Modified interleukin p40 subunit proteins and methods of use thereof

Abstract

The present disclosure relates generally to modified human interleukin-12 (IL12) or IL23 p40 polypeptides having alterations to reduce binding affinity to receptor subunits to generate IL12 or IL23 proteins for the treatment of cancer that are less toxic compared to wild-type IL12 or IL23 proteins.

Description

[Technical field]

[0001] The present disclosure relates to modified interleukin P40 subunit proteins, including fusion proteins, modified to reduce the binding activity of interleukin cytokines, particularly IL12p40 and IL23p40, or any other cytokine that has a p40 subunit protein; compositions comprising them, and methods of using the compositions for the treatment of a variety of diseases, including cancer. [Background technology]

[0002] background Interleukin 12 (IL12) was the first recognized member of a family of heterodimeric cytokines that includes IL12, IL23, IL27, IL35, and IL39. IL12 and IL23 are proinflammatory cytokines important for the development of T helper 1 (Th-1) and T helper 17 (Th-17) T cell subsets, respectively, whereas IL27 and IL35 are potent inhibitory cytokines. IL39 is a cytokine important in controlling innate and / or adaptive immune responses. IL12 can directly enhance the activity of effector CD4 and CD8 T cells as well as natural killer (NK) and NKT cells.

[0003] IL12 is a heterodimeric molecule composed of an alpha chain (p35 subunit) and a beta chain (p40 subunit) covalently linked by disulfide bridges to form a biologically active 70 kDa dimer. IL23 is a member of the IL12 cytokine family and is composed of two subunits: the p40 subunit shared with IL12 and p19. IL-23 has the same chain, IL-12p40, as IL-12. The p40 subunit is also secreted as a free monomer and a disulfide-linked homodimer (p80) whose functions generally antagonize those of IL12 and IL-23. The IL12 receptor, or receptor complex, is composed of IL12Rβ1 and IL12Rβ2. The IL23 receptor complex (IL23R) consists of the IL23R subunit and the IL12Rβ1 subunit, which is a common subunit for the IL12 receptor and interacts with tyrosine kinase 2 (Tyk2). The IL12Rβ1 subunit interacts only with the p40 subunit of IL12 or IL23, whereas the IL12Rβ2 subunit interacts with both the p35 and p40 subunits of IL12, and the IL23R subunit interacts with both the p19 and p40 subunits of IL23. IL23R is primarily expressed on immune cells, especially T cells (e.g., Th17 and gamma delta T cells), macrophages, dendritic cells, and NK cells (Duvallet et al., 2011). It has recently been shown that non-activated neutrophils express basal amounts of IL23R and that IL23R expression increases upon cell activation (Chen et al., 2016).

[0004] Biologically, IL12 is a proinflammatory cytokine produced in response to infection by a variety of cells of the immune system, including phagocytes, B cells, and activated dendritic cells (Colombo and Trinchieri (2002), Cytokine and Growth Factor Reviews, 13:155-168 and Hamza et al., "Interleukin-12 a Key Immunoregulatory Cytokine in Infection Applications" Int. J. Mol. Sci. 11;789-806 (2010). IL12 plays an essential role in mediating the interactions of the innate and adaptive arms of the immune system, acting on T cells and natural killer (NK) cells, and enhancing the proliferation and activity of cytotoxic lymphocytes as well as the production of other proinflammatory cytokines, particularly interferon-gamma (IFN-gamma or IFNγ).

[0005] IL12 has been tested in human clinical trials as an immunotherapeutic agent for the treatment of a variety of cancers, including renal, colon, and ovarian cancer, melanoma, and T-cell lymphoma (Atkins et al. (1997), Clin. Cancer Res., 3:409-17; Gollob et al. (2000), Clin. Cancer Res., 6:1678-92; Hurteau et al. (2001), Gynecol. Oncol., 82:7-10; and Youssoufian, et al. (2013) Surgical Oncology Clinics of North America, 22(4):885-901), and as an adjuvant for cancer vaccines (Lee et al. (2001), J. Clin. Oncol. 19:3836-47). However, IL12 is toxic when administered systemically as a recombinant protein. Trinchieri, Adv. Immunol. 1998;70:83-243. To maximize the antitumor effects of IL12 while minimizing its systemic toxicity, an IL12 gene therapy approach has been proposed to enable production of the cytokine at the tumor site, thereby achieving high local levels of IL12 at low serum concentrations. Qian et al., Cell Research (2006) 16:182-188; US Patent Publication No. 20130195800.

[0006] Since IL12 is a heterodimeric molecule composed of an alpha chain (p35 subunit) and a beta chain (p40 subunit), simultaneous expression of the two subunits is necessary for the generation of a biologically active heterodimer. Recombinant IL12 expression has been achieved by using a bicistronic vector containing the p40 and p35 subunits separated by an IRES (internal ribosome entry site) sequence, allowing independent expression of both subunits from a single vector. However, the use of the IRES sequence may impair protein expression. Mizuguchi et al., Mol Ther (2000); 1: 376-382. Moreover, unequal expression of the p40 and p35 subunits may result in the formation of homodimeric proteins (e.g., p40-p40 dimers) that may have inhibitory effects on IL12 signaling. Gillessen et al. Eur. J. Immunol. 25 (1): 200-6 (1995).

[0007] As an alternative to bicistronic expression of IL12 subunits, functional single-chain IL12 fusion proteins have been generated by connecting the p40 and p35 subunits with (Gly4Ser)3 or Gly6Ser linkers. Lieschke et al., (1997), Nature Biotechnology 15, 35-40; Lode et al., (1998), PNAS 95, 2475-2480. (These forms of p40-linker-p35 or p35-linker-p40 IL12 configurations may be referred to herein as "single-chain IL12 (scIL12).")

[0008] Human IL12 p70 (i.e., a dimer of p35 and p40) has a reported in vivo half-life of 5-19 hours that may result in significant systemic toxicity when administered as a therapeutic compound. See, e.g., Car et al. "The Toxicology of Interleukin-12: A Review" Toxicologic Path. 27:1, 58-63 (1999); Robertson et al. "Immunological Effects of Interleukin 12 Administered by Bolus Intravenous Injection to Patients with Cancer" Clin. Cancer Res. 5:9-16 (1999); Atkins et al. "Phase I Evaluation of Intravenous Recombinant Human Interleukin 12 in Patients with Advance Malignancies" Clin. Cancer Res. 3:409-417 (1997). Preclinical studies in murine tumor treatment models have demonstrated potent antitumor effects following systemic administration of IL12. However, in humans, attempts to administer recombinant IL12 systemically have resulted in significant toxicity, including patient deaths, and limited efficacy. Thus, there remains a need in the art for improved therapeutic control of the form of IL12 delivered in vivo. Summary of the Invention

[0009] overview One aspect of the present disclosure provides cytokine fusion proteins containing modified p40 domains, in particular IL12 and IL23 fusion proteins, also referred to herein as IL12 HetFc fusion proteins. The IL12 fusion proteins described herein may comprise an IL12 polypeptide, an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, and optionally a masking moiety (MM) that reduces, inhibits or blocks IL12 activity.

[0010] One aspect of the present disclosure describes a modified p40 domain comprising one or more amino acid substitutions relative to the wild-type human mature IL12 p40 domain sequence set forth in SEQ ID NO:10, wherein the one or more amino acid substitutions are located at one or more of positions E45, D62 and D161, and wherein the numbering of the amino acid residues is based on the amino acid sequence set forth in SEQ ID NO:10.

[0011] One embodiment of the disclosure is a modified p40 domain comprising one or more amino acid substitutions relative to the wild-type human mature IL12 p40 domain sequence set forth in SEQ ID NO: 10, wherein the one or more amino acid substitutions are selected from the group consisting of W15H, W15K, W15R, D18G, E45K, K58H, K58W, E59D, E59G, E59R, F60D, F60E, F60K, F60R, F60V, D62H, D62I, D62N, K84E, K84I, K84L, K84V, K84W, , K84Y, E86L, E86R, E86S, E86W, D93E, D93H, D93R, D93W, D161R, D161S, K197D, K197E, K197Q, K197T, or K197W, or combinations thereof, wherein the numbering of the amino acid residues is based on the amino acid sequence set forth in SEQ ID NO:10.

[0012] Another embodiment of the present disclosure describes an IL12 fusion protein that includes a modified p40 domain of the present disclosure.

[0013] Another aspect of the present disclosure describes an IL12 fusion protein comprising an IL12 polypeptide, where the IL12 polypeptide comprises a modified p40 domain of the present disclosure linked to a p35 domain.

[0014] Another aspect of the present disclosure describes a masked IL12 HetFc fusion protein comprising: (i) an IL-12 polypeptide comprising a modified p40 domain of the present disclosure linked via a linker (G4S)4 to a p35 domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:11; (ii) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide; and (iii) a masking moiety (MM) comprising an anti-IL12 scFv domain, wherein the IL12 polypeptide is linked either directly or via a second linker to the C-terminus of the first Fc polypeptide, and the masking moiety is linked either directly or via a third linker to the C-terminus of the second Fc polypeptide, and is capable of non-covalently interacting with the IL12 polypeptide, thereby reducing the binding affinity of the IL12 polypeptide to at least one of its cognate receptors.

[0015] Another aspect of the present disclosure describes an unmasked IL12 HetFc fusion protein comprising: (i) an IL-12 polypeptide comprising a modified p40 domain described in this disclosure linked via a linker (G4S)4 to a p35 domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:11; and (ii) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein the IL12 polypeptide is linked either directly or via a second linker to the C-terminus of the first Fc polypeptide.

[0016] Another aspect of the present disclosure describes a pharmaceutical composition comprising (i) a modified p40 domain of the present disclosure, (ii) a masked IL12 HetFc fusion protein of the present disclosure, and / or (iii) an unmasked IL12 HetFc fusion protein of the present disclosure, and a pharma- ceutically acceptable carrier.

[0017] Another aspect of the present disclosure describes a nucleic acid molecule or set of nucleic acid molecules that encodes (i) a modified p40 domain of the present disclosure, (ii) a masked IL12 HetFc fusion protein of the present disclosure, and / or (iii) an unmasked IL12 HetFc fusion protein of the present disclosure.

[0018] Another aspect of the present disclosure describes a vector or a set of vectors comprising a nucleic acid molecule or a set of nucleic acid molecules described herein that encodes (i) a modified p40 domain of the present disclosure, (ii) a masked IL12 HetFc fusion protein of the present disclosure, and / or (iii) an unmasked IL12 HetFc fusion protein of the present disclosure.

[0019] Another aspect of the disclosure is a method for identifying one or more amino acid substitutions in a p40 domain amino acid sequence to generate a modified p40 domain, comprising: performing molecular dynamics and mutagenesis simulations, thereby identifying one or more amino acid substitutions listed in Table C. wherein one or more amino acid substitutions in the modified p40 domain amino acid sequence are relative to the sequence set forth in SEQ ID NO:1, and the one or more amino acid substitutions increase the binding affinity (K D ) is reduced relative to an unmodified p40 domain that does not contain one or more amino acid substitutions.

[0020] The present disclosure provides modifications of human IL12 and IL23 p40 subunit domains D1 and D2, specifically the modifications include engineered amino acid changes in the D1 and D2 domains aimed at reducing binding affinity to the receptor. More specifically, the changes / modifications occur at one or more positions corresponding to amino acid residue positions 15, 18, 45, 58, 59, 60, 62, 84, 86, 93, 161, 195 and 197 of SEQ ID NO: 1. One aspect of the present disclosure provides a p40 domain modified to have reduced binding to the IL12Rβ1 subunit, the modified p40 domain containing at least one amino acid substitution.

[0021] In some embodiments, the modified p40 domain is expressed as a component of an IL12 fusion protein comprising a p35 subunit and a modified p40 subunit, and the IL12 activity of the IL12 fusion protein containing the modified p40 domain is attenuated compared to the IL12 activity of a corresponding IL12 fusion protein containing an unmodified p40 domain. These embodiments also provide for modifications of human IL12 or IL23 p40 D1 and D2 domains. In some embodiments of the IL12 fusion proteins herein, the IL12 polypeptide is a single chain IL12 polypeptide having the orientation p40-linker-p35. In some embodiments of the IL12 fusion proteins herein, the single chain IL12 polypeptide is fused to an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, and the IL12 polypeptide is fused to the first Fc polypeptide by a first linker.

[0022] In some embodiments, the modified p40 domain is expressed as a component of a masked IL12 fusion protein comprising: a) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide; b) a masking moiety (MM); and c) an IL12 polypeptide, wherein the masking moiety is fused to the first Fc polypeptide by a first linker and the IL12 polypeptide is fused to the second Fc polypeptide by a second linker, and wherein the IL12 activity of the masked IL12 fusion protein containing the modified p40 domain is attenuated compared to the IL12 activity of a corresponding IL12 fusion protein containing an unmodified p40 domain.

[0023] In some embodiments of the masked IL12 fusion proteins herein, the masking moiety is a single chain Fv (scFv) antibody fragment. In certain embodiments, the scFv comprises VH CDR1-3 having the amino acid sequences set forth in SEQ ID NOs: 4-6, respectively, and VL CDR1-3 having the amino acid sequences set forth in SEQ ID NOs: 7-9, respectively. In some embodiments, the scFv comprises a VH and a VL comprising the amino acid sequences set forth in SEQ ID NOs: 2 and 3, respectively.

[0024] In some embodiments of the IL12 fusion proteins herein, the fusion protein further comprises a targeting domain, hi some embodiments, the targeting domain specifically binds to a tumor-associated antigen.

[0025] In some embodiments of the IL12 fusion proteins herein, the first Fc polypeptide comprises a first CH3 domain and the second Fc polypeptide comprises a second CH3 domain.

[0026] In some embodiments of the IL12 fusion proteins herein, IL12 activity is determined by measuring the relative cell abundance or cytokine production of a cell or cell line that is sensitive to IL12. In some embodiments, the cell or cell line is selected from PBMC, CD8+ T cells, CTLL-2 cell line, and NK cell line. In some embodiments, IL12 activity is determined by measuring IFNγ release by CD8+ T cells. In some embodiments, IL12 activity is determined by measuring the relative cell abundance of NK cells.

[0027] In some embodiments of the IL12 fusion proteins described herein, the first CH3 domain or the second CH3 domain, or both, comprise asymmetric amino acid modifications, and the first and second CH3 domains preferentially pair to form heterodimers rather than homodimers.

[0028] One aspect of the disclosure provides a composition comprising any of the IL12 or IL23 fusion proteins described herein and a pharma- ceutically acceptable excipient.

[0029] One aspect of the disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising any of the IL12 fusion proteins described herein (e.g., an IL12 HetFc fusion protein) and a pharma- ceutically acceptable excipient.

[0030] One aspect of the disclosure provides an isolated nucleic acid encoding an IL12 fusion protein described herein.

[0031] One aspect of the disclosure provides an expression vector comprising an isolated nucleic acid encoding an IL12 fusion protein described herein.

[0032] One aspect of the disclosure provides an isolated host cell comprising an isolated nucleic acid encoding an IL12 fusion protein described herein or an expression vector comprising such an isolated nucleic acid.

[0033] One aspect of the disclosure provides a method of making an IL12 fusion protein, the method comprising culturing a host cell comprising an isolated nucleic acid encoding an IL12 fusion protein as described herein or an expression vector comprising such an isolated nucleic acid under conditions suitable for expression of the IL12 fusion protein, and optionally recovering the IL12 fusion protein from the host cell culture medium.

[0034] One aspect of the disclosure provides an interleukin 23 (IL23) fusion protein comprising a p19 domain and a modified p40 domain as described herein, wherein the IL23 activity of the IL23 fusion protein containing the modified p40 domain is attenuated compared to the IL23 activity of a corresponding IL23 fusion protein containing an unmodified p40 domain.

[0035] Implementations disclosed herein are illustrated by way of example, and not by way of limitation, in the accompanying drawings, which are for illustrative purposes and aid in understanding only, and are not intended as a definition of the limits of the modified p40 domains, fusion proteins, compositions and methods of the present disclosure. [Brief description of the drawings]

[0036] [Figure 1] FIGS. 5A-5D show schematic diagrams of p40-containing cytokines according to embodiments of the present disclosure, e.g., a monomeric p40 domain (A), a dimeric p40 domain (p80, B), and cytokines containing such p40 domains, e.g., IL12 (C) and IL23 (D). [Diagram 2] AB show schematic diagrams of IL12 HetFc and masked IL12 HetFc fusion protein variants according to embodiments of the present disclosure. [Figure 3A] Representative CE-SDS and UPLC-SEC profiles following Protein A and Prep-SEC purification of IL12 HetFc fusion protein (v30806) are shown. [Figure 3B] Representative CE-SDS and UPLC-SEC profiles following Protein A and Prep-SEC purification of masked IL12 HetFc fusion protein (v35436) are shown. [Figure 4A] 1 shows data from an IL12 reporter gene assay (RGA) experiment using IL12 HetFc and masked IL12 HetFc fusion protein variants. RGA experiments for each set of variants were performed twice (indicated by the designations "replicate 1" and "replicate 2"). Each graph shows a set of unmasked IL12 HetFc fusion proteins with control variant 30806, and a set of masked IL12 HetFc fusion proteins with control variant 35436. Additionally, the RGA response to variants 37172, 37174, 37175, 37485, 37487, and 37488 is shown. [Figure 4B] 1 shows data from an IL12 reporter gene assay (RGA) experiment using IL12 HetFc and masked IL12 HetFc fusion protein variants. RGA experiments for each set of variants were performed twice (indicated by the designations "replicate 1" and "replicate 2"). Each graph shows a set of unmasked IL12 HetFc fusion proteins with control variant 30806, and a set of masked IL12 HetFc fusion proteins with control variant 35436. Additionally, the RGA response to variants 37176, 37178, 37173, 37489, 37491, and 37486 is shown. [Figure 4C]1 shows data from an IL12 reporter gene assay (RGA) experiment using IL12 HetFc and masked IL12 HetFc fusion protein variants. RGA experiments for each set of variants were performed twice (indicated by the designations "replicate 1" and "replicate 2"). Each graph shows a set of unmasked IL12 HetFc fusion proteins with control variant 30806, and a set of masked IL12 HetFc fusion proteins with control variant 35436. Additionally, the RGA response to variants 37157, 37158, 37156, 37470, 37471, and 37469 is shown. [Figure 4D] 1 shows data from an IL12 reporter gene assay (RGA) experiment using IL12 HetFc and masked IL12 HetFc fusion protein variants. RGA experiments for each set of variants were performed twice (indicated by the designations "replicate 1" and "replicate 2"). Each graph shows a set of unmasked IL12 HetFc fusion proteins with control variant 30806, and a set of masked IL12 HetFc fusion proteins with control variant 35436. Additionally, the RGA response to variants 37177, 37154, 37159, 37490, 37467, and 37472 is shown. [Figure 4E] 1 shows data from an IL12 reporter gene assay (RGA) experiment using IL12 HetFc and masked IL12 HetFc fusion protein variants. RGA experiments for each set of variants were performed twice (indicated by the designations "replicate 1" and "replicate 2"). Each graph shows a set of unmasked IL12 HetFc fusion proteins with control variant 30806, and a set of masked IL12 HetFc fusion proteins with control variant 35436. Additionally, the RGA response to variants 37179, 37163, 37492, and 37476 is shown. [Figure 4F]1 shows data from an IL12 reporter gene assay (RGA) experiment using IL12 HetFc and masked IL12 HetFc fusion protein variants. RGA experiments for each set of variants were performed twice (indicated by the designations "replicate 1" and "replicate 2"). Each graph shows a set of unmasked IL12 HetFc fusion proteins with control variant 30806, and a set of masked IL12 HetFc fusion proteins with control variant 35436. Additionally, RGA responses to variants 37181, 37162, 37155, 37494, 37475, and 37468 are shown. [Figure 4G] 1 shows data from an IL12 reporter gene assay (RGA) experiment using IL12 HetFc and masked IL12 HetFc fusion protein variants. RGA experiments for each set of variants were performed twice (indicated by the designations "replicate 1" and "replicate 2"). Each graph shows a set of unmasked IL12 HetFc fusion proteins with control variant 30806, and a set of masked IL12 HetFc fusion proteins with control variant 35436. Additionally, the RGA response to variants 37161, 37182, 37164, 37474, 37495, and 37477 is shown. [Figure 4H] 1 shows data from an IL12 reporter gene assay (RGA) experiment using IL12 HetFc and masked IL12 HetFc fusion protein variants. RGA experiments for each set of variants were performed twice (indicated by the designations "replicate 1" and "replicate 2"). Each graph shows a set of unmasked IL12 HetFc fusion proteins with control variant 30806, and a set of masked IL12 HetFc fusion proteins with control variant 35436. Additionally, the RGA response to variants 37166, 37165, 37180, 37479, 37478, and 37493 is shown. [Figure 4I]1 shows data from an IL12 reporter gene assay (RGA) experiment using IL12 HetFc and masked IL12 HetFc fusion protein variants. RGA experiments for each set of variants were performed twice (indicated by the designations "replicate 1" and "replicate 2"). Each graph shows a set of unmasked IL12 HetFc fusion proteins with control variant 30806, and a set of masked IL12 HetFc fusion proteins with control variant 35436. Additionally, the RGA response for variants 37169, 37171, 37168, 37482, 37484, and 37481 is shown. [Figure 4J] 1 shows data from an IL12 reporter gene assay (RGA) experiment using IL12 HetFc and masked IL12 HetFc fusion protein variants. RGA experiments for each set of variants were performed twice (indicated by the designations "replicate 1" and "replicate 2"). Each graph shows a set of unmasked IL12 HetFc fusion proteins with control variant 30806, and a set of masked IL12 HetFc fusion proteins with control variant 35436. Additionally, the RGA response to variants 37167, 37170, 37480, and 37483 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description The present disclosure relates to p40 domains that have been modified to have reduced binding to the cytokine receptor IL12Rβ1 subunit. In particular, the present disclosure relates to IL12 family member cytokines that contain modified p40 domains, more specifically IL12 and IL23 fusion proteins that contain modified p40 domains. The present disclosure further provides compositions and kits that include cytokines that contain modified p40 domains as described herein, as well as methods of using the compositions for the treatment of various diseases.

[0038] IL12 is an immunostimulatory cytokine capable of inducing antitumor responses by innate and adaptive immune cells. The use of IL12 as a therapeutic has been extensively studied in preclinical models of cancer, including mouse models of melanoma, renal cell carcinoma, breast cancer, and colon cancer. Antitumor activity of IL12 administration has been shown even when IL12 was administered at a later stage with large established tumors in mice. The strong antitumor effect of IL12 in preclinical models led to clinical trials of recombinant IL12. Unfortunately, toxicity, including treatment-related deaths of two patients, led to the halting of clinical trials of recombinant IL12. It is also notable that the recombinant cytokine has poor PK due to its small size. The present disclosure provides IL12 fusion proteins that have reduced affinity for the cytokine receptor IL12Rβ1 subunit, avoiding toxicity by reducing cytokine activity through the use of a modified p40 domain resulting in reduced IL12 binding and / or activity. The reduced cytokine activity of IL12 fusion proteins containing modified p40 domains may allow for increased dosage, exposure, and clinical efficacy compared to IL12 proteins with unmodified p40 domains, where dosage, exposure, and efficacy are limited by toxicity. The present disclosure also provides improved pharmacokinetics of IL12 by fusion to the Fc domain.

[0039] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0040] As used herein, the term "about" refers to approximately a ±10% variation from a given value, unless otherwise indicated. In some embodiments, the term "about" refers to a ±10% variation from a given value. In some embodiments, the term "about" refers to a ±8% variation from a given value. In some embodiments, the term "about" refers to a ±6% variation from a given value. In some embodiments, the term "about" refers to a ±4% variation from a given value. In some embodiments, the term "about" refers to a ±2% variation from a given value. It should be understood that such a variation is always included in any given value provided herein, whether or not it is specifically mentioned.

[0041] The use of the word "a" or "an," when used herein in conjunction with the term "comprising," can mean "one," but it also, in certain embodiments, coincides with the meaning of "one or more," "at least one," or "one or more."

[0042] As used herein, the terms "comprising," "having," "including," and "containing," as well as grammatical variations thereof, are inclusive or open ended and do not exclude additional unrecited elements and / or method steps. When used herein in the context of a use or method, the term "consisting essentially of" indicates that additional elements and / or method steps may be present, but that these additions do not substantially affect the manner in which the recited composition, method, or use functions. When used herein in the context of a composition, use, or method, the term "consisting of" excludes the presence of additional elements and / or method steps. A composition, use, or method described herein as comprising certain elements and / or steps may also consist essentially of those elements and / or steps in certain embodiments, and may consist of those elements and / or steps in other embodiments, whether or not those embodiments are specifically referred to.

[0043] "Fused" means that the components (e.g., a cytokine molecule and an Fc domain polypeptide or a masking moiety and an Fc domain polypeptide) are linked by a peptide bond, either directly or via one or more peptide linkers.

[0044] As used herein, the term "single chain" refers to a molecule that comprises amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the cytokine proteins or domains is a single chain cytokine molecule, i.e., an IL12 molecule in which the p35 and p40 domains are connected by a peptide linker to form a single peptide chain; or an IL23 molecule in which the p19 and p40 domains are connected by a peptide linker to form a single peptide chain.

[0045] As used herein, the terms "IL12 polypeptide," "single-chain (sc) IL12," and "IL12 p70" may be used interchangeably and generally refer to human IL12 comprising a contiguous single-chain amino acid sequence that includes the IL12p40 domain, such as a modified p40 domain described herein, and the IL12p35 domain.

[0046] As used herein, the term "affinity" or "binding affinity" or "binding activity" refers to the strength of binding interaction between a single biomolecule and its ligand / binding partner (e.g., p40 domain and IL12Rβ1). Binding can be characterized by an association constant or equilibrium association constant (KA) or its reciprocal, the equilibrium dissociation constant (KD). For any given equilibrium binding interaction between p40 domain and IL12Rβ1, the higher the KA (or lower KD) value, the more complexes of p40 and IL12Rβ1 will be present at equilibrium, and the less of their free forms. In general, when referring to KA or KD, "tighter" binding means a larger KA or smaller KD value. Proteins such as cytokines can bind much tighter to their receptors, and the more the binding activity increases, the more the binding activity decreases.10 ~10 12 "Close" or "very close" k a It has a value known as the value.

[0047] The strength or affinity of specific binding can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction, where a smaller KD represents a higher affinity and a larger KD represents a lower affinity. Binding properties can be determined by methods well known in the art, such as methods based on biolayer interferometry and surface plasmon resonance, including Biacore and Octet methodologies. Thus, both the association rate constant (ka) and the dissociation rate constant (kd) can be determined, with the ratio of kd / ka being equal to the equilibrium dissociation constant KD (see Nature 361:186-187 (1993) and Davies et al. (1990) Annual Rev Biochem 59:439-473). Both of these are incorporated by reference in their entirety for the methods described therein.

[0048] As used herein, the term "modification" or "mutation" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence, or a change to a moiety chemically linked to a protein. An "amino acid modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. For clarity of explanation, unless otherwise indicated, the amino acid modification is always to an amino acid encoded by DNA, e.g., the 20 amino acids that have codons in DNA and RNA. Thus, a "modified p40 domain" or a "mutated p40 domain" refers to a p40 polypeptide having an amino acid substitution, insertion, and / or deletion.

[0049] As used herein, the term "amino acid substitution" or "substitution" herein refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is a substitution with an amino acid that does not naturally occur at a particular position in the organism or in any organism. For clarity of explanation, a protein that is engineered to change the nucleic acid coding sequence but not the starting amino acid (e.g., replacing CGG (which codes for arginine) with CGA (which still codes for arginine) to increase host organism expression levels) is not an "amino acid substitution". That is, if a protein has the same amino acid at a particular starting position despite the creation of a new gene that codes for the same protein, it is not an amino acid substitution.

[0050] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, use, or composition disclosed herein.

[0051] Particular features, structures and / or characteristics described in connection with an embodiment disclosed herein can be combined in any suitable manner with the features, structures and / or characteristics described in connection with other embodiments disclosed herein to provide one or more additional embodiments.

[0052] It should also be understood that the positive recitation of a feature in one embodiment serves as a basis for excluding features in alternative embodiments. For example, when a list of options is presented for a given embodiment or claim, it should be understood that one or more options may be deleted from the list and that the shortened list may form an alternative embodiment, whether or not such alternative embodiment is specifically referenced.

[0053] Modified p40 domain The present disclosure describes modified p40 domains. Such modified p40 domains may have their amino acid sequences modified, for example, relative to the corresponding wild-type (WT) p40 sequence. As described herein, the modified p40 domains of the present disclosure may have a reduced binding affinity to another protein when compared to an unmodified p40 polypeptide. In some embodiments, such other protein to which the modified p40 domain binds with reduced affinity is one of the cognate IL12 receptors, for example, the receptor IL12Rβ1. The modified p40 proteins of the present disclosure may also have a stability (e.g., thermal stability measured as melting temperature (Tm) and / or chemical stability in the presence of certain reagents) that is about the same (e.g., ±5% variation in Tm compared to the WT p40 domain) or higher (e.g., greater than 5% increase in Tm) than that of the unmodified (e.g., WT) p40 domain.

[0054] The present disclosure provides p40 domains modified to have reduced binding to the IL12Rβ1 subunit, including the following list (numbered according to the mature p40 sequence; see SEQ ID NO: 10 and Table A): W15H, W15K, W15R, D18G, E45K, E45R, K58H, K58S, K58W, E59D, E59G, E59R, E59S, F60D, F60E, F60K, F60R, F60V, D62H, D62I, D62N, K62H, K62I, K ... 84E, K84I, K84L, K84V, K84W, K84Y, E86L, E86R, E86S, E86W, D93E, D93H, D93R, D93W, D161R, D161S, K195D, K197D, K197E , K197Q, K197T, K197W, W15H_K84L, K58H_K84I, E59D_K84W, E59G_K84W, E59R_K84E, E59R_K84W, E59R_E86W, E59D_D93H, E59R_D93R, E59R_K197E, E59R_K197W, F60E_K84W, F60R_K84Y, F60K_K197W, F60R_K197W, K84I_E86R, K84E_D93H, K84I _D93H, K84V_D93H, K84W_D93W, K84I_D161R, K84W_D161R, K84W_K197E, K84W_K197Q, K84W_K197W, E86W_D93E, E86R_K19 7D, E86W_K197W, W15H_K84L_K197Q, K58S_E59S_K195D, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, W15R_E59D_F60D_K197W, E45R_K58S_E59S_K195D,

[0055] As described herein, combinations of amino acid modifications at multiple positions in a single chain are identified using a "_" between each modified position. For example, "15_84" indicates that positions 124 and 186 are both modified in the referenced p40 polypeptide chain. Similarly, "15_84_197" indicates that positions 124, 133, and 180 are all modified in the referenced p40 polypeptide chain.

[0056] The present disclosure provides p40 domains modified to have reduced binding to the IL12Rβ1 subunit, the sequences of which are captured in Table M (SEQ ID NOs: 12-57) and nucleic acid sequences encoding the modified p40 domains (SEQ ID NOs: 137-182) of the following list (numbered according to the mature p40 sequence; see SEQ ID NO: 10 and Table A); W15H, W15K, W15R, E45R, K58H, K58S, E59D, E59R, E59S, F60D, F60K, F60R, K84E, K84I, K84L, K84W, K84Y, E86R, E86W, D93E, D93H, D93R, D161R, K195D, K197 D, K197Q, K197T, K197W, W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_ E86R, K84W_D161R, K84W_K197W, E86R_K197D, E86W_D93E, W15H_K84L_K197Q, K58H_E86R_K19 7D, K58S_E59S_K195D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, W15R_E59D_F60D_K197W, E45R_K58S_E59S_K195D.

[0057] The present disclosure relates to p40 domains modified to have reduced binding to the IL12Rβ1 subunit, the sequences of the modified p40 domains being provided in Table M (SEQ ID NOs: 12, 13, 19, 20, 24, 27, 30, 34, 38, 39, 41, 47, 48, 49, 51, 52, 54, 55, 56, 57, 42, 44, 45, 46, 50, 53, 40, 43), and ... The nucleic acid sequences encoding the modified p40 domains provided in the following list (numbered according to the mature p40 sequence; SEQ ID NOs: 137, 138, 144, 145, 149, 152, 155, 159, 163, 164, 166, 172, 173, 174, 176, 177, 179, 180, 181, 182, 167, 169, 170, 171, 175, 178, 165, 168) are captured in the following list (numbered according to the mature p40 sequence; SEQ ID NOs: 137, 138, 144, 145, 149, 152, 155, 159, 163, 164, 166, 172, 173, 174, 176, 177, 179, 180, 181, 182, 167, 169, 170, 171, 175, 178, 165, 168). See No. 10 and Table A); W15H, W15K, E59R, E59S, K84E, K84W, E86W, D161R, K197T, K197W, W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, E86W_D93E, W15H_ Modified p40 domains are provided that contain at least one amino acid substitution or group of substitutions from: K84L_K197Q, K58H_E86R_K197D, K58S_E59S_K195D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, W15R_E59D_F60D_K197W, E45R_K58S_E59S_K195D.

[0058] In general, the function of the p40 domain modified to have reduced binding to the IL12Rβ1 subunit is to provide a biologically active IL12 family protein with reduced toxicity and a broader therapeutic window. As used herein, "therapeutic window" refers to the range of dosages that can effectively treat a disease without having toxic effects; for example, the area between an adverse response and a desired response is the therapeutic window. Examples of toxic effects of IL12 administration include, but are not limited to, skin toxicity, local inflammation, stomatitis, systemic inflammation, fatigue, weight loss, vomiting, anorexia, hematologic toxicity, such as anemia, lymphopenia, neutropenia, thrombocytopenia, hypoproteinemia, hypophosphatemia, and hypocalcemia, lymph node enlargement, splenomegaly, and bone marrow hyperplasia, bone marrow toxicity, muscle toxicity, neurotoxicity, liver toxicity, such as liver dysfunction, elevated aminotransferases, elevated aspartate aminotransferase (AST), elevated alanine aminotransferase (ALT), elevated alkaline phosphatase, hyperbilirubinemia, and hypoalbuminemia, elevated creatinine, diarrhea, dyspnea, and gastrointestinal bleeding. In some embodiments, toxic effects refer to dose-limiting toxicity. Other toxic effects of IL12 administration are known to those skilled in the art.

[0059] The p40 domain is a secreted protein, either as a monomer, homodimer, or heterodimer with p19 or p35, and thus has an N-terminal secretory signal peptide, or SP (SEQ ID NO:122, also residues 1-22 of SEQ ID NO:123), that targets the newly translated protein to the endoplasmic reticulum (ER) for translocation and subsequent secretion from the cell. Upon translocation to the ER, the SP is cleaved from the remainder of the p40 protein. In this regard, proteins containing the SP are often referred to as "protein precursors" or "precursors," and the protein secreted after SP cleavage is referred to as the "mature" protein. When applying numbering to amino acids within a protein sequence, it is common to follow one of two conventions: 1) numbering the protein beginning with the first residue at the N-terminus of the SP as 1, or 2) numbering the protein beginning with the first residue at the N-terminus of the mature protein after SP cleavage as 1. Table A provides the numbering of the amino acids within the p40 protein precursor and mature protein according to both conventions. In this disclosure, amino acid substitutions made to the p40 domains are numbered according to their position in the mature p40 protein (SEQ ID NO:10).

[0060] [Table A] TIFF2024535925000002.tif237165TIFF2024535925000003.tif237165TIFF2024535925000004.tif237165TIFF2024535925000005.tif237165 TIFF2024535925000006.tif237165TIFF2024535925000007.tif237165TIFF2024535925000008.tif237165TIFF2024535925000009.tif159165

[0061] The iterative structure-guided in silico mutagenesis and structure evaluation approach described herein, specifically in Example 1, allowed for the creation of a concise library of designs with a wide range of predicted affinities between p40 and IL12Rβ1. This in silico approach offers advantages in both efficiency of design and breadth of solutions obtained, compared to other common in vitro approaches to protein mutation and selection known in the art. For example, random or semi-random mutagenesis of individual amino acids followed by expression of the protein library and screening for mutations with the desired properties may require screening of a very large number of samples, as well as multiple rounds of cloning, protein expression, and selection, to repeatedly test combinations of mutations to obtain designs with the desired activity. In contrast, the iterative in silico design, mutagenesis, and selection approach described herein allowed for the pre-preparation of design libraries containing single and multiple amino acid substitutions to achieve a wide range of predicted affinities between p40 and IL12Rβ1.

[0062] Furthermore, the in silico structure-guided approach described herein enabled the discovery of mutations that achieve both a desired reduction in the predicted affinity between p40 and IL12Rβ1 with a positive or minimal negative effect on the predicted stability of the uncomplexed p40 domain, as well as the discovery of pairs or groups of mutations that are complementary or synergistic in reducing the predicted affinity between p40 and IL12Rβ1 and / or maintaining or improving the predicted stability of the uncomplexed p40 domain.

[0063] For example, structural analysis of the p40 domain complexed with IL12Rβ1, as described in Example 1, identified amino acids K84, E86, and K197, among others, as hot spots at the p40-IL12Rβ1 interface, i.e., key residues contributing to stability and affinity. However, in structural analysis of uncomplexed p40, K84, E86, and K197 were found to also contribute significantly to the predicted stability of uncomplexed p40. Therefore, it was important to consider the effect of mutations at these positions on both the predicted affinity between p40 and IL12Rβ1 and the predicted stability of uncomplexed p40. A typical approach to protein mutagenesis known in the art is "alanine scanning," in which all or selected amino acid groups in a protein of interest are individually replaced with alanine and the effect of the replacement is measured by binding or functional assays using the mutated protein. Considering the contribution of K84, E86, and K197 to the predicted stability of p40 through steric and electrostatic interactions with neighboring residues, it is predicted that mutations to alanine at these positions may be detrimental to the stability and / or other biophysical properties of the mutant p40 domain (e.g., increased aggregation tendency caused by increased exposure of hydrophobic residues in the vicinity of the substitution). Thus, the in silico approach to design described herein, specifically in Example 1, identified several amino acid substitutions at these positions that result in a decrease in the predicted affinity between p40 and IL12Rβ1 that is comparable to or greater than that caused by substitution with alanine, but have a smaller negative and sometimes positive effect on the predicted stability of uncomplexed p40. For example, modeling of the amino acid substitution K84W revealed increased steric complementarity with the neighboring amino acid in uncomplexed p40 relative to unsubstituted lysine at the same position, resulting in an improvement in predicted stability.

[0064] It has also been discovered that a substitution identified as favorable at a particular amino acid site when substituted alone may not be a favorable substitution at the same site when substituted in combination with another or multiple other amino acids in the vicinity of the same structure. For example, the substitution K84W described above may or may not be a favorable substitution at amino acid K84 when combined with other amino acid substitutions. In fact, when combined with the substitution E86R, the substitution K84I is preferred over K84W because the smaller size of K84I allows E86R to adopt a conformation in p40 that forms favorable hydrogen bonds that result in predicted improved stability. These favorable hydrogen bonds may be hindered by steric clashes in the combination of E86R and K84W. Similarly, the substitution E86R alone is prevented from adopting this favorable conformation by steric clashes with the unmodified K84. While E86R alone is predicted to reduce the affinity between p40 and IL12Rβ1 as desired, it is predicted to have a significant and detrimental effect on the stability of uncomplexed p40. The structural synergy produced by the combination of K84I and E86R substitutions instead confers a benefit on p40 stability, thus demonstrating the advantage of the in silico modeling approach described herein to discover complementary substitutions.

[0065] In another example, structural analysis of the p40 domain complexed with IL12Rβ1, as described in Example 1, identified amino acid W15 as a hotspot in the p40-IL12Rβ1 interface. Molecular dynamics simulations and analysis of uncomplexed p40 revealed significant conformational changes in the region around W15, such that in the uncomplexed state, W15 was observed to contribute a significantly larger intramolecular p40 contact area and a hydrogen bond between its indole nitrogen and the backbone oxygen of H83 compared to the W15 conformation observed in the p40 domain complexed with IL12Rβ1, allowing for an increased intermolecular contact area between p40 and IL12Rβ1 at the expense of the intramolecular contact area in p40. In silico mutagenesis modeling as described in Example 1 allowed the discovery of mutations and mutations involving W15 that reduce the predicted affinity between p40 and IL12Rβ1 as desired, but also minimize the detrimental effect on the predicted stability of uncomplexed p40 by restoring or restoring intramolecular contacts made by W15 that may be lost upon substitution. For example, the substitution W15H was designed to preserve the hydrogen bond made to the backbone oxygen of H83, minimizing the detrimental effect on the predicted stability of mutations that may occur at position W15. In another example, the mutation group W15R_E59D_F60D_K197W was designed to introduce a strong bidentate hydrogen bonding interaction between W15R and F60D, and further restore intramolecular packing in the uncomplexed state with K197W, resulting in a design that minimizes the deleterious effects of mutations at these positions on the predicted stability of uncomplexed p40, while also achieving a large reduction in the predicted affinity between p40 and IL12Rβ1.

[0066] Using this approach, described in Example 1, preferred substitutions and groups of substitutions at the p40-IL12Rβ1 interface hotspot and adjacent residues were identified to generate a design library that spans a wide range of predicted affinities between p40 and IL12Rβ1 while positively or minimally adversely affecting the predicted stability of the uncomplexed p40 domain. Preferred substitutions and groups of substitutions are provided in Table B.

[0067] [Table B] TIFF2024535925000011.tif222165

[0068] In various embodiments, described herein are modified p40 domains that have reduced binding to the IL12Rβ1 subunit compared to a p40 domain that does not contain such amino acid substitution(s). In some embodiments, the modified p40 domain comprises one or more amino acid substitution(s) selected from Table C.

[0069] [Table C] TIFF2024535925000013.tif197165

[0070] In various embodiments, modified p40 domains are described herein that may contain at least one amino acid substitution or set of amino acid substitutions that have reduced binding affinity to the IL12Rβ1 subunit compared to a p40 domain that does not contain such amino acid substitution(s). In some embodiments, such one or more amino acid substitution(s) or set(s) of substitutions are (numbered according to the mature p40 sequence set forth in SEQ ID NO: 10 and Table A): W15H, W15K, W15R, D18G, E45K, E45R, K58H, K58S, K58W, E59D, E59G, E59R, E59S, F60D, F60E, F60K, F60R, F60V, D62H, D62I, D62N, K8 4E, K84I, K84L, K84V, K84W, K84Y, E86L, E86R, E86S, E86W, D93E, D93H, D93R, D93W, D161R, D161S, K195D, K197D, K19 7E, K197Q, K197T, K197W, W15H_K84L, K58H_K84I, E59D_K84W, E59G_K84W, E59R_K84E, E59R_K84W, E59R_E86W, E59D _D93H, E59R_D93R, E59R_K197E, E59R_K197W, F60E_K84W, F60R_K84Y, F60K_K197W, F60R_K197W, K84I_E86R, K84E _D93H, K84I_D93H, K84V_D93H, K84W_D93W, K84I_D161R, K84W_D161R, K84W_K197E, K84W_K197Q, K84W_K197W, E86W _D93E, E86R_K197D, E86W_K197W, W15H_K84L_K197Q, K58S_E59S_K195D, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, W15R_E59D_F60D_K197W and / or E45R_K58S_E59S_K195D (see, e.g., Table B).

[0071] In certain embodiments, described herein are modified p40 domains that have reduced binding affinity to the IL12Rβ1 subunit compared to a p40 domain that does not contain such amino acid substitution(s), and such modified p40 domains have the following amino acid substitution(s) or set(s) of amino acid substitutions (numbered according to the mature p40 sequence set forth in SEQ ID NO: 10 and Table A): W15H, W15K, W15R, D18G, E4 5K, K58H, K58W, E59D, E59G, E59R, F60D, F60E, F60K, F60R, F60V, D62H, D62I, D62N, K84E, K84I, K84L, K84V, K84W, K84Y, E86L, E86R, E86S, E86W, D93E, D93H, D93R, D93W, D161R, D161S, K197D, K197E, K197Q, K197T, K197W, W15H_K84L, K58H_K84I, E59D_K 84W, E59G_K84W, E59R_K84E, E59R_K84W, E59R_E86W, E59D_D93H, E59R_D93R, E59R_K197E, E59R_K197W, F60E_K84W, F60R_K8 4Y, F60K_K197W, F60R_K197W, K84I_E86R, K84E_D93H, K84I_D93H, K84V_D93H, K84W_D93W, K84I_D161R, K84W_D161R, K84W_K 197E, K84W_K197Q, K84W_K197W, E86W_D93E, E86R_K197D, E86W_K197W, W15H_K84L_K197Q, K58S_E59S_K195D, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H and / or W15R_E59D_F60D_K197W (see, e.g., Table C).

[0072] In some embodiments, the p40 domain has a decreased binding affinity to the IL12Rβ1 subunit (e.g., compared to a p40 domain that does not contain such amino acid substitution(s)) and contains the following amino acid substitution(s) or set(s) of substitutions (numbered according to the mature p40 sequence set forth in SEQ ID NO: 10): W15H, W15K, W15R, E45R, K58H, K58S, E59D, E59R, E59S, F60D, F60K, F60R, K84E, K84I, K84L, K84W, K84Y, E86R, E86W, D93E, D93H, D93R, D161R, K195D, K197D, K197Q, K197T, K197 W, W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D1 61R, K84W_K197W, E86R_K197D, E86W_D93E, W15H_K84L_K197Q, K58H_E86R_K197D, K58 Described herein are modified p40 domains comprising one or more of S_E59S_K195D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, W15R_E59D_F60D_K197W and / or E45R_K58S_E59S_K195D. The amino acid sequences of the 46 modified p40 domains, each containing one of these 46 amino acid substitutions, are provided in Table M herein and comprise or consist of an amino acid sequence set forth in SEQ ID NOs: 12-57, respectively, or a sequence having at least about 90%, 95%, 97%, or at least about 99% sequence identity thereto. In various embodiments, such amino acid sequences of the 46 modified p40 domains are provided in Table M herein and are encoded by nucleic acid sequences corresponding to SEQ ID NOs: 137-182, respectively.

[0073] In some embodiments, the p40 domain has a decreased binding affinity to the IL12Rβ1 subunit (e.g., compared to a p40 domain that does not contain such amino acid substitution(s)), and contains the following amino acid substitution(s) or substitution(s) (numbered according to the mature p40 sequence set forth in SEQ ID NO: 10): W15H, W15K, W15R, K58H, E59D, E59R, F60D, F60K, F60R, K84E, K84I, K84L, K84W, K84Y, E86R, E86W, D93E, D93H, D93R, D161R, K197D, K197Q, K197T, K197W, Described herein are modified p40 domains comprising one or more of W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, E86W_D93E, W15H_K84L_K197Q, K58H_E86R_K197D, K58S_E59S_K195D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, and / or W15R_E59D_F60D_K197W. The amino acid sequences of the 41 modified p40 domains, each containing one of these 41 amino acid substitutions, are set forth in Table M herein and comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 12-14, 16, 18, 19, 21-34, 36-56, respectively, or a sequence having at least about 90%, 95%, 97%, or at least about 99% sequence identity thereto. In various embodiments, such amino acid sequences of the 41 modified p40 domains are set forth in Table M herein and are encoded by nucleic acid sequences corresponding to SEQ ID NOs: 137-139, 141, 143, 144, 146-159, 161-181, respectively.

[0074] In some embodiments, the disclosure provides a modified p40 domain having a reduced binding affinity to the IL12Rβ1 subunit (e.g., compared to a p40 domain that does not contain such amino acid substitution(s)), comprising the following amino acid substitution(s) or substitution(s) (numbered according to the mature p40 sequence; see SEQ ID NO: 10): W15H, W15K, E59R, E59S, K84E, K84W, E86W, D161R, K197T, K197W, W15H_K84L, E59D_D93H, E59R_D93R, F6 0K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, E86W_D93E, W15H_K84L_K197Q, K58H_E86R_K197D, K58S_E59S_K195D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, W15R_E59D_F60D_K197W and / or E45R_K58S_E59S_K195D are described. The amino acid sequences of the modified p40 domains, each containing one of these amino acid substitutions, are provided in Table M and may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 12, 13, 19, 20, 24, 27, 30, 34, 38, 39, 41, 47, 48, 49, 51, 52, 54, 55, 56, 57, 42, 44, 45, 46, 50, 53, 40 and 43, respectively, or a sequence having at least about 90%, 95%, 97%, or at least about 99% sequence identity thereto. In various embodiments, such amino acid sequences are provided in Table M and are encoded by nucleic acid sequences corresponding to SEQ ID NOs: 137, 138, 144, 145, 149, 152, 155, 159, 163, 164, 166, 172, 173, 174, 176, 177, 179, 180, 181, 182, 167, 169, 170, 171, 175, 178, 165, and 168, respectively.

[0075] In some embodiments, the disclosure provides a modified p40 domain having reduced binding affinity to the IL12Rβ1 subunit (e.g., compared to a p40 domain that does not contain such amino acid substitution(s)), comprising the following amino acid substitution(s) or group(s) of substitutions (numbered according to the mature p40 sequence; see SEQ ID NO: 10): W15H, W15K, E59R, K84E, K84W, E86W, D161R, K197T, K197W, W15H_K84L, E59D_D93H, E59R _D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, E86W_D93E, W15H_K84L_K197Q, K58H_E86R_K197D, K58S_E59S_K195D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H and / or W15R_E59D_F60D_K197W. The amino acid sequences of the modified p40 domains, each containing one of these amino acid substitutions, are provided in Table M and may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 12, 13, 19, 24, 27, 30, 34, 38, 39, 41, 47, 48, 49, 51, 52, 54, 55, 56, 57, 42, 44, 45, 46, 50, 53 and 40, respectively, or a sequence having at least about 90%, 95%, 97%, or at least about 99% sequence identity thereto. In various embodiments, such amino acid sequences are provided in Table M and are encoded by nucleic acid sequences corresponding to SEQ ID NOs: 137, 138, 144, 149, 152, 155, 159, 163, 164, 166, 172, 173, 174, 176, 177, 179, 180, 181, 182, 167, 169, 170, 171, 175, 178, and 165, respectively.

[0076] In some embodiments, described herein is a modified p40 domain having reduced binding affinity to one or more cognate IL12 receptor(s) compared to an unmodified p40 domain, the modified p40 domain comprising one or more amino acid substitutions relative to the wild-type human mature IL12 p40 domain sequence set forth in SEQ ID NO: 10, the one or more amino acid substitutions being located at one or more of E45, D62 and / or D161, and the numbering of the amino acid residues being based on the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the one or more amino acid substitutions at the one or more positions are K, H, I, N, R and / or S substitutions. In certain embodiments, the one or more amino acid substitutions are E45K, D62H, D62I, D62N, D161R and / or D161S.

[0077] In yet another embodiment, a modified p40 domain comprises one or more amino acid substitution(s) relative to the wild-type human mature IL12 p40 domain sequence set forth in SEQ ID NO: 10, wherein the one or more amino acid substitution(s) are W15H, W15K, W15R, D18G, E45K, K58H, K58W, E59D, E59G, E59R, F60D, F60E, F60K, F60R, F60V, D62H, D62I ... Described herein are modified p40 domains that are 2N, K84E, K84I, K84L, K84V, K84W, K84Y, E86L, E86R, E86S, E86W, D93E, D93H, D93R, D93W, D161R, D161S, K197D, K197E, K197Q, K197T, K197W, or any combination thereof, where the amino acid residue numbering is based on the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, such modified p40 domains comprise one or more, two or more, or three or more amino acid substitutions. In certain embodiments, the one or more amino acid substitution(s) may be W15H, W15K, D18G, E45K, K58H, K58W, E59G, E59R, F60V, D62H, D62I, D62N, K84E, K84W, E86L, E86S, E86W, D93H, D93W, D161R, D161S, K197E, K197Q, K197T, and / or K197W. In some of these embodiments, the one or more amino acid substitutions may be W15H, W15K, E59R, K84E, K84W, E86W, D161R, K197T, and / or K197W.

[0078] In embodiments in which the modified p40 domain comprises two or more amino acid substitutions relative to the wild-type p40 sequence set forth in SEQ ID NO: 10, the two or more amino acid substitutions are selected from the group consisting of W15H_K84L, K58H_K84I, E59D_K84W, E59G_K84W, E59R_K84E, E59R_K84W, E59R_E86W, E59D_D93H, E59R_D93R, E59R_K197E, E59R_K197W, F60E_K84W, F60R_K84Y, F60K_K197W, F60R_K197W, K84I_E86R, K84E_D93H, K84I_D93H, K84V_D93H, K84W_D93W, K84I_D161R, K84W_D161R, K84W_K197E, K84W_K197Q, K84W_K197W, E86W_D93E, E86R_K197D, E86W_K197W, W15H_K84L_K197Q, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H and / or W15R_E59D_F60D_K197W.

[0079] In certain embodiments, the two or more amino acid substitutions are W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, E86W_D93E, W15H_K84L_K197Q, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, and / or W15R_E59D_F60D_K197W.

[0080] In other embodiments, the two or more amino acid substitutions are K58H_K84I, E59D_K84W, E59G_K84W, E59R_E86W, E59R_K197E, E59R_K197W, E59R_K84E, E59R_K84W, F60E_K84W, F60R_K197W, K84E_D93H, K84I_D161R, K84I_D93H, K84V_D93H, K84W_D93W, K84W_K197E, K84W_K197Q, and / or E86W_K197W.

[0081] In yet other embodiments, the two or more amino acid substitutions are W15H_K84L, K58H_K84I, E59D_K84W, E59G_K84W, E59R_K84E, E59R_K84W, E59R_E86W, E59D_D93H, E59R_D93R, E59R_K197E, E59R_K197W, F60E_K84W, F60R_K84Y, F60K_ K197W, F60R_K197W, K84I_E86R, K84E_D93H, K84I_D93H, K84V_D93H, K84W_D93W, K84I_D161R, K84W_D161R, K84W_K197E, K84W_K197Q, K84W_K197W, E86W_D93E, E86R_K197D, and / or E86W_K197W.

[0082] In certain embodiments, the two or more amino acid substitutions are W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, and / or E86W_D93E.

[0083] In embodiments in which the modified p40 domain comprises three or more amino acid substitutions relative to the wild-type p40 sequence set forth in SEQ ID NO: 10, the three or more amino acid substitutions are W15H_K84L_K197Q, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H and / or W15R_E59D_F60D_K197W.

[0084] In some embodiments, the modified p40 domains of the disclosure include the following: W15R_E59D_F60D_K197W, W15H_K84L_K197Q, E59D_K84W_K197W, K58H_E86R_K197D, W15H_K84L, F60R_K84E_K197W, K84W_K197W, F60K_K197W, E86R_K197D, K84I_E86R_D93H, E59R_D93 R, K84I_E86R, W15K, K84W_D161R, E45R_K58S_E59S_K195D, E59D_D93H, F60R_K84Y, E86W_D93E, K58S_E59S_K195D, K84E, K197T, E59R, W15H, K84W, K197W, E59S, D161R and / or E86W. In some of these embodiments, the modified p40 domain comprises an amino acid substitution or set of amino acid substitutions selected from one or more of the following: W15H_K84L_K197Q, E59D_K84W_K197W, K84W_K197W, F60K_K197W, E59R_D93R, W15K, and / or F60R_K84Y.

[0085] In various embodiments of the present disclosure, the modified p40 domain may include one or more amino acid substitution(s) selected from Table C. In at least some of these embodiments, the modified p40 domain comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 12-14, 16, 18, 19, 21-34, and 36-56. In some of these embodiments, the modified p40 domain comprises or consists of an amino acid sequence having at least about 99% or 100% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 12-14, 16, 18, 19, 21-34, and 36-56. In some embodiments, a modified p40 domain may have at least about a 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or at least about a 600-fold reduction in binding affinity to at least one of its cognate receptors (e.g., IL12Rβ1) when compared to an unmodified p40 domain that does not contain one or more amino acid modifications disclosed herein. Such a reduction in binding affinity may be measured by SPR, relative NK cell abundance, CD8+ T cell IFNγ release assays, reporter gene assays, assays using cells transfected with one or more IL12 receptor types (e.g., IL12Rβ1), assays using stimulated T cells, or a combination of these assays.

[0086] In some embodiments, an IL12 fusion protein comprising an IL12 polypeptide comprising a modified p40 domain as described herein (e.g., an IL12 HetFc fusion protein) may have at least about 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or at least about 600-fold reduced binding affinity for at least one cognate IL12 receptor (e.g., IL12Rβ1) when compared to a corresponding IL12 fusion protein comprising an unmodified p40 domain that does not contain one or more amino acid modifications disclosed herein. Such a decrease in binding affinity may be measured by SPR, relative NK cell abundance, CD8+ T cell IFNγ release assays, reporter gene assays, assays using cells transfected with one or more IL12 receptor types (e.g., IL12Rβ1), assays using stimulated T cells, or a combination of these assays.

[0087] In some embodiments, a modified p40 domain of the present disclosure may have a reduction in binding affinity for at least one of its cognate receptors (e.g., IL12Rβ1) of about 5 to about 2000, about 10 to about 1500, about 15 to about 1000, about 20 to about 1800, about 20 to about 1600, about 20 to about 1400, about 20 to about 1200, about 20 to about 100, about 150 to about 100, about 150 to about 12000, about 100 to about 12000 or less, or about 1500 to about 12000 or less, as compared to an unmodified p40 domain that does not contain one or more amino acid modifications disclosed herein.

[0088] In some embodiments, a modified p40 domain of the disclosure may have a reduction in binding affinity for at least one of its cognate receptors (e.g., IL12Rβ1) of about 5-fold to about 1-1000, about 5-fold to about 1-800, about 5-fold to about 1-600, about 1-10-fold to about 1-500, about 1-10-fold to about 1-300, or about 1-20-fold to about 1-200, relative to the binding affinity of an unmodified wild-type p40 domain, whose sequence is set forth in SEQ ID NO:10, as determined in a reporter gene assay.

[0089] In some embodiments, a modified p40 domain of the disclosure may have a reduction in binding affinity for at least one of its cognate receptors (eg, IL12Rβ1) of about 20-fold to about 200-fold or less.

[0090] As used herein, unless otherwise defined, the terms "reduced binding" and "reduced binding affinity" in the context of a modified p40 domain disclosed herein that contains one or more amino acid modification(s) refer to a measurably reduced binding affinity of the modified p40 domain, or a fusion protein containing such a modified p40 domain, to at least one of its cognate receptors (e.g., IL12Rβ1) relative to a wild-type p40 domain that does not contain such one or more amino acid modification(s), where the reduced binding is determined using any of the analytical methods described herein. Such reduced binding or reduced binding affinity may be measured directly via p40-receptor interaction experiments or indirectly by analyzing changes in downstream processes that are affected by the reduced binding affinity of the modified p40 domain.

[0091] Functional IL12 activity may be measured, for example, by assays measuring NK cell relative abundance, immune, e.g., IFNγ production by NK cells, or a CD8+ T cell IFNγ release assay (e.g., as shown in Example 4). In some embodiments, a modified p40 domain (or a fusion protein comprising such a p40 domain as described herein) may exhibit complete loss of binding affinity for IL12Rβ1, e.g., such that the binding activity of an IL12 polypeptide comprising a modified p40 domain cannot be detected using established detection assays such as SPR, NK, CD8+ T cell or other cell-based assays.

[0092] In some embodiments, a modified p40 domain of the present disclosure and / or a fusion protein comprising such a modified p40 domain may have a thermal stability within ±5° C., ±4° C., ±3° C., ±2° C. or ±1° C. of the thermal stability of a fusion protein containing an unmodified wild-type p40 domain having the sequence set forth in SEQ ID NO: 10, as determined by differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC) as further described herein in the context of the fusion protein. Thus, in various embodiments, one or more amino acid modifications (e.g., substitutions) may not significantly affect (e.g., by at most about ±2° C. or less) the thermal stability of the p40 domain and / or fusion protein comprising the modified p40 domain, respectively, as compared to the unmodified p40 domain or the fusion protein containing the unmodified p40 domain.

[0093] IL12 fusion protein One aspect of the present disclosure provides cytokine fusion proteins containing modified p40 domains, in particular IL12 and IL23 fusion proteins, also referred to herein as IL12 HetFc fusion proteins. The IL12 fusion proteins described herein comprise an IL12 polypeptide, an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, and optionally a masking moiety (MM) that reduces, inhibits or blocks IL12 activity. The IL12 HetFc fusion proteins containing MM are also referred to herein as masked IL12 fusion proteins or masked IL12 HetFc fusion proteins. In general, the function of IL12 fusion proteins with modified p40 domains and masked IL12 fusion proteins with modified p40 domains is to provide biologically active IL12 proteins with reduced toxicity and broader therapeutic window. In some embodiments, the modified p40 domain of such IL12 fusion proteins may comprise one or more amino acid modification(s) listed in Table B herein. In certain embodiments, the modified p40 domain of the IL12 fusion protein may include one or more amino acid modification(s) listed in Table C herein.

[0094] In some embodiments, the function of an IL12 fusion protein comprising a modified p40 domain may be to provide a biologically active IL12 protein that has a reduced binding affinity to at least one of the cognate IL12 receptors, e.g., the IL12Rβ1 subunit, when compared to an IL12 polypeptide that does not comprise a modified p40 domain, and that may provide reduced toxicity in vivo and therefore a broader therapeutic window. The above may be similarly envisaged for an IL23 fusion protein comprising an IL23 polypeptide instead of an IL12 polypeptide, as further described herein.

[0095] In various embodiments, an IL12 HetFc fusion protein herein comprises (i) an IL12 polypeptide comprising a modified p40 domain, (ii) a heterodimeric Fc domain (HetFc) comprising a first Fc polypeptide and a second Fc polypeptide, and, optionally, (iii) a masking moiety (MM) that inhibits or at least reduces IL12 activity compared to an unmasked IL12 polypeptide. In some embodiments, an IL12 HetFc fusion protein comprising a MM may also be referred to herein as a masked IL12 fusion protein or a masked IL12 HetFc fusion protein.

[0096] In some embodiments, an IL12 fusion protein comprising a modified p40 domain of the present disclosure (e.g., IL12 The IL12 fusion protein (HetFc fusion protein) may have a reduction in binding affinity for at least one of its cognate receptors (e.g., IL12Rβ1) of about 5 to about 1 / 2000, about 1 / 10 to about 1 / 1500, about 1 / 15 to about 1 / 1000, about 1 / 20 to about 1 / 800, about 1 / 20 to about 1 / 600, about 1 / 20 to about 1 / 400, about 1 / 20 to about 1 / 200, about 1 / 20 to about 1 / 100, about 1 / 50 to about 1 / 100, about 1 / 50 to about 1 / 2000, about 1 / 100 to about 1 / 2000 or less, or about 1 / 500 to about 1 / 2000 or less, as compared to an IL12 fusion protein comprising an unmodified p40 domain that does not contain one or more amino acid modifications disclosed herein.

[0097] In some embodiments, an IL12 fusion protein comprising a modified p40 domain of the present disclosure (e.g., an IL12 HetFc fusion protein) may have a reduction in binding affinity for at least one of its cognate receptors (e.g., IL12Rβ1) of about 5-fold to about 1 / 1000, about 5-fold to about 1 / 800, about 5-fold to about 1 / 600, about 1 / 10- to about 1 / 500, about 1 / 10- to about 1 / 300, or about 1 / 20- to about 1 / 200, relative to the binding affinity of an IL12 fusion protein comprising an unmodified wild-type p40 domain, the sequence of which is set forth in SEQ ID NO:10, as determined in a reporter gene assay.

[0098] In some embodiments, an IL12 fusion protein comprising a modified p40 domain (e.g., an IL12 HetFc fusion protein) may have about 20-fold to about 200-fold or less reduced binding affinity for at least one of its cognate receptors (e.g., IL12Rβ1) compared to an IL12 fusion protein comprising an unmodified p40 domain that does not contain one or more amino acid modifications disclosed herein.

[0099] Masked IL12 fusion protein constructs "Masked IL12 fusion protein", as used herein, is specifically meant to include fusion proteins described herein that include any cytokine from the IL12 family of heterodimeric cytokines, and thus specifically means to include IL12 and IL23 masked fusion proteins. In certain places, "masked cytokine fusion protein" may be used, which is also meant to include masked IL12 or IL23 fusion proteins. Masked IL12 fusion proteins may also be referred to herein as "masked HetFc IL12 fusion proteins" when the fusion protein is, in some embodiments, made with a modified Fc polypeptide described herein. The terms "masked IL12 fusion protein" and "masked cytokine fusion protein" are also meant to include any masked HetFc IL12 fusion protein.

[0100] It should be noted that the numbering of the linker is for clarity of description only, and the numbers are interchangeable. Any given linker may have different numbers depending on the configuration or geometry. L1 in one geometry is not necessarily the same linker as L1 in a different geometry. Moreover, similar geometries may number the linker differently.

[0101] In certain embodiments, IL12 fusion proteins or masked IL12 fusion proteins containing a modified p40 domain described herein demonstrate a complete reduction in the potency of the IL12 polypeptide, in that IL12 activity is not detectable, for example by NK or other cell-based assays. In this case, a "fold reduction in potency" cannot be calculated since the activity is below the limit of detection.

[0102] Methods for measuring binding or functional IL12 activity are known in the art and described herein.In certain embodiments, binding activity can be measured using surface plasmon resonance (SPR).Functional IL12 activity can be measured, for example, in NK cell relative abundance or CD8+T cell IFNγ release assay (see also, for example, Example 3).

[0103] Thus, in certain embodiments, provided herein are IL12 fusion proteins and masked IL12 fusion proteins containing modified p40 domains that exhibit at least 5-fold, 1 / 10-fold, 1 / 15-fold, 1 / 20-fold, 1 / 30-fold, 1 / 40-fold, 1 / 50-fold, 1 / 100-fold, 1 / 200-fold, 1 / 300-fold, 1 / 400-fold, 1 / 500-fold, 1 / 600-fold, 1 / 700-fold, 1 / 800-fold, 1 / 900-fold, 1 / 1000-fold, 1 / 1200-fold, 1 / 1500-fold, 1 / 2000-fold, 1 / 2500-fold, 1 / 3000-fold, or even greater reduced binding activity, functional IL12 activity, or potency as compared to a suitable control as measured by SPR, NK cell, CD8+ T cell IFNγ release, or other suitable assay.

[0104] In some embodiments, an IL12 polypeptide used in a fusion protein described herein may comprise a modified p40 domain as described herein, e.g., a p40 domain sequence comprising any one or more of the substitutions listed in Tables B and C. Such modified p40 domains may have reduced binding to the IL12 receptor as compared to an unmodified (e.g., WT) p40 domain. In various embodiments, such modified p40 domains may comprise or consist of any one of the amino acid sequences set forth in SEQ ID NOs: 12-57, or a sequence having at least about 90%, 95%, 97%, or at least about 99% sequence identity thereto. In other embodiments, such modified p40 domains may comprise or consist of an amino acid sequence set forth in SEQ ID NOs: 12-14, 16, 18, 19, 21-34, 36-56, respectively, or a sequence having at least about 90%, 95%, 97%, or at least about 99% sequence identity thereto. In yet other embodiments, such modified p40 domains may comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 12, 13, 19, 20, 24, 27, 30, 34, 38, 39, 41, 47, 48, 49, 51, 52, 54, 55, 56, 57, 42, 44, 45, 46, 50, 53, 40 and 43, respectively, or a sequence having at least about 90%, 95%, 97%, or at least about 99% sequence identity thereto.

[0105] The IL12 family of cytokines The present disclosure provides IL12 fusion proteins. Interleukin 12 (IL12) was the first recognized member of a family of heterodimeric cytokines that includes IL12, IL23, IL27, IL35, and IL39. IL12 and IL23 are proinflammatory cytokines important for the development of T helper 1 (Th-1) and T helper 17 (Th-17) T cell subsets, whereas IL27 and IL35 are potent inhibitory cytokines. IL39 is a cytokine important in controlling innate and / or adaptive immune responses. IL12 can directly enhance the activity of effector CD4 and CD8 T cells as well as natural killer (NK) and NKT cells.

[0106] Interleukin-12 (IL12) is a heterodimeric molecule composed of an alpha chain (p35 subunit) and a beta chain (p40 subunit) covalently linked by disulfide bridges to form a biologically active 70 kDa dimer. Exemplary amino acid sequences of the mature p35 and p40 subunits of IL12 are provided in Table M. See SEQ ID NOs: 10 and 11 and variants thereof, e.g., variants of the p40 subunit that contain amino acid substitutions that reduce affinity for IL12Rβ1 (SEQ ID NOs: 12-57).

[0107] IL23 is a member of the IL12 cytokine family and is composed of two subunits: p40 subunit shared with IL12 and p19. An exemplary amino acid sequence of the p19 subunit of IL23 is provided in Table M. See SEQ ID NOs: 127 and 128. The receptor for IL23 (IL23R) consists of the IL23R subunit and the IL12Rβ1 subunit, which is a subunit common to the IL12 receptor and interacts with tyrosine kinase 2 (Tyk2). IL23R is primarily expressed on immune cells, particularly T cells (e.g., Th17 and gamma delta T cells), macrophages, dendritic cells, and NK cells (Duvallet et al., 2011). It has recently been shown that non-activated neutrophils express basal amounts of IL23R and that IL23R expression increases upon cell activation (Chen et al., 2016).

[0108] The terms "protein having the function of IL12" or "protein having the function of IL23" encompass variants of wild-type IL12 or IL23 sequences, respectively, where the wild-type sequence has been altered by one or more of the addition, deletion, or substitution of amino acids. IL12 and IL23 sequences contemplated herein include IL12 and IL23 sequences from any animal, particularly any mammal, including human, mouse, dog, cat, pig, and non-human primates. "Wild type," "wildtype," or "WT," as used herein, refers to an amino acid or nucleotide sequence found in nature, including allelic variations. A WT protein has an amino acid or nucleotide sequence that has not been intentionally modified.

[0109] The biological activities of IL12 are well known and include, but are not limited to, differentiation of naive T cells into Th1 cells, stimulation of T cell growth and function, production of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-a) from T and natural killer (NK) cells, reduction of IL4-mediated suppression of IFN-gamma, and upregulation of NK cells and CD8 +These include enhancing the cytotoxic activity of cytotoxic T lymphocytes, stimulating the expression of IL12Rβ1 and IL12Rβ2, promoting the presentation of tumor antigens via upregulation of MHCI and II molecules, and antiangiogenic activity. IL12 is primarily produced by antigen-presenting cells and promotes cell-mediated immunity by binding to a two-chain receptor complex expressed on the surface of T cells or natural killer (NK) cells. The IL12 receptor beta-1 (IL12Rβ1) chain binds to the p40 subunit of IL12. Ligation of IL12p35 to the second receptor chain, IL12Rβ2, confers intracellular signaling (e.g., STAT4 phosphorylation) and activation of receptor-bearing cells (Presky et al, 1996). Studies have shown comparable cell-based affinity of IL12 for Rβ1 and Rβ2 individually, as well as a higher affinity for their complex (J Immunol. 1998 Mar 1;160(5):2174-9). IL12 also acts on dendritic cells (DCs) leading to increased maturation and antigen presentation, which may enable the initiation of T cell responses against tumor-specific antigens. It also promotes the secretion of IL12 by DCs, generating a positive feedback mechanism to amplify the response.

[0110] Exemplary nucleic acid and amino acid sequences for IL12, IL23 and the masked fusion proteins described herein are provided in Table M.

[0111] Variants of any of the nucleic acid and amino acid sequences provided herein are also contemplated for use in the fusion proteins described herein in the section entitled "Polypeptides and Polynucleotides." In certain embodiments, an IL12 fusion protein polypeptide described herein comprises a p35 amino acid sequence as set forth in SEQ ID NO: 11. In certain embodiments, an IL12 fusion protein described herein comprises a p40 amino acid sequence as set forth in SEQ ID NO: 10. In another embodiment, an IL12 fusion polypeptide described herein comprises a p35 amino acid sequence as set forth in SEQ ID NO: 11 and a p40 amino acid sequence as set forth in SEQ ID NO: 10. In one embodiment, an IL12 fusion protein described herein comprises a scIL12 having the configuration p40-L-p35, where L is a linker moiety, e.g., a peptide-based linker as described herein. In other embodiments, an IL12 polypeptide described herein may comprise a variant of the p35 and / or p40 sequence. In this regard, variants may include variants of nucleic acid sequences encoding p35 or p40 amino acid sequences that encode proteins that retain IL12 functional activity compared to wild-type IL12, or other suitable controls. Variant nucleic acid sequences may comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher percent identity to a polynucleotide sequence encoding p35 and / or p40, e.g., the polynucleotide sequences set forth in SEQ ID NOs: 129 and 130. Exemplary variants of IL12 polynucleotides include codon-optimized polynucleotide sequences.

[0112] In certain embodiments, variants may include variant p35 and / or p40 polypeptides that comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher percent identity to the amino acid sequence of IL12 p35 and / or p40 as set forth in SEQ ID NOs: 11 and 10, respectively, and such variant polypeptides retain IL12 functional activity compared to a suitable comparator molecule, including wild-type IL12.

[0113] In other embodiments, the IL23 polypeptides described herein may include variants of p19 and / or p40 sequences. In this regard, variants may include variants of nucleic acid sequences encoding p19 or p40 amino acid sequences that encode proteins that retain IL23 functional activity compared to wild-type IL23. Variant nucleic acid sequences may include at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher percent identity to the polynucleotide sequences encoding p19 and / or p40 set forth in SEQ ID NOs: 131 and 130, respectively. Exemplary variants of IL23 polynucleotides include codon-optimized polynucleotide sequences.

[0114] Reduced or inhibited binding or activity means that the binding or functional IL12 activity is lower than that of a suitable control, e.g., wild-type IL12, or the corresponding unmasked parent fusion protein. Reduced or inhibited binding or activity can be expressed as reduced potency. In certain embodiments, the potency of the IL12 fusion protein with a modified p40 domain is reduced by about 2-fold to about 2500-fold compared to the IL12 activity of a suitable control, e.g., an IL12 fusion protein with a wild-type p40 domain. The potency of an IL12 fusion protein having a modified p40 domain described herein is, in certain embodiments, reduced by about 5 to about 2000, about 10 to about 1500, about 15 to about 1000, about 20 to about 1800, about 25 to about 1600, about 25 to about 100, about 50 to about 100, about 50 to about 12000, about 100 to about 2000, or about 500 to about 12000, compared to an unmodified p40 domain. In some embodiments, the potency of an IL12 fusion protein having a modified p40 domain described herein is about 1 / 100, 1 / 125, 1 / 150, 1 / 175, 1 / 200, 1 / 225, 1 / 250, 1 / 275, 1 / 300, 1 / 325, 1 / 350, 1 / 375, 1 / 400, 1 / 425, 1 / 450, 1 / 475, 1 / 500, 1 / 525, 1 / 550, 1 / 575, 1 / 6 00, 1 / 625, 1 / 650, 1 / 675, 1 / 700, 1 / 725, 1 / 750, 1 / 775, 1 / 800, 1 / 825, 1 / 850, 1 / 875, 1 / 900, 1 / 925, 1 / 950, 1 / 975, 1 / 1000, 1 / 1100, 1 / 1200, 1 / 1300, 1 / 1400, 1 / 1500, 1 / 1600, 1 / 1700, 1 / 1800, 1 / 1900, 1 / 2000, 1 / 2500, or 1 / 3000. In certain embodiments, the potency is reduced by more than 3500-fold, 4000-fold, 4500-fold, 5000-fold, 5500-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold or 10,000-fold compared to the unmodified p40 domain.In some embodiments, a modified p40 domain containing one or more amino acid modifications described herein, or a fusion protein containing such a modified p40 domain, may have reduced binding by about 2-fold to about 2500-fold compared to an unmodified p40 domain.

[0115] In certain embodiments, the variants may comprise variant p19 and / or p40 polypeptides comprising at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher percent identity to the amino acid sequence of IL23 p19 and / or p40 as set forth in SEQ ID NOs: 128 and 10, respectively, and such variant polypeptides retain IL23 functional activity compared to wild-type IL23.

[0116] The variant cytokine polypeptides described herein or fusion proteins comprising them exhibit functional activity within 2-20-fold of an appropriate control, e.g., the functional activity of a relevant comparative fusion protein comprising a wild-type cytokine (e.g., IL12 or IL23). In certain embodiments, the cytokine variant polypeptides demonstrate equivalent potency compared to the wild-type control, e.g., as measured by relative abundance of NK cells, IFNγ release by CD8+ T cells, or cell signaling following receptor engagement. In other embodiments, the cytokine variant polypeptides demonstrate a maximum attenuation of potency between about 2-fold to about 20-fold, or between about 20-fold to about 200-fold. In certain embodiments, the cytokine variant polypeptides or fusion proteins comprising them demonstrate about 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 11, 1 / 12, 1 / 13, 1 / 14, 1 / 15, 1 / 16, 1 / 17, 1 / 18, 1 / 19 or about 1 / 20 of reduced potency. As noted elsewhere, IL12 is highly toxic. Thus, in certain embodiments, it may be desirable to use variant IL12 polypeptides with reduced potency. In certain embodiments, variants may exhibit reduced functional activity or reduced potency compared to a control, e.g., between about 2-fold to about 100-fold, or about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or 200-fold reduced activity or potency compared to a suitable control. Cytokine functional activity may be measured using assays known in the art and described herein, e.g., splenocyte, NK or CTLL-2 assays or IFNγ release by CD8+ T cells.

[0117] Methods for measuring the functional activity of IL12 family cytokines are known in the art, including assays known in the art, such as assays for determining cellular responsiveness to IL12 or IL23, measuring cytokine production in response to incubation of appropriate cells with IL12 or IL23, measuring receptor binding and signaling activation.

[0118] In certain embodiments, IL12 activity is determined by measuring cell proliferation of cells or cell lines that are sensitive to IL12. Exemplary cells that can be used to test IL12 activity include CTLL-2 or NK or CD8 cells. Such proliferation assays include, for example, the assays described in Khatri A,et al.2007.J Immunol Methods 326(1-2):41-53, Puskas J,et al.2011.Immunology 133(2):206-220, Hodge DL.,et al.J Immunol.2002 Jun 15;168(12):6090-8. Assays known in the art can be modified as desired to suit the particular cytokine being tested, such as IL12 or IL23.

[0119] Briefly, the CTLL-2 assay for measuring IL12 functional activity involves serially diluting the recombinant protein to be tested (e.g., a masked fusion protein described herein) 1:5 into 50 μL of medium, followed by 4×10 dilutions in 100 μL of medium. 4 The incubation period may involve adding 10 CTLL-2 cells per well to a 96-well plate and incubating for 18-22 hours at 37 °C, 5% CO2. At the end of this period, 75 µg / well of thiazolyl blue tetrazolium bromide (MTT; Sigma-Aldrich) is added and the plate is incubated for 8 hours at 37 °C, 5% CO2. Cells are lysed with 100 µL / well of 10% SDS (Gibco) acidified with HCl, incubated overnight at 37 °C, 5% CO2, and absorbance is read at 570 nm.

[0120] Briefly, the NK assay to measure IL12 functional activity can be performed as follows: NK cells are cultured in growth medium without IL2 (assay medium) for 12 hours, harvested, and spun down to pellet the cells. Cells are resuspended in assay medium to 400 million cells / mL and 10,000 cells or 25uL per well are added to the assay plate. Variant test samples are titrated in triplicate at a 1:5 dilution in 25ul directly in 384-well black flat-bottom assay plates. Recombinant cytokines (e.g., human IL12 (Peprotech, Rocky Hill, NJ)) are included as positive controls. Plates are incubated at 37°C and 5% carbon dioxide for 3 days. After incubation, 25uL / well of supernatant is transferred to a non-binding 384-well plate (Greiner-Bio-One, Kremsmunster, Austria) and stored at -80°C. After removing the supernatant, CellTiter-Glo® Luminescent Cell Viability Reagent (Promega, Madison, WI) or equivalent may be added to the plate at 25 uL / well and the plate is incubated at room temperature away from light for 30 minutes. After incubation, the plate luminescence is scanned, such as with a BioTek Synergy H1 plate reader (BioTek, Winooski, VT).

[0121] In one embodiment, IL12 activity may be determined by measuring the cell signaling cascade triggered by the interaction of IL12 with its receptors (e.g., the interaction of IL12Rβ2 and IL12Rβ1 with the IL12 p35-p40 heterodimer). In one embodiment, IL12 activity is determined by measuring STAT4 signaling activity using assays known in the art and commercially available, for example, from Abeomics, San Diego, CA USA.

[0122] In one embodiment, IL12 activity may be determined by measuring IFNγ release from CD8+ T cells following stimulation with IL12 protein, as described in Example 3.

[0123] Masking part The masked IL12 or IL23 fusion proteins described herein comprise a masking moiety (MM) that blocks or reduces the binding of IL12 or IL23 to its native receptor(s) and / or blocks or reduces its functional activity. In some embodiments, the masked IL12 or IL23 fusion proteins described herein comprise (i) a first fusion polypeptide comprising a first Fc polypeptide fused C-terminally to an IL12 polypeptide comprising a modified p40 domain, and (ii) a second fusion polypeptide comprising a second Fc polypeptide fused C-terminally to a MM, wherein the two Fc polypeptides form a dimeric Fc domain complex (e.g., a heterodimeric Fc domain).

[0124] In certain embodiments, the MM specifically binds to IL12. "Specifically binds," "specific binding," or "selective binding" means that the binding is selective for the desired antigen (in the present disclosure, the MM specifically binds to IL12 or IL23) and can be distinguished from unwanted or non-specific interactions. The ability of the MM to bind and block or reduce IL12 / IL23 activity can be measured by either enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the MM to unrelated proteins is less than about 10% of the binding of the MM to IL12 / IL23, as measured, for example, by SPR. In certain embodiments, MM that binds to IL12 / IL23 or a biologically active fragment thereof has a binding affinity of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K d ).

[0125] MM in the present disclosure generally refers to an amino acid sequence that is present in a masked cytokine fusion protein and positioned to reduce the ability of the cytokine, in the context of the masked cytokine fusion protein, to specifically bind to a target and / or function. In some cases, the MM is attached to the masked cytokine fusion protein by a linker.

[0126] When the IL12 fusion protein described herein comprises a MM and is in the presence of a target (e.g., an IL12 receptor), specific binding of the masked IL12 fusion protein to the IL12 receptor is reduced or inhibited compared to specific binding of the unmasked parent IL12 fusion protein.

[0127] When the IL12 fusion proteins described herein include MM and are in the presence of a target (e.g., an IL12 receptor), the potency of the masked IL12 fusion protein is reduced or inhibited compared to the unmasked parent IL12 fusion protein. Thus, the MM functions to block the functional activity of IL12.

[0128] In certain embodiments of IL12 HetFc fusion proteins further described herein, the MM can be bound to the first Fc polypeptide of the dimeric (e.g., heterodimeric) Fc domain either directly or through a linker, and the IL12 polypeptide can be bound to the second Fc polypeptide of the dimeric Fc domain either directly or through a linker. In such a configuration, for example as shown in FIG. 2B herein, the MM can specifically interact or bind to the IL12 portion of the fusion protein. Such binding can include or consist of a non-covalent bond. The interaction of the MM with the IL12 in the fusion protein can mask the IL12 by inhibiting or at least reducing the ability of IL12 to interact with at least one of its cognate receptors when compared to the corresponding "unmasked" IL12 polypeptide. Such masking activity of MM can further inhibit or suppress any downstream events mediated by receptors activated by IL12, and thus MM activity can be measured by various methods described herein and known in the art, such as enzyme-linked immunosorbent assay (ELISA), or other techniques well known to those of skill in the art and described herein.

[0129] In certain embodiments, the dissociation constant (K d ) is generally the K of the same IL12 fusion protein without MM. d Conversely, the binding affinity of the masked IL12 fusion proteins to the IL12 receptor is generally lower than that of the non-MM-modified IL12 fusion proteins.

[0130] In certain embodiments, the K of MM for an IL12 polypeptide d Generally, the K d Conversely, in certain embodiments, the binding affinity of the MM to an IL12 polypeptide is generally lower than the binding affinity of the IL12 polypeptide to the IL12 receptor.

[0131] It should be noted that due to proximity (i.e., when the MM is fused to the IL12 fusion protein by a linker), the apparent "affinity" of the MM for the IL12 polypeptide is higher than when the MM is not fused to the IL12 fusion protein.

[0132] The MM may inhibit the binding of the masked IL12 fusion protein to the IL12 receptor, thereby inhibiting the IL12 functional activity of the fusion protein compared to an IL12 polypeptide that is not modified by MM. The MM may bind to the IL12 polypeptide and inhibit it from binding to its receptor. The MM may sterically inhibit the binding of the masked IL12 fusion protein to the IL12 receptor. The MM may allosterically inhibit the binding of the masked IL12 fusion protein to the IL12 receptor. In those embodiments, when the masked IL12 fusion protein is in the presence of the IL12 receptor, the binding of the masked IL12 fusion protein to the IL12 receptor is absent or substantially absent, or the binding of the masked IL12 fusion protein to the target is in is present at .001 percent, .01 percent, .1 percent, 1 percent, 2 percent, 3 percent, 4 percent, 5 percent, 6 percent, 7 percent, 8 percent, 9 percent, 10 percent, 15 percent, 20 percent, 25 percent, 30 percent, 35 percent, 40 percent, or 50 percent or less compared to the binding of the unmasked IL12 fusion protein, i.e., the parent IL12, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or longer, as measured in vivo or by surface plasmon resonance (SPR).

[0133] MM can inhibit the binding of the masked IL12 fusion protein to the IL12 receptor, thereby inhibiting the IL12 functional activity of the fusion protein compared to an IL12 polypeptide that is not modified by MM. MM can bind to the IL12 polypeptide and inhibit it from binding to its receptor. MM can sterically inhibit the binding of the masked IL12 fusion protein to the IL12 receptor. MM can allosterically inhibit the binding of the masked IL12 fusion protein to the IL12 receptor. In those embodiments, when the masked IL12 fusion protein is in the presence of an IL12 receptor, binding of the masked IL12 fusion protein to the IL12 receptor is absent or substantially absent, or binding of the masked IL12 fusion protein to a target is less than or equal to .001 percent, .01 percent, .1 percent, 1 percent, 2 percent, 3 percent, 4 percent, 5 percent, 6 percent, 7 percent, 8 percent, 9 percent, 10 percent, 15 percent, 20 percent, 25 percent, 30 percent, 35 percent, 40 percent, or 50 percent compared to binding of the unmasked IL12 fusion protein, i.e., parental IL12, for at least about 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or longer, as measured in an in vitro assay, such as a reporter gene assay (see, e.g., Example 4).

[0134] In certain embodiments, the MM is not a natural binding partner of the IL12 polypeptide. The MM can be a modified binding partner of the IL12 polypeptide that contains amino acid changes that at least slightly reduce the affinity and / or avidity of binding to the IL12 polypeptide. In some embodiments, the MM contains no or substantially no homology to the IL12 receptor. In other embodiments, the MM is less than 5 percent, 10 percent, 15 percent, 20 percent, 25 percent, 30 percent, 35 percent, 40 percent, 45 percent, 50 percent, 55 percent, 60 percent, 65 percent, 70 percent, 75 percent, or 80 percent similar to the IL12 receptor.

[0135] When the IL12 fusion protein is in a "masked" state, even in the presence of the IL12 receptor, the MM prevents or inhibits the binding of the masked IL12 fusion protein to the receptor.

[0136] In various embodiments, the MM of the masked IL12 fusion protein herein interacts non-covalently and reversibly with the IL12 polypeptide. Thus, in certain embodiments, masked IL12 fusion proteins are described herein that may include a masked IL12 polypeptide with inhibited or reduced IL12 receptor binding affinity and may become unmasked under certain conditions in an in vitro or in vivo environment, such as pH, protease activity, as further described herein. Thus, in some embodiments, the masked IL12 fusion proteins herein are transiently masked and may become unmasked after a certain period of time under certain conditions, such as pH or protease activity that may be present in a certain tissue or organ of a mammalian subject, such as a tumor microenvironment. Conditionally unmasked IL12 fusion proteins may induce IL-12 activity generally comparable to the IL12 activity of natively unmasked IL12 fusion proteins. However, because the IL12 polypeptide used in the fusion proteins herein contains a modified p40 domain, under certain conditions the IL12 functional activity of the unmasked IL12 fusion protein may still be reduced compared to a fusion protein using an unmasked wild-type IL12 polypeptide.

[0137] The structural characteristics of the MM will vary depending on a variety of factors, including the minimal amino acid sequence required to block cytokine binding and / or activity, the cytokine-cytokine receptor protein binding pair of interest, the size of the cytokine and fusion protein, the length of the protease-cleavable linker (PCL) (whether the PCL is located within the MM, between the Fc and the cytokine, or between the Fc and the mask), the presence or absence of additional linkers, etc.

[0138] MM can be provided in a variety of different forms. In certain embodiments, MM can be selected to be a known binding partner of cytokine. In certain embodiments, MM masks cytokine from target binding when covalently bound in masked cytokine fusion protein, but does not substantially or significantly interfere with or compete with cytokine polypeptide for target binding when not covalently bound in cytokine fusion protein. In certain embodiments, MM does not contain the amino acid sequence of naturally occurring binding partner of cytokine.

[0139] The efficiency of the MM to inhibit cytokine binding or activity when bound can be measured by SPR or cell-based assays as described herein and as detailed elsewhere (see, e.g., NK, CTLL-2 or CD8+ T cell IFNγ release assays) and as described herein in the Examples section of this disclosure. The masking efficiency of the MM can be determined by at least two parameters: the affinity of the MM for the cytokine or a fusion protein containing the cytokine, and the spatial relationship of the MM to the binding interface of the cytokine and its receptor.

[0140] With regard to affinity, as an example, one MM may have high affinity but only partially inhibit the binding of a cytokine to its receptor, whereas another MM may have a lower affinity for the cytokine but fully inhibit target binding. For short periods of time, the lower affinity MM may exhibit sufficient masking; in contrast, over time, that same MM may be displaced by the target (due to insufficient affinity for the cytokine).

[0141] In a similar manner, two MMs with the same affinity may show different degrees of masking based on how much they promote inhibition of cytokine binding to its receptor.In another example, one MM with high affinity may bind to a cytokine or a fusion protein containing a cytokine and change its structure, thereby completely inhibiting its binding to its target, whereas another MM with high affinity may only partially inhibit binding.As a result, the discovery of effective MMs may generally include the measurement of the potency of masked cytokine fusion proteins compared to appropriate controls, rather than being based solely on affinity.

[0142] As described herein, in various embodiments, the MM of an IL12 fusion protein can comprise or consist of an antibody or antigen-binding fragment thereof that specifically binds IL12. Thus, in some embodiments, the MM can be a single chain Fv (scFv) antibody fragment. Exemplary scFv MMs comprise the VH and VL amino acid sequences provided in SEQ ID NOs: 2-3. In certain embodiments, exemplary MMs comprise the VH and VL CDRs set forth in SEQ ID NOs: 4-9.

[0143] Antibodies and antigen-binding fragments thereof In certain embodiments, the masking moiety used in the masked fusion proteins herein comprises an antibody or an antigen-binding fragment of an antibody. The antigen-binding fragment may comprise the light and / or heavy chains (V L , V H ), a variable fragment (Fv), a Fab' fragment, a F(ab')2 fragment, a Fab fragment, a single chain antibody (scAb), a single chain variable region (scFv), a complementarity determining region (CDR), a domain antibody (dAb), a single domain heavy chain immunoglobulin, a single domain light chain immunoglobulin, or other polypeptides known in the art that contain an antigen-binding fragment capable of binding to a target protein or an epitope on a target protein.

[0144] Exemplary antigen binding domains are derived from antibodies that bind to IL12 and / or IL23.

[0145] In one embodiment, the MM comprises an antibody or antigen-binding fragment thereof that specifically binds to IL12. In one embodiment, the MM comprises an antibody or antigen-binding fragment thereof that specifically binds to IL23. In certain embodiments, the MM comprises an scFv that specifically binds to IL12 or IL23.

[0146] In some embodiments, MMs may be identified through screening for antibodies or antigen-binding fragments thereof that bind IL12 or IL23. Candidate MMs may be fused in various configurations in cytokine fusion proteins (see, e.g., Figures 1A-B and Examples herein) and screened for their ability to reduce cytokine binding, reduce IL12 potency, and / or restore cytokine activity after cleavage. Antibodies may be derived from antibodies known in the art that bind IL12 and / or IL23. Such antibodies are known and available, for example, from the literature or can be found in the TABS Therapeutic Antibody Database (see tabs(dot)craic(dot)com). Exemplary antibodies for use in the masked IL12 fusion proteins herein include briakinumab (US6914128; US7504485; US8168760; US8629257; US9035030); ustekinumab (US6902734; US7279157; U8080247; US7736650; US8420081; US7887801; US8361474; US8084233; US9676848), AK101, PMA204 (see, e.g., US8563697), 6F6 (see, e.g., US8563697; Clarke AW et al., 2010 MAbs 2:539-49). The h6F6 antibody binds to a different epitope on p40 than briakinumab or ustekinumab.

[0147] In one embodiment, the MM is derived from an antibody that comprises an antigen-binding domain that binds human IL12 and human IL23. In another embodiment, the antibody binds to human IL12p40, which exists as a monomer (human IL12p40) and as a homodimer (human IL12p80), and the antibody inhibits binding of human IL12 to human IL12Rβ2 and human IL23 to human IL23R, but does not inhibit binding of human IL12 or human IL23, or human IL12p40 or human IL12p80 to human IL12Rβ1.

[0148] Antibodies or antigen-binding fragments thereof that bind IL12 and / or IL23 can be further modified to increase or decrease affinity as necessary and then further tested for their ability to mask and reduce efficacy as described herein.

[0149] In certain embodiments, candidate peptides may be screened to identify MMs capable of binding to IL12 or IL23 using methods such as those described in, for example, WO 2010 / 081173 and U.S. Patent No. 10,118,961. Such methods include providing a library of peptide scaffolds, each peptide scaffold comprising a transmembrane protein (TM); and a candidate peptide, contacting IL12 or IL23 with the library, identifying at least one candidate peptide capable of binding to an IL12 or IL23 polypeptide, and determining the dissociation constant (K) of the candidate peptide for IL12 or IL23. d ) is between 1 and 10 nM.

[0150] In various embodiments, the MM of the IL12 fusion protein comprises or consists of a single chain variable fragment (scFv) of an antibody. In such embodiments, the scFv MM comprises a heavy chain variable domain (V) comprising the complementarity determining region (CDR) sequences set forth in SEQ ID NOs: 4-6. H ), and a light chain variable domain (VL Thus, in some embodiments, the scFv MM may comprise a V that comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:2. H domain, and a V domain comprising or consisting of an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:3. L It may comprise or consist of a domain.

[0151] Linkers and Protease-Cleavable Linkers In certain embodiments of the fusion protein of the present disclosure, one or more different components or domains are directly fused from one to the other without a linker. For example, in certain embodiments, the Fc domain can be directly fused to the MM or directly fused to the p35 or p40 polypeptide. However, in certain embodiments, the masked cytokine fusion construct comprises one or more linkers of various lengths. The peptide linker allows for the arrangement of the fusion protein to form a functional masking portion and a cytokine that retains cytokine activity when cleaved from the larger / complete fusion protein.

[0152] A "linker" is a peptide that connects or joins other peptides or polypeptides, for example, a linker of about 2 to about 150 amino acids. A peptide linker of the present disclosure can comprise or consist of an amino acid sequence of about 2 to about 150 amino acids, about 5 to about 100 amino acids, about 5 to about 75 amino acids, about 5 to about 50 amino acids, about 5 to about 40 amino acids, about 5 to about 30 amino acids, or about 5 to about 20 amino acids. In a masked cytokine fusion protein of the present disclosure, a linker can be used to fuse any of the components of the fusion protein, for example, an Fc polypeptide to the MM, or the linker can connect an Fc polypeptide to a cytokine polypeptide, for example, p35 or p40 of IL12. In certain embodiments, the linker can be present within the MM, for example, when the MM is a scFV and the linker connects the VH and VL.

[0153] Exemplary linkers for use in the fusion proteins described herein include (Gly n Ser) family, e.g., (Gly3Ser) n (Gly4Ser)1, (Gly3Ser)1(Gly4Ser) n , (Gly3Ser) n (Gly4Ser) n , or (Gly4Ser) n (wherein n is an integer from 1 to 5). In certain embodiments, peptide linkers suitable for connecting different domains include sequences that include glycine-serine linkers, such as, but not limited to, (G m S) n -GG, (SG n ) m , (SEG n ) m (where m and n are between 0 and 20).

[0154] In certain embodiments, the linker may be an amino acid sequence obtained, derived, or designed from an antibody hinge region sequence, a sequence that links a binding domain to a receptor, or a sequence that links a binding domain to a cell surface transmembrane region or membrane anchor. In some embodiments, the linker may have at least one cysteine ​​that can participate in at least one disulfide bond under physiological conditions or other standard peptide conditions (e.g., peptide purification conditions, conditions for peptide storage). In certain embodiments, a linker that corresponds to or resembles an immunoglobulin hinge peptide retains a cysteine ​​that corresponds to the hinge cysteine ​​located toward the amino terminus of the hinge. In further embodiments, the linker is from an IgG1 hinge, modified to remove any cysteine ​​residues, or an IgG1 hinge with one or two cysteines that correspond to the hinge cysteines.

[0155] In addition to providing spacing functionality, the linker may provide suitable flexibility or rigidity to properly orient one or more domains of the unmasked or masked cytokine fusion proteins herein, both within the fusion protein and between the fusion protein and their target(s). Furthermore, the linker may support expression of the full-length fusion protein and stability of the purified protein, both in vitro and in vivo after administration to a subject in need thereof, e.g., a human, and is preferably non-immunogenic or low immunogenic in that same subject. In certain embodiments, the linker may comprise part or all of the stalk region of a human immunoglobulin hinge, a C-type lectin, which is a family of type II membrane proteins. The linker ranges in length from about 2 to about 100 amino acids, or from about 5 to about 75 amino acids, or from about 10 to about 50 amino acids, or from about 2 to about 40 amino acids, or from about 8 to about 20 amino acids, about 10 to about 60 amino acids, about 10 to about 30 amino acids, or from about 15 to about 25 amino acids.

[0156] In certain embodiments, a linker for use herein may comprise an "altered wild-type immunoglobulin hinge region" or an "altered immunoglobulin hinge region." Such an altered hinge region refers to (a) a wild-type immunoglobulin hinge region having up to 30 percent amino acid changes (e.g., up to 25 percent, 20 percent, 15 percent, 10 percent, or 5 percent amino acid substitutions or deletions), (b) a portion of a wild-type immunoglobulin hinge region that is at least 10 amino acids in length (e.g., at least 12, 13, 14, or 15 amino acids) and has up to 30 percent amino acid changes (e.g., up to 25 percent, 20 percent, 15 percent, 10 percent, or 5 percent amino acid substitutions or deletions), or (c) a portion of a wild-type immunoglobulin hinge region that includes the core hinge region (which can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length). In certain embodiments, one or more cysteine ​​residues in a wild-type immunoglobulin hinge region, such as an IgG1 hinge, including the upper and core regions, may be replaced by one or more other amino acid residues (e.g., one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally have a proline residue in a wild-type immunoglobulin hinge region, such as an IgG1 hinge, including the upper and core regions, replaced by another amino acid residue (e.g., a serine residue).

[0157] Alternative hinge and linker sequences that may be used as connecting regions may be made from portions of cell surface receptors that connect IgV-like or IgC-like domains. Regions between IgV-like domains where the cell surface receptor contains multiple IgV-like domains in tandem, and regions between IgC-like domains where the cell surface receptor contains multiple tandem IgC-like domains may also be used as connecting regions or linker peptides. In certain embodiments, the hinge and linker sequences are 5-60 amino acids in length and may be primarily flexible, but may also provide more rigid properties and may contain a primarily helical structure with minimal beta sheet structure.

[0158] Certain exemplary linkers are provided in SEQ ID NOs: 132-135. Exemplary linkers are also provided in the context of various masked cytokines and unmasked parent fusion proteins herein, shown in SEQ ID NOs: 58-89 (see also Table M).

[0159] Non-limiting examples of diseases targeted by the masked cytokine fusion proteins herein include all types of cancer, including, but not limited to, breast cancer, including triple negative breast cancer, ER / PR+ breast cancer, and Her2+ breast cancer, lung cancer (e.g., non-small cell squamous cell carcinoma and adenocarcinoma), colorectal cancer, gastric cancer, glioblastoma, ovarian cancer, endometrial cancer, kidney cancer, sarcoma, skin cancer, cervical cancer, liver cancer, bladder cancer, bile duct cancer, prostate cancer, melanoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), esophageal, squamous cell carcinoma, basal cell carcinoma, pancreatic cancer, leukemia, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic diseases, including multiple myeloma, and solid tumors. Indications also include bone disease or metastasis in cancer, regardless of the primary tumor origin. Other exemplary diseases include rheumatoid arthritis, Crohn's disease, SLE, cardiovascular injury, and ischemia. In certain embodiments, the target disease is selected from the group consisting of colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, bladder cancer, cervical cancer, and lung cancer (e.g., non-small cell squamous carcinoma and adenocarcinoma).

[0160] In one embodiment, the two heterologous polypeptides are selected from a cytokine polypeptide or functional fragment thereof, an antibody, an antigen-binding fragment of an antibody, and an Fc domain. In another embodiment, the recombinant polypeptide comprises a cytokine polypeptide or functional fragment thereof, an MM, and an Fc domain. In certain other embodiments, the MM is a single chain Fv (scFv) antibody fragment that binds to a cytokine or a cytokine receptor polypeptide or cytokine-binding fragment thereof. In a further embodiment, the recombinant polypeptide comprises an antibody or antigen-binding fragment thereof that binds to a target, and an MM that binds to the antibody or antigen-binding fragment thereof and blocks binding of the antibody or antigen-binding fragment thereof to the target.

[0161] In some embodiments, the IL12 fusion protein of the present disclosure comprises one or more cleavable peptide linkers. Such one or more cleavable linkers may be cleavable due to reactivity under certain conditions, for example, such linkers may be protease-sensitive, acid-sensitive, or reduction-sensitive. In various embodiments, the cleavable linkers herein comprise an amino acid sequence that is a cleavage recognition sequence for a protease, i.e., a protease-cleavable linker. Many such cleavage recognition sequences are known in the art. In some embodiments, an amino acid sequence that is recognized and cleaved by a protease present in the extracellular matrix in the vicinity of a target cell, such as a cancer cell, can be used. Examples of extracellular tumor-associated proteases include, for example, plasmin, matrix metalloproteases (MMPs), elastase, and kallikrein-related peptidases.

[0162] Fc domain In some embodiments, the masked IL12 fusion proteins described herein comprise an Fc, and in some embodiments, the Fc is a dimeric Fc. The dimeric Fc domain can be a heterodimeric Fc domain, as described herein. Such a dimeric Fc domain of the fusion protein can comprise a first and a second Fc polypeptide, where the first Fc polypeptide can bind to MM and the second Fc polypeptide can bind to an IL12 polypeptide, for example as shown in FIG. 2B.

[0163] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0164] The "Fc polypeptide" of a dimeric Fc, as used herein, refers to one of the two polypeptides that form the dimeric Fc domain, i.e., the polypeptide that comprises the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, the Fc polypeptide of a dimeric IgG Fc comprises the IgG CH2 and IgG CH3 constant domain sequences.

[0165] The Fc domain comprises either a CH3 domain or a CH3 and a CH2 domain. The CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc. The CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc.

[0166] In some embodiments, the Fc comprises at least one or two CH3 sequences. In some embodiments, the Fc is linked to an IL12 polypeptide construct (e.g., linked at the C-terminus to a first Fc polypeptide) and / or linked to a masking moiety (MM) (e.g., linked at the C-terminus to a second Fc polypeptide) with or without one or more linkers. In some embodiments, the Fc is a human Fc. In some embodiments, the Fc is a human IgG or IgG1 Fc. In some embodiments, the Fc is a heterodimeric Fc. In some embodiments, the Fc comprises at least one or two CH2 sequences.

[0167] In some embodiments, the Fc comprises one or more modifications in at least one of the CH3 sequences. In some embodiments, the Fc comprises one or more modifications in at least one of the CH2 sequences. In some embodiments, the Fc is a single polypeptide. In some embodiments, the Fc is multiple peptides, e.g., two polypeptides.

[0168] In some embodiments, the Fc is an Fc described in patent application PCT / CA2011 / 001238 filed November 4, 2011 (WO2012058768; U.S. Patent Nos. 9,562,109 and 10,875,931) or PCT / CA2012 / 050780 filed November 2, 2012 (WO2013063702); U.S. Patent Nos. 9,574,010; 9,732,155; 10,457,742 and U.S. Patent Application No. 2020008741, all of which are incorporated herein by reference in their entirety.

[0169] Modified CH3 domain In some aspects, the masked IL12 fusion proteins described herein comprise an asymmetrically modified heterodimeric Fc ("HetFc") comprising a modified CH3 domain. The heterodimeric Fc may comprise two heavy chain constant domain polypeptides: a first Fc polypeptide and a second Fc polypeptide, which may be used interchangeably as long as the Fc domain comprises one first Fc polypeptide and one second Fc polypeptide. Generally, the first Fc polypeptide comprises a first CH3 sequence and the second Fc polypeptide comprises a second CH3 sequence. In certain figures and elsewhere herein, the first Fc polypeptide and the second Fc polypeptide may be referred to as Fc polypeptide A and Fc polypeptide B (or chain A or chain B for short), which may also be used interchangeably as long as the Fc domain or region comprises one Fc polypeptide A and one Fc polypeptide B. In some cases, an Fc domain comprising one Fc polypeptide A and one Fc polypeptide B may be referred to as a variant, and the variant may be referred to by a specific variant number to distinguish it from other Fc variants.

[0170] Two CH3 sequences that contain one or more amino acid modifications introduced in an asymmetric manner generally result in a heterodimeric Fc rather than a homodimer when the two CH3 sequences dimerize. As used herein, "asymmetric amino acid modification" refers to any modification in which an amino acid at a particular position on a first CH3 sequence is different from an amino acid at the same position on a second CH3 sequence, and the first and second CH3 sequences preferentially pair to form a heterodimer rather than a homodimer. This heterodimerization can be the result of modification of only one of the two amino acids at the same amino acid position on each sequence; or modification of both amino acids on each sequence at the same position on each of the first and second CH3 sequences. The first and second CH3 sequences of the heterodimeric Fc can contain one or more asymmetric amino acid modifications.

[0171] Table D provides the amino acid sequence of the human IgG1 Fc sequence corresponding to amino acids 231 to 447 of the full length human IgG1 heavy chain. The CH3 sequence includes amino acids 341 to 447 of the full length human IgG1 heavy chain.

[0172] Typically, an Fc may comprise two consecutive heavy chain sequences (A and B) capable of dimerizing. In some embodiments, one or both sequences of the Fc comprise one or more mutations or modifications at the following positions: L351, F405, Y407, T366, K392, T394, T350, S400, and / or N390, using EU numbering. In some embodiments, an Fc comprises a variant sequence as shown in Table D. In some embodiments, an Fc comprises a mutation of variant 1A-B. In some embodiments, an Fc comprises a mutation of variant 2A-B. In some embodiments, an Fc comprises a mutation of variant 3A-B. In some embodiments, an Fc comprises a mutation of variant 4A-B. In some embodiments, an Fc comprises a mutation of variant 5A-B.

[0173] [Table D]

[0174] The first and second CH3 sequences may comprise the amino acid mutations described herein with respect to amino acids 231-447 of a full-length human IgG1 heavy chain. In one embodiment, the heterodimeric Fc comprises a modified CH3 domain having a first CH3 sequence with amino acid modifications at positions F405 and Y407 and a second CH3 sequence with an amino acid modification at position T394. In one embodiment, the heterodimeric Fc comprises a modified CH3 domain having a first CH3 sequence with one or more amino acid modifications selected from L351Y, F405A, and Y407V and a second CH3 sequence with one or more amino acid modifications selected from T366L, T366I, K392L, K392M, and T394W.

[0175] In one embodiment, the heterodimeric Fc comprises a modified CH3 domain having a first CH3 sequence having amino acid modifications at positions L351, F405 and Y407 and a second CH3 sequence having amino acid modifications at positions T366, K392 and T394, wherein one of the first or second CH3 sequence further comprises an amino acid modification at position Q347 and the other CH3 sequence further comprises an amino acid modification at position K360. In another embodiment, the heterodimeric Fc comprises a modified CH3 domain having a first CH3 sequence having amino acid modifications at positions L351, F405 and Y407 and a second CH3 sequence having amino acid modifications at positions T366, K392 and T394, wherein one of the first or second CH3 sequences further comprises an amino acid modification at position Q347 and the other CH3 sequence further comprises an amino acid modification at position K360, and one or both of said CH3 sequences further comprises the amino acid modification T350V.

[0176] In one embodiment, the heterodimeric Fc comprises a modified CH3 domain having a first CH3 sequence having amino acid modifications at positions L351, F405 and Y407, and a second CH3 sequence having amino acid modifications at positions T366, K392 and T394, wherein one of the first and second CH3 sequences further comprises an amino acid modification of D399R or D399K, and the other CH3 sequence comprises one or more of T411E, T411D, K409E, K409D, K392E and K392D. In another embodiment, the heterodimeric Fc comprises a modified CH3 domain having a first CH3 sequence having amino acid modifications at positions L351, F405 and Y407, and a second CH3 sequence having amino acid modifications at positions T366, K392 and T394, wherein one of the first and second CH3 sequences further comprises an amino acid modification of D399R or D399K, and the other CH3 sequence comprises one or more of T411E, T411D, K409E, K409D, K392E and K392D, and one or both of the CH3 sequences further comprises the amino acid modification T350V.

[0177] In one embodiment, the heterodimeric Fc comprises a modified CH3 domain having a first CH3 sequence having amino acid modifications at positions L351, F405 and Y407, and a second CH3 sequence having amino acid modifications at positions T366, K392, and T394, wherein one or both of the CH3 sequences further comprises an amino acid modification of T350V.

[0178] In one embodiment, the heterodimeric Fc comprises a modified CH3 domain comprising the following amino acid modifications, where "A" represents an amino acid modification to a first CH3 sequence and "B" represents an amino acid modification to a second CH3 sequence: A: L351Y_F405A_Y407V, B: T366L_K392M_T394W, A: L351Y_F405A_Y407V, B: T366L_K392L _T394W, A:T350V_L351Y_F405A_Y407V, B:T350V_T366L_K392L_T394W, A:T350V_L351Y_F405A_Y407V, B:T350V_T366L_K392M_T394W, A:T350V_L351Y_S400E_F405A_Y407V, and / or B:T350V_T366L_N390R_K392M_T394W.

[0179] The one or more asymmetric amino acid modifications may promote the formation of a heterodimeric Fc in which the heterodimeric CH3 domain has a stability equivalent to that of a wild-type homodimeric CH3 domain. In an embodiment, the one or more asymmetric amino acid modifications promote the formation of a heterodimeric Fc domain with a stability equivalent to that of a wild-type homodimeric Fc domain. In an embodiment, the one or more asymmetric amino acid modifications promote the formation of a heterodimeric Fc domain with a stability observed in a differential scanning calorimetry study by a melting temperature that is within 4°C of the melting temperature (Tm) observed for the corresponding symmetric wild-type homodimeric Fc domain. In some aspects, the Fc comprises one or more modifications in at least one of the CH3 sequences that promote the formation of a heterodimeric Fc with a stability equivalent to that of a wild-type homodimeric Fc.

[0180] Modified CH2 domain In certain embodiments, an Fc domain contemplated for use herein is an Fc with a modified CH2 domain. In some embodiments, an Fc domain contemplated for use herein is an IgG Fc with a modified CH2 domain, where modification of the CH2 domain results in altered binding to one or more Fc receptors (FcR), e.g., receptors of the FcγRI, FcγRII, and FcγRIII subclasses.

[0181] Several amino acid modifications to the CH2 domain that selectively alter the affinity of Fc to different Fcγ receptors are known in the art. Both amino acid modifications that result in increased binding and amino acid modifications that result in decreased binding may be useful in certain indications. For example, increasing the binding affinity of Fc to FcγRIIIa (an activating receptor) may result in increased antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn results in increased lysis of target cells. Decreased binding to FcγRIIb (an inhibitory receptor) may also be beneficial in some situations. In certain indications, reducing or eliminating ADCC and complement-mediated cytotoxicity (CDC) may be desired. In such cases, modified CH2 domains containing amino acid modifications that result in increased binding to FcγRIIb or that reduce or eliminate binding of the Fc region to all of the Fcγ receptors ("knockout" variants) may be useful.

[0182] Examples of amino acid modifications to the CH2 domain that alter binding of Fc by Fcγ receptors include, but are not limited to, the following: S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L (increased affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90); F243L / R292P / Y300L / L235V / P396L (increased affinity for FcγRIIIa) (Nordstrom JL, et al., 2011, Breast Cancer Res, 13(6):R123); F243L (increased affinity for FcγRIIIa) (Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8); S298A / E333A / K334A (increased affinity for FcγRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604); S239D / I332E / A330L and S239D / I332E (increased affinity for FcγRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (increased affinity for FcγRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33).

[0183] Additional modifications that affect the binding of Fc to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 37 9, Oct 2012, page 283).

[0184] In certain embodiments, the masked or unmasked IL12 fusion proteins comprise an IgG Fc-based scaffold with a modified CH2 domain that contains one or more amino acid modifications (i.e., "knockout" variants) that result in reduced or eliminated binding of the Fc region to all of the Fcγ receptors.

[0185] Various publications describe strategies that have been used to engineer antibodies to generate "knockout" variants (see, for example, Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp 225-249). These strategies include reducing effector function via modifying glycosylation (described in more detail below), using an IgG2 / IgG4 scaffold, or introducing mutations into the hinge or CH2 domains of the Fc (see also U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO2006 / 105338, U.S. Patent Publication No. 2012 / 0225058, U.S. Patent Publication No. 2012 / 0251531, and Strop et al., 2012, J. Mol. Biol., 420:204-219).

[0186] Specific non-limiting examples of known amino acid modifications to reduce FcγR and / or complement binding to Fc include those identified in Table E.

[0187] [Table E]

[0188] Additional examples include Fc regions engineered to contain the amino acid modifications L234A / L235A / D265S, and asymmetric amino acid modifications in the CH2 domain that reduce binding of Fc to all Fcγ receptors are described in International Publication No. WO2014 / 190441.

[0189] In additional embodiments, certain amino acid substitutions are introduced into human IgG1 Fc such that the Fc domain of the disclosure abolishes immune effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Mutations in the CH2 region of the antibody heavy chain can include positions 234, 235, and 265 in the EU numbering to reduce or eliminate immune effector function.

[0190] Targeting Domains In certain embodiments, the IL12 fusion proteins described herein may include a "targeting domain" that targets the fusion protein to a site of action (e.g., a site of inflammation, a specific anatomical site such as an organ, or a tumor). As used herein, a "targeted antigen" is an antigen that is recognized and specifically bound by the targeting domain.

[0191] In some embodiments, the targeting domain is specific for (specifically binds to) an antigen found on cells in a protease-rich environment, such as the tumor microenvironment. In some embodiments, the encoded targeting domain is specific for (e.g., specifically binds to or recognizes) regulatory T cells (Tregs), for example targeting CCR4 or CD39 receptors. Other suitable targeting domains include those with alloligands overexpressed in inflamed tissues, such as IL1 receptors, or IL6 receptors. In other embodiments, suitable targeting domains include those with alloligands present on immune cells, such as dendritic cells (DCs), T cells, NK cells, etc. In other embodiments, suitable targeting domains include those with alloligands overexpressed in tumor tissues, such as tumor-associated antigens (TAA).

[0192] TAAs contemplated herein for tumor targeting include, but are not limited to, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, and CEA. In certain embodiments, the masked fusion protein comprises two targeting domains that bind to two different target antigens known to be expressed in diseased cells or tissues. Exemplary pairs of antigen binding domains include, but are not limited to, EGFR / CEA, EpCAM / CEA, and HER-2 / HER-3.

[0193] Suitable targeting domains include antigen-binding domains such as antibodies and fragments thereof, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibody 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 species nanobodies (VHH), dAbs, etc. Other suitable antigen-binding domains include binding domains based on non-immunoglobulin proteins that mimic antibody binding and / or structure, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and other engineered scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include ligands for desired receptors, ligand-binding portions of receptors, lectins, and peptides that bind or associate with one or more target antigens.

[0194] In some embodiments, the targeting domain specifically binds to a cell surface molecule. In some embodiments, the targeting domain specifically binds to a tumor antigen. In some embodiments, the targeting domain specifically and independently binds to a tumor antigen selected from at least one of fibroblast activation protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling agent 2 (Trop2), fibronectin EDB (EDB-FN), fibronectin F.IIIB domain, CGS-2, EpCAM, EGER, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the targeting polypeptide specifically and independently binds to two different antigens, and at least one of the antigens is a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. The TAA targeted by the targeting domain can be a tumor antigen expressed on a tumor cell. Tumor antigens are well known in the art and include, for example, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, PSMA, CD38, BCMA, and CEA, 5T4, AFP, B7-H3, cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 including E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Mucl, Mucl6, NaPi2b, nectin-4, P-cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, FAP, or WT1.

[0195] In some embodiments, the targeted antigen is an immune checkpoint protein. Examples of immune checkpoint proteins include, but are not limited to, CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, 0X40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD80, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDOL, ID02, TDO, KIR, LAG-3, TIM-3, or VISTA. In certain embodiments, the targeting domain is an antibody or antigen-binding fragment thereof that specifically binds to an immune checkpoint protein, or the targeting domain is a ligand or binding fragment thereof that binds to an immune checkpoint protein.

[0196] The targeting domain may specifically bind to a cell surface molecule, such as a protein, lipid, or polysaccharide. In some embodiments, the targeted antigen is an antigen expressed on tumor cells, virus-infected cells, bacteria-infected cells, damaged red blood cells, arterial plaque cells, inflammatory or fibrotic tissue cells. The targeted antigen may include an immune response regulator. Examples of immune response regulators include, but are not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin 2 (IL2), interleukin 3 (IL3), interleukin 12 (IL12), interleukin 15 (IL15), B7-1 (CD80), B7-2 (CD86), GITRL, CD3, or GITR.

[0197] In certain embodiments, the targeting domain specifically binds to a cytokine receptor. Examples of cytokine receptors include type I cytokine receptors, such as GM-CSF receptor, G-CSF receptor, type I IL receptor, Epo receptor, LIF receptor, CNTF receptor, TPO receptor; type II cytokine receptors, such as IFN-alpha receptor (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptor (IFNGR1, IFNGR2), type II IF receptor; chemokine receptors, such as CC chemokine receptor, CXC chemokine receptor, CX3C chemokine receptor, XC chemokine receptor, CX ... kine receptors; tumor necrosis receptor superfamily receptors, e.g., TNFRSF5 / CD40, TNFRSF8 / CD30, TNFRSF7 / CD27, TNFRSFlA / TNFRl / CD120a, TNFRSF1B / TNFR2 / CD120b; TGF-beta receptors, e.g., TGF-beta receptor 1, TGF-beta receptor 2; Ig superfamily receptors, e.g., IF-1 receptor, CSF-1R, PDGFR (PDGFRA, PDGFRB), SCFR, but are not limited to these.

[0198] In some embodiments, the targeting domain is fused to the masked IL12 fusion protein via a linker or PCL (also known as a protease-cleavable linker). In certain embodiments, the linker fusing the targeting domain to the masked IL12 fusion protein is a PCL that is cleaved at the site of action (e.g., by an inflammation or cancer-specific protease). In this regard, the PCL can be the same as or different from any other PCL present in the masked IL12 fusion protein, such as a PCL fusing MM to an Fc polypeptide, a PCL present with MM, or a PCL that links an IL12 polypeptide to an Fc polypeptide. In certain embodiments, the PCL to which the targeting domain is fused is the same as the PCL fusing MM to an Fc polypeptide and / or the PCL fusing IL12 to an Fc polypeptide, such that all of the cleavage sites are cleaved upon reaching the target. In some embodiments, the targeting domain is fused to the masked IL12 fusion protein via a linker that is not cleaved at the site of action (e.g., by an inflammation or cancer-specific protease).

[0199] Certain embodiments relating to IL12 and IL23 fusion proteins In certain embodiments, an IL12 fusion protein of the present disclosure may comprise (i) an IL12 polypeptide having a modified p40 domain as described herein, (ii) an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, linked to a p35 domain. In some embodiments, the p35 domain comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the N-terminus of the p35 domain is linked to the C-terminus of the modified p40 domain, either directly or via a first linker. The first linker is (G4S) xwhere x is 1, 2, 3 or 4. In some embodiments, the IL12 fusion protein is an IL12 HetFc fusion protein, which may further comprise a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide. In such embodiments, the IL12 polypeptide may be linked to the first Fc polypeptide either directly or via a second linker. In some embodiments, the IL12 polypeptide is linked to the C-terminus of the first Fc polypeptide. Thus, in some embodiments, the IL12 polypeptide is linked to the C-terminus of the first Fc polypeptide via the N-terminus of a modified p40 domain.

[0200] In some embodiments, the IL12 HetFc fusion protein can further comprise a masking moiety, which is capable of non-covalently interacting with the modified p40 domain, thereby masking the modified p40 domain and increasing the binding affinity (K) of the modified p40 domain for binding to at least one of its cognate receptors when compared to the unmasked modified p40 domain. D ). In some embodiments, the masking moiety is attached to the C-terminus of the second Fc polypeptide either directly or via a third linker. The third linker may be a protease-cleavable linker. The second and / or third linker(s) may each comprise or consist of an amino acid sequence ranging from 5 to about 50 amino acids. Thus, in some embodiments, the second linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 132 and the third linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 134. In some embodiments, the masking moiety is a V L V linked to the domain either directly or via a fourth linker H In some embodiments, the fourth linker comprises or consists of an scFv domain comprising the V domain. In some embodiments, the fourth linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135, HThe domain comprises or consists of an amino acid sequence having about 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:2, and L The domain comprises or consists of an amino acid sequence having about 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3. In some embodiments, the masking moiety is capable of reducing the binding affinity of the modified p40 domain to at least one cognate receptor by at least about 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, or at least about 300-fold compared to the corresponding fusion protein without the masking moiety. The at least one cognate receptor may comprise or consist of IL12Rβ1.

[0201] In various embodiments, described herein is a fusion protein that is a masked IL12 HetFc fusion protein comprising: (i) an IL-12 polypeptide comprising a modified p40 domain according to any of the embodiments described herein linked via a linker (G4S)4 to a p35 domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11; (ii) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide; and (iii) a masking moiety (MM) comprising an anti-IL12 scFv domain, wherein the IL12 polypeptide is linked either directly or via a second linker to the C-terminus of the first Fc polypeptide, and the masking moiety is linked either directly or via a third linker to the C-terminus of the second Fc polypeptide. In some of these embodiments, the fusion protein comprises or consists of two polypeptide chains, from N-terminus to C-terminus: (i) an Fc-IL12 polypeptide chain and (ii) an Fc-MM polypeptide chain. In some embodiments, the Fc-IL12 polypeptide chain comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 61-89. In some embodiments, the Fc-IL12 polypeptide chain comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 61-89. An Fc-MM polypeptide chain capable of pairing with an Fc-IL12 polypeptide chain via a CH3 domain may comprise or consist of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the Fc-MM polypeptide chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60.Thus, in some embodiments, the masked IL12 HetFc fusion protein comprises: (i) v28046, v28047, v28048, v28049, v28050, v28051, v28053, v28054, v28055, v28056, v28057, v28058, v28059, v28060, v28061, v28062, v28063, v28064, v28065, v28066, v28067, v28068, v28069, v28070, v28071, v28072, v28073, v28074, v28075, v28076, v28077, v28078, v28079, v28080, v28081, v28082, v28083, v28084, v28085, v28086, v28087, v28088, v28089, v28090, v28091, v28092, v28093, v28094, v28095, v28096, v28097, v28098, v28099, v28000, v28001, v28002, v28003, v28004, v28005, v280060, v28004, v28005, v280061, v28003, v28004, v28005, v280062, v280063, v28004 and (ii) an Fc-IL12 polypeptide chain comprising (from N-terminus to C-terminus) an amino acid sequence selected from the group consisting of the sequences v26503, v26504, v26505, v26506, v28067, v28068, v28069, v28070, v28071, v28072, v28074 and v28075.

[0202] Also described herein in some embodiments is a fusion protein that is an unmasked IL12 HetFc fusion protein comprising: (i) an IL-12 polypeptide comprising a modified p40 domain according to any of the embodiments described herein linked via a linker (G4S)4 to a p35 domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:11; and (ii) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein the IL12 polypeptide is linked either directly or via a second linker to the C-terminus of the first Fc polypeptide. In some embodiments, the fusion protein comprises or consists of two polypeptide chains, from N-terminus to C-terminus: (i) an Fc-IL12 polypeptide chain and (ii) an Fc polypeptide chain. In some embodiments, the Fc-IL12 polypeptide chain comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 61 to 89. In some embodiments, the Fc-IL12 polypeptide chain comprises or consists of an amino acid sequence set forth in SEQ ID NOs: 61 to 89. An Fc polypeptide chain capable of pairing with an Fc-IL12 polypeptide chain may comprise or consist of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 58.In some embodiments, the unmasked IL12 HetFc fusion protein is selected from the group consisting of: (i) v28046, v28047, v28048, v28049, v28050, v28051, v28053, v28054, v28055, v28056, v28057, v28058, v28059, v28060, v28061, v28062, v28063, v28064, v28065, v28066, v28067, v28068, v28069, v28070, v28071, v28072, v28073, v28074, v28075, v28076, v28077, v28078, v28079, v28080, v28081, v28082, v28083, v28084, v28085, v28086, v28087, v28088, v28089, v28090, v28091, v28092, v28093, v28094, v28095, v28096, v28097, v28098, v28099, v28000, v28001, v28002, v28003, v28004, v28005, v280060, v28004, v28005, v280061, v280062, v28003, v28004, v28005, v280063, v and (ii) an Fc-IL12 polypeptide chain comprising (from N-terminus to C-terminus) an amino acid sequence selected from the group consisting of the sequences of v12153, v12154, v12155, v12156, v12157, v12158, v12159, v12160, v12161, v12162, v12163, v12164, v12165, v12166, v12167, v12168, v12169, v12170, v12171, v12172, v12174, and v12175.

[0203] Polypeptides and Polynucleotides The cytokine (e.g., IL12 and other members of the IL12 family of cytokines) fusion proteins described herein comprise at least one polypeptide. Polynucleotides encoding the polypeptides described herein are also described. Masked cytokine fusion proteins are typically isolated.

[0204] As used herein, "isolated" refers to an agent (e.g., a polypeptide or polynucleotide) that has been identified and separated and / or recovered from components of its natural cell culture environment. Contaminant components of its natural environment are substances that would interfere with diagnostic or therapeutic uses of the cytokine fusion protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Isolated also refers to agents that have been produced synthetically, for example, by human intervention.

[0205] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers and to amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0206] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, praline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bonded to hydrogen, carboxyl groups, amino groups, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Reference to amino acids includes, for example, naturally occurring proteinogenic L-amino acids; chemically modified amino acids, such as D-amino acids, amino acid variants and derivatives; naturally occurring non-proteinogenic amino acids, such as β-alanine, ornithine, and the like; as well as chemically synthesized compounds having properties known in the art that are characteristic of amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, α-methyl amino acids (e.g., α-methylalanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, β-hydroxy-histidine, homohistidine), amino acids with an additional methylene in the side chain ("homo" amino acids), and amino acids in which the carboxylic acid functionality in the side chain is replaced with a sulfonic acid group (e.g., cysteic acid). The incorporation of synthetic non-natural amino acids, substituted amino acids, or non-natural amino acids, including one or more D-amino acids, into the proteins described herein can be advantageous in a number of different ways. D-amino acid-containing peptides and the like exhibit increased stability in vitro or in vivo compared to their L-amino acid-containing counterparts.Therefore, constructing peptides and the like incorporating D-amino acids can be particularly useful when higher intracellular stability is desired or required.More specifically, D-peptides and the like are resistant to endogenous peptidases and proteases, thereby providing improved bioavailability of the molecule and extended effective time in vivo when such properties are desired.In addition, D-peptides and the like are less likely to induce humoral immune responses in the whole organism, since they cannot be efficiently processed for major histocompatibility complex class II restricted presentation to helper T cells.

[0207] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted one-letter codes.

[0208] Also provided herein is a polynucleotide encoding a masked cytokine fusion protein. The term "polynucleotide" or "nucleotide sequence" is intended to indicate a continuous stretch of two or more nucleotide molecules. The nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic or synthetic origin, or any combination thereof.

[0209] The term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers in either single-stranded or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), analogs of DNA used in antisense technology (phosphorothioates, phosphoramidates, etc.). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (including, but not limited to, degenerate codon substitutions) and complementary sequences in addition to the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0210] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that code for the same or essentially identical amino acid sequences, or to essentially identical sequences if the nucleic acid does not code for an amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are a type of conservatively modified variation. Every nucleic acid sequence herein that codes for a polypeptide also describes every possible silent variation of the nucleic acid. Those skilled in the art will understand that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, every silent variation of a nucleic acid that codes for a polypeptide is implicit in each sequence described.

[0211] With respect to amino acid sequences, one of skill in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," which alterations result in the deletion of an amino acid, the addition of an amino acid, or the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known to those of skill in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles described herein.

[0212] Conservative substitution tables providing functionally similar amino acids are known to those of skill in the art. The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0213] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same. When the sequences are compared and aligned for best correspondence over a comparison window or designated region, they are "substantially identical" if they have a percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity over a particular region) as measured using one of the following sequence comparison algorithms (or other algorithms available to those of skill in the art) or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence. Identity can exist over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 75-100 amino acids or nucleotides in length, or, if not specified, over the entire sequence of the polynucleotide or polypeptide. Polynucleotides encoding the polypeptides described herein, including homologs from species other than human, may be obtained by a process comprising screening libraries under stringent hybridization conditions with a labeled probe having a polynucleotide sequence described herein or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequences. Such hybridization techniques are well known to those skilled in the art.

[0214] For sequence comparison, typically, one sequence serves as a reference sequence, and this sequence is compared with test sequence.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and if necessary, partial sequence coordinates are designated, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0215] A "comparison window," as used herein, includes reference to any segment of a number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for purposes of comparison are known to those of skill in the art. Optimal sequence alignment for comparison can be performed, but is not limited to, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0216] An example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information and available on the World Wide Web at ncbi.nlm.nih.gov. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and an expectation (E) of 10, as well as the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915), alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm is typically run with the "low complexity" filter turned off.

[0217] BLAST algorithm also performs statistical analysis of similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.

[0218] The phrase "selectively (or specifically) hybridizes" refers to the fact that when a particular nucleotide sequence is present in a complex mixture (including, but not limited to, total cellular or library DNA or RNA), a molecule will bind, duplex or hybridize under stringent hybridization conditions only to that sequence.

[0219] The phrase "stringent hybridization conditions" refers to the hybridization of sequences of DNA, RNA, or other nucleic acids, or combinations thereof, under conditions of low ionic strength and high temperature as known in the art. Typically, under stringent conditions, a probe will hybridize to its target subsequence in a complex mixture of nucleic acids (including, but not limited to, total cellular or library DNA or RNA), but not to other sequences in the complex mixture. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to nucleic acid hybridization is provided in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).

[0220] As used herein, the terms "engineer, engineered, engineering" are considered to include any processing of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modification of the amino acid sequence, of the glycosylation pattern, or of individual amino acid side groups, as well as combinations of these approaches. Engineered proteins are expressed and produced by standard molecular biology techniques.

[0221] By "isolated nucleic acid molecule or polynucleotide" is intended a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered to be isolated. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides that have been purified (partially or substantially) in solution. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain the polynucleotide molecule, but the polynucleotide is present extrachromosomally or at a chromosomal location that differs from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts, as well as positive and negative strand forms, and double-stranded forms. The isolated polynucleotides or nucleic acids described herein further include such molecules that have been produced synthetically, e.g., via PCR or chemical synthesis. In certain embodiments, the polynucleotide or nucleic acid also includes a control element, e.g., a promoter, a ribosome binding site, or a transcription terminator.

[0222] The term "polymerase chain reaction" or "PCR" generally refers to a method for the amplification of a desired nucleotide sequence in vitro, for example, as described in U.S. Patent No. 4,683,195. In general, the PCR method involves repeated cycles of primer extension synthesis, using oligonucleotide primers capable of preferentially hybridizing to a template nucleic acid.

[0223] By a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present disclosure, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence.That is, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.These changes in the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence or anywhere between these terminal positions, and may be interspersed individually between the residues in the reference sequence or may be interspersed in one or more consecutive groups within the reference sequence. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present disclosure can be routinely determined using known computer programs, such as those discussed above for polypeptides (e.g., ALIGN-2).

[0224] A derivative or variant of a polypeptide is said to share "homology" or be "homologous" to a peptide if the amino acid sequence of the derivative or variant has at least 50% identity with a 100 amino acid sequence from the original peptide. In certain embodiments, the derivative or variant is at least 75% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 85% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, the amino acid sequence of the derivative is at least 90% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In some embodiments, the amino acid sequence of the derivative is at least 95%, 96%, 97%, or 98% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative. In certain embodiments, the derivative or variant is at least 99% identical to either a peptide or a fragment of a peptide having the same number of amino acid residues as the derivative.

[0225] The term "modified" as used herein refers to any alteration made to a given polypeptide, such as changes to the length, amino acid sequence, chemical structure of the polypeptide, co-translational modifications, or post-translational modifications of the polypeptide. The form of the term "(modified)" means that the polypeptide under discussion is optionally modified, i.e., the polypeptide under discussion may or may not be modified.

[0226] In some embodiments, the cytokine fusion protein construct comprises an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the relevant amino acid sequence, or a fragment thereof, set forth in the table(s) or accession number(s) disclosed herein. In some embodiments, the cytokine fusion protein comprises an amino acid sequence encoded by a polynucleotide that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the relevant nucleotide sequence, or a fragment thereof, set forth in the table(s) or accession number(s) disclosed herein.

[0227] In some embodiments, the one or more amino acid substitutions are one or more non-conservative substitutions. In other embodiments, the one or more amino acid substitutions are one or more conservative substitutions. In general, a "conservative substitution" as used herein is considered to be a substitution of one amino acid with another amino acid having similar physical, chemical and / or structural properties. Common conservative substitutions are listed in the first column of Table F. Those skilled in the art will understand that while the main factors determining what constitutes a conservative substitution are usually the size of the amino acid side chain and its physical / chemical properties, certain environments allow for the substitution of a given amino acid with a broader range of amino acids than those listed in the first column of Table F. These additional amino acids tend to have similar properties to the amino acid being substituted but are more diverse in size, or tend to be similar in size but are more diverse in physical / chemical properties. This broader range of conservative substitutions is listed in the second column of Table F. Those skilled in the art can easily ascertain the most appropriate group of substitutes to select, given the particular protein environment in which the amino acid substitution is to be made.

[0228] [Table F]

[0229] Methods for preparing IL12 fusion proteins / recombinant proteins The IL12 fusion proteins or other recombinant proteins described herein (e.g., recombinant proteins comprising modified p40 domains) can be produced using standard recombinant methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 and "Antibodies: A Laboratory Manual," 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014) and as further described herein.

[0230] Typically, for recombinant production of an IL12 fusion protein or other recombinant protein, a nucleic acid encoding the IL12 fusion protein or other recombinant protein is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using routine procedures (e.g., by using an oligonucleotide probe capable of specifically binding to the IL12 fusion protein or other recombinant protein gene).

[0231] Suitable host cells for cloning or expressing vectors encoding the IL12 fusion protein or other recombinant proteins include prokaryotic or eukaryotic cells described herein.

[0232] "Recombinant host cell" or "host cell" refers to a cell that contains an exogenous polynucleotide, regardless of the method used for insertion, such as direct uptake, transduction, f-mating, or other methods known in the art for making recombinant host cells. The exogenous polynucleotide may be maintained as a non-integrated vector, such as a plasmid, or alternatively, may be integrated into the host genome.

[0233] As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eucarya, such as animals (including but not limited to mammals, insects, reptiles, and birds), ciliates, plants (including but not limited to monocotyledons, dicotyledons, and algae), fungi, yeasts, flagellates, microsporidia, protists, etc.

[0234] As used herein, the term "prokaryote" refers to a prokaryotic organism. For example, non-eukaryotes may be from the Eubacteria (including, but not limited to, Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or Archaea (Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacteria, such as Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, etc.) phylogenetic domain. fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogenetic domain.

[0235] For example, IL12 fusion protein constructs or other recombinant proteins comprising modified p40 domain constructs described herein can be produced in bacteria, particularly where glycosylation and Fc effector functions are not required. For expression of polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes expression of antibody fragments in E. coli.) After expression, the IL12 fusion protein or other recombinant protein described herein can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0236] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding IL12 fusion proteins, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of IL12 fusion proteins with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0237] Suitable host cells for the expression of glycosylated polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0238] Plant cell cultures may also be utilized as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing recombinant proteins in transgenic plants).

[0239] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol Reprod, 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells as described, for example, in Mather et al., Annals NY Acad Sci, 383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc Natl Acad Sci USA, 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for production of fusion proteins, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0240] In some embodiments, an IL12 fusion protein or other recombinant protein described herein is produced in a stable mammalian cell by a method comprising transfecting at least one stable mammalian cell with a nucleic acid encoding an IL12 fusion protein or other recombinant protein described herein in a predetermined ratio, and expressing the nucleic acid in the at least one mammalian cell. In some embodiments, the predetermined ratio of nucleic acids is determined in a transient transfection experiment to determine the relative ratio of introduced nucleic acids that results in the highest percentage of fusion protein in the expression product (see also the Examples section for Protocols 3 and 4, as well as Example 3).

[0241] In some embodiments, in the methods of producing an IL12 fusion protein or other recombinant protein in a stable mammalian cell described herein, the expression product of the stable mammalian cell contains a higher percentage of the desired HetFc IL12 fusion protein compared to the monomeric fusion protein. In certain embodiments, the fusion protein herein is glycosylated.

[0242] In some embodiments, in the methods of producing a fusion protein in a stable mammalian cell, the method further comprises identifying and purifying the desired fusion protein, in some embodiments, the identification is by one or both of liquid chromatography and mass spectrometry (see also the Examples herein).

[0243] If required, IL12 fusion proteins or other recombinant proteins can be purified or isolated after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques including ion exchange, hydrophobic interaction, affinity, size or gel filtration, and reverse phase, performed at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration are also useful. As is well known in the art, various natural proteins bind to Fc and antibodies, and these proteins can be used to purify IL12 fusion proteins. For example, bacterially derived proteins A and G bind to the Fc region. Similarly, bacterially derived protein L binds to the Fab region of some antibodies. In many cases, purification can be made possible by a specific fusion partner. For example, the antibody may be purified using glutathione resin if a GST fusion is used, Ni+2 affinity chromatography if a His tag is used, or immobilized anti-flag antibody if a flag tag is used. For general guidance in suitable purification techniques, see, for example, Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY (1994). The degree of purification required will vary depending on the use of the IL12 fusion protein. In some cases, purification may not be necessary.

[0244] In certain embodiments, an IL12 fusion protein or other recombinant protein may be purified using anion exchange chromatography, including but not limited to, chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAF, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource / Source Q and DEAE, Fractogel Q or DEAE columns or their equivalents or equivalents.

[0245] In some embodiments, IL12 fusion proteins or other recombinant proteins may be purified using cation exchange chromatography, including but not limited to, chromatography on SP-sepharose, CM sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource / Source S or CM, or Fractogel S or CM columns, or their equivalents or equivalents.

[0246] In certain embodiments, the IL12 fusion protein or other recombinant protein herein is substantially pure. The term "substantially pure" (or "substantially purified") refers to a construct described herein, or a variant thereof, which may be substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e., in a natural cell, or in the case of a recombinantly produced construct, in a host cell. In certain embodiments, a construct that is substantially free of cellular material includes preparations of the protein that have less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating proteins. When the construct is recombinantly produced by a host cell, the protein in certain embodiments is present at no more than about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% by dry weight of the cells. When the construct is recombinantly produced by a host cell, the protein in certain embodiments is present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or less than about 1 mg / L.

[0247] In certain embodiments, the term "substantially purified", as applied to a HetFc IL12 fusion protein comprising a heterodimeric Fc as described herein, means that the heterodimeric Fc has a purity level of at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, specifically at least about 75%, 80%, 85%, more specifically at least about 90%, at least about 95%, at least about 99% or more, as determined by suitable methods such as, for example, SDS / PAGE analysis, RP-HPLC, size exclusion chromatography (SEC) and capillary electrophoresis.

[0248] IL12 fusion proteins and other recombinant proteins can also be chemically synthesized using techniques known in the art (see, e.g., Creighton, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., NY (1983), and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, D-isomers of common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, 4 aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as α-methyl amino acids, C α-methyl amino acids, N α-methyl amino acids, etc., and amino acid analogs in general. Furthermore, amino acids can be D (dextrorotatory) or L (levorotatory).

[0249] Certain embodiments of the present disclosure relate to isolated nucleic acids encoding the masked or unmasked HetFc IL12 fusion proteins or other recombinant proteins described herein, such as those that may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of MM, or modified IL12 polypeptides.

[0250] Certain embodiments relate to vectors (e.g., expression vectors) that contain a nucleic acid encoding a HetFc IL12 fusion protein or other recombinant protein described herein. The nucleic acid may be contained in a single vector or may be contained in multiple vectors. In some embodiments, the nucleic acid is contained in a multicistronic vector.

[0251] Certain embodiments relate to host cells comprising such nucleic acids or one or more vectors comprising nucleic acids. In some embodiments, the host cell comprises (e.g., has been transformed with) a vector comprising a nucleic acid encoding an amino acid sequence comprising a first fusion protein described herein (e.g., a first Fc polypeptide fused to MM) and an amino acid sequence comprising a second fusion protein described herein (e.g., a second Fc polypeptide fused to an IL12 or IL23 polypeptide). In some embodiments, the host cell comprises (e.g., has been transformed with) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a first fusion protein described herein (e.g., a first Fc polypeptide fused to MM) and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a second fusion protein described herein (e.g., a second Fc polypeptide fused to an IL12 or IL23 polypeptide). In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, or a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell).

[0252] Certain embodiments relate to methods of making an IL12 fusion protein by culturing a host cell into which a nucleic acid encoding the fusion protein has been introduced under conditions suitable for expression of the IL12 fusion protein, and, optionally, recovering the IL12 fusion protein from the host cell (or host cell culture medium).

[0253] Post-translational modifications In certain embodiments, the IL12 fusion proteins described herein may be differentially modified during or after translation.

[0254] The term "modified," as used herein, refers to any alteration made to a given polypeptide, such as alterations to the length, amino acid sequence, chemical structure of the polypeptide, co-translational modifications, or post-translational modifications of the polypeptide.

[0255] The term "post-translationally modified" refers to any modification of a natural or unnatural amino acid that occurs to such an amino acid after it is incorporated into a polypeptide chain. This term includes, by way of example only, co-translational in vivo modifications, co-translational in vitro modifications (such as in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications.

[0256] In some embodiments, the IL12 fusion protein may include modifications such as glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage or linkage to an antibody molecule or antigen-binding construct or other cellular ligand, or a combination of these modifications. In some embodiments, the IL12 fusion protein may be chemically modified by known techniques, including, but not limited to, specific chemical cleavage with cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation; formylation; oxidation; reduction, or metabolic synthesis in the presence of tunicamycin.

[0257] Any additional post-translational modifications of the IL12 fusion protein or portions thereof, such as termini, attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The IL12 fusion proteins described herein may optionally be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label, to allow for detection and isolation of the protein. Examples of suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, or aequorin; and examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, or fluorine.

[0258] In some embodiments, the IL12 fusion proteins described herein may be conjugated to a macrocyclic chelate that associates with a radioactive metal ion.

[0259] In embodiments where IL12 fusion proteins are modified by natural processes, such as post-translational processing, or by chemical modification techniques, the same type of modification may optionally be present in the same or varying degrees at several sites in a given polypeptide. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, formation of disulfide bonds, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, e.g., Proteins-Structure and Molecular Properties, 2nd Ed., TECreighton, W.H. Freeman and Company, New York (1993), Post-Translational Covalent Modification of See Proteins, B.C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. NY Acad. Sci. 663:48-62 (1992).

[0260] In certain embodiments, the IL12 fusion protein may be bound to a solid support, which may be particularly useful for immunoassays or purification of polypeptides that are bound by, or that bind to, or that associate with the proteins described herein. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0261] Pharmaceutical Compositions Also provided herein is a pharmaceutical composition comprising an IL12 fusion protein described herein. The pharmaceutical composition comprises an IL12 fusion protein and a pharma- ceutically acceptable carrier.

[0262] The term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more specifically in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers are sterile liquids, such as water and oils, and may include those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. In some aspects, the carrier is an artificial carrier not found in nature. Water may be used as a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like. The compositions may also contain small amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions may be formulated as suppositories, using traditional binders and carriers, such as triglycerides. Oral formulations may include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions will contain a therapeutically effective amount of the IL12 fusion protein, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the method of administration.

[0263] In certain embodiments, the composition comprising the IL12 fusion protein is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachette indicating the quantity of active agent. When the composition is administered by injection, it can be dispensed with an infusion bottle containing sterile water or saline of pharmaceutical grade. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0264] In certain embodiments, the compositions described herein are formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0265] How to use The present disclosure provides methods of using IL12 fusion proteins and other recombinant fusion proteins that contain the modified p40 domains described herein.

[0266] In particular, further provided herein are methods of treating a subject having or at risk of developing cancer, an autoimmune disease, an inflammatory disorder, or an infectious disease. Further provided herein are methods of treating a subject having or at risk of developing any type of cancer, for example, but not limited to, a disease selected from the group consisting of breast cancer, including, by way of non-limiting example, triple negative breast cancer, ER / PR+ breast cancer, and Her2+ breast cancer, lung cancer (e.g., non-small cell squamous cell carcinoma and adenocarcinoma), colorectal cancer, gastric cancer, glioblastoma, ovarian cancer, endometrial cancer, kidney cancer, sarcoma, skin cancer, cervical cancer, liver cancer, bladder cancer, bile duct cancer, prostate cancer, melanoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), esophageal, squamous cell carcinoma, basal cell carcinoma, pancreatic cancer, leukemia, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic disorders, including multiple myeloma, solid tumors, bone disease, or metastasis in cancer, regardless of primary tumor origin. Further provided are methods of treating a subject having or at risk of developing rheumatoid arthritis, Crohn's disease, SLE, cardiovascular injury, or ischemia.

[0267] In certain embodiments, the present disclosure provides a method of treating a disease in a subject by administering to the subject a therapeutically effective amount of a cytokine fusion protein disclosed herein, wherein the disease is selected from the group consisting of colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, bladder cancer, cervical cancer, and lung cancer (e.g., non-small cell squamous carcinoma and adenocarcinoma).

[0268] The method includes administering to a subject in need thereof an effective amount of an IL12 fusion protein or other recombinant fusion protein (e.g., comprising a modified p40 domain) disclosed herein (fusion protein), typically administered as a pharmaceutical composition. In some embodiments, the method further includes selecting a subject having or at risk of developing cancer. In some embodiments, the pharmaceutical composition includes an IL12 fusion protein, or a fragment thereof, that is activated at the tumor site. In one embodiment, the tumor is a solid tumor.

[0269] In certain embodiments, methods of treating cancer are provided, comprising administering to a subject in whom such treatment or remission is desired an IL12 fusion protein as described herein in an amount effective to treat or remit cancer. In other embodiments, methods are provided of using an IL12 fusion protein as described herein in the preparation of a medicament for the treatment or remission of cancer in a subject.

[0270] The term "subject" refers to an animal, in some embodiments a mammal, that is the object of treatment, observation or experiment. The animal may be a human, a non-human primate, a companion animal (e.g., dog, cat, etc.), a livestock animal (e.g., cows, sheep, pigs, horses, etc.), or a laboratory animal (e.g., rats, mice, guinea pigs, etc.).

[0271] The term "mammal" as used herein includes, but is not limited to, humans, non-human primates, canine, feline, murine, bovine, equine, and porcine.

[0272] "Treatment" refers to clinical intervention in an attempt to change the natural history of the individual or cells being treated and can occur during the course of clinical pathology. Desirable effects of treatment include prevention of disease recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, remission or palliative disease, and remission or improved prognosis. In some embodiments, the IL12 fusion proteins described herein are used to delay the onset of a disease or disorder. In one embodiment, the IL12 fusion proteins described herein and methods described herein result in tumor regression. In one embodiment, the IL12 fusion proteins described herein and methods described herein result in inhibition of tumor / cancer growth.

[0273] Desirable effects of treatment include, but are not limited to, one or more of: prevention of disease recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, remission or palliation of disease symptoms, improved survival, and remission or improved prognosis. In some embodiments, the IL12 fusion proteins described herein are used to delay disease onset or slow disease progression.

[0274] In some embodiments, the IL12 fusion proteins of the present disclosure can be used to prevent the development of a disease, such as cancer.

[0275] The term "effective amount" as used herein refers to the amount of an IL12 fusion protein described herein or a composition comprising an IL12 fusion protein described herein administered that achieves the goal of the described method, e.g., can alleviate to some extent one or more of the symptoms of the disease, condition or disorder being treated. The amount of a composition described herein that is effective in treating and inhibiting a disease or disorder associated with aberrant expression and / or activity of a therapeutic protein can be determined by standard clinical techniques. In addition, in vitro assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0276] The IL12 fusion protein described herein is administered to a subject. Various delivery systems, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of nucleic acids as part of retroviruses or other vectors, are known and can be used to administer the IL12 fusion protein formulations described herein. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intratumoral, intranasal, epidural, and oral routes. The compound or composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration may be systemic or local. Additionally, in certain embodiments, it may be desirable to introduce the IL12 fusion protein compositions described herein into the central nervous system by any suitable route, including intraventricular and intrathecal injection, which may be facilitated, for example, by an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0277] In certain embodiments, it may be desirable to administer an IL12 fusion protein as described herein, or a composition as described herein, locally to the area in need of treatment, which may be accomplished, for example, but not limited to, by local infusion, topical application during surgery, for example, in combination with a wound dressing after surgery, by injection, by catheter, by suppository, or by implant, which may be a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers. Preferably, when administering a protein, including an IL12 fusion protein as described herein, care must be taken to use a material that does not absorb the protein.

[0278] In another embodiment, the IL12 fusion proteins described herein or compositions comprising same may be delivered in vesicles, in particular liposomes (see Langer, Science 249:1527-1533 (1990); Treat et al., Lopez-Berestein, ibid., pp. 317-327 in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); see ibid. in its entirety).

[0279] In yet another embodiment, the IL12 fusion protein or composition described herein can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra, Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed close to the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson in Medical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)).

[0280] In certain embodiments involving nucleic acids encoding IL12 fusion proteins as described herein, the nucleic acid may be administered in vivo by constructing it as part of an appropriate nucleic acid expression vector and administering it intracellularly (e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of particle bombardment (e.g., gene gun, Biolistic, Dupont) or coating with lipids or cell surface receptors or transfection agents) to promote expression of the encoded protein, or by administering the nucleic acid conjugated to a homeobox-like peptide known to enter the nucleus (see, e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, the nucleic acid may be introduced intracellularly and incorporated into the host cell DNA for expression by homologous recombination.

[0281] The IL12 fusion proteins described herein can be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy, immune checkpoint inhibitors, and anti-tumor agents). Generally, administration of products of species origin or species reactivity (in the case of antibodies) that is allogeneic to the patient's species is preferred.

[0282] The IL12 fusion proteins described herein can be used in the treatment of cancer. In some embodiments, the IL12 fusion proteins described herein can be used in the treatment of patients who have received one or more alternative forms of anti-cancer therapy. In some embodiments, the patient has relapsed or failed to respond to one or more alternative forms of anti-cancer therapy. In other embodiments, the IL12 fusion protein is administered to the patient in combination with one or more alternative forms of anti-cancer therapy. In other embodiments, the IL12 fusion protein is administered to the patient who has become refractory to treatment with one or more alternative forms of anti-cancer therapy.

[0283] Kits and manufactured products Also described herein is a kit that includes one or more fusion protein(s) or another recombinant protein described herein. The individual components of the kit are packaged in separate containers, and such containers may be accompanied by a notice in a format prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products, indicating that the manufacture, use, or sale has been approved by the agency. The kit may optionally contain instructions or directions outlining the method of use or administration regime of the masked IL12 fusion protein.

[0284] Where one or more components of the kit are provided as a liquid solution, e.g., an aqueous solution, or a sterile aqueous solution, the container means may itself be an inhaler, syringe, pipette, eye dropper, or other similar device by which the solution can be administered to a subject or applied to and mixed with the other components of the kit.

[0285] The components of the kit may also be provided in dry or lyophilized form, and the kit may further contain a suitable solvent for reconstitution of the lyophilized components. Regardless of the number or type of container, the kit described herein may also include an instrument to assist in administering the composition to a patient. Such an instrument may be an inhaler, a nasal spray device, a syringe, a pipette, forceps, a measuring spoon, an eye dropper, or a similar medically approved delivery vehicle.

[0286] Certain embodiments relate to an article of manufacture containing materials useful for treating a patient as described herein. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous infusion bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition comprising an IL12 fusion protein alone or in combination with another composition effective for treating a patient, and may have a sterile access port (e.g., the container may be an intravenous infusion bag or a vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used to treat a condition suitable for use of the composition. In some embodiments, the article of manufacture includes (a) a first container containing a composition, the composition comprising an IL12 fusion protein as described herein, and (b) a second container containing a composition, the composition in the container comprising an additional cytotoxic or otherwise therapeutic agent. In such embodiments, the article of manufacture may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may optionally further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0287] Exemplary embodiments Further specific embodiments of the present disclosure are described as follows.

[0288] These embodiments are intended to illustrate the compositions and methods described in this disclosure and are not intended to limit the scope of the disclosure. Additionally, the terms "p40 domain" and "p40 subunit" can be used interchangeably herein.

[0289] Embodiment 1. A modified p40 subunit of an interleukin-12 (IL12) or IL23 protein, comprising a human IL12 p40 subunit polypeptide, said polypeptide comprising at least one amino acid substitution relative to wild-type human IL-12 p40 of SEQ ID NO: 10 at one or more positions corresponding to residues 15, 18, 45, 58, 59, 60, 62, 84, 86, 93, 161, 195, and 197.

[0290] Embodiment 2. The at least one amino acid substitution is selected from the group consisting of W15H, W15K, W15R, D18G, E45K, E45R, K58H, K58S, K58W, E59D, E59G, E59R, E59S, F60D, F60E, F60K, F60R, F60V, D62H, D62I, D62N, K84E, K84I, K84L, K 2. The modified IL12 or IL23 p40 subunit polypeptide of embodiment 1, wherein the substitution is selected from the group consisting of 84V, K84W, K84Y, E86L, E86R, E86S, E86W, D93E, D93H, D93R, D93W, D161R, D161S, K195D, K197D, K197E, K197Q, K197T, and K197W.

[0291] Embodiment 3. The modified IL12 or IL23 p40 subunit polypeptide of embodiment 2, wherein said at least one amino acid substitution is a substitution selected from the group consisting of: W15H, W15K, D18G, E45K, E45R, K58H, K58S, K58W, E59G, E59R, E59S, F60V, D62H, D62I, D62N, K84E, K84W, E86L, E86S, E86W, D93H, D93W, D161R, D161S, K195D, K197E, K197Q, K197T, and K197W.

[0292] Embodiment 4. The modified IL12 or IL23 p40 subunit polypeptide of embodiment 2 or 3, wherein said at least one amino acid substitution comprises a substitution selected from the group consisting of W15H, W15K, E59R, E59S, K84E, K84W, E86W, D161R, K197T, and K197W.

[0293] Embodiment 5. The modified IL12 or IL23 p40 subunit polypeptide of embodiment 2, wherein said at least one amino acid substitution comprises a substitution selected from the group consisting of: W15H, W15R, E45R, K58H, K58S, E59D, E59G, E59R, E59S, F60D, F60E, F60K, F60R, K84E, K84I, K84L, K84V, K84W, K84Y, E86R, E86W, D93E, D93H, D93R, D93W, D161R, K195D, K197D, K197E, K197Q, and K197W.

[0294] Embodiment 6. W15H_K84L, K58H_K84I, E59D_K84W, E59G_K84W, E59R_K84E, E59R_K84W, E59R_E86W, E59D_D93H, E59R_D93R, E59R_K197E, E59R_K197W, F60E_K84W, F60R_K84Y, F60K_K197W, F60R_K197W, K84I_E86R, K84E_D93H, K84I_D93H, K84V_D93H, K84W_D93W, K84I_D161R, K84W_D161R, K8 6. The modified IL12 or IL23 p40 subunit polypeptide of embodiment 2 or 5, comprising 4W_K197E, K84W_K197Q, K84W_K197W, E86W_D93E, E86R_K197D, E86W_K197W, W15H_K84L_K197Q, K58S_E59S_K195D, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, W15R_E59D_F60D_K197W, and E45R_K58S_E59S_K195D.

[0295] Embodiment 7. W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, E86W_D93E, W15H_K84L_K197Q, K58H_E86R_K1 97D, K58S_E59S_K195D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, W15R_E59D_F60D_K197W, and E45R_K58S_E59S_K195D.

[0296] Embodiment 8. The modified IL12 or IL23 p40 subunit polypeptide of embodiment 6, comprising K58H_K84I, E59D_K84W, E59G_K84W, E59R_E86W, E59R_K197E, E59R_K197W, E59R_K84E, E59R_K84W, F60E_K84W, F60R_K197W, K84E_D93H, K84I_D161R, K84I_D93H, K84V_D93H, K84W_D93W, K84W_K197E, K84W_K197Q, and E86W_K197W.

[0297] Embodiment 9. W15H_K84L, K58H_K84I, E59D_K84W, E59G_K84W, E59R_K84E, E59R_K84W, E59R_E86W, E59D_D93H, E59R_D93R, E59R_K197E, E59R_K197W, F60E_K84W, F60R_K84Y, F60K_K197W, F60R_K197W, K84I 9. The modified IL12 or IL23 p40 subunit polypeptide of embodiment 6-8, comprising: K84I_D93H, K84V_D93H, K84W_D93W, K84I_D161R, K84W_D161R, K84W_K197E, K84W_K197Q, K84W_K197W, E86W_D93E, E86R_K197D, and E86W_K197W.

[0298] Embodiment 10. A modified IL12 or IL23 p40 subunit polypeptide according to embodiments 6-9, comprising W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, and E86W_D93E.

[0299] Embodiment 11. A modified IL12 or IL23 p40 subunit polypeptide according to embodiment 7 or 8, comprising W15H_K84L_K197Q, K58H_E86R_K197D, K58S_E59S_K195D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, W15R_E59D_F60D_K197W, and E45R_K58S_E59S_K195D.

[0300] Embodiment 12. A modified IL12 or IL23 p40 subunit polypeptide according to any one of embodiments 1 to 11, comprising a single amino acid substitution relative to the human IL12 p40 subunit of SEQ ID NO:10.

[0301] Embodiment 13. An IL12 protein comprising a modified IL12 p40 subunit polypeptide according to any one of embodiments 1 to 12. 14. An IL23 protein comprising a modified IL23 p40 subunit polypeptide according to any one of embodiments 1 to 12.

[0302] Embodiment 14. A fusion protein comprising a modified IL12 or IL23 p40 subunit polypeptide according to any one of embodiments 1 to 14 and a heterologous polypeptide.

[0303] Embodiment 15 The fusion protein of embodiment 14, wherein the heterologous polypeptide is an antibody or a fragment thereof.

[0304] Embodiment 16 The fusion protein of embodiment 15, wherein the heterologous polypeptide is a heterodimeric Fc protein.

[0305] Embodiment 17. A composition comprising a modified IL12 or IL23p 40 subunit polypeptide according to any one of embodiments 1 to 14 or a fusion protein according to embodiment 15 or 16, and a pharma- ceutically acceptable carrier.

[0306] Embodiment 18. The composition of embodiment 17 for use in treating cancer in a subject.

[0307] Embodiment 19. A nucleic acid molecule encoding a modified IL12 or IL23 p40 subunit polypeptide according to any one of embodiments 1 to 14, or a fusion protein according to embodiment 15 or 16.

[0308] Embodiment 20. A vector comprising the nucleic acid molecule of embodiment 19.

[0309] Embodiment 21. A composition comprising the nucleic acid molecule of embodiment 19 or the vector of embodiment 20 and a pharma- ceutically acceptable carrier.

[0310] Embodiment 22. A modified p40 domain comprising one or more amino acid substitutions relative to the wild-type human mature IL12 p40 domain sequence set forth in SEQ ID NO:10, wherein said one or more amino acid substitutions are located at one or more of positions E45, D62 and D161, and wherein the numbering of the amino acid residues is based on the amino acid sequence set forth in SEQ ID NO:10.

[0311] Embodiment 23. The modified p40 domain of embodiment 22, wherein said one or more amino acid substitutions at said one or more positions are K, H, I, N, R or S substitutions, or a combination thereof.

[0312] Embodiment 24. The modified p40 domain of embodiment 22 or 23, wherein said one or more amino acid substitutions comprises E45K.

[0313] Embodiment 25. The modified p40 domain of any one of embodiments 22 to 24, wherein the one or more amino acid substitutions comprises D62H.

[0314] Embodiment 26. The modified p40 domain of any one of embodiments 22 to 24, wherein the one or more amino acid substitutions comprises D62I.

[0315] Embodiment 27. The modified p40 domain of any one of embodiments 22 to 24, wherein the one or more amino acid substitutions comprises D62N.

[0316] Embodiment 28. The modified p40 domain of any one of embodiments 22 to 27, wherein the one or more amino acid substitutions comprises D161R.

[0317] Embodiment 29. The modified p40 domain of any one of embodiments 22 to 27, wherein the one or more amino acid substitutions comprises D161S.

[0318] Embodiment 30. The modified p40 domain of any one of embodiments 22 to 29, wherein the one or more amino acid substitutions include a combination of two or more of the following substitutions: E45K, D62H, D62I, D62N, D161R or D161S.

[0319] Embodiment 31. A modified p40 domain comprising one or more amino acid substitutions relative to the wild-type human mature IL12 p40 domain sequence set forth in SEQ ID NO: 10, wherein the one or more amino acid substitutions are selected from the group consisting of W15H, W15K, W15R, D18G, E45K, K58H, K58W, E59D, E59G, E59R, F60D, F60E, F60K, F60R, F60V, D62H, D62I, D62N, K84E, K84I, K84L, K84V, K84H ... W, K84Y, E86L, E86R, E86S, E86W, D93E, D93H, D93R, D93W, D161R, D161S, K197D, K197E, K197Q, K197T, or K197W, or a combination thereof, wherein the numbering of the amino acid residues is based on the amino acid sequence set forth in SEQ ID NO:10.

[0320] Embodiment 32. The modified p40 domain of embodiment 31, comprising one or more, two or more, or three or more amino acid substitutions.

[0321] Embodiment 33. The modified p40 domain of embodiment 31 or 32, wherein the one or more amino acid substitutions are W15H, W15K, D18G, E45K, K58H, K58W, E59G, E59R, F60V, D62H, D62I, D62N, K84E, K84W, E86L, E86S, E86W, D93H, D93W, D161R, D161S, K197E, K197Q, K197T, or K197W, or a combination thereof.

[0322] Embodiment 34. The modified p40 domain of any one of embodiments 31 to 33, wherein the one or more amino acid substitutions are W15H, W15K, E59R, K84E, K84W, E86W, D161R, K197T, or K197W, or a combination thereof.

[0323] Embodiment 35. The two or more amino acid substitutions are selected from the group consisting of W15H_K84L, K58H_K84I, E59D_K84W, E59G_K84W, E59R_K84E, E59R_K84W, E59R_E86W, E59D_D93H, E59R_D93R, E59R_K197E, E59R_K197W, F60E_K84W, F60R_K84Y, F60K_K197W, F60R_K197W, K84I_E86R, K84E_D93H, K84I_D93H, K84V_D93H, K84W_D93W, K84 33. The modified p40 domain of embodiment 31 or 32, which is I_D161R, K84W_D161R, K84W_K197E, K84W_K197Q, K84W_K197W, E86W_D93E, E86R_K197D, E86W_K197W, W15H_K84L_K197Q, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, or W15R_E59D_F60D_K197W, or a combination thereof.

[0324] Embodiment 36. The modified p40 domain of embodiment 35, wherein the two or more amino acid substitutions are W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, E86W_D93E, W15H_K84L_K197Q, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, or W15R_E59D_F60D_K197W, or a combination thereof.

[0325] Embodiment 37. The modified p40 domain of embodiment 35, wherein the two or more amino acid substitutions are K58H_K84I, E59D_K84W, E59G_K84W, E59R_E86W, E59R_K197E, E59R_K197W, E59R_K84E, E59R_K84W, F60E_K84W, F60R_K197W, K84E_D93H, K84I_D161R, K84I_D93H, K84V_D93H, K84W_D93W, K84W_K197E, K84W_K197Q, or E86W_K197W, or a combination thereof.

[0326] Embodiment 38. The two or more amino acid substitutions are selected from the group consisting of W15H_K84L, K58H_K84I, E59D_K84W, E59G_K84W, E59R_K84E, E59R_K84W, E59R_E86W, E59D_D93H, E59R_D93R, E59R_K197E, E59R_K197W, F60E_K84W, F60R_K84Y, F60K_K197W, F60R_K197W, K 36. The modified p40 domain of embodiment 35, which is 84I_E86R, K84E_D93H, K84I_D93H, K84V_D93H, K84W_D93W, K84I_D161R, K84W_D161R, K84W_K197E, K84W_K197Q, K84W_K197W, E86W_D93E, E86R_K197D, or E86W_K197W, or a combination thereof.

[0327] Embodiment 39. The modified p40 domain of embodiment 35, wherein the two or more amino acid substitutions are W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, or E86W_D93E, or a combination thereof.

[0328] Embodiment 40. The modified p40 domain of embodiment 31 or 32, wherein the three or more amino acid substitutions are W15H_K84L_K197Q, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H or W15R_E59D_F60D_K197W, or a combination thereof.

[0329] Embodiment 41 The modified p40 domain of embodiment 31 or 32, wherein said one or more amino acid substitutions are selected from Table C.

[0330] Embodiment 42. A modified p40 domain according to embodiment 31 or 32, comprising or consisting of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 12 to 14, 16, 18, 19, 21 to 34, and 36 to 56.

[0331] Embodiment 43. W15R_E59D_F60D_K197W, W15H_K84L_K197Q, E59D_K84W_K197W, K58H_E86R_K197D, W15H_K84L, F60R_K84E_K197W, K84W_K197W, F60K_K197W, E86R_K197D, K84I_E86R_D93H, E59R_D93R, K84I_E86R, W15K, K84W_D161R, 33. The modified p40 domain of embodiment 31 or 32, comprising an amino acid substitution or set of amino acid substitutions selected from E45R_K58S_E59S_K195D, E59D_D93H, F60R_K84Y, E86W_D93E, K58S_E59S_K195D, K84E, K197T, E59R, W15H, K84W, K197W, E59S, D161R or E86W, or a combination thereof.

[0332] Embodiment 44. The modified p40 domain of embodiment 43, comprising an amino acid substitution or set of amino acid substitutions selected from W15H_K84L_K197Q, E59D_K84W_K197W, K84W_K197W, F60K_K197W, E59R_D93R, W15K, or F60R_K84Y, or a combination thereof.

[0333] Embodiment 45. The modified p40 domain according to any one of embodiments 22 to 44, having a binding affinity for at least one of its cognate receptors that is reduced by about 5 to about 1000, about 5 to about 1800, about 5 to about 1600, about 10 to about 1500, about 10 to about 1300, or about 20 to about 1200 fold, relative to the binding affinity of the unmodified wild-type p40 domain having the sequence set forth in SEQ ID NO:10, as determined in a reporter gene assay (RGA).

[0334] Embodiment 46. A modified p40 domain according to embodiment 45, having a binding affinity for at least one of its cognate receptors that is reduced by about 20-fold to about 200-fold.

[0335] Embodiment 47. The modified p40 domain of embodiment 45 or 46, wherein said at least one cognate receptor comprises IL12Rβ1.

[0336] Embodiment 48. The modified p40 domain of any one of embodiments 22 to 47, having thermal stability as measured by a melting temperature within ±5°C, ±4°C, ±3°C, ±2°C or ±1°C of the melting temperature of the unmodified wild-type p40 domain having the sequence set forth in SEQ ID NO:10, wherein the melting temperature is determined by differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC).

[0337] Embodiment 49. An IL12 fusion protein comprising a modified p40 domain according to any one of embodiments 22 to 48.

[0338] Embodiment 50. An IL12 fusion protein comprising an IL12 polypeptide, The modified p40 domain of any one of embodiments 22 to 48, linked to a p35 domain. The IL12 fusion protein comprising:

[0339] Embodiment 51. An IL12 fusion protein according to embodiment 50, wherein the p35 domain comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:11.

[0340] Embodiment 52. An IL12 fusion protein according to embodiment 50 or 51, wherein the N-terminus of the p35 domain is linked to the C-terminus of the modified p40 domain either directly or via a first linker.

[0341] Embodiment 53. The first linker is (G4S) x and x is 1, 2, 3 or 4.

[0342] Embodiment 54. An IL12 fusion protein according to any one of embodiments 50 to 53, further comprising a heterodimeric Fc (HetFc) domain comprising a first Fc polypeptide and a second Fc polypeptide, thereby forming an IL12 HetFc fusion protein.

[0343] Embodiment 55. An IL12 HetFc fusion protein according to embodiment 54, wherein said IL12 polypeptide is linked to said first Fc polypeptide either directly or via a second linker.

[0344] Embodiment 56 The IL12 HetFc fusion protein of embodiment 55, wherein said IL12 polypeptide is linked to the C-terminus of said first Fc polypeptide.

[0345] Embodiment 57. An IL12 HetFc fusion protein according to embodiment 55 or 56, wherein said IL12 polypeptide is linked to the C-terminus of said first Fc polypeptide via the N-terminus of said modified p40 domain.

[0346] Embodiment 58. The method further comprises the steps of: The masking moiety is capable of non-covalently interacting with the modified p40 domain, thereby masking the modified p40 domain and increasing the binding affinity (K) of the modified p40 domain for binding to at least one of its cognate receptors when compared to an unmasked modified p40 domain. D ) to reduce 58. An IL12 HetFc fusion protein according to any one of embodiments 55 to 57.

[0347] Embodiment 59. The masked IL12 HetFc fusion protein of embodiment 58, wherein the masking moiety is attached to the C-terminus of the second Fc polypeptide either directly or via a third linker.

[0348] Embodiment 60 The masked IL12 HetFc fusion protein of embodiment 59, wherein said third linker is a protease-cleavable linker.

[0349] Embodiment 61. A masked IL12 HetFc fusion protein according to any one of embodiments 58 to 60, wherein the second linker and the third linker each comprise or consist of an amino acid sequence spanning from 5 to about 50 amino acids.

[0350] Embodiment 62. The masked IL12 HetFc fusion protein of embodiment 61, wherein said second linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 132 and said third linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 134.

[0351] Embodiment 63. The masking portion is V L V linked to the domain either directly or via a fourth linker H 63. A masked IL12 HetFc fusion protein according to any one of embodiments 58 to 62, comprising or consisting of an scFv domain comprising the domain.

[0352] Embodiment 64. The masked IL12 HetFc fusion protein of embodiment 63, wherein said fourth linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 135.

[0353] Embodiment 65. The V H The V domain comprises or consists of an amino acid sequence having about 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:2, L 65. The masked IL12 HetFc fusion protein of embodiment 63 or 64, wherein the domain comprises or consists of an amino acid sequence having about 95%, 97%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:3.

[0354] Embodiment 66. A masked IL12 HetFc fusion protein according to any one of embodiments 58 to 65, wherein the masking moiety is capable of reducing the binding affinity of the modified p40 domain to the at least one cognate receptor by at least about 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, or at least about 300-fold compared to a corresponding polypeptide construct not comprising the masking moiety.

[0355] Embodiment 67. The masked IL12 HetFc fusion protein of embodiment 66, wherein said at least one cognate receptor comprises IL12Rβ1.

[0356] Embodiment 68. An IL-12 polypeptide comprising: (i) a modified p40 domain according to any one of embodiments 22 to 48 linked via a linker (G4S)4 to a p35 domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:11; (ii) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide; (iii) a masking moiety (MM) comprising an anti-IL12 scFv domain; 1. A masked IL12 HetFc fusion protein comprising: the IL12 polypeptide is linked to the C-terminus of the first Fc polypeptide either directly or via a second linker; The masked IL12 HetFc fusion protein, wherein the masking moiety is attached either directly or via a third linker to the C-terminus of the second Fc polypeptide and is capable of non-covalently interacting with the IL12 polypeptide, thereby reducing the binding affinity of the IL12 polypeptide to at least one of its cognate receptors.

[0357] Embodiment 69. A masked IL12 HetFc fusion protein according to embodiment 68, comprising or consisting of two polypeptide chains, from N-terminus to C-terminus: (i) an Fc-IL12 polypeptide chain and (ii) an Fc-MM polypeptide chain.

[0358] Embodiment 70. The masked IL12 HetFc fusion protein of embodiment 69, wherein the Fc-IL12 polypeptide chain comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 61-89.

[0359] Embodiment 71. A masked IL12 HetFc fusion protein according to embodiment 69 or 70, wherein the Fc-IL12 polypeptide chain comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs: 61 to 89.

[0360] Embodiment 72. The masked IL12 HetFc fusion protein of any one of embodiments 69 to 71, wherein the Fc-MM polypeptide chain comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:60.

[0361] Embodiment 73. A masked IL12 HetFc fusion protein according to any one of embodiments 69 to 72, wherein the Fc-MM polypeptide chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60.

[0362] 74. (i) The amino acid sequence of the Fc-IL12 polypeptide chain is selected from the group consisting of v28046, v28047, v28048, v28049, v28050, v28051, v28053, v28054, v28055, v28056, v28057, v28058, v28059, v28060, v28061, v28062, v28063, v28064, 74. The masked IL12 HetFc fusion protein of any one of embodiments 69-73, wherein (i) said Fc-MM polypeptide chain comprises or consists of the amino acid sequence of v26503; and (ii) said Fc-MM polypeptide chain comprises or consists of the amino acid sequence of v26503.

[0363] Embodiment 75. An IL-12 polypeptide comprising: (i) a modified p40 domain according to any one of embodiments 22 to 48 linked via a linker (G4S)4 to a p35 domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:11; (ii) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide; wherein said IL12 polypeptide is linked either directly or via a second linker to the C-terminus of said first Fc polypeptide.

[0364] Embodiment 76. An unmasked IL12 HetFc fusion protein according to embodiment 75, comprising or consisting of two polypeptide chains, from N-terminus to C-terminus: (i) an Fc-IL12 polypeptide chain and (ii) an Fc polypeptide chain.

[0365] Embodiment 77. The unmasked IL12 HetFc fusion protein of embodiment 76, wherein the Fc-IL12 polypeptide chain comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 61-89.

[0366] Embodiment 78. The unmasked IL12 HetFc fusion protein of embodiment 76 or 77, wherein the Fc-IL12 polypeptide chain comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 61 to 89.

[0367] Embodiment 79. The unmasked IL12 HetFc fusion protein of any one of embodiments 76 to 78, wherein the Fc polypeptide chain comprises or consists of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:58.

[0368] (i) the amino acid sequence of the IL12 polypeptide chain is selected from the group consisting of v28046, v28047, v28048, v28049, v28050, v28051, v28053, v28054, v28055, v28056, v28057, v28058, v28059, v28060, v28061, v28062, v28063, v28064, v 80. The unmasked IL12 HetFc fusion protein of any one of embodiments 76-79, wherein (i) said Fc polypeptide chain comprises or consists of the amino acid sequence of v12153; and (ii) said Fc polypeptide chain comprises or consists of the amino acid sequence of v12153.

[0369] Embodiment 81. A pharmaceutical composition comprising (i) a modified p40 domain according to any one of embodiments 22 to 48, (ii) a masked IL12 HetFc fusion protein according to any one of embodiments 58 to 74, and / or (iii) an unmasked IL12 HetFc fusion protein according to embodiments 75 to 80, and a pharma- ceutically acceptable carrier.

[0370] Embodiment 82. A nucleic acid molecule or a set of nucleic acid molecules encoding (i) a modified p40 domain according to any one of embodiments 22 to 48, (ii) a masked IL12 HetFc fusion protein according to any one of embodiments 58 to 74, and / or (iii) an unmasked IL12 HetFc fusion protein according to embodiments 75 to 80.

[0371] Embodiment 83. A vector or a set of vectors comprising a nucleic acid molecule or a set of nucleic acid molecules according to embodiment 82.

[0372] Embodiment 84. A method for identifying one or more amino acid substitutions in a p40 domain amino acid sequence to generate a modified p40 domain, comprising: performing molecular dynamics and mutagenesis simulations, thereby identifying one or more amino acid substitutions listed in Table C. wherein the one or more amino acid substitutions in the modified p40 domain amino acid sequence are relative to the sequence set forth in SEQ ID NO:1, and the one or more amino acid substitutions increase the binding affinity (K D ) is reduced compared to an unmodified p40 domain not containing the one or more amino acid substitutions.

[0373] Embodiment 85. The method of embodiment 84, wherein said at least one cognate receptor comprises IL12Rβ1.

[0374] Embodiment 86. The method of embodiment 84 or 85, wherein the binding affinity of the modified p40 domain is reduced by at least about 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, or at least about 600-fold compared to the unmodified p40 domain, as determined in a reporter gene assay.

[0375] Embodiment 87. A method of treating a disease in a subject in need of such treatment, comprising administering to said subject: administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of (i) a modified p40 domain according to any one of embodiments 22 to 48, (ii) a masked IL12 HetFc fusion protein according to any one of embodiments 58 to 74, and / or (iii) an unmasked IL12 HetFc fusion protein according to embodiments 75 to 80, thereby treating said disease in said subject. The method comprising:

[0376] Embodiment 88. The method of embodiment 87, wherein the polypeptide construct is a masked IL12 HetFc fusion protein according to any one of embodiments 58 to 74.

[0377] Embodiment 89. The method of embodiment 88, wherein the masking portion of the masked IL12 HetFc fusion protein is cleaved from the fusion protein in the diseased tissue or organ.

[0378] Embodiment 90. The method of any one of embodiments 87 to 89, wherein the disease is cancer.

[0379] Embodiment 91. A modified p40 domain according to any one of embodiments 22 to 48, a masked IL12 HetFc fusion protein according to any one of embodiments 58 to 74, or an unmasked IL12 HetFc fusion protein according to any one of embodiments 75 to 80, for use in therapy.

[0380] Embodiment 92. A modified p40 domain according to any one of embodiments 22 to 48, a masked IL12 HetFc fusion protein according to any one of embodiments 58 to 74, or an unmasked IL12 HetFc fusion protein according to any one of embodiments 75 to 80, for use in the treatment of cancer.

[0381] Embodiment 93. Use of a modified p40 domain according to any one of embodiments 22 to 48, a masked IL12 HetFc fusion protein according to any one of embodiments 58 to 74, or an unmasked IL12 HetFc fusion protein according to any one of embodiments 75 to 80 in the manufacture of a medicament for the treatment of cancer.

[0382] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the claimed subject matter belongs. In the event that there are multiple definitions for terms herein, the definitions in this section shall prevail. When referring to a URL or other such identifier or address, it is understood that such identifiers may change and specific information on the Internet may change, but equivalent information may be found by searching the Internet. The references evidence the availability and general dissemination of such information. Terms understood by those skilled in the art of antibody technology are each given the meaning acquired in the art, unless otherwise expressly defined herein as different.

[0383] It is to be understood that the general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed.

[0384] In this application, the use of the singular includes the plural unless specifically stated otherwise.

[0385] In this description, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer in the range recited, as well as fractions thereof (such as tenths and hundredths of integers), if appropriate, unless otherwise indicated. As used herein, "about" means ±10% of the indicated range, value, sequence, or structure, unless otherwise indicated. The terms "a" and "an" as used herein should be understood to refer to "one or more" of the recited components, unless otherwise indicated or indicated by the context. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include" and "comprise" are used interchangeably. It should also be understood that the individual single chain polypeptides or immunoglobulin constructs derived from the various combinations of structures and substituents described herein are disclosed by this disclosure to the same extent as if each single chain polypeptide or heterodimer were individually set forth, and thus the selection of the particular components to form the individual single chain polypeptides or heterodimers is within the scope of this disclosure.

[0386] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0387] It is to be understood that the methods and compositions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein, as such may vary. It is also to be understood that the terminology used herein is used to describe particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which are limited only by the scope of the appended claims. EXAMPLES

[0388] Example 1: Design of modified p40 domains with reduced affinity for IL12Rβ1 by molecular modeling and computer-guided engineering The clinical usefulness of IL12 as an antitumor agent is limited by its high toxicity driven by its immune stimulatory effect. To reduce the immune stimulatory potency of IL12, the affinity of IL12 for its receptors, IL12Rβ1 and / or IL12Rβ2, can be engineered to decrease its affinity.

[0389] This example describes the in silico design, by molecular modeling and computer-guided engineering, of a library of modified p40 domains with reduced affinity for IL12Rβ1.

[0390] method: Preparation and analysis of structures As a starting point for the work, the structural coordinates of the p40 domain in complex with IL12Rβ1 were extracted from the crystal structure of IL12 in complex with IL12Rβ1 (PDB ID 6wdq). The structural model was optimized using a proprietary in silico modeling tool. This procedure reconstructs missing atoms and corrects poorly modeled regions in the structure, including amino acid side chains and backbone conformations that may be affected by crystal packing restrictions.

[0391] The optimized structures were analyzed using several proprietary molecular modeling tools (e.g., ResidueContacts™ tool and AffinityDecomposition™ tool) to visually identify hotspot positions in the p40-IL12Rβ1 interface, i.e., key residues that contribute to stability and affinity.

[0392] The same procedure was used to prepare and evaluate structures of the p40 domain in complex with the antibody antigen-binding fragment (Fab) of the antibodies briakinumab (from PDB ID 5njd) and ustekinumab (from PDB ID 3hmx).

[0393] Comparison of multiple published crystal structures containing the p40 domain (e.g., 1f45, 3duh, 3hmx, 3qrw, 4grw, 5mxa, 5mzv, 5njd, 6wdq, etc.) revealed that the p40 domain has a significant degree of conformational flexibility. To evaluate the effect of potential mutations on the stability of uncomplexed p40, it was desirable to obtain a structure of the uncomplexed p40 domain that represents the most common conformations in the vicinity of the IL12Rβ1 binding site where mutations may be designed. Explicit molecular dynamics simulations of the isolated p40 domain structure were performed, and the trajectory coordinates were linked and clustered using alignments of only the immunoglobulin domain and the first fibronectin type III domain of p40 to generate structures representing five conformational clusters.

[0394] Mutation design using computer-guided manipulation Potential mutations at selected positions in p40 were simulated by in silico mutagenesis and modeling using the ZymeCAD® platform. The ZymeCAD® platform is a software suite that, given an input structure and a set of mutations, changes the residue types in the input structure according to the supplied mutations to generate a new structure that approximates the physical structure of the mutant protein. The ZymeCAD® platform also evaluates the properties of the mutant protein relative to the unmutated parent protein by calculating a variety of physics-based and knowledge-based quantitative indices. These indices include, for example, measures of steric and electrostatic complementarity calculated based on energy factors, e.g., van der Waals packing, cavitation effects, close contacts of hydrophobic groups, Coulombic interactions between charges, hydrogen bonding, desolvation effects, etc., that may correlate with the stability, binding affinity, or binding specificity of the mutant protein(s).

[0395] This procedure was performed using prepared structures of p40 in complex with IL12Rβ1, as well as with prepared structures of p40 in complex with the antibody antigen-binding fragment (Fab) of the antibodies briakinumab and ustekinumab to assess the effect of potential mutations on the affinity and stability of each complex.

[0396] In addition to the effects on complex stability and affinity described above, potential p40 mutations were also evaluated for their effect on the stability of the isolated, uncomplexed p40 domain. This was accomplished by using the ZymeCAD® platform to simulate potential mutations in representative structures of each of the top three conformational clusters of the uncomplexed p40 domain generated by molecular dynamics simulations and assessing their effect on predicted stability.

[0397] Potential mutations were designed in an iterative process starting with the simulation and evaluation of mutations of individual hotspot residues using the ZymeCAD™ platform, the selection of promising mutations based on stability and affinity indices and visual inspection, followed by the simulation, evaluation, and selection of additional mutation sets comprising the selected parent mutation(s) and additional mutations of nearby residues or alternative hotspots that may have a synergistic or otherwise desirable effect on the predicted stability and / or affinity indices of the modified p40 domain containing the resulting mutation set.

[0398] Potential mutations and mutation sets were further evaluated for their effect on the predicted T cell epitope content of the resulting modified p40 domains using a proprietary in silico tool that calculates per-residue and total T cell epitope scores for a protein sequence based on MHC class II peptide binding affinity predictions for a representative set of MHC alleles from the Immune Epitope Database (IEDB; https: / / www.iedb.org / ).

[0399] result: Potential mutations and mutation sets generated by an iterative process of in silico simulated mutagenesis and structure evaluation using the ZymeCAD® platform were filtered based on several in silico metrics and by visual inspection to obtain a library of mutations and mutation sets predicted to provide a wide range of affinity between the modified p40 domain and IL12Rβ1, to have a positive or minimally adverse effect on the stability of the uncomplexed modified p40 domain, to have a minimal effect on the affinity between the modified p40 domain and briakinumab, and to have a minimal effect on the predicted T cell epitope content of the modified p40 domain.

[0400] The resulting library is provided in Table G along with selected in silico metrics generated by the ZymeCAD® platform that can be correlated to the following: affinity of the modified p40 domain with IL12Rβ1 ("ΔA Rβ1" and "ΔS Rβ1", physics-based affinity and stability metrics, respectively; calculated as the difference between the unmodified and modified p40 domains in complex with IL12Rβ1; a negative ΔA value indicates improved or enhanced affinity, a positive ΔA value indicates worsening or reduced affinity; a negative ΔS value indicates improved or increased stability, a positive ΔS value indicates worsening or reduced stability), stability of the uncomplexed modified p40 domain ("ΔS", the difference in stability score between the uncomplexed unmodified and modified p40 domains). Apo"), the affinity between the modified p40 domain and briakinumab and ustekinumab ("ΔA Bria" and "ΔA Uste", which are the difference in affinity scores between the unmodified and modified p40 domains in complex with briakinumab and ustekinumab, respectively), and the predicted T-cell epitope content of the modified p40 domain ("TCES", which is the total T-cell epitope score of the modified p40 domain; a positive TCES value indicates an increase in predicted T-cell epitope content relative to unmodified p40).

[0401] [Table G]

[0402] Example 2: Generation, purification, and biophysical characterization of an IL12 HETFC fusion protein with a p40 domain modified for reduced affinity for IL12Rβ1 The biophysical and functional impact of modified p40 domains engineered for reduced affinity for IL12Rβ1 can be assessed in the context of any p40 domain-containing fusion protein by comparing the properties of the fusion protein containing the modified p40 domain to a corresponding fusion protein with an unmodified p40 domain. This example describes the expression, purification, and biophysical characterization of IL12 HetFc fusion proteins and masked IL12 HetFc fusion proteins containing modified and unmodified p40 domains.

[0403] method: Design of IL12 HetFc and masked IL12 HetFc fusion proteins: IL12 fusion proteins to heterodimeric Fc ("HetFc") were constructed using the geometry of variant 30806 shown in Figures 2A-B. Briefly, fusion proteins were designed using single chain IL12 ("scIL12") consisting of a p35 domain fused C-terminally to a p40 domain by a peptide linker, and scIL12 fused C-terminally to one of the two chains of HetFc by a polypeptide linker. Masked IL12 fusion proteins were similarly constructed with the addition of a briakinumab-derived scFv fused C-terminally to a second HetFc chain by a peptide linker, as shown in Figure 2B as variant v35436. IL12 HetFc and masked IL12 HetFc fusion proteins were constructed with mutated and unmutated p40 domains as outlined in Table H.

[0404] Cloning: The designed polypeptide sequences of IL12 HetFc fusion proteins were reverse translated into DNA, codon-optimized for mammalian cell expression, and the genes were synthesized. All sequences were preceded by an artificially designed signal peptide of the sequence MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 1) (Barash S et al., Biochem and Biophys Res. Comm. 2002; 294, 835-842). For all sequences, vector inserts consisting of a 5'-EcoR1 restriction site, the signal peptide described above, the codon-optimized DNA sequence corresponding to the clone presented in this example, a TGA or TAA stop codon, and a BamH1 restriction site-3' were ligated into the pTT5 vector to generate expression vectors (Durocher Y et al., Nucl. Acids Res. 2002; 30, No. 2 e9). The resulting expression vectors were sequenced to confirm the correct reading frame and sequence of the coding DNA.

[0405] Mammalian cell transient transfection and protein expression: To generate recombinant IL12 HetFc fusion protein, 1.5-2.2 x 10 6 HEK293-6E cells at a density of 10000 cells / ml are grown in FreeStyle™ F17 medium (GIBCO Cat# A13835-01) supplemented with G418 (Wisent bioproducts cat# 400-130-IG), 4 mM glutamine, and 0.1% Pluronic F-68 (Gibco Cat# 24040-032) at 37° C. Cells are transfected with 1 μg of DNA (DNA consisting of a 1:1 w / w ratio of variant expression vector DNA mix and GFP / AKT / stuffer DNA) per mL of cells using PEI-max (Polyscience, Philadelphia, PA) at a DNA:PEI ratio of 1:2.5 (w / w). 24 hours after addition of the DNA-PEI mixture, 0.5 mM valproic acid (final concentration) and 0.5% w / v tryptone N1 (final concentration) are added to the cells, which are then transferred to 37° C. and incubated for 7 days before being harvested.

[0406] Protein purification by Protein A affinity chromatography: The supernatant from the transient transfection is applied to a slurry containing 50% mAb Select SuRe™ resin (GE Healthcare, Chicago, IL) and incubated overnight at 2-8 °C on an orbital shaker at 150 rpm. The slurry is transferred to a chromatography column and the flow-through is collected. The resin is then washed with 5 bed volumes (BV) of resin equilibration buffer (PBS). To elute the targeted protein, 5.5 BV of acidic elution buffer (100 mM sodium citrate buffer pH 3.5) is added to the column and collected in fractions. The elution fractions are then neutralized by adding 10% (v / v) 1 M Tris pH 9 to reach a final pH of 6-7. The protein content of each elution fraction is determined by 280 nm absorbance measurements using a Nanodrop™ or with a relative colorimetric protein assay. The most concentrated fractions are pooled, which represent at least 80% of the total eluted protein.

[0407] Protein purification by preparative size-exclusion chromatography (Prep-SEC): Samples are loaded onto a Superdex 200 Increase 10 / 300 column (#28-9909-44, GE Healthcare Life Sciences, Marlborough, MA) of an Akta pure 25 chromatography system (GE Healthcare Life Sciences, Marlborough, MA) at a flow rate of 0.8 mL / min in PBS. Fractions of eluted protein were collected based on A280 nm, and their purity was analyzed by non-reduced CE-SDS using a LabChip™ GXII Touch (PerkinElmer, Waltham, MA). High purity protein-containing fractions were pooled, and the protein in the final pool was quantified based on A280 nm (Nanodrop™) after SEC.

[0408] Purity assessment by capillary electrophoresis (CE) using LabChip™ The purity of the fusion protein samples is assessed by non-reduced and reduced LabChip™ CE-SDS. LabChip™ GXII Touch (PerkinElmer, Waltham, MA) assays are performed according to the Protein Express Assay User Guide (PerkinElmer, Waltham, MA) with the following modifications. Samples ranging in concentration from 5 to 2000 ng / μl are added to separate wells of a 96-well plate (#MSP9631, BioRad, Hercules, CA) with 7 μl of HT Protein Express Sample Buffer (#CLS920003, PerkinElmer) and denatured at 90° C. for 5 minutes. The LabChip™ instrument is run using the HT Protein Express 200 assay setting with the LabChip™ HT Protein Express Chip (PerkinElmer #760528).

[0409] Purity assessment by UPLC-SEC Fusion protein samples are evaluated by UPLC-SEC to determine their percentage of high molecular weight species. UPLC-SEC is performed using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6×150 mm, stainless steel, 1.7 μm particles) (Waters LTD, Mississauga, ON) mounted on an Agilent Technologies 1260 infinity II system set at 30° C. and equipped with a PDA detector. At 0.4 mL / min, the run time consisted of 7 minutes with a total volume of 2.8 mL per injection, containing running buffers of either 150 mM sodium phosphate (pH 6.95), DPBS+0.02% Tween 20, or 200 mM KPO4, 200 mM KCl (pH 7), or 200 mM KPO4, 200 mM KCl, 0.02% Tween 20 (pH 7). The elution is monitored by UV absorbance in the range 210-500 nm and the chromatograms are extracted at 280 nm. Peak integration is performed using OpenLAB™ CDS ChemStation™ software.

[0410] Stability assessment by differential scanning fluorimetry (DSF) The stability of the fusion protein samples is assessed by DSF using a CFX Connect real-time PCR detection system (BioRad). 25 μl of buffer, 5 μl of 40xSYPRO Orange dye, and 10 μl of variant are added to the wells of a 96-well PCR plate. The plate is sealed with an optical PCR plate seal, centrifuged at 1000 rpm for 1 minute, placed in a thermocycler, and read at a gradient of 0.5°C / min or 1°C / min from 25°C to 95°C.

[0411] result: It is expected that samples of IL12 HetFc and masked IL12 HetFc fusion proteins containing modified and unmodified p40 domains can be expressed from transiently transfected mammalian cells and purified to homogeneity by a two-step purification process consisting of Protein A affinity chromatography followed by Prep-SEC or IEX. It is expected that the expression titers, purification yields, and stability of IL12 HetFc and masked IL12 HetFc fusion proteins containing modified p40 domains may differ from those of IL12 HetFc and masked IL12 HetFc fusion proteins containing unmodified p40 domains in terms of the tendency to correlate with the stability of the modified p40 domain as predicted in silico as described in Example 1.

[0412] [Table H] TIFF2024535925000019.tif90165

[0413] Example 3: Generation, purification, and biophysical characterization of an IL12 HETFC fusion protein with a p40 domain modified for reduced affinity for IL12Rβ1 The biophysical and functional impact of modified p40 domains engineered for reduced affinity for IL12Rβ1 was assessed in the context of any p40 domain-containing fusion protein by comparing the properties of fusion proteins containing modified p40 domains to corresponding fusion proteins with unmodified p40 domains. This example describes the expression, purification, and biophysical characterization of IL12 HetFc fusion proteins and masked IL12 HetFc fusion proteins containing modified and unmodified p40 domains.

[0414] method: Design of IL12 HetFc and masked IL12 HetFc fusion proteins: IL12 fusion proteins to heterodimeric Fc ("HetFc") were constructed using the geometry of variant 30806 shown in Figures 2A-B. Briefly, fusion proteins were designed using single chain IL12 ("scIL12") consisting of a p35 domain fused C-terminally to a p40 domain by a peptide linker, and scIL12 fused C-terminally to one of the two chains of HetFc by a polypeptide linker. Masked IL12 fusion proteins were similarly constructed with the addition of a briakinumab-derived scFv fused C-terminally to a second HetFc chain by a peptide linker, as shown in Figure 2B as variant v35436. IL12 HetFc and masked IL12 HetFc fusion proteins were constructed with mutated and unmutated p40 domains as outlined in Table I.

[0415] Cloning: The designed polypeptide sequences of IL12 HetFc fusion proteins were reverse translated into DNA, codon-optimized for mammalian cell expression, and the genes were synthesized. All sequences were preceded by an artificially designed signal peptide of the sequence MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 1) (Barash S et al., Biochem and Biophys Res. Comm. 2002; 294, 835-842). For all sequences, vector inserts consisting of a 5'-EcoR1 restriction site, the signal peptide described above, the codon-optimized DNA sequence corresponding to the clone presented in this example, a TGA or TAA stop codon, and a BamH1 restriction site-3' were ligated into the pTT5 vector to generate expression vectors (Durocher Y et al., Nucl. Acids Res. 2002; 30, No. 2 e9). The resulting expression vectors were sequenced to confirm the correct reading frame and sequence of the coding DNA.

[0416] Mammalian cell transient transfection and protein expression: To generate recombinant IL12 HetFc fusion protein, 1.5-2.2 x 106 HEK293-6E cells at a density of 10000 cells / ml were grown in FreeStyle™ F17 medium (GIBCO Cat# A13835-01) supplemented with G418 (Wisent bioproducts cat# 400-130-IG), 4 mM glutamine, and 0.1% Pluronic F-68 (Gibco Cat# 24040-032) at 37° C. Cells were transfected with 1 μg of DNA (DNA consisting of a 1:1 w / w ratio of variant expression vector DNA mix and GFP / AKT / stuffer DNA) per mL of cells using PEI-max (Polyscience, Philadelphia, PA) at a DNA:PEI ratio of 1:2.5 (w / w). 24 hours after addition of the DNA-PEI mixture, 0.5 mM valproic acid (final concentration) and 0.5% w / v tryptone N1 (final concentration) were added to the cells, which were then transferred to 37°C and incubated for 7 days before being harvested.

[0417] Protein purification by Protein A affinity chromatography: The supernatant from the transient transfection was applied to a slurry containing 50% mAb Select SuRe™ resin (GE Healthcare, Chicago, IL) and incubated overnight at 2-8 °C on an orbital shaker at 150 rpm. The slurry was transferred to a chromatography column and the flow-through was collected. The resin was then washed with 5 bed volumes (BV) of resin equilibration buffer (PBS). To elute the targeted protein, 5.5 BV of acidic elution buffer (100 mM sodium citrate buffer pH 3.5) was added to the column and collected in fractions. The elution fractions were then neutralized by adding 10% (v / v) 1 M Tris pH 9 to reach a final pH of 6-7. The protein content of each elution fraction was determined by 280 nm absorbance measurements using a Nanodrop™ or with a relative colorimetric protein assay. The most concentrated fractions were pooled, representing at least 80% of the total eluted protein.

[0418] Protein purification by preparative size-exclusion chromatography (Prep-SEC): Samples were loaded onto a Superdex 200 Increase 10 / 300 column (#28-9909-44, GE Healthcare Life Sciences, Marlborough, MA) of an Akta pure 25 chromatography system (GE Healthcare Life Sciences, Marlborough, MA) at a flow rate of 0.8 mL / min in PBS. Fractions of eluted protein were collected based on A280 nm, and their purity was analyzed by non-reducing CE-SDS using a LabChip™ GXII Touch (PerkinElmer, Waltham, MA). High purity protein-containing fractions were pooled, and the protein in the final pool was quantified based on A280 nm (Nanodrop™) after SEC.

[0419] Purity assessment by capillary electrophoresis (CE) using LabChip™ The purity of the fusion protein samples was assessed by non-reduced and reduced LabChip™ CE-SDS. LabChip™ GXII Touch (PerkinElmer, Waltham, MA) assays were performed according to the Protein Express Assay User Guide (PerkinElmer, Waltham, MA) with the following modifications. Samples ranging in concentration from 5 to 2000 ng / μl were added to separate wells of a 96-well plate (#MSP9631, BioRad, Hercules, CA) with 7 μl of HT Protein Express Sample Buffer (#CLS920003, PerkinElmer) and denatured at 90° C. for 5 minutes. The LabChip™ instrument was run using the HT Protein Express 200 assay setup with a LabChip™ HT Protein Express Chip (PerkinElmer #760528).

[0420] Purity assessment by UPLC-SEC Fusion protein samples were evaluated by UPLC-SEC to determine their percentage of high molecular weight species. UPLC-SEC was performed using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6×150 mm, stainless steel, 1.7 μm particles) (Waters LTD, Mississauga, ON) mounted on an Agilent Technologies 1260 infinity II system set at 30° C. and equipped with a PDA detector. At 0.4 mL / min, the run time consisted of 7 min, the total volume per injection was 2.8 mL, and contained running buffers of either 150 mM sodium phosphate (pH 6.95), DPBS+0.02% Tween 20, or 200 mM KPO4, 200 mM KCl (pH 7), or 200 mM KPO4, 200 mM KCl, 0.02% Tween 20 (pH 7). Elution was monitored by UV absorbance in the range 210-500 nm and chromatograms were extracted at 280 nm. Peak integration was performed using OpenLAB™ CDS ChemStation™ software.

[0421] Stability assessment by differential scanning fluorimetry (DSF) The stability of the fusion protein samples was assessed by DSF using a CFX Connect real-time PCR detection system (BioRad). 25 μl of buffer, 5 μl of 40xSYPRO Orange dye, and 10 μl of variant were added to wells of a 96-well PCR plate. The plate was sealed with an optical PCR plate seal, centrifuged at 1000 rpm for 1 minute, placed in a thermocycler, and read at a gradient of 0.5°C / min or 1°C / min from 25°C to 95°C.

[0422] result: IL12 HetFc and masked IL12 HetFc fusion proteins containing modified and unmodified p40 domains were expressed from transiently transfected mammalian cells and purified to homogeneity by a two-step purification process consisting of Protein A affinity chromatography followed by Prep-SEC. After Protein A affinity purification, the yields of IL12 HetFc and masked IL12 HetFc fusion proteins containing modified p40 domains were between 50% and 138% of the corresponding fusion proteins with unmodified p40 domains (Tables J-K). All fusion proteins containing modified p40 domains showed similar UPLC-SEC and CE-SDS profiles after Protein A affinity purification compared to the corresponding fusion proteins with unmodified p40 domains (Figures 3A and 3B), with contaminants being dimer / trimer high molecular weight (HMW) species and low molecular weight (LMW) species consisting of single chains or homodimers of unfused HetFc chains for unmasked IL12 HetFc fusion proteins or scFv-fused HetFc chains for masked IL12 HetFc fusion proteins. Monodispersity after Protein A purification of the desired heterodimeric IL12 HetFc fusion protein species was between 44% and 75% (Tables J-K). After Prep-SEC purification, all samples showed >93% monodispersity.

[0423] The thermal stability of the tested fusion proteins containing the modified p40 domains listed in Table I, as measured by melting temperature using DSF as described herein, was nearly identical to that of the fusion proteins containing the wild-type p40 domain, i.e., the p40 domain substitutions did not substantially affect the stability of the fusion proteins described herein.

[0424] [Table I] TIFF2024535925000021.tif92165

[0425] [Table J]

[0426] [Table K]

[0427] Example 4: Cellular activity of IL12 HETFC fusion protein CD8+ T cells are an important target population for IL12. The potency of selected variants on CD8+ T cells can be assessed by IFNγ release.

[0428] method: CD8+ T cell IFNγ release assay CD8+ T cells are thawed and stimulated with anti-CD3 / CD28 Dynabeads (ThermoFisher, Waltham, MA) at a cell to bead ratio of 10:1 and seeded at 30,000 cells / well in 30ul of RPMI1640 (Gibco) + 10% FBS (ThermoFisher) + 1% Pen-Strep (Gibco) in 384-well black flat-bottom assay plates (ThermoFisher, Waltham, MA). Plates are incubated overnight at 37°C and 5% carbon dioxide. The next day, samples are prepared as described below and 30ul is added to the CD8+ T cells. Plates are incubated for 3 days at 37°C and 5% carbon dioxide. After incubation, 30uL / well of supernatant is transferred to a non-binding 384-well plate (Greiner-Bio-One, Kremsmunster, Austria) and stored at -80°C.

[0429] Aliquots of variants or control samples are thawed from -80°C storage on the day of the assay. Samples are titrated in quadruplicate at a dilution of 1:10 in 100ul in non-binding 384-well plates (Greiner-Bio-One, Kremsmunster, Austria). Recombinant human IL12 (Peprotech, Rocky Hill, NJ) is included as a positive control. 30ul of the titrated variants are then transferred into CD8+ T cells stimulated as described above.

[0430] IFNγ is quantified using an MSD (Mesoscale Discovery, Piscataway, NJ). MSD plates are blocked and coated with capture antibody according to manufacturer's instructions. Plates are washed 3 times in PBS-T and 5ul of assay diluent is added to each plate. IFNγ standards are titrated down from 500ng / mL to 0.5pg / mL. 5uL of supernatant is transferred to the MSD plate and incubated at room temperature for 1 hour. Plates are washed 3 times in PBS-T and 10ul of detection antibody at the appropriate dilution is added to each sample and standard well. Plates are sealed with adhesive plate seals for 1 hour. Plates are washed 3 times in PBS-T and 40uL of MSD Gold Read Buffer B is added to each well. Plates are read on a MESO SECTOR 6000 and cytokine concentrations are determined using MSD software. Using the data from the standard curve and samples, a non-linear curve fit is performed with x-interpolation to obtain IFNγ concentrations (pg / mL). Three independent experiments are performed and data from each are analyzed with a nonlinear mixed effects model to generate curve fits and 95% confidence intervals.

[0431] result: A control IL12 HetFc fusion protein containing an unmodified p40 domain is expected to stimulate comparable levels of IFNγ release from CD8+ T cells (within about 10-fold) as recombinant IL12 in a CD8+ T cell IFNγ release assay.

[0432] As a control, a masked IL12 HetFc fusion protein containing an unmodified p40 domain is expected to stimulate significantly reduced levels (less than 10-fold reduction) of IFNγ release from CD8+ T cells compared to the control IL12 HetFc fusion protein containing an unmodified p40 domain.

[0433] IL12 HetFc fusion proteins and masked IL12 HetFc fusion proteins containing modified p40 domains are expected to stimulate reduced levels of IFNγ release from CD8+ T cells compared to corresponding control IL12 HetFc and masked IL12 HetFc fusion proteins containing unmodified p40 domains. The degree of reduction in IFNγ release from CD8+ T cells is expected to correlate with the affinity of the modified p40 domains for IL12Rβ1, as predicted in silico, as described in Example 1.

[0434] It is expected that the reduced activity of the modified p40 domain may be synergistic with the activity of the masked IL12 HetFc fusion protein, such that the fold reduction in the level of IFNγ release stimulated by the masked IL12 HetFc fusion protein containing the modified p40 domain compared to a masked IL12 HetFc fusion protein containing an unmodified p40 domain as a control may be greater than the fold reduction in the level of IFNγ release stimulated by the corresponding IL12 HetFc fusion protein containing the modified p40 domain compared to the control IL12 HetFc fusion protein containing the unmodified p40 domain.

[0435] Example 5: Cellular activity of IL12 fusion proteins As described herein, IL12 can exert its biological activity by binding to the IL12 receptor heterodimer, which can initiate a signaling pathway that leads to the activation of STAT4. This example describes the characterization of IL12 fusion proteins and masked IL12 fusion proteins containing a heterodimeric Fc domain (HetFc) and modified or unmodified p40 domains using an IL12-responsive colorimetric reporter gene assay (RGA) in which HEK cells stably transfected with genes for the IL12 receptor, the IL12 signaling pathway, and a STAT4-inducible reporter responded to IL12 by secreting phosphatase that was detected in a colorimetric assay.

[0436] IL12 receptor transfected reporter cells suspended in assay buffer (DMEM 4.5g / l glucose, 2mM L-glutamine, 10% heat-inactivated FBS, Pen-Strep (100U / ml-100μg / ml)) were seeded into 384-well white flat bottom plates (Thermo Scientific Nunc Cat# 164610) at a concentration of 10,000 cells in 30ul per well. IL12 HetFc fusion protein variants were serially diluted separately in assay buffer from 8nM to 0.14pM (for variant 30806) or 100nM to 1.7pM (all other variants) in 96-well V-bottom plates (Sarstedt 82.1583001). 30ul of variants were transferred and mixed with 30ul of IL12R transfected reporter cells in a 384-well flat bottom plate and the plate was transferred to a 37°C incubator with 5% CO2 and humidity for 18-24 hours. 90ul of Quanti-Blue solution was added per well and 10ul of supernatant was transferred to a new 384-well clear flat bottom plate, the plate was then incubated at 37°C with 5% CO2 for 1 hour and read at 620nm in a Synergy H1.

[0437] IL12 HetFc fusion proteins containing a modified p40 domain (e.g., any one of variants 37467-37495) showed reduced potency compared to a control IL12 HetFc fusion protein containing an unmodified p40 domain (e.g., variant 30806) when assessed by IL12 RGA (Table L, Figures 4A-4J), and EC 50 Values ​​were increased up to approximately 570-fold in variant 37173 versus variant 30806 (average of n=2 replicates).

[0438] Masked IL12 HetFc fusion proteins containing modified p40 domains showed reduced potency compared to the corresponding unmasked IL12 HetFc fusion proteins (Table L, Figures 4A-4J). The potency of the masked IL12 HetFc fusion proteins ranged from unchanged compared to a masked IL12 HetFc fusion protein containing an unmodified p40 domain (variant 35436) as a control, to a complete loss of detectable activity (e.g., variants 37468, 37471, 37475, 37486).

[0439] [Table L] TIFF2024535925000025.tif192165

[0440] These data demonstrate that at least some of the one or more amino acid substitutions determined using the in silico methods described in Example 1 successfully generated modified p40 domains with reduced affinity for the cognate IL12 receptor.

[0441] Sequence Listing [Table M] TIFF2024535925000027.tif226156TIFF2024535925000028.tif225156TIFF2024535925000029.tif225156TIFF2024535925000030.tif221156TIFF2024535925000031.tif225156TIFF2024535925000032.tif225156TIFF2024535925000033.tif221156TIFF2024535925000034.tif225156TIFF2024535925000035.tif224156TIFF2024535925000036.tif220156TIFF2024535925000037.tif224156TIFF2024535925000038.tif224156TIFF2024535925000039.tif224156TIFF2024535925000040.tif224156TIFF2024535925000041.tif224156TIFF2024535925000042.tif224156TIFF2024535925000043.tif220156TIFF2024535925000044.tif224156TIFF2024535925000045.tif224156TIFF2024535925000046.tif224156TIFF2024535925000047.tif224156TIFF2024535925000048.tif224156TIFF2024535925000049.tif224156TIFF2024535925000050.tif224156TIFF2024535925000051.tif224156TIFF2024535925000052.tif224156TIFF2024535925000053.tif224156TIFF2024535925000054.tif228156TIFF2024535925000055.tif224156TIFF2024535925000056.tif224156TIFF2024535925000057.tif224156TIFF2024535925000058.tif224156TIFF2024535925000059.tif224156TIFF2024535925000060.tif224156TIFF2024535925000061.tif224156TIFF2024535925000062.tif224156TIFF2024535925000063.tif224156TIFF2024535925000064.tif228156TIFF2024535925000065.tif224156TIFF2024535925000066.tif224156TIFF2024535925000067.tif224156TIFF2024535925000068.tif224156TIFF2024535925000069.tif224156TIFF2024535925000070.tif224156TIFF2024535925000071.tif224156TIFF2024535925000072.tif224156TIFF2024535925000073.tif224156TIFF2024535925000074.tif224156TIFF2024535925000075.tif224156TIFF2024535925000076.tif224156TIFF2024535925000077.tif224156TIFF2024535925000078.tif224156TIFF2024535925000079.tif220156TIFF2024535925000080.tif220156TIFF2024535925000081.tif226156TIFF2024535925000082.tif224156TIFF2024535925000083.tif224156TIFF2024535925000084.tif224156TIFF2024535925000085.tif224156TIFF2024535925000086.tif224156TIFF2024535925000087.tif224156TIFF2024535925000088.tif224156TIFF2024535925000089.tif224156TIFF2024535925000090.tif224156TIFF2024535925000091.tif224156TIFF2024535925000092.tif220156TIFF2024535925000093.tif224156TIFF2024535925000094.tif224156TIFF2024535925000095.tif224156TIFF2024535925000096.tif224156TIFF2024535925000097.tif224156TIFF2024535925000098.tif224156TIFF2024535925000099.tif224156TIFF2024535925000100.tif224156TIFF2024535925000101.tif224156TIFF2024535925000102.tif119156.

Claims

1. 1. A modified p40 domain comprising one or more amino acid substitutions relative to the wild-type human mature IL12 p40 domain sequence set forth in SEQ ID NO: 10, wherein the one or more amino acid substitutions are located at one or more of positions D62, D161 and E45, and comprise one or more substitutions of D62H, D62I, D161R and D161S, wherein the numbering of the amino acid residues is based on the amino acid sequence set forth in SEQ ID NO:

10.

2. The modified p40 domain of claim 1 , wherein the one or more amino acid substitutions further include one or more of E45K and D62N.

3. A modified p40 domain of wild-type human mature IL12 as set forth in SEQ ID NO:

10.

10. The modified p40 domain comprising one or more amino acid substitutions relative to the p40 domain sequence, wherein the one or more amino acid substitutions are W15H, W15K, W15R, D18G, K58H, K58W, E59D, E59G, E59R, F60D, F60E, F60K, F60R, F60V, D62H, D62I, K84E, K84I, K84L, K84V, K84W, K84Y, E86L, E86R, E86S, E86W, D93E, D93H, D93R, D93W, D161R, D161S, K197D, K197E, K197Q, K197T, or K197W, or a combination thereof, and wherein the numbering of the amino acid residues is based on the amino acid sequence set forth in SEQ ID NO:

10.

4. 4. The modified p40 domain of claim 3, comprising one or more, two or more, or three or more amino acid substitutions.

5. 5. The modified p40 domain of claim 4, wherein the one or more amino acid substitutions are W15H, W15K, D18G, K58H, K58W, E59G, E59R, F60V, D62H, D62I, K84E, K84W, E86L, E86S, E86W, D93H, D93W, D161R, D161S, K197E, K197Q, K197T, or K197W, or a combination thereof.

6. 6. The modified p40 domain of claim 5, wherein the one or more amino acid substitutions are W15H, W15K, E59R, K84E, K84W, E86W, D161R, K197T, or K197W, or a combination thereof.

7. The two or more amino acid substitutions are W15H_K84L, K58H_K84I, E59D_K84W, E59G_K84W, E59R_K84E, E59R_K84W, E59R_E86W, E59D_D93H, E59R_D93R, E59R_K197E, E59R_K197W, F60E_K84W, F60R_K84Y, F60K_K197W, F60R_K197W, K84I_E86R, K84E_D93H, K84I_D93H, K84V_D93H, K84W_D93W, K84 5. The modified p40 domain of claim 4, which is I_D161R, K84W_D161R, K84W_K197E, K84W_K197Q, K84W_K197W, E86W_D93E, E86R_K197D, E86W_K197W, W15H_K84L_K197Q, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, or W15R_E59D_F60D_K197W, or a combination thereof.

8. 8. The modified p40 domain of claim 7, wherein the two or more amino acid substitutions are W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, E86W_D93E, W15H_K84L_K197Q, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H, or W15R_E59D_F60D_K197W, or a combination thereof.

9. 8. The modified p40 domain of claim 7, wherein the two or more amino acid substitutions are K58H_K84I, E59D_K84W, E59G_K84W, E59R_E86W, E59R_K197E, E59R_K197W, E59R_K84E, E59R_K84W, F60E_K84W, F60R_K197W, K84E_D93H, K84I_D161R, K84I_D93H, K84V_D93H, K84W_D93W, K84W_K197E, K84W_K197Q, or E86W_K197W, or a combination thereof.

10. The two or more amino acid substitutions are W15H_K84L, K58H_K84I, E59D_K84W, E59G_K84W, E59R_K84E, E59R_K84W, E59R_E86W, E59D_D93H, E59R_D93R, E59R_K197E, E59R_K197W, F60E_K84W, F60R_K84Y, F60K_K197W, F60R_K197W, K84 8. The modified p40 domain of claim 7, which is I_E86R, K84E_D93H, K84I_D93H, K84V_D93H, K84W_D93W, K84I_D161R, K84W_D161R, K84W_K197E, K84W_K197Q, K84W_K197W, E86W_D93E, E86R_K197D, or E86W_K197W, or a combination thereof.

11. 8. The modified p40 domain of claim 7, wherein the two or more amino acid substitutions are W15H_K84L, E59D_D93H, E59R_D93R, F60K_K197W, F60R_K84Y, K84I_E86R, K84W_D161R, K84W_K197W, E86R_K197D, or E86W_D93E, or a combination thereof.

12. 5. The modified p40 domain of claim 4, wherein the three or more amino acid substitutions are W15H_K84L_K197Q, K58H_E86R_K197D, E59D_K84W_K197W, F60R_K84E_K197W, K84I_E86R_D93H or W15R_E59D_F60D_K197W, or a combination thereof.

13. 5. The modified p40 domain of claim 4, comprising or consisting of an amino acid sequence having at least about 90%, 95%, 97%, 99%, or 100% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 12-14, 16, 18, 19, 21-34, and 36-56.

14. W15R_E59D_F60D_K197W, W15H_K84L_K197Q, E59D_K84W_K197W, K58H_E86R_K197D, W15H_K84L, F60R_K84E_ K197W, K84W_K197W, F60K_K197W, E86R_K197D, K84I_E86R_D93H, E59R_D93R, K84I_E86R, W15K, K84W_D161R, 5. The modified p40 domain of claim 4, comprising an amino acid substitution or set of amino acid substitutions selected from E45R_K58S_E59S_K195D, E59D_D93H, F60R_K84Y, E86W_D93E, K58S_E59S_K195D, K84E, K197T, E59R, W15H, K84W, K197W, E59S, D161R or E86W, or a combination thereof.

15. 15. The modified p40 domain of claim 14, comprising an amino acid substitution or set of amino acid substitutions selected from W15H_K84L_K197Q, E59D_K84W_K197W, K84W_K197W, F60K_K197W, E59R_D93R, W15K, or F60R_K84Y, or a combination thereof.

16. 16. An IL12 fusion protein comprising a modified p40 domain according to any one of claims 1 to 15.

17. 1. An IL12 fusion protein comprising an IL12 polypeptide, A modified p40 domain according to any one of claims 1 to 15, linked to a p35 domain. The IL12 fusion protein comprising:

18. 18. The IL12 fusion protein of claim 17, further comprising a heterodimeric Fc (HetFc) domain comprising a first Fc polypeptide and a second Fc polypeptide, thereby forming an IL12 HetFc fusion protein.

19. (i) a p35 domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11, and a linker (G ４ S) ４ an IL-12 polypeptide comprising the modified p40 domain of any one of claims 1 to 15 linked via (ii) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide; (iii) a masking moiety (MM) comprising an anti-IL12 scFv domain; and 1. A masked IL12 HetFc fusion protein comprising: the IL12 polypeptide is linked to the C-terminus of the first Fc polypeptide either directly or via a second linker; The masked IL12 HetFc fusion protein, wherein the masking moiety is attached to the C-terminus of the second Fc polypeptide either directly or via a third linker and is capable of non-covalently interacting with the IL12 polypeptide, thereby reducing the binding affinity of the IL12 polypeptide to at least one of its cognate receptors.

20. (i) a p35 domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 11, and a linker (G ４ S) ４ an IL-12 polypeptide comprising the modified p40 domain of any one of claims 1 to 15 linked via (ii) a heterodimeric Fc domain comprising a first Fc polypeptide and a second Fc polypeptide; wherein said IL12 polypeptide is linked to the C-terminus of said first Fc polypeptide either directly or via a second linker.

21. A pharmaceutical composition comprising a modified p40 domain according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

22. A nucleic acid molecule or a set of nucleic acid molecules encoding a modified p40 domain described in any one of claims 1 to 15.

23. 23. A vector or set of vectors comprising the nucleic acid molecule or set of nucleic acid molecules of claim 22.

24. 1. A method for identifying one or more amino acid substitutions in a p40 domain amino acid sequence to generate a modified p40 domain, comprising: performing molecular dynamics and mutagenesis simulations to identify one or more amino acid substitutions listed in Table C; Including, The one or more amino acid substitutions in the modified p40 domain amino acid sequence are relative to the sequence set forth in SEQ ID NO: 1, and the one or more amino acid substitutions increase the binding affinity (K Ｄ ) is reduced compared to an unmodified p40 domain that does not contain the one or more amino acid substitutions.

25. A pharmaceutical for the treatment of cancer, comprising a modified p40 domain described in any one of claims 1 to 15.

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