two heavy chain molecules and two light chain molecules. Those skilled in the art will recognize that other forms of antibodies exist (e.g., camel and shark antibodies).

[0055] As used herein, the term "attenuated" refers to an IL-12 receptor agonist or IL-23 receptor agonist that has reduced receptor agonist activity compared to the natural agonist of the IL-12 receptor or IL-23 receptor. An attenuated IL-12 agonist or an attenuated IL-23 agonist may have agonist activity that is at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 1000-fold, or less, compared to the natural agonist of the receptor. When an IL-12 polypeptide complex containing IL-12 described herein is described as having "attenuated" or "attenuated activity," it means that the IL-12 polypeptide complex is an attenuated IL-12 receptor agonist. When an IL-23 polypeptide complex comprising IL-23 as described herein is described as having "attenuated" or "attenuated activity," it means that the IL-23 polypeptide complex is an attenuated IL-23 receptor agonist.

[0056] The term "cancer" refers to a physiological condition in mammals in which a population of cells is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and / or specific morphological features. Cancers often take the form of a tumor or mass, but may exist alone within a subject or circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. The term cancer includes all types of cancer and metastases, including hematological malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., triple-negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. Triple-negative breast cancer refers to breast cancer that is negative for expression of the estrogen receptor (ER), progesterone receptor (PR), and Her2 / neu genes.

[0057] As used herein, "conservative" amino acid substitutions generally refer to the substitution of one amino acid residue for another within a recognized group, which may alter the structure of the peptide but substantially retain the biological activity of the peptide. Conservative amino acid substitutions are known to those skilled in the art. Conservative amino acid substitutions include, but are not limited to, substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. For example, those skilled in the art would reasonably expect that a single substitution of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or a similar amino acid substitution with a structurally related amino acid, would not significantly affect the biological activity of the resulting molecule.

[0058] As used herein, the term "half-life extending element" with respect to the polypeptide conjugates disclosed herein refers to a chemical element, preferably a polypeptide, that extends blood half-life and improves pK, for example, by modifying its size (e.g., above the renal filtration cutoff), shape, hydrodynamic radius, charge, or by modifying the parameters of absorption, biodistribution, metabolism, and elimination.

[0059] As used herein, the term "operably linked" with respect to a polypeptide complex refers to an orientation of the components of the polypeptide complex that allows them to function in their intended manner. For example, a polypeptide comprising an IL-12 subunit and an IL-12 blocking element is operably linked by a protease-cleavable linker in the polypeptide complex if the IL-12 blocking element can inhibit the IL-12 receptor-activating activity of the IL-12 polypeptide; however, upon cleavage of the protease-cleavable linker, the blocking element can diffuse away from the IL-12, thereby reducing or eliminating inhibition of the IL-12 receptor-activating activity of the IL-12 polypeptide by the IL-12 blocking element.

[0060] As used herein, the terms "peptide," "polypeptide," or "protein" are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used interchangeably herein to refer to an amino acid sequence. It should be recognized that the term polypeptide is not used herein to imply a specific size or number of amino acids comprising the molecule; the peptides of the present invention can contain up to a few or more amino acid residues.

[0061] The term "subject" as used herein refers to any animal, e.g., any mammal, including, but not limited to, humans, non-human primates, rodents, etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0062] As used herein, the term "therapeutically effective amount" refers to an amount of a compound described herein (i.e., an IL-12 polypeptide complex) sufficient to achieve the desired pharmacological or physiological effect under the conditions of administration. For example, a "therapeutically effective amount" can be an amount sufficient to alleviate the signs or symptoms of a disease or condition (e.g., a tumor). Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject. A therapeutically effective amount of a pharmaceutical composition may vary according to factors such as the individual's disease state, age, sex, and weight, as well as the ability of the pharmaceutical composition to elicit a desired response in an individual. Those skilled in the art can determine the appropriate dosage to achieve the desired therapeutic effect based on these and other considerations.

[0063] A. IL-12 Polypeptide Complex The present disclosure relates to an inducible IL-12 polypeptide complex comprising at least two polypeptide chains, and optionally three or more polypeptide chains. The two or more polypeptide chains disclosed herein may be different, i.e., the complex may be a heterodimer, heterotrimer, or the like. The inducible IL-12 polypeptide complex comprises a p35 IL-12 subunit, a p40 IL-12 subunit, a half-life-extending element, an IL-12 blocking element, and a protease-cleavable linker. The p35 and p40 subunits associate to form an IL-12 heterodimer, which possesses intrinsic IL-12 receptor agonist activity. The IL-12 polypeptide complex exhibits attenuated IL-12 receptor agonist activity and prolonged circulating half-life. The IL-12 receptor agonist activity is attenuated by the blocking element. The half-life-extending element may also contribute to attenuation, for example, through steric effects. The blocking element can block all or part of the receptor agonist activity of IL-12 by sterically blocking it and / or by non-covalently binding to IL-12 (e.g., p35, p40, or the p35p40 complex). Cleavage of the protease-cleavable linker liberates an active (e.g., more active) form of IL-12 from the IL-12 polypeptide complex. Typically, the free IL-12 is at least 10-fold more active than the IL-12 polypeptide complex. Preferably, the free IL-12 is at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 500-fold, at least 1000-fold, or at least about 10,000-fold more active than the IL-12 polypeptide complex.

[0064] The form of IL-12 released upon cleavage of the IL-12 polypeptide complex generally has a short half-life, often substantially similar to that of native IL-12. Although the half-life of the IL-12 polypeptide complex is extended, toxicity is reduced or eliminated because circulating IL-12 polypeptide complexes are attenuated and active IL-12 is targeted to the desired site (e.g., the tumor microenvironment).

[0065] Those skilled in the art will understand that the number of polypeptide chains and the location of the p35 and p40 subunits, half-life extending elements, protease-cleavable linker(s), and blocking elements (and components of such elements, e.g., VH or VL domains) on the polypeptide chains can vary and are often a matter of design preference. All such variations are encompassed by the present disclosure.

[0066] In embodiments, the IL-12 polypeptide complex comprises two distinct polypeptide chains. Typically, the first polypeptide chain comprises p35 and the second polypeptide chain comprises p40. The p35 and p40 subunits combine to form a biologically active heterodimer. The p35p40 heterodimer complex can be covalently linked, for example, via a disulfide bond.

[0067] In embodiments, either the first or second polypeptide may comprise an IL-12 blocking element (e.g., an scFV that binds IL-12) operably linked to an IL-12 subunit via a protease-cleavable linker. The other polypeptide chain may further comprise a half-life extending element operably linked to an IL-12 subunit via a protease-cleavable linker. Preferably, the complex comprises one functional blocking element and one functional half-life extending element. For example, if the first polypeptide chain comprises an IL-12 blocking element, the second polypeptide chain does not comprise an IL-12 blocking element. In other embodiments, one polypeptide chain comprises either p35 or p40 and further comprises a half-life extending element and a blocking element, each of which is operably linked to p35 or p40 via a protease-cleavable linker (e.g., one or more protease-cleavable linkers), and the other polypeptide comprises the complementary IL-12 subunit (e.g., either p40 or p35). The IL-12 blocking element on the second polypeptide can be operably linked to the IL-12 subunit via a protease-cleavable linker. Alternatively, the IL-12 blocking element can be operably linked to the half-life extending element via an optional protease-cleavable linker. The protease-cleavable linkers on the first and second polypeptide chains can be the same or different. Preferably, the protease-cleavable linkers on the first and second polypeptide chains are the same. The blocking element in this IL-12 polypeptide complex can be a single-chain antibody. Any single-chain antibody having binding specificity for IL-12 can be the blocking element. Preferably, the blocking element is an scFv.

[0068] The conjugates disclosed herein preferably contain one half-life extending element and one blocking element, although such elements may contain two or more components present on the same or different polypeptide chains. As shown, and as disclosed and exemplified herein, the components of a blocking element can be present on separate polypeptide chains. For example, a first polypeptide chain can contain an antibody light chain (VL+CL) or a light chain variable domain (VL), and a second polypeptide chain can contain an antibody heavy chain Fab fragment (VH+CH1) or a heavy chain variable domain (VH) that is complementary to the VL+CL or VL on the first polypeptide. In such a situation, these components can associate in the peptide conjugate to form an antigen-binding site, such as a Fab, that binds to IL-12 and attenuates IL-12 activity.

[0069] In embodiments, the p35 and p40 subunits are located on the same polypeptide chain, optionally linked via a protease-cleavable linker. In such embodiments of a two-chain or multi-chain complex, the half-life extending element, the blocking element, or at least one component of the half-life extending element or blocking element is located on a separate polypeptide. For example, a first polypeptide may include p35 and p40, optionally linked via a cleavable polypeptide chain, with the other elements of the IL-12 polypeptide complex located on a second polypeptide chain. In another example, the first polypeptide chain includes a p35 subunit, a p40 subunit, a half-life extending element, and a portion of an antibody light chain. The second polypeptide includes a portion of an antibody heavy chain complementary to the antibody light chain. The portion of the antibody light chain associates with the complementary heavy chain in the complex to form a binding site for IL-12. In another example, the first polypeptide includes a p35 subunit, a p40 subunit, a half-life extending element, and a portion of an antibody heavy chain. In this example, the second polypeptide comprises a portion of an antibody light chain that is complementary to the antibody heavy chain. The portion of the antibody heavy chain associates with the complementary light chain in a complex to form a binding site for IL-12. In these complexes, the p35 and p40 subunits may be operably linked via an optional protease-cleavable linker. Preferably, the p35 and p40 subunits are operably linked by a non-cleavable linker.

[0070] In the complexes disclosed herein, the half-life extending element is preferably operably linked to either the p35 subunit or the p40 subunit via a protease-cleavable linker. For example, the complex may comprise a first polypeptide in which p35 or p40 is operably linked to a half-life extending element via a protease-cleavable linker. In another example, the complex may comprise a first polypeptide in which p35 or p40 is operably linked to a half-life extending element via a protease-cleavable linker, and the half-life extending element is further operably linked to a blocking element (or a component of a blocking element) optionally via a protease-cleavable linker. In such exemplary embodiments, the complex comprises at least one additional polypeptide comprising an IL-12 subunit (p40 or p35) that is not present in the first polypeptide. Additional arrangements of the elements of the complex are contemplated and encompassed by the present disclosure. For example, the blocking element can be operably linked to either the p35 subunit or the p40 subunit via a protease-cleavable linker. One of the half-life extending element or the blocking element can be operably linked to the p35 subunit, and the other of the half-life extending element or the blocking element can be operably linked to the p40 subunit. When the half-life extending element is operably linked to the p35 subunit, the blocking element can be operably linked to the p40 subunit. When the half-life extending element is operably linked to the p40 subunit, the blocking element can be operably linked to the p35 subunit. The blocking element in this complex is preferably Fab.

[0071] An inducible IL-12 polypeptide complex can comprise three polypeptide chains. Typically, one polypeptide chain contains either the p35 or p40 IL-12 subunit, but not both; a second polypeptide contains the other IL-12 subunit; and a third polypeptide contains at least a portion (component) of a blocking element. If the IL-12 subunit on the first polypeptide is p35, the IL-12 subunit on the second polypeptide is p40. If the IL-12 subunit on the first polypeptide is p40, the IL-12 subunit on the second polypeptide is p35. Upon polypeptide expression and folding, the p35 and p40 subunits can associate to form a biologically active heterodimer. The p35p40 heterodimer complex can be covalently linked, for example, via a disulfide bond.

[0072] In some embodiments, the first polypeptide, if present, may further comprise a half-life extending element operably linked to the IL-12 subunit via a protease-cleavable linker. The second polypeptide may further comprise a portion of the blocking element, and the third polypeptide may comprise the remainder of the blocking element. In such a complex, the IL-12 blocking element may be an antigen-binding fragment of an antibody, such as a Fab fragment, formed by the interaction of polypeptide 2 and polypeptide 3. In embodiments, the second polypeptide may comprise at least an antigen-binding portion of an antibody light chain. Alternatively, the second polypeptide may comprise at least an antigen-binding portion of an antibody heavy chain. The antigen-binding portion of the antibody light chain or the antigen-binding portion of the heavy chain may be operably linked to the IL-12 subunit via a protease-cleavable linker. In some embodiments, the second polypeptide may comprise a half-life extending element. If the second polypeptide comprises a half-life extending element, the first polypeptide does not comprise a half-life extending element. The half-life extending element may be operably linked to the IL-12 subunit via a protease-cleavable linker. Alternatively, or in addition, the half-life extending element may be operably linked to a portion of the blocking element (e.g., the antigen-binding portion of the antibody light chain or the antigen-binding portion of the antibody heavy chain) via an optional protease-cleavable linker. When a half-life extending element is present and operably linked to an IL-12 subunit, the antibody heavy or light chain may be operably linked to the IL-12 subunit via a protease-cleavable linker. Alternatively, when a half-life extending element is present and operably linked to an IL-12 subunit, the antibody heavy or light chain may be operably linked to the IL-12 subunit via an optional cleavable linker. The protease-cleavable linkers on the first, second, and / or polypeptide chains may be the same or different.

[0073] In some embodiments, an IL-12 polypeptide complex comprises a first polypeptide chain comprising an amino acid sequence selected from SEQ ID NOs: 95-110, 119-126, and 135-143. Particularly preferred IL-12 polypeptide complexes comprise the amino acid sequence of SEQ ID NO: 104 or SEQ ID NO: 136. In some embodiments, an IL-12 polypeptide complex comprises a first polypeptide sequence comprising an amino acid sequence selected from SEQ ID NOs: 119-126 and 135-143, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 18. A preferred IL-12 polypeptide complex comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 104 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18. Another preferred IL-12 polypeptide comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 136 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18.

[0074] In some embodiments, the first polypeptide chain of the IL-12 polypeptide complex comprises 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 98%, or at least about 99% identical to an amino acid sequence selected from SEQ ID NOs: 95-110, 119-126, and 135-143. In some embodiments, the second polypeptide chain of the IL-12 polypeptide complex comprises 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 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 18.

[0075] As described above, IL-12 can be a mutein, if desired. An IL-12 mutein retains IL-12 activity, e.g., intrinsic IL-12 receptor agonist activity. The IL-12 subunits, p35 and / or p40, can be muteins. Preferably, the IL-12 mutein has an altered glycosylation pattern. For example, the IL-12 mutein can be partially aglycosylated or fully aglycosylated. For example, a partially or fully aglycosylated IL-12 polypeptide can comprise a polypeptide selected from the group consisting of SEQ ID NOs: 104, 434, or 442-445, or an amino acid sequence having at least 80% identity to SEQ ID NOs: 104, 434, or 442-445.

[0076] The p35 and / or p40 subunits can contain one or more amino acid modifications, e.g., substitutions. For example, the p35 and / or p40 subunits can contain about one, about two, about three, about four, about five, or more amino acid substitutions. Typically, the p35 and / or p40 subunits contain one or two amino acid substitutions. The substitutions can be conservative or non-conservative, but are preferably conservative. A common modification alters the glycosylation pattern of the p35 and / or p40 subunits, thereby rendering the p35 and / or p40 subunits partially or fully aglycosylated. Preferably, the amino acid modification includes the substitution of an asparagine amino acid, e.g., asparagine to glutamine. In a particular example, the asparagine at amino acid position 16, 75, 85, 133, 151, 158, 201, 206, 221, 250, 267, 280, 282, 326, 400, 404, 425, 555, 572, 575, 582, or 602 of IL-12 p35 of SEQ ID NO: 434 can be mutated. In a particular example, the asparagine at amino acid position 103, 114, 163, 219, 227, or 282 of IL-12 p40 of SEQ ID NO: 18 can be mutated.

[0077] The present invention also relates to specific single-chain IL-12-inducing polypeptides. The single-chain IL-12 polypeptides disclosed herein comprise IL-12, a blocking element, a half-life extending element, and a protease-cleavable linker. IL-12 has receptor agonist activity against its cognate IL-12 receptor. When the blocking element binds to IL-12, IL-12 receptor activation activity is attenuated. Cleavage of the protease-cleavable linker releases the active IL-12 polypeptide. Single-chain inducible IL-12 polypeptides are disclosed in International Application Nos. PCT / US2019 / 032320 and PCT / US2019 / 032322.

[0078] The single-chain IL-12-inducing polypeptides disclosed herein comprise an amino acid sequence selected from SEQ ID NOs: 7, 9, 10, 18, 24-94, 110-118, and 127-134. In some embodiments, the single-chain IL-12-inducing polypeptides comprise a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, 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: 7, 9, 10, 18, 24-94, 110-118, and 127-134.

[0079] B. IL-23 Polypeptide Complex The present disclosure relates to inducible IL-23 polypeptide complexes that contain at least two polypeptide chains, and can optionally contain three or more polypeptide chains. The two or more polypeptide chains disclosed herein can be different, i.e., the complexes can be heterodimers, heterotrimers, etc. The inducible IL-23 polypeptide complexes contain a p19 IL-23 subunit, a p40 IL-23 subunit, a half-life-extending element, an IL-23 blocking element, and a protease-cleavable linker. The p19 and p40 subunits associate to form an IL-23 heterodimer, which possesses intrinsic IL-23 receptor agonist activity. As will be appreciated by those skilled in the art, IL-23 and IL-12 share the same p40 subunit. The IL-23 polypeptide complexes exhibit attenuated IL-23 receptor agonist activity and prolonged circulating half-life. The IL-23 receptor agonist activity is attenuated by the blocking element. Half-life extending elements may also contribute to attenuation, for example, through steric effects. A blocking element can block all or part of the receptor agonist activity of IL-23 by sterically blocking and / or by non-covalently binding to IL-23 (e.g., p19, p40, or p19p40 complex). Cleavage of the protease-cleavable linker liberates an active (e.g., more active) form of IL-23 from the IL-23 polypeptide complex. Typically, the free IL-23 is at least 10-fold more active than the IL-23 polypeptide complex. Preferably, the free IL-23 is at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 500-fold, at least 1000-fold, or at least about 10,000-fold more active than the IL-23 polypeptide complex.

[0080] The form of IL-23 released upon cleavage of the fusion protein typically has a short half-life, often substantially similar to that of naturally occurring IL-23, further restricting IL-23 cytokine activity to the tumor microenvironment. Although the half-life of the IL-23 polypeptide complex is extended, toxicity is reduced or eliminated because circulating IL-23 polypeptide complexes are attenuated and active IL-23 is targeted to the desired site (e.g., the tumor microenvironment).

[0081] Those skilled in the art will understand that the number of polypeptide chains and the location of the p19 and p40 subunits, half-life extending elements, protease-cleavable linker(s), and blocking elements (and components of such elements, e.g., VH or VL domains) on the polypeptide chains can vary and are often a matter of design preference. All such variations are encompassed by the present disclosure.

[0082] In embodiments, an IL-23 polypeptide complex comprises two distinct polypeptide chains. Typically, a first polypeptide chain comprises p19 and a second polypeptide chain comprises p40. The p19 and p40 subunits combine to form a biologically active heterodimer. The p19p40 heterodimer complex can be covalently linked, for example, via a disulfide bond.

[0083] In embodiments, either the first or second polypeptide may comprise an IL-23 blocking element (e.g., an scFV that binds IL-23) operably linked to an IL-23 subunit via a protease-cleavable linker. The other polypeptide chain may further comprise a half-life extending element operably linked to an IL-23 subunit via a protease-cleavable linker. Preferably, the complex comprises one functional blocking element and one functional half-life extending element. For example, if the first polypeptide chain comprises an IL-23 blocking element, the second polypeptide chain does not comprise an IL-23 blocking element. In other embodiments, one polypeptide chain comprises either p19 or p40 and further comprises a half-life extending element and a blocking element, each of which is operably linked to p19 or p40 via a protease-cleavable linker (e.g., one or more protease-cleavable linkers), and the other polypeptide comprises the complementary IL-23 subunit (e.g., either p40 or p19). The IL-23 blocking element on the second polypeptide can be operably linked to the IL-23 subunit via a protease-cleavable linker. Alternatively, the IL-23 blocking element can be operably linked to the half-life extending element via an optional protease-cleavable linker. The protease-cleavable linkers on the first and second polypeptide chains can be the same or different. Preferably, the protease-cleavable linkers on the first and second polypeptide chains are the same. The blocking element in this IL-23 polypeptide complex can be a single-chain antibody. Any single-chain antibody having binding specificity for IL-23 can be the blocking element. Preferably, the blocking element is an scFv.

[0084] The conjugates disclosed herein preferably contain one half-life extending element and one blocking element, although such elements may contain two or more components present on the same or different polypeptide chains. As shown, and as disclosed and exemplified herein, the components of a blocking element can be present on separate polypeptide chains. For example, a first polypeptide chain can contain an antibody light chain (VL+CL) or a light chain variable domain (VL), and a second polypeptide chain can contain an antibody heavy chain Fab fragment (VH+CH1) or a heavy chain variable domain (VH) that is complementary to the VL+CL or VL on the first polypeptide. In such a situation, these components can associate in the peptide conjugate to form an antigen-binding site, such as a Fab, that binds to IL-23 and attenuates IL-23 activity.

[0085] In embodiments, the p19 and p40 subunits are located on the same polypeptide chain, optionally linked via a protease-cleavable linker. In such embodiments of a two-chain or multi-chain complex, the half-life extending element, the blocking element, or at least one component of the half-life extending element or blocking element is located on a separate polypeptide. For example, a first polypeptide may include p19 and p40, optionally linked via a cleavable polypeptide chain, with the other elements of the IL-23 polypeptide complex located on a second polypeptide chain. In another example, the first polypeptide chain includes a p19 subunit, a p40 subunit, a half-life extending element, and a portion of an antibody light chain. The second polypeptide includes a portion of an antibody heavy chain complementary to the antibody light chain. The portion of the antibody light chain associates with the complementary heavy chain in the complex to form a binding site for IL-23. In another example, the first polypeptide includes a p19 subunit, a p40 subunit, a half-life extending element, and a portion of an antibody heavy chain. In this example, the second polypeptide comprises a portion of an antibody light chain that is complementary to the antibody heavy chain. The portion of the antibody heavy chain associates with the complementary light chain in a complex to form a binding site for IL-23. In these complexes, the p19 and p40 subunits may be operably linked via an optional protease-cleavable linker. Preferably, the p19 and p40 subunits are operably linked by a non-cleavable linker.

[0086] In the complexes disclosed herein, the half-life extending element is preferably operably linked to either the p19 or p40 subunit via a protease-cleavable linker. For example, the complex may comprise a first polypeptide in which p19 or p40 is operably linked to a half-life extending element via a protease-cleavable linker. In another example, the complex may comprise a first polypeptide in which p19 or p40 is operably linked to a half-life extending element via a protease-cleavable linker, and the half-life extending element is further operably linked to a blocking element (or a component of a blocking element) optionally via a protease-cleavable linker. In such exemplary embodiments, the complex comprises at least one additional polypeptide comprising an IL-23 subunit (p40 or p19) that is not present in the first polypeptide. Additional arrangements of the elements of the complex are contemplated and encompassed by the present disclosure. For example, the blocking element can be operably linked to either the p19 or p40 subunit via a protease-cleavable linker. One of the half-life extending element or the blocking element can be operably linked to the p19 subunit, and the other of the half-life extending element or the blocking element can be operably linked to the p40 subunit. When the half-life extending element is operably linked to the p19 subunit, the blocking element can be operably linked to the p40 subunit. When the half-life extending element is operably linked to the p40 subunit, the blocking element can be operably linked to the p19 subunit. The blocking element in this complex is preferably Fab.

[0087] An inducible IL-23 polypeptide complex can comprise three polypeptide chains. Typically, one polypeptide chain contains either the p19 or p40 IL-23 subunit, but not both; a second polypeptide contains the other IL-23 subunit; and a third polypeptide contains at least a portion (component) of a blocking element. If the IL-23 subunit on the first polypeptide is p19, then the IL-23 subunit on the second polypeptide is p40. If the IL-23 subunit on the first polypeptide is p40, then the IL-23 subunit on the second polypeptide is p19. Upon polypeptide expression and folding, the p19 and p40 subunits can associate to form a biologically active heterodimer. The p19p40 heterodimer complex can be covalently linked, for example, via a disulfide bond.

[0088] In some embodiments, the first polypeptide, if present, may further comprise a half-life extending element operably linked to the IL-23 subunit via a protease-cleavable linker. The second polypeptide may further comprise a portion of the blocking element, and the third polypeptide may comprise the remainder of the blocking element. In such a complex, the IL-23 blocking element may be an antigen-binding fragment of an antibody, such as a Fab fragment, formed by the interaction of polypeptide 2 and polypeptide 3. In embodiments, the second polypeptide may comprise at least an antigen-binding portion of an antibody light chain. Alternatively, the second polypeptide may comprise at least an antigen-binding portion of an antibody heavy chain. The antigen-binding portion of the antibody light chain or the antigen-binding portion of the heavy chain may be operably linked to the IL-23 subunit via a protease-cleavable linker. In some embodiments, the second polypeptide may comprise a half-life extending element. If the second polypeptide comprises a half-life extending element, the first polypeptide does not comprise a half-life extending element. The half-life extending element may be operably linked to the IL-23 subunit via a protease-cleavable linker. Alternatively, or in addition, the half-life extending element may be operably linked to a portion of the blocking element (e.g., the antigen-binding portion of the antibody light chain or the antigen-binding portion of the heavy chain) via an optional protease-cleavable linker. When a half-life extending element is present and operably linked to an IL-23 subunit, the antibody heavy or light chain may be operably linked to the IL-23 subunit via a protease-cleavable linker. Alternatively, when a half-life extending element is present and operably linked to an IL-23 subunit, the antibody heavy or light chain may be operably linked to the IL-23 subunit via an optional cleavable linker. The protease-cleavable linkers on the first, second, and / or polypeptide chains may be the same or different.

[0089] In embodiments, the IL-23 polypeptide complex comprises a first polypeptide selected from the group consisting of SEQ ID NOs: 423-428, or an amino acid sequence having at least 80% identity to SEQ ID NOs: 423-428. In embodiments, the IL-23 polypeptide complex comprises a second polypeptide selected from the group consisting of SEQ ID NOs: 18 or 433.

[0090] In some embodiments, the first polypeptide chain of the IL-23 polypeptide complex comprises 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 98%, or at least about 99% identical to an amino acid sequence selected from SEQ ID NOs: 423-428. In some embodiments, the second polypeptide chain of the IL-23 polypeptide complex comprises 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 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 18 or 433.

[0091] As described above, IL-23 can be a mutein, if desired. IL-23 muteins retain IL-23 activity, e.g., intrinsic IL-23 receptor agonist activity. IL-23 subunits, p19 and / or p40, can be muteins. Preferably, IL-23 muteins have an altered glycosylation pattern. For example, IL-23 muteins can be partially aglycosylated or fully aglycosylated.

[0092] The p19 and / or p40 subunits can contain one or more amino acid modifications, e.g., substitutions. For example, the p19 and / or p40 subunits can contain about one, about two, about three, about four, about five, or more amino acid substitutions. Typically, the p19 and / or p40 subunits contain one or two amino acid substitutions. The substitutions can be conservative or non-conservative, but are preferably conservative. A common modification alters the glycosylation pattern of the p19 and / or p40 subunits, thereby rendering the p19 and / or p40 subunits partially or fully aglycosylated. Preferably, the amino acid modification includes a substitution of an asparagine amino acid, e.g., asparagine to glutamine. For example, asparagine to glutamine. In a specific example, the asparagine at amino acid position 47 or 66 on IL-12 p19 of SEQ ID NO: 424 can be mutated. In a particular example, the asparagine at amino acid position 103, 114, 163, 219, 227, or 282 of IL-12 p40 of SEQ ID NO: 18 can be mutated.

[0093] The present invention also relates to specific single-chain IL-23-inducing polypeptides. The single-chain IL-23 polypeptides disclosed herein comprise IL-23, a blocking element, a half-life extending element, and a protease-cleavable linker. IL-23 has receptor agonist activity against its cognate IL-23 receptor. Binding of the blocking element to IL-23 attenuates IL-23 receptor activation activity. Cleavage of the protease-cleavable linker liberates the active IL-23 polypeptide.

[0094] The single-chain IL-23-inducing polypeptides disclosed herein comprise an amino acid sequence selected from SEQ ID NOs: 422 or 429-432. In some embodiments, the single-chain IL-23-inducing polypeptide comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, 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: 422 or 429-432.

[0095] C. Half-life extending elements Contemplated herein are domains that extend the half-life of IL-12 polypeptide complexes. Also contemplated herein are domains that extend the half-life of IL-23 polypeptides. Increasing the in vivo half-life of therapeutic molecules that naturally have short half-lives allows for more tolerable and manageable dosing regimens without sacrificing efficacy.

[0096] Half-life extending elements increase the in vivo half-life and provide modified pharmacodynamics and pharmacokinetics of IL-12 or IL-23 polypeptide complexes. Without being bound by theory, half-life extending elements alter the pharmacodynamic properties of IL-12 or IL-23 polypeptide complexes, including altering tissue distribution, penetration, and diffusion. In some embodiments, half-life extending elements can improve tissue targeting, tissue penetration, and diffusion within tissues, resulting in enhanced efficacy, compared to proteins lacking a half-life extending element. Without being bound by theory, an exemplary method for improving the pharmacokinetics of polypeptides is through the expression of elements in the polypeptide chain that bind to receptors that recycle to the plasma membrane of cells rather than being degraded in lysosomes, such as the FcRn receptor and transferrin receptor on endothelial cells. Three proteins, such as human IgG, HSA (or fragments), and transferrin, persist in human serum much longer than would be predicted by their size alone, a function of their ability to bind to receptors that recycle rather than being degraded in lysosomes. These proteins or fragments retain FcRn binding and can be linked to other polypeptides in a routine manner to extend their serum half-life. HSA can also be directly linked to pharmaceutical compositions or linked via a short linker. Fragments of HSA can also be used. HSA and its fragments can function as both blocking elements and half-life extension elements. Human IgG and Fc fragments can also perform similar functions.

[0097] The serum half-life extending element can also be an antigen-binding polypeptide that binds to proteins with long serum half-lives, such as serum albumin or transferrin. Examples of such polypeptides include polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies, such as antibodies and fragments thereof, including heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid nanobodies (VHH), and dAbs. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, 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 ligands for a desired receptor, ligand-binding portions of a receptor, lectins, and peptides that bind to or associate with one or more target antigens.

[0098] The half-life extending elements provided herein are preferably human serum albumin (HSA) binding domains, and antigen-binding polypeptides that bind to human serum albumin, or immunoglobulin Fc or fragments thereof.

[0099] The half-life extending element of an IL-12 or IL-23 polypeptide complex extends the half-life of the IL-12 or IL-23 polypeptide complex by at least about 2 days, about 3 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days or more. In some embodiments, the half-life extending element extends the half-life of the IL-12 or IL-23 polypeptide complex by at least 2-3 days, 3-4 days, 4-5 days, 5-6 days, 6-7 days, 7-8 days or more.

[0100] D. Cut-off element A blocking element can be any element that binds to IL-12 or IL-23 and inhibits the ability of an IL-12 polypeptide complex or an IL-23 polypeptide complex to bind to and activate its receptor. A blocking element can inhibit the ability of IL-12 or IL-23 to bind to and / or activate its receptor, for example, by sterically blocking and / or by covalently binding to the IL-12 polypeptide complex. The blocking elements disclosed herein can bind to p19, p35, p40, a p35p40 heterodimer complex, or a p19p40 heterodimer complex.

[0101] Examples of suitable blocking elements include the full-length or IL-12-binding fragments or muteins of the cognate receptor for IL-12. Other examples of suitable blocking elements include the full-length or IL-23-binding fragments or muteins of the cognate receptor for IL-23. Polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. Antibodies and antibody-binding fragments that bind IL-12 or IL-23 can also be used, including single-chain variable fragments (scFv), single-domain antibodies, e.g., heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid nanobodies (VHH), dAbs, etc. Other suitable antigen-binding domains that bind to IL-12 or IL-23 can also be used, including non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, 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 the binding of IL-12 or IL-23 to their cognate receptors. Advantageously, such moieties also function as half-life-extending elements. For example, peptides modified by conjugation to water-soluble polymers such as PEG can sterically inhibit or prevent the binding of cytokines to their receptors. Polypeptides or fragments thereof with a long serum half-life, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin, and the like, as well as fragments and muteins of such polypeptides, can also be used.

[0102] A preferred IL-12 blocking element is a single chain variable fragment (scFv) or a Fab fragment. A preferred IL-23 blocking element is a single chain variable fragment (scFv) or a Fab fragment. An scFv blocking element comprises the amino acid sequence set forth in SEQ ID NOs: 145-188. Alternatively, an Fab blocking element comprises the amino acid sequence set forth in SEQ ID NOs: 189-194. The IL-12 antibody fragments encompassed by SEQ ID NOs: 145-194 have been optimized to enhance the developability of the IL-12 polypeptide complexes disclosed herein.

[0103] Preferred antibody light chain blocking elements include SEQ ID NOs: 192-193. These preferred components can be located on one polypeptide chain, with the complementary antigen-binding portion of the heavy chain located on a second polypeptide chain. Preferred heavy chain blocking elements include SEQ ID NOs: 189-191 and 194. These preferred components can be located on one polypeptide chain, with the complementary light chain located on a second polypeptide chain. The antibody light chain and antibody heavy chain together form the binding site for IL-12.

[0104] In some embodiments, the IL-12 blocking element comprises 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 SEQ ID NOs: 145-194, e.g., over the entire length of SEQ ID NOs: 145-194. Generally, the amino acid sequences of the CDRs are unaltered, with amino acid substitutions occurring in the framework regions.

[0105] The present disclosure also relates to functional variants of IL-12 blocking elements comprising SEQ ID NOs: 145-194. Functional variants of IL-12 blocking elements comprising SEQ ID NOs: 145-194 generally differ from SEQ ID NOs: 145-194 by one or several amino acids (including substitutions, deletions, insertions, or any combination thereof) and retain the ability to substantially bind to an IL-12 polypeptide (e.g., the p35 subunit, the p40 subunit, or the p35p40 complex) and inhibit IL-12 from binding to its cognate receptor.

[0106] Functional variants may contain at least one or more amino acid substitutions, deletions, or insertions compared to IL-12 blocking elements comprising SEQ ID NOs: 145-194. Functional variants may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to IL-12 blocking elements comprising SEQ ID NOs: 145-194. In some preferred embodiments, functional variants differ from IL-12 blocking elements comprising SEQ ID NOs: 145-194 by fewer than 10, fewer than 8, fewer than 5, fewer than 4, fewer than 3, fewer than 2, or one amino acid change, e.g., an amino acid substitution or deletion. In other embodiments, functional variants may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NOs: 145-194. Amino acid substitutions may be conservative or non-conservative, but are preferably conservative.

[0107] In other embodiments, functional variants of the IL-12 blocking element may contain one, two, three, four, five, or more non-conservative amino acid substitutions compared to the IL-12 blocking element comprising SEQ ID NOs: 145-194. Non-conservative amino acid substitutions are recognizable by those skilled in the art. Functional variants of the separation portion preferably contain no more than one, two, three, four, or five amino acid deletions.

[0108] Also disclosed herein are inducible IL-12 polypeptides that comprise a blocking element with specificity for IL-12 and that comprise a half-life extending element. Also disclosed herein are inducible IL-12 polypeptides that comprise a blocking element with specificity for IL-23 and that comprise a half-life extending element. The blocking element is an antibody or antigen-binding fragment that has binding specificity for IL-12, particularly the IL-12 subunit beta precursor (p40) defined by SEQ ID NO:421 as disclosed herein. The antibody or antigen-binding fragment comprises an antigen-binding domain that binds to residues set forth in Table 1 of SEQ ID NO:421. The present disclosure relates to antibodies or antigen-binding fragments that bind to an IL-12 epitope defined by the amino acid residues set forth in Table 1, inducible IL-12 polypeptide complexes comprising such antibodies or antigen-binding fragments, and the use of such antibodies or antigen-binding fragments to prepare inducible IL-12 polypeptide complexes or medicaments comprising such inducible IL-12 polypeptide complexes. [Table 1]

[0109] E. Protease-Cleavable Linkers As disclosed herein, an IL-12 or IL-23 polypeptide complex comprises one or more linker sequences. The linker sequence serves to provide flexibility between the polypeptides, allowing, for example, a blocking element to inhibit the activity of IL-12 or IL-23. The linker can be disposed between the IL-12 or IL-23 subunit, the half-life extending element, and / or the blocking element. As described herein, an IL-12 polypeptide complex comprises a protease-cleavable linker. As described herein, an IL-23 polypeptide complex 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 proteases are concentrated or selectively expressed in the desired target site of IL-12 or IL-23 activity (e.g., the tumor microenvironment). Thus, the IL-12 or IL-23 polypeptide complex is preferentially or selectively cleaved at the target site of the desired IL-12 or IL-23 activity.

[0110] Suitable linkers are generally less than about 100 amino acids. Such linkers can vary in length, such as from 1 amino acid (e.g., Gly) to 30 amino acids, 1 to 40 amino acids, 1 to 50 amino acids, 1 to 60 amino acids, 1 to 70 amino acids, 1 to 80 amino acids, 1 to 90 amino acids, and 1 to 100 amino acids. In some embodiments, the linker is at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. Preferred linkers are generally from about 5 to about 30 amino acids.

[0111] Preferably, the length of the linker can vary between 2 and 30 amino acids, optimized for each condition, so that the linker does not impose any constraints on the conformation or interaction of the linking domains. In a preferred embodiment, the linker is cleavable by a cleavage agent, such as an enzyme. Preferably, the separation portion includes a protease cleavage site. In some cases, the separation portion includes one or more cleavage sites. The separation portion may include a single protease cleavage site. The separation portion may also include two or more protease cleavage sites. For example, two cleavage sites, three cleavage sites, four cleavage sites, five cleavage sites, or more. When the separation portion includes two or more protease cleavage sites, the cleavage sites may be cleaved by the same protease or different proteases. A separation portion including two or more cleavage sites is called 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 followed by another cleavage site with an intervening amino acid between the two cleavage sites.

[0112] Particularly interesting in the present invention is disease-specific protease-cleavable linker.Also preferred is protease-cleavable linker that is preferentially cleaved in desired location in the body, such as tumor microenvironment, compared with peripheral circulation.For example, the cleavage rate of protease-cleavable linker in 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 desired location in the body, such as tumor microenvironment, compared with peripheral circulation (e.g., in plasma).

[0113] Proteases known to be associated with affected cells or tissues include serine proteases, cysteine ​​proteases, aspartic acid proteases, threonine proteases, glutamic acid proteases, metalloproteases, aspartic peptide lyases, serum proteases, cathepsin, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, cathepsin L, kallikrein, hKl, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidain, bromelain, calpain, caspases, and caspase- 3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAPα), dipeptidyl peptidase, meprin, granzyme, and dipeptidyl peptidase IV (DPPIV / CD26). The protease capable of cleaving the linker amino acid sequence (which may be encoded by the chimeric nucleic acid sequence provided herein) may be selected from the group consisting of, for example, prostate-specific antigen (PSA), matrix metalloproteinase (MMP), A distigrin and metalloproteinase (ADAM), plasminogen activator, cathepsin, caspase, tumor cell surface protease, and elastase. The MMP may be, for example, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), or matrix metalloproteinase 14 (MMP14).Additionally or alternatively, the linker may be cleaved by a cathepsin, such as cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, and / or cathepsin L. Preferably, the linker is cleavable by MMP14 or cathepsin L.

[0114] Proteases useful for cleaving linkers and for use in the IL-12 polypeptide complexes disclosed herein are listed in Table 2, and exemplary proteases and their cleavage sites are listed in Table 3. [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2]

[0115] 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, calpain-cleavable linkers, matrix metalloproteinase (MMP)-cleavable linkers, plasminogen activator-cleavable linkers, caspase-cleavable linkers, tryptase-cleavable linkers, or tumor cell surface proteases. In particular, MMP9-cleavable linkers, ADAM-cleavable linkers, CTSL1-cleavable linkers, FAPα-cleavable linkers, and cathepsin-cleavable linkers. Some preferred protease-cleavable linkers are cleaved by MMPs and / or cathepsins.

[0116] Separating moieties disclosed herein are generally less than 100 amino acids. Such separating moieties can vary in length, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 to 40 amino acids, from 1 to 50 amino acids, from 1 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 generally from about 5 to about 30 amino acids.

[0117] Preferably, the length of the linker may vary between 2 and 30 amino acids, optimized for each condition, so that the linker does not impose any constraints on the conformation or interactions of the linking domains.

[0118] In some embodiments, the separating portion has 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); KG GGPAGLYAQGPAGLYAQGPAGLYAQGSR (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); RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (SEQ ID NO: 212); GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (SEQ ID NO: 213); GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (SEQ ID NO: 214); RGGALFKSSFPLAQKLKS SGPAGLYAQGGK (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).

[0119] Certain preferred separating portions comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). The separating portions disclosed herein may comprise one or more cleavage motifs or functional variants, which may be the same or different. A separating portion may comprise one, two, three, four, five, or more cleavage motifs or functional variants. A separating portion comprising 30 amino acids may comprise two cleavage motifs or functional variants, three cleavage motifs or functional variants, or more. A "functional variant" of a separating portion retains the ability to be cleaved with high efficiency at the target site (e.g., a tumor microenvironment expressing high levels of proteases) and is not cleaved or is cleaved with low efficiency in the periphery (e.g., serum). For example, a functional variant retains at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a separation portion comprising any one of SEQ ID NOs: 195-220 or 447-448.

[0120] A separation portion comprising multiple cleavage motifs may be selected from SEQ ID NOs: 195-201 or 447-448 and combinations thereof. Preferred separation portions comprising multiple cleavage motifs comprise amino acids selected from SEQ ID NOs: 202-220.

[0121] The separating portion may comprise both ALFKSSFP (SEQ ID NO: 198) and GPAGLYAQ (SEQ ID NO: 195). The separating portion may comprise two cleavage motifs, each having the sequence GPAGLYAQ (SEQ ID NO: 195). Alternatively, or in addition, the separating portion may comprise two cleavage motifs, each having the sequence ALFKSSFP (SEQ ID NO: 198). The separating portion may comprise a third cleavage motif, which may be the same or different.

[0122] In some embodiments, the separated portion 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 NO:195-220 or 447-448 over the entire length of SEQ ID NO:195-220 or SEQ ID NO:447-448.

[0123] The present disclosure also relates to functional variants of isolated portions comprising SEQ ID NOs: 195-220 or 447-448. Functional variants of isolated portions comprising SEQ ID NOs: 195-220 or 447-448 generally differ from SEQ ID NOs: 195-220 or 447-448 by one or several amino acids (including substitutions, deletions, insertions, or any combination thereof) and substantially retain the ability to be cleaved by a protease.

[0124] A functional variant may contain at least one or more amino acid substitutions, deletions, or insertions relative to the discrete portion comprising SEQ ID NOs: 195-220 or 447-448. A functional variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications subdivided into discrete portions comprising SEQ ID NOs: 195-220 or 447-448. In some preferred embodiments, a functional variant differs from a discrete portion comprising SEQ ID NOs: 195-220 by fewer than 10, fewer than 8, fewer than 5, fewer than 4, fewer than 3, fewer than 2, or even one amino acid change, e.g., an amino acid substitution or deletion. In other embodiments, a functional variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to SEQ ID NOs: 195-220 or 447-448. The amino acid substitutions may be conservative or non-conservative, but are preferably conservative.

[0125] In other embodiments, functional variants of the separated portion may contain one, two, three, four, five, or more non-conservative amino acid substitutions compared to the separated portion comprising SEQ ID NOs: 195-220 or 447-448. Non-conservative amino acid substitutions can be recognized by those skilled in the art. Functional variants of the separated portion preferably contain no more than one, two, three, four, or five amino acid deletions.

[0126] The amino acid sequences disclosed in the separating portion can be described by the relative linear position of the separating portion with respect to the scissile bond. As will be appreciated by those skilled in the art, a separating portion containing an eight amino acid protease substrate (e.g., SEQ ID NOS: 195-201 or 447-448) is at positions P4, P3, P2, P1, P1', P2', P3', P4', with the scissile bond between P1 and P1'. For example, the amino acid positions of a separating portion containing the sequence GPAGLYAQ (SEQ ID NO: 195) can be described as follows: [Table 4]

[0127] The amino acid positions of the detached portion comprising the sequence ALFKSSFP (SEQ ID NO: 198) can be written as follows: [Table 5]

[0128] Preferably, the amino acids surrounding the cleavage site (eg, P1 and P1' positions of SEQ ID NOs: 195-201 or 447-448) are not substituted.

[0129] In embodiments, the separated portion comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), or a functional variant of SEQ ID NO: 195 or a functional variant of SEQ ID NO: 198. As described herein, functional variants of PAGLYAQ (SEQ ID NO: 447) or ALFKSSFP (SEQ ID NO: 198) may contain one or more amino acid substitutions and substantially retain the ability to be cleaved by a protease. Specifically, functional variants of GPAGLYAQ (SEQ ID NO: 195) are cleaved by MMP14, and functional variants of ALFKSSFP (SEQ ID NO: 198) are cleaved by capthepsin L (CTSL1). The functional variants also retain the ability to be cleaved with high efficiency at target sites (e.g., tumor microenvironments expressing high levels of proteases). For example, a functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retains at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a separation portion comprising the amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.

[0130] Preferably, a functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) contains 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 positions P1 and P1' are not substituted. The amino acids at positions P1 and P1' in SEQ ID NO: 195 are G and L, and the amino acids at positions P1 and P1' in SEQ ID NO: 198 are K and S.

[0131] Functional variants of GPAGLYAQ (SEQ ID NO: 195) may preferably include 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) an asparagine amino acid substitution at position P2; d) a histidine, asparagine, or glycine amino acid substitution at position P1; e) an 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) a serine, glutamine, or lysine amino acid substitution at position P4'. The following amino acid substitutions are not preferred 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 P4'. In some embodiments, functional variants of GPAGLYAQ (SEQ ID NO: 195) do not include amino acid substitutions at positions P1 and / or P1'.

[0132] The amino acid substitutions of functional variants of GPAGLYAQ (SEQ ID NO: 195) preferably include amino acid substitutions at positions P4 and / or P4'. For example, a functional variant of GPAGLYAQ (SEQ ID NO: 195) may include a leucine at position P4, or a serine, glutamine, lysine, or phenylalanine at position P4. Alternatively, or in addition, a functional variant of GPAGLYAQ (SEQ ID NO: 195) may include a glycine, phenylalanine, or proline at position P4'.

[0133] In some embodiments, amino acid substitutions at positions P2 or P2' of GPAGLYAQ (SEQ ID NO: 195) are not preferred.

[0134] In some embodiments, a functional variant of GPAGLYAQ (SEQ ID NO: 195) comprises an amino acid sequence selected from SEQ ID NOs: 221-295. Particular functional variants of GPAGLYAQ (SEQ ID NO: 195) include GPLGLYAQ (SEQ ID NO: 259) and GPAGLKGA (SEQ ID NO: 249).

[0135] Functional variants of LFKSSFP (SEQ ID NO: 448) preferably contain hydrophobic amino acid substitutions. Functional variants of LFKSSFP (SEQ ID NO: 448) may preferably contain one or more of the following: (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at position P4; (b) lysine, histidine, glycine, proline, asparagine, or phenylalanine at position P3; (c) arginine, leucine, alanine, glutamine, or histatine at position P2; (d) phenylalanine, histidine, threonine, alanine, or glutamine at position P1; (e) P1' (f) histidine, leucine, lysine, alanine, isoleucine, arginine, phenylalanine, asparagine, glutamic acid, or glycine at position P2'; (g) phenylalanine, leucine, isoleucine, lysine, alanine, glutamine, or proline at position P3'; and phenylalanine, histidine, glycine, alanine, serine, valine, glutamine, lysine, or leucine.

[0136] Inclusion of aspartic acid and / or glutamic acid in functional variants of SEQ ID NO: 448 is generally not preferred and is avoided. The following amino acid substitutions are also not preferred 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'.

[0137] Amino acid substitutions in functional variants of LFKSSFP (SEQ ID NO: 448) preferably include amino acid substitutions at positions P4 and / or P1. In some embodiments, amino acid substitutions at position P4' in functional variants of LFKSSFP (SEQ ID NO: 448) are not preferred.

[0138] In some embodiments, a functional variant of LFKSSFP (SEQ ID NO:448) comprises an amino acid sequence selected from SEQ ID NOs: 296 to 374. Particular 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).

[0139] The detached moieties disclosed herein can form stable complexes with the amino acid sequences (e.g., domains) to which they are linked under physiological conditions, but can be cleaved by proteases. For example, the detached moieties can be stable in circulation (e.g., not cleaved or cleaved with low efficiency) and cleaved with higher efficiency at the target site (i.e., tumor microenvironment). Thus, a fusion polypeptide comprising a linker disclosed herein can, if desired, have an extended circulating half-life and / or reduced biological activity in circulation compared to the components of the fusion polypeptide as separate molecular entities. However, when in the desired location (e.g., tumor microenvironment), the linker can be efficiently cleaved to release the components connected by the linker, restoring or nearly restoring the half-life and biological activity of the components as separate molecular entities.

[0140] The separated portion desirably remains stable in circulation for at least 2 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or longer.

[0141] In some embodiments, the separation moiety is cleaved in circulation at less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% of the target site. The separation moiety is also stable in the absence of an enzyme that can cleave the linker. However, when exposed to a suitable enzyme (i.e., a protease), the separation moiety is cleaved, separating the linked domains.

[0142] F. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions comprising the IL-12 or IL-23 polypeptide complexes described herein, vectors comprising a polynucleotide encoding the IL-12 or IL-23 polypeptide complexes, or host cells transformed with the vectors, and at least one pharmaceutically acceptable carrier.

[0143] Provided herein are pharmaceutical formulations or compositions comprising an IL-12 polypeptide complex or an IL-23 polypeptide complex described herein and a pharmaceutically acceptable carrier. The compositions comprising the IL-12 polypeptide complex or the IL-23 polypeptide complex described herein are suitable for in vitro or in vivo administration. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the components and is not toxic to the subject to which it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and administered to a subject in an appropriate dosage. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservatives, emulsifying agents, and dispersing agents. Prevention of microbial attack can be ensured by including various antibacterial and antifungal agents.

[0144] 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). Generally, an appropriate amount of a pharmacologically acceptable salt is used in the formulation to render the formulation isotonic; however, if desired, the formulation can be hypertonic or hypotonic. Examples of pharmacologically acceptable carriers include, but are not limited to, sterile water, physiological saline, buffers such as Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or about 7 to 7.5. Other carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptide. The matrices are in the form of shaped articles, such as films, liposomes, or microparticles. Depending, for example, on the route of administration and the concentration of the composition being administered, certain carriers may be more preferable. The carrier is one suitable for administration of an IL-12 or IL-23 polypeptide complex or a nucleic acid sequence encoding an IL-12 or IL-23 polypeptide complex to a human or other subject.

[0145] In some embodiments of the pharmaceutical composition, the IL-12 or IL-23 polypeptide complex described herein is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a fullerene, a liquid crystal, a liposome, a quantum dot, a superparamagnetic nanoparticle, a dendrimer, or a nanorod. In other embodiments of the pharmaceutical composition, the IL-12 or IL-23 polypeptide complex is bound to a liposome. In some examples, the IL-12 or IL-23 polypeptide complex is complexed to the surface of a liposome. In some examples, the IL-12 or IL-23 polypeptide complex is encapsulated within the shell of a liposome. In some examples, the liposome is a cationic liposome.

[0146] The IL-12 polypeptide complexes or IL-23 polypeptide complexes described herein are intended for use as pharmaceuticals. Administration can be accomplished by a variety of methods, including intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In some embodiments, the route of administration depends on the type of treatment and the type of compound included in the pharmaceutical composition. The administration regimen will be determined by the attending physician and other clinical factors. The dose for a single patient will vary depending on many factors, including the patient's size, body surface area, age, sex, the specific compound being administered, the time and route of administration, the type of treatment, general health, and other drugs being administered concomitantly. An "effective dose" refers to the amount of active ingredient sufficient to affect the course and severity of the disease, resulting in the reduction or remission of such pathology, and can be determined using known methods.

[0147] Optionally, the nucleic acid sequence encoding the IL-12 polypeptide complex or the IL-12 polypeptide is administered via a vector. Optionally, the nucleic acid sequence encoding the IL-23 polypeptide complex or the IL-23 polypeptide is administered via a vector. Numerous compositions and methods exist that can be used to deliver nucleic acid molecules and / or polypeptides to cells, either in vitro or in vivo, e.g., via expression vectors. These methods and compositions can be broadly divided into two categories: viral-based delivery systems and non-viral-based delivery systems. Such methods are well known in the art and can be readily adapted 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, e.g., to produce cell lines that express, and preferably secrete, the encoded chimeric polypeptide, or to therapeutically deliver nucleic acids to a subject. The components of the IL-12 or IL-23 polypeptide disclosed herein are typically operably linked in-frame to encode a fusion protein.

[0148] As used herein, a plasmid or viral vector is a substance that transports the disclosed nucleic acid to a cell without degradation and contains a promoter that drives expression of the nucleic acid molecule and / or polypeptide in the cell. Viral vectors include, for example, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, Sindbis, and other RNA viruses, including those with an HIV backbone. Also preferred are any virus families that share the properties of these viruses and are suitable for use as vectors. Retroviral vectors, reviewed and described for their production, are described in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997). The construction of replication-deficient 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 advantage and use of these viruses as vectors is that they can replicate within the initially infected cell but are unable to form new infectious viral particles, limiting the extent to which they can spread to other cell types. Recombinant adenoviruses have been shown to achieve high efficiency after direct in vivo delivery to respiratory epithelia, hepatocytes, vascular endothelium, CNS parenchyma, and many other tissue sites. Other useful systems include, for example, replicating vaccinia virus vectors and host-restricted non-replicating vaccinia virus vectors.

[0149] The provided IL-12 polypeptide complexes and / or nucleic acid molecules can be delivered via virus-like particles. The provided IL-23 polypeptide complexes and / or nucleic acid molecules can be delivered via virus-like particles. Virus-like particles (VLPs) consist of viral protein(s) derived from viral structural proteins. Methods for producing and using virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0150] The IL-12 or IL-23 polypeptide complexes disclosed herein can be delivered by subviral dense bodies (DBs). DBs transport proteins to target cells by membrane fusion. Methods for producing and using DBs are described, for example, in Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003). The provided polypeptides can be delivered by exoskeleton aggregates. Methods for producing and using exoskeleton aggregates are described in International Publication No. WO 2006 / 110728.

[0151] Non-viral delivery methods can include expression vectors containing nucleic acid sequences encoding nucleic acid molecules and polypeptides, where the nucleic acid is operably linked to an expression control sequence. Suitable vector backbones include those routinely used in the art, such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from companies such 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, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to produce IL-12 or IL-23 polypeptide complexes by expression in suitable host cells, such as CHO cells.

[0152] Preferred promoters for controlling transcription from vectors in mammalian host cells may be obtained from a variety of sources, for example, from the genomes of viruses such as polyoma virus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and most preferably, cytomegalovirus (CMV), or from heterologous mammalian promoters, such as the β-actin promoter or EF1α promoter, or from hybrid or chimeric promoters (e.g., the CMV promoter fused to the β-actin promoter). Of course, promoters from the host cell or related species are also useful herein.

[0153] Enhancers generally refer to DNA sequences that function at variable distances from the transcription start site and can be 5' or 3' to the transcription unit. Furthermore, enhancers can be located within introns or within the coding sequence itself. They are typically 10 to 300 base pairs (bp) in length and function in cis. Enhancers typically function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate transcriptional regulation. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), and enhancers from eukaryotic viruses are commonly used for general expression. Preferred examples include 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.

[0154] The promoter and / or enhancer may be inducible (e.g., chemically or physically regulated). Chemically regulated promoters and / or enhancers may be regulated, for example, by the presence of alcohol, tetracycline, steroids, or metals. Physically regulated promoters and / or enhancers may be regulated by environmental factors such as temperature and light. Optionally, the promoter and / or enhancer region may act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region may be active in a cell-type-specific manner. Optionally, in certain vectors, the promoter and / or enhancer region may be active in all eukaryotic cells, regardless of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the β-actin promoter, the EF1α promoter, and retroviral long terminal repeats (LTRs).

[0155] Vectors can also include, for example, an origin of replication and / or a marker. Marker genes can confer a selectable phenotype, such as antibiotic resistance, to cells. The marker product is used to determine whether the vector has been delivered to a cell and, once delivered, whether it is expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into mammalian host cells, the transformed mammalian host cells can survive when placed under selective pressure. Other examples of markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. Furthermore, expression vectors can include tag sequences 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™ tags (Kodak; New Haven, Conn.), are typically expressed as fusions with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.

[0156] G. Therapeutic Applications Also provided herein are methods and uses for treating a disease, disorder, or condition associated with a target antigen, comprising administering an IL-12 polypeptide complex or IL-23 polypeptide complex described herein to a subject in need thereof. The disease, disorder, or condition includes, but is not limited to, cancer, inflammatory disease, immune disease, autoimmune disease, and infectious disease (i.e., bacterial, viral, or parasitic disease). Preferably, the disease, disorder, or condition is cancer.

[0157] Any suitable cancer can be treated with the IL-12 or IL-23 polypeptide complexes provided herein. Exemplary suitable cancers include, for example, 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, cancer of unknown primary, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrohistiocytoma, and fibrohistiocytoma. tumor, Ewing's 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 carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma , melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary origin, midline duct carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal and paranasal cancer, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, These include pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, 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, gastric cancer, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor. In embodiments, the cancer is melanoma or breast cancer.

[0158] In some embodiments, provided herein are methods for enhancing an immune response in a subject in need thereof by administering to the subject an effective amount of an IL-12 or IL-23 polypeptide complex provided herein. The enhanced immune response may prevent, delay, or treat the onset of cancer, tumors, or viral diseases. Without being bound by theory, the IL-12 or IL-23 polypeptide complex enhances the immune response by activating innate and adaptive immunity. In some embodiments, the methods described herein increase the activity of natural killer cells and T lymphocytes. In some embodiments, the IL-12 or IL-23 polypeptide complex provided herein can induce IFNγ release from natural killer cells and CD4+ and CD8+ T cells.

[0159] The method may further include administering one or more additional agents to treat the cancer, such as a chemotherapeutic agent (e.g., Adriamycin, Cervidine, Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine), an immunotherapy agent (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), a cellular therapy agent (e.g., CAR-T, T-cell therapy), an oncolytic virus, etc. Non-limiting examples of anticancer drugs that may be used include acivicin; aclarubicin; acodazole hydrochloride; acronine; adzelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacytidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carze Resin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexorumaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziconazole; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidin; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; EstramustineEstramustine sodium phosphate; Etanidazole; Etoposide; Etoposide phosphate; Etoprine; Fadrozole hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine phosphate; Fluorouracil; Flurocitabine; Foskidone; Fostriecin sodium; Gemcitabine; Gemcitabine hydrochloride; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Ilmofosine; Interleukin II (including recombinant interleukin II, i.e., rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-nl Interferon alpha-n3; Interferon beta-Ia; Interferon gamma-Ib; 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; Mitochromin; Mitodilin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole; Nogalamycin; O Lumaplatin; Oxisuran; Paclitaxel; Pegaspargase; Periomycin; Pentamustine; Peplomycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone hydrochloride; Plicamycin; Promestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofurin; Ribopurin; Rogletimide; Safingol; Safingol hydrochloride; Semustine; Simtrazene; Sparfosate sodium; Sparsomycin; Spirogermanium hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; SulofenurTalisomycin; Tecogalan sodium; Tegafur; Teroxantrone hydrochloride; Temoporfin; Teniposide; Teloxiron; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan hydrochloride; Toremifene citrate; Trestrone acetate; Triciribine phosphate; Trimetrexate; Trimetrexate glucuronate; Triptorelin; Tubrozole hydrochloride; Uracil mustard; Uredepa; Bubreotide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Binepidine sulfate; Vinglycinate sulfate Sulfate); vinleurosine sulfate; vinorelbine tartrate; vinzolidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and antitumor drugs such as zorubicin hydrochloride.

[0160] In some embodiments of the methods described herein, the IL-12 or IL-23 polypeptide complex is administered in combination with an agent to treat a particular disease, disorder, or condition. The agent may include, but is not limited to, antibodies, small molecules (e.g., chemotherapeutic agents), hormones (steroids, peptides, etc.), radiation therapy (direct delivery of gamma rays, C-rays, and / or radioisotopes, microwaves, ultraviolet light, etc.), gene therapy (e.g., antisense, retroviral therapy, etc.), and other immunotherapies. In some embodiments, the IL-12 or IL-23 polypeptide complex is administered in combination with an antidiarrheal, antiemetic, analgesic, and / or nonsteroidal anti-inflammatory agent.

[0161] 6. Equivalents It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present invention described herein will be obvious and may be made using suitable equivalents without departing from the scope of the disclosure or embodiments. While certain compounds and methods have been described in detail herein, the same will be more clearly understood by reference to the following examples, which are introduced for purposes of illustration only and are not intended to be limiting. [Example]

[0162] 7. Working Example The present invention is further illustrated by the following examples, which are not intended to be limiting in any way.

[0163] Example 1: HEK-Blue assay HEK-Blue IL-12 cells (InvivoGen) were seeded at 50,000 cells / well in suspension in medium with or without 15 or 40 mg / ml human serum albumin (HSA) and stimulated with serial dilutions of recombinant hIL-12, chimeric IL-12 (mouse p35 / human p40), activatable chimeric IL-12, or activatable hIL-12 for 20–24 h at 37°C and 5% CO2. The activity of uncleaved and cleaved activatable hIL-12 was tested. Inducible cleaved hIL-12 was generated by incubation with activated MMP9 or CTSL-1. IL-12 activity was assessed by quantitating secreted alkaline phosphatase (SEAP) activity using the QUANTI-Blue reagent (InvivoGen), a colorimetric assay. Results confirm that the IL-12 fusion proteins are active and inducible. The results are shown in Figures 2A to 2S.

[0164] Example 2: IL-12 luciferase reporter assay IL-12 luciferase reporter cells (Promega), purchased from the manufacturer in "thaw and use" format, were plated according to the manufacturer's instructions and stimulated with serial dilutions of recombinant hIL-12 or activatable hIL-12 for 6 hours at 37°C, 5% CO2. The activity of uncleaved and cleaved forms of activatable hIL-12 was tested. Inducible cleaved IL-12 was generated by incubation with active MMP9 or CTSL-1. IL-12 activity was assessed by quantification of luciferase activity using Bio-Glo™ reagent (Promega), which allows for measurement of luciferase activity with a luminescent readout. The results confirm that the IL-12 protein fusion proteins are active and inducible. The results are shown in Figures 3A-3F.

[0165] Example 3: Human T-Blast assay T-Blasts were induced from human PBMCs by PHA stimulation for 72 hours. T-Blasts were then washed and frozen before use. For the assay, T-Blasts were thawed, suspended in medium containing human albumin, plated at 100,000 cells / well, and stimulated with recombinant hIL-12, chimeric activatable IL-12 (mouse p35 / human p40), or activatable human IL-12 for 72 hours at 37°C and 5% CO2. The activity of uncleaved and truncated IL-12 fusion proteins was tested. Inducible truncated hIL-12 was generated by incubation with activated MMP9 or CTSL-1 enzymes. IL-12 activity was assessed by quantitating IFNγ production in the supernatant using the hIFNγ Alpha-LISA kit. The results confirm that the IL-12 fusion proteins are active and inducible. The results are shown in Figures 4A-4G.

[0166] Example 4: Protease cleavage of fusion proteins by MMP9 protease Those skilled in the art will be familiar with how to set up a protein cleavage assay. 100 μg of protein in 1×PBS (pH 7.4) was cleaved with 1 μg of active MMP9 (Sigma catalog number SAE0078-50 or Enzo catalog number BML-SE360) and incubated at room temperature for up to 16 hours. The digested protein was then used in functional analysis or stored at −80° C. before testing. The extent of cleavage was monitored by SDS-PAGE using methods well known in the art. Complete cleavage of the fusion protein by MMP9 was observed.

[0167] Example 5: Expression comparison in mammalian host cell lines The expression plasmid for WW0663, an IL-12 fusion protein in which the human p40 and p35 subunits are connected by a non-cleavable linker, was transiently transfected into a mammalian expression host cell line and purified from the cell supernatant by protein A chromatography. Similarly, the expression plasmids for WW0750 and WW0636 were transiently co-transfected into the same parent mammalian host cell line to express the IL-12 fusion protein, but the human p40 and p35 subunits are not connected by a linker sequence but are assembled via native disulfide bonds. WW0750 / WW0636 was purified from the cell supernatant by protein A chromatography. Both WW0663 and WW0750 / WW0636 were run on non-reducing and reducing SDS-PAGE gels to compare correct assembly and any unintended cleavage products (Figure 5). WW0663 exhibits two unintended molecular weight fragments (cleavage products). Furthermore, the intact band of WW0663 was reduced under reducing conditions, suggesting unintended cleavage at or near the linker between the p40 and p35 subunits, generating two equally sized products (the smallest molecular weight shown in lane 4), and separation of p40 and p35 by reduction of the p40 / p35 disulfide band. WW0750 / WW0636 under reducing and nonreducing conditions (lanes 6 and 7, respectively) show the expected sizes.

[0168] Example 6: MC38 Experiment (Study MC38-e493) The MC38 cell line, a rapidly growing colon adenocarcinoma cell line, was used. This tumor model was used to examine the ability of the fusion protein to affect tumor growth and body weight. [Table 6]

[0169] Mice were anesthetized with isoflurane for transplantation of ulcer-reducing cells. 326 CR female C57BL / 6 mice were implanted with 5 × 10 subcutaneously in the flank. 5 The mice were prepared with 0% Matrigel containing MC38 tumor cells. The cell injection volume was 0.1 mL per mouse. Mice were 8–12 weeks old on the day of initiation. Pair-matching was performed and treatment began when tumors reached an average size of 100–150 mm3. This was Day 1 of the study. Body weight was measured at baseline and twice weekly thereafter until termination. Caliper measurements were performed twice weekly until termination. Any adverse reactions were reported immediately. Individual animals were euthanized if a single weight loss of >25% or three consecutive weight loss measurements of >20% were observed. Treatment was discontinued for any group with a mean weight loss of >20% or mortality of >10%; the group was not euthanized and allowed to recover. Individuals within a group with >20% weight loss who reached their individual weight loss endpoint were euthanized. If a group treatment associated with weight loss recovered to within 10% of its original body weight, dosing was resumed at a lower dose or less frequent dosing schedule. Exceptions to the % weight recovery of non-treated animals were allowed on a case-by-case basis. The endpoint was tumor growth delay (TGD). Animals were monitored individually. The experimental endpoint was a tumor volume of 1500 mm 3 or day 40, whichever came first. When the endpoint was reached, the animals were euthanized.

[0170] Example 7: MC38 Experiment (Study MC38-e495) The MC38 cell line, a rapidly growing colon adenocarcinoma cell line, was used. This tumor model was used to examine the ability of the fusion protein to affect tumor growth and body weight. [Table 7]

[0171] Mice were anesthetized with isoflurane for transplantation of ulcer-reducing cells. 326 CR female C57BL / 6 mice were implanted with 5 × 10 subcutaneously in the flank. 5 The tumors were prepared with 0% Matrigel containing MC38 tumor cells. The cell injection volume was 0.1 mL per mouse. Mice were 8-12 weeks old on the day of initiation. The tumors had an average size of 100-150 mm. 3 Once the weight loss reached 10%, pair-matching was performed and treatment was initiated. This marked study day 1. Body weights were measured at baseline and twice weekly thereafter until termination. Caliper measurements were taken twice weekly until termination. Any adverse reactions were reported immediately. Individual animals were euthanized if a single body weight loss of >25% or three consecutive body weight loss measurements of >20% were observed. Treatment was discontinued for any group with a mean body weight loss of >20% or a mortality rate of >10%; the group was not euthanized but allowed to recover. Individuals within groups with >20% weight loss that reached an individual weight loss endpoint were euthanized. If the group treatment associated with weight loss recovered to within 10% of its original body weight, treatment was resumed at a lower dose or a less frequent dosing schedule. Exceptions to the non-treated % weight recovery were permitted on a case-by-case basis. The endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1500 mm 3 or day 40, whichever came first. When the endpoint was reached, the animals were euthanized.

[0172] Example 8: MC38 Experiment (Study MC38-e503) The MC38 cell line, a rapidly growing colon adenocarcinoma cell line, was used. This tumor model was used to examine the ability of the fusion protein to affect tumor growth and body weight. [Table 8]

[0173] Mice were anesthetized with isoflurane for transplantation of ulcer-reducing cells. 326 CR female C57BL / 6 mice were implanted with 5 × 10 subcutaneously in the flank. 5 The tumors were prepared with 0% Matrigel containing MC38 tumor cells. The cell injection volume was 0.1 mL per mouse. Mice were 8-12 weeks old on the day of initiation. The tumors had an average size of 100-150 mm. 3 Pair-matching was performed and treatment began when the weight loss reached 10%. This marked study day 1. Body weights were measured at baseline and twice weekly thereafter until termination. Caliper measurements were taken twice weekly until termination. Any adverse reactions were reported immediately. Individual animals were euthanized if a single body weight loss of >25% or three consecutive body weight loss measurements of >20% were observed. Treatment was discontinued for any group with a mean body weight loss of >20% or a mortality rate of >10%; the group was not euthanized but allowed to recover. Individuals within groups with >20% weight loss who reached an individual weight loss endpoint were euthanized. If the group treatment associated with weight loss recovered to within 10% of its original body weight, treatment was resumed at a lower dose or a less frequent dosing schedule. Exceptions to the non-treated % weight recovery were permitted on a case-by-case basis. The endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1500 mm 3 or day 40, whichever came first. When the endpoint was reached, the animals were euthanized.

[0174] Example 9: Octet binding kinetics assay KD measurements were performed using multi-concentration kinetics with scFv. Binding affinity to human IL-12 was measured using an Octet QKe instrument (ForteBio). A strategy was used in which 6xHis-tagged (SEQ ID NO: 446) scFv was captured on the sensor, followed by IL-12 binding / dissociation. BLI analysis was performed at 30°C using 1x kinetics buffer (ForteBio) as the assay buffer. Ni-NTA (NTA) biosensors (ForteBio) were first soaked in assay buffer for >5 min. Test scFv (5 μg / mL) was captured on the sensor for 300 s. The sensor was then soaked in assay buffer for 120 s to establish a baseline, after which binding to IL-12 was measured. The sensor was then soaked for 300 s with various concentrations of IL-12 (50–0.78 nM, diluted 2-fold in assay buffer) and a blank buffer well for reference subtraction correction to measure association. The dissociation of IL-12 was then measured by immersing the sensor in assay buffer for 300 seconds. Agitation at all steps was 1000 rpm. Kinetic parameters were generated using Octet Data Analysis Software version 8.2 using a reference subtraction correction (scFv "binding" to buffer), dissociation based on step-to-step correction, a one-to-one binding model, and a global fit (Rmax not linked by the sensor). KD values ​​are shown in Table 7. Table 7. Summary of scFv IL-12 blocking factor kinetics [Table 9-1] [Table 9-2]

[0175] Example 10: HEKBlue IL-23 reporter assay HEK-Blue IL23 cells (InvivoGen) were seeded at a density of 50,000 cells / well in culture medium with or without 15 mg / ml human serum albumin (HSA) and stimulated with serial dilutions of recombinant murine IL-23 or half-life-extended murine IL-23 (anti-HSA-L-mIL23) for 20–24 h at 37°C, 5% CO. IL-23 activity was assessed by quantification of secreted alkaline phosphatase (SEAP) activity using the QUANTI-Blue (InvivoGen) reagent, a colorimetric assay. Results are shown in Figures 40A and 40B.

[0176] Example 11: MC38 Efficacy Study Using Half-Life Extended IL-23 Protein WW5009 The MC38 cell line, a rapidly growing colon adenocarcinoma cell line, was used to investigate the ability of the fusion protein to affect tumor growth. [Table 10]

[0177] Mice were anesthetized with isoflurane for transplantation of ulcer-reducing cells. Charles River female C57BL / 6 mice were treated with 5 x 10 subcutaneous injections of the cells into the flank. 5 The tumors were prepared with 0% Matrigel containing MC38 tumor cells. The cell injection volume was 0.1 mL per mouse. Mice were 8-12 weeks old on the day of initiation. The tumors had an average size of 100-150 mm. 3Once the group reached 100%, pair-matching was performed and treatment was initiated. Body weights were measured at baseline and then twice weekly until termination. Caliper measurements were taken twice weekly until termination. Any adverse reactions were reported immediately. Individual animals were euthanized if a single body weight loss of more than 30% or three consecutive body weight loss measurements of more than 25% were observed. Treatment was discontinued for any group with a mean body weight loss of more than 20% or a mortality rate of more than 10%; the group was not euthanized but allowed to recover. Individuals within a group with a weight loss of more than 20% that reached an individual weight loss endpoint were euthanized. If the group treatment associated with weight loss recovered to within 10% of its original body weight, treatment was resumed at a lower dose or less frequent dosing schedule. Exceptions to the non-treated % weight recovery were allowed on a case-by-case basis. The endpoint was tumor growth delay (TGD). Animals were monitored individually. The experimental endpoint was a tumor volume of 1500 mm 3 Responders were followed for 14 days or 45 days, whichever came first. Responders were followed thereafter. When the endpoint was reached, the animals were euthanized. The results are shown in Figures 49A, 49B, and 50A-50D.

[0178] Example 12: CT26 Experiment (Study CT26-e676) The CT26 cell line, a rapidly growing colon adenocarcinoma cell line, was used as a tumor model to examine the ability of the fusion protein to affect tumor growth. [Table 11]

[0179] Thirty CR female BALB / c mice were treated with 3 × 10 subcutaneous injections into the flank. 5 The tumors were prepared with 0% Matrigel containing CT26 tumor cells. The cell injection volume was 0.1 mL per mouse. Mice were 8-12 weeks old on the day of initiation. The tumors had an average size of 30-60 mm. 3Once the weight loss reached 10%, pair-matching was performed and treatment was initiated. This marked study day 1. Caliper measurements were taken twice weekly until termination. Any adverse reactions were reported immediately. Individual animals were euthanized if a single weight loss of more than 25% or three consecutive weight loss measurements of more than 20% were observed. Treatment was discontinued for any group with a mean weight loss of more than 20% or a mortality rate of more than 10%; the group was not euthanized but allowed to recover. Individuals within a group with a weight loss of more than 20% who reached an individual weight loss endpoint were euthanized. If the group treatment associated with weight loss recovered to within 10% of its original weight, treatment was resumed at a lower dose or less frequent dosing schedule. Exceptions to the non-treated % weight recovery were allowed on a case-by-case basis. The endpoint was tumor growth delay (TGD). Animals were monitored individually. The experimental endpoint was a tumor volume of 2000 mm 3 The endpoint was 2 days or 22 days, whichever came first. When the endpoint was reached, the animals were euthanized. The results are shown in Figure 41 and Figures 42A-42C.

[0180] Example 13: B16F10 Experiment (Study B16F10-ITAA-0215) We used the B16F10 cell line, a rapidly growing melanoma cell line, to investigate the ability of the fusion protein to affect tumor growth. [Table 12]

[0181] Thirty CR female C57Bl / 6 mice were treated with 1 × 10 subcutaneous injections into the flank. 5 The tumors were prepared using 50% Matrigel containing B16F10 tumor cells. The cell injection volume was 0.1 mL per mouse. Mice were 8-12 weeks old on the day of initiation. The tumors had an average size of 100 mm. 3Once the weight loss reached 10%, pair-matching was performed and treatment was initiated. This marked study day 1. Caliper measurements were taken twice weekly until termination. Any adverse reactions were reported immediately. Individual animals were euthanized if a single weight loss of more than 25% or three consecutive weight loss measurements of more than 20% were observed. Treatment was discontinued for any group with a mean weight loss of more than 20% or a mortality rate of more than 10%; the group was not euthanized but allowed to recover. Individuals within a group with a weight loss of more than 20% who reached an individual weight loss endpoint were euthanized. If the group treatment associated with weight loss recovered to within 10% of its original weight, treatment was resumed at a lower dose or less frequent dosing schedule. Exceptions to the non-treated % weight recovery were allowed on a case-by-case basis. The endpoint was tumor growth delay (TGD). Animals were monitored individually. The experimental endpoint was a tumor volume of 2000 mm 3 The endpoint was 2 days or 22 days, whichever came first. When the endpoint was reached, the animals were euthanized. The results are shown in Figure 43 and Figures 44A-44C.

[0182] Example 14: EMT6 Experiment (Study EMT6-ITAA-0216) The EMT6 cell line, a rapidly growing breast adenocarcinoma cell line, was used to investigate the ability of the fusion protein to affect tumor growth. [Table 13]

[0183] Thirty CR female BALB / c mice were treated with 1 × 10 subcutaneous injections into the flank. 5 The tumors were prepared using 50% Matrigel containing EMT6 tumor cells. The cell injection volume was 0.1 mL per mouse. Mice were 8-12 weeks old on the day of initiation. The tumors had an average size of 100 mm. 3Once the weight loss reached 10%, pair-matching was performed and treatment was initiated. This marked study day 1. Caliper measurements were taken twice weekly until termination. Any adverse reactions were reported immediately. Individual animals were euthanized if a single weight loss of more than 25% or three consecutive weight loss measurements of more than 20% were observed. Treatment was discontinued for any group with a mean weight loss of more than 20% or a mortality rate of more than 10%; the group was not euthanized but allowed to recover. Individuals within a group with a weight loss of more than 20% who reached an individual weight loss endpoint were euthanized. If the group treatment associated with weight loss recovered to within 10% of its original weight, treatment was resumed at a lower dose or less frequent dosing schedule. Exceptions to the non-treated % weight recovery were allowed on a case-by-case basis. The endpoint was tumor growth delay (TGD). Animals were monitored individually. The experimental endpoint was a tumor volume of 2000 mm 3 The endpoint was 2 days or 22 days, whichever came first. When the endpoint was reached, the animals were euthanized. The results are shown in Figure 45 and Figures 46A-46C.

[0184] Example 15: Nanostring analysis of total tumor RNA Mouse tumors were harvested from treated animals and dissociated into single cell suspensions. Briefly, tumors were divided into 5 mm sections before enzymatic digestion. 3The cells were minced to less than 1 / 4 of a millimeter. Samples were shaken with 3 mg / mole collagenase IV at 37°C for 35 minutes and then mechanically separated through a 70 μM nylon mesh filter. Samples were then washed and counted, and a total of 3–5 e5 viable cells from each sample were spun down and frozen in RLT+ buffer for subsequent RNA extraction. RNA isolation and NanoString processing were performed by LakePharma. RNA was isolated using the RNEasy Micro kit according to the manufacturer's protocol, and 100 ng of total RNA was run on an nCounter system using the Mouse PanCancer Immune Profiling Codeset. Data analysis was performed by Werewolf Therapeutics using nSolver software with the Advanced Analysis module installed. All statistical analyses were performed using nSolver software (see nCounter Advanced Analysis 2.0 Plugin for nSolver Software, User Manual, NanoString Technologies, 2018). Heatmaps and other graphs were generated using Prism software.

[0185] Example 16: Mouse tumor treatment and flow cytometry analysis MC38 tumors were implanted into C57BL / 6 mice and reached an average size of 150 mm 3 After growth to 10 days, mice were randomly assigned to treatment groups (day 0). Mice were treated with vehicle or attenuated IL-12 by intraperitoneal injection on days 1 and 4, and tumors were harvested 24 hours after the second dose (day 5). Tumors were harvested and divided into 5 mm sections before enzymatic digestion in phenol-free RPMI. 3 The cells were minced to less than 5 × 10 per well. Samples were shaken with 3 mg / m collagenase IV at 37°C for 35 minutes and then mechanically dissociated through a 70 μM nylon mesh filter. Samples were then washed, counted, and plated for flow cytometry analysis. Up to 5 × 10 per well of a 96-well round-bottom plate was used. 6Cells were seeded onto the cells. For intracellular cytokine staining, samples were stimulated with phorbol 12-myristate 13-acetate (PMA), ionomycin, and brefeldin A for 4 hours before staining. For cell staining, FC receptors were first blocked, followed by staining for extracellular markers. After extracellular staining, cells were washed, fixed, and permeabilized, and then stained for intracellular markers. Samples were run on a Cytek Aurora system running SpectroFlo® software, and data were analyzed using FlowJo™ software. All graphs and statistical analyses were performed using GraphPad Prism software.

[0186] 8. Permutation of constructs The elements of the polypeptide constructs shown in Table 8 include the following abbreviations: "L," "X," "LX," and "XL," each referring to a linker. "X" refers to a cleavable linker. "L" refers to an optional cleavable linker. If L is the only linker in the polypeptide, L is cleavable. "LX" or "XL" each refer to a cleavable linker with an extended non-cleavable sequence adjacent to it. Linker 1 refers to a linker containing an MMP9 substrate motif sequence, Linker 2 refers to a linker containing an MMP14 substrate motif sequence, and Linker 3 refers to a linker containing a CTSL-1 substrate motif sequence. [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] Table 14-7 Table 14-8 Table 14-9 Table 15-1 Table 15-2 Table 15-3 Table 15-4 Table 15-5 Table 15-6 Table 15-7 Table 15-8 Table 15-9 Table 15-10 Table 15-11 Table 15-12 Table 15-13 Table 15-14 Table 15-15 Table 15-16 Table 15-17 Table 15-18 Table 15-19 Table 15-20 Table 15-21 Table 15-22 Table 15-23 Table 15-24 Table 15-25 Table 15-26 Table 15-27 Table 15-28 Table 15-29 Table 15-30 Table 15-31 Table 15-32 Table 15-33 Table 15-34 Table 15-35 Table 15-36 Table 15-37 Table 15-38 Table 15-39 Table 15-40 Table 15-41 Table 15-42 Table 15-43 Table 15-44 Table 15-45 Table 15-46 Table 15-47 Table 15-48 Table 15-49 Table 15-50 Table 15-51 Table 15-52 Table 15-53 Table 15-54 Table 15-55 Table 15-56 Table 15-57 Table 15-58 Table 15-59 Table 15-60 Table 15-61 Table 15-62 Table 15-63 Table 15-64 Table 15-65 Table 15-66 Table 15-67 Table 15-68 Table 15-69 Table 15-70 Table 15-71 Table 15-72 Table 15-73 Table 15-74 Table 15-75 Table 15-76 Table 15-77 Table 15-78 Table 15-79 Table 15-80 Table 15-81 Table 15-82 Table 15-83 Table 15-84 Table 15-85 Table 15-86 Table 15-87 Table 15-88 Table 15-89 Table 15-90 Table 15-91 Table 15-92 Table 15-93 Table 15-94 Table 15-95 Table 15-96 Table 15-97 Table 15-98 Table 15-99 Table 15-100 Table 15-101 Table 15-102 Table 15-103 Table 15-104 Table 15-105 Table 15-106 Table 15-107 Table 15-108 Table 15-109 Table 15-110 Table 15-111 Table 15-112 Table 15-113 Table 15-114 Table 15-115 Table 15-116 Table 15-117 Table 15-118 Table 15-119

Table 15-120

Claims

[Claim 1] The invention described in the specification.

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