TGF-β polypeptides

Abstract

The present disclosure provides transforming growth factor beta (TGF-β) polypeptide constructs and complexes for use in the therapeutic treatment of diseases, including, for example, autoimmune diseases and metabolic diseases and disorders. Nucleic acids encoding the constructs and complexes, as well as methods for preparing the constructs and complexes in cell-based expression systems, are also described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 250,990, filed September 30, 2021, U.S. Provisional Patent Application No. 63 / 195,673, filed June 1, 2021, U.S. Provisional Patent Application No. 63 / 177,935, filed April 21, 2021, and U.S. Provisional Patent Application No. 63 / 117,307, filed November 23, 2020.

[0002] Incorporation by Reference of Sequence Listing This application contains an electronically submitted Sequence Listing, which serves as both a paper copy and a computer readable form (CRF), consisting of a file entitled "2910-25PCT_seqlist.txt", which was created on November 22, 2021, is 351,169 bytes in size, and is incorporated herein by reference in its entirety.

[0003] I. Introduction A. TGF-β and its effects Transforming growth factor beta (TGF-β) is a cytokine belonging to the transforming growth factor superfamily, which includes three mammalian (human) isoforms: TGF-β1, TGF-β2, and TGF-β3. TGF-β is synthesized as a precursor molecule containing the TGF-β sequence, which homodimerizes to form the active form of TGF-β, plus a propeptide region. TGF-β is secreted by macrophages and other cell types as a latent complex in association with two other polypeptides—latent TGF-β binding protein (LTBP) and latent-associated peptide (LAP). The latent TGF-β complex is stored in the extracellular matrix (ECM) and is bound to the cell surface by CD36, for example, via thrombospondin-1 (which can be activated by plasmin), or by latent transforming growth factor beta binding proteins 1, 2, 3, and / or 4 (LTBP1-4).

[0004] The biological functions of TGF-β are observed after activation of latent TGF-β, which is tightly regulated in response to ECM perturbations. TGF-β can be activated by specific pathways in various cells or tissues, or pathways observed in multiple cell or tissue types, although the complete mechanisms behind such activation pathways are not fully known. Activators include, but are not limited to, proteases, integrins, pH, and reactive oxygen species (ROS). Upon activation, the latent TGF-β complex bound to cells / tissues functions to sense and respond to environmental perturbations and release active TGF-β in a spatial and / or temporal manner. Released TGF-β acts to promote or inhibit cell proliferation, depending on the context of its release. It also recruits stem / progenitor cells to participate in tissue regeneration / remodeling processes. Abnormalities in TGF-β ligand expression, bioavailability, activation, receptor function, or post-transcriptional modification can interfere with normal function and lead to pathological consequences associated with many diseases, such as excessive precursor mobilization (e.g., in osteoarthritis or Kamrači-Engelman disease) or transdifferentiation of resident cells into undesired lineages (e.g., in epithelial-to-mesenchymal transition during cancer metastasis or tissue / organ fibrosis). Xu et al. Bone Research, 6 (Article No. 2) (2018).

[0005] 1. Integrin-independent and integrin-dependent activation a. Integrin-independent activation Integrin-independent means of TGF-β activation include those that act through the action of proteases and / or metalloproteases, reactive oxygen species (ROS), and thrombospondin-1 (TSP-1), among others.

[0006] Plasmin and several matrix metalloproteinases (MMPs) promote tumor invasion and tissue remodeling through the proteolysis of ECM components. TGF-β can be activated by the action of such proteases, which release latent complexes from the matrix, followed by proteolysis of LAP and release of TGF-β to its receptor. Both MMP-9 and -2 are known to cleave latent TGF-β.

[0007] TGF-β has been shown to be rapidly activated in vivo after radiation exposure, which induces ROS release. ROS are thought to alter the interaction between LAP and TGF-β, leading to its activation.

[0008] TSP-1, a glycoprotein found in the plasma of healthy individuals, is known to increase in response to injury. TSP-1 is thought to activate latent TGF-β by directly interacting with the latent TGF-β complex and preventing its binding to mature TGF-β. Thrombospondin-mediated activation is thought to be involved in wound (e.g., skin wound) healing.

[0009] b. Activation by α(V)-containing integrins Integrins, particularly β6-, αV-, and β8-containing integrins, are understood to contribute to the activation of latent TGF-β (e.g., TGF-β1). Activation is thought to occur by inducing a conformational change in the latent TGF-β1 complex, thus releasing active TGF-β1, or by an integrin-protease-dependent mechanism. In particular, in epithelial cells, the conformational change leading to proteolytically uninvolved TGF-β1 activation is understood to occur through integrin binding to the arginyl-glycyl-asparagine cell adhesion motif (RGD motif) present in LAP-β1 or LAP-β3. The RGD motif-containing LAP is recognized by most αV-containing integrins. For example, αVβ6 integrin can activate / release TGF-β1 by binding to the RGD motif present in LAP-β1 and LAP-β3. Additionally, integrin-protease-dependent activation of TGF-β can occur by forming links between the latent TGF-β complex and MMPs, such as MMP-2 and MMP-9, which can activate TGF-β by proteolysis of the latent TGF-β complex.

[0010] 2. TGF-β signal transduction and action Activated TGF-β plays an important role in cell differentiation and T cell regulation. See, for example, Cold Spring Harbor Perspect. Biol. 2017;9:a022236 and citations therein. TGF-β promotes the development of several T cell lineages in the thymus by supporting the survival of thymus-derived Tregs (tTregs), invariant natural killer T (iNKT), and CD8α+ T cell precursors, thereby promoting T cell development induced by potent agonist ligands. TGF-β supports conventional CD8+ T cells by promoting thymocyte expression of interleukin (IL)-7Rα. TGF-β also regulates peripheral T cell homeostasis by promoting the IL-7-dependent survival of low-affinity T cells, regulating IL-7Rα expression in thymocytes, and inhibiting T cell receptor (TCR)-driven activation of autoreactive or high-affinity T cells. During early CD8+ T cell differentiation, TGF-β inhibits the formation of cytotoxic T lymphocytes (CTLs) and promotes the apoptosis of short-lived effector cells (SLECs), while promoting the differentiation of CD103-expressing tissue-resident memory (TRM) cells. TGF-β inhibits the differentiation of T helper 1 and 2 (Th1 and Th2) cells, but TGF-β, acting in conjunction with other factors, promotes the development of various T cell types. TGF-β, together with IL-2, promotes the generation of peripheral Tregs (pTregs), together with IL-6, promotes the generation of Th-17 cells, together with IL-4, promotes the generation of Th9 cells, and together with IL-21 and / or -23, promotes the generation of T follicular helper (Tfh) cells.

[0011] In addition to its effects on T cells, various other cells, including B lymphocytes or "B cells," monocytes, and macrophages, are regulated by TGF-β. TGF-β generally has inhibitory effects on B cells (Li et al., Annual Review of Immunology. 24(1):99-146 (2006) and Roes et al., PNAS USA, 100(12):7241-7246 (2003)), inhibiting B cell proliferation and inducing apoptosis of immature or resting B cells (Arsura, et al., Immunity 5(1):31-40 (1996)). At least some of the effects of TGF-β on B cells can be attributed to the induction of IKBa, an inhibitor of NF-κB, which regulates the production of cytokines including IL-1, TNF-α, and defensins. See, e.g., Cold Spring Harbor Perspect. Biol. 2017;9:a022236 and citations therein.

[0012] In addition to its effects on B cells, TGF-β stimulates resting monocytes and inhibits activated macrophages. TGF-β exhibits inhibitory effects, such as inhibiting the proinflammatory response of macrophages stimulated by Toll-like receptor ("TLR") ligands. TGF-β stimulation promotes the production of several proinflammatory cytokines by myeloid cells in the absence of TLR ligands or other cytokines. TGF-β has been shown to attract peripheral blood monocytes and macrophages into tissues and enhance the adhesive properties of monocytes. TGF-β can induce chemotaxis and enhance the adhesive properties of mast cells. See, e.g., Cold Spring Harbor Perspect. Biol. 2017;9:a022236 and citations therein.

[0013] It is understood that upon activation, TGF-β acts through signaling receptors on the cell surface. Signaling is initiated when an activated TGF-β ligand binds to transforming growth factor β receptor II ("TβRII") on the cell surface. This interaction can result in the recruitment of transforming growth factor β receptor I ("TβRI"). While TβRII can bind to TGF-β1 alone, TβRI can only bind to the ligand in cooperation with TβRII. TβRI is activated by phosphorylation of TβRII, leading to signaling via the classical signaling pathway through the recruitment and phosphorylation of R-Smad proteins (Smad2 and Smad3). These Smads then bind to co-Smad (Smad4), and together the complex drives the transcription of several genes. See Smith et al., Clin. Cancer Res.; 18(17):4514-21 (2012). TGF-β can also signal through non-canonical (non-Smad) pathways, including various branches of the MAP kinase pathway, the Rho-like GTPase signaling pathway, and the phosphatidylinositol-3-kinase / AKT pathway, which are activated by ligand-occupied receptors. Signaling through non-canonical pathways can enhance, attenuate, or otherwise modulate downstream cellular responses. See Zhang Ye, Cell Res. 19(1):128-39 (2009). In contrast to TβRI and TβRII, transforming growth factor beta receptor III ("TβRIII" receptor or "betaglycan") is not involved in TGF-β signaling but rather functions as a reservoir for TGF-β.

[0014] Perturbations in activators, abnormal levels of activated TGF-β, and / or altered TGF-β signaling can lead to uncontrolled TGF-β signaling levels, which can result in several diseases or complex disease states. Indeed, TGF-β has been shown to have beneficial effects on a variety of conditions, including inflammation, autoimmune diseases, fibrosis, cancer, and cataracts.

[0015] TGF-β plays a pivotal role in maintaining hemostasis in the immune system as a factor involved in the induction of tolerance, thereby influencing autoimmune responses. One mechanism by which TGF-β can exert such effects is through the activation of T cells (e.g., CD4 + , CD8 + , CD4 - CD8 + and CD4 - CD8 - TGF-β induces tolerance to antigens, including self-antigens, by driving the differentiation of T cells (e.g., T cell regulatory subtypes) into regulatory T cells or "T-Reg" cells (e.g., in the presence of IL-2). See, e.g., Bettini and Vagnali, Ann. N.Y. Acad. Sci., 1183:1-12 (2010). T-Reg cells are essential for maintaining immune tolerance. Josefowicz et al., Annu Rev Immunol, 30:531-564 (2012). The role of TGF-β in inducing tolerance to antigens, including self-antigens, is an important factor in preventing the development of diseases such as arthritis (rheumatoid arthritis or "RA"), type 1 diabetes ("T1D"), multiple sclerosis ("MS"), and systemic lupus erythematosus ("SLE"). For example, a major function of TGF-β is the regulation of autoimmune diseases and associated inflammatory processes. This is particularly true in the intestine, where it is thought to suppress cytokine production by macrophages and mucosal inflammation in conditions such as inflammatory bowel disease or "IBD." Sanjab et al. Cold Spring Harbor Perspect. Biol. 2017;9:a022236. RA, like IBD, is an autoimmune disease of the joints with an inflammatory component. RA results from an abnormal response in T cells and / or B cells. TGF-β is thought to provide systemic protection from the development of RA. See Schramm et al., Arthritis Res. Ther. 6:R114-R119 (2004) and Sanjab et al. Cold Spring Harbor Perspect. Biol. 2017;9:a022236) and references cited therein.

[0016] Many approaches have been reported in the literature to regulate TGF-β activity at the protein level by capturing the protein and effectively neutralizing its activity. For example, monoclonal antibodies such as methelimumab (CAT192) against TGF-β1 and fresolimumab against multiple isoforms of TGF-β have been developed to bind to, capture, and neutralize TGF-β in vivo. In addition, receptor traps have also been developed that tightly bind to, capture, and neutralize TGF-β (see, for example, Swagrtra, et al., Mol Cancer Ther; 11(7): 1477-87 (2012) and U.S. Patent Publication No. 2018 / 0327477).

[0017] Unlike the molecules described above that are designed to bind and capture TGF-β and function as antagonists of TGF-β action, the masked TGF-β complexes described herein provide an active TGF-β polypeptide (e.g., a TGF-β signaling pathway agonist) and a masking polypeptide (e.g., a TGF-β receptor fragment) that interact with each other to reversibly mask the TGF-β polypeptide sequence. The masked TGF-β complexes may contain sequence variations in the TGF-β and / or masking polypeptide that reduce their mutual affinity, contributing to the unmasking of TGF-β and allowing its binding signaling through heteromeric cell surface receptors (e.g., binding to TβRII followed by TβRI forming a heteromeric receptor). Once formed, heteromeric TβRI-TβRII polypeptide complexes with high affinity for TGF-β can effectively compete with the masking polypeptide. Sequence variations in the TGF-β and / or its masking polypeptide can also prevent undesired interactions between unmasked TGF-β polypeptides and other molecules. Such sequence mutations include deletion of a portion of the N-terminus of TβRII that attenuates binding to TβRI, and / or TGF-β sequence mutations (e.g., C77S substitutions) that prevent its dimerization, relieving the target binding restriction to a reservoir of non-signaling TβRIII molecules. In addition to the above, masked TGF-β constructs and complexes can contain additional wild-type (wt) and / or mutant immunomodulatory polypeptide sequences (MODs) that can substantially affect the consequences of TGF-β binding to target cells, including in vitro and in vivo effects, such as therapeutic effects. Summary of the Invention

[0018] II. Summary of the Invention The present disclosure describes the preparation of constructs in which TGF-β is masked by another polypeptide ("masked TGF-β constructs," e.g., see Structure A in Figure 1, which has a single polypeptide chain) and complexes in which TGF-β is masked by another polypeptide ("TGF-β polypeptide complexes," e.g., see Structures B-F in Figure 1, which show complexes comprising two polypeptide chains). TGF-β constructs and complexes, which may also include additional elements, are collectively referred to herein as "masked TGF-β constructs and complexes." Masked TGF-β constructs and complexes are constructed around a scaffold polypeptide (e.g., an immunoglobulin Fc region) and contain a masking polypeptide sequence ("masking polypeptide sequence," "masking polypeptide," or "masking sequence") that binds to TGF-β. Masked TGF-β constructs and complexes may also contain one or more independently selected immunomodulatory polypeptide sequences (singular "MOD," plural "MODs"), such as wild-type or mutant IL-2 polypeptide sequences.

[0019] Masked TGF-β constructs and complexes can be expressed in many types of mammalian cells because the masked, non-targeted TGF-β activity does not have as adverse effects within the cell as observed with unmasked TGF-β.

[0020] The masked TGF-β construct comprises, as a first polypeptide: i) a scaffold polypeptide sequence; and ii) a TGF-β polypeptide sequence; and iii) a masking polypeptide sequence optionally comprising a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; iv) optionally, one or more independently selected MOD polypeptide sequences; v) optionally, one or more independently selected linker polypeptide sequences; Constructs comprising these elements are collectively referred to herein as "masked TGF-β constructs," in which the masking polypeptide sequence and the TGF-β polypeptide sequence are linked to each other. The masked TGF-β construct comprises, in order (from N-terminus to C-terminus), e.g., i) a scaffold polypeptide sequence, a masking polypeptide sequence, and a TGF-β polypeptide sequence; or ii) a first MOD polypeptide sequence, a scaffold polypeptide sequence, a masking polypeptide sequence, and a TGF-β polypeptide sequence; or iii) a first independently selected MOD polypeptide sequence, a second independently selected MOD polypeptide sequence, optionally one or more additional MOD polypeptide sequences, a scaffold polypeptide sequence, a masking polypeptide sequence, and a TGF-β polypeptide sequence; The masked TGF-β construct optionally includes one or more independently selected linker polypeptide sequences.

[0021] The scaffold polypeptides of the above-described masked TGF-β constructs may contain interspecific or non-interspecific dimerization sequences that lead to the formation of homodimers, wherein the scaffold polypeptide sequences optionally have one or more covalent attachments to each other.

[0022] The scaffold polypeptide of the masked TGF-β construct described above may also include a mutually specific dimerization sequence and may further include a second polypeptide that dimerizes with the first polypeptide (described above) via a complementary mutually specific dimerization sequence to form a masked TGF-β complex heterodimer. The second polypeptide may have one of the following structures: (i) a scaffold polypeptide sequence including a complementary mutually specific dimerization sequence, (ii) a scaffold polypeptide sequence including one or two (or more) independently selected MOD sequences and a complementary mutually specific dimerization sequence, (iii) a scaffold polypeptide sequence including a complementary mutually specific dimerization sequence and an independently selected MOD sequence, or (iv) a scaffold polypeptide sequence including one or two (or more) independently selected MOD sequences and a complementary mutually specific dimerization sequence, wherein the first and / or second polypeptides optionally include one or more independently selected linker polypeptide sequences. Thus, the second polypeptide may comprise one of the following structures: (i) a scaffold polypeptide sequence comprising a complementary mutually specific dimerization sequence, (ii) a scaffold polypeptide sequence comprising one or two (or more) independently selected MOD sequences and a complementary mutually specific dimerization sequence, (iii) a scaffold polypeptide sequence comprising a complementary mutually specific dimerization sequence and one or two (or more) independently selected MOD sequences, or (iv) a scaffold polypeptide sequence comprising one or two (or more) independently selected MOD sequences and a complementary mutually specific dimerization sequence, wherein the first and / or second polypeptide optionally comprises one or more independently selected linker polypeptide sequences.Alternatively, the masked TGF-β complex heterodimer may comprise, in order from N-terminus to C-terminus, (i) a scaffold polypeptide sequence comprising a complementary mutually specific dimerization sequence, a masking polypeptide sequence, and a TGF-β polypeptide sequence; (ii) a first MOD polypeptide sequence, a scaffold polypeptide sequence comprising a complementary mutually specific dimerization sequence, a masking polypeptide sequence, and a TGF-β polypeptide sequence; or (iii) a first independently selected MOD polypeptide sequence, a second independently selected MOD polypeptide sequence, a scaffold polypeptide sequence comprising a complementary mutually specific dimerization sequence, a masking polypeptide sequence, and a TGF-β polypeptide sequence.

[0023] The masked TGF-β complex can also comprise a first polypeptide and a second polypeptide as a masked TGF-β complex heterodimer, (i) the first polypeptide is a) a scaffold polypeptide sequence comprising a mutually specific dimerization sequence; b) a masking polypeptide sequence optionally comprising a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; c) optionally, one or more independently selected MOD polypeptide sequences; d) optionally, one or more independently selected linker polypeptide sequences; (ii) the second polypeptide is a) a scaffold polypeptide sequence comprising a mutually specific dimerization sequence that is complementary to a mutually specific dimerization sequence in a first polypeptide; b) a TGF-β polypeptide sequence; and c) optionally, one or more independently selected MOD polypeptide sequences; d) optionally, one or more independently selected linker polypeptide sequences; A complex containing these elements is collectively referred to as a "masked TGF-β complex," in which the masking polypeptide sequence and the TGF-β polypeptide sequence are provided on different polypeptide chains and are linked to each other; The mutually specific binding sequence and the counterpart mutually specific binding sequence interact with each other in the heterodimer, The masked TGF-β first polypeptide and / or second polypeptide optionally comprise one or more independently selected linker polypeptide sequences.

[0024] The TGF-β polypeptide sequence can be derived from any of the TGF-β isoforms and can contain substitutions that limit the ability of the TGF-β sequence to dimerize. The masking sequence can be, for example, an antigen-binding amino acid sequence from an anti-TGF-β antibody sequence or a TGF-β receptor (TβR) ectodomain sequence. When the TβR ectodomain is used to mask the TGF-β sequence, it can be modified to avoid inadvertent signal transduction by the masked molecule (e.g., by deleting all or part of the ectodomain that is not necessary for interaction with the TGF-β sequence).

[0025] The present disclosure also describes and provides methods for producing masked TGF-β constructs and complexes and their use in various cell types to treat various diseases / disorders, including autoimmune and inflammatory diseases. The therapeutic methods described herein may involve co-administration of masked TGF-β constructs and complexes with other molecules, such as, but not limited to, immunomodulators (e.g., interleukins, cytokines, chemokines, etc.), antibodies and antibody fragments (e.g., scFvs, nanobodies, etc.), small molecule therapeutics (e.g., vitamin D or retinoic acid), and combinations thereof that may be beneficial in achieving a desired laboratory or therapeutic result. [Brief explanation of the drawings]

[0026] III. BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figures 1A-1D show several formats of masked TGF-β constructs and complexes that use TGF-β receptor sequences to mask TGF-β polypeptides. Structure A shows a monomeric construct with one or more independently selected MODs (e.g., a set of independently selected tandem MODs) in one location. Structure B shows a symmetric homodimer in which the polypeptides interact through their respective IgFc sequences, where disulfide bonds linking the two polypeptides can spontaneously form. Structures C-F show heterodimeric structures in which the TGF-β and TGF-β receptor sequences are in "cis" (on the same polypeptide) or "trans" (on different polypeptides) in the heterodimer. Positions where one or more independently selected MODs can be placed are indicated by diagonal or vertical lines or checkered filled circles. Mutually specific binding pairs are represented by knob-in-hole sequences, but may be any other, as discussed below. Constructs may contain no MOD, or may contain one, two, or more independently selected MOD sequences, including tandem MOD sequences, where MODs can be provided where indicated. Exemplary MODs include, for example, wild-type or mutant or variant (e.g., with reduced and / or selective affinity for a particular receptor or receptors) PD-L1, FAS-L, 4-1BBL, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, and IL-23 MOD sequences. In each case where a TGF-β receptor sequence is used to mask a TGF-β polypeptide, the receptor polypeptide can be replaced with another masking polypeptide, such as an antibody polypeptide (e.g., an scFV or nanobody) with affinity for the TGF-β polypeptide. Scaffold sequences can be linked (e.g., by disulfide bonds) to form covalent homodimers or covalent heterodimers. Any of the illustrated constructs may have a sequence variation in the TGF-β polypeptide that limits its ability to dimerize (eg, a C77S substitution).

[0027] [Figure 2A]The amino acid sequence of the immunoglobulin Fc polypeptide (SEQ ID NO:68) is provided. [Figure 2B] The amino acid sequence of the immunoglobulin Fc polypeptide (SEQ ID NO:69) is provided. [Figure 2C] The amino acid sequence of the immunoglobulin Fc polypeptide (SEQ ID NO:70) is provided. [Figure 2D] The amino acid sequences of immunoglobulin Fc polypeptides (SEQ ID NOs: 71-78) are provided. [Figure 2E] The amino acid sequence of the immunoglobulin Fc polypeptide (SEQ ID NO:79) is provided. [Figure 2F] The amino acid sequence of the immunoglobulin Fc polypeptide (SEQ ID NO:80) is provided. [Figure 2G] The amino acid sequence of the immunoglobulin Fc polypeptide (SEQ ID NOs: 81 and 82) is provided. [Figure 2H] The amino acid sequence of the immunoglobulin Fc polypeptide (SEQ ID NO:83) is provided.

[0028] [Figure 2I] The amino acid sequence of human J chain (SEQ ID NO: 84) is provided, with the signal peptide aas 1-22 underlined.

[0029] [Figure 2J] The sequence of the IgG1 heavy chain constant region CH1 domain is provided. The serine residues at positions 70 and 72 can be replaced by glutamic acid and valine, respectively (S70E and S72V), to form MD13-like constructs.

[0030] [Figure 2K] The sequences of the light chain constant region "CL" domains from Igκ and Igλ chains are provided. Serine at position 68 and threonine at position 70 can be replaced by leucine and serine, respectively (S68L and T70S) to form MD13-like constructs.

[0031] [Figure 3]The sequences of three different TGF-β isoforms as preproproteins and the mature form of TGF-β3 are provided, along with the C77S mutant of the mature protein.

[0032] [Figure 4] An alignment of TGF-β isoforms 1 to 3 is provided, in which residues corresponding to the mature form of TGF-β2, except for aa residues Lys25, Cys77, Ile92, and / or Lys94 of TGF-β2, are shown in bold, and their corresponding residues in the other forms of TGF-β isoforms 1 and 3 are shown underlined and not bold.

[0033] [Figure 5A] The sequence of the type 1 TGF-β receptor (TβRI) and its ectodomain is provided.

[0034] [Figure 5B]

[0023] Figure 1 provides the sequence of the type 2 TGF-β receptor (TβRII), its ectodomain, and fragments of the ectodomain. In isoform B, the positions shown in bold and underlined are aas F30, D32, S52, E55, and D118 of the mature polypeptide, any of which may be substituted with an aa other than the naturally occurring aa.

[0035] [Figure 5C] The sequence of the type 3 TGF-β receptor (TβRIII) is provided.

[0036] [Figure 6A] Figure 1 shows a plot depicting the ability of different concentrations of various masked TGF-β constructs and complexes to stimulate the expression of FoxP3 on naive CD4+ T cells based on fluorescence cytometry analysis. Shown is the induction of FoxP3 (as a percentage of CD4+ cells) based on the indicated concentrations of TGF-β3 or masked TGF-β3WT constructs (see Figure 1, Structure A) in the absence and presence of added 50 U / ml of IL-2 after 5 days in cell culture. [Figure 6B]Figure 7 shows plots demonstrating the ability of various masked TGF-β constructs and complexes at different concentrations to stimulate FoxP3 expression on naive CD4+ T cells, based on fluorescence cytometry analysis. The distribution of FoxP3+ cells (as a percentage of CD4+ cells) within a population of naive T cells treated for 5 days with various concentrations of TGF-β3, or one of three masked TGF-β3 constructs, or a masked TGF-β3 complex bearing at least one N-terminal wt or mutant IL-2 MOD (see Figures 7G-7I for structures). [Figure 6C] 6 shows a plot illustrating the ability of various masked TGF-β constructs and complexes at different concentrations to stimulate the expression of FoxP3 on naive CD4+ T cells based on fluorescence cytometry analysis. Induction of FoxP3+ CD4+ cells is shown in the presence of masked TGF-β3 polypeptide (structure (i) in part B of FIG. 6) at concentrations of 0.1 nM or 1000 nM.

[0037] [Figure 7A]

[0023] Provided is the aa sequence of a representative masked TGF-β (construct number 3470), SEQ ID NO: 146, having the overall structure of Structure A in Figure 1. The polypeptide comprises, from N- to C-terminus, a wt. human IL-2 (hIL2) sequence, three repeats of a G4S linker, a human mono IgGFc with an LALA substitution, three repeats of a G4S linker, a human TβRII (hTβRII) Δ25 sequence with a D118A substitution, five repeats of a G4S linker, and a human TGF-β3 (hTGF-β3) sequence with a C77S substitution.

[0038] [Figure 7B]Provided is the aa sequence of a representative masked TGF-β (construct number 3334, SEQ ID NO: 147) having the overall structure of Structure B in Figure 1. The polypeptides that form homodimers include, from N- to C-terminus, hIL2 with H16T and F42A substitutions, three repeats of G4S, human IgG1Fc with LALA substitutions, two repeats of G5S and G4S linkers, hTβRIIΔ25, D118A, five repeats of G4S, and an hTGF-β3 sequence.

[0039] [Figure 7C]

[0039] Figure 1 provides an aa sequence of a representative masked TGF-β construct having the overall structure of Structure D in Figure 1, comprising a first and second polypeptide. The first polypeptide (construct number 3618, SEQ ID NO: 148) comprises, from N- to C-terminus, wt. hIL-2, three repeats of the G4S linker, human IgG1 Fc knob-in-hole (KiH) polypeptide chain A with a LALA substitution, five repeats of the G4S linker sequence, and an hTGF-β3 sequence with a C77S substitution. The second polypeptide (construct number 3619, SEQ ID NO: 149) comprises, from N- to C-terminus, wt. hIL-2, three repeats of the G4S linker, human IgG1 Fc KiH polypeptide chain B with a LALA substitution, a G5S linker and two repeats of the G4S linker, and an hTβRIIΔ25, D118A sequence.

[0040] [Figure 7D]

[0033] Figure 1 provides aa sequences of representative masked TGF-β constructs having the overall structure of Structure E in Figure 1, including first and second polypeptides. The first polypeptide described above (Construct No. 3618, SEQ ID NO: 150) comprises, from N- to C-terminus, wild-type hIL-2, three repeats of a G4S linker, human IgG1 Fc knob-in-hole (KiH) polypeptide chain A with a LALA substitution, five repeats of a G4S linker sequence, and an hTGF-β3 sequence with a C77S substitution. The second polypeptide (Construct No. 3855, SEQ ID NO: 151) comprises, from N- to C-terminus, human IgG1 Fc KiH polypeptide chain B with a LALA substitution, T366S, L368A, and Y407V substitutions, three repeats of a G4S linker, and an hTβRIIΔ25, D118A sequence.

[0041] [Figure 7E]

[0039] The aa sequences of representative masked TGF-β constructs having the overall structure of Structure F in Figure 1, including first and second polypeptides, are provided. The first polypeptide (Construct No. 3891), SEQ ID NO: 152, comprises, from N- to C-terminus, hIL-2 with H16A, F42A, three repeats of a G4S linker sequence, a human IgG1 Fc knob-in-hole (KiH) polypeptide chain A with a LALA and T366W substitution, two repeats of a G5S and G4S linker, hTβRIIΔ25, D118A, five repeats of a G4S linker, and a C77S substitution. The second polypeptide (Construct No. 3664), SEQ ID NO: 153, comprises, from N- to C-terminus, a human IgG1 Fc KiH polypeptide chain B with a LALA substitution.

[0042] [Figure 7F]

[0039] Figure 1 provides the aa sequences of representative masked TGF-β constructs having the overall structure of Structure F in Figure 1, including first and second polypeptides. The first polypeptide (construct number 3715, SEQ ID NO: 155) comprises, from N- to C-terminus, human IgG1 Fc KiH polypeptide chain A with a LALA substitution, three repeats of a G4S linker, and a human wt. IL2 sequence. The second polypeptide (construct number 3714, SEQ ID NO: 156) comprises, from N- to C-terminus, human IgG1 Fc KiH polypeptide chain B with a LALA substitution, three repeats of a G4S linker, hTβRIIΔ25 with D32N and D118A substitutions, five repeats of a G4S linker sequence, and an hTGF-β3 sequence with a C77S substitution.

[0043] Any of the IL-2 sequences in Figures 7A-7F may be replaced with a MOD other than IL-2 or a mutant MOD, such as replaced with wt. IL-2 or replaced with an IL-2 sequence having substitutions at N88, H16 and / or F42 (e.g., a substitution at H16 selected from N88R, H16A or H16T, and / or a substitution at F42 selected from F42A and F42T).

[0044] [Figure 7G] Provided is the aa sequence of a representative masked TGF-β construct (construct number 3472, SEQ ID NO: 157) having the overall structure of Structure A in Figure 1. The polypeptide comprises, from N- to C-terminus, wt. hIL2, three repeats of G4S linkers, a human mono IgG Fc with a LALA substitution, three repeats of G4S linkers, a human TβRII (hTβRII) Δ25 sequence with D32N and D118A substitutions, five repeats of G4S linkers, and a human TGF-β3 (hTGF-β3) sequence with a C77S substitution.

[0045] [Figure 7H]Provided is the aa sequence of a representative masked TGF-β construct (construct number 3466, SEQ ID NO: 158) having the overall structure of Structure A in Figure 1. The polypeptide comprises, from N- to C-terminus, wt. hIL2, three repeats of G4S linkers, a human mono IgG Fc with a LALA substitution, three repeats of G4S linkers, a human TβRII (hTβRII) Δ25 sequence with a D118A substitution, five repeats of G4S linkers, and a human TGF-β3 (hTGF-β3) sequence with a C77S substitution.

[0046] [Figure 7I] Provided is the aa sequence of a representative masked TGF-β construct (construct number 3468, SEQ ID NO: 159) having the overall structure of Structure A in Figure 1. The polypeptide comprises, from N- to C-terminus, hIL2 with H16T F42A substitutions, three repeats of G4S linkers, a human mono IgG Fc with a LALA substitution, three repeats of G4S linkers, a human TβRII (hTβRII) Δ25 sequence with D32N and D118A substitutions, five repeats of G4S linkers, and a human TGF-β3 (hTGF-β3) sequence with a C77S substitution.

[0047] [Figure 7J]

[0039] Figure 1 provides an aa sequence of a representative masked TGF-β construct having the overall structure of Structure D in Figure 1, comprising a first and second polypeptide. The first polypeptide (construct number 3618, SEQ ID NO: 148) comprises, from N- to C-terminus, wt. hIL-2, three repeats of the G4S linker, human IgG1 Fc knob-in-hole (KiH) polypeptide chain A with a LALA substitution, five repeats of the G4S linker sequence, and an hTGF-β3 sequence with a C77S substitution. The second polypeptide (construct number 3621, SEQ ID NO: 160) comprises, from N- to C-terminus, wt. hIL-2, three repeats of the G4S linker, human IgG1 Fc KiH polypeptide chain B with a LALA substitution, three repeats of the G4S linker, and an hTβRIIΔ25 sequence with D32N and D118A substitutions.

[0048] [Figure 8] A masked TGF-β construct (left) and two masked TGF-β complexes (center and right) are shown. Complex samples were prepared by constructing a nucleic acid vector encoding the polypeptide, transfecting it into ExpiCHO cells, and expressing the polypeptide. The polypeptide was purified by protein A chromatography followed by size-exclusion chromatography. The purified protein was subjected to SDS-PAGE, and the resulting gel was stained with Coomassie blue. NR = non-reduced sample; R = reduced sample (reduced with a disulfide-reducing agent).

[0049] [Figure 9]The effect of various mutations (aa substitutions) in the masked TβRII polypeptide sequence (see Figure 5B) on affinity for TGF-β3 or TGF-β1 is shown (Table, top left). Figure 9 also demonstrates the relatively large effect of these three substitutions, E55A, D32N, and S52L, on the binding interaction between the masked TβRII aa sequence and masked TGF-β (TGF-β1 or TGF-β3), as reflected in the affinity of the masked TGF-β construct to the immobilized TβRII-Fc receptor construct. On the right side of Figure 9, the assay format is depicted in two microtiter wells. In the left well, the masked TGF-β construct is in a closed configuration, engaged or unengaged with an immobilized TβRII-FC fusion (the shaded box labeled "TβRII-FC" attached to the bottom of the assay well) with its TGF-β and TβRII sequences. In the right microtiter well, the masked TGF-β construct is in the open configuration, shown in a capture assay format with its TGF-β3 sequence above, interacting with the immobilized TβRII-Fc receptor. The Ig scaffold of the masked construct employed does not form cross-specific bonds to other scaffolds, and therefore the masked construct remains monomeric. Detection of the specifically immobilized masked TGF-β construct shown in the right microtiter well is achieved using anti-IL-2 labeled biotin as the antibody ("B"), and streptavidin and horseradish peroxidase ("Strep" and "HRP") for detection. Capture curves generated for each of the masked constructs prepared as in Example 3 were used to determine the reported Kd values.

[0050] [Figure 10A] 1 shows the structure of the polypeptides that form the masked TGF-β3 complex PSM-4033-4039. [Figure 10B] 1 shows the amino acid sequence of the polypeptide that forms the masked TGF-β3 complex PSM-4033. [Figure 10C]1 shows the amino acid sequence of the polypeptide that forms the masked TGF-β3 complex PSM-4039.

[0051] [Figure 11]

[0023] Figure 1 shows the results of an experiment in which PSM-4033-4039 was used to induce Foxp3+ iTregs from human peripheral naive CD4+ T cells. See Example 4.

[0052] [Figure 12]

[0039] Figure 1 shows the results of an experiment in which Foxp3+ iTregs induced by PSM-4033-4039 were used to suppress T cell proliferation. See Example 4.

[0053] [Figure 13]

[0023] Figure 1 shows the results of an experiment in which PSM-4033-4039 was used to induce Foxp3+ iTreg expression from human peripheral CD4+ T cells, including naive and memory CD4+ T cells. See Example 4.

[0054] [Figure 14A]

[0023] Figure 1 shows the results of an experiment in which PSM-4033-4039 was used to induce Foxp3+ iTregs from CD4+ T cells activated by an allogeneic lymphocyte reaction. See Example 4. [Figure 14B]

[0023] Figure 1 shows the results of an experiment in which PSM-4033-4039 was used to induce Foxp3+ iTregs from CD4+ T cells activated by an allogeneic lymphocyte reaction. See Example 4.

[0055] [Figure 15]

[0023] Figure 1 shows the results of an experiment in which PSM-4033-4039 was administered intravenously to mice at various concentrations and serum concentrations in the mice were determined at various intervals up to 72 hours after injection. See Example 4.

[0056] [Figure 16]In A, induction of FoxP3 expression in CD4+ T cells by PBS vehicle control, by combined treatment with recombinant TGF-β3 and recombinant IL-2, or by PSM-4033-4039 (see Figure 10A) is shown. In B, the dose-response curve (EC50 measurement) for PSM-4033-4039 shows an EC50 value of approximately 30 nM.

[0057] [Figure 17] 1 shows inhibition of T effector cell proliferation by co-culture with CD4+ T cells induced to express FoxP3 by treatment with PSM-4033-4039.

[0058] [Figure 18] Shown is the viability of FoxP3+ T cells upon treatment with control medium, IL-2, recombinant TGF-β3 in combination with recombinant IL-2, and PSM-4033-4039.

[0059] [Figure 19] Figure 19 shows the induction of FoxP3+CD4+ cells in the blood of TxA23 mice treated with PBS (A) or PSM-4033-4039 (B). This figure also shows the induction of FoxP3+CD4+ cells in the gastric lymph nodes of TxA23 mice treated with PBS (C) or PSM-4033-4039 (D). Panel E of Figure 19 compares the levels of FoxP3+CD4+ cells in the gastric lymph nodes of three TxA23 mice treated with PBS and three TxA23 mice treated with PSM-4033-4039. A comparison of the data in panels A and B is provided in the histogram in panel G, and a comparison of the data in panels C and D is provided in the histogram in panel H. The numbers of T effector cells and Treg cells in gastric lymph nodes bearing TCR specific for the gastric epithelial cell marker Ki67 in mice treated with PBS or PSM-4033-4039 in PBS are provided in panels H and I.

[0060] [Figure 20]A-G provide a comparison of the expression of phenotypic markers CD25, CTL4, PD1, GITR, CD38, CD73, and GARP by conventional and PSM-4033-4039-induced iTregs.

[0061] [Figure 21] Employing keyhole limpet hemocyanin to induce a delayed-type hypersensitivity (DTH) response, a schematic timeline for testing the effect of PSM-4033-4039 in a mouse model of DTH is provided, as well as plots of the effect of PSM-4033-4039 along with an injected vehicle control and a cyclosporine positive control. DETAILED DESCRIPTION OF THE INVENTION

[0062] IV. Detailed Description A.Definition As used herein, amino acids (abbreviated as "aa" in the singular and "aas" in the plural unless the context requires otherwise) refer to the naturally occurring proteinogenic alpha amino acids that are incorporated into polypeptides and proteins during translation in mammalian cells. Unless otherwise specified, the following abbreviations are used: L (Leu, leucine), A (Ala, alanine), G (Gly, glycine), S (Ser, serine), V (Val, valine), F (Phe, phenylalanine), Y (Tyr, tyrosine), H (His, histidine), R (Arg, arginine), N (Asn, asparagine), E (Glu, glutamic acid), D (Asp, asparagine), C (Cys, cysteine), Q (Gln, glutamine), I (Ile, isoleucine), M (Met, methionine), P (Pro, proline), T (Thr, threonine), K (Lys, lysine), and W (Trp, tryptophan). Amino acids also include the amino acids hydroxyproline and selenocysteine, which appear in several proteins found in mammalian cells.

[0063] As used herein, the terms "polypeptide," "polypeptide sequence," and "protein" are synonymous and refer to a sequence of aas linked by peptide bonds between their C-1 carboxyl groups and their alpha amines to form the backbone of the polypeptide. Thus, each polypeptide (e.g., a first polypeptide) comprising any one or more of a MOD polypeptide sequence, a scaffold polypeptide sequence, a TGF-β polypeptide sequence, and / or a masking polypeptide sequence (e.g., a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence) comprises any one or more of those polypeptide sequences as a polypeptide chain having a single continuous backbone. Such a polypeptide (e.g., a first polypeptide) may be linked to other polypeptides by covalent bonds (e.g., disulfide bonds between the side chains of cysteine ​​residues). Furthermore, as used herein, the terms "polypeptide," "polypeptide sequence," and "protein" include modifications to the native sequence (generally conservative in nature, as known to those skilled in the art) such as deletions, additions, and substitutions, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins, or errors resulting from PCR amplification or other recombinant DNA techniques.

[0064] When an embodiment, claim, or aspect is directed to a particular type of mammal (e.g., a human or non-human subject), the nucleic acid and polypeptide sequences may be limited to sequences derived from that mammal. Unless otherwise specified, polypeptide sequences for proteins (e.g., TGF-β, TβR, immunoglobulins, and MOD) are human (Homo Sapiens) sequences.

[0065] As used herein, "masked" means that a molecule (e.g., a masked polypeptide or masked protein) is bound to or otherwise engaged with a masking molecule (e.g., a polypeptide, protein, or protein fragment) where the availability of the masked molecule by other proteins (e.g., cell surface receptors) that also have affinity for the former molecule is limited.

[0066] As used herein, the term masked TGF-β construct refers to a single polypeptide that includes both a TGF-β (e.g., TGF-β1, TGF-β2, or TGF-β3) polypeptide sequence and a masking polypeptide sequence that binds to or otherwise interacts with the TGF-β polypeptide. Unless otherwise specified, a masked TGF-β construct includes a scaffold polypeptide sequence and, optionally, one or more independently selected immunomodulatory (MOD) polypeptide sequences.

[0067] As used herein, the term masked TGF-β complex refers to two or more polypeptides (two polypeptides designated as a first and a second polypeptide, typically arranged as a homodimer or heterodimer, but which may also be higher order multimers). A masked TGF-β complex includes a TGF-β (e.g., TGF-β1, TGF-β2, or TGF-β3) polypeptide sequence, a masking polypeptide sequence that binds to or otherwise interacts with the TGF-β polypeptide, and a scaffold polypeptide that includes a dimerization or multimerization sequence through which the polypeptides of the TGF-β complex associate. Any one or more of the TGF-β complex polypeptides optionally includes one or more independently selected MOD polypeptide sequences.

[0068] The term "masked TGF-β construct or complex" is an abbreviation for masked TGF-β construct or masked TGF-β complex. The abbreviation may be used in its plural form, "masked TGF-β construct or complex."

[0069] As used herein, dimerization and multimerization sequences are polypeptide sequences that allow for the association of polypeptide sequences (e.g., separate polypeptides) as dimers (e.g., heterodimers or homodimers) or multimers (homo- or heteromultimers of three, four, five or more polypeptide sequences). Dimerization and multimerization sequences allow for non-covalent association of the sequences, which in some circumstances can be converted to covalent complexes (e.g., disulfide bond formation between the polypeptides).

[0070] Mutual specific binding sequences are dimerization sequences that allow asymmetric pairing (heterodimerization) of polypeptides. Mutual specific binding sequences favor the formation of heterodimers with their counterpart mutual specific binding sequence(s), i.e., their cognate binding partner (as opposed to forming homodimers). A key-in-hole (or key-into-hole) Fc polypeptide pair represents an example of a mutual specific binding sequence and its counterpart mutual specific binding sequence.

[0071] Nanobodies (or nanobody) as used herein refers to antibody fragments consisting of a single monomeric antibody variable domain that, like a whole antibody, is capable of selectively binding to a specific antigen.

[0072] Wild-type (abbreviated "wt") with respect to an aa sequence means a naturally occurring aa sequence, or a contiguous portion of a naturally occurring aa sequence as understood from the context, that is unchanged (having no substitutions, deletions, or insertions therein) compared to a sequence found naturally in an organism. A particular naturally occurring sequence may be designated as a wt sequence for reference purposes.

[0073] As used herein, "T cells" encompass all types of immune cells that express CD3, such as helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Tregs), and NK-T cells.

[0074] The term "binding," as used herein, refers to a non-covalent interaction between two molecules, e.g., a non-covalent interaction between a MOD and its co-MOD. Non-covalent binding refers to a direct association between two molecules, e.g., through electrostatic, hydrophobic, ionic, and / or hydrogen-bonding interactions, including, for example, interactions through salt bridges and water bridges. Non-covalent interactions generally occur in the 10 -6 Under M, 10 -7 Under M, 10 -8 Under M, 10 -9 Under M, 10 -10 Under M, 10 -11 Under M, 10 -12 Under M, 10 -13 Under M, 10 -14 Less than M, also 10 -15 A dissociation constant (K D "Specific binding" generally refers to binding of at least about 10 -7 M or more, e.g., 5 x 10 -7 M, 10 -8 M, 5 x 10 -8 M, 10 -9 "Non-specific binding" generally refers to binding with an affinity of about 10 M or greater. -7 Binding with an affinity of less than M (e.g., binding of a ligand to a moiety other than its designated binding site or receptor) (e.g., 10 -6 M, 10 -5 M, 10 -4 "Covalent binding" or "covalent bond" as used herein refers to the formation of one or more covalent chemical bonds between two different molecules.

[0075] "Affinity" refers to the strength of non-covalent binding; increased binding affinity indicates a lower K DAs used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen), and is related to the dissociation constant (K D )

[0076] As used herein, the term "immunomodulatory polypeptide" or MOD includes a polypeptide, or a portion of a polypeptide, on an antigen-presenting cell (APC) (e.g., a dendritic cell, a B cell, etc.) that specifically binds to a cognate co-immunomodulatory polypeptide ("co-MOD") on a T cell, thereby generating a signal. For example, binding of an interleukin, such as IL-2, or a fragment thereof (MOD) to a cell surface IL-2 receptor (co-MOD) provides a signal to the cell. MODs include, but are not limited to, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, IL-23, PD-L1, 4-1BBL, and Fas Ligand (FAS-L). MODs also encompass, inter alia, antibodies or antibody sequences (e.g., nanobodies) that specifically bind to a co-MOD molecule present on a T cell, resulting in signal transduction by the co-MOD. As discussed herein, MOD also includes mutant forms of wt. MOD, including, for example, mutant MODs with reduced binding affinity for co-MODs. Such reduced affinity can take multiple forms. For example, a mutant IL-2 MOD may have reduced affinity for one or more of the α, β, and / or γ chains of the IL-2R. As discussed herein, a mutant IL-2 MOD containing mutations at positions 16 and 42 may exhibit substantially no binding to the α chain of the IL-2R and reduced affinity for the β chain of the IL-2R. Additionally, in the case of a MOD with two or more co-MODs (e.g., CD80 binds to both CD28 and CTLA-4), the mutant MOD may have reduced affinity for one co-MOD and preferentially or selectively bind to another co-MOD. For purposes of this disclosure, TGF-β (e.g., TGF-β1, TGF-β2, or TGF-β3) and fragments thereof are not considered MODs.

[0077] Unless otherwise indicated, the term "substantially" is intended to encompass both "completely" and "largely but not completely." For example, a mutant IL-2 MOD that exhibits substantially no binding to the α-chain of the IL-2R is an IL-2 mutant MOD that does not bind to the α-chain of the IL-2R at all or that barely binds to the α-chain of the IL-2R.

[0078] As used herein, the term "in vivo" refers to any process or procedure that occurs within the body, for example, in an autoimmune patient.

[0079] As used herein, "in vitro" refers to any process or procedure that occurs outside the body, including procedures that may be referred to as ex vivo.

[0080] "Tandem," as used herein to describe the arrangement of MOD polypeptide sequences, means having two or more MODs arranged adjacent to each other on a polypeptide, separated by at most a linker (e.g., no intervening scaffold, masking polypeptide, or TGF-β sequence).

[0081] As used herein, the term "ectodomain" means the portion (domain) of a membrane protein that extends into the extracellular space and does not contain a sufficient portion of the transmembrane domain to anchor it within the cell membrane.

[0082] As used herein, "sequence identity" is a measure of the aa or nucleotide identity between two polynucleotide sequences or two polypeptide sequences. Describing a polynucleotide or polypeptide sequence as having a particular percentage of "sequence identity" with another polynucleotide or polypeptide means that the percentage of bases or amino acids are the same and in the same relative positions when comparing the two sequences. Sequence identity can be determined using a number of different methods. To measure sequence identity, sequences may be aligned using a variety of convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) available on the world wide web at sites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , and mafft.cbrc.jp / alignment / software / . See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. Unless otherwise stated, sequence identity is determined using alignments performed with the NCBI BLAST algorithm, version BLAST+ 2.9.0, released April 1, 2019 (BLASTP 2.9.0+ for proteins and BLASTN 2.9.0+ for nucleic acids).

[0083] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps that result in a construct having structural coding or non-coding sequences distinguishable from endogenous nucleic acids present in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides to provide synthetic nucleic acids that can be expressed from recombinant transcription units contained in a cell or a cell-free transcription and translation system. Recombinant, when used with respect to peptides, polynucleotides, or proteins, indicates that they have been prepared by expression from a recombinant nucleic acid.

[0084] As used herein, the term "about" when used in connection with a quantity indicates that the quantity may vary by 10% of the stated amount. For example, "about 100" means an amount of 90 to 110. When "about" is used in the context of a range, "about" when used with respect to the lower limit of a range means that the lower limit includes an amount 10% lower than the lower limit of the range, and "about" when used with respect to the upper limit of a range means that the upper limit includes an amount 10% higher than the upper limit of the range. For example, about 100 to about 1000 means that the range spans from 90 to 1100.

[0085] As used herein, the terms "treatment," "treating," and the like broadly refer to obtaining a desired pharmaceutical and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of a disease or condition in a mammal, including (a) preventing a disease or condition from occurring in a subject who may be predisposed to the disease or condition but has not yet been diagnosed with it; (b) inhibiting a disease or condition, i.e., arresting its progression; and / or (c) alleviating the disease, i.e., causing regression of the disease. Therapeutic agents (e.g., masked TGF-β constructs or complexes) can be administered before, during, or after the onset of disease or injury. Treatment of ongoing disease (wherein treatment stabilizes or suppresses undesirable clinical symptoms in the patient) is particularly advantageous. Such treatment is desirably performed before complete loss of function of the affected tissue. Therapeutic treatment is administered during, and in some cases after, the symptomatic stage of the disease.

[0086] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. Mammals include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cattle, rats, goats, sheep, horses, pigs, etc.), canines (e.g., dogs), felines (e.g., cats), etc. (e.g., humans, cattle, dogs, cats, rodents, murines, goats, simians, sheep, horses, lappines, pigs, etc.).

[0087] It should be noted that, as used herein and in the accompanying embodiments and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a Treg" includes a plurality of such Tregs, reference to a "TGF-β polypeptide" includes reference to one or more TGF-β polypeptides and equivalents thereof known to those skilled in the art, and so forth. Furthermore, it should be noted that the claims may be drafted to either include or exclude any optional element. That is, this is intended to serve as a basis for either the use (inclusion) of such elements in the claims (e.g., using terms such as "solely," "only," etc.) or their omission from the claims, or as a basis for a "negative" limitation excluding particular optional elements.

[0088] It will be understood that certain features of the invention, which are, for clarity, described in the context of individual aspects or embodiments, may also be provided in combination in a single aspect or embodiment, including the invention as set forth in the dependent claims. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments belonging to the invention are expressly embraced by the invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also expressly embraced by the invention and are disclosed herein as if all such subcombinations were individually and explicitly disclosed herein.

[0089] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0090] B. Description 1. Introduction The TGF-β superfamily encompasses endogenous proteins with growth-inhibitory functions. Increased expression of TGF-β or defects in cellular mechanisms that inhibit the action of TGF-β are correlated with the malignancies of many cancers, particularly due to the immunosuppressive effects of TGF-β. Dysregulation of the immunosuppressive function of TGF-β is also involved in autoimmune diseases. Because TGF-β is a key regulator of immune function, it has been the target of extensive research, and both TGF-β and its signaling pathways are considered therapeutic targets for the treatment of various diseases, including inflammatory processes and autoimmune disorders.

[0091] Effective preparation and delivery of TGF-β as a therapeutic agent is challenging due to the toxicity of the molecule and the complexity of the TGF-β receptor system. Large-scale production of TGF-β in mammalian cell expression systems is limited by the toxicity of the protein to many mammalian cells. Cells where TGF-β cytotoxicity is an issue include many used for the production of biomolecules, such as Chinese hamster ovary cells or "CHO" cells, which are among the most robust and commonly used cells for industrial protein production. The use of TGF-β as a therapeutic agent is also complicated by the high isoelectric point (pI) of TGF-β1, which has a pI of approximately 8.59 (as opposed to the pI of approximately 6.1 for TGF-β3), and its limited stability / solubility under non-acidic conditions (such acidic conditions are generally not suitable for therapeutic use). Additionally, the abundance of high-affinity TβRIII receptors present in mammalian systems (e.g., on the order of 5 nM for TGF-β2) represents a significant pharmacodynamic sink (on the order of 1-2 μM) compared to the affinity of TβRII for TGF-β, limiting the access of TGF-β-based biologics to target tissues. Similarly, the role of TβRIII in recruiting TGF-β to the TβRII / TβRI complex can result in significant off-target delivery of TGF-β, leading to unintended, unwanted, and even toxic effects. Such off-target delivery can lead to nonspecific activation and, particularly when TGF-β signaling is under feedforward control, to the further production of active TGF-β, thereby causing further unintended and undesirable effects. See, e.g., Jiang et al., Redox Biol. 2:267-272 (2014).

[0092] The effective use of TGF-β as a therapeutic agent is further complicated by the need to provide additional stimuli to cells to achieve the results of TGF-β stimulation. As noted above, cytokines such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, IL-23, PD-L1, 4-1BBL, and Fas Ligand (FAS-L) can profoundly affect the action of TGF-β. Thus, the ability to deliver additional stimuli in the form of TGF-β and cytokines can be advantageous in achieving specific therapeutic or cell-mediated (e.g., in vitro or in vivo) effects.

[0093] As discussed in more detail below, the present disclosure describes the use of TGF-β polypeptides that can interact with cells bearing TβRI and TβRII proteins and stimulate signal transduction. Advantageously, the TGF-β polypeptide is part of a masked TGF-β construct or complex, which is a fusion protein (single polypeptide chain) or fusion protein complex (two or more polypeptide chains) that also contains a polypeptide that binds to and masks the TGF-β polypeptide sequence (e.g., a TβRII sequence that functions as a masking sequence) constructed around a scaffold (e.g., one or two polypeptides, such as an immunoglobulin Fc polypeptide). An example of such a fusion protein or fusion protein complex is depicted in FIG. 1. When the TGF-β polypeptide of the fusion protein interacts with, for example, cellular TβRIII, the portion of the fusion protein that binds and masks TGF-β competes with TβRIII and prevents TGF-β from being sequestered in the TβRIII reservoir. Additionally, the affinity of a masking polypeptide for a TGF-β polypeptide, which can be intentionally varied by up to three orders of magnitude using aa substitutions, controls the overall potency of the masking polypeptide for its target receptor (e.g., TβRII), as shown in Figure 9. Because TGF-β polypeptides bind primarily to TβRIII as disulfide-linked dimers, interactions with the receptor that lead to sequestration can be attenuated by including aa substitutions (e.g., C77S) that limit dimerization or dimer stability, as discussed below. Interaction of a TGF-β fusion protein or fusion protein complex with TβRII displaces the masking sequence, forming a cell surface-bound TGF-β / TβRII complex. Subsequent recruitment of TβRII to form a heterodimeric TGF-β receptor provides a high-affinity complex (e.g., picomolar affinity) that tightly binds to TGF-β, even in the presence of the masking polypeptide sequence. Thus, masked TGF-β can still bind to the heterodimeric TβRI / TβRII receptor complex and signal through the canonical Smad protein pathway, the non-canonical Jun kinase pathway, and the p38 signaling pathway.In effect, the masking polypeptide delivers TGF-β into the cell and delivers it to cell surface TβRII molecules that subsequently recruit TβRI proteins to form a functional and active signaling complex that effectively holds the TGF-β polypeptide in place.

[0094] In addition to delivering TGF-β masked by a TGF-β binding protein (e.g., a fragment of the TGF-β receptor containing all or part of its TGF-β-binding ectodomain) in a form that avoids undesirable off-target interactions, the masked TGF-β construct or complex may also contain one or more polypeptides that function as immunomodulators ("MOD" polypeptides) that can influence the outcome of TGF-β action on target cells. The ability to deliver both TGF-β and an immunomodulator together not only allows the action of the TGF-β activation signal to be directed, but also reduces the amount of immunomodulator required to produce the same effect on target cells compared to administration of the immunomodulator alone. This is a result of the increased affinity (due to enhanced avidity) obtained by having two polypeptide sequences with affinity for a receptor on the same target cell. Increased avidity (resulting from an increase in the free energy of binding, ΔG, due to MOD interaction with their receptors) between masked TGF-β constructs or complexes bearing one or more MODs and target cells bearing receptors for both TGF-β polypeptides and one or more MODs provides improved selectivity in activating target cells bearing both types of receptors, provided that subsaturating amounts of the masked TGF-β construct or complex are present. As an example, a masked TGF-β construct, such as that of structure A in Figure 1 or Figure 7G, bearing IL-2 MOD is a more potent inducer of iTreg differentiation of naive CD4+ cells than an otherwise identical masked TGF-β construct lacking IL-2 MOD, even in the presence of an equivalent (equimolar) amount of IL-2 MOD polypeptide. Additionally, at sub-saturating doses and when similar numbers of cell types are present, constructs such as that of Structure A in Figure 1 with IL-2 MOD shown in Figure 7G selectively bind to cells with both TGF-β and IL-2 receptors compared to constructs lacking IL-2 MOD.

[0095] The effect of masked TGF-β constructs or complexes can be further characterized by incorporating modifications that alter the effect of individual polypeptide sequences. In some embodiments, aa substitutions that alter the dimerization ability of TGF-β (e.g., C77S substitution in TGF-β3) can be incorporated. Although the monomeric form of TGF-β exhibits little affinity for TβRIII, incorporating mutations that limit TGF-β's ability to dimerize limits off-target binding to TβRIII, which can draw the complex into the TGF-β "reservoir," limiting its ability to stimulate target cells.

[0096] Modifications (eg, substitutions, deletions, insertions, etc.) can also be made to polypeptide sequences other than the TGF-β polypeptide sequence, including masking polypeptide sequences and immunomodulatory polypeptide sequences.

[0097] Modifications to polypeptides that bind and mask TGF-β can alter the availability of TGF-β polypeptide sequences by changing the affinity of the masking polypeptide for TGF-β and the respiratory rates (on and off rates) of the masking and TGF-β polypeptides. Two different TGF-β polypeptide / masking polypeptide complexes may have the same binding association constant (ratio of k to k), but a complex with a higher k and k may be effectively more available for cell surface TβRII binding, depending on the rate constants. Figure 9 provides a list of several aa substitutions in TβRII that alter affinity for TGF-β1 and TGF-β3, along with a table showing the affinity of otherwise identical TβRII-masked TGF-β constructs with and without various substitutions, each of which differentially affects TβRII binding to TGF-β3.

[0098] Although various polypeptides can be used to mask TGF-β, including single-chain antibody sequences (e.g., humanized single-chain antibody sequences), minimal TGF-β receptor sequences that bind to TGF-β (e.g., the ectodomains of TβRI, TβRII, or TβRIII) may be employed. The ectodomain of TβRIII may be used as a masking polypeptide for dimeric TGF-β, but its high affinity for TGF-β may antagonize the binding of TGF-β polypeptide sequences to TβRII. Nevertheless, the TβRIII masking sequence can be effectively displaced by cell surface TβRI / TβRII complexes, which have a higher affinity for TGF-β polypeptides, thereby enabling activation of their cell surface receptors.

[0099] The ectodomain of TβRII can be used as a masking polypeptide. Deletion of the N-terminal amino acids of TβRII (e.g., Δ14 or Δ25), with or without the D118A substitution, can produce a protein (or polypeptide) suitable for masking TGF-β with a calculated pI of about 4.5 to about 5.0 (e.g., about 4.7 to 4.85). Combination of a TGF-β1 polypeptide (having a high pI) with a TβRII masking polypeptide (e.g., including one with an N-terminal deletion) can potentially neutralize the charge of the TGF-β1 polypeptide. The calculated pI of TβRII(Δ25, D118A) / TGF-β1 is approximately 6.23, while TGF-β1 has a pI of about 8.59. In contrast, complexes of Δ14 or Δ25 TβRII and TGF-β3 (with or without the D118A and / or C77S substitutions) are calculated to have pI values ​​of about 4.9 to about 5.3 (pIs of about 5.06 to 5.17). Additionally, combining the TβRII ectodomain with an active TGF-β polypeptide results in a complex capable of interacting with TβRI on the cell surface, thereby affecting TGF-β signaling (e.g., acting as a TGF-β agonist, partial agonist, antagonist, or partial antagonist), whereas incorporation of aa substitutions that limit interaction with TβRI limits or blocks the ability of the masked complex to participate in active signaling. Thus, as discussed below, incorporating TβRII ectodomain sequences with N-terminal deletions (e.g., deletions of 14-25 aa, Δ14-Δ25) or substitutions (e.g., substitutions at D118 with aa other than aspartic acid, such as D118A, D118R) that reduce or eliminate binding to TβRI can be used to mask TGF-β and prevent stimulation of cells by TβRII ectodomain-masked TGF-β polypeptides in which the TβRII N-terminus is intact.Consequently, masked TGF-β complexes, including those in which TGF-β is masked by N-terminal deletion mutants of TβRII, can act by unmasking TGF-β (dissociating from the masked peptide or opening the folded molecule) and binding to TβRII and TβRI on target cells to form an active heterodimeric TβRI / TβRII signaling complex.

[0100] Masking TGF-β allows its expression at high levels in mammalian cells (e.g., CHO cells) without reducing cell viability. This is particularly true when the masked TGF-β polypeptide is blocked from binding to TβRI by N-terminal α deletions, substitutions, and / or other mutations. Blocking TβRIII interactions (e.g., by blocking dimerization) can further reduce problems associated with cellular expression.

[0101] C. Masked TGF-β constructs and masked TGF-β complexes The present disclosure describes the preparation of masked TGF-β constructs (e.g., having a single polypeptide chain, see Structure A in Figure 1 ) and masked TGF-β complexes (e.g., having a complex of two polypeptide chains, see Structures B-F in Figure 1 ). Masked TGF-β constructs and masked TGF-β complexes include, as their components, at least one TGF-β polypeptide sequence, at least one polypeptide that binds to and masks the TGF-β polypeptide(s), and optionally, one or more (e.g., one, two, or three) MODs, all of which are assembled on a scaffold structure. Masked TGF-β constructs and masked TGF-β complexes include portions of membrane-bound proteins (e.g., TGF-β receptors), but, unless otherwise specified, do not include portions of membrane-anchoring domains (e.g., transmembrane domains sufficient to anchor the majority of the expressed protein within a cell membrane (e.g., CHO cells in which they are expressed)).

[0102] Non-limiting examples of TGF-β constructs and complexes, including those in the form shown in FIG. 1, are described below.

[0103] Components of masked TGF-β constructs and complexes, including MODs, scaffolds, linkers, TGF-β polypeptides, and TGF-β masking polypeptides (e.g., single-chain antibodies or TGF-β receptor ectodomains), are each described in the sections below.

[0104] D. Immunomodulatory Polypeptide Sequences ("MODs") 1. Masked TGF-β Constructs and Incorporation of MOD into Complexes As discussed above, although a MOD is not required for delivery of a masked TGF-β polypeptide or its ability to activate cells through the TβRI and TβRII heterodimeric receptor, a MOD can substantially affect the outcome of TGF-β receptor activation. Thus, it may be desirable to incorporate a wild-type (wt) or mutant MOD (e.g., exhibiting reduced affinity, increased affinity, or selectivity for a particular receptor, also referred to as a "co-MOD," "co-immunomodulatory polypeptide," or "cognate costimulatory receptor" or subtypes thereof). While TGF-β is also an immunomodulatory polypeptide, because it is a central element in the masked TGF-β constructs and complexes described herein, the term "MOD(s)" as used herein does not include TGF-β or its polypeptides.

[0105] MODs suitable for inclusion in any of the masked TGF-β constructs and complexes (e.g., homodimeric or heterodimeric complexes) include, but are not limited to, PD-L1, FAS-L, 4-1BBL, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, and IL-23. In some examples, the MOD is independently selected from mature PD-L1, FAS-L, 4-1BBL, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, and IL-23 polypeptides, or fragments of any thereof. The MOD polypeptide sequence(s) incorporated into the masked TGF-β constructs and complexes can include only the secreted MOD polypeptide portion, or, if membrane-anchored, the extracellular portion of the full-length mature MOD protein. Thus, for example, the MOD polypeptide sequence within a masked TGF-β construct or complex may, in some instances, exclude one or more (e.g., each) of the signal peptide, transmembrane domain, and / or intracellular domain typically found within some native MODs.

[0106] In some examples, a MOD polypeptide sequence suitable for inclusion in the masked TGF-β constructs and complexes of the present disclosure comprises all or a portion (e.g., the extracellular portion) of the amino acid sequence of a naturally occurring MOD. In other examples, a MOD suitable for inclusion in the masked TGF-β constructs and complexes of the present disclosure includes at least one (e.g., one, two, or three independently selected) mutant MOD that comprises at least one amino acid insertion, substitution, and / or deletion compared to the amino acid sequence of a naturally occurring MOD.

[0107] In some instances, a mutant MOD exhibits a lower binding affinity for the co-MOD than the affinity of a corresponding native MOD (e.g., a MOD that does not contain the amino acid substitution(s) present in the mutant) for the co-MOD. In response to the reduced affinity, the use of a MOD with reduced affinity for its co-MOD allows the TGF-β polypeptide to have more influence on or dominate the binding interaction. When the binding affinity of a TGF-β polypeptide is higher than that of the MOD, it can drive the masked TGF-β construct or complex to associate with cells that have a TGF-β receptor system (e.g., TGF-βR1 and TGF-βR2, which form a high-affinity TGF-β-binding heterodimer), while restricting target binding to cells that lack or have few TGF-β receptors, even in the presence of a given amount of the co-MOD. Essentially, if the affinity of a TGF-β polypeptide for its cellular receptor (e.g., the ΔG of binding) is greater than the affinity of the MOD for their co-MOD (e.g., the ΔG of their binding), TGF-β drives the binding and specificity of the masked TGF-β construct or complex.

[0108] In one embodiment, any one or more MODs associated with the masked TGF-β constructs and complexes are independently selected from the group consisting of wt. or mutant PD-L1, FAS-L, 4-1BBL, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, IL-23, and combinations thereof.

[0109] In some instances, such as when it is desirable to stimulate the production of Treg cells, at least one MOD polypeptide (e.g., one, two, or three independently selected MODs) present in the masked TGF-β constructs and complexes is an IL-2 polypeptide or an IL-2 variant polypeptide. Sequence variations in IL-2 can be selected to bias the binding of the IL-2 polypeptide and masked TGF-β constructs or complexes to target cells with different combinations of IL-2R receptor subunits. The IL-2 receptor consists of a common IL-2Rγ and two additional IL-2Rα and / or IL-2Rβ subunits, forming a trimer (IL-2Rα). 2- IL-2Rγ, (IL-2Rβ) 2- They form the IL-2Rγ, or high-affinity (Kd of approximately 10 picomolar) IL-2Rα-IL-2Rβ-IL-2Rγ receptor. While the α chain (CD25) is unique to IL-2, the β chain (CD122) is shared with the IL-15 receptor, and the γ chain (CD132), which is important for signal transduction, can partner with other cytokine receptor chains. Substitutions at H16 (e.g., H16A or H16T) or F42 (e.g., F42A or F42T) can bias binding in favor of receptors bearing the IL-2Rβ subunit, and thus their incorporation can favor the β-γ receptor (IL-2Rβ). 2-Substitution at N88 (e.g., N88R) biases binding to memory T cells and NK cells that exhibit high-affinity α-β-γ (IL-2Rα-IL-2Rβ-IL-2Rγ) receptors, or activated T cells and Tregs that exhibit high-affinity α-β-γ (IL-2Rα-IL-2Rβ-IL-2Rγ) receptors. In contrast, substitution at N88 (e.g., N88R) can reduce binding to IL-2Rβ and bias binding in favor of receptors with IL-2Rα subunits; thus, substitution at N88 biases binding to cells with α-γ ((IL-2Rα)2-IL-2Rγ)) and α-β-γ (IL-2Rα-IL-2Rβ-IL-2Rγ) receptors while avoiding activation of cells with β-γ receptors. See, e.g., Skrombolas and Frelinger Expert Rev Clin Immunol., 10(2):207-217 (2014). Biasing, when used in the context of binding a substituted MOD (such as an IL-2 polypeptide having an aa substitution) to its co-MOD or cells exhibiting the co-MOD, means that the presence of the substitution alters the extent of interaction of the substituted MOD with the same co-MOD compared to the interaction between the wt. MOD and the co-MOD. For example, an IL-2 sequence having a substitution at N88 (e.g., N88R), which has a lower side effect profile (e.g., is safer) and is better tolerated by human subjects, can be included along with the above-mentioned substitutions at H16 and / or F42.

[0110] In some instances, such as when it is desirable to stimulate the production of iTreg cells (CD4+FoxP3+ cells) (e.g., when it is desirable to actively suppress effector T cells and / or induce peripheral tolerance to inhibit immune-mediated tissue damage), at least one MOD polypeptide (e.g., one, two, or three independently selected MODs) present in the masked TGF-β construct or complex is an independently selected wt or mutant PD-L1 MOD polypeptide. See, e.g., Francisco et al., J. Exp. Med., 206(13):3015-3029 (2009). In addition to a wt or mutant PD-L1 sequence, the masked TGF-β construct or complex can include one or more independently selected wt or mutant IL-2 polypeptides. Sequence variations in IL-2 can be selected to bias the binding of the IL-2 polypeptide and the masked TGF-β construct to target cells bearing different combinations of IL-2R receptor subunits. As discussed above, IL-2 variants include substitutions at H16 (e.g., H16A or H16T) and / or F42 (e.g., F42A or F42T), which can bias binding in favor of receptors having the IL-2Rβ subunit, and / or at N88 (e.g., N88R), which reduces binding to IL-2Rβ and is better tolerated in human subjects. In one embodiment, a masked TGF-β construct / -β complex includes both H16T and F42A, or both H16A and F42A, substitutions, either pair of which may be combined with an N88 (e.g., N88R) substitution.

[0111] In some instances, such as when it is desirable to stimulate the production of Th17 cells, at least one MOD (e.g., one, two, or three independently selected MODs) present in the masked TGF-β constructs and complexes is an IL-6 polypeptide or an IL-6 variant polypeptide. In some instances, such as when it is desirable to stimulate the production of Th9 cells, at least one MOD (e.g., one, two, or three independently selected MODs) present in the masked TGF-β constructs and complexes is an IL-4 polypeptide or an IL-4 variant polypeptide. See, e.g., Elyaman et al., Immunity, 36(4):623-634, Immunity. (2012). In some instances, such as when it is desirable to promote IL-7-dependent survival of low-affinity T cells by regulating thymocyte IL-7Ra expression, at least one MOD (e.g., one, two, or three independently selected MODs) present in the masked TGF-β constructs and complexes is an IL-7 polypeptide or an IL-7 variant polypeptide. In some instances, such as when it is desirable to stimulate the production of T follicular helper (Tfh) cells, at least one MOD (e.g., one, two, or three independently selected MODs) present in the masked TGF-β constructs and complexes is an IL-21 or IL-23 polypeptide, or a variant of an IL-21 or IL-23 polypeptide. In some instances, such as when it is desirable to induce tolerance, at least one MOD (e.g., one, two, or three independently selected MODs) present in the masked TGF-β constructs and complexes is a variant of a Fas Ligand (FasL) polypeptide.

[0112] In some instances, such as when it is desirable to inhibit type II innate lymphoid cells (ILC2 cells) (e.g., to suppress asthma and allergic inflammation), at least one MOD (e.g., one, two, or three independently selected MODs) present in the masked TGF-β constructs and complexes is an IL-10 polypeptide or a variant of an IL-10 polypeptide. See, e.g., Rajas et al., J Allergy Clin Immunol, 139(5):1468 (2017) and Ogasawara, et al., J Allergy Clin Immunol, 141(3):1147-1151 (2018). Inhibition of ILC2 cells can be assessed by reduced production of the type 2 cytokines IL-5 and IL-13 in vivo (in tissues or body fluids) or in vitro (in culture medium). The IL-10 polypeptide can be a monomeric isomer, such as the IL-10M1 molecule described by Josephson et al., J. Biol. Chem. 275:13552-13557 (2000), or a variant thereof, both of which are discussed below. In contrast to wt. IL-10, which is an intertwined pair of IL-10 peptides whose biologically active form forms a complex consisting of two IL-10 molecules and four IL-10Ra receptor chains, IL-10M1 forms a 1:1 complex with soluble IL-10Ra with a dissociation constant of 30 nM that is biologically active in cell proliferation assays. (Id.)

[0113] 2. Low-affinity mods and variant mods Suitable MODs that exhibit reduced affinity for co-MOD may have between one amino acid (aa) and 20 aa differences from the wild-type (wt) MOD sequence. For example, in some instances, a mutant MOD polypeptide sequence present in a masked TGF-β construct or complex may differ in amino acid sequence by 1 aa, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa from the corresponding wt. MOD sequence. As another example, in some instances, a mutant MOD polypeptide present in a masked TGF-β construct or complex may differ in amino acid sequence by 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, or 20 aa from the corresponding wt. MOD sequence. By way of example, in some cases, a mutant MOD polypeptide present in a masked TGF-β construct or complex comprises a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aa substitution compared to a corresponding reference (e.g., wt.) MOD sequence. In some cases, a mutant MOD polypeptide present in a masked TGF-β construct or complex comprises a single aa substitution compared to a corresponding reference (e.g., wt.) MOD sequence. In some cases, a mutant MOD polypeptide present in a masked TGF-β construct or complex comprises a 2 aa substitution (e.g., a substitution of 2 aa or less) compared to a corresponding reference (e.g., wt.) MOD sequence. In some cases, a mutant MOD polypeptide present in a masked TGF-β construct or complex comprises a 3 aa substitution (e.g., a substitution of 3 aa or less) compared to a corresponding reference (e.g., wt.) MOD sequence. In some cases, a mutant MOD polypeptide present in a masked TGF-β construct or complex comprises a 4 aa or 5 aa substitution compared to a corresponding reference (e.g., wt.) MOD sequence. In some examples, the mutant MOD present in the masked TGF-β construct or complex comprises a 6 aa or 7 aa substitution compared to the corresponding reference (e.g., wt.) MOD sequence, hi some examples, the mutant MOD present in the masked TGF-β construct or complex comprises an 8 aa or 9 aa substitution compared to the corresponding reference (e.g., wt.) MOD sequence.In some examples, the mutant MOD present in the masked TGF-β construct or complex comprises a 10 aa or 11 aa substitution (e.g., a substitution of 10 aa or less) compared to the corresponding reference (e.g., wt.) MOD sequence.

[0114] In some examples, a mutant MOD present in a masked TGF-β construct or complex comprises an 11 aa or 12 aa substitution compared to a corresponding reference (e.g., wt.) MOD sequence. In some examples, a mutant MOD present in a masked TGF-β construct or complex comprises a 13 aa or 14 aa substitution compared to a corresponding reference (e.g., wt.) MOD sequence. In some examples, a mutant MOD present in a masked TGF-β construct or complex comprises a 15 aa or 16 aa substitution compared to a corresponding reference (e.g., wt.) MOD sequence. In some examples, a mutant MOD present in a masked TGF-β construct or complex comprises a 17 aa or 18 aa substitution compared to a corresponding reference (e.g., wt.) MOD sequence. In some examples, a mutant MOD present in a masked TGF-β construct or complex comprises a 19 aa or 20 aa substitution compared to a corresponding reference (e.g., wt.) MOD sequence.

[0115] As discussed above, mutant MODs suitable for inclusion in masked TGF-β constructs or complexes may exhibit lower affinity for their cognate co-MOD compared to the affinity of the corresponding wt. MOD for the cognate co-MOD.

[0116] In some examples, the mutant MOD present in the masked TGF-β construct or complex has a binding affinity for the cognate co-MOD of 1 nM to 100 μM. For example, in some examples, the mutant MOD present in the masked TGF-β construct or complex has a binding affinity for the cognate co-MOD of about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 50 nM, about 50 nM to about 100 nM, about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, It has a binding affinity of about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0117] 3. Determining Binding Affinity The binding affinity between a MOD (e.g., a MOD polypeptide) and its cognate co-MOD can be determined by biolayer interferometry (BLI) using purified MOD and purified cognate co-MOD. The binding affinity between a masked TGF-β construct or complex containing a MOD and its cognate co-MOD can also be measured by BLI using purified masked TGF-β constructs or complexes and its cognate co-MOD. BLI methods are well known to those skilled in the art. See, for example, Lad et al. (2015) J. Biomol. Screen. 20(4):498-507 and Shah and Duncan (2014) J. Vis. Exp. 18:e51383. The specific and relative binding affinities described in this disclosure between a MOD and its cognate co-MOD, or between a masked TGF-β construct or complex with a MOD and its cognate co-MOD, can be determined using the following procedure.

[0118] To determine the binding affinity between a masked TGF-β construct or complex containing MOD and its cognate co-MOD, a BLI assay can be performed using an Octet RED96 (Pal ForteBio) instrument or a similar instrument, as follows: A control masked TGF-β construct or complex (e.g., a masked TGF-β construct or complex containing wt. MOD) is immobilized on an insoluble support ("biosensor"). The immobilized masked TGF-β construct or complex is the "target." Immobilization can be performed by immobilizing a capture antibody on the insoluble support, which immobilizes the masked TGF-β construct or complex. For example, if the masked TGF-β construct or complex contains an IgFc scaffold, immobilization can be performed by immobilizing an anti-IgFc (e.g., anti-human IgGFc) antibody on an insoluble support, where the immobilized anti-IgFc antibody binds to and immobilizes the masked TGF-β construct or complex. Co-MODs are applied to the immobilized masked TGF-β construct or complex at several different concentrations, and the device response is recorded. The assay is performed in a liquid medium containing 25 mM HEPES (pH 6.8), 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% Tween 20 non-ionic surfactant. Binding of the co-MOD to the immobilized masked TGF-β construct or complex is performed at 30°C.

[0119] Antibodies (e.g., monoclonal antibodies) can be used as positive controls for binding and binding affinity. Antibodies can be selected based on the specific structure of the masked TGF-β construct or complex (see, e.g., Figure 1). For example, monoclonal antibodies (mAbs) against TGF-β, TGF-β receptor, scaffold, or MOD polypeptide sequences can be used as positive controls, as long as the antibodies do not dissociate the masked TGF-β construct or complex from the support (biosensor). A standard curve can be generated using serial dilutions of anti-MHC class I or class II monoclonal antibodies. The co-MOD or anti-MHC mAb is the "analyte." BLI analyzes the interference pattern of white light reflected from two surfaces: i) the immobilized polypeptide ("target"), and ii) an internal reference layer. Changes in the number of molecules ("analytes"; e.g., co-MOD; anti-HLA antibodies) bound to the biosensor tip cause a shift in the interference pattern, and this shift in the interference pattern can be measured in real time. Two kinetic terms that describe the affinity of a target / analyte interaction are the association constant (k a ) and dissociation constant (k d ) These two terms (k d / a ) is the affinity constant K D This results in:

[0120] As described above, the binding affinity between a MOD (e.g., IL-2 or IL-2 variant) and its cognate co-MOD (e.g., IL-2R) can also be determined by BLI. This assay is similar to that described above for a masked TGF-β construct or complex. A BLI assay can be performed using an Octet RED 96 (Pal ForteBio) instrument, or a similar instrument, as follows: A MOD (e.g., a variant IL-2 polypeptide of the present disclosure) that is a component of a masked TGF-β construct or complex, and a control MOD (the control MOD comprises a wt. MOD, e.g., wt. IL-2) are separately immobilized on an insoluble support (a "biosensor"). Each MOD is a "target." Immobilization can be performed by immobilizing a capture antibody on the insoluble support, where the capture antibody immobilizes the MOD. For example, if the target is fused to an immunoaffinity tag (e.g., FLAG, human IgG Fc, etc.), immobilization can be performed by immobilizing an appropriate antibody to the immunoaffinity tag on an insoluble support; the immobilized antibody binds to and immobilizes the MOD (e.g., if the MOD contains an IgFc polypeptide, an anti-human IgG Fc can be immobilized). The co-MOD is applied to the immobilized MOD at several different concentrations, and the device response is recorded. Alternatively, the co-MOD (e.g., for the IL-2 receptor heterotrimer as a monomeric subunit plus heterodimeric subcomplex, or as a complete heterotrimer) is immobilized on a biosensor, and the MOD is applied to the immobilized co-MOD(s) at several different concentrations, and the device response is recorded. The assay is performed in a liquid medium containing 25 mM HEPES (pH 6.8), 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% Tween 20 non-ionic surfactant. Binding of the co-MOD to the immobilized MOD is carried out at 30° C. BLI analyzes the interference pattern of white light reflected from two surfaces: i) the immobilized polypeptide (the "target") and ii) an internal reference layer.Changes in the number of molecules ("analytes"; e.g., co-MODs) bound to the biosensor tip cause a shift in the interference pattern, and this shift in the interference pattern can be measured in real time. Two kinetic terms that describe the affinity of the target / analyte interaction are the association constant (k). a ) and dissociation constant (k d ) The ratio of these two terms (k d / a ) is the affinity constant K D Thus, determining both the binding affinity of a wt. MOD (e.g., IL-2) to its co-MOD (e.g., its cognate binding partner or receptor, IL-2R in the case of IL-2), as well as the binding affinity of a mutant MOD (e.g., an IL-2 variant disclosed herein) to its co-MOD (e.g., IL-2R in the case of an IL-2 variant), allows one to determine the relative binding affinity of the mutant co-MOD for the MOD, as compared to the wt. co-MOD. That is, one can determine whether the binding affinity of the mutant MOD to its co-MOD is reduced compared to the binding affinity of the wt. MOD to the same cognate co-MOD, and if so, what the percentage reduction from the binding affinity of the wt. co-MOD is.

[0121] BLI assays are performed in multiwell plates. To run the assay, the plate layout is defined, the assay steps are defined, and the biosensors are assigned in the Octet Data Acquisition software. The biosensor assembly is hydrated. The hydrated biosensor assembly and assay plate are equilibrated on the Octet instrument for 10 minutes. Once the data is acquired, it is loaded into the Octet Data Analysis software. The data is processed in the Processing window, specifying the methods for reference subtraction, y-axis alignment, interstep correction, and Savitzky-Golay filtering. The data is analyzed in the Analysis window by specifying the analysis steps (association and dissociation) and selecting the curve fit model (1:1), fitting method (global), and window of interest (in seconds). The quality of the fit is assessed. The K for each data trace (analyte concentration) is calculated. D Values ​​can be averaged within a factor of three. D The error value must be within one order of magnitude of the affinity constant value, R 2 The value should be greater than 0.95. See, e.g., Abdiche et al. (2008) J. Anal. Biochem. 377:209.

[0122] In some examples, the ratio of i) the binding affinity of a control masked TGF-β construct or complex (where the control masked TGF-β construct or complex comprises wt. MOD) to its cognate co-MOD to ii) the binding affinity of a masked TGF-β construct or complex comprising a mutant form of wt. MOD to its cognate co-MOD, as measured by BLI (as described above), is at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least 500:1, at least 10 2 :1, at least 5 × 10 2 :1, at least 10 3 :1, at least 5 × 103 :1, at least 10 4 :1, at least 10 5 :1, or at least 10 6 In some instances, the ratio of i) the binding affinity of a control masked TGF-β construct or complex (where the control masked TGF-β construct or complex includes wt. MOD) to its cognate co-MOD to ii) the binding affinity of a masked TGF-β construct or complex including a mutant form of wt. MOD to its cognate co-MOD is 1.5:1 to 10, as measured by BLI. 6 :1, for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 :1, 10 2 :1~10 3 :1, 10 3 :1~10 4 :1, 10 4 :1~10 5 :1, or 10 5 :1~10 6 :1 range.

[0123] In some instances, the mutant MOD present in the masked TGF-β construct or complex has a binding affinity for its cognate co-MOD of 1 nM to 100 nM or 100 nM to 100 μM. For example, in some instances, the mutant MOD present in the masked TGF-β construct or complex has a binding affinity for its cognate co-MOD of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM. In some examples, the mutant MOD present in the masked TGF-β construct or complex has a binding affinity for its cognate co-MOD of between 1 nM and 5 nM, between 5 nM and 10 nM, between 10 nM and 50 nM, or between 50 nM and 100 nM.

[0124] 4.PD-L1 and its mutations As one non-limiting example, the MOD or mutant MOD present in the masked TGF-β construct or complex is a PD-L1 or mutant PD-L1 polypeptide. Wild-type PD-L1 binds to PD1.

[0125] A wt. human PD-L1 polypeptide may comprise the following aa sequence: MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKICLT LSPST (SEQ ID NO: 1), in which aas 1-18 form the signal sequence, aas 19-127 form the Ig-like V-type or IgV domain, and aas 133-225 form the Ig-like C2-type domain.

[0126] The wt. human PD-L1 ectodomain may comprise the following aa sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO: 2), in which aas 1-109 form an Ig-like V-type or IgV domain, and aas 115-207 form an Ig-like C2-type domain.

[0127] A wt. PD-L1 IgV domain suitable for use as a MOD may include aas 18-127 or 19-127 of SEQ ID NO: 1, and a carboxyl-terminal stabilizing sequence, for example, the last 7 amino acids of the sequence (shown in bold and italics): A FTVTVPKDLY VVEYGSNMTI ECKFPVEKQL DLAALIVYWE MEDKNIIQFV HGEEDLKTQH SSYRQRARLL KDQLSLGNAA LQITDVKLQD AGVYRCMISY GGADYKRITV KVNAPYAAAL HEH (SEQ ID NO: 138). If the carboxyl stabilizing sequence includes a histidine at about aa 122 (e.g., the histidine about five residues C-terminal to Tyr (Y), appearing as aa 117 in SEQ ID NO: 138), the histidine can form a stabilizing electrostatic bond with the backbone amide at aas 82 and 83 (shown in bold and italics in SEQ ID NO: 138 (Q107 and L106 in SEQ ID NO: 1)). Alternatively, a stabilizing disulfide bond can be formed by substituting one of aas 82 or 83 (Q107 and L106 in SEQ ID NO: 1) and one of aa residues 121, 122, or 123 (equivalent to aa positions 139-141 in SEQ ID NO: 1).

[0128] The wt. PD-1 polypeptide can comprise the following aa sequence: PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL (SEQ ID NO: 3).

[0129] In some examples, a mutant PD-L1 polypeptide (e.g., a mutant version of SEQ ID NO: 2 or the IgV domain of PD-1) exhibits reduced binding affinity to PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) compared to the binding affinity of a PD-L1 polypeptide comprising the aa sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. For example, in some examples, a mutant PD-L1 polypeptide binds to PD-1 (e.g., a PD-1 polypeptide comprising the aa sequence set forth in SEQ ID NO: 3) with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of a PD-L1 polypeptide comprising the aa sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0130] In some examples, the mutant PD-L1 polypeptide (e.g., SEQ ID NO: 2 or an IgV domain variant thereof) has a binding affinity to PD-1 (e.g., of SEQ ID NO: 3) that is between 1 nM and 1 mM (e.g., between 1 nM and 10 nM, between 10 nM and 100 nM, between 100 nM and 1 μM, between 1 μM and 10 μM, between 10 μM and 100 μM, or between 100 μM and 1 mM). For another example, in some cases, a mutant PD-L1 polypeptide (e.g., a mutant of SEQ ID NO: 2) may have a potency of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, or about 900 nM to about 1000 nM. nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0131] The PD-L1 ectodomain sequence used as a MOD may have several aa substitutions, including substitutions with sequences having greater than 90% (95%, 98%, or 99%) sequence identity to at least 85 contiguous aas (e.g., at least 90, at least 95, at least 100, or at least 105 contiguous aas) of any one of SEQ ID NO: 1, SEQ ID NO: 2, aas 19-127 of SEQ ID NO: 1 (IgV domain), and SEQ ID NO: 138. The substitutions may include (a) disulfide bond substitutions D103C and G33C, or V104 and S34C, (b) salt-bridge forming substitutions Q107D and K62R, or Q107D and S80R, and / or (c) Pi-stacking substitutions M36Y or M36F (based on SEQ ID NO: 1). A PD-L1 MOD sequence may comprise a sequence having at least 85 contiguous aas (e.g., at least 90, at least 95, at least 100, or at least 105 contiguous aas) of SEQ ID NO: 2, and at least one disulfide, salt bridge, or Pi-stacking substitution. A PD-L1 MOD sequence may comprise a sequence having at least 85 contiguous aas (e.g., at least 90, at least 95, at least 100, or at least 105 contiguous aas) of aas 19-127 (IgV domain) of SEQ ID NO: 1, and at least one disulfide, salt bridge, or Pi-stacking substitution. A PD-L1 MOD sequence may comprise a sequence having at least 85 contiguous aas (e.g., at least 90, at least 95, at least 100, or at least 105 contiguous aas) of aas SEQ ID NO: 138, and at least one disulfide, salt bridge, or Pi-stacking substitution.

[0132] In some examples, the mutant PD-L1 polypeptide has a single aa substitution compared to the PD-L1 aa sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1. In some examples, the mutant PD-L1 polypeptide has a 2-10 aa substitution compared to the PD-L1 aa sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1. In some examples, the mutant PD-L1 polypeptide has a 2-aa substitution compared to the PD-L1 aa sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1. In some examples, the mutant PD-L1 polypeptide has a 3-aa or 4-aa substitution compared to the PD-L1 aa sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1. In some examples, the mutant PD-L1 polypeptide has a 5-aa or 6-aa substitution compared to the PD-L1 aa sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1. In some examples, the mutant PD-L1 polypeptide has a 7 aa or 8 aa substitution compared to the PD-L1 aa sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1. In some examples, the mutant PD-L1 polypeptide has a 9 aa or 10 aa substitution compared to the PD-L1 aa sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1.

[0133] Suitable mutant PD-L1 polypeptide sequences include polypeptide sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 170 contiguous aa (e.g., at least 180, 190, or 200 contiguous aa) of SEQ ID NO: 2 (e.g., with at least one aa insertion, deletion, or substitution). Suitable mutant PD-L1 IgV polypeptide sequences include polypeptide sequences that have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 70 contiguous aa (e.g., at least 80, 90, 100, or 105 contiguous aa) of aa 1-109 of SEQ ID NO: 2 (e.g., with at least one aa insertion, deletion, or substitution).

[0134] In some examples, mutant PD-L1 polypeptide sequences include polypeptide sequences that have at least 90% (e.g., at least 95%, 98%, or 99%) or 100% aa sequence identity to SEQ ID NO:2, and wherein the residue at position 8 is an aa other than D, and in one such example, the residue is A and in another example, R. Mutant PD-L1 polypeptide sequences include polypeptide sequences that have at least 90% (e.g., at least 95%, 98%, or 99%) or 100% aa sequence identity to SEQ ID NO:2, and wherein the residue at position 36 is an aa other than I, and in one such example, the residue is A and in another example, D. Mutant PD-L1 polypeptide sequences also include polypeptide sequences that have at least 90% (e.g., at least 95%, 98%, or 99%) or 100% aa sequence identity to SEQ ID NO:2, and wherein the residue at position 54 is an aa other than E, and in one such example, the residue is A and in another example, R.

[0135] 5. IL-1 and its variants As one non-limiting example, the MOD or mutant MOD present in the masked TGF-β construct or complex is an IL-1 or mutant IL-1 polypeptide. Wild-type IL-1 has two isoforms, IL-1α and IL-1β, both of which bind to the IL-1 receptor.

[0136] The wt. human IL-1α precursor polypeptide may comprise the following aa sequence: MAKVPDMFED LKNCYSENEE DSSSIDHLSL NQKSFYHVSY GPLHEGCMDQ SVSLSISETS KTSKLTFKES MVVVATNGKV LKKRRLSLSQ SITDDDLEAI ANDSEEEIIK PRSAPFSFLS NVKYNFMRII KYEFILNDAL NQSIIRANDQ YLTAAALHNL DEAVKFDMGA YKSSKDDAKI TVILRISKTQ LYVTAQDEDQ PVLLKEMPEI PKTITGSETN LLFFWETHGT KNYFTSVAHP NLFIATKQDY WVCLAGGPPS ITDFQILENQ A (SEQ ID NO: 4) UniProtKB-P01583, NCBI reference sequence NP_000566.3, which may have one or more of the following naturally occurring variations: R85Q, A114S, N125D, D138N, and D176H.

[0137] The mature wt. human IL-1α polypeptide can comprise the following aa sequence: PRSAPFSFLS NVKYNFMRII KYEFILNDAL NQSIIRANDQ YLTAAALHNL DEAVKFDMGA YKSSKDDAKI TVILRISKTQ LYVTAQDEDQ PVLLKEMPEI PKTITGSETN LLFFWETHGT KNYFTSVAHP NLFIATKQDY WVCLAGGPPS ITDFQILENQ A (SEQ ID NO: 5).

[0138] The wt. human IL-1β precursor polypeptide can comprise the following aa sequence: MAEVPELASE MMAYYSGNED DLFFEADGPK QMKCSFQDLD LCPLDGGIQL RISDHHYSKG FRQAASVVVA MDKLRKMLVP CPQTFQENDL STFFPFIFEE EPIFFDTWDN EAYVHDAPVR SLNCTLRDSQ QKSLVMSGPY ELKALHLQGQ DMEQQVVFSM SFVQGEESND KIPVALGLKE KNLYLSCVLK DDKPTLQLES VDPKNYPKKK MEKRFVFNKI EINNKLEFES AQFPNWYIST SQAENMPVFL GGTKGGQDIT DFTMQFVSS (SEQ ID NO: 6) UniProtKB-P0158, NCBI Reference Sequence NP_000567.1.

[0139] A mature wt. human IL-1β polypeptide can comprise the following aa sequence: APVRSLNCTL RDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS (SEQ ID NO: 7).

[0140] Both IL-1α and IL-1β bind to the IL-1 receptor, which has the following sequence:MKVLLRLICF IALLISSLEA DKCKEREEKI ILVSSANEID VRPCPLNPNE HKGTITWYKD DSKTPVSTEQ ASRIHQHKEK LWFVPAKVED SGHYYCVVRN SSYCLRIKIS AKFVENEPNL CYNAQAIFKQ KLPVAGDGGL VCPYMEFFKN ENNELPKLQW YKDCKPLLLD NIHFSGVKDR LIVMNVAEKH RGNYTCHASY TYLGKQYPIT RVIEFITLEE NKPTRPVIVS PANETMEVDL GSQIQLICNV TGQLSDIAYW KWNGSVIDED DPVLGEDYYS VENPANKRRS TLITVLNISE IESRFYKHPF TCFAKNTHGI DAAYIQLIYP VTNFQKHMIG ICVTLTVIIV CSVFIYKIFK IDIVLWYRDS CYDFLPIKAS DGKTYDAYIL YPKTVGEGST SDCDIFVFKV LPEVLEKQCG YKLFIYGRDD YVGEDIVEVI NENVKKSRRL IIILVRETSG FSWLGGSSEE QIAMYNALVQ DGIKVVLLEL EKIQDYEKMP ESIKFIKQKH GAIRWSGDFT QGPQSAKTRF WKNVRYHMPV QRRSPSSKHQ LLSPATKEKL QREAHVPLG (SEQ ID NO: 8), NCBI Reference Sequence NP_000868.1, aas 21 to 569 form the mature polypeptide, the ectodomain of which can be used to determine binding affinity for IL-1α and IL-1β.

[0141] In some examples, the variant IL-1α or IL-1β polypeptide exhibits reduced binding affinity to an IL-1 receptor or ectodomain thereof having the amino acid sequence set forth in SEQ ID NO: 8 compared to the binding affinity of an IL-1 polypeptide comprising the aa sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7. For example, in some examples, the variant IL-1α and IL-1β polypeptides bind to the IL-1 receptor (or ectodomain of the mature protein) set forth in SEQ ID NO: 8 with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of an IL-1α or IL-1β polypeptide comprising the aa sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7.

[0142] In some examples, the mutant IL-1α or IL-1β polypeptide (e.g., a mutant of SEQ ID NO: 5 or 7) has a binding affinity for an IL-1 receptor having the sequence set forth in SEQ ID NO: 8, or an ectodomain thereof, of 1 nM to 1 mM (e.g., 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1 μM, 1 μM to 10 μM, 10 μM to 100 μM, or 100 μM to 1 mM). As another example, in some instances, a mutant IL-1α or IL-1β polypeptide (e.g., a mutant of SEQ ID NO: 5 or 7) may have a cytotoxicity of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about The antibody has a binding affinity for the IL-1 receptor shown in SEQ ID NO: 8 or an ectodomain thereof of 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0143] In some examples, the mutant IL-1α or IL-1β polypeptide has a single aa substitution compared to the IL-1α or IL-1β aa sequence set forth in SEQ ID NO:5 or SEQ ID NO:7. In some examples, the mutant IL-1α or IL-1β polypeptide has 2 to 10 aa substitutions compared to the IL-1α or IL-1β aa sequence set forth in SEQ ID NO:5 or SEQ ID NO:7. In some examples, the mutant IL-1α or IL-1β polypeptide has a 2 aa substitution compared to the IL-1α or IL-1β aa sequence set forth in SEQ ID NO:5 or SEQ ID NO:7. In some examples, the mutant IL-1α or IL-1β polypeptide has a 3 or 4 aa substitution compared to the IL-1α or IL-1β aa sequence set forth in SEQ ID NO:5 or SEQ ID NO:7. In some examples, the mutant IL-1α or IL-1β polypeptide has a 5 or 6 aa substitution compared to the IL-1α or IL-1β aa sequence set forth in SEQ ID NO:5 or SEQ ID NO:7. In some examples, the mutant IL-1α or IL-1β polypeptide has 7 aa or 8 aa substitutions compared to the IL-1α or IL-1β aa sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7. In some examples, the mutant IL-1α or IL-1β polypeptide has 9 aa or 10 aa substitutions compared to the IL-1α or IL-1β aa sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7.

[0144] Suitable mutant IL-1α or IL-1β polypeptide sequences include polypeptide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 100 contiguous aa of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:7 (e.g., having at least one aa substitution, deletion, or insertion).

[0145] 6. IL-2 and its variants As one non-limiting example, the MOD or mutant MOD present in the masked TGF-β construct or complex is IL-2 or a mutant IL-2 polypeptide. In some examples, the mutant MOD present in the masked TGF-β construct or complex is a mutant IL-2 polypeptide. Wild-type IL-2 binds to the IL-2 receptor (IL-2R). The wt. IL-2 aa sequence can be as follows: APTSSSTKKT QLQL EH LLL D LQMILNGINN YKNPKLTRML T F KF Y MPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLIS N IN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC Q SIIS TLT (aa 21 to 153 of UniProt P60568, SEQ ID NO: 9).

[0146] Wild-type IL-2 binds to the IL-2 receptor (IL-2R) on the surface of cells. IL-2 receptors are, in some instances, heterotrimeric polypeptides comprising an α chain (IL-2Rα; also referred to as CD25), a β chain (IL-2Rβ; also referred to as CD122), and a γ chain (IL-2Rγ; also referred to as CD132). The amino acid sequences of human IL-2Rα, IL-2Rβ, and IL-2Rγ may be as follows:

[0147] Human IL-2Rα: ELCDDDPPE IPHATFKAMA YKEGTMLNCE CKRGFRRIKS GSLYMLCTGN SSHSSWDNQC QCTSSATRNT TKQVTPQPEE QKERKTTEMQ SPMQPVDQAS LPGHCREPPP WENEATERIY HFVVGQMVYY QCVQGYRALH RGPAESVCKM THGKTRWTQP QLICTGEMET SQFPGEEKPQ ASPEGRPESE TSCLVTTTDF QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL ISVLLLSGLT WQRRQRKSRR TI (SEQ ID NO: 10)

[0148] Human IL-2Rβ:VNG TSQFTCFYNS RANISCVWSQ DGALQDTSCQ VHAWPDRRRW NQTCELLPVS QASWACNLIL GAPDSQKLTT VDIVTLRVLC REGVRWRVMA IQDFKPFENL RLMAPISLQV VHVETHRCNI SWEISQASHY FERHLEFEAR TLSPGHTWEE APLLTLKQKQ EWICLETLTP DTQYEFQVRV KPLQGEFTTW SPWSQPLAFR TKPAALGKDT IPWLGHLLVG LSGAFGFIIL VYLLINCRNT GPWLKKVLKC NTPDPSKFFS QLSSEHGGDV QKWLSSPFPS SSFSPGGLAP EISPLEVLER DKVTQLLLQQ DKVPEPASLS SNHSLTSCFT NQGYFFFHLP DALEIEACQV YFTYDPYSEE DPDEGVAGAP TGSSPQPLQP LSGEDDAYCT FPSRDDLLLF SPSLLGGPSP PSTAPGGSGA GEERMPPSLQ ERVPRDWDPQ PLGPPTPGVP DLVDFQPPPE LVLREAGEEV PDAGPREGVS FPWSRPPGQG EFRALNARLP LNTDAYLSLQ ELQGQDPTHL V (SEQ ID NO: 11)

[0149] Human IL-2Rγ: LNTTILTP NGNEDTTADF FLTTMPTDSL SVSTLPLPEV QCFVFNVEYM NCTWNSSSEP QPTNLTLHYW YKNSDNDKVQ KCSHYLFSEE ITSGCQLQKK EIHLYQTFVV QLQDPREPRR QATQMLKLQN LVIPWAPENL TLHKLSESQL ELNWNNRFLN HCLEHLVQYR TDWDHSWTEQ SVDYRHKFSL PSVDGQKRYT FRVRSRFNPL CGSAQHWSEW SHPIHWGSNT SKENPFLFAL EAVVISVGSM GLIISLLCVY FWLERTMPRI PTLKNLEDLV TEYHGNFSAW SGVSKGLAES LQPDYSERLC LVSEIPPKGG ALGEGPGASP CNQHSPYWAP PCYTLKPET (SEQ ID NO: 12)

[0150] In some examples, when the masked TGF-β construct or complex comprises a mutant IL-2 polypeptide, the cognate co-MOD is an IL-2R comprising a polypeptide comprising the aa sequence of any one of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0151] In some examples, the variant IL-2 polypeptide exhibits reduced binding affinity to IL-2R compared to the binding affinity of an IL-2 polypeptide comprising the aa sequence set forth in SEQ ID NO: 9. For example, in some examples, the variant IL-2 polypeptide binds to IL-2R with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of an IL-2 polypeptide comprising the aa sequence set forth in SEQ ID NO: 9 to IL-2R (e.g., an IL-2R comprising a polypeptide comprising the aa sequence set forth in SEQ ID NOs: 10-12) when assayed under the same conditions.

[0152] In some examples, a mutant IL-2 polypeptide (e.g., a mutant of SEQ ID NO: 9) has a binding affinity to IL-2R (e.g., of SEQ ID NOs: 10 to 12) of 100 nM to 100 μM. In other examples, a mutant IL-2 polypeptide (e.g., a mutant of SEQ ID NO: 9) has a binding affinity to IL-2R of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1000 nM, or about 1000 nM to about 1200 nM. The polypeptide has a binding affinity to IL-2R (e.g., an IL-2R comprising a polypeptide comprising the aa sequences set forth in SEQ ID NOs: 10 to 12) of 00 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0153] In some examples, the mutant IL-2 polypeptide has a single aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO: 9. In some examples, the mutant IL-2 polypeptide has 2 to 10 aa substitutions compared to the IL-2 aa sequence set forth in SEQ ID NO: 9. In some examples, the mutant IL-2 polypeptide has a 2 aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO: 9. In some examples, the mutant IL-2 polypeptide has a 3 aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO: 9. In some examples, the mutant IL-2 polypeptide has a 4 aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO: 9. In some examples, the mutant IL-2 polypeptide has a 5 aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO: 9. In some examples, the mutant IL-2 polypeptide has a 6 aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO: 9. In some examples, the mutant IL-2 polypeptide has a 7 aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO: 9. In some examples, the variant IL-2 polypeptide has an 8 aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO: 9. In some examples, the variant IL-2 polypeptide has a 9 aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO: 9. In some examples, the variant IL-2 polypeptide has a 10 aa substitution compared to the IL-2 aa sequence set forth in SEQ ID NO: 9.

[0154] Suitable variant IL-2 polypeptide sequences include polypeptide sequences comprising an aa sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 80 (e.g., 90, 100, 110, 120, 130, or 133) consecutive aas of SEQ ID NO: 9. In addition, IL-2 variants include polypeptides comprising an aa sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 80 (e.g., 90, 100, 110, 120, 130, or 133) consecutive aas of any one of the following variant IL-2 aa sequences (see SEQ ID NOs: 13-27).

[0155] APTSSSTKKT QLQL X HLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 13), wherein X is any aa except Glu. In some examples, X is Ala.

[0156] APTSSSTKKT QLQLEHLLL X LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 14), wherein X is any aa except Asp. In some examples, X is Ala.

[0157] APTSSSTKKT QLQLE XLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 15), wherein X (H16) is any aa other than His. In some examples, X is Ala (H16A). In some examples, X is Arg. In some examples, X is Asn. In some examples, X is Asp. In some examples, X is Cys. In some examples, X is Glu. In some examples, X is Gln. In some examples, X is Gly. In some examples, X is Ile. In some examples, X is Lys. In some examples, X is Leu. In some examples, X is Met. In some examples, X is Phe. In some examples, X is Pro. In some examples, X is Ser. In some examples, X is Thr(H16T). In some examples, X is Tyr. In some examples, X is Trp. In some examples, X is Val.

[0158] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML T X KFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 16), wherein X(F42) is any aa other than Phe. In some examples, X is Ala (F42A). In some examples, X is Thr (F42T).

[0159] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKF XMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 17), wherein X is any aa except Tyr. In some examples, X is Ala.

[0160] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLIS X IN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 18), wherein X(N88) is any aa other than Asn. In some examples, X is Ala, and in some examples, X is Arg.

[0161] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X SIIS TLT (SEQ ID NO: 19), wherein X is any aa other than Gln. In some examples, X is Ala.

[0162] APTSSSTKKT QLQLE X 1LLLD LQMILNGINN YKNPKLTRML T X2KFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 20), wherein X1 (H16) is any aa other than His, and X2 (F42) is any aa other than Phe. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X1 is Ala and X2 is Ala (H16A, F42A). In some examples, X1 is Thr and X2 is Ala (H16T, F42A). In some examples, X1 is Ala and X2 is Thr (H16A, F42T). In some examples, X1 is Thr and X2 is Thr (H16T, F42T).

[0163] APTSSSTKKT QLQLE, containing the N88R substitution X 1LLLD LQMILNGINN YKNPKLTRML T X 2KFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLIS R IN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 21), wherein X1 (H16) is any aa other than His, and X2 (F42) is any aa other than Phe. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X1 is Ala and X2 is Ala. In some examples, X1 is Thr and X2 is Ala. In some examples, X1 is Ala and X2 is Thr. In some examples, X1 is Thr and X2 is Thr.

[0164] APTSSSTKKT QLQLEHLLL X 1LQMILNGINN YKNPKLTRML T X2KFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 22), wherein X1 is any aa other than Asp, and X2 is any aa other than Phe. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X1 is Ala, and X2 is Ala.

[0165] APTSSSTKKT QLQL X 1HLLL X 2LQMILNGINN YKNPKLTRML T X 3KFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 23), wherein X1 is any aa other than Glu, X2 is any aa other than Asp, and X3 is any aa other than Phe. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, any two or all three of X1 is Ala, X2 is Ala, and X3 is Ala.

[0166] APTSSSTKKT QLQLE X 1LLL X 2LQMILNGINN YKNPKLTRML T X 3KFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 24), wherein X1 is any aa other than His, X2 is any aa other than Asp, and X3 is any aa other than Phe. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X1 is Ala, X2 is Ala, and X3 is Ala.

[0167] APTSSSTKKT QLQLEHLLL X 1LQMILNGINN YKNPKLTRML T X 2KFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X 3SIIS TLT (SEQ ID NO: 25), wherein X1 is any aa other than Asp, X2 is any aa other than Phe, and X3 is any aa other than Gln. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X1 is Ala, X2 is Ala, and X3 is Ala.

[0168] APTSSSTKKT QLQLEHLLL X 1LQMILNGINN YKNPKLTRML T X 2KF X 3MPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 26), wherein X1 is any aa other than Asp, X2 is any aa other than Phe, and X3 is any aa other than Tyr. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X1 is Ala, X2 is Ala, and X3 is Ala.

[0169] APTSSSTKKT QLQLE X 1LLL X 2LQMILNGINN YKNPKLTRML T X 3KF X4MPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 27), wherein X1 is any aa other than His, X2 is any aa other than Asp, X3 is any aa other than Phe, and X4 is any aa other than Tyr. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X4 is Ala. In some examples, X1 is Ala, X2 is Ala, X3 is Ala, and X4 is Ala.

[0170] APTSSSTKKT QLQLEHLLL X 1LQMILNGINN YKNPKLTRML T X 2KF X 3MPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X 4SIIS TLT (SEQ ID NO: 139), wherein X1 is any aa other than Asp, X2 is any aa other than Phe, X3 is any aa other than Tyr, and X4 is any aa other than Gln. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X4 is Ala. In some examples, X1 is Ala, X2 is Ala, X3 is Ala, and X4 is Ala.

[0171] APTSSSTKKT QLQLE X 1LLL X 2LQMILNGINN YKNPKLTRML T X 3KF X 4MPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X5SIIS TLT (SEQ ID NO: 140), wherein X1 is any aa other than His, X2 is any aa other than Asp, X3 is any aa other than Phe, X4 is any aa other than Tyr, and X5 is any aa other than Gln. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, X4 is Ala. In some examples, X5 is Ala. In some examples, any two, three, four, or all five of X1 are Ala, X2 is Ala, X3 is Ala, X4 is Ala, and / or X5 is Ala.

[0172] APTSSSTKKT QLQLE X 1LLLD LQMILNGINN YKNPKLTRML T X 2KFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC X 3SIIS TLT (SEQ ID NO: 141), wherein X1 is any aa other than His, X2 is any aa other than Phe, and X3 is any aa other than Gln. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Ala. In some examples, any two or all three of X1 are Ala, X2 is Ala, and / or X3 is Ala.

[0173] 7. IL-4 and its variants As one non-limiting example, the MOD or mutant MOD present in the masked TGF-β construct or complex is an IL-4 or mutant IL-4 polypeptide. Wild-type IL-4 has two isoforms, IL-4α and IL-4β, both of which bind to the membrane-bound IL-4 receptor (isoform 1) or its soluble counterpart (isoform 2).

[0174] The wt. human IL-4 isoform 1 precursor polypeptide can comprise the following aa sequence: MGLTSCLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKTLCTE LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKYSK CSS (SEQ ID NO: 28) NCBI Reference Sequence NP_000580.1.

[0175] A mature wt. human IL-4 isoform 1 polypeptide can comprise the following aa sequence: KCDIT LQEIIKTLNS LTEQKTLCTE LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCL GATAQQFHRH KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKYSK CSS (SEQ ID NO: 29).

[0176] A wt. human IL-4 isoform 2 precursor polypeptide can comprise the following aa sequence: MGLTSCLLPP LFFLLACAGN FVHGHKCDIT LQEIIKTLNS LTEQKNTTEK ETFCRAATVL RQFYSHHEKD TRCLGATAQQ FHRHKQLIRF LKRLDRNLWG LAGLNSCPVK EANQSTLENF LERLKTIMRE ​​KYSKCSS, NCBI Reference Sequence: NP_758858.1 (SEQ ID NO: 30).

[0177] A mature wt. human IL-4 isoform 2 polypeptide can comprise the following aa sequence: KCDIT LQEIIKTLNS LTEQKNTTEK ETFCRAATVL RQFYSHHEKD TRCLGATAQQ FHRHKQLIRF LKRLDRNLWG LAGLNSCPVK EANQSTLENF LERLKTIMRE ​​KYSKCSS (SEQ ID NO: 31).

[0178] Both IL-4 isoform 1 and isoform 2 bind to the membrane-bound IL-4 receptor (IL-4R) and / or its soluble isoform 2. Membrane-bound IL-4 has the sequence MGWLCSGLLF PVSCLVLLQV ASSGNMKVLQ EPTCVSDYMS ISTCEWKMNG PTNCSTELRL LYQLVFLLSE AHTCIPENNG GAGCVCHLLM DDVVSADNYT LDLWAGQQLL WKGSFKPSEH VKPRAPGNLT VHTNVSDTLL LTWSNPYPPD NYLYNHLTYA VNIWSENDPA DFRIYNVTYL EPSLRIAAST LKSGISYRAR VRAWAQCYNT TWSEWSPSTK WHNSYREPFE QHLLLGVSVS CIVILAVCLL CYVSITKIKK EWWDQIPNPA RSRLVAIIIQ DAQGSQWEKR SRGQEPAKCP HWKNCLTKLL PCFLEHNMKR DEDPHKAAKE MPFQGSGKSA WCPVEISKTV LWPESISVVR CVELFEAPVE CEEEEEVEEE KGSFCASPES SRDDFQEGRE GIVARLTESL FLDLLGEENG GFCQQDMGES CLLPPSGSTS AHMPWDEFPS AGPKEAPPWG KEQPLHLEPS PPASPTQSPD NLTCTETPLV IAGNPAYRSF SNSLSQSPCP RELGPDPLLA RHLEEVEPEM PCVPQLSEPT TVPQPEPETW EQILRRNVLQ HGAAAAPVSA PTSGYQEFVH AVEQGGTQAS AVVGLGPPGE AGYKAFSSLL ASSAVSPEKC GFGASSGEEG YKPFQDLIPG CPGDPAPVPV PLFTGFLDRE PPRSPQSSHL PSSSPEHLGL EPGEKVEDMP KPPLPQEQAT DPLVDSLGSG IVYSALTCHL CGHLKQCHGQ EDGGQTPVMA SPCCGCCCGD RSSPPTTPLR APDPSPGGVP LEASLCPASL APSGISEKSK SSSSFHPAPG NAQSSSQTPK IVNFVSVGPTYMRVS (SEQ ID NO: 32), which can have the NCBI reference sequence NP_000409.1, where aas 26 to 825 form the mature polypeptide and aas 233 to 256 form the transmembrane region, the ectodomain of the protein can be used to determine binding affinity to IL-4 isoform 1 or 2.

[0179] Soluble isoform 2 having the sequence MGWLCSGLLF PVSCLVLLQV ASSGNMKVLQ EPTCVSDYMS ISTCEWKMNG PTNCSTELRL LYQLVFLLSE AHTCIPENNG GAGCVCHLLM DDVVSADNYT LDLWAGQQLL WKGSFKPSEH VKPRAPGNLT VHTNVSDTLL LTWSNPYPPDN YLYNHLTYAVN IWSENDPADF RIYNVTYLEP SLRIAASTLK SGISYRARVRA WAQCYNTTWSE WSPSTKWHNS NIC (SEQ ID NO: 33), UniProtKB-P24394, can also be used to measure the binding affinity of both IL-4 isoforms.

[0180] In some examples, a variant IL-4 isoform 1 or 2 polypeptide (e.g., a variant of SEQ ID NO: 29 or 31) exhibits reduced binding affinity to the mature IL-4 receptor sequence set forth in SEQ ID NO: 32 or the ectodomain thereof, or the soluble IL-4 receptor set forth in SEQ ID NO: 33, compared to the binding affinity of an IL-4 polypeptide comprising the aa sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 31. For example, in some examples, a variant IL-4 isoform 1 or 2 polypeptide binds to the mature IL-4 receptor set forth in SEQ ID NO: 32 (or the ectodomain thereof) or SEQ ID NO: 33 with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of an IL-4 isoform 1 or 2 polypeptide comprising the aa sequence set forth in SEQ ID NO: 29 or 31.

[0181] In some examples, a mutant IL-4 isoform 1 or 2 polypeptide (e.g., a mutant of SEQ ID NO: 29 or 31) has a binding affinity of 1 nM to 1 mM (e.g., 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1 μM, 1 μM to 10 μM, 10 μM to 100 μM, or 100 μM to 1 mM) for the mature IL-4 receptor set forth in SEQ ID NO: 32 (or the ectodomain thereof) or SEQ ID NO: 33. As another example, in some instances, a mutant IL-4 isoform 1 or 2 polypeptide (e.g., a mutant of SEQ ID NO: 29 or 31) may have a IL-4 concentration of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM, The binding affinity for the mature IL-4 receptor shown in SEQ ID NO: 32 (or the ectodomain thereof) or SEQ ID NO: 33 is about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0182] In some examples, the mutant IL-4 isoform 1 or 2 polypeptide has a single aa substitution compared to the IL-4 isoform 1 or 2 aa sequence set forth in SEQ ID NO:29 or SEQ ID NO:31. In some examples, the mutant IL-4 isoform 1 or 2 polypeptide has a 2-10 aa substitution compared to the IL-4 isoform 1 or 2 aa sequence set forth in SEQ ID NO:29 or SEQ ID NO:31. In some examples, the mutant IL-4 isoform 1 or 2 polypeptide has a 2 aa substitution compared to the IL-4 isoform 1 or 2 aa sequence set forth in SEQ ID NO:29 or SEQ ID NO:31. In some examples, the mutant IL-4 isoform 1 or 2 polypeptide has a 3 aa or 4 aa substitution compared to the IL-4 isoform 1 or 2 aa sequence set forth in SEQ ID NO:29 or SEQ ID NO:31. In some examples, the mutant IL-4 polypeptide has a 5 aa or 6 aa substitution compared to the IL-4 isoform 1 or 2 aa sequence set forth in SEQ ID NO:29 or SEQ ID NO:31. In some examples, the variant IL-4 isoform 1 or 2 polypeptide has a 7 aa or 8 aa substitution compared to the IL-4 isoform 1 or 2 aa sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 31. In some examples, the variant IL-4 isoform 1 or 2 polypeptide has a 9 aa or 10 aa substitution compared to the IL-4 isoform 1 or 2 aa sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 31.

[0183] Suitable mutant IL-4 isoform 1 or 2 polypeptide sequences include polypeptide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 80 contiguous aa (e.g., at least 100 or 110 contiguous aa) of SEQ ID NO:29 or SEQ ID NO:31 (e.g., having at least one aa substitution, deletion, or insertion).

[0184] 8. IL-6 and its variants As one non-limiting example, the MOD or mutant MOD present in the masked TGF-β construct or complex is IL-6 or a mutant IL-6 polypeptide. Wild-type IL-6 binds to the IL-6 receptor, which is composed of α and β (gp130) subunits, forming a signaling hexamer of two trimers with an IL-6 molecule and each subunit. See, e.g., Lacroix et al., J. Biol. Chem. 290(45)26943-953 (2015).

[0185] The wt. human IL-6 precursor polypeptide can comprise the following aa sequence: MNSFSTSAFG PVAFSLGLLL VLPAAFPAPV PPGEDSKDVA APHRQPLTSS ERIDKQIRYI LDGISALRKE TCNKSNMCES SKEALAENNL NLPKMAEKDG CFQSGFNEET CLVKIITGLL EFEVYLEYLQ NRFESSEEQA RAVQMSTKVL IQFLQKKAKN LDAITTPDPT TNASLLTKLQ AQNQWLQDMT THLILRSFKE FLQSSLRALR QM (SEQ ID NO: 34) NCBI Reference Sequence NP_000591.1.

[0186] The mature wt. human IL-6 polypeptide can comprise the following aa sequence: VPPGEDSKDVA APHRQPLTSS ERIDKQIRYI LDGISALRKE TCNKSNMCES SKEALAENNL NLPKMAEKDG CFQSGFNEET CLVKIITGLL EFEVYLEYLQ NRFESSEEQA RAVQMSTKVL IQFLQKKAKN LDAITTPDPT TNASLLTKLQ AQNQWLQDMT THLILRSFKE FLQSSLRALR QM (SEQ ID NO: 35).

[0187] IL-6 binds to the membrane-bound IL-6 receptor, which is composed of α and β subunits. The human IL-6R alpha subunit has the sequence MLAVGCALLA ALLAAPGAAL APRRCPAQEV ARGVLTSLPG DSVTLTCPGV EPEDNATVHW VLRKPAAGSH PSRWAGMGRR LLLRSVQLHD SGNYSCYRAG RPAGTVHLLV DVPPEEPQLS CFRKSPLSNV VCEWGPRSTP SLTTKAVLLV RKFQNSPAED FQEPCQYSQE SQKFSCQLAV PEGDSSFYIV SMCVASSVGS KFSKTQTFQG CGILQPDPPA NITVTAVARN PRWLSVTWQD PHSWNSSFYR LRFELRYRAE RSKTFTTWMV KDLQHHCVIH DAWSGLRHVV QLRAQEEFGQ GEWSEWSPEA MGTPWTESRS PPAENEVSTP MQALTTNKDD DNILFRDSAN ATSLPVQDSS SVPLPTFLVA GGSLAFGTLL CIAIVLRFKK TWKLRALKEG KTSMHPPYSL GQLVPERPRP TPVLVPLISP PVSPSSLGSD NTSSHNRPDA RDPRSPYDIS NTDYFFPR (SEQ ID NO: 36), NCBI Reference Sequence: NP_000556.1, where aas 26 to 825 form the mature polypeptide and aas 233 to 256 form the transmembrane region. After binding to IL-6, the IL-6α subunit binds to the IL-6β subunit.

[0188] The human IL-6R β subunit has the sequence MLTLQTWLVQ ALFIFLTTES TGELLDPCGY ISPESPVVQL HSNFTAVCVL KEKCMDYFHV NANYIVWKTN HFTIPKEQYT IINRTASSVT FTDIASLNIQ LTCNILTGFQ LEQNVYGITI ISGLPPEKPK NLSCIVNEGK KMRCEWDGGR ETHLETNFTL KSEWATHKFA DCKAKRDTPT SCTVDYSTVY FVNIEVWVEA ENALGKVTSD HINFDPVYKV KPNPPHNLSV INSEELSSIL KLTWTNPSIK SVIILKYNIQ YRTKDASTWS QIPPEDTAST RSSFTVQDLK PFTEYVFRIR CMKEDGKGYW SDWSEEASGI TYEDRPSKAP SFWYKIDPSH TQGYRTVQLV WKTLPPFEAN GKILDYEVTL TRWKSHLQNY TVNATKLTVN LTNDRYLATL TVRNLVGKSD AAVLTIPACD FQATHPVMDL KAFPKDNMLW VEWTTPRESV KKYILEWCVL SDKAPCITDW QQEDGTVHRT YLRGNLAESK CYLITVTPVY ADGPGSPESI KAYLKQAPPS KGPTVRTKKV GKNEAVLEWD QLPVDVQNGF IRNYTIFYRT IIGNETAVNV DSSHTEYTLS SLTSDTLYMV RMAAYTDEGG KDGPEFTFTT PKFAQGEIEA IVVPVCLAFL LTTLLGVLFC FNKRDLIKKH IWPNVPDPSK SHIAQWSPHT PPRHNFNSKD QMYSDGNFTD VSVVEIEAND KKPFPEDLKS LDLFKKEKIN TEGHSSGIGG SSCMSSSRPS ISSSDENESS QNTSSTVQYS TVVHSGYRHQ VPSVQVFSRS ESTQPLLDSE ERPEDLQLVD HVDGGDGILP RQQYFKQNCS QHESSPDISH FERSKQVSSV NEEDFVRLKQ QISDHISQSC GSGQMKMFQE VSAADAFGPG TEGQVERFETVGMEAATDEG MPKSYLPQTV RQGGYMPQ (SEQ ID NO: 37), UniProtKB-P40189, where aas 23 to 918 form the mature polypeptide and aas 620 to 641 form the transmembrane region.

[0189] As an alternative to IL-6 binding to the membrane-bound IL-6R α subunit, it has a mature polypeptide containing aas 20 to 468, the sequence MLAVGCALLA ALLAAPGAAL APRRCPAQEV ARGVLTSLPG DSVTLTCPGV EPEDNATVHW VLRKPAAGSH PSRWAGMGRR LLLRSVQLHD SGNYSCYRAG RPAGTVHLLV DVPPEEPQLS CFRKSPLSNV VCEWGPRSTP SLTTKAVLLV RKFQNSPAED FQEPCQYSQE SQKFSCQLAV PEGDSSFYIV SMCVASSVGS KFSKTQTFQG CGILQPDPPA NITVTAVARN PRWLSVTWQD PHSWNSSFYR LRFELRYRAE RSKTFTTWMV KDLQHHCVIH DAWSGLRHVV QLRAQEEFGQ GEWSEWSPEA MGTPWTESRS It can bind to the mature soluble form of the IL-6R α subunit, which has the sequence PPAENEVSTP MQALTTNKDD DNILFRDSAN ATSLPVQDSS SVPLPTFLVA GGSLAFGTLL CIAIVLRFKK TWKLRALKEG KTSMHPPYSL GQLVPERPRP TPVLVPLISP PVSPSSLGSD NTSSHNRPDA RDPRSPYDIS NTDYFFPR (SEQ ID NO: 38), UniProtKB-P08887.1. The soluble subunit can replace the membrane-bound IL-6R α subunit and can be used in binding affinity assays.

[0190] In some examples, a mutant IL-6 polypeptide (e.g., a mutant of SEQ ID NO: 35) exhibits reduced binding affinity to the mature IL-6 receptor shown in SEQ ID NOs: 36 and 37, or SEQ ID NOs: 37 and 38, compared to the binding affinity of an IL-6 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 35. For example, in some examples, the mutant IL-6 polypeptide binds to the mature IL-6 receptor shown in SEQ ID NOs: 36 and 37, or SEQ ID NOs: 37 and 38 with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of an IL-6 polypeptide comprising the aa sequence set forth in SEQ ID NO: 35.

[0191] In some examples, the mutant IL-6 polypeptide (e.g., a mutant of SEQ ID NO: 35) has a binding affinity for the mature IL-6 receptor shown in SEQ ID NOs: 36 and 37, or SEQ ID NOs: 37 and 38, of 1 nM to 1 mM (e.g., 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1 μM, 1 μM to 10 μM, 10 μM to 100 μM, or 100 μM to 1 mM). In some examples, the mutant IL-6 polypeptide (e.g., a mutant of SEQ ID NO: 35) has a binding affinity for the mature IL-6 receptor shown in SEQ ID NOs: 36 and 37, or SEQ ID NOs: 37 and 38, of 100 nM to 100 μM (e.g., 100 nM to 1 μM, 1 μM to 10 μM, or 10 μM to 100 μM). As another example, in some examples, the mutant IL-6 polypeptide (e.g., the mutant of SEQ ID NO: 35) has a potency of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, or about 100 nM to about 200 nM. The binding affinity for the mature IL-6 receptor shown in SEQ ID NOs: 36 and 37, or SEQ ID NOs: 37 and 38, is about 900 nM, about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0192] In some examples, the mutant IL-6 polypeptide has a single aa substitution compared to the IL-6 aa sequence set forth in SEQ ID NO: 35. In some examples, the mutant IL-6 polypeptide has a 2-10 aa substitution compared to the IL-6 aa sequence set forth in SEQ ID NO: 35. In some examples, the mutant IL-6 polypeptide has a 2 aa substitution compared to the IL-6 aa sequence set forth in SEQ ID NO: 35. In some examples, the mutant IL-6 polypeptide has a 3 or 4 aa substitution compared to the IL-6 aa sequence set forth in SEQ ID NO: 35. In some examples, the mutant IL-6 polypeptide has a 5 or 6 aa substitution compared to the IL-6 aa sequence set forth in SEQ ID NO: 35. In some examples, the mutant IL-6 polypeptide has a 7 or 8 aa substitution compared to the IL-6 aa sequence set forth in SEQ ID NO: 35. In some examples, the mutant IL-6 polypeptide has a 9 or 10 aa substitution compared to the IL-6 aa sequence set forth in SEQ ID NO: 35.

[0193] Suitable mutant IL-6 polypeptide sequences include polypeptide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 80 contiguous aa (e.g., at least 100 or 110 contiguous aa) of SEQ ID NO: 35 (e.g., having at least one aa substitution, deletion, or insertion).

[0194] 9. IL-7 and its variants As one non-limiting example, the MOD or mutant MOD present in the masked TGF-β construct or complex is an IL-7 or mutant IL-7 polypeptide. Wild-type IL-7 has four isoforms, all of which bind to the membrane-bound IL-7 receptor, which has two subunits, the alpha (α) and common gamma (γ) chains.

[0195] A wt. human IL-7 isoform 1 precursor polypeptide can comprise the following aa sequence: MFHVSFRYIF GLPPLILVLL PVASSDCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEGMF LFRAARKLRQ FLKMNSTGDF DLHLLKVSEG TTILLNCTGQ VKGRKPAALG EAQPTKSLEE NKSLKEQKKL NDLCFLKRLL QEIKTCWNKI LMGTKEH (SEQ ID NO: 39), UniProtKB-P13232, NCBI Reference Sequence NP_000871.1.

[0196] A mature wt. human IL-7 isoform 1 polypeptide can comprise the following aa sequence: DCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEGMF LFRAARKLRQ FLKMNSTGDF DLHLLKVSEG TTILLNCTGQ VKGRKPAALG EAQPTKSLEE NKSLKEQKKL NDLCFLKRLL QEIKTCWNKI LMGTKEH (SEQ ID NO: 40).

[0197] A wt. human IL-7 isoform 2 precursor polypeptide can comprise the following aa sequence: MFHVSFRYIF GLPPLILVLL PVASSDCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKVKGR KPAALGEAQP TKSLEENKSL KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH (SEQ ID NO: 41) NCBI Reference Sequence: NP_001186815.1.

[0198] A mature wt. human IL-7 isoform 2 polypeptide can comprise the following aa sequence: SDCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKVKGR KPAALGEAQP TKSLEENKSL KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH (SEQ ID NO: 42).

[0199] A wt. human IL-7 isoform 3 precursor polypeptide can comprise the following aa sequence: MFHVSFRYIF GLPPLILVLL PVASSDCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEGMF LFRAARKLRQ FLKMNSTGDF DLHLLKVSEG TTILLNCTGQ EENKSLKEQK KLNDLCFLKR LLQEIKTCWN KILMGTKEH (SEQ ID NO: 43), NCBI Reference Sequence: NP_001186816.1.

[0200] A mature wt. human IL-7 isoform 3 polypeptide can comprise the following aa sequence: CDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEGMF LFRAARKLRQ FLKMNSTGDF DLHLLKVSEG TTILLNCTGQ EENKSLKEQK KLNDLCFLKR LLQEIKTCWN KILMGTKEH (SEQ ID NO: 44).

[0201] A wt. human IL-7 isoform 4 precursor polypeptide can comprise the following aa sequence: MFHVSFRYIF GLPPLILVLL PVASSDCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEENK SLKEQKKLND LCFLKRLLQE IKTCWNKILM GTKEH, NCBI Reference Sequence: NP_001186817.1 (SEQ ID NO: 45).

[0202] A mature wt. human IL-7 isoform 4 polypeptide can comprise the following aa sequence: SDCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEENK SLKEQKKLND LCFLKRLLQE IKTCWNKILM GTKEH (SEQ ID NO: 46).

[0203] The IL-7 receptor alpha subunit has the following sequence: MTILGTTGFM VFSLLQVVSG ESGYAQNGDL EDAELDDYSF SCYSQLEVNG SQHSLTCAFE DPDVNITNLE FEICGALVEV KCLNFRKLQE IYFIETKKFL LIGKSNICVK VGEKSLTCKK IDLTTIVKPE APFDLSVVYR EGANDFVVTF NTSHLQKKYV KVLMHDVAYR QEKDENKWTH VNLSSTKLTL LQRKLQPAAM YEIKVRSIPD HYFKGFWSEW SPSYYFRTPE INNSSGEMDP ILLTISILSF FSVALLVILA CVLWKKRIKP IVWPSLPDHK KTLEHLCKKP RKNLNVSFNP ESFLDCQIHR VDDIQARDEV EGFLQDTFPQ QLEESEKQRL GGDVQSPNCP SEDVVITPES FGRDSSLTCL AGNVSACDAP ILSSSRSLDC RESGKNGPHV YQDLLLSLGT TNSTLPPPFS LQSGILTLNP VAQGQPILTS LGSNQEEAYV TMSSFYQNQ (SEQ ID NO: 47, NCBI Reference Sequence NP_002176.2), where aas 21-459 form the mature polypeptide and aas 240-264 form the transmembrane region. All or a portion of the receptor subunit of the protein (e.g., the ectodomain (aas 21-239)) can be used to determine binding affinity for IL-7 isoforms along with the IL-7 receptor gamma subunit.

[0204] The common gamma subunit (IL-7RG or IL-R7γ) has the sequence MLKPSLPFTS LLFLQLPLLG VGLNTTILTP NGNEDTTADF FLTTMPTDSL SVSTLPLPEV QCFVFNVEYM NCTWNSSSEP QPTNLTLHYW YKNSDNDKVQ KCSHYLFSEE ITSGCQLQKK EIHLYQTFVV QLQDPREPRR QATQMLKLQN LVIPWAPENL TLHKLSESQL ELNWNNRFLN HCLEHLVQYR TDWDHSWTEQ SVDYRHKFSL PSVDGQKRYT FRVRSRFNPL CGSAQHWSEW SHPIHWGSNT SKENPFLFAL EAVVISVGSM GLIISLLCVY FWLERTMPRI PTLKNLEDLV TEYHGNFSAW SGVSKGLAES LQPDYSERLC LVSEIPPKGG ALGEGPGASP CNQHSPYWAP PCYTLKPET (NCBI Reference Sequence NP_000197.1, SEQ ID NO:48), where aas 23-369 form the mature polypeptide and aas 263-283 form the transmembrane region. All or part of the receptor subunit of the protein (e.g., the ectodomain (aas 23-262)) can be used, along with the α subunit, to determine binding affinity to IL-7.

[0205] In some examples, a variant IL-7 isoform 1, 2, 3, or 4 polypeptide (e.g., of SEQ ID NO: 40, 42, 44, or 46) exhibits reduced binding affinity to a mature IL-7 receptor sequence (e.g., an IL-7 receptor comprising all or a portion of the polypeptide set forth in SEQ ID NO: 47 and 48, including the ectodomain thereof) compared to the binding affinity of an IL-7 polypeptide comprising the aa sequence set forth in SEQ ID NO: 40, 42, 44, or 46. For example, in some examples, a variant IL-7 isoform 1, 2, 3, or 4 polypeptide binds to an IL-7 receptor comprising all or a portion of the polypeptide set forth in SEQ ID NO: 47 or SEQ ID NO: 48, including the ectodomain thereof, with a binding affinity that is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, or more than 95% less than the binding affinity of an IL-7 isoform 1, 2, 3, or 4 polypeptide comprising the aa sequence set forth in SEQ ID NO: 40, 42, 44, or 46.

[0206] In some examples, a variant of an IL-7 isoform 1, 2, 3, or 4 polypeptide (e.g., a variant of SEQ ID NO: 40, 42, 44, or 46) has a binding affinity for an IL-7 receptor comprising all or a portion of a polypeptide set forth in SEQ ID NOs: 47 and 48, including its ectodomain, of 1 nM to 1 mM. In some examples, a variant IL-7 isoform 1, 2, 3, or 4 polypeptide (e.g., a variant of SEQ ID NO: 40, 42, 44, or 46) has a binding affinity for a mature IL-7 receptor comprising all or a portion of a polypeptide set forth in SEQ ID NOs: 47 and 48, including its ectodomain, of 100 nM to 100 μM (e.g., 100 nM to 1 μM, 1 μM to 10 μM, or 10 μM to 100 μM). As another example, in some examples, the mutant IL-7 isoform 1, 2, 3, or 4 polypeptide may have a cytotoxicity of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1000 nM, about 1000 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 1000 nM to about 2000 nM, about 1000 nM to about 2500 nM, about 1000 nM to about 3500 nM, about 1000 nM to about 3500 nM, about 1000 nM to about 3500 nM, about 1000 nM to about 4500 nM, about 1000 nM to about 4500 nM, about 1000 nM to about 5500 nM, about 1000 nM to about 5500 nM, about 1000 nM to about 6500 nM, about 1000 nM to about 6500 nM, The polypeptides have a binding affinity for the mature IL-7 receptor comprising all or a part of the polypeptides shown in SEQ ID NOs: 47 and 48, such as the ectodomain, of from about 100 nM to about 1 μM, from about 1 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 20 μM, from about 20 μM to about 30 μM, from about 30 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM.

[0207] In some examples, a mutant IL-7 isoform 1, 2, 3, or 4 polypeptide has a single aa substitution compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequence set forth in SEQ ID NO: 40, 42, 44, or 46. In some examples, a mutant IL-7 isoform 1, 2, 3, or 4 polypeptide has a 2 aa to 10 aa substitution compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequence set forth in SEQ ID NO: 40, 42, 44, or 46. In some examples, a mutant IL-7 isoform 1, 2, 3, or 4 polypeptide has a 2 aa substitution compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequence set forth in SEQ ID NO: 40, 42, 44, or 46. In some examples, a mutant IL-7 isoform 1, 2, 3, or 4 polypeptide has a 3 aa or 4 aa substitution compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequence set forth in SEQ ID NO: 40, 42, 44, or 46. In some examples, the mutant IL-7 isoform 1, 2, 3, or 4 polypeptide has a 5 aa or 6 aa substitution compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequence set forth in SEQ ID NO: 40, 42, 44, or 46. In some examples, the mutant IL-7 isoform 1, 2, 3, or 4 polypeptide has a 7 aa or 8 aa substitution compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequence set forth in SEQ ID NO: 40, 42, 44, or 46. In some examples, the mutant IL-7 isoform 1, 2, 3, or 4 polypeptide has a 9 aa or 10 aa substitution compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequence set forth in SEQ ID NO: 40, 42, 44, or 46.

[0208] Suitable mutant IL-7 isoform 1, 2, 3, or 4 polypeptide sequences include those having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity (e.g., having at least one aa substitution, deletion, or insertion) to at least 50 contiguous aa (e.g., at least 60, at least 70, at least 80, at least 90, at least 100, or at least 110 contiguous aa) of SEQ ID NO: 40, 42, 44, or 46.

[0209] 10. IL-10 and its variants As one non-limiting example, the MOD or mutant MOD present in the masked TGF-β construct or complex is an IL-10 or mutant IL-10 polypeptide, such as the monomeric IL-10 mutant having an insertion in the hinge region between the D and E helices described in Josephson et al., J. Biol. Chem. 275:13552-13557 (2000). Wild-type IL-10 has isoforms, all of which bind to the membrane-bound IL-10 receptor, which has both the alpha (α) IL-10RA and beta (β) IL-10RB subunits. The receptor is present as a tetramer on the surface of cells (e.g., B cells, T cells, NK cells, mast cells, and dendritic cells).

[0210] A wt. human IL-10 isoform 1 precursor polypeptide can comprise the following aa sequence: MHSSALLCCL VLLTGVRASP GQGTQSENSC THFPGNLPNM LRDLRDAFSR VKTFFQMKDQ LDNLLLKESL LEDFKGYLGC QALSEMIQFY LEEVMPQAEN QDPDIKAHVN SLGENLKTLR LRLRRCHRFL PCENKSKAVE QVKNAFNKLQ EKGIYKAMSE FDIFINYIEA YMTMKIRN (SEQ ID NO: 49, UniProtKB-P22301, NCBI Reference Sequence NP_000563.1), which can have a H227L sequence variation.

[0211] The mature wt. human IL-10 polypeptide can comprise the following aa sequence: SP GQGTQSENSC THFPGNLPNM LRDLRDAFSR VKTFFQMKDQ LDNLLLKESL LEDFKGYLGC QALSEMIQFY LEEVMPQAEN QDPDIKAHVN SLGENLKTLR LRLRRCHRFL PCENKSKAVE QVKNAFNKLQ EKGIYKAMSE FDIFINYIEA YMTMKIRN (SEQ ID NO: 50).

[0212] The human IL-10 polypeptide can comprise the following aa sequence: MIQFYLEEVM PQAENQDPDI KAHVNSLGEN LKTLRLRLRR CHRFLPCENK SKAVEQVKNA FNKLQEKGIY KAMS, UniProtKB-A0A286YEX3 1 (SEQ ID NO: 51).

[0213] An IL-10 polypeptide can include an insertion within the hinge region between the D helix and E helix of the IL-10 polypeptide (e.g., a 5-7 aa insertion adjacent to any of E48, N49, K50, or S51 of SEQ ID NO:51, or at an equivalent location within SEQ ID NO:49 or 50), resulting in a monomeric form. A monomeric IL-10 polypeptide can include a 5-7 aa insertion between N49 and K50 of SEQ ID NO:51 (or an equivalent location within SEQ ID NO:49 or 50). In examples, the 5-7 aa include Ala, Gly, and / or Ser. In some examples, the 5-7 aa are selected from Ala or Ser. In some examples, the 5-7 aa are selected from Gly and Ser. In one example, the insertion includes an IL-10M1 aa insertion (GGGGSGGG, SEQ ID NO:142) between N49 and K50 of SEQ ID NO:51 (or an equivalent location within SEQ ID NO:49 or 50). In one example, the IL-10 variant consists of IL-10M1 (SEQ ID NO: 189)GGGSGG inserted into SEQ ID NO: 51 between aa 49 and 50. See, e.g., Josephson et al., J. Biol. Chem. 275:13552-13557 (2000).

[0214] The IL-10 receptor alpha subunit has the following sequence: MLPCLVVLLA ALLSLRLGSD AHGTELPSPP SVWFEAEFFH HILHWTPIPN QSESTCYEVA LLRYGIESWN SISNCSQTLS YDLTAVTLDL YHSNGYRARV RAVDGSRHSN WTVTNTRFSV DEVTLTVGSV NLEIHNGFIL GKIQLPRPKM APANDTYESI FSHFREYEIA IRKVPGNFTF THKKVKHENF SLLTSGEVGE FCVQVKPSVA SRSNKGMWSK EECISLTRQY FTVTNVIIFF AFVLLLSGAL AYCLALQLYV RRRKKLPSVL LFKKPSPFIF ISQRPSPETQ DTIHPLDEEA FLKVSPELKN LDLHGSTDSG FGSTKPSLQT EEPQFLLPDP HPQADRTLGN REPPVLGDSC SSGSSNSTDS GICLQEPSLS PSTGPTWEQQ VGSNSRGQDD SGIDLVQNSE GRAGDTQGGS ALGHHSPPEP EVPGEEDPAA VAFQGYLRQT RCAEEKATKT GCLEEESPLT DGLGPKFGRC LVDEAGLHPP ALAKGYLKQD PLEMTLASSG APTGQWNQPT EEWSLLALSS CSDLGISDWS FAHDLAPLGC VAAPGGLLGS FNSDLVTLPL ISSLQSSE (SEQ ID NO: 52, NCBI Reference Sequence NP_001549.2), in which aas 21 to 587 form the mature polypeptide and aas 236 to 256 form the transmembrane region. All or a portion of the receptor subunit of the protein (e.g., the ectodomain (aas 21 to 235)) can be used to determine binding affinity to IL-10 isoforms along with the IL-10 receptor beta subunit.

[0215] The IL-10 receptor β subunit has the sequence MAWSLGSWLG GCLLVSALGM VPPPENVRMN SVNFKNILQW ESPAFAKGNL TFTAQYLSYR IFQDKCMNTT LTECDFSSLS KYGDHTLRVR AEFADEHSDW VNITFCPVDD TIIGPPGMQV EVLADSLHMR FLAPKIENEY ETWTMKNVYN SWTYNVQYWK NGTDEKFQIT PQYDFEVLRN LEPWTTYCVQ VRGFLPDRNK AGEWSEPVCE QTTHDETVPS WMVAVILMAS VFMVCLALLG CFALLWCVYK KTKYAFSPRN SLPQHLKEFL GHPHHNTLLF FSFPLSDEND VFDKLSVIAE DSESGKQNPG DSCSLGTPPG QGPQS (SEQ ID NO: 53, NCBI Reference Sequence NP_000619.3), where aas 20-325 form the mature polypeptide and aas 221-242 form the transmembrane region. All or a portion of the receptor subunit of the protein (e.g., the ectodomain (aas 20-220)) can be used to determine binding affinity to IL-10 along with the α subunit.

[0216] In some examples, variant IL-10 isoform polypeptides (e.g., variants of SEQ ID NOs: 50, 51, or monomeric IL-10 variants of those sequences having a 5-7 aa insertion in the hinge between the D and E helices as set forth above) exhibit reduced binding affinity to a mature IL-10 receptor sequence (e.g., an IL-10 receptor comprising all or a portion of the polypeptide set forth in SEQ ID NO: 52 or 53, such as the ectodomain thereof) compared to the binding affinity of an IL-10 polypeptide comprising the aa sequence set forth in SEQ ID NO: 50 or 51. For example, in some examples, a variant of a mutant IL-10 polypeptide (e.g., a variant of SEQ ID NO: 50 or 51) binds to an IL-10 receptor comprising all or a portion of a polypeptide set forth in SEQ ID NO: 52 or 53 with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of an IL-10 isoform (e.g., of SEQ ID NO: 50 or 51) to an IL-10 receptor comprising all or a portion of the polypeptide set forth in SEQ ID NO: 52 or 53 (such as the ectodomain thereof).

[0217] In some examples, a variant IL-10 polypeptide (e.g., a variant of SEQ ID NO: 50 or 51, which may include one of the above-described insertions in the hinge region between the D helix and the E helix) has a binding affinity for an IL-10 receptor (e.g., comprising all or a portion of the polypeptide set forth in SEQ ID NOs: 52 and 53, such as the ectodomain thereof) that is 1 nM to 1 mM (e.g., 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1 μM, 1 μM to 10 μM, 10 μM to 100 μM, or 100 μM to 1 mM). In some examples, a variant IL-10 polypeptide (e.g., a variant of SEQ ID NO: 50 or 51) has a binding affinity for a mature IL-10 receptor comprising all or a portion of the polypeptide set forth in SEQ ID NOs: 52 and 53, including its ectodomain, of 100 nM to 100 μM (e.g., 100 nM to 1 μM, 1 μM to 10 μM, or 10 μM to 100 μM). As another example, in some examples, a mutant IL-10 polypeptide (e.g., a mutant of SEQ ID NO: 50 or 51) may have a cytotoxicity of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1000 nM, about 1000 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 1000 nM to about 2000 nM, about 1000 nM to about 2000 nM, about 1000 nM to about 300 nM, about 1000 nM to about 2000 nM, about 1000 nM to about 300 nM, about 1000 nM to about 400 nM, about 1000 nM to about 500 nM, about 1000 nM to about 2000 nM, about 1000 nM to about 300 nM, about 1000 nM to about 2000 nM, about 1000 nM to about 300 n The polypeptides have a binding affinity for the mature IL-10 receptor (e.g., comprising all or a portion of the polypeptides set forth in SEQ ID NOs: 52 and 53, such as the ectodomain thereof) of about 1 μM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0218] In some examples, the mutant IL-10 polypeptide has a single aa substitution compared to the IL-10 amino acid sequence set forth in SEQ ID NO: 50 or 51. In some examples, the mutant IL-10 polypeptide has a 2-10 aa substitution compared to the IL-10 amino acid sequence set forth in SEQ ID NO: 50 or 51. In some examples, the mutant IL-10 polypeptide has a 2-aa substitution compared to the IL-10 amino acid sequence set forth in SEQ ID NO: 50 or 51. In some examples, the mutant IL-10 polypeptide has a 3-aa or 4-aa substitution compared to the IL-10 amino acid sequence set forth in SEQ ID NO: 50 or 51. In some examples, the mutant IL-10 polypeptide has a 5-aa or 6-aa amino acid substitution compared to the IL-10 amino acid sequence set forth in SEQ ID NO: 50 or 51. In some examples, the mutant IL-10 polypeptide has a 7-aa or 8-aa amino acid substitution compared to the IL-10 amino acid sequence set forth in SEQ ID NO: 50 or 51. In some examples, the mutant IL-10 polypeptide has a 9 aa or 10 aa amino acid substitution compared to the IL-10 amino acid sequence set forth in SEQ ID NO: 50 or 51. In some examples, the mutant IL-10 polypeptide has a 1-10 aa substitution compared to the IL-10 polypeptide sequence set forth in SEQ ID NO: 50 or 51, and includes a 5-7 aa substitution in the hinge region between the D helix and E helix (e.g., between K49 and N50 of SEQ ID NO: 50).

[0219] Suitable mutant IL-10 polypeptide sequences include polypeptide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity over at least 50 (e.g., at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 160) consecutive aas of SEQ ID NO: 50 or 51 (e.g., having at least one aa substitution, deletion, or insertion).

[0220] 11. IL-15 and its variants In some examples, the MOD present in a masked TGF-β construct or complex of the present disclosure is an IL-15 polypeptide. The sequence of IL-15 polypeptides, including two isoforms formed by alternative splicing to give rise to different precursor proteins, is known in the art. In one embodiment, the wt. IL-15 polypeptide has the following sequence: MRISKPHLRS ISIQCYLCLL LNSHFLTEAG IHVFILGCFS AGLPKTEANW VNVISDLKKI EDLIQSMHID ATLYTESDVH PSCKVTAMKC FLLELQVISL ESGDASIHDT VENLIILANN SLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS (SEQ ID NO: 54, UniProtKB-P40933, NCBI reference NP_000576.1), an IL-15 preprotein having aa 1-29 as a signal peptide and aa 30-48 as a propeptide.

[0221] A mature IL-15 polypeptide, designated for purposes of this disclosure as isoform 1, can have the form NW VNVISDLKKI EDLIQSMHID ATLYTESDVH PSCKVTAMKC FLLELQVISL ESGDASIHDT VENLIILANN SLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS (SEQ ID NO: 55).

[0222] IL-15 is structurally similar to IL-2 and signals through a cell surface trimeric receptor with the same β and γ chains as the IL-2 receptor, but with a distinct IL-15 receptor α (IL-15Rα) subunit. The aa sequence of human IL-15Rα isoform 1 precursor protein can be MAPRRARGCR TLGLPALLLL LLLRPPATRG ITCPPPMSVE HADIWVKSYS LYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPE SLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTEISSHES SHGTPSQTTA KNWELTASAS HQPPGVYPQG HSDTTVAIST STVLLCGLSA VSLLACYLKS RQTPPLASVE MEAMEALPVT WGTSSRDEDL ENCSHHL (SEQ ID NO: 56, NCBI reference NP_002180), with aas 1 to 30 forming the signal sequence and aas 31 to 267 forming the mature polypeptide. The sequences of IL-2Rβ and IL-2Rγ are provided as SEQ ID NOs: 11 and 12.

[0223] In some examples, a variant IL-15 isoform 1 polypeptide (e.g., a variant of SEQ ID NO: 55) exhibits reduced binding affinity to a mature IL-15 receptor sequence (e.g., an IL-15 receptor comprising all or a portion of the polypeptides set forth in SEQ ID NOs: 11, 12, and 56, including the ectodomain thereof) compared to the binding affinity of an IL-15 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 55. For example, in some examples, a variant IL-15 polypeptide (e.g., a variant of SEQ ID NO: 55) binds to an IL-15 receptor comprising all or a portion of the polypeptides set forth in SEQ ID NOs: 11, 12, and 56, including the ectodomain thereof, with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of an IL-15 polypeptide comprising the aa sequence set forth in SEQ ID NO: 55.

[0224] In some examples, a mutant IL-15 polypeptide (e.g., a mutant of SEQ ID NO: 55) has a binding affinity of 1 nM to 1 mM (e.g., 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1 μM, 1 μM to 10 μM, 10 μM to 100 μM, or 100 μM to 1 mM) to an IL-15 receptor comprising all or a portion of the polypeptides set forth in SEQ ID NOs: 11, 12, and 56, e.g., the ectodomains thereof. As another example, in some examples, a mutant IL-15 polypeptide (e.g., a mutant of SEQ ID NO: 55) has a binding affinity of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, or about 400 nM to about 500 nM to a mature IL-15 receptor comprising all or a portion of the polypeptides set forth in SEQ ID NOs: 11, 12, and 56, e.g., the ectodomains thereof. The antibody has a binding affinity of about 0 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0225] In some examples, a mutant IL-15 polypeptide (e.g., a variant of SEQ ID NO: 55) has a single aa substitution compared to the IL-15 polypeptide sequence set forth in SEQ ID NO: 55. In some examples, a mutant IL-15 polypeptide (e.g., a variant of SEQ ID NO: 55) has 2 to 10 aa substitutions compared to the IL-15 polypeptide sequence set forth in SEQ ID NO: 55. In some examples, a mutant IL-15 polypeptide has a 2 aa substitution compared to the IL-15 amino acid sequence set forth in SEQ ID NO: 55. In some examples, a mutant IL-15 polypeptide has a 3 or 4 aa substitution compared to the IL-15 amino acid sequence set forth in SEQ ID NO: 55. In some examples, a mutant IL-15 polypeptide has a 5 or 6 aa substitution compared to the IL-15 amino acid sequence set forth in SEQ ID NO: 55. In some examples, a mutant IL-15 polypeptide has a 7 or 8 aa substitution compared to the IL-15 amino acid sequence set forth in SEQ ID NO: 55. In some examples, the mutant IL-15 polypeptide has a 9 aa or 10 aa substitution compared to the IL-15 amino acid sequence set forth in SEQ ID NO:55.

[0226] Suitable mutant IL-15 polypeptide sequences include polypeptide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 50 contiguous aa (e.g., at least 60, at least 70, at least 80, at least 90, at least 100, or at least 110 contiguous aa) of SEQ ID NO: 55, and having at least one aa substitution, deletion, or insertion.

[0227] 12. IL-21 and its variants In some examples, the MOD present in a masked TGF-β construct or complex of the present disclosure is an IL-21 polypeptide. The sequence of the IL-21 polypeptide, including two isoforms formed by alternative splicing that give rise to different precursor proteins, is known in the art.

[0228] In one embodiment, the wt. IL-21 isoform 1 polypeptide has the sequence MRSSPGNMER IVICLMVIFL GTLVHKSSSQ GQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DS (SEQ ID NO: 57, UniProtKB-Q9HBE4, NCBI reference NP_068575.1), an IL-21 protein with aa 1-29 as a signal peptide.

[0229] A mature IL-21 isoform 1 polypeptide can have the aa sequence Q GQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DS (SEQ ID NO: 58).

[0230] In one embodiment, the wt. IL-21 isoform 2 polypeptide has the sequence MRSSPGNMER IVICLMVIFL GTLVHKSSSQ GQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DS (SEQ ID NO: 59), NP_001193935.1, an IL-21 protein with aa 1-29 as the signal peptide.

[0231] A mature IL-21 isoform 2 polypeptide can have the aa sequence MRSSPGNMER IVICLMVIFL GTLVHKSSSQ GQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKVSTL SFI (SEQ ID NO: 60).

[0232] IL-21 signals through a dimeric cell surface receptor with the same gamma chain as the IL-2 receptor, but with distinct IL-21R receptor subunits. The aa sequence of the human IL-21R isoform 1 precursor protein is: MPRGWAAPLL LLLLQGGWGC PDLVCYTDYL QTVICILEMW NLHPSTLTLT WQDQYEELKD EATSCSLHRS AHNATHATYT CHMDVFHFMA DDIFSVNITD QSGNYSQECG SFLLAESIKP APPFNVTVTF SGQYNISWRS DYEDPAFYML KGKLQYELQY RNRGDPWAVS PRRKLISVDS RSVSLLPLEF RKDSSYELQV RAGPMPGSSY QGTWSEWSDP VIFQTQSEEL KEGWNPHLLL LLLLVIVFIP AFWSLKTHPL WRLWKKIWAV PSPERFFMPL YKGCSGDFKK WVGAPFTGSS LELGPWSPEV PSTLEVYSCH PPRSPAKRLQ LTELQEPAEL VESDGVPKPS FWPTAQNSGG SAYSEERDRP YGLVSIDTVT VLDAEEGPCTW PCSCEDDGYP ALDLDAGLEP SPGLEDPLLD AGTTVLSCGC VSAGSPGLGG PLGSLLDRLK PPLADGEDWA GGLPWGGRSP GGVSESEAGS PLAGLDMDTF DSGFVGSDCS SPVECDFTSP GDEGPPRSYL RQWVVIPPPL SSPGPQAS (SEQ ID NO: 61, NCBI Reference Sequence NP_068570.1), where aas 1-19 form the signal sequence, aas 20-538 form the mature polypeptide, aas 233-253 form the transmembrane domain, and aas 20-232 form the ectodomain.

[0233] In some examples, variant IL-21 polypeptides exhibit reduced binding affinity to a mature IL-21 receptor sequence (e.g., an IL-21 receptor comprising all or a portion of the polypeptides set forth in SEQ ID NOs: 12 and 61, such as the ectodomain thereof) compared to the binding affinity of a wt. IL-21 polypeptide comprising the aa sequence set forth in SEQ ID NO: 58 or 60. For example, in some examples, a variant of an IL-21 polypeptide comprising SEQ ID NO: 58 or 60 binds to an IL-21 receptor comprising all or a portion of the polypeptides set forth in SEQ ID NOs: 12 and 61, such as the ectodomain thereof, with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of a wt. IL-21 polypeptide comprising the aa sequence set forth in SEQ ID NO: 58 or 60.

[0234] In some examples, a mutant IL-21 polypeptide (e.g., a variant of SEQ ID NO: 58 or 60) has a binding affinity for an IL-21 receptor (e.g., including all or a portion of the polypeptides set forth in SEQ ID NOs: 12 and 61, such as the ectodomain thereof) that is 1 nM to 1 mM (e.g., 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1 μM, 1 μM to 10 μM, 10 μM to 100 μM, or 100 μM to 1 mM). In some examples, a mutant IL-21 polypeptide (e.g., a variant of SEQ ID NO: 58 or 60) has a binding affinity for all or a portion of an IL-21 receptor (e.g., including all or a portion of the polypeptides set forth in SEQ ID NOs: 12 and 61, such as the ectodomain thereof) that is 100 nM to 100 μM (e.g., 100 nM to 1 μM, 1 μM to 10 μM, or 10 μM to 100 μM). As another example, in some examples, a mutant IL-21 polypeptide (e.g., a mutant of SEQ ID NO: 58 or 60) may have a cytotoxicity of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1000 nM, about 10 ...0 nM, about 1000 nM to about 2000 nM, about 1000 nM to about 3000 nM, about 1000 nM to about 2000 nM, about 1000 nM to about 3000 nM, about 1000 nM to about 3000 nM, about 1000 nM to about 4000 nM, about 1000 nM to about 2000 nM, about 1000 nM to about 2000 nM, about 1000 nM to about The antibody has a binding affinity for all or a portion of the mature IL-21 receptor (including all or a portion of the polypeptides set forth in SEQ ID NOs: 12 and 61, such as the ectodomain thereof) of about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0235] In some examples, a mutant IL-21 polypeptide (e.g., a variant of SEQ ID NO: 58 or 60) has a single aa substitution compared to the IL-21 polypeptide sequence set forth in SEQ ID NO: 58 or 60. In some examples, a mutant IL-21 polypeptide (e.g., a variant of SEQ ID NO: 58 or 60) has a 2-10 aa substitution compared to the IL-21 polypeptide sequence set forth in SEQ ID NO: 58 or 60. In some examples, a mutant IL-21 polypeptide has a 2 aa substitution compared to the IL-21 polypeptide sequence set forth in SEQ ID NO: 58 or 60. In some examples, a mutant IL-21 polypeptide has a 3 or 4 aa substitution compared to the IL-21 polypeptide sequence set forth in SEQ ID NO: 58 or 60. In some examples, a mutant IL-21 polypeptide has a 5 or 6 aa substitution compared to the IL-21 polypeptide sequence set forth in SEQ ID NO: 58 or 60. In some examples, the mutant IL-21 polypeptide has a 7 aa or 8 aa substitution compared to the IL-21 polypeptide sequence set forth in SEQ ID NO: 58 or 60. In some examples, the mutant IL-21 polypeptide has a 9 aa or 10 aa substitution compared to the IL-21 polypeptide sequence set forth in SEQ ID NO: 58 or 60.

[0236] Suitable IL-21 polypeptide sequences include polypeptide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 50 contiguous aa (e.g., at least 60, at least 70, at least 80, at least 90, at least 100, or at least 110 contiguous aa) of SEQ ID NO: 58 or 60, and having at least one aa substitution, deletion, or insertion.

[0237] 13. IL-23 and its variants In some examples, the MOD present in a masked TGF-β construct or complex of the present disclosure is an IL-23 polypeptide. IL-23 is a heterodimeric cytokine composed of the IL-23A (IL-23p19) subunit and the IL-12B (IL-12p40) subunit (shared with IL-12).

[0238] In one embodiment, the wt. IL-23A polypeptide has the sequence: MLGSRAVMLL LLLPWTAQGR AVPGGSSPAW TQCQQLSQKL CTLAWSAHPL VGHMDLREEG DEETTNDVPH IQCGDGCDPQ GLRDNSQFCL QRIHQGLIFY EKLLGSDIFT GEPSLLPDSP VGQLHASLLG LSQLLQPEGH HWETQQIPSL SPSQPWQRLL LRFKILRSLQ AFVAVAARVF AHGAATLSP (SEQ ID NO: 62, NCBI Reference Sequence NP_057668.1), with aa 1-19 as the signal peptide and 20-189 as the mature peptide.

[0239] A mature IL-23A polypeptide can have the aa sequence: R AVPGGSSPAW TQCQQLSQKL CTLAWSAHPL VGHMDLREEG DEETTNDVPH IQCGDGCDPQ GLRDNSQFCL QRIHQGLIFY EKLLGSDIFT GEPSLLPDSP VGQLHASLLG LSQLLQPEGH HWETQQIPSL SPSQPWQRLL LRFKILRSLQ AFVAVAARVF AHGAATLSP (SEQ ID NO: 63).

[0240] In one embodiment, the wt IL-12B polypeptide has the sequence: MCHQQLVISW FSLVFLASPL VAIWELKKDV YVVELDWYPD APGEMVVLTC DTPEEDGITW TLDQSSEVLG SGKTLTIQVK EFGDAGQYTC HKGGEVLSHS LLLLHKKEDG IWSTDILKDQ KEPKNKTFLR CEAKNYSGRF TCWWLTTIST DLTFSVKSSR GSSDPQGVTC GAATLSAERV RGDNKEYEYS VECQEDSACP AAEESLPIEV MVDAVHKLKY ENYTSSFFIR DIIKPDPPKN LQLKPLKNSR QVEVSWEYPD TWSTPHSYFS LTFCVQVQGK SKREKKDRVF TDKTSATVIC RKNASISVRA QDRYYSSSWS It has EWASVPCS (SEQ ID NO: 64, UniProtKB-P29460), a signal peptide of aa 1 to 22, and a mature peptide of aa 23 to 328.

[0241] A mature IL-12B polypeptide can have the aa sequence: IWELKKDV YVVELDWYPD APGEMVVLTC DTPEEDGITW TLDQSSEVLG SGKTLTIQVK EFGDAGQYTC HKGGEVLSHS LLLLHKKEDG IWSTDILKDQ KEPKNKTFLR CEAKNYSGRF TCWWLTTIST DLTFSVKSSR GSSDPQGVTC GAATLSAERV RGDNKEYEYS VECQEDSACP AAEESLPIEV MVDAVHKLKY ENYTSSFFIR DIIKPDPPKN LQLKPLKNSR QVEVSWEYPD TWSTPHSYFS LTFCVQVQGK SKREKKDRVF TDKTSATVIC RKNASISVRA QDRYYSSSWS EWASVPCS (SEQ ID NO: 65).

[0242] IL-23 signals through a dimeric cell surface receptor consisting of the IL-23 receptor polypeptide (IL-23R) and a subunit (designated 12RB1 or 12Rβ1) common to the IL-12 receptor.

[0243] The IL-23R isoform 1 precursor protein sequence is MNQVTIQWDA VIALYILFSW CHGGITNINC SGHIWVEPAT IFKMGMNISI YCQAAIKNCQ PRKLHFYKNG IKERFQITRI NKTTARLWYK NFLEPHASMY CTAECPKHFQ ETLICGKDIS SGYPPDIPDE VTCVIYEYSG NMTCTWNAGK LTYIDTKYVV HVKSLETEEE QQYLTSSYIN ISTDSLQGGK KYLVWVQAAN ALGMEESKQL QIHLDDIVIP SAAVISRAET INATVPKTII YWDSQTTIEK VSCEMRYKAT TNQTWNVKEF DTNFTYVQQS EFYLEPNIKY VFQVRCQETG KRYWQPWSSL FFHKTPETVP QVTSKAFQHD TWNSGLTVAS ISTGHLTSDN RGDIGLLLGM IVFAVMLSIL SLIGIFNRSF RTGIKRRILL LIPKWLYEDI PNMKNSNVVK MLQENSELMN NNSSEQVLYV DPMITEIKEI FIPEHKPTDY KKENTGPLET RDYPQNSLFD NTTVVYIPDL NTGYKPQISN FLPEGSHLSN NNEITSLTLK PPVDSLDSGN NPRLQKHPNF AFSVSSVNSL SNTIFLGELS LILNQGECSS PDIQNSVEEE TTMLLENDSP SETIPEQTLL PDEFVSCLGI VNEELPSINT YFPQNILESH FNRISLLEK (SEQ ID NO: 66, NCBI Reference Sequence NP_653302.2), where aas 1-23 form the signal sequence, aas 24-629 form the mature polypeptide, aas 356-376 form the transmembrane domain, and aas 24-355 form the ectodomain.

[0244] The 12RB1 isoform 1 precursor protein aa sequence is MEPLVTWVVP LLFLFLLSRQ GAACRTSECC FQDPPYPDAD SGSASGPRDL RCYRISSDRY ECSWQYEGPT AGVSHFLRCC LSSGRCCYFA AGSATRLQFS DQAGVSVLYT VTLWVESWAR NQTEKSPEVT LQLYNSVKYE PPLGDIKVSK LAGQLRMEWE TPDNQVGAEV QFRHRTPSSP WKLGDCGPQD DDTESCLCPL EMNVAQEFQL RRRQLGSQGS SWSKWSSPVC VPPENPPQPQ VRFSVEQLGQ DGRRRLTLKE QPTQLELPEG CQGLAPGTEV TYRLQLHMLS CPCKAKATRT LHLGKMPYLS GAAYNVAVIS SNQFGPGLNQ TWHIPADTHT EPVALNISVG TNGTTMYWPA RAQSMTYCIE WQPVGQDGGL ATCSLTAPQD PDPAGMATYS WSRESGAMGQ EKCYYITIFA SAHPEKLTLW STVLSTYHFG GNASAAGTPH HVSVKNHSLD SVSVDWAPSL LSTCPGVLKE YVVRCRDEDS KQVSEHPVQP TETQVTLSGL RAGVAYTVQV RADTAWLRGV WSQPQRFSIE VQVSDWLIFF ASLGSFLSIL LVGVLGYLGL NRAARHLCPP LPTPCASSAI EFPGGKETWQ WINPVDFQEE ASLQEALVVE MSWDKGERTE PLEKTELPEG APELALDTEL SLEDGDRCKA KM (SEQ ID NO: 67, NCBI Reference Sequence NP_005526.1), where aas 1 to 23 form the signal sequence, aas 24 to 662 form the mature polypeptide, aas 546 to 570 form the transmembrane domain, and aas 24 to 545 form the ectodomain.

[0245] In some examples, mutant IL-23 (e.g., comprising a variant of SEQ ID NO: 63 and / or 65) polypeptides exhibit reduced binding affinity to a mature IL-23 receptor sequence (e.g., an IL-23 receptor comprising all or a portion of the polypeptides set forth in SEQ ID NOs: 66 and 67, including the ectodomain thereof) compared to the binding affinity of an IL-23 polypeptide comprising the aa sequence set forth in SEQ ID NO: 63 and / or 65. For example, in some examples, a variant of a mutant IL-23 polypeptide (e.g., a variant of SEQ ID NO: 63 and / or 65) binds to an IL-23 receptor (e.g., comprising all or a portion of the polypeptide set forth in SEQ ID NOs: 66 and 67, such as the ectodomain thereof) with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of an IL-23 polypeptide comprising the aa sequence set forth in SEQ ID NO: 63 and / or 65.

[0246] In some examples, a variant IL-23 polypeptide (e.g., comprising a variant of SEQ ID NO: 63 and / or 65) has a binding affinity for an IL-23 receptor (e.g., comprising all or a portion of the polypeptide set forth in SEQ ID NOs: 66 and 67, including the ectodomain thereof) that is between 1 nM and 1 mM (e.g., between 1 nM and 10 nM, 10 nM and 100 nM, 100 nM and 1 μM, 1 μM and 10 μM, 10 μM and 100 μM, or 100 μM and 1 mM). As another example, in some examples, mutant IL-23 polypeptides (e.g., including mutants of SEQ ID NOs: 63 and / or 65) may have a potency of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 90 The binding affinity for the mature IL-23 receptor (e.g., comprising all or a part of the polypeptides shown in SEQ ID NOs: 66 and 67, such as the ectodomain thereof) is 0 nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0247] In some examples, a mutant IL-23 polypeptide (e.g., comprising a variant of SEQ ID NO: 63 and / or 65) has a single aa substitution compared to the IL-23 polypeptide sequence set forth in SEQ ID NO: 63 and / or 65. In some examples, a mutant IL-23 polypeptide (e.g., comprising a variant of SEQ ID NO: 63 and / or 65) has 2 to 10 aa substitutions compared to the IL-23 polypeptide sequence set forth in SEQ ID NO: 63 and / or 65. In some examples, a mutant IL-23 polypeptide has a 2 aa substitution compared to the IL-23 polypeptide sequence set forth in SEQ ID NO: 63 and / or 65. In some examples, a mutant IL-23 polypeptide has a 3 or 4 aa substitution compared to the IL-23 polypeptide sequence set forth in SEQ ID NO: 63 and / or 65. In some examples, a mutant IL-23 polypeptide has a 5 or 6 aa substitution compared to the IL-23 polypeptide sequence set forth in SEQ ID NO: 63 and / or 65. In some examples, the mutant IL-23 polypeptide has a 7 aa or 8 aa substitution compared to the IL-23 polypeptide sequence set forth in SEQ ID NOs: 63 and / or 65. In some examples, the mutant IL-23 polypeptide has a 9 aa or 10 aa substitution compared to the IL-23 polypeptide sequence set forth in SEQ ID NOs: 63 and / or 65.

[0248] Suitable variant IL-23 polypeptide sequences include polypeptide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 50 contiguous aa (e.g., at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 320, or at least 340 contiguous aas) of SEQ ID NOs: 63 and / or 65, and having at least one aa substitution, deletion, or insertion.

[0249] 14. Fas Ligand (FasL) and its Mutants In some examples, the MOD present in the masked TGF-β construct or complex of the present disclosure is Fas ligand (FasL). FasL is a homomeric type II transmembrane protein in the tumor necrosis factor (TNF) family. FasL transmits a signal by trimerizing the Fas receptor in target cells, which forms a cell death-inducing complex that leads to apoptosis of the target cells. Soluble FasL is produced by cleavage of membrane-bound FasL at a conserved site by matrix metalloproteinase-7 (MMP-7).

[0250] In one embodiment, the wt. Homo sapiens FasL protein has the sequence MQQPFNYPYP QIYWVDSSAS SPWAPPGTVL PCPTSVPRRP GQRRPPPPPP PPPLPPPPPP PPLPPLPLPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG MFQLFHLQKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L (SEQ ID NO: 143, NCBI Reference Sequence NP_000630.1, UniProtKB-P48023), where residues 1-80 are cytoplasmic, aas 81-102 are the transmembrane domain, and aas 103-281 are extracellular (ectodomain).

[0251] A suitable FasL polypeptide comprises all or part of the ectodomain of FasL QLFHLQKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L (SEQ ID NO: 144).

[0252] The Fas receptor has the sequence MLGIWTLLPL VLTSVARLSS KSVNAQVTDI NSKGLELRKT VTTVETQNLE GLHHDGQFCH KPCPPGERKA RDCTVNGDEP DCVPCQEGKE YTDKAHFSSK CRRCRLCDEG HGLEVEINCT RTQNTKCRCK PNFFCNSTVC EHCDPCTKCE HGIIKECTLT SNTKCKEEGS RSNLGWLCLL LLPIPLIVWV KRKEVQKTCR KHRKENQGSH ESPTLNPETV AINLSDVDLS KYITTIAGVM TLSQVKGFVR KNGVNEAKID EIKNDNVQDT AEQKVQLLRN WHQLHGKKEA YDTLIKDLKK ANLCTLAEKI QTIILKDITS DSENSNFRNE IQSLV (SEQ ID NO: 145, NCBI Reference Sequence: NP_000034.1, UniProtKB-P25445), where aas 26 to 173 form the ectodomain (extracellular domain), aas 174 to 190 form the transmembrane domain, and aas 191 to 335 form the cytoplasmic domain. The ectodomain can be used to determine binding affinity to FasL.

[0253] In some instances, a mutant FasL polypeptide (e.g., comprising a variant of SEQ ID NO: 144) exhibits reduced binding affinity to a mature Fas receptor sequence (e.g., a FasL receptor comprising all or a portion of the polypeptide set forth in SEQ ID NO: 145, such as the ectodomain thereof) compared to the binding affinity of a FasL polypeptide comprising the aa sequence set forth in SEQ ID NO: 144. For example, in some instances, a mutant FasL polypeptide (e.g., comprising a variant of SEQ ID NO: 144) binds to a Fas receptor (e.g., comprising all or a portion of the polypeptide set forth in SEQ ID NO: 145, such as the ectodomain thereof) with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of a FasL polypeptide comprising the aa sequence set forth in SEQ ID NO: 144.

[0254] In some examples, a mutant FasL polypeptide (e.g., comprising a variant of SEQ ID NO: 144) has a binding affinity for a Fas receptor (e.g., comprising all or a portion of the polypeptide set forth in SEQ ID NO: 145, such as the ectodomain thereof) that is 1 nM to 1 mM (e.g., 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1 μM, 1 μM to 10 μM, 10 μM to 100 μM, or 100 μM to 1 mM). As another example, in some instances, mutant FasL polypeptides (including, for example, the mutant of SEQ ID NO: 144) have a potency of about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 900 nM to about 1000 nM, about 1000 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM to about 500 nM, about 500 nM to about 600 nM, about 600 nM to about 700 nM, about 700 nM to about 800 nM, about 800 nM to about 900 nM, about 1000 nM to about 2000 nM, about 1000 nM to about 2500 nM, about 1000 nM to about 3500 nM, about 1000 nM to about 3500 nM, about 1000 nM to about 4500 nM, about 1000 nM to about 5500 nM, about 1000 nM to about 2500 nM, about 1000 nM to about 2500 nM, about 1000 nM to about 3500 nM, about 1000 nM to about 3500 nM, about 1000 nM to about 4500 nM, about The polypeptide has a binding affinity for the mature Fas receptor (e.g., comprising all or a portion of the polypeptide set forth in SEQ ID NO: 145, such as the ectodomain thereof) of nM to about 1 μM, about 1 μM to about 5 μM, about 5 μM to about 10 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0255] In some cases, a mutant FasL polypeptide (e.g., including a variant of SEQ ID NO: 144) has a single aa substitution compared to the FasL polypeptide sequence set forth in SEQ ID NO: 144. In some cases, a mutant FasL polypeptide (e.g., including a variant of SEQ ID NO: 144) has 2-10 aa substitutions compared to the FasL polypeptide sequence set forth in SEQ ID NO: 144. In some cases, a mutant FasL polypeptide has a 2 aa substitution compared to the FasL polypeptide sequence set forth in SEQ ID NO: 144. In some cases, a mutant FasL polypeptide has a 3- or 4-aa substitution compared to the FasL polypeptide sequence set forth in SEQ ID NO: 144. In some cases, a mutant FasL polypeptide has a 5- or 6-aa substitution compared to the FasL polypeptide sequence set forth in SEQ ID NO: 144. In some cases, a mutant FasL polypeptide has a 7- or 8-aa substitution compared to the FasL polypeptide sequence set forth in SEQ ID NO: 144. In some examples, the mutant FasL polypeptide has a 9 aa or 10 aa substitution compared to the FasL polypeptide sequence shown in SEQ ID NO:144.

[0256] Suitable mutant FasL polypeptide sequences include polypeptide sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity (e.g., having at least one aa substitution, deletion, or insertion) to at least 50 contiguous aa of SEQ ID NO: 144 (e.g., at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 140, at least 160, or at least 180 contiguous aa).

[0257] 15 4-1BBL and its variants In some instances, the wild-type and / or mutant 4-1BBL polypeptide sequence is present as MOD. Wild-type 4-1BBL binds to 4-1BB (CD137).

[0258] The wt. 4-1BBL aa sequence can be: MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA CPWAVSGARA SPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 193, NCBI Reference Sequence: NP_003802.1), where aas 29-49 are the transmembrane region.

[0259] In some examples, the mutant 4-1BBL polypeptide is a mutant of the tumor necrosis factor (TNF) homology domain (THD) of human 4-1BBL. The wt. aa sequence of the THD of human 4-1BBL can include one of SEQ ID NOs: 194-196, for example, as follows:

[0260] PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 194),

[0261] D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 195), and

[0262] D PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPA (SEQ ID NO: 196).

[0263] The wt. 4-1BB aa sequence can be as follows: MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL (SEQ ID NO: 197).

[0264] A mutant 4-1BBL polypeptide exhibits reduced binding affinity for 4-1BB compared to the binding affinity of a 4-1BBL polypeptide comprising an aa sequence set forth in one of SEQ ID NOs: 194 to 196. For example, in some examples, a mutant 4-1BBL polypeptide may bind to 4-1BB with a binding affinity that is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower than the binding affinity of a 4-1BBL polypeptide comprising an aa sequence set forth in one of SEQ ID NOs: 194 to 196 for a 4-1BB polypeptide (e.g., a 4-1BB polypeptide comprising the aa sequence set forth in SEQ ID NO: 197) when assayed under the same conditions.

[0265] 4-1BBL variants suitable for use as MODs include polypeptides having at least one aa substitution that have at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to one of SEQ ID NOs: 194, 195, or 196.

[0266] 4-1BBL variants suitable for use as MODs include those having at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity to at least 140 (e.g., at least 160, 175, 180, or 181) consecutive aa of SEQ ID NO: 193.

[0267] E. Scaffolding The scaffold functions, inter alia, as a structural element providing a framework around which other structural components of the masked TGF-β construct or complex are organized (see, e.g., Structure A in Figure 1, with an IgFc scaffold). When the polypeptide sequence masking the TGF-β polypeptide and the TGF-β polypeptide are located in trans (on different polypeptides of the complex), the scaffold sequence, which forms mutually specific and non-mutually specific duplexes (or higher-order structures), can keep the masking polypeptide sequence associated with the TGF-β polypeptide, even during periods when the complex is in an open form and the TGF-β polypeptide sequence can interact with other molecules (not in direct contact with the masking sequence). Depending on the nature of the scaffold, it can also function as an organizing element providing higher-order structure in terms of protein folding and dimerization or multimerization (e.g., homodimerization or heterodimerization achieved through a dimerization sequence). The scaffold can also contribute to stability in serum, particularly in the case of an immunoglobulin heavy chain constant region (e.g., IgFc). A suitable scaffold polypeptide, in some instances, is a polypeptide that enhances half-life. For example, in some instances, a suitable scaffold polypeptide increases the in vivo half-life (e.g., serum half-life) of a masked TGF-β construct or complex by at least about 10%, at least about 15%, at least about 25%, at least about 50%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold, compared to a control masked TGF-β construct or complex having a scaffold polypeptide with a different, non-immunoglobulin sequence. For example, in some instances, an Ig Fc polypeptide sequence (e.g., comprising a mutually specific Ig sequence such as a knob-in-hole sequence pair) increases the stability and / or in vivo half-life (e.g., serum half-life) of a masked TGF-β construct or complex compared to a control masked TGF-β construct or complex having an Ig Fc polypeptide sequence (e.g., GGGS aa repeats of equal sequence length) replaced by a linker.The increase in in vivo half-life can be at least about 10%, at least about 15%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold. When an Ig Fc polypeptide is employed in a masked TGF-β construct, the Ig Fc can include mutations that prevent spontaneous formation of dimers of the masked TGF-β construct (see, e.g., Tianlei Ying et al., J. Biol. Chem., 287(23), pp 19399-19408 (June 1, 2012)), and, in addition, can include mutations (e.g., the LALA mutations discussed below) that substantially reduce or eliminate the ability of the Ig polypeptide to induce cell lysis, for example, through complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC).

[0268] When the scaffold polypeptide sequence of a masked TGF-β construct contains one or more sequences that enable the scaffold to interact (specifically bind) with other scaffold molecules, the masked TGF-β construct can form a homodimeric complex (see, e.g., Figure 1, structure B) or a heterodimeric complex (see, e.g., Figure 1, structures C-F). The masked TGF-β construct can also contain one or more aa sequences that enable the scaffold to interact (specifically bind) with other scaffold molecules to form higher-order structures. Sequences that form higher-order multimeric structures enable the formation of higher-order masked TGF-β constructs or complexes (e.g., trimers, tetramers, pentamers, etc.). As an example, a scaffold polypeptide containing an IgM Fc region (e.g., SEQ ID NO: 83) enables the formation of pentameric (e.g., SEQ ID NO: 84), or hexameric masked TGF-β constructs or complexes, particularly when a J chain sequence is also expressed. Petrusic et al., Med Hypotheses. 77(6):959-61(2011). Masked TGF-β constructs in the form of homo- and hetero-dimers, trimers, tetramers, pentamers, etc. are referred to as masked TGF-β complexes.

[0269] When it is desired to combine the masked TGF-β with one or more other polypeptide sequences (such as one or more specific MODs or copies of MODs), the scaffold polypeptide sequence may include mutually specific dimerization sequences that tend to form heterodimers with their counterpart mutually specific binding (dimerization) sequences. Mutual specific binding sequences may, in some instances, form homodimers, but preferentially dimerize (bind more strongly) with their counterpart mutually specific binding sequences. Thus, specific heterodimers can form (tend to form) when a mutually specific dimerization sequence and its counterpart mutually specific binding sequence are incorporated into a pair of polypeptides. By way of example, when a mutually specific dimerization sequence and its counterpart mutually specific binding sequence combine into a pair of polypeptides to preferentially form heterodimers, more than 60%, 70%, 80%, 90%, 95%, 98%, or 99% of an equimolar mixture of peptides participates in heterodimer formation, with the remaining peptides existing as monomers or homodimers.

[0270] A variety of aa sequences that specifically bind to each other or to themselves with sufficient affinity can be utilized as dimerization sequences in masked TGF-β constructs or complexes (see, e.g., U.S. Patent Publication No. 2003 / 0138440). The sequences can be of a relatively compact size (e.g., less than about 300, 250, 225, 200, 175, 150, 125, 100, 75, or 50 aa, etc.). Dimerization / multimerization sequences include, but are not limited to, immunoglobulin heavy chain constant region (Ig Fc) polypeptide sequences (sequences including the CH2 to CH3 regions of immunoglobulins, e.g., see Figures 2A to 2H and SEQ ID NOs: 68 to 83), Fc knob-in-hole sequences (e.g., SEQ ID NOs: 77 and 78), collectin family polypeptides (e.g., ACRP30 or ACRP30-like proteins) including a collagen domain consisting of collagen repeats Gly-Xaa-Yaa, coiled-coil domains, leucine zipper domains, Fos / Jun binding pairs, and Ig heavy chain region 1 (CH1) and light chain constant region CL sequences (CH1 / CL pairs, such as a CH1 sequence paired with a κ or λ Ig light chain constant region sequence).

[0271] In some embodiments, the scaffold polypeptide sequence comprises an immunoglobulin heavy chain constant region (CH2-CH3) polypeptide sequence that functions as a dimerization or multimerization sequence (see, e.g., Figures 2A-2H and SEQ ID NOs: 68-83). In embodiments, the Ig polypeptide does not substantially induce cell lysis, e.g., through activation of CDC and / or ADCC, and thus may comprise mutations that substantially reduce or eliminate the ability of the Ig polypeptide to induce cell lysis. In some examples, the Fc sequence has at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity to the aa sequence of the Fc region shown in Figures 2A-2H. Such immunoglobulin sequences can covalently link polypeptides of a masked TGF-β complex by forming one or two interchain disulfide bonds. As discussed below, additional disulfide bonds can be introduced to stabilize the dimer, particularly when a pair of mutually specific Ig sequences is used, such as a knob-in-hole polypeptide pair.

[0272] In one embodiment, the scaffold polypeptide sequence of the masked TGF-β complex comprises at least 150 contiguous aa (at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, or at least 350 contiguous aa) of the IgA Fc sequence shown in Figure 2A (SEQ ID NO: 68), or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity for all aa. In one embodiment, the scaffold polypeptide sequence comprises at least 150 contiguous aa (at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, or at least 350 contiguous aa) of the IgD Fc sequence shown in Figure 2B (SEQ ID NO: 69), or a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity for all aa. In one embodiment, the scaffold polypeptide sequence comprises a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity for at least 125 contiguous aa (at least 150, at least 175, or at least 200 contiguous aa), or for all aa, of the IgE Fc sequence shown in Figure 2C (SEQ ID NO:70). In one embodiment, the scaffold polypeptide sequence comprises a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity for at least 125 contiguous aa (at least 150, at least 175, or at least 200 contiguous aa), or for all aa, of a wt. IgG Fc polypeptide sequence, such as the IgG1 Fc sequence shown in Figure 2D (SEQ ID NOs:71-78).In one embodiment, the scaffold polypeptide sequence comprises a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity for at least 125 contiguous aa (at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, or at least 300), or all aa, of the IgG2 polypeptide sequence shown in Figure 2E (SEQ ID NO: 79). In one embodiment, the scaffold polypeptide sequence comprises a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity for at least 125 contiguous aa (at least 150, at least 175, at least 200, or at least 225), or all aa, of the IgG3 Fc sequence shown in Figure 2F (SEQ ID NO: 80). In one embodiment, the scaffold polypeptide sequence comprises a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity for at least 125 contiguous aa (at least 150, at least 175, at least 200, at least 225, or at least 250), or all aa, of the IgG4 Fc sequence shown in Figure 2G (SEQ ID NO: 81 or 82). In one embodiment, the scaffold polypeptide sequence comprises a sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity for at least 125 contiguous aa (at least 150, at least 175, at least 200, at least 225, or at least 250), or all aa, of the IgM Fc polypeptide sequence shown in Figure 2H (SEQ ID NO: 83). The above-described polypeptides of the masked TGF-β complex, including immunoglobulin scaffold polypeptide sequences (e.g., as shown in Figures 2A-2H), can be covalently linked together by the formation of one or two interchain disulfide bonds between cysteines adjacent to their hinge regions.

[0273] In some examples, the dimerization sequence of a scaffold polypeptide present in a masked TGF-β construct or complex has at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, 99%, or 100%) aa sequence identity to the human IgG1 Fc polypeptide shown in Figure 2D and includes a substitution of N297 with an alanine (the N297A substitution as numbered in Figure 2D, SEQ ID NO: 74, or N77). In some examples, the dimerization sequence of a scaffold polypeptide present in a masked TGF-β construct or complex includes the aa sequence shown in Figure 2D (human IgG1 Fc), except for the substitution of N297 with an aa other than asparagine (N77 in the aa sequence shown in Figure 2D). The substitution at N297 leads to the removal of the carbohydrate modification, resulting in an antibody sequence with reduced binding to complement component 1q ("C1q") compared to the wt. protein, and therefore, reduced CDC.

[0274] In some examples, the dimerization sequence of a scaffold polypeptide present in a masked TGF-β construct or complex comprises the aa sequence shown in FIG. 2D (human IgG1 Fc), except for the substitution of an aa other than leucine at L234 (L14 in the aa sequence shown in FIG. 2D). L234 and other aaas within the lower hinge region of IgG (e.g., aas234-LLGGPS-239, corresponding to aas 14-19 of SEQ ID NO: 71) are involved in binding to the Fc lambda receptor (FcλR), and thus mutations at that position reduce receptor binding (compared to the wt protein). In some examples, the dimerization sequence of a scaffold polypeptide present in a masked TGF-β construct or complex comprises the aa sequence shown in FIG. 2D (human IgG1 Fc), except for the substitution of an aa other than leucine at L235 (L15 in the aa sequence shown in FIG. 2D). In some examples, the dimerization sequence of a scaffold polypeptide present in a masked TGF-β construct or complex comprises the aa sequence shown in Figure 2D (e.g., a wt. human IgG1 sequence) with L234A and L235A ("LALA") substitutions (positions corresponding to positions 14 and 15 of the wt. aa sequence shown in Figure 2D, see e.g., SEQ ID NO: 75).

[0275] In some examples, the dimerization sequence of the scaffold polypeptide present in the masked TGF-β construct or complex comprises the amino acid sequence shown in FIG. 2D (human IgG1Fc) containing a substitution of P331 (P111 of the amino acid sequence shown in FIG. 2D) with an amino acid other than proline, in some examples, the substitution is a P331S substitution. Substitution at P331, like substitution at N297, leads to reduced binding to C1q relative to the wt. protein, and thus reduced CDC. For example, substitution of D270, K322, and / or P329 (corresponding to D50, K122, and P119 of SEQ ID NO: 71 in FIG. 2D) with alanine can be used alone or in place of the P331 substitution to reduce binding to C1q. In some examples, the dimerization sequence of a scaffold polypeptide present in a masked TGF-β construct or complex is an IgG1Fc polypeptide comprising an L234A and / or L235A substitution (a substitution of Ala for leucine at L14 and / or L15 of the aa sequence shown in Figure 2D). In some examples, the dimerization sequence of a scaffold polypeptide present in a masked TGF-β construct or complex comprises the aa sequence shown in Figure 2D (wt. human IgG1Fc), except for the substitution of an aa other than leucine at L234 and / or L235 (L14 and / or L15 of the aa sequence shown in Figure 2D) and the substitution of an aa other than proline at P331 (P111 of the aa sequence shown in Figure 2D). In some examples, the dimerization sequence of the scaffold polypeptide present in the masked TGF-β construct or complex comprises the "triple mutant" aa (SEQ ID NO: 73) shown in FIG. 2D (human IgG1 Fc), which comprises the L234F, L235E, and P331S substitutions (corresponding to aa positions 14, 15, and 111 of the aa sequence shown in FIG. 2D).

[0276] When asymmetric pairing between two polypeptides in a masked TGF-β complex is desired, the dimerization sequence of the scaffold polypeptide present in the masked TGF-β construct or complex comprises, consists essentially of, or consists of a mutually specific binding sequence. Mutual specific binding sequences preferentially form heterodimers with their cognate polypeptide sequences (i.e., a mutually specific sequence and its partner mutually specific sequence), particularly those based on variants of immunoglobulin Fc sequences. Such mutually specific polypeptide sequences include the knobs-in-holes (KiH) without stabilizing disulfide bonds or the KiHs-s sequences with stabilizing disulfide bonds, HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 sequences. One mutually specific binding pair includes the T366Y and Y407T mutant pair at the CH3 domain interface of IgG1 or corresponding residues in other immunoglobulins. See Ridgway et al., Protein Engineering 9:7, 617-621 (1996) (substitutions are indicated by the EU numbering scheme of Kabat et al. (1991)). A second mutually specific binding pair involves the formation of a knob by a T366W substitution and a hole by the triple substitutions T366S, L368A, and Y407V on the complementary Fc sequence. See Xu et al., mAbs 7:1, 231-242 (2015). Another mutually specific binding pair has a first Fc polypeptide with Y349C, T366S, L368A, and Y407V substitutions and a second Fc polypeptide with S354C and T366W substitutions (a disulfide bond can form between the Y349C and S354C substitutions). See Brinkmann and Konthermann, mAbs 9:2, 182-212 (2015). Fc polypeptides, with or without knob-in-hole modifications, can be stabilized by the formation of disulfide bonds (e.g., hinge region disulfide bonds) between Fc polypeptides.Some interspecific polypeptide binding sequences are summarized in Table 1, with cross-reference to the aa position numbering as it appears in the wt. IgG1 sequence (SEQ ID NO: 71) set forth in Figure 2D, shown in brackets "{}". [Table 1] TIFF2023550626000003.tif62170

[0277] In addition to the interspecific pairs of sequences in Table 1, interspecific pairs of sequences include a "SEED" sequence having 45 residues derived from IgA within the IgG1 CH3 domain of the interspecific sequence and 57 residues derived from IgG1 within the IgA CH3 domain on the counterpart interspecific sequence. See Ha et al., Frontiers in Immunol. 7:1-16 (2016).

[0278] In one embodiment, the scaffold sequence found in the masked TGF-β construct or complex comprises a mutually specific binding sequence or its counterpart mutually specific binding sequence selected from the group consisting of knobs-in-holes (KiH), knobs-in-holes with stabilizing disulfides (KiHs-s), HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, A107, and a seed sequence.

[0279] In one embodiment, the masked TGF-β complex comprises a first polypeptide comprising an IgG1 scaffold with a T146W KiH sequence substitution and a second polypeptide comprising an IgG1 scaffold with T146W, L148A, and Y187V KiH sequence substitutions, wherein the scaffold comprises a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity for at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 contiguous aa of the IgG1 of SEQ ID NO: 71. One or both scaffold aa sequences optionally include substitutions at one or more of L234 and L235 (e.g., L234A / L235A "LALA" or L234F / L235E), N297 (e.g., N297A), P331 (e.g., P331S), L351 (e.g., L351K), T366 (e.g., T366S), P395 (e.g., P395V), F405 (e.g., F405R), Y407 (e.g., Y407A), and K409 (e.g., K409Y), using Kabat numbering. These substitutions occur at positions L14 and L15 (e.g., L14A / L15A "LALA" or L14F / L15E), N77 (e.g., N77A), P111 (e.g., P111S), L131 (e.g., L131K), T146 (e.g., T146S), P175 (e.g., P175V), F185 (e.g., F185R), Y187 (e.g., Y187A), and K189 (e.g., K189Y) in the IgG1 sequence of SEQ ID NO: 71.

[0280] In one embodiment, the masked TGF-β complex comprises a first polypeptide comprising an IgG1 scaffold with a T146W KiH sequence substitution and a second polypeptide comprising an IgG1 scaffold with a T146S, L148A, and Y187V KiH sequence substitution. and a second polypeptide comprising an IgG1 scaffold with KiH sequence substitutions, wherein the scaffold comprises a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 contiguous aas of the IgG1 of SEQ ID NO: 71, and wherein zero, one, or both of the scaffold aa sequences comprise L14 and L15 substitutions (e.g., L234A and L235A "LALA") and / or N77 substitutions (N297, e.g., N297A or N297G in Kabat numbering) that eliminate effector function by blocking interaction with Fcγ receptors. See, e.g., Figure 2D, SEQ ID NOs: 77 and 78.

[0281] In one embodiment, the first and second polypeptides of the masked TGF-β complex comprise T146W and S134C KiHs-s substitutions in the first scaffold sequence and T146S, L148A, Y187V and Y129C KiHs-s substitutions in the second scaffold sequence. and KiHs-s substitutions, wherein the scaffold comprises a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 contiguous aas of an IgG1 of SEQ ID NO: 71, and wherein 0, one, or both of the scaffold aa sequences comprise L14 and L15 substitutions (e.g., L234A and L235A "LALA"), and / or N77 substitutions (N297, e.g., N297A or N297G) that eliminate effector function by blocking interaction with Fcγ receptors.

[0282] In one embodiment, the first and second polypeptides of the masked TGF-β complex comprise S144H and F185A HA-TF substitutions in a first scaffold sequence and Y129T and T174F HA-TF substitutions in a second scaffold sequence, wherein the scaffold comprises a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 contiguous aas of an IgG1 of SEQ ID NO: 71, and wherein zero, one, or both of the scaffold aa sequences comprise an L14 and L15 substitution (e.g., L234A and L235A "LALA"), and / or an N77 substitution (N297, e.g., N297A or N297G), that eliminate effector function by blocking interaction with Fcγ receptors.

[0283] In one embodiment, the first and second polypeptides of the masked TGF-β complex comprise T130V, L131Y, F185A, and Y187V ZW1 substitutions in the first scaffold sequence and T130V, T146L, K172L, and T174W in the second scaffold sequence. and a ZW1 substitution, wherein the scaffold comprises a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 contiguous aas of an IgG1 of SEQ ID NO: 71, and wherein 0, one, or both of the scaffold aa sequences comprise an L14 and L15 substitution (e.g., L234A and L235A "LALA"), and / or an N77 substitution (N297, e.g., N297A or N297G) that eliminate effector function by blocking interaction with Fcγ receptors.

[0284] In one embodiment, the first and second polypeptides of the masked TGF-β complex comprise K140D, D179M, and Y187A 7.8.60 substitutions in the first scaffold sequence and E125R, Q127R, T146V, and K189V in the second scaffold sequence. 7.8.60 substitutions, wherein the scaffold comprises a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 contiguous aas of an IgG1 of SEQ ID NO: 71, and wherein 0, one, or both of the scaffold aa sequences comprise L14 and L15 substitutions (e.g., L234A and L235A "LALA"), and / or N77 substitutions (N297, e.g., N297A or N297G) that eliminate effector function by blocking interaction with Fcγ receptors.

[0285] In one embodiment, the first and second beta polypeptides of the masked TGF-β complex contain K189D and K172D DD-KK substitutions in the first scaffold sequence and D179K and E136K in the second scaffold sequence. and DD-KK substitutions, wherein the scaffold comprises a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 contiguous aas of an IgG1 of SEQ ID NO: 71, and wherein 0, one, or both of the scaffold aa sequences comprise L14 and L15 substitutions (e.g., L234A and L235A "LALA"), and / or N77 substitutions (N297, e.g., N297A or N297G) that eliminate effector function by blocking interaction with Fcγ receptors.

[0286] In one embodiment, the first and second polypeptides of the masked TGF-β complex comprise K140E and K189W EW-RVT substitutions in the first scaffold sequence and Q127R, D179V, and F185T in the second scaffold sequence. EW-RVT substitutions, wherein the scaffold comprises a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 contiguous aas of an IgG1 of SEQ ID NO: 71, and wherein 0, one, or both of the scaffold aa sequences comprise L14 and L15 substitutions (e.g., L234A and L235A "LALA"), and / or N77 substitutions (N297, e.g., N297A or N297G) that eliminate effector function by blocking interaction with Fcγ receptors.

[0287] In one embodiment, the first and second polypeptides of the masked TGF-β complex comprise K140E, K189W, and Y129C EW-RVTs-s substitutions in the first scaffold sequence and Q127R, D179V, F185T, and S134C in the second scaffold sequence. EW-RVTs-s substitutions, wherein the scaffold comprises a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 contiguous aas of an IgG1 of SEQ ID NO: 71, and wherein 0, one, or both of the scaffold aa sequences comprise L14 and L15 substitutions (e.g., L234A and L235A "LALA"), and / or N77 substitutions (N297, e.g., N297A or N297G) that eliminate effector function by blocking interaction with Fcγ receptors.

[0288] In one embodiment, the first and second polypeptides of the masked TGF-β complex comprise K150E and K189W A107 substitutions in the first scaffold sequence and E137N, D179V, and F185T in the second scaffold sequence. and an A107 substitution, wherein the scaffold comprises a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 contiguous aas of an IgG1 of SEQ ID NO: 71, and wherein 0, one, or both of the scaffold aa sequences comprise an L14 and L15 substitution (e.g., L234A and L235A "LALA"), and / or an N77 substitution (N297, e.g., N297A or N297G) that eliminate effector function by blocking interaction with Fcγ receptors.

[0289] As an alternative to using an immunoglobulin heavy chain constant region as a scaffold, an immunoglobulin light chain constant region can be used to pair with a heavy chain CH1 sequence as a dimerization sequence that forms a scaffold polypeptide sequence or is part of a scaffold polypeptide sequence. In one embodiment, the first and second polypeptides of the masked TGF-β complex comprise an Ig CH1 domain (e.g., the polypeptide of SEQ ID NO: 85) in the first scaffold sequence and an Ig κ chain constant region sequence (SEQ ID NO: 86) in the second scaffold sequence, wherein the scaffolds each comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 70, at least 80, at least 90, at least 100, or at least 110 consecutive aa of SEQ ID NO: 85 and / or 86, respectively. See Figures 2J and 2K. The CH1 and Igκ sequences can be modified to increase their affinity for each other and, therefore, the stability of any heterodimers formed using them as dimerization sequences. Substitutions that increase the stability of CH1-Igκ heterodimers include those identified as the MD13 combination in Chen et al., MAbs, 8(4):761-774 (2016). In MD13, two substitutions are introduced into each of the CH1 and Igκ sequences. The CH1 sequence is modified to include S64E and S66V substitutions (S70E and S72V in SEQ ID NO: 85 shown in Figure 2J). The Igκ sequence is modified to include S69L and T71S substitutions (S68L and T70S in SEQ ID NO: 86 shown in Figure 2K).

[0290] In another embodiment, the first and second polypeptides of the masked TGF-β complex comprise an Ig CH1 domain (e.g., the polypeptide of SEQ ID NO: 85) in a first scaffold sequence and an Ig kappa chain constant region sequence (SEQ ID NO: 87) in a second scaffold sequence, wherein the scaffolds each comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to at least 70, at least 80, at least 90, at least 100, or at least 110 contiguous aa of SEQ ID NO: 85 and / or 87, respectively. See Figures 2J and 2K.

[0291] In some instances, the scaffold polypeptide sequences of the first and second polypeptides of the masked TGF-β complex each include a leucine zipper polypeptide as a dimerization sequence that binds to one another to form a dimer (e.g., a homodimer). Non-limiting examples of leucine zipper polypeptides include, for example, peptides having any one of the following aa sequences: RMKQIEDKIEEILSKIYHIENEIARIKKLIGER (SEQ ID NO: 88), LSSIEKKQEEQTSWLI WISNELTLIRNELAQS (SEQ ID NO: 89), LSSIEKKLEEITSQLIQISNELTLIRNELAQ (SEQ ID NO: 90), LSSIEKKLEEITSQLIQIRNELTLIRNELAQ (SEQ ID NO: 91), LSSIEKKLEEITSQLQQIRN ELTLIRNELAQ (SEQ ID NO: 92), LSSLEKKLEELTSQLIQLRNELTLLRNELAQ (SEQ ID NO: 93), ISSLEKKIEELTSQIQQLRNEITLLRNEIAQ (SEQ ID NO: 94). In some examples, the leucine zipper polypeptide comprises the following aa sequence: LEIEAAFLERENTALETRVAELRQRVQRLRNRV SQYRTRYGPLGGGK (SEQ ID NO: 95). Additional leucine-zipper polypeptides are known in the art and may be suitable for use as a scaffold or incorporation into a scaffold as a dimerization sequence.

[0292] In some examples, the scaffold polypeptide sequences of the first and second polypeptides of the masked TGF-β complex each comprise a coiled-coil peptide that forms a dimer (e.g., a homodimer). Non-limiting examples of coiled-coil polypeptides include, for example, peptides having any one of the following aa sequences: LKSVENRLAVVENQLKTVIEELKTVKDLLSN (SEQ ID NO: 96), LARIEEKLKTIKA QLSEIASTLNMIREQLAQ (SEQ ID NO: 97), VSRLEEKVKTLKSQVTELASTVSLLREQVAQ (SEQ ID NO: 98), IQSEKKIEDISSLIGQIQSEITLIRNEIAQ (SEQ ID NO: 99), LMSLEKKLEELTQTLMQL QNELSMLKNELAQ (SEQ ID NO: 100).

[0293] In some instances, the scaffold polypeptide sequences that enable dimerization (homodimerization) of the first and second polypeptides of the masked TGF-β complex each comprise a polypeptide sequence having at least one cysteine ​​residue capable of forming a disulfide bond. Examples of such polypeptide sequences include the human FasL polypeptides VDLEGSTSNGRQCAGIRL (SEQ ID NO: 101), EDDVTTTEELAPALVPPPKGTCAGWMA (SEQ ID NO: 102), and GHDQETTTQGPGVLLPLP KGACTGQMA (SEQ ID NO: 103).

[0294] Peptides suitable as oligomerization sequences that allow for the formation of masked TGF-β complexes larger than dimers (e.g., trimers, tetramers, pentamers, hexamers, etc.) include, but are not limited to, the IgM constant region (see, e.g., Figure 2H), which forms hexamers or pentamers (particularly when combined with a mature J chain peptide lacking a signal sequence, such as that provided in Figure 2I). Trimer-forming collagen domains can also be employed. The collagen domain can include the sequence Gly-Xaa-Xaa, which can be repeated 10 to 40 times, where Xaa and Yaa are independently any aa. In the Gly-Xaa-Yaa sequence, Xaa and Yaa are frequently proline and hydroxyproline, respectively, in more than 25%, 50%, 75%, 80%, 90%, or 95% of the occurrences of Gly-Xaa-Yaa, or in each occurrence of Gly-Xaa-Yaa. In some cases, the collagen domain comprises the sequence Gly-Xaa-Pro, which may be repeated 10 to 40 times. The collagen oligomerization peptide may comprise the following aa sequence: VTAFSNMDDMLQKAHLVIE GTFIYLRDSTEFFIRVRDGWKKLQLGELIPIPADSPPPP ALSSNP (SEQ ID NO: 104).

[0295] While the scaffolds discussed above are described as polypeptides, the masked TGF-β constructs and complexes described herein provide a general solution for the delivery and immobilization of active TGF-β, which can act as agonists in vitro and in vivo using a variety of scaffolds. The masked TGF-β construct or complex can be in the form of a fusion protein(s) in which the scaffold acts as a carrier, but the scaffold need not be an essentially inert carrier; it can have little or no biological activity and, at most, function as a structural support that may increase the biological half-life of the molecule. The scaffold can be protein / polypeptide or non-proteinaceous in nature, and the scaffold can be biologically active (e.g., have enzymatic activity) or biologically inactive.

[0296] When the construct has the mask and TGF-β aa sequence (arranged in cis, see Figure 1A-C and F) as part of the same polypeptide, any suitable molecule can function as the scaffold, including, but not limited to, enzymatically active proteins (enzymes) and non-enzymatically active proteins (e.g., structural proteins such as collagen, antibodies, antibody-related molecules (e.g., Fabs, nanobodies, scFvs), protein A, or all or part of a class I or class II MHC (e.g., HLA) molecule). The scaffold can also be non-proteinaceous in nature, including, but not limited to, organic and / or inorganic polymers (e.g., nucleic acids, polyethylene glycol (PEG), peptide nucleic acids, aptamers, carbohydrates, siloxanes, etc.). The scaffold can also be selected from non-polymeric materials, including, but not limited to, liposomes, vesicles, microparticles, and nanoparticles (e.g., gold nanoparticles).

[0297] When the mask and TGF-β aa sequences are arranged in trans as part of separate polypeptides (see Figure 1, D and E), any molecule that brings the mask and TGF-β aa sequences into close proximity in a manner that allows them to interact can function as a scaffold. In addition to mutually specific proteins / polypeptides, non-protein molecules (e.g., complementary nucleic acids that specifically bind to each other or a pair of aptamers) can also be used as a scaffold in which the mask and TGF-β aa sequences are located in trans. As an example, a pair of complementary nucleic acid sequences can be used as a mutually specific non-protein scaffold, for example, by using a first nucleic acid containing a masking aa sequence at its 5' end that hybridizes with a second nucleic acid having a TGF-β sequence at its 3' end.

[0298] F. TGF-β polypeptide As discussed above, masked TGF-β constructs and complexes comprise at least one TGF-β polypeptide reversibly masked by a polypeptide ("masking polypeptide") that binds to the TGF-β polypeptide. The masking polypeptide can be, for example, a TGF-β receptor polypeptide or an antibody that functions to reversibly mask the TGF-β polypeptide present in the masked TGF-β construct or complex, where the TGF-β polypeptide can otherwise act as an agonist of a cellular TGF receptor. The masked TGF-β MOD provides an active TGF-β polypeptide (e.g., a TGF-β signaling pathway agonist). The TGF-β polypeptide and the masking polypeptide (e.g., a TGF-β receptor fragment) interact with each other to reversibly mask the TGF-β polypeptide, thereby allowing the TGF-β polypeptide to interact with its cellular receptor. In addition, the masking sequence competes with cellular receptors that can capture TGF-β, such as non-signaling TβRIII, thereby allowing the masked TGF-β construct or complex to effectively deliver active TGF-β agonists.

[0299] Furthermore, while the masked TGF-β constructs or complexes of the present disclosure can include both one or more masked TGF-β polypeptide sequences and one or more additional MODs, as discussed above, if desired, the masked TGF-β constructs or complexes of the present disclosure can include only one or more masked TGF-β polypeptide sequences. That is, one or more additional MODs need not be included in the masked TGF-β construct or complex. The masked TGF-β constructs and complexes of the present disclosure can function as a means to generate a TGF-β-driven T cell response. For example, TGF-β itself can inhibit the development of effector cell function in T cells, activate macrophages, and / or promote tissue repair after local immune and inflammatory effects have subsided.

[0300] Masked TGF-β constructs and complexes contain a masked TGF-β polypeptide, but the TGF-β polypeptide can still act as a TβR agonist because the TGF-β polypeptide-mask complex is reversible, "breathing" between an open state in which the TGF-β polypeptide is available to the cellular receptor and a closed state in which the mask engages the TGF-β polypeptide. Thus, the masking polypeptide functions to bind to the TGF-β polypeptide and prevent it from forming a tight complex with, for example, ubiquitous non-signaling TβR3 molecules that could otherwise capture free TGF-β. Furthermore, because the active form of TGF-β is a dimer with higher affinity for TβR3, substitutions that limit dimerization (e.g., C77S substitution of cysteine ​​at position 77 with serine) can be incorporated into the TGF-β sequence to prevent capture by the receptor.

[0301] One effect of the masking sequence is to reduce the effective affinity of TGF-β1, TGF-β2, and TGF-β3 polypeptides for TβR. At the same time, the affinity of the masking polypeptide for the TGF-β polypeptide can be altered so that it can more easily dissociate from the TGF-β polypeptide, making the TGF-β polypeptide available to cellular TβR proteins. That is, if the affinity of the masking polypeptide for the TGF-β polypeptide is reduced, the reversibly masked TGF-β polypeptide sequence will spend more time in the open state. In the open state, where TGF-β polypeptide is available for binding to the cellular receptor, TβRII protein is generally the first peptide in the heterologous TβR1 / TβR2 signaling complex that interacts with TGF-β, so controlling the affinity of the TGF-β polypeptide for TβRII effectively controls the entry of TGF-β into the active signaling complex. For example, incorporation of substitutions at one or more, two or more, or all three of Lys25, Ile92, and / or Lys94 of TGF-β2 (or the corresponding positions in TGF-β1, TGF-β3) reduces affinity for the TβRII polypeptide, which allows control over masked TGF-β polypeptide sequence signaling.

[0302] If it is desired to block / limit signaling by the masked TGF-β polypeptide sequence through TβRI and / or to modify (e.g., reduce) the affinity of the masked TβRII polypeptide for TGF-β, N-terminal deletions and / or aa substitutions can be incorporated into the masked TβRII polypeptide. Modifications that can be made include N-terminal aa deletions (e.g., N-terminal Δ14 or Δ25 deletions) and / or substitutions at one or more of L27, F30, D32, S49, 150, T51, S52, 153, E55, V77, D118, and / or E119. Some specific TβRII modifications that result in reduced TβRI association with TβRII and reduced affinity for TGF-β include any one or more of L27A, F30A, D32A, D32N, S49A, I50A, T51A, S52A, S52L, I53A, E55A, V77A, D118A, D118R, E119A, and / or E119Q.

[0303] The TGF-β polypeptide sequence present in the masked TGF-β construct or complex is, in some instances, a mutant TGF-β polypeptide sequence that has a lower affinity for at least one class of TGF-β receptor or is a mutant that is selective for at least one class of TGF-β receptor compared to the wt. TGF-β polypeptide.

[0304] While TGF-β1, TGF-β2, or TGF-β3 polypeptide sequences can be incorporated into masked TGF-β constructs or complexes, various factors may influence the selection of a particular TGF-β polypeptide and the specific sequence and aa substitutions employed. For example, TGF-β1 and TGF-β3 polypeptides undergo "clipping" of their aa sequences when expressed in certain mammalian cell systems (e.g., CHO cells). In addition, dimerized TGF-β (e.g., TGF-β2) has a higher affinity for TβR3 (betaglycan receptor) than for the TβR2 receptor, which can result in off-target binding and loss of biologically active masked proteins to the large in vivo pool of non-signaling TβR3 molecules. To minimize high-affinity off-target binding to TβR3, it may be desirable to replace residues that connect dimeric TGF-β molecules joined by disulfide bonds. Thus, cysteine ​​77 (C77) can be substituted by an aa other than cysteine ​​(eg, serine to form a C77S substitution).

[0305] Suitable TGF-β polypeptides can have from about 70 aas to about 125 aas, for example, suitable TGF-β polypeptides can have a length of from about 70 aas to about 80 aas, from about 80 aas to about 90 aas, from about 90 aas to about 100 aas, from about 100 aas to about 105 aas, from about 105 aas to about 110 aas, from about 110 aas to about 112 aas, from about 113 aas to about 120 aas, or from about 120 aas to about 125 aas. A suitable TGF-β polypeptide sequence can include an aa sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 80, at least 90, at least 100, or at least 110 consecutive aas of the mature form of a human TGF-β1 polypeptide, a human TGF-β2 polypeptide, or a human TGF-β3 polypeptide (e.g., an ectodomain portion thereof).

[0306] 1. TGF-β1 polypeptide A suitable TGF-β1 polypeptide can comprise an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 aas of the following TGF-β1 amino acid sequence: AL DTNYCFSSTE KNCCVRQLYI DFRKDLGWKW IHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA SAAPCCVPQA LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS (SEQ ID NO: 105, 112 aas in length), wherein the TGF-β1 polypeptide has a length of approximately 112 aas. The TGF-β1 preprotein is provided in FIG. 3 as SEQ ID NO: 106. Amino acids R25, C77, V92, and R94 are shown in bold and italic (see Figure 4).

[0307] In some examples, a suitable TGF-β1 polypeptide comprises a C77S substitution. Thus, in some examples, a suitable TGF-β1 polypeptide comprises an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 aas of the following TGF-β1 aa amino acid sequence: AL DTNYCFSSTE KNCCVRQLYI DFRKDLGWKW IHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA SAAPSCVPQA LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS (SEQ ID NO: 107), in which amino acid 77 is Ser. Positions 25, 77, 92, and 94 are shown in bold and italic.

[0308] 2. TGF-β2 polypeptide A suitable TGF-β2 polypeptide can comprise an aa sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 aas of the following TGF-β2 amino acid sequence: ALDAAYCFR NVQDNCCLRP LYIDFKRDLG WKWIHEPKGY NANFCAGACP YLWSSDTQHS RVLSLYNTIN PEASASPCCV SQDLEPLTIL YYIGKTPKIE QLSNMIVKSC KCS (SEQ ID NO: 108), wherein the TGF-β2 polypeptide has a length of approximately 112 aas. The TGF-β2 preprotein is provided in FIG. 3 as SEQ ID NO: 109. Residues Lys 25, Ile 92, and / or Lys 94 are shown in bold and italic.

[0309] In some examples, a suitable TGF-β2 polypeptide comprises a C77S substitution. Thus, in some examples, a suitable TGF-β2 polypeptide comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 aas of the following TGF-β2 amino acid sequence: ALDAAYCFR NVQDNCCLRP LYIDFKRDLG WKWIHEPKGY NANFCAGACP YLWSSDTQHS RVLSLYNTIN PEASASPSCV SQDLEPLTIL YYIGKTPKIE QLSNMIVKSC KCS (SEQ ID NO: 110), wherein amino acid 77 is Ser.

[0310] 3. TGF-β3 polypeptide A suitable TGF-β3 polypeptide can include an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 aas of the following TGF-β3 amino acid sequence: ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQ LSNMVVKSCK CS (SEQ ID NO: 111), wherein the TGF-β3 polypeptide has a length of approximately 112 aas. TGF-β3 isoform 1 preproprotein is provided in FIG. 3 as SEQ ID NO: 112. Positions 25, 92, and 94 are shown in bold and italic.

[0311] In some examples, a suitable TGF-β3 polypeptide comprises a C77S substitution. In some examples, a suitable TGF-β3 polypeptide comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, or 112 aas of the following TGF-β3 amino acid sequence: ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPSCVP QDLEPLTILY YVGRTPKVEQ LSNMVVKSCK CS (SEQ ID NO: 113), in which amino acid 77 is Ser. Positions 25, 92, and 94 are shown in bold and italic.

[0312] 4. Additional TGF-β Polypeptide Sequence Variations In addition to sequence variants that alter the dimerization of TGF-β molecules (e.g., a cysteine ​​77 substitution, such as C77S), TGF-β1-3 polypeptides with sequence variants that affect affinity and other properties can be incorporated into masked TGF-β constructs or complexes. When masked TGF-β constructs or complexes contain TGF-β variants with reduced affinity for a masking polypeptide (e.g., a TβR polypeptide, such as a TβRII polypeptide), these components dissociate more readily, making the masked TGF-β polypeptide more accessible to cellular TβR proteins. Because the TβRII protein is generally the first peptide in heteromeric TβR signaling complexes that interact with TGF-β, interaction with TβRII effectively controls the entry of TGF-β into the active signaling complex. Therefore, variants that control the affinity of TGF-β for TβRII effectively control the entry of masked TGF-β constructs and complexes into the active signaling complex.

[0313] The present disclosure encompasses and provides masked TGF-β constructs and complexes comprising mutant masked TβR (e.g., TβRII) polypeptide sequences and / or mutant TGF-β polypeptides that have altered (e.g., decreased) affinity for each other (compared to an otherwise identical masked TGF-β construct or complex lacking the sequence variation(s)). The affinity between a TGF-β polypeptide and a TβR (e.g., TβRII) polypeptide can be determined using BLI (blood flow spectrometry) as described above for MODs and their co-MODs.

[0314] a. Additional TGF-β2 sequence variants The present disclosure includes and provides masked TGF-β2 constructs and complexes comprising a masked TβR (e.g., TβRII) polypeptide sequence and either a wt. or mutant TGF-β2 polypeptide, where the mutant polypeptide has reduced affinity for the masked TβR (compared to an otherwise identical wt. TGF-β polypeptide sequence without the sequence variation).

[0315] The present disclosure provides a masked TGF-β construct or complex comprising a masked TβRII receptor sequence and a mutant TβRII polypeptide having greater than 85% (e.g., greater than 90%, greater than 95%, greater than 98%, or greater than 99%) sequence identity to at least 100 consecutive aa of SEQ ID NO: 108 and including substitutions that reduce the affinity of the mutant TGF-β2 polypeptide for the TβRII receptor sequence.

[0316] In some examples, the masked TGF-β construct or complex comprises a masked TβRII polypeptide and a mutant TGF-β (e.g., TGF-β2) polypeptide comprising substitutions at one or more, two or more, or all three of Lys25, Ile92, and / or Lys94 (see SEQ ID NO: 108 for residue locations and Figure 4 for corresponding residues in TGF-β1 and TGF-β3). These aa residues have been shown to affect the affinity of TGF-β2 for TβRII polypeptides (see Crescenzo et al., J. Mol. Biol. 355:47-62 (2006)). The masked TGF-β polypeptide optionally comprises one or more independently selected MODs, such as IL-2 or variants thereof. In one example, a masked TGF-β construct or complex comprises a masked TβRII polypeptide and a TGF-β2 polypeptide having an aa other than Lys or Arg at position 25 of SEQ ID NO: 108, and optionally comprises one or more independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). In one example, a masked TGF-β construct or complex with a masked TβRII polypeptide can comprise a TGF-β2 polypeptide having an aa other than Ile or Val at position 92 of SEQ ID NO: 108 (or an aa other than Ile, Val, or Leu at position 92), and optionally comprises one or more independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). In one example, a masked TGF-β construct or complex having a masked TβRII polypeptide can include a TGF-β2 polypeptide having an aa other than Lys or Arg at position 94 of SEQ ID NO: 108, and optionally include one or more independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). A masked TGF-β construct or complex having a masked TβRII polypeptide can include a TGF-β2 polypeptide that includes substitutions of one or more, two or more, or all three of Lys25, Ile92, and / or Lys94, and can further include one or more independently selected MODs.A masked TGF-β construct or complex having a masking TβRII polypeptide may include a TGF-β2 polypeptide containing substitutions of one or more, two or more, or all three of Lys25, Ile92, and / or Lys94, and may further include one or more independently selected IL-2 MODs or affinity-reduced variants thereof.

[0317] b. Additional TGF-β1 and TGF-β3 sequence variants In some examples, the masked TGF-β construct or complex comprises a masked TβRII polypeptide and a mutant TGF-β1 or TGF-β3 polypeptide containing substitutions at one or more, two or more, or all three aa positions corresponding to Lys25, Ile92, and / or Lys94 in SEQ ID NO: 108 of TGF-β2. In TGF-β1 or TGF-β3, the corresponding aa positions are as follows: Lys25 is Arg25, Ile92 is Val92, and Lys94 is Arg94, each of which is a conservative substitution. See, for example, SEQ ID NOs: 106 and 107 for TGF-β1, and SEQ ID NOs: 112 and 113 for TGF-β3.

[0318] As described above, a masked TGF-β construct or complex optionally includes one or more independently selected MODs, such as IL-2 or variants thereof. In one example, a masked TGF-β construct or complex with a masked TβRII polypeptide includes a TGF-β1 or β3 polypeptide with an aa other than Arg or Lys at position 25, and optionally includes one or more independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). In one example, a masked TGF-β construct or complex with a masked TβRII polypeptide includes a TGF-β1 or β3 polypeptide with an aa other than Val or Ile at position 92 (or an aa other than Ile, Val, or Leu at position 92), and optionally includes one or more independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). In another example, a masked TGF-β construct or complex having a masked TβRII polypeptide comprises a TGF-β2 polypeptide having an aa other than Arg or Lys, and optionally comprises one or more independently selected MODs (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). In one particular example, a masked TGF-β construct or complex having a masked TβRII polypeptide comprises a TGF-β1 or β3 polypeptide comprising one or more, two or more, or all three substitutions of Arg25, Val92, and / or Arg94, and further comprises one or more independently selected MODs. In another particular example, a masked TGF-β construct or complex having a masked TβRII polypeptide comprises a TGF-β1 or β3 polypeptide comprising one or more, two or more, or all three substitutions of Arg25, Val92, and / or Arg94, and further comprises one or more independently selected IL-2 MODs or affinity-reduced variants thereof.

[0319] G. TGF-β Receptor Polypeptides and Other Polypeptides that Bind and Mask TGF-β In any of the TGF-β polypeptides or polypeptide complexes described above, the polypeptide that binds to and masks the TGF-β polypeptide (the "masking polypeptide") can take a variety of forms, including TβRI, TβRII, TβRIII, and anti-TGF-β antibodies, or fragments thereof (e.g., Fab, single-chain antibodies, etc.).

[0320] 1. TGF-β receptor polypeptide Masking of TGF-β in masked TGF-β constructs and complexes can be achieved by utilizing a TGF-β receptor fragment (e.g., the ectodomain sequence of TβRI, TβRII, or TβRIII) that contains sufficient polypeptide sequence to bind to a TGF-β polypeptide (e.g., TGF-β1, TGF-β2, or TGF-β3). In one embodiment, the masking sequence comprises all or a portion of the TβRI, TβRII, or TβRIII ectodomain.

[0321] TGF-β receptor I (TβRI) In one embodiment, the polypeptide sequence that masks TGF-β within a masked TGF-β construct or complex can include all or a portion of the TβRI ectodomain (aas 34-126), which can be derived from TβRI (e.g., isoform 1, SEQ ID NO: 114). In some examples, a suitable TβRI polypeptide for masking TGF-β comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, or 103 aas of the following TβRI ectodomain aa sequence: LQCFCHL CTKDNFTCVT DGLCFVSVTE TTDKVIHNSM CIAEIDLIPR DRPFVCAPSS KTGSVTTTYC CNQDHCNKIE LPTTVKSSPG LGPVEL (SEQ ID NO: 115).

[0322] b. TGF-β receptor II (TβRII) In embodiments, the polypeptide sequence that masks TGF-β in a masked TGF-β construct or complex may be derived from TβRII (e.g., sequence number 116 of isoform A) and may include all or part of the TβRII ectodomain sequence (aa 24-177). In one embodiment, a suitable TβRII isoform A polypeptide for masking TGF-β comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, or 154 aas of the following TβRII isoform A ectodomain aa sequence: IPPHVQK SDVEMEAQKD EIICPSCNRT AHPLRHINND MIVTDNNGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCV AVWRKNDENI TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSS D ECND NIIFSEE (SEQ ID NO: 117). The position of the aspartic acid residue corresponding to D118 in the B isoform is shown in bold, underlined, and italicized.

[0323] In one embodiment, the polypeptide sequence that masks TGF-β in the masked TGF-β construct or complex may be derived from TβRII isoform B (sequence number 118) and may include all or part of the TβRII ectodomain sequence (aa 24-166). In one embodiment, a suitable TβRII isoform B polypeptide for masking TGF-β comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, or 103 aas of the following aa sequence of the TβRII isoform B ectodomain: IPPHVQKSVN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEEY NTSNPDLLLV IFQ (SEQ ID NO: 119). As discussed below, any one or more of F30, D32, S52, E55, or D118 (italicized and bolded) may be substituted with an amino acid other than the naturally occurring aa at those positions (e.g., alanine). A polypeptide sequence that masks TGF-β may include the polypeptide of SEQ ID NO: 119 having a D118A or D118R substitution. A sequence that masks TGF-β may include the peptide of SEQ ID NO: 119 having a D118A or D118R substitution and one or more of F30A, D32N, S52L, and / or E55A substitutions.

[0324] The ectodomain of TβRII can be used as a masking polypeptide; however, regions of the protein contain charged and hydrophobic patches that confer an unfavorable pI and may be toxic to cells expressing the polypeptide. Additionally, combining the TβRII ectodomain with an active TGF-β polypeptide can result in a complex that combines with cell-surface TβRI and can activate its signaling receptor (e.g., signaling through the Smad pathway). Modifying the TβRII ectodomain sequence used to mask TGF-β by removing or altering sequences involved in TβRI association can prevent unintended intracellular stimulation by the masked TGF-β other than through its own cell-surface heterodimeric TβRI / TβRII complex. Modification of TβRII can also alter (e.g., decrease) the affinity of TβRII for TGF-β (e.g., TGF-β3), thereby allowing for controlled unmasking of TGF-β and increasing the availability of TGF-β as a signaling molecule. Masked TGF-β constructs or complexes containing TβR (e.g., TβRII) peptides with the highest affinity for TGF-β (e.g., TGF-β3) mask the TGF-β sequence most tightly and require higher doses to achieve the same effect. In contrast, affinity-reducing aa substitutions in TβRII unmask the TGF-β polypeptide and are biologically effective at lower doses. See, for example, Example 4.

[0325] Thus, if it is desired to block / limit signaling by a masked TGF-β polypeptide via TβRI and / or modify (e.g., reduce) the affinity of a masked TβRII polypeptide for TGF-β, a number of modifications to TβRII can be incorporated into the TβRII polypeptide sequence. Modifications that can be made include deletion of up to 25 N-terminal amino acids (e.g., 1-25 aa in length, such as a 14 aa deletion (Δ14) or a 25 aa deletion (Δ25)) as described above, and / or substitution with one or more of L27, F30, D32, S49, 150, T51, S52, 153, E55, V77, D118, and / or E119. Some specific modifications in the masked TβRII sequence that result in reduced TβRI association with masked TβRII and reduced / altered affinity for TGF-β include any one or more of L27A, F30A, D32A, D32N, S49A, I50A, T51A, S52A, S52L, I53A, E55A, V77A, D118A, D118R, E119A, and / or E119Q, based on SEQ ID NO: 119. See, e.g., J. Groppe et al. Mol Cell 29, 157-168, (2008) and De Crescenzo et al. JMB 355, 47-62 (2006). See Figure 9 for the effects of these substitutions on TGF-β3-TβRII and TβRI-TβRII complexes. Modifications of TβRII, including N-terminal Δ25 deletions and / or substitutions at F24 (e.g., F24A substitutions), substantially or completely block signaling through the canonical SMAD signaling pathway. In one embodiment, the aspartic acid at position 118 (D118) of the mature TβRIIB isoform (SEQ ID NO: 119) is substituted with an amino acid other than Asp or Glu, such as Ala, resulting in a "D118A" substitution, or a D118R substitution with Arg. The Asp residue corresponding to D118 is shown in SEQ ID NOs: 117-123 (bold and underlined in Figure 5B). N-terminal deletions of 1-25 aa in length (e.g., Δ25 deletions) and / or substitutions at F24 (e.g., F24A substitutions) can be combined with D118 substitutions (e.g., D118A or D118R).N-terminal deletions of 1 to 25 aa in length (e.g., deletion of Δ25) and / or substitutions at F24 (e.g., substitution of F24A) may also be combined with substitutions at any of the L27, F30, D32, S49, 150, T51, S52, I53, E55, V77, D118, and / or E119 substitutions (e.g., D118A), and in particular any of the specific substitutions listed for those positions in SEQ ID NO: 119 above to modify affinity.

[0326] N-terminal deletion of the TβRII polypeptide can also result in loss of interaction with TβRI, preventing masked TGF-β constructs and complexes containing the TβRII polypeptide from acting as constitutively active complexes that participate in and activate TβRI signaling. A 14-aa deletion (Δ14) of the TβRII polypeptide substantially reduces the protein's interaction with TβRI, and a Δ25-aa deletion of TβRII is believed to completely block its interaction with TβRI. N-terminal deletions also substantially alter the protein's pI, with the Δ14 TβRII ectodomain mutant exhibiting a pI of approximately 4.5 to 5.0 (e.g., approximately 4.74). Thus, a TGF-β construct or complex can include a TβRII ectodomain polypeptide (e.g., the polypeptide of SEQ ID NO: 117 or 118) having an N-terminal deletion, such as 14 to 25 aa (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aa). Modified ectodomain sequences, including those that limit interaction with TβRI, that can be utilized to mask TGF-β polypeptides in a masked TGF-β construct or complex are described in the following paragraphs.

[0327] In one embodiment, the sequence that masks TGF-β in the masked TGF-β construct or complex comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, or 103 aas of the following TβRII isoform B ectodomain sequence: IPPHVQKSVN NDMIVTDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEE (SEQ ID NO: 120). Any one or more of F30, D32, S52, E55, or D118 (italicized and bolded) may be substituted with an amino acid other than the aa naturally occurring at those positions (e.g., alanine). In one embodiment, the TGF-β masking sequence comprises the peptide of SEQ ID NO: 120 having a D118A substitution. In one embodiment, the TGF-β masking sequence comprises the polypeptide of SEQ ID NO: 120 having a D118A substitution and one or more of F30A, D32N, S52L, and / or E55A substitutions.

[0328] Combinations of N-terminal deletions of TβRII, e.g., 14-25 aa (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aa), that block undesired intracellular signaling resulting from a masked TGF-β / TβRII complex interacting with TβRI, can be combined with other TβRII ectodomain substitutions, including TβRII ectodomain substitutions at any one or more of positions F30, D32, S52, E55, and / or D118. The combination of deletions and substitutions ensures that the masked TGF-β construct or complex does not trigger cell signaling other than through the membrane-bound TβRI and TβRII receptors in the cell.

[0329] In one embodiment, the sequence that masks TGF-β in the masked TGF-β construct or complex comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, or 103 aas of the following TβRII isoform B ectodomain sequence: TDNNG AVKFPQLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEE (SEQ ID NO: 121), with aas 1 to 14 (Δ14) deleted. Any one or more of F30, D32, S52, E55, or D118 (italicized and bolded) may be substituted with an amino acid other than the aa naturally occurring at those positions (e.g., alanine). In one embodiment, the TGF-β masking sequence comprises the peptide of SEQ ID NO: 120 having a D118A substitution. In one embodiment, the TGF-β masking sequence comprises the polypeptide of SEQ ID NO: 121 having a D118A substitution and one or more of F30A, D32N, S52L, and / or E55A substitutions.

[0330] In one embodiment, the sequence masking TGF-β in the masked TGF-β construct or complex comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, or 103 aa of the sequence (SEQ ID NO: 122) with aas 1 to 25 (Δ25) deleted: QLCKF CDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSSDEC NDNIIFSEE (SEQ ID NO: 122). Any one or more of F30, D32, S52, E55, or D118 (italicized and bolded) may be substituted with an amino acid other than the aa naturally occurring at those positions (e.g., alanine). In one embodiment, the TGF-β masking sequence comprises the polypeptide of SEQ ID NO: 122 having a D118A substitution (shown as SEQ ID NO: 123 in FIG. 5B). In one embodiment, the TGF-β masking sequence in a masked TGF-β construct or complex comprises the peptide of SEQ ID NO: 122 having a D118A substitution and one or more of F30A, D32N, S52L, and / or E55A substitutions. In one embodiment, the TGF-β masking sequence in a masked TGF-β construct or complex comprises the peptide of SEQ ID NO: 122 having a D118A and F30A substitutions (see FIG. 5B). In one embodiment, the sequence that masks TGF-β in a masked TGF-β construct or complex comprises the peptide of SEQ ID NO: 122 with D118A and D32N substitutions (see Figure 5B). In one embodiment, the sequence that masks TGF-β in a masked TGF-β construct or complex comprises the peptide of SEQ ID NO: 122 with D118A and S52L substitutions (see Figure 5B).In one embodiment, the sequence that masks TGF-β in the masked TGF-β construct or complex comprises the peptide of SEQ ID NO: 122 having D118A and E55A (see Figure 5B).

[0331] c. TGF-β receptor III (TβRIII) In one embodiment, the polypeptide sequence that masks TGF-β in a masked TGF-β construct or complex can be derived from TβRIII (e.g., isoform A, SEQ ID NO: 124 and isoform B, 125) and can include all or a portion of the TβRIII ectodomain (aas 27-787 of the A isoform or 27-786 of the B isoform). In some examples, a TβRIII polypeptide suitable for masking TGF-β comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity to at least 70, at least 80, at least 90, at least 100, or 120 aas of the ectodomain sequence of the TβRIII A or B isoform (e.g., as provided in FIG. 5C as SEQ ID NO: 124 or SEQ ID NO: 125).

[0332] 2. Antibodies Although TGF-β receptor polypeptides (e.g., ectodomain sequences) can function to bind to and mask TGF-β polypeptides in masked TGF-β constructs or complexes, other polypeptide sequences (protein sequences) that bind to TGF-β sequences can also be used as masking polypeptides. Suitable polypeptide or protein sequences that can be used to mask TGF-β include antibodies with affinity for TGF-β (e.g., antibodies specific for one or more of TGF-β1, TGF-β2, or TGF-β3), or fragments thereof, nanobodies with affinity for TGF-β polypeptides, and particularly single-chain anti-TGF-β antibodies (e.g., any of which may be humanized). Several antibodies, including scFV antibodies, that bind to and neutralize TGF-β have been reported. See, e.g., U.S. Patent No. 9,090,685. In consideration of the embodiments and / or aspects of the invention described in this disclosure, the TβR (e.g., TβRII) sequence used to mask the TGF-β polypeptide may be replaced with a masking antibody sequence (e.g., scFV or nanobody) having affinity for the TGF-β polypeptide. For example, in each of the masked TGF-β constructs or complexes of FIG. 1 in which a TGF-β receptor sequence is used to mask the TGF-β polypeptide, the receptor polypeptide may be replaced with a masking antibody polypeptide (e.g., scFV or nanobody) having affinity for the TGF-β polypeptide.

[0333] One potential advantage of using an antibody (e.g., a single-chain antibody) as a masking polypeptide is its ability to limit the isoform of the TGF-β polypeptide(s) being masked. As an example, a single-chain antibody sequence based on methelimumab (CAT192) against TGF-β1 (e.g., Lord et al., mAbs 10(3):444-452(2018)) can be used to mask that TGF-β isoform when present in a TGF-β construct or complex. In another embodiment, a single-chain antibody sequence specific for TGF-β2 is used to mask that TGF-β isoform when present in a TGF-β construct or complex. In another embodiment, a single-chain antibody sequence specific for TGF-β3 is used to mask that TGF-β isoform when present in a TGF-β construct or complex. Single chain antibodies can also be specific for combinations of TGF-β isoforms (e.g., ectodomain sequences present in a masked TGF-β construct or complex selected from the group consisting of TGF-β1 & TGF-β2, TGF-β1 & TGF-β3, and TGF-β2 & TGF-β3). Single chain antibodies can also be generally specific for TGF-β1, TGF-β2, and TGF-β3 ectodomain sequences present in a masked TGF-β construct or complex. See, e.g., WO 2014 / 164709. Antibodies and single chain antibodies with desired specificity and affinity for TGF-β isoforms can be prepared by a variety of methods, including screening hybridomas and / or modifying (e.g., combinatorially modifying) variable region sequences of antibodies with affinity for the target TGF-β polypeptide sequence.

[0334] In one embodiment, the masked TGF-β construct or complex comprises a single chain antibody for masking a TGF-β sequence (e.g., a TGF-β3 sequence). In one such embodiment, the single chain amino acid sequence is specific for TGF-β3 as set forth in SEQ ID NO: 111, which contains a C77S substitution (see SEQ ID NO: 112).

[0335] H. Linker As noted above, a masked TGF-β construct or complex may include a linker peptide / polypeptide sequence interposed between any two elements of the masked TGF-β construct or complex. Although the term "linker" is used, sequences similar to the linker below may also be placed at the N- and / or C-termini of the polypeptide of the masked TGF-β construct or complex, e.g., as protection against proteolysis.

[0336] Suitable linkers (also referred to as "spacers") can be readily selected and can be any of several suitable lengths, such as 1 aa to 25 aa, 3 aa to 20 aa, 2 aa to 15 aa, 3 aa to 12 aa, 4 aa to 10 aa, 5 aa to 9 aa, 6 aa to 8 aa, or 7 aa to 8 aa. Suitable linkers can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aa in length. Suitable linkers can be 25 to 35 aa in length. Suitable linkers can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 aa in length. Suitable linkers may also be 35-45 aa in length. Suitable linkers may be 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 aa in length. Suitable linkers may be 45-50 aa in length. Suitable linkers may be 45, 46, 47, 48, 49, or 50 aa in length.

[0337] Exemplary linkers include those that contain glycine, or polyglycine-containing sequences of about 2 to about 50 (e.g., 2-4, 4-7, 7-10, 10-20, 20-35, or 35-50) consecutive glycine residues, glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 126), and (GGGS) n(SEQ ID NO: 127), where n is at least one integer (e.g., 1-10, 10-20, or 20-30), glycine-alanine polymers, or alanine-serine polymers (e.g., having a length of 1-10, 10-20, or 20-30 aa), as well as other flexible linkers known in the art. Glycine or glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore function as neutral tethers between components. Glycine polymers can be used; glycine recognizes significantly more phi-psi space than alanine, and is significantly less restricted than residues with long side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers may include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 128), GGSGG (SEQ ID NO: 129), GSGSG (SEQ ID NO: 130), GSGGG (SEQ ID NO: 131), GGGSG (SEQ ID NO: 132), GSSSG (SEQ ID NO: 133), and the like. Exemplary linkers may include, for example, GGSG (SEQ ID NO: 134), which may be repeated 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some examples, the linker includes the amino acid sequence (GSSSS) (SEQ ID NO: 135), which may be repeated 2, 3, or 4 times. In some examples, the linker includes the amino acid sequence (GSSSS) (SEQ ID NO: 135), which is repeated 4 or 5 times. Exemplary linkers include, for example, (GGGGS) (SEQ ID NO: 136), which may be repeated 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some examples, the linker comprises the amino acid sequence (GGGGS) (SEQ ID NO: 136) repeated once or twice. In some examples, the linker comprises the amino acid sequence (GGGGS) (SEQ ID NO: 136) repeated three or four times. In some examples, the linker comprises the amino acid sequence (GGGGS) (SEQ ID NO: 136) repeated five, six, or seven times. In some examples, the linker comprises the amino acid sequence (GGGGS) (SEQ ID NO: 136) repeated eight, nine, or ten times.

[0338] In some instances, a linker polypeptide present in a first polypeptide of a masked TGF-β complex comprises a cysteine ​​residue capable of forming a disulfide bond with a cysteine ​​residue present in a second polypeptide of the masked TGF-β construct or complex. In some instances, for example, a suitable linker comprises the amino acid sequence G C Contains GASGGGGSGGGGS (SEQ ID NO: 137).

[0339] I. Exemplary Masked TGF-β Constructs and Complexes As discussed above, in any of the masked TGF-β constructs and complexes described in this disclosure, the masking polypeptide that binds to and masks the TGF-β polypeptide sequence can take a variety of forms. The masking peptide can be an antibody, a binding fragment of an antibody, a single-chain antibody or portion thereof that binds to TGF-β (e.g., an scFv), or a nanobody, any of which can be humanized. The masking polypeptide can also be a TGF-β receptor fragment (e.g., the ectodomain sequence of TβRI, TβRII, or TβRIII) that contains sufficient polypeptide sequence to bind to a TGF-β polypeptide (e.g., TGF-β1, TGF-β2, or TGF-β3).

[0340] In any of the masked TGF-β constructs and complexes described above, the TGF-β polypeptide sequence used can be based on TGF-β1, TGF-β2, or TGF-β3. In one embodiment, the TGF-β polypeptide comprises a TGF-β3 sequence. The full-length mature TGF-β protein sequence is not required for masked TGF-β constructs and complexes; only the portion of TGF-β necessary to interact with cell surface TβRII and for the masked TGF-β complex to recruit TβRI along with cell surface TβRII, thereby initiating signal transduction (e.g., signaling through Smad and non-Smad pathways).

[0341] As discussed above, an immunomodulatory polypeptide (MOD) is not required for the delivery of masked TGF-β or its ability to activate TβRI- and TβRII-bearing cells, although the presence of a MOD can substantially affect the outcome of TGF-β cell activation. Thus, incorporation of a MOD into any of the masked TGF-β constructs and complexes described above can be used to achieve various effects, including therapeutic benefits, from the use of the masked TGF-β constructs and complexes described herein. In one embodiment, the MOD present in the masked TGF-β construct or complex is selected from the group consisting of PD-L1, Fas-L, 4-1BBL, IL-2, IL-4, IL-6, IL-7, IL-21, IL-23, and mutant forms of any of these, including those with reduced affinity for their co-MODs.

[0342] While it may be desirable to incorporate a MOD into masked TGF-β constructs and complexes, its presence is not required in all cases, particularly when the masked TGF-β constructs and complexes are administered with other materials, including cytokines (e.g., one or more independently selected interleukins, lymphokines, interferons, chemokines, and / or tumor necrosis factors). For example, where it is desirable to support the development of conventional CD8+ T cells (or the survival of low-affinity CD8+ T cells) by promoting thymocyte expression of the interleukin-7 receptor (e.g., IL-7Ra), masked TGF-β constructs and complexes that do not contain a MOD polypeptide ("MOD-free") may be used. Similarly, where it is desirable to promote the development of T cell populations induced by potent agonist ligands, masked TGF-β constructs and complexes, with or without a MOD, may be used to support the survival of thymus-derived Tregs (tTregs), invariant natural killer T (iNKT), and CD8αα+ T cell precursors.

[0343] The following are non-limiting examples of masked TGF-β constructs and complexes.

[0344] 1. Masked TGF-β Construct In the case of a masked TGF-β construct, all of the components (e.g., TGF-β, scaffold, masking polypeptide such as a TβRII sequence, and optionally one or more MODs) are part of a single polypeptide chain (see, e.g., Figure 1, structure A). In such embodiments, the scaffold polypeptides do not form dimers or higher order structures with other scaffold polypeptides, and thus the masked TGF-β construct is not in the form of a homodimer, heterodimer, or higher order multimeric structure (such as a trimer).

[0345] In the case of the masked TGF-β construct of Figure 1, in structure A, the polypeptide may optionally include, from N-terminus to C-terminus, one or more MODs, a scaffold polypeptide (not including a mutually specific binding sequence), a polypeptide that binds to and masks the TGF-β polypeptide, and a TGF-β polypeptide sequence. Such masked TGF-β constructs include the following: (i) a polypeptide comprising, from N-terminus to C-terminus, any one or more independently selected wt or affinity-reduced mutant MODs, a scaffold polypeptide (not including a mutually specific binding sequence), a TβR polypeptide that binds to and masks a TGF-β polypeptide, and a TGF-β polypeptide sequence; (ii) a polypeptide comprising, from N-terminus to C-terminus, any one or more independently selected wt or affinity-reduced mutant MODs, a scaffold polypeptide (not including a mutually specific binding sequence), a TβRII polypeptide that binds to and masks a TGF-β polypeptide, and a TGF-β polypeptide sequence; (iii) a polypeptide comprising, from N-terminus to C-terminus, one or more independently selected wt or affinity-reduced mutant MODs, a scaffold polypeptide (not including a mutually specific binding sequence), a TβR polypeptide that binds to and masks a TGF-β polypeptide, and a TGF-β polypeptide sequence; (iv) a polypeptide comprising, from N-terminus to C-terminus, one or more independently selected wt or affinity-reduced mutant IL-2 MOD, a scaffold polypeptide (not including a mutually specific binding sequence), a TβR polypeptide that binds to and masks a TGF-β polypeptide, and a TGF-β polypeptide sequence; (v) a polypeptide comprising, from N-terminus to C-terminus, one or more independently selected wt or affinity-reduced mutant MODs, a scaffold polypeptide (not including a mutually specific binding sequence), a TβR polypeptide that binds to and masks a TGF-β3 polypeptide, and a TGF-β3 polypeptide sequence; (vi) a polypeptide comprising, from N-terminus to C-terminus, one or more independently selected wt or affinity-reduced mutant MODs, a scaffold polypeptide (not including a mutually specific binding sequence), a TβRII polypeptide that binds to and masks a TGF-β3 polypeptide, and a TGF-β3 polypeptide sequence; and (vii) A polypeptide comprising, from N-terminus to C-terminus, one or more independently selected wt or affinity-reduced mutant IL-2 MOD, a scaffold polypeptide (not including a mutually specific binding sequence), a TβRII polypeptide that binds to and masks a TGF-β3 polypeptide, and a TGF-β3 polypeptide sequence.

[0346] In any of the examples of masked TGF-β constructs described herein, C77 of the TGF-β polypeptide sequence may be substituted to prevent dimerization (e.g., a C77S substitution), and the TGF-β polypeptide may further include variations to reduce affinity for the masked TβR polypeptide (e.g., one, two, or all three of aas 25, 92, and / or 94) along with modifications to the MOD and TβR polypeptide sequences. Exemplary TβR polypeptide sequences that may be incorporated into masked TGF-β constructs include Δ14 or Δ25 TβRII polypeptides, optionally with a D118A or D118R substitution to attenuate TβRI engagement. MOD variants are described along with their polypeptide sequences, and additional modifications of TβRI, TβRII, and TβRIII are described above.

[0347] In one embodiment, the masked TGF-β construct has the sequence set forth in SEQ ID NO: 146 (see Figure 7A). In one embodiment, the masked TGF-β construct has the sequence set forth in SEQ ID NO: 147 (see Figure 7B). In one embodiment, the masked TGF-β construct has the sequence set forth in SEQ ID NO: 157 (see Figure 7G). In one embodiment, the masked TGF-β construct has the sequence set forth in SEQ ID NO: 158 (see Figure 7H). In one embodiment, the masked TGF-β construct has the sequence set forth in SEQ ID NO: 159 (see Figure 71).

[0348] 2. Masked TGF-β complex A masked TGF-β complex comprises at least two polypeptides, a first and a second polypeptide, each of which comprises a scaffold polypeptide that associates with another scaffold polypeptide to bring the first and second polypeptides together into a complex, such that the TGF-β polypeptide complex forms a homodimer, heterodimer, or higher order multimeric structure. (i) In a first example, a masked TGF-β complex includes at least one TGF-β polypeptide sequence, at least one polypeptide that binds to and masks one or more TGF-β polypeptides (e.g., a masking sequence for each TGF-β polypeptide sequence), and, optionally, one or more immunomodulatory polypeptides (MODs), assembled on a scaffold structure that can dimerize to form a homodimer (e.g., a symmetric dimer), such as structure B in Figure 1. In such a homodimer, the IgFc polypeptide can include a mutation (e.g., the LALA mutation discussed herein) that allows for the spontaneous formation of disulfide bonds between the IgFc polypeptides within the scaffold of each construct and that substantially reduces or eliminates the ability of the Ig polypeptide to induce cell lysis through, for example, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). (ii) In a second example, the masked TGF-β complex is (a) a first polypeptide comprising at least one TGF-β polypeptide sequence, at least one polypeptide that binds to and masks one or more TGF-β polypeptides (e.g., a masking sequence for each TGF-β polypeptide sequence), and, optionally, one or more immunomodulatory polypeptides (MODs), assembled on a scaffold comprising mutually specific dimerization sequences; and (b) a second polypeptide comprising at least one TGF-β polypeptide sequence, at least one polypeptide that binds and masks the at least one TGF-β polypeptide, and optionally one or more immunomodulatory polypeptides (MODs), assembled on a scaffold comprising a sequence that is a counterpart to the mutually specific dimerization sequence of the first polypeptide; Here, the first and second polypeptides form a heterodimer through the interaction of mutually specific dimerization sequences, such as structure C in FIG. (iii) In a third example, the masked TGF-β complex is (a) a first polypeptide comprising at least one TGF-β polypeptide sequence, at least one polypeptide that binds to and masks at least one or more TGF-β polypeptides (e.g., a masking sequence of each TGF-β polypeptide sequence), and, optionally, one or more immunomodulatory polypeptides (MODs), assembled on a scaffold comprising mutually specific dimerization sequences; and (b) a second polypeptide comprising a scaffold comprising a sequence that is a counterpart to the mutually specific dimerization sequence of the first polypeptide and, optionally, one or more immunomodulatory polypeptides (MODs); Here, the first and second polypeptides form a heterodimer through the interaction of mutually specific dimerization sequences, such as structure F in FIG. (iv) In a fourth example, the masked TGF-β complex is (a) a first polypeptide comprising at least one TGF-β polypeptide sequence and, optionally, one or more immunomodulatory polypeptides (MODs), assembled on a scaffold comprising mutually specific dimerization sequences; and (b) a second polypeptide comprising at least one polypeptide that binds to and masks at least one or more TGF-β polypeptides, and optionally one or more immunomodulatory polypeptides (MODs), assembled on a scaffold comprising a sequence that is a counterpart to the mutually specific dimerization sequence of the first polypeptide; Here, the first and second polypeptides form heterodimers via mutually specific dimerization sequences, such as structures D and E in FIG.

[0349] In some examples, in a masked TGF-β complex (FIG. 1, Structures B, C, and F), the sequence comprising a TGF-β polypeptide (first polypeptide) can optionally include, from N- to C-terminus, one or more MODs, a scaffold polypeptide (with or without a mutually specific binding sequence), a polypeptide that binds to and masks the TGF-β polypeptide, and a TGF-β polypeptide sequence. The polypeptide without the TGF-β sequence in FIG. 1, Structure F (second polypeptide), includes a scaffold polypeptide with a mutually specific binding sequence and optionally includes a MOD at the N-terminus, C-terminus, or both the N-terminus and C-terminus.

[0350] In some examples, in the masked TGF-β complexes of structures D and E in Figure 1, the TGF-β polypeptide-containing polypeptide (first polypeptide) can comprise, from N-terminus to C-terminus, one or more optional MODs, a scaffold polypeptide (with mutually specific binding sequences), and a TGF-β polypeptide sequence. The TGF-β sequence-free polypeptide (second polypeptide) of structures D and E in Figure 1 can comprise, from N-terminus to C-terminus, optionally one or more MODs, a scaffold polypeptide with mutually specific binding sequences, and a polypeptide that binds to and masks the TGF-β polypeptide. Although not shown in Figure 1, the first polypeptide comprising the TGF-β polypeptide sequence can be free of one or more MODs, and the second polypeptide comprising the masking sequence can comprise one or more MODs.

[0351] Examples of the masked TGF-β complexes described above include those in which the first polypeptide comprises, from N-terminus to C-terminus: (i) optionally, one or more MOD, a scaffold polypeptide (having mutually specific binding sequences), and a TGF-β polypeptide sequence; (ii) optionally, one or more independently selected wt or affinity-reduced mutant MOD, scaffold polypeptide (having mutually specific binding sequences), and TGF-β polypeptide sequence; (iii) one or more independently selected wt or affinity-reduced mutant MODs, scaffold polypeptides (having mutually specific binding sequences), and TGF-β1 or 2 polypeptide sequences; (iv) one or more independently selected wt or affinity-reduced mutant IL-2 MOD, a scaffold polypeptide (without mutually specific binding sequences), and a TGF-β polypeptide sequence; (v) one or more independently selected wt or affinity-reduced mutant MOD, a scaffold polypeptide (having mutually specific binding sequences), and a TGF-β3 polypeptide sequence; (vi) one or more independently selected wt or affinity-reduced mutant MOD, a scaffold polypeptide (having mutually specific binding sequences), and a TGF-β3 polypeptide sequence; (vii) one or more independently selected wt or affinity-reduced mutant IL-2 MOD, a scaffold polypeptide (having a mutually specific binding sequence), and a TGF-β3 polypeptide sequence. In each example, the second polypeptide comprises, from N- to C-terminus, a scaffold polypeptide comprising a mutually specific binding (dimerization sequence) partner of the first polypeptide, followed by a TβR (e.g., TβRII) polypeptide that binds to and masks the TGF-β polypeptide of the first polypeptide. In the case of a masked TGF-β complex such as Figure 1, Structure F, the TβR (e.g., TβRII) polypeptide may be positioned between the N-terminal MOD (if present) and the scaffold of the first polypeptide, and the second polypeptide comprises a mutually specific binding (dimerization sequence) partner of the first polypeptide, to which one or more independently selected wt or affinity-reduced mutant MODs (e.g., wt or mutant IL-2 MODs) may be bound at the N- or C-terminus.

[0352] In any of the examples of masked TGF-β complexes described herein, C77 of the TGF-β polypeptide sequence may be substituted to prevent dimerization (e.g., a C77S substitution), and the TGF-β polypeptide may further include variations to reduce affinity for the masked TβR polypeptide (e.g., one, two, or all three of aas 25, 92, and / or 94), along with modifications to the MOD and TβR polypeptide sequences. Exemplary TβR polypeptide sequences that may be incorporated into masked TGF-β constructs include a Δ14 or Δ25 TβRII polypeptide, optionally with a D118A substitution. MOD variants are described along with their polypeptide sequences, and additional modifications of TβRI, TβRII, and TβRIII are described above.

[0353] In one embodiment, the masked TGF-β complex comprises polypeptides having the sequences set forth in SEQ ID NOs: 148 and 149 (see FIG. 7C). In one embodiment, the masked TGF-β complex comprises polypeptides having the sequences set forth in SEQ ID NOs: 150 and 151 (see FIG. 7D). In one embodiment, the masked TGF-β complex comprises polypeptides having the sequences set forth in SEQ ID NOs: 152 and 153 (see FIG. 7E). In one embodiment, the masked TGF-β complex comprises polypeptides having the sequences set forth in SEQ ID NOs: 155 and 156 (see FIG. 7F). In one embodiment, the masked TGF-β complex comprises polypeptides having the sequences set forth in SEQ ID NOs: 148 and 160 (see FIG. 7J).

[0354] J. Tissue-specific targeting moieties The constructs and complexes disclosed herein can further comprise a moiety capable of targeting the constructs and complexes to a particular tissue in the body. Such a targeting moiety can comprise, for example, an antibody or antigen-binding fragment / portion thereof (e.g., an scFv, a nanobody such as a heavy chain nanobody or a light chain nanobody, a Fab, a diabody, or a multimeric scFv). The targeting moiety can be incorporated directly into the masked TGF-β constructs or complexes described herein in the same manner and at the same position as the MOD polypeptide sequence. For example, the nanobody sequence can be located at one of the positions where the MOD appears in the structure shown in Figure 1. Alternatively, tissue-specific targeting can be achieved using a bivalent molecule that recognizes both a molecule specific for the target tissue and the masked TGF-β construct or complex. For example, masked TGF-β constructs or complexes (e.g., scaffolds, linkers, or added amino acid sequences such as 6X His tags) and bivalent and bispecific antibody constructs such as diabodies that are specific for target tissue-specific molecules (see, e.g., Holliger et al. PNAS 90:6444-48 (1993)) can achieve tissue-specific targeting.

[0355] The molecules specific to tissues involved in autoimmune diseases / disorders that can be used for targeting are specific to each individual autoimmune disease / condition. For example, joint-specific targeting in rheumatoid arthritis can be achieved using extracellular matrix components expressed in inflamed joint tissue. These RA-related extracellular matrix components include the extra domain-A of fibronectin (ED-A), for which a targeting antibody fragment (F8) has already been identified. See, for example, Bruijnen et al., Mol. Pharm., 6(1):273-281 (2019). Tenascin represents another RA-related target molecule.

[0356] Mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is primarily expressed in intestinal mucosal tissue and represents a tissue-specific target for localization of masked TGF-β constructs or complexes for use in treating inflammatory bowel disease (IBD) and / or Crohn's disease. Similarly, blood-brain barrier (BBB)-specific proteins and neural tissue-specific proteins represent tissue-specific target proteins for the treatment of MS. Pancreatic islet β cell-specific proteins represent tissue-specific targets for the treatment of type 1 diabetes (T1D). Additional tissue-specific target proteins associated with various autoimmune diseases are known in the art.

[0357] K. Nucleic acids The present disclosure provides nucleic acids comprising nucleotide sequences encoding masked TGF-β constructs or complexes. In some examples, the nucleic acids are recombinant expression vectors, and thus the present disclosure provides recombinant expression vectors comprising nucleotide sequences encoding masked TGF-β constructs or complexes. In some examples, the nucleic acids are recombinant expression vectors, and thus the present disclosure provides recombinant expression vectors comprising nucleotide sequences encoding masked TGF-β constructs and complexes. The discussion of nucleic acids below deals with nucleic acids encoding the masked TGF-β constructs and complexes of the present disclosure.

[0358] Nucleic Acids Encoding Single-Chain Antigen-Presenting Polypeptides As described above, the masked TGF-β construct comprises a single-chain polypeptide. Thus, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the single-chain masked TGF-β construct. The nucleic acid comprising a nucleotide sequence encoding the single-chain masked TGF-β construct can be operably linked to a transcriptional control element(s), such as a promoter.

[0359] Nucleic acid(s) encoding the masked TGF-β complex As described above, in some examples, a masked TGF-β complex comprises at least two distinct polypeptide chains (a first polypeptide chain and a second polypeptide chain). The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a masked TGF-β complex. In some examples, each individual polypeptide chain of a masked TGF-β complex is encoded within an individual nucleic acid. In some examples, all polypeptide chains of a masked TGF-β construct or complex are encoded within a single nucleic acid. In some examples, a first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a masked TGF-β complex, and a second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of a masked TGF-β complex. In some examples, a single nucleic acid comprises nucleotide sequences encoding the first and second polypeptides of a masked TGF-β complex, which may be operably linked under the transcriptional control of a single promoter or two independently selected promoters.

[0360] Individual nucleic acids encoding individual polypeptide chains of a masked TGF-β construct or complex As described above, in some cases, the individual polypeptide chains of the masked TGF-β complex are encoded within individual nucleic acids. In some cases, the nucleotide sequences encoding the individual polypeptide chains of the masked TGF-β complex are operably linked to a transcriptional control element, such as a promoter, e.g., a promoter that is functional in eukaryotic cells, and the promoter can be a constitutive promoter or an inducible promoter.

[0361] For example, the present disclosure provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of a masked TGF-β complex, and the second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of the masked TGF-β complex. In some examples, the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide are operably linked to a transcriptional control element. In some examples, the transcriptional control element is a promoter functional in eukaryotic cells. In some examples, the nucleic acids are present in individual expression vectors.

[0362] In some cases, the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide are operably linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that is functional in eukaryotic cells. In some cases, the nucleic acids are present in individual expression vectors.

[0363] Nucleic acids encoding two or more polypeptides present in a masked TGF-β complex The present disclosure provides nucleic acids comprising nucleotide sequences encoding at least a first polypeptide and a second polypeptide of a masked TGF-β complex. In some examples, the masked TGF-β complex comprises a first, second, and third polypeptide, and the nucleic acid comprises nucleotide sequences encoding the first, second, and third polypeptides. In some examples, the nucleotide sequences encoding the first and second polypeptides of the masked TGF-β complex encode a proteolytic cleavable site or linker disposed between the encoded first and second polypeptides. In some examples, the nucleotide sequences encoding the first and second polypeptides of the masked TGF-β complex comprise nucleotides encoding an internal ribosome entry site (IRES) disposed between the encoded first and second polypeptides. In some examples, the nucleotide sequences encoding the first and second polypeptides of the masked TGF-β complex comprise a sequence encoding a ribosome skipping signal (or cis-acting hydrolase element, CHYSEL) disposed between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide.

[0364] In some examples, a first nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) comprises a nucleotide sequence encoding a first polypeptide chain of a masked TGF-β complex, and a second nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) comprises a nucleotide sequence encoding a second polypeptide chain of a masked TGF-β complex. In some examples, the nucleotide sequence encoding the first polypeptide and the second nucleotide sequence encoding the second polypeptide are each operably linked to an independently selected transcriptional control element, such as a promoter, such as a promoter functional in eukaryotic cells, which can be a constitutive promoter or an inducible promoter.

[0365] Recombinant Expression Vectors The present disclosure provides a recombinant expression vector that comprises nucleic acid.In some examples, the recombinant expression vector is a non-viral vector.In some examples, the recombinant expression vector is a viral construct, for example, recombinant adeno-associated viral construct (see, for example, U.S. Patent No. 7,078,387), recombinant adenoviral construct, recombinant lentiviral construct, recombinant retroviral construct, non-integral viral vector, etc.

[0366] Suitable expression vectors include viral vectors (e.g., vaccinia virus, poliovirus, adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649; WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 00655), adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86,1998, Flannery et al.,PNAS 94:6916 6921,1997,Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863,1997,Jomary et al.,Gene Ther 4:683 690,1997,Rolling et al.,Hum Gene Ther 10:641 648,1999, Ali et al., Hum Mol Genet 5:591 594,1996, Srivastava in WO93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828, Mendelson et al., Virol. (1988) 166:154-165, and Flotte et al. al., PNAS (1993) 90:10613-10617), SV40, herpes simplex virus, human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997, Takahashi et al.Examples of suitable vectors include, but are not limited to, viral vectors based on human leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus. Many suitable expression vectors are known to those of skill in the art, and many are commercially available.

[0367] Depending on the host / vector system utilized, any of a number of suitable transcriptional and translational control elements, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, etc., may be used in the expression vector (see, e.g., Bitter et al (1987) Methods in Enzymology, 153:516-544).

[0368] In some examples, the nucleotide sequence encoding the polypeptide of the masked TGF-β constructs and complexes is operably linked to a control element, e.g., a transcription control element, such as a promoter. The transcription control element may be functional in either eukaryotic cells (e.g., mammalian cells) or prokaryotic cells (e.g., bacterial or archaeal cells). In some examples, the nucleotide sequence encoding the DNA-targeting RNA and / or site-directed modifying polypeptide is operably linked to multiple control elements that allow expression of the nucleotide sequence encoding the DNA-targeting RNA and / or site-directed modifying polypeptide in both prokaryotic and eukaryotic cells.

[0369] Non-limiting examples of suitable eukaryotic promoters (promoters functional in eukaryotic cells) include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retroviruses, and mouse metallothionein-I. Selection of appropriate vectors and promoters is well within the level of ordinary skill in the art. Expression vectors may also contain a ribosome binding site for translation initiation and a transcription terminator. Expression vectors may also include appropriate sequences for amplifying expression.

[0370] Preparation of genetically modified host cells expressing masked TGF-β constructs and complexes and purification of masked TGF-β constructs and complexes The present disclosure provides genetically engineered host cells, which are genetically engineered with one or more nucleic acid(s) encoding a masked TGF-β construct or complex.

[0371] Suitable host cells include eukaryotic cells such as yeast cells, insect cells, and mammalian cells. In some cases, the host cells are cells of a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

[0372] Genetically engineered host cells can be used to produce masked TGF-β constructs or complexes. For example, genetically engineered host cells can be used to produce masked TGF-β complexes or single-chain masked TGF-β constructs by introducing into the host cell an expression vector(s) as described above containing a nucleotide sequence encoding a polypeptide(s), thereby producing a genetically engineered host cell. In the host cell, the masked TGF-β construct or complex can be constitutively expressed, or it can be expressed in response to exposure to an inducible inducer using a promoter to drive expression (e.g., a CMV promoter and a tetracycline resistance operon that is induced by exposure to tetracycline).

[0373] The masked TGF-β construct or complex is obtained from cells or, if the polypeptide(s) are designed to enter the secretory pathway by incorporating a signal sequence, from the cell culture medium. The protein can be purified by any means known in the art, including, for example, one or more of precipitation (with ammonium sulfate or ethanol), isoelectric focusing, and one or more types of chromatography. Suitable chromatographic methods include, but are not limited to, size-based chromatographic separation (e.g., size exclusion or gel permeation), hydrophobic interaction chromatography, ion exchange chromatography, and affinity chromatography. If the masked TGF-β construct or complex contains an immunoglobulin polypeptide sequence (e.g., as a scaffold), Protein A or Protein G can be used to affinity purify the masked TGF-β construct or complex. In the absence of immunoglobulin polypeptides, the complex may be affinity purified using an antibody that recognizes the masked TGF-β construct or a polypeptide present in the complex, or alternatively, by incorporating an affinity tag such as a myc epitope (CEQKLISEEDL SEQ ID NO: 154), a "HIS" tag (for divalent metal ion resin binding), or a "FLAG" tag. Purification and / or concentration steps, which may be combined with any of the foregoing methods, employ size-limiting semi-permeable membranes (e.g., dialysis membranes or pressure cells) that can be used to remove contaminants having substantially different molecular weights and / or to concentrate the purified protein.

[0374] In one embodiment, the masked TGF-β construct or complex is expressed from a nucleic acid sequence introduced into mammalian cells (e.g., CHO cells) and targeted to the secretory pathway for secretion from the cells into their culture medium (e.g., serum-free medium). The masked TGF-β construct or complex is purified from the cell culture medium using affinity chromatography alone or in combination with size-based separation (e.g., size-based chromatography or membrane separation). In a particular example of such an embodiment, the masked TGF-β construct or complex comprises an immunoglobulin scaffold (e.g., an IgG polypeptide sequence), and the purification is carried out using affinity chromatography (e.g., protein A or G) alone or in combination with size-based separation (size-based chromatography).

[0375] L. Composition The present disclosure provides compositions (including pharmaceutical compositions) comprising a masked TGF-β construct or complex. The present disclosure also provides compositions (including pharmaceutical compositions) comprising a nucleic acid or recombinant expression vector of the present disclosure.

[0376] 1. Compositions Comprising Masked TGF-β Constructs or Complexes In addition to the masked TGF-β construct or complex, the compositions of the present disclosure may include one or more of the following: a salt, e.g., NaCl, MgCl, KCl, MgSO, etc.; a buffer, e.g., Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a surfactant, e.g., a non-ionic surfactant such as Tween-20; a protease inhibitor; glycerol, etc.

[0377] The composition may include a pharmaceutically acceptable excipient, the types of which are well known in the art and need not be discussed in detail here. Pharmaceutically acceptable excipients are described, for example, in "Remington: The Science and Practice of Pharmacy," 1999. th Ed. (1995) or latest edition, Mack Publishing Co., A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins, Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Cansel et al., eds. 7 th ed., Lippincott, Williams, & Wilkins, and Handbook of Pharmaceutical Excipients (2000) AHKibbe et al., eds.,3 rd ed.Amer.Pharmaceutical Assoc.

[0378] The pharmaceutical composition can include i) a masked TGF-β construct or complex, and ii) a pharmaceutically acceptable excipient. In some examples, the pharmaceutical composition is suitable for administration to a subject, e.g., sterile. For example, in some embodiments, the pharmaceutical composition is suitable for administration to a human subject, e.g., the composition is sterile and substantially free of detectable pyrogens and / or other toxins, or such detectable pyrogens and / or other toxins are present at levels within acceptable limits set by applicable regulatory agencies, e.g., USF&DA.

[0379] Protein compositions may also contain other ingredients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonates, etc. The compositions may also contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, hydrochloride salts, sulfate salts, solvates (e.g., mixed ionic salts, water, organic materials), hydrates (e.g., water), etc.

[0380] For example, compositions may include aqueous solutions, powder forms, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc. Compositions may be formulated for various routes of administration, as described below.

[0381] When a masked TGF-β construct or complex is utilized (e.g., introduced into a cell culture system) or administered directly to a tissue as an injection (e.g., subcutaneously, intraperitoneally, intramuscularly, intralymphaticly, and / or intravenously), the formulation can be provided in a ready-to-use form, or in a non-aqueous form (e.g., a reconstitutable, shelf-stable powder) or an aqueous form (e.g., a liquid comprised of a pharmaceutically acceptable carrier and excipients). Protein-containing formulations can also be provided...

Claims

1. For the treatment of a metabolic disease or disorder, one or more independently selected masked TGF-β constructs; (ii) one or more independently selected masked TGF-β complexes, and / or (iii) one or more nucleic acids encoding said one or more independently selected masked TGF-β constructs or complexes.

2. 2. The one or more masked TGF-β constructs or the one or more masked TGF-β complexes of claim 1, wherein the metabolic disease or disorder is selected from the group consisting of familial hypercholesterolemia, Gaucher's disease, Hunter syndrome, Krabbe's disease, maple syrup urine disease, metachromatic leukodystrophy, cystic fibrosis, mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS), Niemann-Pick disease, phenylketonuria (PKU), porphyria, sickle cell anemia, Tay-Sachs disease, Wilson's disease, type 2 diabetes (T2D), pre-T2D, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).

3. A) the masked TGF-β construct comprises: i) a scaffold polypeptide sequence; ii) a TGF-β polypeptide sequence; iii) a masking polypeptide sequence optionally selected from a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; iv) optionally, one or more independently selected immunomodulatory polypeptide sequences (MODs); and v) optionally, a first polypeptide comprising one or more independently selected linker polypeptide sequences, the masking polypeptide sequence and the TGF-β polypeptide sequence are linked to each other; and B) the masked TGF-β complex comprises a first polypeptide and a second polypeptide linked to each other as a heterodimer; (i) the first polypeptide comprises: a) a scaffold polypeptide sequence comprising a mutually specific dimerization sequence; b) a masking polypeptide sequence optionally selected from a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; c) optionally, one or more independently selected MODs, and d) optionally, one or more independently selected linker polypeptide sequences; and (ii) the second polypeptide is a) a scaffold polypeptide sequence comprising a mutually specific dimerization sequence that corresponds to the mutually specific dimerization sequence in the first polypeptide; b) a TGF-β polypeptide sequence; c) optionally, one or more independently selected MODs; and d) optionally, one or more independently selected linker polypeptide sequences, the masking polypeptide sequence and the TGF-β polypeptide sequence reversibly bind to each other; the mutually specific binding sequence and the corresponding mutually specific binding sequence interact with each other in the heterodimer; and 2. The one or more masked TGF-β constructs or one or more masked TGF-β complexes of claim 1, wherein the first polypeptide and / or the second polypeptide of the masked TGF-β optionally comprise one or more independently selected linker polypeptide sequences.

4. one or more independently selected masked TGF-β complexes; (i) the first polypeptide comprises, from the N-terminus to the C-terminus: a) one or more independently selected MOD sequences, the scaffold polypeptide sequence comprising the mutually specific dimerization sequence, and the masking polypeptide sequence; or b) the scaffold polypeptide sequence comprising the mutually specific dimerization sequence, and the masking polypeptide sequence; and 4. The one or more masked TGF-β constructs or one or more masked TGF-β complexes of claim 3, wherein (ii) the second polypeptide comprises, from N-terminus to C-terminus, one or two independently selected MOD sequences, the scaffold polypeptide sequence comprising the corresponding mutually specific dimerization sequences, the TGF-β polypeptide sequence.

5. (i) the scaffold polypeptide sequences of the first and second polypeptides comprise an Ig Fc polypeptide sequence comprising a mutation that substantially reduces or eliminates the ability of the Ig polypeptide to induce cell lysis through complement dependent cytotoxicity (CDC) and / or antibody dependent cellular cytotoxicity (ADCC); and (ii) the masking polypeptide sequence is a TGF-β receptor ("TβR") polypeptide sequence comprising an ectodomain fragment of type I (TβRI), type II (TβRII), or type III (TβRIII) TβR, 5. The one or more masked TGF-β constructs or one or more masked TGF-β complexes of claim 4, wherein the MOD is independently selected from the group consisting of PD-L1, FAS-L, 4-1BBL, IL-2, IL-10 MOD, and mutant forms thereof.

6. (i) the scaffold polypeptide sequences of the first and second polypeptides comprise a mutually specific dimerization sequence and a corresponding mutually specific dimerization sequence pair selected from the group consisting of KiH, KiHs-s, HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 sequence pairs; (ii) the masking polypeptide sequence comprises a TβRII polypeptide having at least 90% or at least 95% aa sequence identity to a TβRII polypeptide sequence provided in SEQ ID NO: 117 or 119-123; and (iii) the TGF-β polypeptide sequence comprises a TGF-β3 polypeptide sequence having at least 90% or at least 95% aa sequence identity to SEQ ID NO: 112 or 113, wherein:

6. The one or more masked TGF-β constructs or one or more masked TGF-β complexes of claim 5, wherein said MOD is independently selected from the group consisting of IL-2 MOD and mutant forms thereof.

7. (i) the scaffold polypeptide sequences of the first and second polypeptides comprise a KIH or KIHs-s mutually specific dimerization sequence and a corresponding mutually specific dimerization sequence pair; (ii) the masking polypeptide sequence comprises a TβRII polypeptide having at least 90% or at least 95% aa sequence identity to the TβRII polypeptide sequences provided in SEQ ID NOs: 121-123; and (iii) the TGF-β polypeptide sequence comprises a TGF-β3 polypeptide sequence having at least 90% or at least 95% aa sequence identity to SEQ ID NO: 113, and comprises a C77S substitution, the MOD is independently selected from the group consisting of an IL-2 MOD and an IL-2 mutant MOD comprising an H16A and / or an F42A sequencing substitution; 7. One or more masked TGF-β constructs or one or more masked TGF-β complexes according to claim 6.

8. the one or more independently selected masked TGF-β complexes comprise a polypeptide of SEQ ID NO: 191 (construct 4033) and / or a polypeptide of SEQ ID NO: 103 (construct 4039); or 8. The one or more masked TGF-β complexes of claim 7, wherein the one or more independently selected masked TGF-β complexes comprise an amino acid sequence having at least 90% or 95% aa sequence identity to SEQ ID NO: 191 or 192.

9. The first polypeptide comprises, in order from the N-terminus to the C-terminus: i) the scaffold polypeptide sequence, the masking polypeptide sequence, and the TGF-β polypeptide sequence; ii) a first MOD, the scaffold polypeptide sequence, the masking polypeptide sequence, and the TGF-β polypeptide sequence; or iii) a first independently selected MOD, a second independently selected MOD, the scaffold polypeptide sequence, the masking polypeptide sequence, and the TGF-β polypeptide sequence; 4. The one or more masked TGF-β constructs of claim 3, wherein the masked TGF-β constructs optionally comprise one or more independently selected linker polypeptide sequences.

10. The one or more masked TGF-β constructs or the one or more masked TGF-β complexes of claim 3, further comprising a targeting moiety.

11. The one or more masked TGF-β constructs or the one or more masked TGF-β complexes according to claim 10, wherein the targeting moiety is an antibody or an antigen-binding fragment / portion thereof.

12. A pharmaceutical composition for the treatment of a metabolic disease or disorder comprising one or more masked TGF-β constructs or one or more masked TGF-β complexes according to any one of claims 1 to 11.

13. A pharmaceutical composition for use in the treatment of a metabolic disease or disorder comprising one or more masked TGF-β constructs or one or more masked TGF-β complexes according to any one of claims 1 to 11.

14. Use of one or more masked TGF-β constructs or one or more masked TGF-β complexes according to any one of claims 1 to 11 in the manufacture of a pharmaceutical composition for the treatment of a metabolic disease or disorder.

15. A) A masked TGF-β construct comprising: i) a scaffold polypeptide sequence; ii) a TGF-β polypeptide sequence; iii) a masking polypeptide sequence optionally selected from a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; iv) optionally, one or more independently selected immune modulating polypeptide sequences (MODs); v) optionally, one or more independently selected linker polypeptide sequences, and vi) a targeting moiety, wherein a first polypeptide, wherein the masking polypeptide sequence and the TGF-β polypeptide sequence bind to each other; and B) the masked TGF-β complex comprises a first polypeptide and a second polypeptide linked to each other as a heterodimer; (i) the first polypeptide comprises: a) a scaffold polypeptide sequence comprising a mutually specific dimerization sequence; b) a masking polypeptide sequence optionally selected from a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; c) optionally, one or more independently selected MODs, and d) optionally, one or more independently selected linker polypeptide sequences; and (ii) the second polypeptide is a) a scaffold polypeptide sequence comprising a mutually specific dimerization sequence that corresponds to the mutually specific dimerization sequence in the first polypeptide; b) a TGF-β polypeptide sequence; c) optionally, one or more independently selected MODs, and d) optionally, one or more independently selected linker polypeptide sequences, the masking polypeptide sequence and the TGF-β polypeptide sequence are linked to each other; the mutually specific binding sequence and the corresponding mutually specific binding sequence interact with each other in the heterodimer; and The masked TGF-β construct or the masked TGF-β complex, wherein the first polypeptide and / or the second polypeptide of the masked TGF-β optionally comprises one or more independently selected linker polypeptide sequences, and wherein the first or second polypeptide comprises a targeting moiety.

16. The masked TGF-β construct or masked TGF-β complex of claim 15, wherein the targeting moiety is an antibody or an antigen-binding fragment / portion thereof.

17. one or more independently selected masked TGF-β complexes; (i) the first polypeptide comprises, from the N-terminus to the C-terminus: a) one or more independently selected MOD sequences, the scaffold polypeptide sequence comprising the mutually specific dimerization sequence, and the masking polypeptide sequence; or b) the scaffold polypeptide sequence comprising the mutually specific dimerization sequence, and the masking polypeptide sequence; (ii) the second polypeptide comprises, from N-terminus to C-terminus, one or two independently selected MOD sequences, the scaffold polypeptide sequence comprising the corresponding mutually specific dimerization sequences, and the TGF-β polypeptide sequence; (iii) the scaffold polypeptide sequence of the first and second polypeptides comprises an Ig Fc polypeptide sequence comprising a mutation that substantially reduces or eliminates the ability of the Ig polypeptide to induce cell lysis through complement dependent cytotoxicity (CDC) and / or antibody dependent cellular cytotoxicity (ADCC); and (iv) the masking polypeptide sequence is a TGF-β receptor ("TβR") polypeptide sequence comprising an ectodomain fragment of type I (TβRI), type II (TβRII), or type III (TβRIII) TβR, 16. The masked TGF-β construct or masked TGF-β complex of claim 15, wherein said MOD is independently selected from the group consisting of PD-L1, FAS-L, 4-1BBL, IL-2, IL-10 MOD, and mutant forms thereof.

18. (i) the scaffold polypeptide sequences of the first and second polypeptides comprise a mutually specific dimerization sequence and a corresponding mutually specific dimerization sequence pair selected from the group consisting of KiH, KiHs-s, HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 sequence pairs; (ii) the masking polypeptide sequence comprises a TβRII polypeptide having at least 90% or at least 95% aa sequence identity to a TβRII polypeptide sequence provided in SEQ ID NO: 117 or 119-123; and (iii) the TGF-β polypeptide sequence comprises a TGF-β3 polypeptide sequence having at least 90% or at least 95% aa sequence identity to SEQ ID NO: 112 or 113, wherein:

18. The masked TGF-β construct or masked TGF-β complex of claim 17, wherein said MOD is independently selected from the group consisting of IL-2 MOD and mutant forms thereof.

19. (i) the scaffold polypeptide sequences of the first and second polypeptides comprise a KIH or KIHs-s mutually specific dimerization sequence and a corresponding mutually specific dimerization sequence pair; (ii) the masking polypeptide sequence comprises a TβRII polypeptide having at least 90% or at least 95% aa sequence identity to the TβRII polypeptide sequences provided in SEQ ID NOs: 121-123; and (iii) the TGF-β polypeptide sequence comprises a TGF-β3 polypeptide sequence having at least 90% or at least 95% aa sequence identity to SEQ ID NO: 113, and comprises a C77S substitution, 20. The masked TGF-β construct or masked TGF-β complex of claim 18, wherein said MOD is independently selected from the group consisting of an IL-2 MOD polypeptide sequence and an IL-2 mutant MOD comprising an H16A and / or an F42A sequencing substitution.

20. The masked TGF-β complex comprises the polypeptide amino acid sequence of SEQ ID NO: 191 (construct 4033) and / or the polypeptide amino acid sequence of SEQ ID NO: 103 (construct 4039), or 16. The masked TGF-β construct or masked TGF-β complex of claim 15, wherein the masked TGF-β complex comprises an amino acid sequence having at least 90% or 95% aa sequence identity to SEQ ID NO: 191 and / or 192.

21. The first polypeptide comprises, in order from the N-terminus to the C-terminus: i) the scaffold polypeptide sequence, the masking polypeptide sequence, and the TGF-β polypeptide sequence; ii) a first MOD, the scaffold polypeptide sequence, the masking polypeptide sequence, and the TGF-β polypeptide sequence; or iii) a first independently selected MOD, a second independently selected MOD, the scaffold polypeptide sequence, the masking polypeptide sequence, and the TGF-β polypeptide sequence; Optionally, the masked TGF-β construct comprises one or more independently selected linker polypeptide sequences.

22. 22. The masked TGF-β construct or masked TGF-β complex of any one of claims 15 to 21, wherein, if one or more MODs are present, at least one of the one or more MODs comprises an IL-2 polypeptide sequence selected from SEQ ID NOs: 13-27 or SEQ ID NOs: 139-141.

23. 23. The masked TGF-β construct or masked TGF-β complex of claim 22, wherein, when one or more MODs are present, at least one of the one or more MODs comprises an IL-2 polypeptide sequence of SEQ ID NO:20, or an IL-2 mutant polypeptide sequence comprising an H16A and / or an F42A sequence substitution.

24. A pharmaceutical composition comprising a masked TGF-β construct or a masked TGF-β complex according to any one of claims 15 to 21.

25. 25. The pharmaceutical composition of claim 24, comprising one or more doses of the masked TGF-β construct or masked TGF-β complex, and a pharma- ceutically acceptable excipient, wherein the one or more doses comprise an amount of the TGF-β construct or TGF-β complex independently selected in the range of 0.1 mg / kg body weight to 10 mg / kg body weight.

26. Use of a masked TGF-β construct or a masked TGF-β complex according to any one of claims 15 to 21 in the manufacture of a pharmaceutical composition for the treatment of an autoimmune disease or disorder, a metabolic disease or disorder, or graft-versus-host disease (GVHD).

27. A masked TGF-β construct or a masked TGF-β complex according to any one of claims 15 to 21 for use in the treatment of an autoimmune disease or disorder, a metabolic disease or disorder, or graft-versus-host disease (GVHD).

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