TGF-beta polypeptides

Abstract

This disclosure provides transforming growth factor β (TGF-β) peptide constructs and complexes that can be used for the therapeutic treatment of diseases, including autoimmune diseases. Nucleic acids encoding the constructs and complexes, as well as methods for preparing the constructs and complexes in a cell-based expression system, are also described.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080074437.3, the original application being the Chinese national phase application of international application PCT / US2020 / 056937 filed on October 22, 2020.

[0002] Cross-reference to related applications This application claims the benefit of U.S. Provisional Application No. 62 / 925,227, filed October 23, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0003] This application belongs to the field of biomedical technology, specifically relating to transforming growth factor β (TGF-β) polypeptide. Background Technology

[0004] A. TGF-β and its effects Transforming growth factor β (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, which contains a propeptide region in addition to the homodimeric TGF-β sequence, the active form of TGF-β. TGF-β is secreted by macrophages and other cell types in the form of a latent complex, in which it is combined with two other peptides: latent TGF-β binding protein (LTBP) and latent-related peptide (LAP). The latent TGF-β complex is stored in the extracellular matrix (ECM) and, for example, is bound to the cell surface via CD36 through platelet-reactive protein-1 (wherein the complex can be activated by cytoplasm) or to latent transforming growth factor β binding proteins 1, 2, 3, and / or 4 (LTBP1-4).

[0005] Following activation of latent TGF-β, the biological functions of TGF-β were observed, with the activation response to ECM perturbations being tightly regulated. TGF-β can be activated via a variety of cell- or tissue-specific pathways or pathways observed in multiple cell or tissue types; however, the complete mechanisms underlying such activation pathways are not fully understood. Activators include, but are not limited to, proteases, integrins, pH, and reactive oxygen species (ROS). In fact, cell / tissue-bound latent TGF-β complexes act as environmental perturbations, sensing and responding to them to release active TGF-β spatially and / or temporally. The released TGF-β promotes or inhibits cell proliferation, depending on the environment in which it is released. It also recruits stem cells / progenitor cells to participate in tissue regeneration / remodeling processes. Abnormalities in TGF-β ligand expression, bioavailability, activation, receptor function, or post-transcriptional modifications can disrupt normal function and lead to pathological consequences associated with many diseases, such as by recruiting excessive progenitor cells (e.g., in osteoarthritis or Camurati–Engelmann disease) or by transdifferentiating resident cells into unfavorable lineages (e.g., the epithelial-mesenchymal transition during cancer metastasis or tissue / organ fibrosis). Xu et al. Bone Research , 6 (Paper No. 2) (2018).

[0006] 1. Integrin-independent and integrin-dependent activation a. Integrin-independent activation Integrin-independent TGF-β activation occurs through mechanisms such as proteases and / or metalloproteinases, reactive oxygen species (ROS), and platelet-reactive protein-1.

[0007] Cytoplasmin and several matrix metalloproteinases (MMPs) promote tumor invasion and tissue remodeling through the proteolysis of extracellular matrix components. TGF-β can be activated by these proteases, which release latent complexes from the matrix, followed by proteolysis of the extracellular matrix protein (LAP) to release TGF-β to its receptor. Matrix metalloproteinases -9 and -2 are known to cleave latent TGF-β.

[0008] It has been demonstrated that TGF-β can be rapidly activated in vivo following radiation exposure to induce ROS release. It is believed that ROS alters the interaction between LAP and TGF-β, leading to its activation.

[0009] Thrombospondin-1 (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 forming a direct interaction with the latent TGF-β complex and preventing its binding to mature TGF-β. Thrombospondin-mediated activation is believed to be involved in wound healing (e.g., skin wounds).

[0010] b. Activation via integrins containing α(V) Integrins, particularly those containing β6, αV, and β8, are understood to facilitate the activation of latent TGF-β (e.g., TGF-β1). Activation appears to occur either by inducing a conformational change in the latent TGF-β1 complex, thereby releasing active TGF-β1, or via an integrin-protease-dependent mechanism. It is understood that, in the absence of proteolysis, the conformational changes leading to TGF-β1 activation (especially in epithelial cells) occur through integrin binding to the arginyl-glycyl-aspartate cell adhesion motif (RGD motif) present in LAP-β1 or LAP-β3. LAPs containing the RGD motif are recognized by most αV-containing integrins. For example, the αVβ6 integrin can activate / release TGF-β1 by binding to the RGD motif present in both LAP-β1 and LAP-β3. In addition, integrin-protease-dependent TGF-β activation can occur by forming a link between the latent TGF-β complex and MMPs such as MMP-2 and MMP-9, which can activate TGF-β through proteolytic degradation of the latent TGF-β complex.

[0011] 2 TGF-β signal transduction and function Activated TGF-β plays a crucial role in cell differentiation and T-cell regulation. See, for example... Cold Spring HarborPerspect. Biol.2017;9:a022236 and its citations. TGF-β promotes thymic development in several T-cell lineages by supporting the survival of thymus-derived Tregs (tTregs), invariant natural killer T cells (iNKTs), and CD8α+ T-cell precursors, and thus promoting the development of T-cells 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 IL-7-dependent survival of low-affinity T cells, by controlling IL-7Rα expression in thymocytes, and by inhibiting the activation of T-cell receptor (TCR)-driven autoreactive or high-affinity T cells. In early CD8+ T-cell differentiation, TGF-β inhibits the formation of cytotoxic T lymphocytes (CTLs) and promotes apoptosis of short-lived effector cells (SLECs), while simultaneously promoting the differentiation of tissue-resident memory (TRM) cells expressing CD103. Although TGF-β inhibits the differentiation of T helper 1 and 2 (Th1 and T helper 2, Th2) cells, it promotes the development of various T-cell types along with other factors. TGF-β binds to IL-2 to promote the production of peripheral Tregs (pTregs), binds to IL-6 to promote the production of Th-17 cells, binds to IL-4 to promote the production of Th9 cells, and binds to IL-21 and / or IL-23 to promote the production of follicular helper (Tfh) cells.

[0012] In addition to its effects on T cells, TGF-β also regulates a variety of other cell types, including B lymphocytes or “B cells,” monocytes, and macrophages. TGF-β typically has an inhibitory effect 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); inhibits B cell proliferation and induces apoptosis in immature or resting B cells (Arsura et al., Immunity 5(1): 31–40.(1996)). At least part of the effect of TGF-β on B cells can be attributed to the induction of NF-κB, an inhibitor of NF-κB that regulates the production of cytokines including IL-1, TNF-α, and defensins. See, for example, Cold Spring Harbor Perspect. Biol. 2017; 9:a022236 and its citations.

[0013] In addition to its effects on B cells, TGF-β stimulates resting monocytes and inhibits activated macrophages. TGF-β exhibits the same inhibitory effects on macrophages stimulated by Toll-like receptor ("TLR") ligands, such as pro-inflammatory responses. In the absence of TLR ligands or other cytokines, TGF-β stimulation promotes the production of several inflammatory cytokines by myeloid cells. TGF-β has been shown to induce peripheral blood monocytes and macrophages into tissues and enhance monocyte adhesion properties. TGF-β can induce chemotaxis and enhance mast cell adhesion properties. See, for example... Cold Spring Harbor Perspect. Biol. 2017; 9:a022236 and its citations.

[0014] Once TGF-β is activated, it should be understood that it functions via cell surface signaling receptors. Signaling begins when an active TGF-β ligand binds to transforming growth factor β receptor II (“TβRII”) on the cell surface. This interaction induces the recruitment of transforming growth factor β receptor I (“TβRI”). TβRII can bind TGF-β1 alone, while TβRI can only bind ligands in cooperation with TβRII. TβRI is phosphorylated and activated by TβRII, thereby signaling via the typical signaling pathway of recruitment and phosphorylation of R-Smad proteins (Smad2 and Smad3). These Smads then bind to co-Smad (Smad4), and the complex collectively drives the transcription of several genes. See Smith et al., Clin. Cancer Res. ; 18(17): 4514–21 (2012). TGF-β can also signal via ligand-occupied receptor-activated atypical (non-Smad) pathways, including various branches of the MAP kinase pathway, the Rho-like GTPase signaling pathway, and the phosphatidylinositol-3-kinase / AKT pathway. Signaling via atypical pathways can enhance, attenuate, or otherwise regulate downstream cellular responses. Zhang Ye, Cell Res. 19(1):128-39 (2009). Compared with TβRI and TβRII, transforming growth factor beta receptor III ("transforming growth factor beta receptor III", TβRIII "receptor" or "β glycan") does not participate in TGF-β signal transduction, but instead acts as a reservoir for TGF-β.

[0015] Perturbations of activating factors, abnormal levels of activated TGF-β, and / or changes in TGF-β signaling can produce unregulated TGF-β signaling levels, leading to several diseases or complex disease conditions. In fact, TGF-β has been shown to have effects on various conditions such as inflammation, autoimmune diseases, fibrosis, cancer, and cataracts.

[0016] TGF-β plays a crucial role in maintaining immune system homeostasis and thereby influencing autoimmune responses as a factor involved in inducing tolerance. One mechanism by which TGF-β can exert such effects is by driving T cells (e.g., CD4+) to... + CD8 + CD4 - CD8 + and CD4 - CD8 - Cells differentiate into regulatory T cells or "T-Reg" cells (e.g., in the presence of IL-2). See, for example, Bettini and Vagnali, Ann. NY Acad. Sci. , 1183:1-12 (2010). T-Reg cells are crucial 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 autoantigens, makes it a key factor in preventing diseases such as arthritis (rheumatoid arthritis, RA), type 1 diabetes mellitus (T1D), multiple sclerosis (MS), and systemic lupus erythematosus (SLE). For example, a key function of TGF-β is to regulate autoimmune diseases and related inflammatory processes. This is especially true in the gut, where TGF-β is thought to inhibit macrophage cytokine production and mucosal inflammation in diseases such as inflammatory bowel disease (IBD). Sanjab et al. Cold Spring Harbor Perspect. Biol. 2017; 9:a022236. Like IBD, RA is an autoimmune disease with an inflammatory component targeting the joints. RA is caused by an abnormal response of T and / or B cells. Systemic TGF-β appears to prevent 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 the references cited therein.

[0017] Numerous methods for modulating the action of TGF-β at the protein level by chelating it to effectively neutralize its effects have been described in the literature. For example, monoclonal antibodies that bind, chelate, and neutralize TGF-β in vivo have been developed, such as metelimumab (CAT192) against TGF-β1 and various isoforms of non-hematoxylinumab (Fresolimumab) against TGF-β. In addition, receptor traps have been developed that tightly bind to and chelate TGF-β, thereby chelating and neutralizing it (see, for example, Swagrtra et al., Mol Cancer Ther; 11(7): 1477-87 (2012) and U.S. Patent Publication No. 2018 / 0327477).

[0018] Unlike the molecules described above, which are designed to bind and chelate TGF-β and act as antagonists of TGF-β, 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 complex may include sequence variations of TGF-β and / or the masking polypeptide that reduce their affinity for each other and facilitate the demasking of TGF-β, allowing it to bind to heterogeneous cell surface receptors via signaling (e.g., binding to TβRII, followed by binding to TβRI to form a heterogeneous receptor). Once formed, the heterogeneous TβRI with high affinity for TGF-β... TβRII peptide complexes can effectively compete with masking peptides. Sequence variations of TGF-β and / or its masking peptides can also allow for the avoidance of undesired interactions between the demasked TGF-β peptide and other molecules. Such sequence variations include deletions of the N-terminal portion of TβRII that weakens binding to TβRI and / or TGF-β sequence variations that prevent its dimerization (e.g., C77S substitution), which restrict off-target binding to reservoirs of non-signaling TβRIII molecules. In addition to the foregoing, masked TGF-β constructs and complexes may also contain additional wild-type (wt.) and / or variant immunomodulatory peptide sequences (MODs) that can substantially affect the outcome of TGF-β binding to target cells, including in vitro and in vivo effects, such as therapeutic outcomes. Summary of the Invention

[0019] This disclosure describes a construct in which TGF-β is masked by another polypeptide (“masked TGF-β construct”, see, for example, one having a single polypeptide chain). Figure 1Structure A), and a complex in which TGF-β is masked by another polypeptide (“TGF-β polypeptide complex”, see, for example, a complex containing two polypeptide chains). Figure 1 The preparation of the structure (BF), and the constructs and complexes that 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 (“masked polypeptide sequence,” “masked polypeptide,” or “masked sequence”) that binds to TGF-β. Masked TGF-β constructs and complexes may also contain one or more independently selected immunomodulatory polypeptide sequences, such as wild-type or variant IL-2 polypeptide sequences.

[0020] Masked TGF-β constructs and complexes can be expressed in many mammalian cell types because masked non-targeted TGF-β activity does not adversely affect cells as much as is observed under demasked TGF-β.

[0021] The masked TGF-β construct may contain the following as the first polypeptide: i) Scaffold polypeptide sequence; ii) TGF-β polypeptide sequence; iii) A masking polypeptide sequence, wherein the masking polypeptide sequence optionally comprises a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; iv) Optionally, one or more independently selected MOD polypeptide sequences; as well as v) Optionally, one or more independently selected adapter polypeptide sequences; The constructs containing these elements are collectively referred to herein as “masked TGF-β constructs”, wherein the masking polypeptide sequence is bound to the TGF-β polypeptide sequence. The masked TGF-β constructs may be organized, for example, in the following order (from N-terminus to C-terminus): i) Scaffold polypeptide sequence, masking polypeptide sequence, and TGF-β polypeptide sequence; or ii) The first MOD polypeptide sequence, the scaffold polypeptide sequence, the masking polypeptide sequence, and the TGF-β polypeptide sequence; or iii) A first independently selected MOD peptide sequence, a second independently selected MOD peptide sequence, optionally one or more additional MOD peptide sequences, scaffold peptide sequences, masking peptide sequences, and TGF-β peptide sequences; The masked TGF-β construct optionally includes one or more independently selected adapter polypeptide sequences.

[0022] The scaffold polypeptide of the masked TGF-β construct may contain interspecies or non-interspecies dimerizing sequences that lead to the formation of homodimers, wherein the scaffold polypeptide sequences optionally have one or more covalently linked to each other.

[0023] The scaffold polypeptide of the masked TGF-β construct described above may further include an interspecies dimerizing sequence and, more particularly, a second polypeptide that dimers with the first polypeptide (as described above) via a corresponding interspecies dimerizing sequence to form a masked TGF-β complex heterodimer. The second polypeptide may comprise one of the following structures: (i) a scaffold polypeptide sequence containing a corresponding interspecies dimerizing sequence; (ii) one or two (or more) independently selected MOD sequences and a scaffold polypeptide sequence containing a corresponding interspecies dimerizing sequence; (iii) a scaffold polypeptide sequence containing a corresponding interspecies dimerizing sequence and an independently selected MOD sequence; or (iv) one or two (or more) independently selected MOD sequences and a scaffold polypeptide sequence containing a corresponding interspecies dimerizing sequence, wherein the first and / or second polypeptide optionally comprises one or more independently selected linker polypeptide sequences. Therefore, the second polypeptide may comprise, from the N-terminus to the C-terminus, one of the following structures: (i) a scaffold polypeptide sequence comprising a corresponding interspecies dimerizing sequence; (ii) one or two (or more) independently selected MOD sequences and a scaffold polypeptide sequence comprising a corresponding interspecies dimerizing sequence; (iii) a scaffold polypeptide sequence comprising a corresponding interspecies dimerizing sequence and one or two (or more) independently selected MOD sequences; or (iv) one or two (or more) independently selected MOD sequences and a scaffold polypeptide sequence comprising a corresponding interspecies dimerizing sequence; wherein the first and / or second polypeptide optionally comprises one or more independently selected adapter polypeptide sequences. Optionally, the masked TGF-β complex heterodimer may comprise, from the N-terminus to the C-terminus, the following sequence: (i) a scaffold polypeptide sequence containing the corresponding interspecies dimerization sequence, a masking polypeptide sequence, and a TGF-β polypeptide sequence; (ii) a first MOD polypeptide sequence, a scaffold polypeptide sequence containing the corresponding interspecies 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 containing the corresponding interspecies dimerization sequence, a masking polypeptide sequence, and a TGF-β polypeptide sequence.

[0024] The masked TGF-β complex may also contain a first polypeptide and a second polypeptide as a masked TGF-β complex heterodimer, wherein: (i) The first polypeptide contains a) Scaffold polypeptide sequences containing interspecies dimerization sequences. b) A masking polypeptide sequence, wherein the masking polypeptide sequence optionally comprises a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence. c) Optionally, one or more independently selected MOD polypeptide sequences, and d) Optionally, one or more independently selected adapter polypeptide sequences; (ii) The second polypeptide contains a) A scaffold polypeptide sequence, wherein the scaffold polypeptide sequence comprises the corresponding interspecies dimer sequence of the interspecies dimer sequence in the first polypeptide. b) TGF-β polypeptide sequence, c) Optionally, one or more independently selected MOD polypeptide sequences, and d) Optionally, one or more independently selected adapter polypeptide sequences; Complexes containing these elements are collectively referred to as “masked TGF-β complexes”, in which the masking polypeptide sequence and the TGF-β polypeptide sequence are provided on different polypeptide chains and bind to each other; The interspecific binding sequence interacts with its corresponding interspecific binding sequence in the heterodimer; and The masked TGF-β first and / or second polypeptides optionally include one or more independently selected adapter polypeptide sequences.

[0025] The TGF-β polypeptide sequence can be derived from any of the TGF-β isoforms and may contain substitutions that restrict the ability of the TGF-β sequence to dimerize. The masking sequence can be, for example, an anti-TGF-β antibody sequence or a TGF-β receptor (TβR) extracellular domain sequence. When the TβR extracellular domain is used to mask the TGF-β sequence, it can be modified to avoid unintentional signaling of the masked molecule (e.g., by deleting all or part of the extracellular domain that is not needed to interact with the TGF-β sequence).

[0026] This disclosure also describes and provides methods for generating masked TGF-β constructs and complexes, and methods for using them to influence various cell types and treat a variety of diseases / conditions, including autoimmune and inflammatory diseases. The therapeutic methods described herein may include co-administration of the masked TGF-β constructs and complexes with other molecules that may facilitate the achievement of desired laboratory or therapeutic outcomes, including but not limited to: immunomodulators (e.g., interleukins, cytokines, chemokines, etc.); antibodies and antibody fragments (e.g., scFv, nanobodies, etc.); small molecule therapeutic agents (e.g., vitamin D or retinoic acid); and combinations thereof. Attached Figure Description

[0027] Figure 1Some forms of masked TGF-β constructs and complexes are depicted, wherein the TGF-β receptor sequence is used to mask the TGF-β peptide. Structure A depicts a monomeric construct having a single position for one or more independently selected MODs (e.g., a set of tandemly selected MODs). Structure B depicts a symmetrical homodimer in which the peptides interact via their respective Ig Fc sequences, which can spontaneously form disulfide bonds connecting the two peptides. Structure CF depicts a heterodimer structure in which TGF-β is heterodimerized with the TGF-β receptor sequence in either a "cis" (same peptide) or "trans" (different peptide) configuration. The positions where one or more independently selected MODs can be placed are indicated by circles filled with diagonal or vertical lines or a checkered pattern. Interspecific binding pairs are represented by knock-in-hole sequences, but can be any of the other sequences discussed below. The construct may not include MODs, or may include one, two, or more independently selected MOD sequences, including tandem MOD sequences, which may be provided at the indicated positions. Exemplary MODs include, for example, wild-type or mutant (e.g., with reduced affinity and / or selective affinity for one or more specific receptors) PD-L1, FAS-L, 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 the TGF-β peptide, the receptor peptide can be replaced with another masking peptide, such as an antibody peptide (e.g., scFV or nanobody) with affinity for the TGF-β peptide. Scaffold sequences can be linked (e.g., via disulfide bonds) to form covalently linked homodimers or covalently linked heterodimers. Any of the constructs shown in the figure may have sequence variations in the TGF-β peptide that limit its dimerization ability (e.g., C77S substitution).

[0028] Figures 2A to 2H The amino acid sequence (aas) of the immunoglobulin Fc polypeptide is provided (SEQ ID NO:68-83).

[0029] Figure 2I The amino acid sequences of the human J-chain of signal peptide aas 1-22 with underlined parts are provided (SEQ ID NO:84).

[0030] Figure 2J The sequence of the CH1 domain of the Ig G1 heavy chain constant region is provided. Serine residues at positions 70 and 72 can be replaced by glutamic acid and valine, respectively (S70E and S72V), to form an MD13-like construct.

[0031] Figure 2KThe sequences of the light chain constant region “CL” domains from the Igκ and Igλ chains were provided. The serine at position 68 and the threonine at position 70 can be replaced by leucine and serine (S68L and T70S), respectively, to form the MD13-like construct.

[0032] Figure 3 Three different isoforms of TGF-β as a precursor protein and the mature form of TGF-β3, as well as the sequence of the C77S mutant of the mature protein, are provided.

[0033] Figure 4 A comparison of TGF-β isoforms 1-3 is provided, where residues corresponding to the mature form of TGF-β2 are in bold, while the aa residues of TGF-β2 Lys 25, Cys 77, Ile 92 and / or Lys 94 and their corresponding residues in other forms of TGF-β isoforms 1 and 3 are underlined but not in bold.

[0034] Figure 5A The sequence of type 1 TGF-β receptor (TβRI) and its extracellular domain is provided.

[0035] Figure 5B The sequences of type 2 TGF-β receptor (TβRII), its extracellular domain, and extracellular domain fragments are provided. In isoform B, the positions indicated by bold and underline are the aas of the mature polypeptide at F30, D32, S52, E55, and D118, any one of which can be substituted by aa other than the naturally occurring aa.

[0036] Figure 5C The sequence of type 3 TGF-β receptor (TβRIII) was provided.

[0037] Figure 6 shows a plot illustrating 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 cell counting analysis. Part A shows the expression of TGF-β3 or masked TGF-β3 at indicated concentrations after 5 days of cell culture, in the absence and presence of 50 U / ml of added IL-2. WT Constructor (see) Figure 1 FoxP3 (as CD4) of structure A) + Induction of TGF-β3 (percentage of cells). Part B shows TGF-β3 complexes induced with various concentrations of TGF-β3 or one of the three masked TGF-β3 constructs or masked TGF-β3 complexes with at least one N-terminal wt. or variant IL-2MOD (for structure, see [link]). Figures 7G to 7I FoxP3 in a 5-day-old naive T cell population +Distribution of cells (as a percentage of CD4 cells). Part C shows the distribution of FoxP3 in the presence of masked TGF-β3 peptide (structure (i) in part B of Figure 6) at concentrations of 0.1 nM or 1000 nM. + CD4 + Cell induction.

[0038] Figure 7A Provided is the aa sequence SEQ ID NO:146 of a representative masked TGF-β (construction number: 3470), which has Figure 1 The overall structure of structure A. The polypeptide, from the N-terminus to the C-terminus, comprises: wt. human IL-2 (hIL2), three repeat sequences of the G4S linker, human single IgG Fc with LALA substitution, three repeat sequences of the G4S linker, and human TβRII (hTβRII) with D118A substitution. Δ25 The sequence, five repeating sequences of the G4S connector, and the human TGF-β3 (hTGF-β3) sequence with C77S substitution.

[0039] Figure 7B The aa sequence SEQ ID NO:147 of the representative masked TGF-β (construction number: 3334) is provided, which has Figure 1 The overall structure of structure B. The homodimer-forming polypeptide contains, from the N-terminus to the C-terminus: hIL2 with H16T and F42A substitutions, three repeat sequences of G4S, human IgG1 Fc with LALA substitutions, two repeat sequences of G5S and G4S linkers, and hTβRII. Δ25 The five repeat sequences of D118A and G4S, as well as the hTGF-β3 sequence.

[0040] Figure 7C Provided having a first and second polypeptide Figure 1 The representative masked TGF-β construct aa sequence of structure D. The first polypeptide construct, number 3618 (SEQ ID NO: 148), from N-terminus to C-terminus, comprises: wt. hIL-2, three repeat sequences of the G4S linker, human IgG1 Fc knock-in-hole (KiH) polypeptide chain A with LALA substitution, five repeat sequences of the G4S linker sequence, and an hTGF-β3 sequence with C77S substitution. The second polypeptide (construction number: 3619), SEQ ID NO: 149, from N-terminus to C-terminus, comprises: wt. hIL-2, three repeat sequences of the G4S linker, human IgG1 Fc KiH polypeptide chain B with LALA substitution, two repeat sequences of the G5S and G4S linksers, and hTβRII. Δ25 D118A sequence.

[0041] Figure 7D Provided having a first and second polypeptide Figure 1 The aa sequence of the TGF-β construct representing the overall structure of structure E is a masked representation. The first polypeptide (construction number: 3618) SEQ ID NO: 150, as described above, comprises from the N-terminus to the C-terminus: wt hIL-2, three repeat sequences of the G4S linker, human IgG1 Fc KiH polypeptide chain A with LALA substitution, five repeat sequences of the G4S linker sequence, and an hTGF-β3 sequence with C77S substitution. The second polypeptide (construction number: 3855) SEQ ID NO: 151, from the N-terminus to the C-terminus, comprises: human IgG1 Fc KiH polypeptide chain B with LALA, T366S, L368A, and Y407V substitutions, three repeat sequences of the G4S linker, and hTβRII. Δ25 , D118A sequence.

[0042] Figure 7E Provided having a first and second polypeptide Figure 1 The aa sequence of the TGF-β construct, which is a representative mask of the overall structure of structure F. The first polypeptide (construction number: 3891) SEQ ID NO: 152 comprises, from the N-terminus to the C-terminus: three repeat sequences of hIL-2 with H16A and F42A, a G4S linker sequence, two repeat sequences of human IgG1 Fc mortis (KiH) polypeptide chain A with LALA and T366W substitutions, G5S and G4S linkers, and hTβRII. Δ25 The second polypeptide (construction number: 3664) SEQ ID NO: 153 comprises, from the N-terminus to the C-terminus: human IgG1 Fc KiH polypeptide chain B with LALA substitution, five repeat sequences of D118A, G4S linker, and hTGF-β3 with C77S substitution.

[0043] Figure 7F Provided having a first and second polypeptide Figure 1 The aa sequence of the TGF-β construct representing the overall structure of structure F is a masked representation. The first polypeptide (construction number: 3715) SEQ ID NO: 155 comprises, from the N-terminus to the C-terminus: a human IgG1 Fc KiH polypeptide chain A with LALA substitution, three repeat sequences of the G4S linker, and a human wt IL2 sequence. The second polypeptide (construction number: 3714) SEQ ID NO: 156 comprises, from the N-terminus to the C-terminus: a human IgG1 Fc KiH polypeptide chain B with LALA substitution, three repeat sequences of the G4S linker, and an hTβRII with D32N and D118A substitutions. Δ25Five repeating sequences of the G4S connector sequence and the hTGF-β3 sequence with C77S substitution.

[0044] Figures 7A to 7F Any of the IL-2 sequences may be replaced by a MOD or variant MOD other than IL-2, by wtIL-2, or by an IL-2 sequence having substitutions at N88, H16, and / or F42 (e.g., N88R, a substitution at H16 selected from H16A or H16T, and / or a substitution at F42 selected from F42A and F42T).

[0045] Figure 7G Provided is the aa sequence SEQ ID NO:157 of a representative masked TGF-β construct (construction number: 3472), which has Figure 1 The overall structure of structure A. The polypeptide, from the N-terminus to the C-terminus, comprises: wt.hIL-2, three repeat sequences of the G4S linker, a human single IgG Fc with LALA substitution, three repeat sequences of the G4S linker, and a human TβRII (hTβRII) with D32N and D118A substitutions. Δ25 The sequence, five repeating sequences of the G4S connector, and the human TGF-β3 (hTGF-β3) sequence with C77S substitution.

[0046] Figure 7H Provided is the aa sequence SEQ ID NO:158 of a representative masked TGF-β construct (construction number: 3466), which has Figure 1 The overall structure of structure A. The polypeptide, from the N-terminus to the C-terminus, comprises: wt.hIL2, three repeat sequences of the G4S linker, a human single IgG Fc with LALA substitution, three repeat sequences of the G4S linker, and a human TβRII (hTβRII) with D118A substitution. Δ25 The sequence, five repeating sequences of the G4S connector, and the human TGF-β3 (hTGF-β3) sequence with C77S substitution.

[0047] Figure 7I The aa sequence SEQ ID NO:159 of a representative masked TGF-β construct (construction number: 3468) is provided, which has Figure 1 The overall structure of structure A. The polypeptide, from the N-terminus to the C-terminus, comprises: hIL-2 with an H16T F42A substitution, three repeat sequences of the G4S linker, a human single IgG Fc with an LALA substitution, three repeat sequences of the G4S linker, and a human TβRII (hTβRII) with D32N and D118A substitutions. Δ25The sequence, five repeating sequences of the G4S connector, and the human TGF-β3 (hTGF-β3) sequence with C77S substitution.

[0048] Figure 7J Provided having a first and second polypeptide Figure 1 The representative masked TGF-β construct aa sequence of structure D. First polypeptide (construction number: 3618) SEQ ID NO: 148 comprises, from the N-terminus to the C-terminus: wt. hIL-2, three repeat sequences of the G4S linker, human IgG1 Fc KiH polypeptide chain A with LALA substitution, five repeat sequences of the G4S linker sequence, and an hTGF-β3 sequence with C77S substitution. Second polypeptide (construction number: 3621) SEQ ID NO: 160 comprises, from the N-terminus to the C-terminus: wt. hIL-2, three repeat sequences of the G4S linker, human IgG1 Fc KiH polypeptide chain B with LALA substitution, three repeat sequences of the G4S linker, and an hTβRII with D32N and D118A substitutions. Δ25 sequence.

[0049] Figure 8 A masked TGF-β construct (left) and two masked TGF-β complexes (center and right) are shown. Samples of the complexes were prepared by constructing a nucleic acid vector encoding the peptide, transfecting it into ExpiCHO cells, and expressing the peptide. The peptide was purified by protein A chromatography followed by size exclusion chromatography. The purified protein underwent SDS-PAGE, and the resulting gel was stained with Coomassie blue. NR = unreduced or non-reduced sample, and R = reduced sample (reduced with a disulfide reducing agent).

[0050] Figure 9 Various aa substitutions in the mature TβRII polypeptide sequence are shown (see [link]). Figure 5B Regarding its affinity for TβRII and TGF-β3 or TGF-β1 (table, upper left). Figure 9 The effects of the three substitutions, E55A, D32N, and S52L, on the binding interaction between the TβRII-masked TGF-β construct and the immobilized TβRII-Fc receptor construct are also shown. The receptor construct is shown in the capture assay format of TGF-β3 in the upper right of the figure, where: the single Fc is an Ig scaffold that does not form interspecies bonds with other scaffolds, and anti-IL-2 is a biotinylate (B)-labeled antibody against IL-2. Strep and HRP are streptavidin and horseradish peroxidase used for detection. The binding effect in the capture assay was detected as binding curves developed for each of the four masked constructs (bottom). See Example 3.

[0051] Figure 10 shows the structure and amino acid sequence of the polypeptide that forms the masked TGF-β3 complex PSM-4033-4039.

[0052] Figure 11 The example shown is PSM-4033-4039 used to induce naive CD4 from human peripheral blood. + Foxp3 T cells + Results of the iTreg experiment. See Example 4.

[0053] Figure 12 This shows the Foxp3 induced by PSM-4033-4039. + Results of experiments using iTreg to inhibit T cell proliferation. See Example 4.

[0054] Figure 13 shows that PSM-4033-4039 was used to induce CD4 from human peripheral blood. + T cells (including naive and memory CD4) + Foxp3 (T cells) + Experimental results regarding iTreg expression. See Example 4.

[0055] Figure 14A and Figure 14B This demonstrates that PSM-4033-4039 is used to induce CD4+ activation via allogeneic lymphocyte response. + Foxp3 T cells + Results of the iTreg experiment. See Example 4.

[0056] Figure 15 The results of an experiment in which PSM-4033-4039 was administered intravenously to mice at various concentrations to determine serum concentrations in mice at different time intervals up to 72 hours post-injection are shown. See Example 4. Detailed Implementation

[0057] A. Definition As used herein, amino acids (abbreviated as "aa" in the singular, and as "aas" in the plural unless the context specifies otherwise) refer to naturally occurring proteinogenic α-amino acids incorporated into polypeptides and proteins during mammalian cell translation. Unless otherwise stated: L (Leu), A (Ala), G (Gly), S (Ser), V (Val), F (Phe), Y (Tyr), H (His), R (Arg), N (Asn), E (Glu), D (Asp), C (Cys), Q (Gln), I (Ile), M (Met), P (Pro), T (Thr), K (Lys), and W (Trp). Amino acids also include the amino acids hydroxyproline and selenocysteine, which are found in some proteins in mammalian cells.

[0058] As used herein, the terms “peptide,” “peptide sequence,” and “protein” are synonyms and refer to an aas sequence in which the polypeptide backbone is linked together by a peptide bond between its C-1 carboxyl group and its α-amine. Therefore, each polypeptide (e.g., a first polypeptide) comprising any one or more of MOD polypeptide sequences, scaffold polypeptide sequences, TGF-β polypeptide sequences, and / or masking polypeptide sequences (e.g., TGF-β receptor polypeptide sequences or anti-TGF-β polypeptide sequences) comprises any one or more of those polypeptide sequences as a polypeptide chain having a single continuous backbone. Such polypeptides (e.g., the first polypeptide) can be covalently linked to other polypeptides via disulfide bonds, such as those between side chains of cysteine ​​residues. Furthermore, as used herein, the terms “peptide,” “peptide sequence,” and “protein” include modifications to the native sequence, such as deletions, additions, and substitutions (which are generally conserved in nature, as known to those skilled in the art), provided that the protein retains the desired activity. These modifications can be intentional, such as by site-directed mutagenesis, or can be accidental, such as by mutations in the host that produces the protein, or due to errors in PCR amplification or other recombinant DNA methods.

[0059] When an embodiment, claim, or aspect is directed to a specific type of mammal (e.g., human or non-human subject), the nucleic acid and polypeptide sequences may be limited to sequences derived from those subjects. Unless otherwise stated, the polypeptide sequences of proteins (e.g., TGF-β, TβR, immunoglobulins, and MOD) are human ( Homo sapiens )sequence.

[0060] As used herein, “masked” means that a molecule (e.g., a masked polypeptide or a masked protein) is bound or otherwise linked by a masking molecule (e.g., a polypeptide, protein, or protein fragment) that limits the availability of the masked molecule relative to other proteins (e.g., cell surface receptors) that also have an affinity for the molecule.

[0061] As used herein, the term masked TGF-β construct refers to a single polypeptide comprising both a TGF-β (e.g., TGF-β1, TGF-β2, or TGF-β) polypeptide sequence and a masking polypeptide sequence that binds to or otherwise interacts with the TGF-β polypeptide. Unless otherwise stated, a masked TGF-β construct comprises a scaffold polypeptide sequence and optionally comprises one or more independently selected immunomodulatory (MOD) polypeptide sequences.

[0062] As used herein, the term masked TGF-β complex refers to two or more polypeptides (typically arranged as homodimers or heterodimers, referred to as the first polypeptide and the second polypeptide, but may be higher-order multimers). A masked TGF-β complex comprises a TGF-β (e.g., TGF-β1, TGF-β2, or TGF-β) polypeptide sequence, a masking polypeptide sequence that binds to or otherwise interacts with the TGF-β polypeptide, and a scaffold polypeptide comprising the TGF-β complex polypeptide through its associated dimerized or multiplied sequence. Any one or more of the TGF-β complex polypeptides optionally comprises one or more independently selected MOD polypeptide sequences.

[0063] The phrase "masked TGF-β construct or complex" is an abbreviation for masked TGF-β construct or masked TGF-β complex. It can be used as an abbreviation in its plural form, "masked TGF-β construct or complex".

[0064] The phrase “masked TGF-β constructs and complexes” is an abbreviation for both “masked TGF-β constructs” and “masked TGF-β complexes”.

[0065] As used herein, dimerizing and multimerizing sequences are polypeptide sequences that allow polypeptide sequences (e.g., single polypeptides) to associate as dimers (e.g., heterodimers or homodimers) or multimers (homo- or hetero-multimers of three, four, five, or more polypeptide sequences). Dimerizing and multimerizing sequences allow sequences to associate in a non-covalent manner, which can be converted into covalent complexes in some cases (e.g., the formation of disulfide bonds between polypeptides).

[0066] Interspecies binding sequences are dimerizing sequences that allow for asymmetric pairing (heterodimerization) of polypeptides. Interspecies binding sequences favor the formation of heterodimers (as opposed to homodimerization) with their corresponding interspecies binding sequences, which are their homologous binding partners. A key-in-hole (or key-into-hole) Fc polypeptide pair represents an example of an interspecies binding sequence and its corresponding interspecies binding sequence.

[0067] As used in this article, Nanobodies® or nanobodies refer to antibody fragments composed of a single monomeric variable antibody domain. Like complete antibodies, these antibody domains can selectively bind to specific antigens.

[0068] The wild-type (abbreviated as "wt.") of an aa sequence refers to a naturally occurring aa sequence, or a continuous portion of a naturally occurring aa sequence, as understood from the context, which has not been altered relative to a sequence naturally found in living organisms (without any substitutions, deletions, or insertions). A specific naturally occurring sequence may be referred to as a wt. sequence for reference.

[0069] As used in this article, “T cells” include all types of CD3-expressing immune cells, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), regulatory T cells (Tregs), and NK-T cells.

[0070] As used herein, the term "bonding" refers to a non-covalent interaction between two molecules, such as the non-covalent interaction between a MOD and its co-MOD. Non-covalent bonding refers to the direct association between two molecules due to interactions such as electrostatics, hydrophobicity, ionic and / or hydrogen bonding (including interactions such as salt bridges and water bridges). Non-covalent bonding interactions are typically characterized by their dissociation constant (K0). D Less than 10 -6 M, less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less than 10 -11 M, less than 10 -12 M, less than 10 -13 M, less than 10 -14 M or less than 10 -15 M. "Specific binding" generally refers to binding with at least about 10 -7 M or larger, such as 5 x 10 -7 M, 10 -8 M, 5 x 10 -8 M, 10 -9M and greater affinity binding. "Non-specific binding" generally refers to binding with less than about 10... -7 Affinity binding of M (e.g., binding of the ligand to a portion other than its designated binding site or receptor) (e.g., at 10) -6 M, 10 -5 M, 10 -4 (Affinity binding of M). As used in this article, "covalent bonding" or "covalent bond" refers to the formation of one or more covalent chemical bonds between two different molecules.

[0071] "Affinity" refers to the strength of non-covalent bonding; an increase in bonding affinity is associated with a lower K0. D Related. As used herein, the term "affinity" refers to the equilibrium constant of the reversible binding of two agents (e.g., antibody and antigen) and is expressed as the dissociation constant (K). D ).

[0072] As used herein, the term "immunomodulatory polypeptide" or MOD includes polypeptides on antigen-presenting cells (APCs) (e.g., dendritic cells, B cells, etc.) or portions of polypeptides on APCs that specifically bind to homologous co-immunomodulatory polypeptides ("co-MODs") on T cells, thereby providing signaling. For example, the binding of interleukins such as IL-2 or fragments thereof (MODs) to the cell surface IL-2 receptor (co-MOD) provides signaling 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 and IL-23, PD-L1, and Fas ligand (FAS-L). MODs also particularly encompass antibodies or antibody sequences (e.g., nanobodies) that specifically bind to co-MOD molecules present on T cells, thereby causing signaling by co-MODs. As discussed herein, MODs also include variants of wt. MODs, including, for example, variant MODs with reduced binding affinity to co-MODs. This reduced affinity can take many forms. For example, variant IL-2 MOD can affect the α, β, and γ of IL-2R. One or more of the α and / or γ chains have reduced affinity. As discussed herein, the variant IL-2MOD containing mutations at positions 16 and 42 can substantially not bind to the α chain of IL-2R and is resistant to IL-2R. The chain exhibits reduced affinity. Furthermore, for MODs having more than one co-MOD (e.g., CD80 binds to both CD28 and CTLA-4), variant MODs may have reduced affinity for one of the co-MODs, allowing it to preferentially or selectively bind to other co-MODs. For the purposes of this disclosure, TGF-β (e.g., TGF-β1, TGF-β2, or TGF-β3) and fragments thereof are not considered MODs.

[0073] Unless otherwise specified, the term “substantially” is intended to cover both “completely” and “substantially but not completely”. For example, a variant IL-2 MOD that exhibits substantial non-binding to the α chain of IL-2R is an IL-2 variant MOD that does not bind to IL-2R at all or substantially does not bind to the α chain of IL-2R.

[0074] As used in this article, the term "in vivo" refers to any process or procedure that occurs in, for example, the body of an autoimmune patient.

[0075] As used in this article, “in vitro” means any process or procedure that occurs outside the body, including procedures that may be referred to as ex vivo.

[0076] As used herein to describe the placement of MOD polypeptide sequences, “tandem” means that two or more MODs are arranged adjacent to each other on the polypeptide, the MODs being separated at most by a linker (e.g., without intervening in a scaffold, masking polypeptide, or TGF-β sequence).

[0077] As used herein, the term “extracellular domain” refers to a portion (domain) of a membrane protein that extends into the extracellular space and does not include a transmembrane domain portion sufficient to anchor it in the cell membrane.

[0078] As used herein, “sequence identity” is a measure of the aa or nucleotide identity between two polynucleotide sequences or two polypeptide sequences. To state that a protein or polypeptide sequence has a certain percentage of “sequence identity” with another polynucleotide or polypeptide means that, when aligned, the percentages of bases or amino acids are the same and in the same relative positions when comparing the two sequences. Sequence identity can be determined in many different ways. To determine sequence identity, various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) available on websites on the World Wide Web, including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , and mafft.cbrc.jp / alignment / software / , are used, for example. See, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-10. Unless otherwise stated, sequence identity was determined using alignments performed with the NCBI BLAST algorithm version BLAST+ 2.9.0 (BLASTP 2.9.0+ for proteins and BLASTN 2.9.0+ for nucleic acids) published on April 1, 2019.

[0079] As used herein, “recombinant” means that a specific nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps, which produce a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or a series of synthetic oligonucleotides to provide synthetic nucleic acids capable of expression from recombinant transcription units contained in cellular or cell-free transcription and translation systems. The use of “recombinant” in relation to peptides, polynucleotides, or proteins indicates that it has been prepared by expression from recombinant nucleic acids.

[0080] As used herein, the term “about” with respect to quantity indicates that the quantity may vary by 10% of the indicated quantity. For example, “about 100” means a quantity of 90-110. When “about” is used in the context of a range, “about” with respect to the lower quantity of the range means that the lower quantity includes quantities that are 10% lower than the lower quantity of the range, and “about” with respect to the higher quantity of the range means that the higher quantity includes quantities that are 10% higher than the higher quantity of the range. For example, about 100 to about 1000 means a range that extends from 90 to 1100.

[0081] The terms “treatment” and “treating” are used herein to generally refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects caused by the disease. As used herein, “treatment” covers any treatment of a disease or symptom in mammals and includes: (a) preventing the occurrence of a disease or symptom in a subject who may be predisposed to acquiring said disease or symptom but has not yet been diagnosed with said disease; (b) suppressing the disease or symptom, i.e., preventing its development; and / or (c) alleviating the disease, i.e., causing disease remission. Therapeutic agents (e.g., masked TGF-β constructs or complexes) may be administered before, during, or after the occurrence of the disease or lesion. Particular attention is paid to the treatment of developing diseases in which said treatment stabilizes or alleviates undesirable clinical symptoms in the patient. Such treatment is ideally administered before complete loss of function in the diseased tissue. Therapeutic treatment may be administered during the symptomatic phase of the disease and, in some cases, after the symptomatic phase of the disease.

[0082] The terms “individual,” “subject,” “host,” and “patient” are used interchangeably in this document and refer to any mammalian subject requiring diagnosis, treatment, or therapy. Mammals include, for example, humans, non-human primates, rodents (e.g., rats; mice), rabbits (e.g., rabbits), ungulates (e.g., cows, rats, goats, sheep, horses, pigs, etc.), canines (e.g., dogs), felines (e.g., cats), etc. (e.g., humans, cattle, dogs, cats, rodents, rats, goats, apes, sheep, horses, rabbits, pigs, etc.).

[0083] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a” and “the” as used herein and in the appended aspects and claims include plural indicators. Thus, for example, reference to “a Treg” includes a plural of such Tregs and reference to “the TGF-β polypeptide” includes reference to one or more TGF-β polypeptides and their equivalents known to those skilled in the art. It should also be noted that claims may be drafted to include or exclude any element that may optionally be present. Therefore, this statement is intended to serve as a premise for the use (including) of such elements in the claims (e.g., the use of terms such as “alone,” “only,” etc.), or for the removal of such elements from the claims, or as a basis for a “negative” limitation excluding any particular optional element.

[0084] It should be understood that certain features of the invention described in a single aspect or embodiment for clarity may also be provided in combination in a single aspect or embodiment, including those subsequently claimed. Conversely, various features of the invention described in a single embodiment for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments relevant to this invention are expressly included by this invention and are disclosed herein as if each combination were individually and expressly disclosed herein. Furthermore, all sub-combinations of multiple embodiments and their elements are also expressly included by this invention and are disclosed herein as if each such sub-combination were individually and expressly disclosed herein.

[0085] The publications discussed herein refer only to those disclosed prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to any prior invention prior to such publication. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.

[0086] B. Description 1 Introduction The TGF-β superfamily comprises endogenous proteins with growth-inhibiting functions. Defects in the cellular mechanisms of increased or decreased TGF-β expression, or inhibition of TGF-β action, are particularly associated with malignant tumors in many cancers resulting from the immunosuppressive effects of TGF-β. Dysregulation of TGF-β's immunosuppressive function is also involved in autoimmune diseases. Because TGF-β is a key regulator of immune function, it is a target of numerous studies, and both TGF-β and its signaling pathways are considered therapeutic targets for treating a variety of diseases, including inflammatory processes and autoimmune disorders.

[0087] The ability to efficiently prepare and deliver TGF-β as a therapeutic agent is complicated by molecular toxicity and the complexity of the TGF-β receptor system. The large-scale production of TGF-β in mammalian cell expression systems is limited by the toxicity of the protein to many mammalian cells. Cells experiencing TGF-β cytotoxicity include many cells used for biomolecule production, such as Chinese hamster ovaries or “CHO” cells, which are among the most robust and commonly used cells for commercial protein production. The use of TGF-β as a therapeutic agent is further complicated by the high pI of TGF-β1 (approximately 8.59 pI, compared to approximately 6.1 pI for TGF-β3), whose stability / solubility is limited under non-acidic conditions (such acidic conditions are generally unsuitable for therapeutic use). Furthermore, the large number of high-affinity TβRIII receptors present in mammalian systems (e.g., approximately 5 nM for TGF-β2) represent a significant pharmacokinetic reservoir relative to the affinity of TβRIIs for TGF-β (approximately 1–2 μM), limiting the accessibility of TGF-β-based biologics to target tissues. Similarly, the role of TβRIII in recruiting TGF-β to the TβRII / TβRI complex can lead to significant off-target delivery of TGF-β, with unintended, unwanted, and even toxic effects. Such off-target delivery can cause nonspecific activation and can also lead to further production of active TGF-β, especially when TGF-β signaling is under feedforward control, resulting in further unintended and unwanted effects. See, for example, Jiang et al., Redox Biol. 2: 267–272 (2014).

[0088] The effective use of TGF-β as a therapeutic agent is further complicated by the need to provide additional stimulation to cells to guide the results of TGF-β stimulation. As shown above, cytokines such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21 and IL-23, PD-L1, and Fas ligand (FAS-L) can have a profound impact on the effects of TGF-β. Therefore, the ability to deliver TGF-β and additional stimulation in the form of cytokines can be advantageous for achieving specific therapeutic or cell-mediated outcomes (e.g., in vitro or in vivo).

[0089] As discussed in more detail below, this disclosure describes the use of TGF-β polypeptides capable of interacting with cells carrying TβRI and TβRII proteins and stimulating signal transduction on said cells. Advantageously, the TGF-β polypeptide is part of a masked TGF-β construct or complex, which is a fusion protein (a single polypeptide chain) or a fusion protein complex (two or more polypeptide chains), said fusion protein or fusion protein complex further containing a polypeptide that binds to and masks a TGF-β polypeptide sequence (e.g., a TβRII sequence acting as a masking sequence) surrounding a scaffold (e.g., one or two polypeptides such as immunoglobulin Fc polypeptide). Examples of such fusion proteins or fusion protein complexes are depicted in Figure 1 In this context, when the TGF-β peptide of a fusion protein interacts with, for example, cellular TβRIII, the fusion protein binds to and masks a portion of the TGF-β peptide, competing with TβRIII and thus preventing TGF-β from chelating in the TβRIII reservoir. Additionally, the affinity of the masking peptide for the TGF-β peptide can be intentionally altered using aa substitutions to control the overall potency of the masking peptide against its target receptor (e.g., TβRII). Figure 9As shown in the diagram. Because the TGF-β peptide primarily binds to TβRIII as a disulfide-linked dimer, the chelating interaction with the receptor can be attenuated by aa substitutions, including those restricting dimerization (e.g., C77S) or dimer stability, as discussed below. The interaction between the TGF-β fusion protein or fusion protein complex and TβRII replaces the masking sequence, thereby forming a cell-surface-bound TGF-β / TβRII complex. TβRII is subsequently recruited to form a heterodimeric TGF-β receptor, providing a high-affinity complex that tightly binds to TGF-β (e.g., picomolar affinity), even in the presence of the masking peptide sequence. Thus, the masked TGF-β can still bind to the heterodimeric TβRI / TβRII receptor complex and signal via the typical Smad protein pathway, the atypical Jun kinase pathway, and the p38 signaling pathway. In fact, the masking peptide delivers TGF-β to the cell and transfers it to the TβRII molecule on the cell surface, which then recruits the TβRI protein to form a functional and active signaling complex that effectively holds the TGF-β peptide in place.

[0090] In addition to delivering TGF-β masked by TGF-β-binding proteins (e.g., fragments containing all or part of their TGF-β-binding extracellular domain in the TGF-β receptor) in a manner that avoids undesirable off-target interactions, masked TGF-β constructs or complexes may also contain one or more polypeptides (“MOD” polypeptides) that act as immunomodulators, capable of influencing the effect of TGF-β on target cells. The ability to deliver TGF-β and an immunomodulator together not only allows for the guidance of TGF-β activation signals but also reduces the amount of immunomodulator required to produce an effect on target cells compared to the administration of the immunomodulator alone. This is a result of increased affinity (through affinity enhancement) achieved by making both polypeptide sequences affinityy for receptors on the same target cells. Increased affinity between masked TGF-β constructs or complexes with one or more MODs and target cells containing both TGF-β peptides and one or more MODs (due to the increased binding free energy ΔG resulting from the interaction of MODs with their receptors) provides enhanced selectivity in the activation of target cells with both types of receptors, provided that a subsaturated amount of the masked TGF-β construct or complex is present. For example, compared to otherwise identical masked TGF-β constructs lacking IL-2 MODs, those with IL-2 MODs, such as… Figure 1 Structure A or Figure 7GThe masked TGF-β construct is a more effective inducer of iTReg differentiation in naive CD4+ cells, even in the presence of an equivalent (equimolar) amount of IL-2 MOD peptide. Furthermore, at subsaturated doses, and when similar numbers of cell types are present, it exhibits, for example, the effect of masked TGF-β constructs compared to constructs lacking IL-2 MOD. Figure 7G The IL-2 MOD shown includes, for example, Figure 1 The construct in structure A selectively binds to cells that have both TGF-β and IL-2 receptors.

[0091] The function of the masked TGF-β construct or complex can be further specified by incorporating modifications that alter the function of individual polypeptide sequences. In some embodiments, an aa substitution (e.g., C77S substitution in TGF-β3) that alters the ability of TGF-β to dimerize can be incorporated. Because the monomeric form of TGF-β exhibits minimal (if any) affinity for TβRIII, incorporating a mutation that restricts the dimerization ability of TGF-β, along with off-target binding to TβRIII, can drag the complex into the TGF-β “reservoir” and limit its ability to stimulate target cells.

[0092] It is also possible to modify polypeptide sequences other than the TGF-β polypeptide sequence (e.g., substitution, deletion, insertion, etc.), including masking polypeptide sequences and immunomodulatory polypeptide sequences.

[0093] In addition to modifications to the TGF-β peptide sequence, peptides that bind to and mask TGF-β can also be modified. Such modifications can alter the availability of the TGF-β peptide sequence by changing the affinity of the masking peptide for TGF-β and the respiration rate (association and dissociation rate) of the masking and TGF-β peptides. Although two different TGF-β peptide / masking peptide complexes can have the same binding association constant (k... Association With k dissociation (ratio), but with a higher k Association and k dissociation The complex can be used more effectively for binding to TβRII on the cell surface, depending on the rate constant, especially k. dissociation Rate. Figure 9 Provides a list of aa substitutions in TβRII that alter the affinity for TGF-β1 and TGF-β3, and a graph showing the affinity of TGF-β constructs masked by the otherwise identical TβRII, in the absence of or presence of one of the three substitutions, each having a different effect on the binding of TβRII to TGF-β3.

[0094] While various peptides can be used to mask TGF-β, including two single-chain antibody sequences (e.g., humanized single-chain antibody sequences), the minimal TGF-β receptor sequence that binds to TGF-β (e.g., the extracellular domain of TβRI, TβRII, or TβRIII) can be employed. The extracellular domain of TβRIII can be used as a masking peptide for dimer TGF-β; however, its high affinity for TGF-β makes it antagonistic to the binding of TGF-β peptide sequences to TβRII. Nevertheless, the TβRIII masking sequence can be efficiently replaced by a cell surface TβRI / TβRII complex with a higher affinity for the TGF-β peptide, thereby allowing activation of those cell surface receptors.

[0095] The extracellular domain of TβRII can be used as a masking peptide. With or without D118A substitution, deletion of the N-terminal amino acid (e.g., δ 14 or 25) of TβRII can produce a protein (or peptide) suitable for masking TGF-β with a pI calculated to be from about 4.5 to about 5.0 (e.g., about 4.7 to 4.85). Combining a TGF-β1 peptide (which has a high pI value) with a TβRII masking peptide (e.g., a peptide comprising an N-terminal deletion) can potentially neutralize the charge of the TGF-β1 peptide. The calculated pI for TβRII (δ 25, D118A) / TGFβ1 is approximately 6.23, with TGFβ1 having a pI of approximately 8.59. In contrast, complexes of δ 14 or δ 25 TβRII and TGF-β3 (with or without D118A and / or C77S substitutions) were calculated to have pI values ​​of approximately 4.9 to approximately 5.3 (approximately 5.06 to 5.17). Furthermore, while combining the extracellular domain of TβRII with an active TGF-β peptide can produce a complex capable of interacting with cell surface TβRIs, thereby influencing TGF-β signaling (e.g., acting as an agonist, partial agonist, antagonist, or partial antagonist), incorporation of aa substitutions that restrict interaction with TβRIs limits or blocks the masked complex's ability to participate in active signaling. Therefore, as discussed below, incorporation of TβRII extracellular domain sequences with N-terminal deletions (e.g., deletions of 14 to 25 aa, Δ14 to Δ25) or substitutions (e.g., substitution of aa other than aspartic acid at D118, such as D118A, D118R, etc.) that reduce or eliminate binding to TβRI can be used to mask TGF-β and prevent stimulation of cells by TGF-β peptides where the N-terminus of TβRII is an intact TβRII extracellular domain. Thus, masked TGF-β complexes, including those where TGF-β is masked by an N-terminal deletion mutant of TβRII, can function by demasking TGF-β (dissociating from the masking peptide or unfolding the molecule), binding TβRII and TβRI to target cells to form an active heterodimeric TβRI / TβRII signaling complex.

[0096] The masking of TGF-β allows it to be expressed at high levels in mammalian cells (e.g., CHO cells) without a decrease in cell viability. This is especially true when the masked TGF-β peptide is blocked from connecting to TβRI via N-terminal aa deletion, substitution, and / or other mutations. Blocking TβRIII interactions (e.g., by blocking dimerization) can further reduce problems associated with cell expression.

[0097] C. Masked TGF-B constructs and masked TGF-B complexes This disclosure describes a masked TGF-β construct (see, for example, Figure 1 Structure A (with a single polypeptide chain) and a masked TGF-β complex (see, for example, Figure 1 Preparation of a masked TGF-β construct and a masked TGF-β complex (a complex with two polypeptide chains). The masked TGF-β construct and the masked TGF-β complex comprise at least one TGF-β polypeptide sequence, at least one polypeptide that binds to and masks the TGF-β polypeptide, and optionally one or more (e.g., one, two, or three) immunomodulatory polypeptides (MODs) as its components, all assembled on a scaffold structure. Although the masked TGF-β construct and the masked TGF-β complex contain portions of membrane-binding proteins (e.g., TGF-β receptors), they do not contain membrane anchoring domain portions (e.g., transmembrane domains sufficient to cause most of the expressed protein to become anchored in the cell membrane (e.g., expressed CHO cells) unless otherwise stated.

[0098] Non-limiting examples of TGF-β constructs and complexes (including) Figure 1 Those in the forms shown are described below.

[0099] The components of the masked TGF-β construct and complex, including MOD, scaffold, linker, TGF-β peptide and TGF-β masking peptide (e.g., single-chain antibody or TGF-β receptor extracellular domain), are each described in the following sections.

[0100] D. Immunomodulatory polypeptide sequence (“MOD”) 1. MOD incorporation into masked TGF-β constructs and complexes As discussed above, while immunomodulatory peptides (MODs) are not essential for the delivery of masked TGF-β peptides or their ability to activate cells via TβRI and TβRII heterodimer receptors, MODs can substantially influence the outcome of TGF-β receptor activation. Therefore, incorporation of wild-type (wt.) or variant MODs (e.g., exhibiting reduced affinity, increased affinity, or selectivity for specific receptors also referred to as “co-MODs,” “co-immunomodulatory peptides,” or homologous costimulatory receptors or their subtypes) may be necessary. Although TGF-β is an immunomodulatory peptide, the term “MOD” as used herein does not include TGF-β or its peptides because it is a central element in the masked TGF-β constructs and complexes described herein.

[0101] MODs suitable for inclusion in either a masked TGF-β construct or a complex (e.g., a homodimer or a heterodimer complex) include, but are not limited to, PD-L1, FAS-L, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, and IL-23.

[0102] In some cases, MODs are independently selected from mature PD-L1, FAS-L, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, and IL-23 peptides or fragments of any one thereof. The MOD peptide sequence incorporated into the masked TGF-β construct and complex may contain only a portion of the secreted MOD peptide or the extracellular portion of the full-length mature MOD protein, provided it is membrane-anchored. Therefore, for example, the MOD peptide sequence in the masked TGF-β construct or complex may, in some cases, exclude one or more (e.g., each) of the signal peptide, transmembrane domain, and / or intracellular domain typically found in some naturally occurring MODs.

[0103] In some cases, the MOD polypeptide sequence suitable for inclusion in the masked TGF-β constructs and complexes of this disclosure comprises all or a portion (e.g., the extracellular portion) of the amino acid sequence of a naturally occurring MOD. In other cases, the MOD suitable for inclusion in the masked TGF-β constructs and complexes of this disclosure comprises at least one (e.g., one, two, or three independently selected) variant MOD that contains at least one amino acid insertion, substitution, and / or deletion compared to the amino acid sequence of a naturally occurring MOD.

[0104] In some cases, variant MODs exhibit lower binding affinity to co-MODs compared to the affinity of the corresponding naturally occurring MOD (e.g., MODs without amino acid substitutions present in variants). Based on this reduced affinity, using MODs with reduced affinity to their co-MODs allows TGF-β peptides to have a greater influence on, or even dominate, the binding interactions. When the binding affinity of a TGF-β peptide is higher than that of the MOD, it can drive the association of masked TGF-β constructs or complexes with cells possessing a TGF-β receptor system (e.g., TGF-βR1 and TGF-βR2 forming high-affinity TGF-β binding heterodimers), while limiting off-target binding to cells with even abundant co-MODs but lacking or having very few TGF-β receptors. Essentially, TGF-β drives the binding and specificity of masked TGF-β constructs or complexes when the affinity of the TGF-β peptide for its cellular receptor (e.g., the binding ΔG) is greater than the affinity of MOD for its co-MOD (e.g., its binding ΔG).

[0105] In one embodiment, any one or more MODs associated with the masked TGF-β construct and complex are independently selected from the group consisting of: wt. or variants: PD-L1; FAS-L; IL-1; IL-2; IL-4; IL-6; IL-7; IL-10; IL-15; IL-21; IL-23; and combinations thereof.

[0106] In some cases, such as when stimulation of Treg cell production is required, at least one MOD polypeptide (e.g., one, two, or three independently selected MODs) present in the masked TGF-β construct and complex 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 to the masked TGF-β construct or complex towards target cells with different combinations of IL-2R receptor subunits. The IL-2 receptor contains a shared IL-2Rγ and two additional IL-2Rα and / or IL-2Rβ subunits to form a trimer (IL-2Rα). 2- IL-2Rγ, (IL-2Rβ) 2- IL-2Rγ or high-affinity (approximately 10 picomol Kd) IL-2Rα-IL-2Rβ-IL-2Rγ receptors. The α chain (CD25) is unique to IL-2, while the β chain (CD122) is shared with the IL-15 receptor, and the γ chain (CD132), crucial for signal transduction, can bind to other cytokine receptor chains. Substitution at H16 (e.g., H16A or H16T) or F42 (e.g., F42A or F42T) can bias binding towards receptors with the IL-2Rβ subunit; and therefore its incorporation biases binding towards receptors exhibiting the β-γ receptor ((IL-2Rβ)). 2- Memory T cells and NK cells with IL-2Rγ, or activated T cells and Tregs exhibiting high affinity for the α-β-γ (IL-2Rα-IL-2Rβ-IL-2Rγ) receptor. In contrast, substitution at N88 (e.g., N88R) reduces binding to IL-2Rβ and can bias binding towards receptors with IL-2Rα subunits; and thus substitution at N88 biases binding towards cells with α-γ ((IL-2Rα)2-IL-2Rγ) and α-β-γ (IL-2Rα-IL-2Rβ-IL-2Rγ) receptors, while avoiding binding and activation of cells with β-γ receptors. See, for example, Skrombolas and Frelinger. Expert Rev Clin Immunol., 10(2): 207–217 (2014). As used in cases where a substituted MOD (such as an IL-2 polypeptide with an aa substitution) binds to its co-MOD or to cells exhibiting the co-MOD, the bias indicates that the presence of substitution alters the degree of interaction between the substituted MOD and the co-MOD relative to the interaction between the wt. MOD and the same co-MOD. For example, an IL-2 sequence with a substitution at N88 (e.g., N88R) that has the aforementioned substitution at H16 and / or F42 may be included, which has lower side effect characteristics (e.g., safer) and is better tolerated by human subjects.

[0107] In some cases, such as when stimulation of iTreg cell (CD4+FoxP3+ cells) production is required (e.g., to induce peripheral tolerance, thereby actively suppressing effector T cells and / or inhibiting immune-mediated tissue destruction), at least one MOD peptide (e.g., one, two, or three independently selected MODs) present in the masked TGF-β construct or complex is an independently selected wt. or variant PD-L1 MOD peptide. See, for example, Francisco et al. J. Exp. Med. , 206(13): 3015-3029 (2009). In addition to the wt. or variant PD-L1 sequence, the masked TGF-β construct or complex may also contain one or more independently selected wt. or variant IL-2 peptides. Sequence variations in IL-2 can be selected to bias the binding of the IL-2 peptide to the masked TGF-β construct towards target cells with different combinations of IL-2R receptor subunits. As discussed above, IL-2 variants include substitutions at H16 (e.g., H16A or H16T) or F42 (e.g., F42A or F42T) that can bias binding towards receptors with IL-2Rβ subunits; and / or IL-2 with substitutions at N88 (e.g., N88R) that reduce binding to IL-2Rβ is better tolerated in human subjects. In one embodiment, the masked TGF-β construct / -β complex comprises both H16T and F42A, or H16A and F42 substituted for both, wherein either pair may be combined with N88 (e.g., N88R) substituted.

[0108] In some cases, such as when it is necessary to stimulate the production of Th17 cells, at least one MOD peptide (e.g., one, two, or three independently selected MODs) present in the masked TGF-β construct and complex is an IL-6 peptide or an IL-6 variant peptide.

[0109] In some cases, such as when stimulation of Th9 cell production is required, at least one MOD peptide (e.g., one, two, or three independently selected MODs) present in the masked TGF-β construct and complex is an IL-4 peptide or an IL-4 variant peptide. See, for example, Elyaman et al. Immunity ., 36(4): 623–634, Immunity (2012).

[0110] In some cases, such as when it is necessary to promote IL-7-dependent survival of low-affinity T cells by controlling thymocyte IL-7Ra expression, at least one MOD peptide (e.g., one, two, or three independently selected MODs) present in the masked TGF-β construct and complex is an IL-7 peptide or an IL-7 variant peptide.

[0111] In some cases, such as when it is necessary to stimulate the production of T follicular helper (Tfh) cells, at least one MOD polypeptide (e.g., one, two, or three independently selected MODs) present in the masked TGF-β construct and complex is an IL-21 or IL-23 polypeptide, or a variant of the IL-21 or IL-23 polypeptide.

[0112] In some cases, such as when tolerance needs to be induced, a variant of at least one MOD peptide (e.g., one, two, or three independently selected MODs) and / or Fas ligand (FasL) peptide present in the masked TGF-β construct may be used.

[0113] In some cases, such as when it is necessary to suppress type II innate lymphocytes (ILC2 cells) (e.g., to suppress asthma and allergic inflammation), at least one MOD peptide (e.g., one, two, or three independently selected MODs) present in the masked TGF-β construct and complex is an IL-10 peptide or a variant of the IL-10 peptide. See, for example, Rajas et al., J Allergy Clin Immunol, 139(5):1468 (2017); and Ogasawara et al., J Allergy Clin Immunol, 141(3): 1147–1151 (2018). Suppression of ILC2 cells can be assessed by a reduction in the production of their type II cytokines IL-5 and IL-13 in vivo (in tissues or body fluids) or in vitro (in culture medium). IL-10 peptides can be monomeric isomers, such as the IL-10M1 molecule described in Josephson et al., J. Biol. Chem. 275:13552-13557 (2000), or variants thereof, both of which are discussed below. Compared to wild-type IL-10, which forms a tangled IL-10 peptide pair 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 and is bioactive in cell proliferation assays. Ibid.

[0114] 2. Mods and variant mods with reduced affinity Suitable MODs exhibiting reduced affinity for their co-MODs can differ from wild-type MOD sequences by 1 to 20 amino acids (aa). For example, in some cases, variant MOD peptide sequences present in masked TGF-β construct complexes may differ from the corresponding wild-type MOD peptide sequences by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aa in amino acid sequence. As another example, in some cases, variant MOD peptides present in masked TGF-β constructs or complexes may differ from the corresponding wild-type MOD peptide sequences by 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aa in amino acid sequence. For example, in some cases, the variant MOD peptide present in the masked TGF-β construct or complex contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD sequence. In some cases, the variant MOD present in the masked TGF-β construct or complex contains a single aa substitution compared to the corresponding reference (e.g., wild-type) MOD sequence. In some cases, the variant MOD present in the masked TGF-β construct or complex contains two aa substitutions (e.g., no more than two aa substitutions) compared to the corresponding reference (e.g., wild-type) MOD sequence. In some cases, the variant MOD present in the masked TGF-β construct or complex contains three aa substitutions (e.g., no more than three aa substitutions) compared to the corresponding reference (e.g., wild-type) MOD sequence. In some cases, variant MODs present in the masked TGF-β construct or complex contain 4 or 5 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD sequence. In some cases, variant MODs present in the masked TGF-β construct or complex contain 6 or 7 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD sequence. In some cases, variant MODs present in the masked TGF-β construct or complex contain 8 or 9 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD sequence. In some cases, variant MODs present in the masked TGF-β construct or complex contain 10 or 11 aa substitutions (e.g., no more than 10 aa substitutions) compared to the corresponding reference (e.g., wild-type) MOD sequence.

[0115] In some cases, variant MODs present in the masked TGF-β construct or complex contain 11 or 12 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD sequence. In some cases, variant MODs present in the masked TGF-β construct or complex contain 13 or 14 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD sequence. In some cases, variant MODs present in the masked TGF-β construct or complex contain 15 or 16 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD sequence. In some cases, variant MODs present in the masked TGF-β construct or complex contain 17 or 18 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD sequence. In some cases, variant MODs present in the masked TGF-β construct or complex contain 19 or 20 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD sequence.

[0116] As discussed above, variant MODs suitable for inclusion in masked TGF-β constructs or complexes exhibit reduced affinity for their corresponding wild-type MODs to their homologous coMODs, compared to the affinity of wild-type MODs for their homologous coMODs.

[0117] In some cases, variant MOD peptide sequences present in masked TGF-β constructs or complexes have binding affinity for homologous coMOD ranging from 1 nM to 100 μM. For example, in some cases, variant MOD peptide sequences present in the masked TGF-β construct or complex have the following binding affinity to homologous co-MOD: 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, 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. μM, approximately 50 μM to approximately 75 μM, or approximately 75 μM to approximately 100 μM.

[0118] 3. Determination of binding affinity The binding affinity between a MOD (e.g., a MOD peptide) and its homologous coMOD can be determined by biomembrane interference (BLI) using purified MOD and purified homologous coMOD. The binding affinity between a masked TGF-β construct or complex containing a MOD and its homologous coMOD can also be determined by BLI using purified masked TGF-β construct or complex and the MOD's homologous coMOD. 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 specificity and relative binding affinity of the MOD between itself and its homologous co-MOD, or between a masked TGF-β construct or complex with MOD and its homologous co-MOD, as described in this disclosure, can be determined using the following procedure.

[0119] To determine the binding affinity between a masked TGF-β construct or complex containing a MOD and a homologous co-MOD of the MOD, the BLI assay can be performed using an Octet RED 96 (Pal FortéBio) instrument or a similar instrument. A control masked TGF-β construct or complex (e.g., a masked TGF-β construct or complex containing a wild-type MOD) is immobilized on an insoluble support (“biosensor”). The immobilized masked TGF-β construct or complex is the “target”. Immobilization can be achieved by immobilizing a capture antibody onto the insoluble support, wherein the capture antibody immobilizes the masked TGF-β construct or complex. For example, when the masked TGF-β construct or complex contains an Ig Fc scaffold, immobilization can be achieved by immobilizing an anti-Ig Fc antibody (e.g., anti-human IgG Fc) onto the insoluble support, wherein the immobilized anti-Ig Fc antibody binds to and immobilizes the masked TGF-β construct or complex. Co-MOD was applied at several different concentrations to immobilized masked TGF-β constructs or complexes, and the instrumental reactions were recorded. Measurements were performed in a liquid medium containing 25 mM HEPES (pH 6.8), 5% polyethylene glycol 6000, 50 mM KCl, 0.1% bovine serum albumin, and 0.02% Tween 20 nonionic detergent. Binding of co-MOD to immobilized masked TGF-β constructs or complexes was performed at 30 °C.

[0120] 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, for example,...). Figure 1For example, monoclonal antibodies (mAbs) targeting TGF-β, TGF-β receptor, scaffold, or MOD polypeptide sequences can be used as positive controls, provided that the antibody does not cause the masked TGF-β construct or complex to dissociate from the support (biosensor). Standard curves can be generated using serially diluted anti-class I or class II MHC monoclonal antibodies. Co-MOD or anti-MHC mAb is the "analyte". BLI analysis measures the interference pattern of white light reflected from two surfaces: i) the immobilized polypeptide ("target"); and ii) the internal reference layer. Changes in the number of molecules bound to the tip of the biosensor ("analyte"; e.g., co-MOD; anti-HLA antibody) cause a shift in the interference pattern; this shift can be measured in real time. Two kinetic terms describing 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 The affinity constant K is generated. D .

[0121] As shown above, the binding affinity between an MOD (e.g., IL-2 or an IL-2 variant) and its homologous co-MOD (e.g., IL-2R) can also be determined by BLI. The determination is similar to that described above for masked TGF-β constructs or complexes. BLI determinations can be performed using an Octet RED 96 (Pal FortéBio) instrument or a similar instrument. Component MODs of the masked TGF-β construct or complex (e.g., variant IL-2 peptides of this disclosure); and control MODs (wherein the control MOD contains a wild-type MOD, such as wild-type IL-2) are individually immobilized on an insoluble support (“biosensor”). Each MOD is a “target”. Immobilization can be achieved by immobilizing a capture antibody onto the insoluble support, wherein 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 achieved by immobilizing a suitable antibody to the immunoaffinity tag (e.g., anti-human IgG Fc) onto an insoluble support, wherein the immobilized antibody binds to and immobilizes the MOD (wherein the MOD contains an IgFc peptide). A co-MOD (or peptide) is applied to the immobilized MOD at several different concentrations, and the instrument's response is recorded. Alternatively, a co-MOD (or peptide) is immobilized to a biosensor (e.g., for an IL-2 receptor heterotrimer, as a monomeric subunit, a heterodimeric subcomplex, or a complete heterotrimer), and the MOD is applied to the immobilized co-MOD at several different concentrations, and the instrument's response is recorded. Assays are 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 nonionic detergent. The binding of co-MOD to immobilized MOD was performed at 30 °C. BLI analysis was performed on the interferogram of white light reflected from two surfaces: i) the immobilized peptide (“target”); and ii) the internal reference layer. Changes in the number of molecules (“analytes”; e.g., co-MOD) bound to the tip of the biosensor cause a shift in the interferogram; this shift in the interferogram can be measured in real time. Two kinetic terms describing the affinity of the target / analyte interaction are the association constant (…). k a ) and dissociation constant ( k d The ratio of the two terms (). k d / a The affinity constant K is generated. DTherefore, determining the binding affinity of a wild-type MOD (e.g., IL-2) to its co-MOD (e.g., its homologous binding partner or receptor; in the case of IL-2, IL-2R), and a variant MOD (e.g., an IL-2 variant as disclosed herein) to its co-MOD (e.g., IL-2R in the case of an IL-2 variant), allows for the determination of the relative binding affinity of the variant co-MOD to the co-MOD compared to the wild-type co-MOD. That is, it is possible to determine whether the binding affinity of the variant MOD to its co-MOD is reduced compared to the binding affinity of the wild-type MOD to the same homologous co-MOD, and if so, to determine the percentage reduction compared to the binding affinity of the wild-type co-MOD.

[0122] BLI assays were performed in multi-well plates. To perform the assay, the plate layout, assay steps, and biosensor assignment were defined in the Octet Data Acquisition software. The biosensor assembly was hydrated. The hydrated biosensor assembly and assay plate were equilibrated on the Octet instrument for 10 minutes. Once data were acquired, they were loaded into the Octet Data Analysis software. In the processing window, data were processed by specifying methods for reference subtraction, y-axis alignment, inter-step correction, and Savitzky-Golay filtering. In the analysis window, data were analyzed by specifying the steps for analysis (association and dissociation), selecting the curve fitting model (1:1), fitting method (global), and target window (in seconds). The fit quality was evaluated. If within a 3-fold range, K was used to track (analyte concentration) for each data point. D The value is taken as the average. K D The error value should be within one order of magnitude of the affinity constant; R 2 The value should be higher than 0.95. See, for example, Abdiche et al. (2008). J. Anal. Biochem 377:209.

[0123] In some cases, i) the binding affinity of the control-masked TGF-β construct or complex (wherein the control-masked TGF-β construct or complex contains wild-type MOD) to the homologous co-MOD is equal to ii) the binding affinity ratio of the masked TGF-β construct or complex containing wild-type MOD variants to the homologous co-MOD, when 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 x 10 2 1. At least 10 31. At least 5 x 10 3 1. At least 10 4 1. At least 10 5 :1 or at least 10 6 1. In some cases, i) the binding affinity of the control-masked TGF-β construct or complex (wherein the control-masked TGF-β construct or complex contains wild-type MOD) to the homologous co-MOD is equal to ii) the binding affinity ratio of the masked TGF-β construct or complex containing wild-type MOD variants to the homologous co-MOD, when measured by BLI, is between 1.5:1 and 10. 6 Within the range of :1, for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 1, 10 2 :1 to 10 3 1, 10 3 :1 to 10 4 1, 10 4 :1 to 10 5 :1 or 10 5 :1 to 10 6 :1.

[0124] In some cases, variant MODs present in masked TGF-β constructs or complexes have binding affinity of 1 nM to 100 μM or 100 nM to homologous coMODs. For example, in some cases, variant MODs present in the masked TGF-β construct or complex have the following binding affinity to homologous coMODs: 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 cases, variant MODs present in the masked TGF-β construct or complex have binding affinity for homologous coMODs of about 1 nM to about 5 nM, about 5 nM to about 10 nM, about 10 nM to about 50 nM, or about 50 nM to about 100 nM.

[0125] 4 PD-L1 and its variants As a non-limiting example, the MOD or variant present in the masked TGF-β construct or complex is PD-L1 or a variant PD-L1 peptide. Wild-type PD-L1 binds to PD1.

[0126] Wild-type human PD-L1 polypeptide may contain the following amino acid sequence: MRIFAVFIFM TYWHLLNAFTVTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKDQLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGYPKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNILNVSIKICLT LSPST (SEQ ID NO:1); wherein aas 1-18 form the signal sequence, aas 19-127 form the Ig-like V-type or IgV domain, and 133-225 are the Ig-like C2-type domain.

[0127] The extracellular domain of wild-type human PD-L1 may contain the following amino acid sequence: FT VTVPKDLYVV EYGSNMTIECKFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAGVYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTTTTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO:2); wherein aas 1-109 form an Ig-like V-type or "IgV" domain, and aas 115-207 are an Ig-like C2-type domain.

[0128] Wild-type PD-L1 IgV domains suitable for use as MODs may include aa 18, aas IgVaas 19-127 of SEQ ID No. 1, and a carboxyl-terminal stabilizing sequence, such as, for example, the last seven amino acids of the sequence described (bold and italic): AFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKTQHSSYRQRARLLKDQLSLGNAA LQ ITDVKLQDAGVYRCMISYGGADYKRITVKVNAPY AAALHEH SEQ ID NO:138. When the carboxyl stabilizing sequence includes a histidine at about aa 122 (e.g., a histidine at about 5 residues on the C-terminal side of tyr(Y) appearing as aa 117 in SEQ ID NO:138), the histidine can form a stable electrostatic bond with the main chain amide at aas 82 and 83 (in bold and italicized form in SEQ ID NO:138 (Q107 and L106 of SEQ ID NO:1)). Alternatively, the stable disulfide bond can be formed by substituting one of aas 82 or 83 (Q107 and L106 of SEQ ID NO:1) and one of aa residues 121, 122, or 123 (corresponding to aa positions 139-141 of SEQ ID NO:1).

[0129] Wild-type PD-1 peptides may contain the following amino acid sequence: PGWFLDSPDR PWNPPTFSPA LLVVTEGDNATFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGTYLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAVICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTSSPARRGSADG PRSAQPLRPE DGHCSWPL (SEQ ID NO:3).

[0130] In some cases, variant PD-L1 peptides (e.g., variants of SEQ ID NO:2 or the IgV domain of PD-L1) exhibit a lower binding affinity to PD-1 (e.g., PD-1 peptides comprising the amino acid sequence listed in SEQ ID NO:3) compared to PD-L1 peptides comprising the amino acid sequence listed in SEQ ID NO:1 or SEQ ID NO:2. For example, in some cases, the binding affinity of variant PD-L1 peptides to PD-1 (e.g., PD-1 peptides comprising the amino acid sequence listed in SEQ ID NO:3) is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% lower than that of PD-L1 peptides comprising the amino acid sequence listed in SEQ ID NO:1 or SEQ ID NO:2.

[0131] In some cases, variant PD-L1 peptides (e.g., variants of SEQ ID NO:2 or their IgV domain) have 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 PD-1 (e.g., SEQ ID NO:3). As another example, in some cases, the binding affinity of the variant PD-L1 peptide (e.g., the variant of SEQ ID NO:2) to PD1 (e.g., the PD1 peptide comprising the amino acid sequence listed in SEQ ID NO:3) is 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.

[0132] Numerous aa substitutions can be created in the PD-L1 extracellular domain sequence used as a MOD, including substitutions of at least 85 consecutive aas (e.g., at least 90, at least 95, at least 100, or at least 105 consecutive aas) having greater than 90% (95%, 98%, or 99%) sequence identity with any of aas 19-127 (IgV domain) of SEQ ID NO:1 and SEQ ID NO:138. Substitutions may include (a) disulfide bond substitutions for D103C and G33C, or V104 and S34C; (b) salt bridge formation substitutions for Q107D and K62R or Q107D and S80R; and / or (c) Pi stacking substitutions for M36Y or M36F (based on SEQ ID NO:1). The PD-L1 MOD sequence may comprise at least 85 consecutive aas (e.g., at least 90, at least 95, at least 100, or at least 105 consecutive aas) having SEQ ID NO:2 and at least one sequence with disulfide, salt bridge, or Pi stack substitution. The PD-L1 MOD sequence may comprise at least 85 consecutive aas (e.g., at least 90, at least 95, at least 100, or at least 105 consecutive aas) having aas 19-127 (IgV domain) having SEQ ID NO:1 and at least one sequence with disulfide, salt bridge, or Pi stack substitution. The PD-L1 MOD sequence may comprise at least 85 consecutive aas (e.g., at least 90, at least 95, at least 100, or at least 105 consecutive aas) having aas SEQ ID NO:138 and at least one sequence with disulfide, salt bridge, or Pi stack substitution.

[0133] In some cases, the variant PD-L1 polypeptide has a single aa substitution compared to the PD-L1 amino acid sequence or the IgV domain of PD-L1 listed in SEQ ID NO:1 and SEQ ID NO:2. In some cases, the variant PD-L1 polypeptide has 2 to 10 aa substitutions compared to the PD-L1 amino acid sequence or the IgV domain of PD-L1 listed in SEQ ID NO:1 and SEQ ID NO:2. In some cases, the variant PD-L1 polypeptide has 2 aa substitutions compared to the PD-L1 amino acid sequence or the IgV domain of PD-L1 listed in SEQ ID NO:1 and SEQ ID NO:2. In some cases, the variant PD-L1 polypeptide has 3 or 4 aa substitutions compared to the PD-L1 amino acid sequence or the IgV domain of PD-L1 listed in SEQ ID NO:1 and SEQ ID NO:2. In some cases, the variant PD-L1 peptide has 5 or 6 amino acid substitutions compared to the PD-L1 amino acid sequence or the IgV domain of PD-L1 listed in SEQ ID NO:1 and SEQ ID NO:2. In some cases, the variant PD-L1 peptide has 7 or 8 amino acid substitutions compared to the PD-L1 amino acid sequence or the IgV domain of PD-L1 listed in SEQ ID NO:1 and SEQ ID NO:2. In some cases, the variant PD-L1 peptide has 9 or 10 amino acid substitutions compared to the PD-L1 amino acid sequence or the IgV domain of PD-L1 listed in SEQ ID NO:1 and SEQ ID NO:2.

[0134] Suitable variant PD-L1 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% sequence identity with at least 170 consecutive aa (e.g., at least 180, 190, or 200 consecutive aa) of SEQ ID NO:2 (e.g., having at least one aa insertion, deletion, or substitution). Suitable variant PD-L1IgV 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% sequence identity with at least 70 consecutive aa (e.g., at least 80, 90, 100, or 105 consecutive aas) of SEQ ID NO:2 (e.g., having at least one aa insertion, deletion, or substitution).

[0135] In one example, the variant PD-L1 polypeptide sequence comprises a polypeptide sequence having at least 90% (e.g., at least 95%, 98%, or 99%) or 100% aa sequence identity with SEQ ID NO:2, wherein the residue at position 8 is an aa other than D; in one such example, the residue is A, and in another example, the residue is R. The variant PD-L1 polypeptide sequence comprises a polypeptide sequence having at least 90% (e.g., at least 95%, 98%, or 99%) or 100% aa sequence identity with SEQ ID NO:2, wherein the residue at position 36 is an aa other than I; in one such example, the residue is A, and in another example, the residue is D. The variant PD-L1 polypeptide sequence further comprises a polypeptide sequence having at least 90% (e.g., at least 95%, 98%, or 99%) or 100% aa sequence identity with SEQ ID NO:2, wherein the residue at position 54 is an aa other than E; in one such example, the residue is A, and in another example, the residue is R.

[0136] 5. IL-1 and its variants As a non-limiting example, the MOD or variant MOD present in the masked TGF-β construct or complex is IL-1 or a variant IL-1 peptide. Wild-type IL-1 has two isoforms, IL-1α and IL-1β, both of which bind to the IL-1 receptor.

[0137] Wild-type human IL-1α precursor polypeptide may contain the following amino acid sequence: MAKVPDMFEDL KNCYSENEEDSSSIDHLSLNQK SFYHVSYGPLH EGCMDQSVSLS ISETSKTSKLT FKESMVVVATN GKVLKKRRLSLSQSITDDDLEA IANDSEEEIIK PRSAPFSFLSN VKYNFMRIIKY EFILNDALNQS IIRANDQYLTAAALHNLDEAVK FDMGAYKSSKD DAKITVILRIS KTQLYVTAQDE DQPVLLKEMPE IPKTITGSETNLLFFWETHGTK NYFTSVAHPNL FIATKQDYWVC LAGGPPSITDF QILENQA (SEQ ID NO:4)UniProtKB - P01583, The NCBI reference sequence NP_000566.3 may have one or more of the following naturally occurring variants: R85Q, A114S, N125D, D138N, and D176H.

[0138] Mature wild-type human IL-1α polypeptide may contain the following amino acid sequence: PRSAPFSFLS NVKYNFMRIIKYEFILNDAL NQSIIRANDQ YLTAAALHNL DEAVKFDMGA YKSSKDDAKI TVILRISKTQ LYVTAQDEDQPVLLKEMPEI PKTITGSETN LLFFWETHGT KNYFTSVAHP NLFIATKQDY WVCLAGGPPS ITDFQILENQA (SEQ ID NO:5).

[0139] Wild-type human IL-1β precursor polypeptide may contain the following amino acid sequence: MAEVPELASE MMAYYSGNEDDLFFEADGPK QMKCSFQDLD LCPLDGGIQL RISDHHYSKG FRQAASVVVA MDKLRKMLVP CPQTFQENDLSTFFPFIFEE EPIFFDTWDN EAYVHDAPVR SLNCTLRDSQ QKSLVMSGPY ELKALHLQGQ DMEQQVVFSMSFVQGEESND KIPVALGLKE KNLYLSCVLK DDKPTLQLES VDPKNYPKKK MEKRFVFNKI EINNKLEFESAQFPNWYIST SQAENMPVFL GGTKGGQDIT DFTMQFVSS (SEQ ID NO:6) UniProtKB - P0158, NCBI reference sequence NP_000567.1.

[0140] Mature wild-type human IL-1β polypeptide may contain the following amino acid sequence: APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS (SEQ ID NO:7).

[0141] IL-1α and IL-1β bind to the IL-1 receptor, which may have the following sequence: MKVLLRLICF IALLISSLEADKCKEREEKI ILVSSANEID VRPCPLNPNE HKGTITWYKD DSKTPVSTEQ ASRIHQHKEK LWFVPAKVEDSGHYYCVVRN SSYCLRIKIS AKFVENEPNL CYNAQAIFKQ KLPVAGDGGL VCPYMEFFKN ENNELPKLQWYKDCKPLLLD NIHFSGVKDR LIVMNVAEKH RGNYTCHASY TYLGKQYPIT RVIEFITLEE NKPTRPVIVSPANETMEVDL GSQIQLICNV TGQLSDIAYW KWNGSVIDED DPVLGEDYYS VENPANKRRS TLITVLNISEIESRFYKHPF TCFAKNTHGI DAAYIQLIYP VTNFQKHMIG ICVTLTVIIV CSVFIYKIFK IDIVLWYRDSCYDFLPIKAS DGKTYDAYIL YPKTVGEGST SDCDIFVFKV LPEVLEKQCG YKLFIYGRDD YVGEDIVEVINENVKKSRRL IIILVRETSG FSWLGGSSEE QIAMYNALVQ DGIKVVLLEL EKIQDYEKMP ESIKFIKQKHGAIRWSGDFT QGPQSAKTRF WKNVRYHMPV QRRSPSSKHQ LLSPATKEKL QREAHVPLG, (SEQ ID NO:8), NCBI reference sequence NP_000868.1, wherein aas 21 to 569 form a mature polypeptide, whose extracellular domains can be used to determine the binding affinity with IL-1α and IL-1β.

[0142] In some cases, the variant IL-1α and IL-1β peptides exhibit reduced binding affinity to the IL-1 receptor or its extracellular domain having the sequence listed in SEQ ID NO:8, compared to the binding affinity of the IL-1 peptide containing the amino acid sequence listed in SEQ ID NO:5 or SEQ ID NO:7. For example, in some cases, the variant IL-1α and IL-1β peptides bind to the mature protein containing the IL-1 receptor or its extracellular domain listed in SEQ ID NO:8 with a binding affinity at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% lower than the binding affinity of the IL-1α or IL-1β peptide containing the amino acid sequence listed in SEQ ID NO:5 or SEQ ID NO:7.

[0143] In some cases, the variant IL-1α or IL-1β peptide (e.g., variants of SEQ ID NO:5 or 7) has a binding affinity of 1 nM to 1 mM to the IL-1 receptor having the sequence listed in SEQ ID NO:8 (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 cases, variant IL-1α or IL-1β peptides (e.g., variants of SEQ ID NO:5 or 7) have binding affinity for the IL-1 receptor or its extracellular domain listed in SEQ ID NO:8 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. μM.

[0144] In some cases, the variant IL-1α or IL-1β polypeptide has a single aa substitution compared to the IL-1α or IL-1β amino acid sequence listed in SEQ ID NO:5 or SEQ ID NO:7. In some cases, the variant IL-1α or IL-1β polypeptide has 2 to 10 aa substitutions compared to the IL-1α or IL-1β amino acid sequence listed in SEQ ID NO:5 or SEQ ID NO:7. In some cases, the variant IL-1α or IL-1β polypeptide has 2 aa substitutions compared to the IL-1α or IL-1β amino acid sequence listed in SEQ ID NO:5 or SEQ ID NO:7. In some cases, the variant IL-1α or IL-1β polypeptide has 3 or 4 aa substitutions compared to the IL-1α or IL-1β amino acid sequence listed in SEQ ID NO:5 or SEQ ID NO:7. In some cases, the variant IL-1α or IL-1β polypeptide has 5 or 6 amino acid substitutions compared to the IL-1α or IL-1β amino acid sequence listed in SEQ ID NO:5 or SEQ ID NO:7. In some cases, the variant IL-1α or IL-1β polypeptide has 7 or 8 amino acid substitutions compared to the IL-1α or IL-1β amino acid sequence listed in SEQ ID NO:5 or SEQ ID NO:7. In some cases, the variant IL-1α or IL-1β polypeptide has 9 or 10 amino acid substitutions compared to the IL-1α or IL-1β amino acid sequence listed in SEQ ID NO:5 or SEQ ID NO:7.

[0145] Suitable variant 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% sequence identity with at least 100 consecutive aa sequences of the amino acid sequence listed in SEQ ID NO:5 or SEQ ID NO:7 (e.g., having at least one aa substitution, deletion, or insertion).

[0146] 6. IL-2 and its variants As a non-limiting example, the MOD or variant MOD present in the masked TGF-β construct or complex is IL-2 or a variant IL-2 peptide. In some cases, the variant MOD present in the masked TGF-β construct or complex is a variant IL-2 peptide. Wild-type IL-2 binds to the IL-2 receptor (IL-2R). The amino acid sequence of wild-type IL-2 may be as follows: APTSSSTKKT QLQL EH LLL D LQMILNGINN YKNPKLTRML TF KF Y MPKKA TELKHLQCLE EELKPLEEVLNLAQSKNFHL RPRDLIS N IN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFC Q SIIS TLT (UniProtP60568, aa 21-153 of SEQ ID NO:9).

[0147] Wild-type IL-2 binds to the IL-2 receptor (IL-2R) on the cell surface. In some cases, the IL-2 receptor is a heterotrimeric polypeptide comprising an α chain (IL-2Rα; also known as CD25), a β chain (IL-2Rβ; also known as CD122), and a γ chain (IL-2Rγ; also known as CD132). The amino acid sequences of human IL-2Rα, IL-2Rβ, and IL-2Rγ are as follows.

[0148] Human IL-2Rα: ELCDDDPPE IPHATFKAMA YKEGTMLNCE CKRGFRRIKS GSLYMLCTGNSSHSSWDNQC QCTSSATRNT TKQVTPQPEE QKERKTTEMQ SPMQPVDQAS LPGHCREPPP WENEATERIYHFVVGQMVYY QCVQGYRALH RGPAESVCKM THGKTRWTQP QLICTGEMET SQFPGEEKPQ ASPEGRPESETSCLVTTTDF QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL ISVLLLSGLT WQRRQRKSRR TI (SEQ IDNO:10).

[0149] Human IL-2Rβ: VNG TSQFTCFYNS RANISCVWSQ DGALQDTSCQ VHAWPDRRRW NQTCELLPVSQASWACNLIL GAPDSQKLTT VDIVTLRVLC REGVRWRVMA IQDFKPFENL RLMAPISLQV VHVETHRCNISWEISQASHY FERHLEFEAR TLSPGHTWEE APLLTLKQKQ EWICLETLTP DTQYEFQVRV KPLQGEFTTWSPWSQPLAFR TKPAALGKDT IPWLGHLLVG LSGAFGFIIL VYLLINCRNT GPWLKKVLKC NTPDPSKFFSQLSSEHGGDV QKWLSSPFPS SSFSPGGLAP EISPLEVLER DKVTQLLLQQ DKVPEPASLS SNHSLTSCFTNQGYFFFHLP DALEIEACQV YFTYDPYSEE DPDEGVAGAP TGSSPQPLQP LSGEDDAYCT FPSRDDLLLFSPSLLGGPSP PSTAPGGSGA GEERMPPSLQ ERVPRDWDPQ PLGPPTPGVP DLVDFQPPPE LVLREAGEEVPDAGPREGVS FPWSRPPGQG EFRALNARLP LNTDAYLSLQ ELQGQDPTHL V (SEQ ID NO:11).

[0150] Human IL-2Rγ: LNTTILTP NGNEDTTADF FLTTMPTDSL SVSTLPLPEV QCFVFNVEYMNCTWNSSSEP QPTNLTLHYW YKNSDNDKVQ KCSHYLFSEE ITSGCQLQKK EIHLYQTFVV QLQDPREPRRQATQMLKLQN LVIPWAPENL TLHKLSESQL ELNWNNRFLN HCLEHLVQYR TDWDHSWTEQ SVDYRHKFSLPSVDGQKRYT FRVRSRFNPL CGSAQHWSEW SHPIHWGSNT SKENPFLFAL EAVVISVGSM GLIISLLCVYFWLERTMPRI PTLKNLEDLV TEYHGNFSAW SGVSKGLAES LQPDYSERLC LVSEIPPKGG ALGEGPGASPCNQHSPYWAP PCYTLKPET (SEQ ID NO:12).

[0151] In some cases, when the masked TGF-β construct or complex contains a variant IL-2 peptide, the homologous co-MOD is an IL-2R containing an amino acid sequence of any one of SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0152] In some cases, the binding affinity of the variant IL-2 peptide to IL-2R is reduced compared to that of the IL-2 peptide containing the amino acid sequence listed in SEQ ID NO:9. For example, in some cases, when measured under the same conditions, the binding affinity of the variant IL-2 peptide to IL-2R is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% lower than that of the IL-2 peptide containing the amino acid sequence listed in SEQ ID NO:9 to IL-2R (e.g., IL-2R containing the amino acid sequence listed in SEQ ID NO:10-12).

[0153] In some cases, variant IL-2 peptides (e.g., variants of SEQ ID NO:9) have binding affinity for IL-2R (e.g., SEQ ID NO:10-12) ranging from 100 nM to 100 μM. As another example, in some cases, variant IL-2 peptides (e.g., variants of SEQ ID NO:9) have binding affinity for IL-2Rs (e.g., IL-2Rs comprising peptides containing the amino acid sequences listed in SEQ ID NO:10-12) 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. μM to approximately 100 μM.

[0154] In some cases, the variant IL-2 polypeptide has a single aa substitution compared to the IL-2 amino acid sequence listed in SEQ ID NO:9. In some cases, the variant IL-2 polypeptide has 2 to 10 aa substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:9. In some cases, the variant IL-2 polypeptide has 2 aa substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:9. In some cases, the variant IL-2 polypeptide has 3 aa substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:9. In some cases, the variant IL-2 polypeptide has 4 aa substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:9. In some cases, the variant IL-2 polypeptide has 5 aa substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:9. In some cases, the variant IL-2 polypeptide has 6 aa substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:9. In some cases, the variant IL-2 polypeptide has 7 aa substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:9. In some cases, the variant IL-2 polypeptide has 8 aa substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:9. In some cases, the variant IL-2 polypeptide has 9 aa substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:9. In some cases, the variant IL-2 polypeptide has 10 aa substitutions compared to the IL-2 amino acid sequence listed in SEQ ID NO:9.

[0155] Suitable variant IL-2 polypeptide sequences include polypeptide sequences comprising at least 80 (e.g., 90, 100, 110, 120, 130, or 133) consecutive aas sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with the aa sequence of SEQ ID NO:9. Additionally, IL-2 variants include polypeptide sequences comprising at least 80 (e.g., 90, 100, 110, 120, 130, or 133) consecutive aas sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% aa sequence identity with the aa sequence of any of the following variant IL-2 aa sequences (see SEQ ID NO: 13-27).

[0156] APTSSSTKKT QLQL X HLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKATELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADETATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:13), where X is any amino acid except Glu. In some cases, X is Ala.

[0157] APTSSSTKKT QLQLEHLLL X LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT (SEQ ID NO:14), where X is any amino acid other than Asp. In some cases, X is Ala.

[0158] APTSSSTKKT QLQLE X LLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIISTLT (SEQ ID NO:15), where X (H16) is any amino acid other than His. In some cases, X is Ala(H16A). In some cases, X is Arg. In some cases, X is Asn. In some cases, X is Asp. In some cases, X is Cys. In some cases, X is Glu. In some cases, X is Gln. In some cases, X is Gly. In some cases, X is Ile. In some cases, X is Lys. In some cases, X is Leu. In some cases, X is Met. In some cases, X is Phe. In some cases, X is Pro. In some cases, X is Ser. In some cases, X is Thr(H16T). In some cases, X is Tyr. In some cases, X is Trp. In some cases, X is Val.

[0159] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML T X KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIISTLT (SEQ ID NO:16), where X (F42) is any amino acid except Phe. In some cases, X is Ala (F42A). In some cases, X is Thr (F42T).

[0160] APTSSSTKKT QLQLEHLLLLD LQMILNGINN YKNPKLTRML TFKF X MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIISTLT (SEQ ID NO:17), where X is any amino acid except Tyr. In some cases, X is Ala; APTSSSTKKT QLQLEHLLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLIS X IN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIISTLT (SEQ ID NO:18), where X (N88) is any amino acid other than Asn. In some cases, X is Ala; in others, X is Arg.

[0161] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFC X SIISTLT (SEQ ID NO:19), where X is any amino acid except Gln. In some cases, X is Ala.

[0162] APTSSSTKKT QLQLE X 1 LLLD LQMILNGINN YKNPKLTRML T X 2KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIISTLT (SEQ ID NO:20), wherein X1 (H16) is any amino acid except His, and X2 (F42) is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X1 is Ala; and X2 is Ala (H16A, F42A). In some cases, X1 is Thr; and X2 is Ala (H16T, F42A). In some cases, X1 is Ala; and X2 is Thr (H16A, F42T). In some cases, X1 is Thr; and X2 is Thr (H16T, F42T).

[0163] APTSSSTKKT QLQLE X 1 LLLD LQMILNGINN YKNPKLTRML T X 2 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLIS R IN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIISTLT (SEQ ID NO:21), which contains an N88R substitution, wherein X1 (H16) is any amino acid other than His, and wherein X2 (F42) is any amino acid other than Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X1 is Ala; and X2 is Ala. In some cases, X1 is Thr; and X2 is Ala. In some cases, X1 is Ala; and X2 is Thr. In some cases, X1 is Thr; and X2 is Thr.

[0164] APTSSSTKKT QLQLEHLLL X 1 LQMILNGINN YKNPKLTRML T X 2KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIISTLT (SEQ ID NO:22), wherein X1 is any amino acid except Asp; and X2 is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X1 is Ala; and X2 is Ala.

[0165] APTSSSTKKT QLQL X 1 HLLL X 2 LQMILNGINN YKNPKLTRML T X 3 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIISTLT (SEQ ID NO:23), wherein X1 is any amino acid except Glu; X2 is any amino acid except Asp; and X3 is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, any two or all three... X1 is Ala ; X2 is Ala ;and X3 It's Ala .

[0166] APTSSSTKKT QLQLE X 1 LLL X 2 LQMILNGINN YKNPKLTRML T X 3 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIISTLT (SEQ ID NO:24), wherein X1 is any amino acid except His; X2 is any amino acid except Asp; and X3 is any amino acid except Phe. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala.

[0167] APTSSSTKKT QLQLEHLLL X 1LQMILNGINN YKNPKLTRML T X 2 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFC X 3 SIISTLT (SEQ ID NO:25), wherein X1 is any amino acid except Asp; wherein X2 is any amino acid except Phe; and wherein X3 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala.

[0168] APTSSSTKKT QLQLEHLLL X 1 LQMILNGINN YKNPKLTRML T X 2 KF X 3 MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIISTLT (SEQ ID NO:26), wherein X1 is any amino acid except Asp; X2 is any amino acid except Phe; and X3 is any amino acid except Tyr. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X1 is Ala; X2 is Ala; and X3 is Ala.

[0169] APTSSSTKKT QLQLE X 1 LLL X 2 LQMILNGINN YKNPKLTRML T X 3 KF X 4MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIISTLT (SEQ ID NO:27), wherein X1 is any amino acid except His; X2 is any amino acid except Asp; X3 is any amino acid except Phe; and X4 is any amino acid except Tyr. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X4 is Ala. In some cases, X1 is Ala; X2 is Ala; X3 is Ala; and X4 is Ala.

[0170] APTSSSTKKT QLQLEHLLL X 1 LQMILNGINN YKNPKLTRML T X 2 KF X 3 MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFC X 4 SIISTLT (SEQ ID NO: 139), wherein X1 is any amino acid except Asp; X2 is any amino acid except Phe; X3 is any amino acid except Tyr; and X4 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X4 is Ala. In some cases, X1 is Ala; X2 is Ala; X3 is Ala; and X4 is Ala.

[0171] APTSSSTKKT QLQLE X 1 LLL X 2 LQMILNGINN YKNPKLTRML T X 3 KF X 4 MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFC X 5SIISTLT (SEQ ID NO: 140), wherein X1 is any amino acid except His; wherein X2 is any amino acid except Asp; wherein X3 is any amino acid except Phe; wherein X4 is any amino acid except Tyr; and wherein X5 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, X4 is Ala. In some cases, X5 is Ala. In some cases, any two, three, four, or all five X1 are Ala; X2 are Ala; X3 are Ala; X4 are Ala; and / or X5 are Ala.

[0172] APTSSSTKKT QLQLE X 1 LLLD LQMILNGINN YKNPKLTRML T X 2 KFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFC X 3 SIISTLT (SEQ ID NO: 141), wherein X1 is any amino acid except His; wherein X2 is any amino acid except Phe; and wherein X3 is any amino acid except Gln. In some cases, X1 is Ala. In some cases, X2 is Ala. In some cases, X3 is Ala. In some cases, any two or all three X1 are Ala; X2 is Ala; and / or X3 is Ala.

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

[0174] Wild-type human IL-4 isoform 1 precursor polypeptide may contain the following amino acid sequence: MGLTSQLLPP LFFLLACAGNFVHGHKCDIT LQEIIKTLNS LTEQKTLCTE LTVTDIFAAS KNTTEKETFC RAATVLRQFY SHHEKDTRCLGATAQQFHRH KQLIRFLKRL DRNLWGLAGL NSCPVKEANQ STLENFLERL KTIMREKYSK CSS (SEQ ID NO:28) NCBI reference sequence NP_000580.1.

[0175] Mature wild-type human IL-4 isoform 1 polypeptide may contain the following amino acid sequence: KCDIT LQEIIKTLNSLTEQKTLCTE LTVTDIFAAS KNTTEKETFC RAATVLRQFYSHHEKDTRCL GATAQQFHRH KQLIRFLKRLDRNLWGLAGLNSCPVKEANQ STLENFLERL KTIMREKYSK CSS (SEQ ID NO:29).

[0176] Wild-type human IL-4 isoform 2 precursor polypeptide may contain the following amino acid sequence: MGLTSQLLPP LFFLLACAGNFVHGHKCDIT LQEIIKTLNS LTEQKNTTEK ETFCRAATVL RQFYSHHEKD TRCLGATAQQ FHRHKQLIRFLKRLDRNLWG LAGLNSCPVK EANQSTLENF LERLKTIMRE ​​KYSKCSS, NCBI reference sequence: NP_758858.1, (SEQ ID NO:30).

[0177] Mature wild-type human IL-4 isoform 2 polypeptide may contain the following amino acid sequence: KCDIT LQEIIKTLNSLTEQKNTTEK ETFCRAATVL RQFYSHHEKD TRCLGATAQQ FHRHKQLIRF LKRLDRNLWG LAGLNSCPVKEANQSTLENFLERLKTIMRE ​​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 can have the sequence MGWLCSGLLF PVSCLVLLQV ASSGNMKVLQ EPTCVSDYMSISTCEWKMNG PTNCSTELRL LYQLVFLLSE AHTCIPENNG GAGCVCHLLM DDVVSADNYT LDLWAGQQLLWKGSFKPSEH VKPRAPGNLT VHTNVSDTLL LTWSNPYPPD NYLYNHLTYA VNIWSENDPA DFRIYNVTYLEPSLRIAAST LKSGISYRAR VRAWAQCYNT TWSEWSPSTK WHNSYREPFE QHLLLGVSVS CIVILAVCLLCYVSITKIKK EWWDQIPNPA RSRLVAIIIQ DAQGSQWEKR SRGQEPAKCP HWKNCLTKLL PCFLEHNMKRDEDPHKAAKE MPFQGSGKSA WCPVEISKTV LWPESISVVR CLEFEAPVE CEEEEEVEEE KGSFCASPESSRDDFQEGRE GIVARLTESL FLDLLGEENG GFCQQDMGES CLLPPSGSTS AHMPWDEFPS AGPKEAPPWGKEQPLHLEPS PPASPTQSPD NLTCTETPLV IAGNPAYRSF SNSLSQSPCP RELGPDPLLA RHLEEVEPEMPCVPQLSEPT TVPQPEPETW EQILRRNVLQ HGAAAAPVSA PTSGYQEFVH AVEQGGTQAS AVVGLGPPGEAGYKAFSSLL ASSAVSPEKC GFGASSGEEG YKPFQDLIPG CPGDPAPVPV PLFTGFLDRE PPRSPQSSHLPSSSPEHLGL EPGEKVEDMP KPPLPQEQAT DPLVDSLGSG IVYSALTCHL CGHLKQCHGQ EDGGQTPVMASPCCGCCCGD RSSPPTTPLR APDPSPGGVP LEASLCPASL APSGISEKSK SSSSFHPAPG NAQSSSQTPKIVNFVSVGPT YMRVS, (SEQ ID NO:32), NCBI reference sequence NP_000409.1. Among them, aas 26 to 825 form the mature polypeptide, and aas 233-256 are transmembrane regions; the extracellular domains of the protein can be used to determine the binding affinity with IL-4 isoforms 1 or 2.

[0179] Soluble isoform 2, having the sequence MGWLCSGLLF PVSCLVLLQV ASSGNMKVLQ EPTCVSDYMSISTCEWKMNG PTNCSTELRL LYQLVFLLSE AHTCIPENNG GAGCVCHLLM DDVVSADNYT LDLWAGQQLLWKGSFKPSEH VKPRAPGNLT VHTNVSDTLL LTWSNPYPPDN YLYNHLTYAVN IWSENDPADFRIYNVTYLEP SLRIAASTLK SGISYRARVRA WAQCYNTTWSE WSPSTKWHNS NIC, (SEQ ID NO:33), UniProtKB - P24394, can also be used to determine the binding affinity of the two IL-4 isoforms.

[0180] In some cases, variant IL-4 isoform 1 or 2 peptides (e.g., variants of SEQ ID NO:29 or 31) exhibit reduced binding affinity to the mature IL-4 receptor sequence listed in SEQ ID NO:32 or its extracellular domain, or the soluble IL-4 receptor listed in SEQ ID NO:33, compared to the binding affinity of IL-4 peptides containing the amino acid sequences listed in SEQ ID NO:29 or SEQ ID NO:31. For example, in some cases, the binding affinity of the variant IL-4 isoform 1 or 2 polypeptide to the mature IL-4 receptor listed in SEQ ID NO:32 (or its extracellular domain) or SEQ ID NO:33 is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% lower than the binding affinity of the IL-4 isoform 1 or 2 polypeptide containing the amino acid sequence listed in SEQ ID NO:29 or SEQ ID NO:31.

[0181] In some cases, the variant IL-4 isoform 1 or 2 peptide (e.g., variants 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) to the mature IL-4 receptor listed in SEQ ID NO:32 (or its extracellular domain) or SEQ ID NO:33. As another example, in some cases, variant IL-4 isoform 1 or 2 peptides (e.g., variants of SEQ ID NO:29 or 31) have binding affinity for the mature IL-4 receptor listed in SEQ ID NO:32 (or its extracellular domain) 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. μM, or approximately 75 μM to approximately 100 μM.

[0182] In some cases, the variant IL-4 isoform 1 or 2 polypeptide has a single aa substitution compared to the amino acid sequence of IL-4 isoform 1 or 2 listed in SEQ ID NO:29 or SEQ ID NO:31. In some cases, the variant IL-4 isoform 1 or 2 polypeptide has 2 to 10 aa substitutions compared to the amino acid sequence of IL-4 isoform 1 or 2 listed in SEQ ID NO:29 or SEQ ID NO:31. In some cases, the variant IL-4 isoform 1 or 2 polypeptide has 2 aa substitutions compared to the amino acid sequence of IL-4 isoform 1 or 2 listed in SEQ ID NO:29 or SEQ ID NO:31. In some cases, the variant IL-4 isoform 1 or 2 polypeptide has 3 or 4 aa substitutions compared to the amino acid sequence of IL-4 isoform 1 or 2 listed in SEQ ID NO:29 or SEQ ID NO:31. In some cases, the variant IL-4 polypeptide has 5 or 6 amino acid substitutions compared to the amino acid sequence of IL-4 isoform 1 or 2 listed in SEQ ID NO:29 or SEQ ID NO:31. In some cases, the variant IL-4 isoform 1 or 2 polypeptide has 7 or 8 amino acid substitutions compared to the amino acid sequence of IL-4 isoform 1 or 2 listed in SEQ ID NO:29 or SEQ ID NO:31. In some cases, the variant IL-4 isoform 1 or 2 polypeptide has 9 or 10 amino acid substitutions compared to the amino acid sequence of IL-4 isoform 1 or 2 listed in SEQ ID NO:29 or SEQ ID NO:31.

[0183] Suitable variant 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% sequence identity with at least 80 consecutive aa (e.g., at least 100 or 110 consecutive aa) of at least 80 consecutive aa (e.g., having at least one aa substitution, deletion, or insertion) of SEQ ID NO:29 or SEQ ID NO:31.

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

[0185] Wild-type human IL-6 precursor polypeptide may contain the following amino acid sequence: MNSFSTSAFG PVAFSLGLLLVLPAAFPAPV PPGEDSKDVA APHRQPLTSS ERIDKQIRYI LDGISALRKE TCNKSNMCES SKEALAENNLNLPKMAEKDG CFQSGFNEET CLVKIITGLL EFEVYLEYLQ NRFESSEEQA RAVQMSTKVL IQFLQKKAKNLDAITTPDPT TNASLLTKLQAQNQWLQDMT THLILRSFKE FLQSSLRALR QM (SEQ ID NO:34) NCBI reference sequence NP_000591.1.

[0186] Mature wild-type human IL-6 polypeptide may contain the following amino acid sequence: VPPGEDSKDVA APHRQPLTSSERIDKQIRYI LDGISALRKE TCNKSNMCES SKEALAENNL NLPKMAEKDG CFQSGFNEET CLVKIITGLLEFEVYLEYLQ NRFESSEEQA RAVQMSTKVL IQFLQKKAKN LDAITTPDPT TNASLLTKLQ AQNQWLQDMTTHLILRSFKE FLQSSLRALR QM (SEQ ID NO:35).

[0187] IL-6 binds to a membrane-bound IL-6 receptor, which contains α and β subunits. The human IL-6R alpha subunit may have the sequence MLAVGCALLA ALLAAPGAAL APRRCPAQEV ARGVLTSLPG DSVTLTCPGV EPEDNATVHWVLRKPAAGSH PSRWAGMGRR LLLRSVQLHD SGNYSCYRAG RPAGTVHLLV DVPPEEPQLS CFRKSPLSNVVCEWGPRSTP SLTTKAVLLV RKFQNSPAED FQEPCQYSQE SQKFSCQLAV PEGDSSFYIV SMCVASSVGSKFSKTQTFQG CGILQPDPPA NITVTAVARN PRWLSVTWQD PHSWNSSFYR LRFELRYRAE RSKTFTTWMVKDLQHHCVIH DAWSGLRHVV QLRAQEEFGQ GEWSEWSPEA MGTPWTESRS PPAENEVSTP MQALTTNKDDDNILFRDSAN ATSLPVQDSS SVPLPTFLVA GGSLAFGTLL CIAIVLRFKK TWKLRALKEG KTSMHPPYSLGQLVPERPRP TPVLVPLISP PVSPSSLGSD NTSSHNRPDA RDPRSPYDIS NTDYFFPR, (SEQ ID NO:36), NCBI reference sequence: NP_000556.1, wherein aas 26 to 825 form the mature polypeptide, and aas 233-256 are transmembrane regions. After binding to IL-6, the IL-6 α subunit binds to the IL-6 β subunit.

[0188] Human IL-6R β subunit can have sequence MLTLQTWLVQ ALFIFLTTES TGELLDPCGY ISPESPVVQL HSNFTAVCVL KEKCMDYFHVNANYIVWKTN HFTIPKEQYT IINRTASSVT FTDIASLNIQ LTCNILTGFQ LEQNVYGITI ISGLPPEKPKNLSCIVNEGK KMRCEWDGGR ETHLETNFTL KSEWATHKFA DCKAKRDTPT SCTVDYSTVY FVNIEVWVEAENALGKVTSD HINFDPVYKV KPNPPHNLSV INSEELSSIL KLTWTNPSIK SVIILKYNIQ YRTKDASTWSQIPPEDTAST RSSFTVQDLK PFTEYVFRIR CMKEDGKGYW SDWSEEASGI TYEDRPSKAP SFWYKIDPSHTQGYRTVQLV WKTLPPFEAN GKILDYEVTL TRWKSHLQNY TVNATKLTVN LTNDRYLATL TVRNLVGKSDAAVLTIPACD FQATHPVMDL KAFPKDNMLW VEWTTPRESV KKYILEWCVL SDKAPCITDW QQEDGTVHRTYLRGNLAESK CYLITVTPVY ADGPGSPESI KAYLKQAPPS KGPTVRTKKV GKNEAVLEWD QLPVDVQNGFIRNYTIFYRT IIGNETAVNV DSSHTEYTLS SLTSDTLYMV RMAAYTDEGG KDGPEFTFTT PKFAQGEIEAIVVPVCLAFL LTTLLGVLFC FNKRDLIKKH IWPNVPDPSK SHIAQWSPHT PPRHNFNSKD QMYSDGNFTDVSVVEIEAND KKPFPEDLKS LDLFKKEKIN TEGHSSGIGG SSCMSSSRPS ISSSDENESS QNTSSTVQYSTVVHSGYRHQ VPSVQVFSRS ESTQPLLDSE ERPEDLQLVD HVDGGDGILP RQQYFKQNCS QHESSPDISHFERSKQVSSV NEEDFVRLKQ QISDHISQSC GSGQMKMFQE VSAADAFGPG TEGQVERFET VGMEAATDEGMPKSYLPQTV RQGGYMPQ,(SEQ ID NO:37), UniProtKB - P40189, wherein aas 23 to 918 form the mature polypeptide, and aas 620-641 are transmembrane regions.

[0189] As an alternative to IL-6 binding to the membrane-bound IL-6R α subunit, it can bind to the mature, soluble form of the IL-6R α subunit having the following sequences: MLAVGCALLA ALLAAPGAAL APRRCPAQEV ARGVLTSLPGDSVTLTCPGV EPEDNATVHW VLRKPAAGSH PSRWAGMGRR LLLRSVQLHD SGNYSCYRAG RPAGTVHLLVDVPPEEPQLS CFRKSPLSNV VCEWGPRSTP SLTTKAVLLV RKFQNSPAED FQEPCQYSQE SQKFSCQLAVPEGDSSFYIV SMCVASSVGS KFSKTQTFQG CGILQPDPPA NITVTAVARN PRWLSVTWQD PHSWNSSFYRLRFELRYRAE RSKTFTTWMV KDLQHHCVIH DAWSGLRHVV QLRAQEEFGQ GEWSEWSPEA MGTPWTESRSPPAENEVSTP MQALTTNKDD DNILFRDSAN ATSLPVQDSS SVPLPTFLVA GGSLAFGTLL CIAIVLRFKKTWKLRALKEG KTSMHPPYSL GQLVPERPRP TPVLVPLISP PVSPSSLGSD NTSSHNRPDA RDPRSPYDISNTDYFFPR, (SEQ ID NO:38), wherein the mature peptide contains aas 20 to 468, UniProtKB - P08887.1. The soluble subunit can replace the membrane-bound IL-6Rα subunit and can be used for binding affinity assays.

[0190] In some cases, variant IL-6 peptides (e.g., variants of SEQ ID NO:35) exhibit reduced binding affinity to the mature IL-6 receptors listed in SEQ ID NO:36 and 37 or SEQ ID NO:37 and 38 compared to the binding affinity of the IL-6 peptide containing the amino acid sequence listed in SEQ ID NO:35. For example, in some cases, the binding affinity of the variant IL-6 peptide to the mature IL-6 receptors listed in SEQ ID NO:36 and 37 or SEQ ID NO:37 and 38 is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% lower than the binding affinity of the IL-6 peptide containing the amino acid sequence listed in SEQ ID NO:35.

[0191] In some cases, the variant IL-6 peptide (e.g., a variant of SEQ ID NO:35) has a binding affinity of 1 nM to 1 mM for the mature IL-6 receptor listed in SEQ ID NO:36 and 37 or SEQ ID NO:37 and 38 (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 cases, the variant IL-6 peptide (e.g., a variant of SEQ ID NO:35) has an affinity of 100 nM to 100 μM for the mature IL-6 receptor listed in SEQ ID NO:36 and 37 or SEQ ID NO:37 and 38 (e.g., 100 nM to 1 μM, 1 μM to 10 μM, or 10 μM to 100 μM). As another example, in some cases, the variant IL-6 peptide (e.g., the variant of SEQ ID NO:35) has a binding affinity to the mature IL-6 receptor listed in SEQ ID NO:36 and 37 or SEQ ID NO:37 and 38 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. μM, or approximately 75 μM to approximately 100 μM.

[0192] In some cases, the variant IL-6 polypeptide has a single aa substitution compared to the IL-6 amino acid sequence listed in SEQ ID NO:35. In some cases, the variant IL-6 polypeptide has 2 to 10 aa substitutions compared to the IL-6 amino acid sequence listed in SEQ ID NO:35. In some cases, the variant IL-6 polypeptide has 2 aa substitutions compared to the IL-6 amino acid sequence listed in SEQ ID NO:35. In some cases, the variant IL-6 polypeptide has 3 or 4 aa substitutions compared to the IL-6 amino acid sequence listed in SEQ ID NO:35. In some cases, the variant IL-6 polypeptide has 5 or 6 aa substitutions compared to the IL-6 amino acid sequence listed in SEQ ID NO:35. In some cases, the variant IL-6 polypeptide has 7 or 8 aa substitutions compared to the IL-6 amino acid sequence listed in SEQ ID NO:35. In some cases, the variant IL-6 peptide has 9 or 10 amino acid substitutions compared to the IL-6 amino acid sequence listed in SEQ ID NO:35.

[0193] Suitable variant 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% sequence identity with at least 80 consecutive aa (e.g., at least 100 or 110 consecutive 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 a non-limiting example, the MOD or variant MOD present in the masked TGF-β construct or complex is IL-7 or a variant IL-7 polypeptide. Wild-type IL-7 has four isoforms, all of which bind to a membrane-bound IL-7 receptor having two subunits, namely an alpha (α) chain and a shared gamma (shared-γ) chain.

[0195] Wild-type human IL-7 isoform 1 precursor polypeptide may contain the following amino acid sequence: MFHVSFRYIF GLPPLILVLLPVASSDCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEGMF LFRAARKLRQFLKMNSTGDF DLHLLKVSEG TTILLNCTGQ VKGRKPAALG EAQPTKSLEE NKSLKEQKKL NDLCFLKRLLQEIKTCWNKI LMGTKEH (SEQ ID NO:39) UniProtKB - P13232, NCBI reference sequence NP_000871.1.

[0196] Mature wild-type human IL-7 isoform 1 polypeptide may contain the following amino acid sequence: DCDIE GKDGKQYESVLMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEGMF LFRAARKLRQ FLKMNSTGDF DLHLLKVSEGTTILLNCTGQ VKGRKPAALG EAQPTKSLEE NKSLKEQKKL NDLCFLKRLL QEIKTCWNKI LMGTKEH (SEQ ID NO:40).

[0197] The wild-type human IL-7 isoform 2 precursor polypeptide may contain the following amino acid sequence: MFHVSFRYIF GLPPLILVLLPVASSDCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKVKGR KPAALGEAQPTKSLEENKSL KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH, (SEQ ID NO:41) NCBI reference sequence: NP_001186815.1.

[0198] Mature wild-type human IL-7 isoform 2 polypeptide may contain the following amino acid sequence: SDCDIE GKDGKQYESVLMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKVKGR KPAALGEAQP TKSLEENKSL KEQKKLNDLCFLKRLLQEIK TCWNKILMGT KEH (SEQ ID NO:42).

[0199] Wild-type human IL-7 isoform 3 precursor polypeptide may contain the following amino acid sequence: MFHVSFRYIF GLPPLILVLLPVASSDCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEGMF LFRAARKLRQFLKMNSTGDF DLHLLKVSEG TTILLNCTGQ EENKSLKEQK KLNDLCFLKR LLQEIKTCWN KILMGTKEH, (SEQ ID NO:43) NCBI reference sequence: NP_001186816.1.

[0200] Mature wild-type human IL-7 isoform 3 polypeptide may contain the following amino acid sequence: CDIE GKDGKQYESVLMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEGMF LFRAARKLRQ FLKMNSTGDF DLHLLKVSEGTTILLNCTGQ EENKSLKEQK KLNDLCFLKR LLQEIKTCWN KILMGTKEH (SEQ ID NO:44).

[0201] The wild-type human IL-7 isoform 4 precursor polypeptide may contain the following amino acid sequence: MFHVSFRYIF GLPPLILVLLPVASSDCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEENK SLKEQKKLNDLCFLKRLLQE IKTCWNKILM GTKEH, NCBI reference sequence: NP_001186817.1 (SEQ ID NO:45).

[0202] Mature wild-type human IL-7 isoform 4 polypeptide may contain the following amino acid sequence: SDCDIE GKDGKQYESVLMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEENK SLKEQKKLND LCFLKRLLQE IKTCWNKILMGTKEH (SEQ ID NO:46).

[0203] The IL-7 receptor alpha subunit may have the sequence: MTILGTTGFM VFSLLQVVSG ESGYAQNGDL EDAELDDYSFSCYSQLEVNG SQHSLTCAFE DPDVNITNLE FEICGALVEV KCLNFRKLQE IYFIETKKFL LIGKSNICVKVGEKSLTCKK IDLTTIVKPE APFDLSVVYR EGANDFVVTF NTSHLQKKYV KVLMHDVAYR QEKDENKWTHVNLSSTKLTL LQRKLQPAAM YEIKVRSIPD HYFKGFWSEW SPSYYFRTPE INNSSGEMDP ILLTISILSFFSVALLVILA CVLWKKRIKP IVWPSLPDHK KTLEHLCKKP RKNLNVSFNP ESFLDCQIHR VDDIQARDEVEGFLQDTFPQ QLEESEKQRL GGDVQSPNCP SEDVVITPES FGRDSSLTCL AGNVSACDAP ILSSSRSLDCRESGKNGPHV YQDLLLSLGT TNSTLPPPFS LQSGILTLNP VAQGQPILTS LGSNQEEAYV TMSSFYQNQ (SEQ ID NO:47), NCBI reference sequence NP_002176.2, wherein aas 21 to 459 form the mature polypeptide, and aas240-264 are transmembrane regions. All or part of the receptor subunit of the protein (e.g., the extracellular domain (aas 21-239)) can be used together with the IL-7 receptor γ subunit to determine binding affinity to IL-7 isoforms.

[0204] The common gamma subunit (IL-7RG or IL-R7γ) can have the sequence MLKPSLPFTS LLFLQLPLG VGLNTTILTPNGNEDTTADF FLTTMPTDSL SVSTLPLPEV QCFVFNVEYM NCTWNSSSEP QPTNLTLHYW YKNSDNDKVQKCSHYLFSEE ITSGCQLQKK EIHLYQTFVV QLQDPREPRR QATQMLKLQN LQPDYSERLC LVSEIPPKGG ALGEGPGASP CNQHSPYWAP PCYTLKPET, NCBI reference sequence NP_000197.1, (SEQ ID NO:48), wherein aas 23 to 369 form the mature polypeptide, and aas 263-283 are transmembrane regions. All or part of the receptor subunit of the protein (e.g., the extracellular domain (aas 23-262)) can be used together with the α subunit to determine the binding affinity to IL-7.

[0205] In some cases, variant IL-7 isoforms 1, 2, 3, or 4 peptides (e.g., SEQ ID NO: 40, 42, 44, or 46) exhibit reduced binding affinity to mature IL-7 receptor sequences (e.g., IL-7 receptors containing all or part of the peptides listed in SEQ ID NO: 47 and 48, such as their extracellular domains) compared to the binding affinity of IL-7 peptides containing the amino acid sequences listed in SEQ ID NO: 40, 42, 44, or 46. For example, in some cases, the binding affinity of the variant IL-7 isoform 1, 2, 3, or 4 polypeptide to an IL-7 receptor containing all or part of the polypeptide listed in SEQ ID NO: 47 and 48, such as its extracellular domain, is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% lower than the binding affinity of the IL-7 isoform 1, 2, 3, or 4 polypeptide containing the amino acid sequence listed in SEQ ID NO: 40, 42, 44, or 46.

[0206] In some cases, the binding affinity of variant IL-7 isoforms 1, 2, 3, or 4 polypeptides (e.g., variants of SEQ ID NO:40, 42, 44, or 46) to IL-7 receptors containing all or a portion of the polypeptides listed in SEQ ID NO:47 and 48, such as their extracellular domains, is from 1 nM to 1 mM. In some cases, the binding affinity of variant IL-7 isoforms 1, 2, 3, or 4 polypeptides (e.g., variants of SEQ ID NO:40, 42, 44, or 46) to mature IL-7 receptors containing all or a portion of the polypeptides listed in SEQ ID NO:47 and 48, such as their extracellular domains, is from 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 cases, the binding affinity of variant IL-7 isoforms 1, 2, 3, or 4 peptides to mature IL-7 receptors containing all or part of the peptides listed in SEQ ID NO: 47 and 48, such as their extracellular domains, is 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. μM, or approximately 75 μM to approximately 100 μM.

[0207] In some cases, the variant IL-7 isoform 1, 2, 3, or 4 polypeptides have a single aa substitution compared to the amino acid sequences of IL-7 isoform 1, 2, 3, or 4 listed in SEQ ID NO:40, 42, 44, or 46. In some cases, the variant IL-7 isoform 1, 2, 3, or 4 polypeptides have 2 to 10 aa substitutions compared to the amino acid sequences of IL-7 isoform 1, 2, 3, or 4 listed in SEQ ID NO:40, 42, 44, or 46. In some cases, the variant IL-7 isoform 1, 2, 3, or 4 polypeptides have 2 aa substitutions compared to the amino acid sequences of IL-7 isoform 1, 2, 3, or 4 listed in SEQ ID NO:40, 42, 44, or 46. In some cases, the variant IL-7 isoform 1, 2, 3, or 4 polypeptides have 3 or 4 amino acid substitutions compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequences listed in SEQ ID NO:40, 42, 44, or 46. In some cases, the variant IL-7 polypeptides have 5 or 6 amino acid substitutions compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequences listed in SEQ ID NO:40, 42, 44, or 46. In some cases, the variant IL-7 isoform 1, 2, 3, or 4 polypeptides have 7 or 8 amino acid substitutions compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequences listed in SEQ ID NO:40, 42, 44, or 46. In some cases, the variant IL-7 isoform 1, 2, 3, or 4 polypeptides have 9 or 10 amino acid substitutions compared to the amino acid sequences of IL-7 isoform 1, 2, 3, or 4 listed in SEQ ID NO:40, 42, 44, or 46.

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

[0209] 10 IL-10 and its variants As a non-limiting example, the MOD or variant present in the masked TGF-β construct or complex is IL-10 or a variant IL-10 peptide, such as a monomeric IL-10 variant with an insertion in the hinge region between the D and E helices, as described by Josephson et al. J. Biol. ChemAs described in 275:13552-13557 (2000). Wild-type IL-10 has isoforms that bind entirely to membrane-bound IL-10 receptors having two alpha (α) IL-10RA and beta (β) IL-10RB subunits. The receptors exist as tetramers on the surface of cells (e.g., B cells, T cells, NK cells, mast cells, and dendritic cells).

[0210] Wild-type human IL-10 isoform 1 precursor polypeptide may contain the following amino acid sequence: MHSSALLCCLVLLTGVRASP GQGTQSENSC THFPGNLPNM LRDLRDAFSR VKTFFQMKDQ LDNLLLKESL LEDFKGYLGCQALSEMIQFY LEEVMPQAEN QDPDIKAHVN SLGENLKTLR LRLRRCHRFL PCENKSKAVE QVKNAFNKLQEKGIYKAMSE FDIFINYIEA YMTMKIRN (SEQ ID NO:49) UniProtKB - P22301, NCBI reference sequence NP_000563.1, which may have H227L sequence variation.

[0211] Mature wild-type human IL-10 peptides may contain the following amino acid sequence: SP GQGTQSENSC THFPGNLPNMLRDLRDAFSR VKTFFQMKDQ LDNLLLKESL LEDFKGYLGC QALSEMIQFY LEEVMPQAEN QDPDIKAHVNSLGENLKTLR LRLRRCHRFL PCENKSKAVE QVKNAFNKLQ EKGIYKAMSE FDIFINYIEA YMTMKIRN (SEQ ID NO:50).

[0212] Human IL-10 polypeptide may contain the following amino acid sequence: MIQFYLEEVM PQAENQDPDI KAHVNSLGENLKTLRLRLRR CHRFLPCENK SKAVEQVKNA FNKLQEKGIY KAMS, UniProtKB - A0A286YEX3 1 (SEQ ID NO:51).

[0213] The IL-10 polypeptide may comprise an insertion in the hinge region between the D and E helices of the IL-10 polypeptide (e.g., 5-7 amino acid insertions adjacent to any one of E48, N49, K50, or S51 of SEQ ID NO: 51 or at an equivalent position in SEQ ID NO: 49 or 50), said insertion causing it to be in monomeric form. The monomeric IL-10 polypeptide may comprise 5-7 amino acid insertions between N49 and K50 of SEQ ID NO: 51 (or at an equivalent position in SEQ ID NO: 49 or 50). In an example, the 5-7 amino acids comprise Ala, Gly, and / or Ser. In one example, the 5-7 amino acids are selected from Ala or Ser. In one example, the 5-7 amino acids are selected from Gly and Ser. In one instance, the insertion comprises an IL-10M1 aa insertion (GGGGSGGG SEQ ID NO:142) between N49 and K50 of SEQ ID NO:51 (or an equivalent position of SEQ ID NO:49 or 50). In another instance, the IL-10 variant consists of an IL-10M1 (SEQ ID NO:189) GGGSGG inserted between aa 49 and 50 of SEQ ID NO:51. See, for example, Josephson et al. J. Biol. Chem 275:13552-13557 (2000).

[0214] The α subunit of the IL-10 receptor may have the following sequence: MLPCLVVLLA ALLSLRLGSD AHGTELPSPP SVWFEAEFFH HILHWTPIPN QSESTCYEVALLRYGIESWN SISNCSQTLS YDLTAVTLDL YHSNGYRARV RAVDGSRHSN WTVTNTRFSV DEVTLTVGSVNLEIHNGFIL GKIQLPRPKM APANDTYESI FSHFREYEIA IRKVPGNFTF THKKVKHENF SLLTSGEVGEFCVQVKPSVA SRSNKGMWSK EECISLTRQY FTVTNVIIFF AFVLLLSGAL AYCLALQLYV RRRKKLPSVLLFKKPSPFIF ISQRPSPETQ DTIHPLDEEA FLKVSPELKN LDLHGSTDSG FGSTKPSLQT EEPQFLLPDPHPQADRTLGN REPPVLGDSC SSGSSNSTDS GICLQEPSLS PSTGPTWEQQ VGSNSRGQDD SGIDLVQNSEGRAGDTQGGS ALGHHSPPEP EVPGEEDPAA VAFQGYLRQT RCAEEKATKT GCLEEESPLT DGLGPKFGRCLVDEAGLHPP ALAKGYLKQD PLEMTLASSG APTGQWNQPT EEWSLLALSS CSDLGISDWS FAHDLAPLGCVAAPGGLLGS FNSDLVTLPL ISSLQSSE, (SEQ ID NO:52), NCBI reference sequence NP_001549.2, wherein aas 21 to 587 form the mature polypeptide, and aas 236-256 is a transmembrane region. All or part of the receptor subunit of the protein (e.g., the extracellular domain (aas 21-235)) can be used together with the IL-10 receptor β subunit to determine binding affinity to IL-10 isoforms.

[0215] The IL-10 receptor β subunit may have sequence MAWSLGSWLG GCLLVSALGM VPPPENVRMN SVNFKNILQW ESPAFAKGNLTFTAQYLSYRIFQDKCMNTT LTECDFSSLS KYGDHTLRVR AEFADEHSDW VNITFCPVDDTIIGPPGMQVEVLADSLHMR FLAPKIENEY ETWTMKNVYN SWTYNVQYWK NGTDEKFQITPQYDFEVLRNLEPWTTYCVQ VRGFLPDRNK AGEWSEPVCE QTTHDETVPS WMVAVILMASVFMVCLALLGCFALLWCVYK KTKYAFSPRN SLPQHLKEFL GHPHHNTLLF FSFPLSDENDVFDKLSVIAEDSESGKQNPG DSCSLGTPPG QGPQS, NCBI reference sequence NP_000619.3, (SEQ ID NO:53), wherein aas 20 to 325 form the mature polypeptide, and aas 221-242 are transmembrane regions. All or part of the receptor subunit of a protein (e.g., the extracellular domain (aas 20-220)) can be used together with the α subunit to determine the binding affinity to IL-10.

[0216] In some cases, variant IL-10 isoform peptides (e.g., variants of SEQ ID NO: 50, 51, or monomeric IL-10 variants with 5-7 aa inserts in the hinge between the D and E helices as described above) exhibit reduced binding affinity to mature IL-10 receptor sequences (e.g., IL-10 receptors containing all or part of the peptides listed in SEQ ID NO: 52 or 53, such as their extracellular domains). For example, in some cases, a variant of the IL-10 peptide (e.g., a variant of SEQ ID NO: 50 or 51) binds to an IL-10 receptor containing all or a portion of the peptide listed in SEQ ID NO: 52 and 53, such as its extracellular domain, with a binding affinity at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% lower than the binding affinity of an IL-10 isoform (e.g., SEQ ID NO: 50 or 51) to an IL-10 receptor containing all or a portion of the peptide listed in SEQ ID NO: 52 or 53, such as its extracellular domain.

[0217] In some cases, the variant IL-10 peptide (e.g., a variant of SEQ ID NO: 50 or 51, which may contain one of the above-described insertions in the hinge region between the D and E helices) has a binding affinity of 1 nM to 1 mM to the IL-10 receptor (e.g., containing all or part of the peptides listed in SEQ ID NO: 52 and 53, such as their extracellular domains). In some cases, the variant IL-10 peptide (e.g., variants of SEQ ID NO: 50 or 51) has a binding affinity of 100 nM to 100 μM (e.g., 100 nM to 1 μM, 1 μM to 10 μM, or 10 μM to 100 μM) to the mature IL-10 receptor (e.g., containing all or part of the peptides listed in SEQ ID NO: 52 and 53, such as their extracellular domains). As another example, in some cases, variant IL-10 peptides (e.g., variants of SEQ ID NO: 50 or 51) have binding affinity to mature IL-10 receptors (e.g., peptides containing all or part of the peptides listed in SEQ ID NO: 52 and 53, such as their extracellular domains) 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 1 μM, about 50 μM to about 1 μM, about 50 μM to about 1 μM, about 50 μM to about 1 μM, about 1 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 μM, about 50 μM to about 1 μM, about 50 μM to about 1 μM, about 50 μM to about 1 μM, about 1 ... μM to approximately 75 μM, or approximately 75 μM to approximately 100 μM.

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

[0219] Suitable variant IL-10 polypeptide sequences include polypeptide sequences having at least 50 consecutive aa sequences (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) with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity with 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 cases, the MOD present in the masked TGF-β construct or complex of this disclosure is an IL-15 polypeptide. The sequence of the IL-15 polypeptide is known in the art, including two isoforms formed by alternative splicing of different precursor proteins. In one embodiment, wt. IL-15 polypeptide has the sequence: MRISKPHLRS ISIQCYLCLLLNSHFLTEAG IHVFILGCFS AGLPKTEANW VNVISDLKKIEDLIQSMHID ATLYTESDVH PSCKVTAMKCFLLELQVISL ESGDASIHDT VENLIILANNSLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFINTS, (SEQ ID NO. 54), UniProtKB - P40933, NCBI Ref. NP_000576.1, IL-15 precursor protein, wherein aa 1 to 29 are signal peptides, and 30-48 are proteptides.

[0221] For the purposes of this disclosure, the mature IL-15 polypeptide represented as isoform 1 may have the form NWVNVISDLKKIEDLIQSMHID ATLYTESDVH PSCKVTAMKC FLLELQVISL ESGDASIHDTVENLIILANNSLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS, (SEQ ID NO:55).

[0222] IL-15 is structurally similar to IL-2 and signals through a cell surface trimer receptor that has the same β and γ chains as the IL-2 receptor but different IL-15 receptor α (IL-15Rα) subunits. The amino acid sequence of the human IL-15Rα isoform 1 precursor protein can be MAPRARGCR TLGLPALLLL LLLRPPATRG ITCPPPMSVE HADIWVKSYSLYSRERYICN SGFKRKAGTS SLTECVLNKA TNVAHWTTPS LKCIRDPALV HQRPAPPSTV TTAGVTPQPESLSPSGKEPA ASSPSSNNTA ATTAAIVPGS QLMPSKSPST GTTEISSHES SHGTPSQTTA KNWELTASASHQPPGVYPQG HSDTTVAIST STVLLCGLSA VSLLACYLKS RQTPPLASVE MEAMEALPVT WGTSSRDEDLENCSHHL (SEQ ID NO:56) NCBI Ref NP_002180, where aas 1-30 form the signal sequence, and aas peptides 31-267 are mature polypeptides. The sequences of IL-2Rβ and IL-2Rγ are provided as SEQ ID NO: 11 and 12.

[0223] In some cases, variant IL-15 isoform 1 peptides (e.g., variants of SEQ ID NO:55) exhibit reduced binding affinity to mature IL-15 receptor sequences (e.g., IL-15 receptors containing all or part of the peptides listed in SEQ ID NO: 11, 12 and 56, such as their extracellular domains) compared to the binding affinity of IL-15 peptides containing the amino acid sequence listed in SEQ ID NO: 55. For example, in some cases, a variant of the IL-15 peptide (e.g., a variant of SEQ ID NO:55) binds to an IL-15 receptor containing all or part of the peptides listed in SEQ ID NO:11, 12 and 56, such as their extracellular domains, with a binding affinity at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% lower than the binding affinity of the IL-15 peptide containing the amino acid sequence listed in SEQ ID NO:55.

[0224] In some cases, the variant IL-15 peptide (e.g., the variant of SEQ ID NO:55) has a binding affinity of 1 nM to 1 mM for IL-15 receptors containing all or part of the peptides listed in SEQ ID NO:11, 12 and 56, such as their extracellular domains. As another example, in some cases, variant IL-15 peptides (e.g., variants of SEQ ID NO: 55) have binding affinity to mature IL-15 receptors containing all or part of the peptides listed in SEQ ID NO: 11, 12, and 56, such as their extracellular domains, in the following quantities: 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. μM, or approximately 75 μM to approximately 100 μM.

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

[0226] Suitable variant 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% sequence identity with at least 50 consecutive aa (e.g., at least 60, at least 70, at least 80, at least 90, at least 100, or at least 110 consecutive aa) of ...80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity with SEQ ID NO:55 (and which has at least one aa substitution, deletion, or insertion).

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

[0228] In one embodiment, the wild-type (wt.) IL-21 isoform 1 polypeptide has the sequence MRSSPGNMERIVICLMVIFL GTLVHKSSSQ GQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQKAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQHLSSRTHGSE DS, (SEQ ID NO:57), UniProtKB - Q9HBE4, NCBI Ref. NP_068575.1, IL-21 protein, wherein aa 1 to 29 are signal peptides.

[0229] Mature IL-21 isoform 1 peptide may have the following sequence: Q GQDRHMIRMR QLIDIVDQLK NYVNDLVPEFLPAPEDVETN CEWSAFSCFQ KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEKKPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DS, (SEQ ID NO:58).

[0230] In one embodiment, the wild-type (wt.) IL-21 isoform 2 polypeptide has the sequence MRSSPGNMERIVICLMVIFL GTLVHKSSSQ GQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQKAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQHLSSRTHGSE DS, (SEQ ID NO:59), NP_001193935.1, IL-21 protein, wherein aa 1 to 29 are signal peptides.

[0231] Mature IL-21 isoform 2 peptide may have the following sequence: MRSSPGNMER IVICLMVIFL GTLVHKSSSQGQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG NNERIINVSIKKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKVSTL SFI, (SEQ ID NO:60).

[0232] IL-21 signals through a dimeric cell surface receptor with the same γ chain as the IL-2 receptor but different IL-21R receptor subunits. The aa sequence of the human IL-21R isoform 1 precursor protein can be... MPRGWAAPLL LLLLQGGWGC PDLVCYTDYL QTVICILEMW NLHPSTLTLT WQDQYEELKDEATSCSLHRS AHNATHATYT CHMDVFHFMA DDIFSVNITD QSGNYSQECG SFLLAESIKP APPFNVTVTFSGQYNISWRS DYEDPAFYML KGKLQYELQY RNRGDPWAVS PRRKLISVDS RSVSLLPLEF RKDSSYELQVRAGPMPGSSY QGTWSEWSDP VIFQTQSEEL KEGWNPHLLL LLLLVIVFIP AWSLKTHPL WRLWKKIWAVPSPERFFMPL YKGCSGDFKK WVGAPFTGSS LELGPWSPEV PSTLEVYSCH PPRSPAKRLQ LTELQEPAELVESDGVPKPS FWPTAQNSGG SAYSEERDRP YGLVSIDTVT VLDAEGPCTW PCSCEDDGYP ALDLDAGLEPSPGLEDPLLD AGTTVLSCGC VSAGSPGLGG PLGSLLDRLK PPLADGEDWA GGLPWGGRSP GGVSESEAGSPLAGLDMDTF DSGFVGSDCS SPVECDFTSP GDEGPPRSYL RQWVVIPPPL SSPGPQAS (SEQ ID NO:61) NCBI reference sequence NP_068570.1, in which aas 1-19 form the signal sequence, aas 20-538 are the mature polypeptide, aas233..253 are the transmembrane domains, and aas 20-232 are the extracellular domains.

[0233] In some cases, variant IL-21 peptides exhibit reduced binding affinity to mature IL-21 receptor sequences (e.g., IL-21 receptors containing all or a portion of the peptides listed in SEQ ID NO: 58 or 60, such as their extracellular domains) compared to the binding affinity of wt. IL-21 peptides containing the amino acid sequences listed in SEQ ID NO: 58 or 60. For example, in some cases, variants of the IL-21 peptide containing SEQ ID NO: 58 or 60 exhibit binding affinity to IL-21 receptors containing all or a portion of the peptides listed in SEQ ID NO: 12 and 61, such as their extracellular domains, that are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% lower than the binding affinity of wt. IL-21 peptides containing the amino acid sequences listed in SEQ ID NO: 58 or 60.

[0234] In some cases, the variant IL-21 peptide (e.g., variants of SEQ ID NO:58 or 60) has a binding affinity of 1 nM to 1 mM to the IL-21 receptor (e.g., containing all or part of the peptides listed in SEQ ID NO:12 and 61, such as their extracellular domains). In some cases, the variant IL-21 peptide (e.g., variants of SEQ ID NO:58 or 60) has a binding affinity of 100 nM to 100 μM (e.g., 100 nM to 1 μM, 1 μM to 10 μM, or 10 μM to 100 μM) to all or part of the mature IL-21 receptor (e.g., containing all or part of the peptides listed in SEQ ID NO:12 and 61, such as their extracellular domains). As another example, in some cases, variant IL-21 peptides (e.g., variants of SEQ ID NO: 58 or 60) have binding affinity for all or part of the IL-21 receptor (e.g., containing all or part of the peptides listed in SEQ ID NO: 12 and 61, such as their extracellular domains) 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 1 μM, about 50 μM to about 1 μM, about 50 μM to about 1 μM, about 1 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 ... μM to approximately 75 μM, or approximately 75 μM to approximately 100 μM.

[0235] In some cases, the variant 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 listed in SEQ ID NO: 58 or 60. In some cases, the variant IL-21 polypeptide (e.g., a variant of SEQ ID NO: 58 or 60) has 2 to 10 aa substitutions compared to the IL-21 polypeptide sequence listed in SEQ ID NO: 58 or 60. In some cases, the variant IL-21 polypeptide has 2 aa substitutions compared to the IL-21 polypeptide sequence listed in SEQ ID NO: 58 or 60. In some cases, the variant IL-21 polypeptide has 3 or 4 aa substitutions compared to the IL-21 polypeptide sequence listed in SEQ ID NO: 58 or 60. In some cases, the variant IL-21 polypeptide has 5 or 6 aa substitutions compared to the IL-21 polypeptide sequence listed in SEQ ID NO: 58 or 60. In some cases, the variant IL-21 polypeptide has 7 or 8 aa substitutions compared to the IL-21 polypeptide sequence listed in SEQ ID NO: 58 or 60. In some cases, the variant IL-21 polypeptide has 9 or 10 aa substitutions compared to the IL-21 polypeptide sequence listed 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% sequence identity with at least 50 consecutive aa (e.g., at least 60, at least 70, at least 80, at least 90, at least 100, or at least 110 consecutive aa) of ...80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity with 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 cases, the MOD present in the masked TGF-β construct or complex of this disclosure is the IL-23 polypeptide. IL-23 is a heterodimeric cytokine composed of the IL-23A (IL-23p19) subunit and the IL-12B (IL-12p40) subunit (which is shared with IL-12).

[0238] In one embodiment, the wild-type (wt.) IL-23A polypeptide has the sequence: MLGSRAVMLL LLLPWTAQGRAVPGGSSPAW TQCQQLSQKL CTLAWSAHPL VGHMDLREEGDEETTNDVPH IQCGDGCDPQ GLRDNSQFCLQRIHQGLIFY EKLLGSDIFT GEPSLLPDSPVGQLHASLLG LSQLLQPEGH HWETQQIPSL SPSQPWQRLLLRFKILRSLQ AFVAVAARVFAHGAATLSP, (SEQ ID NO:62), NCBI reference sequence NP_057668.1 protein, wherein aa 1 to 19 are signal peptides and 20-189 are mature peptides.

[0239] Mature IL-23A polypeptides may have the aa sequence: RAVPGGSSPAW TQCQQLSQKL CTLAWSAHPLVGHMDLREEGDEETTNDVPH IQCGDGCDPQ GLRDNSQFCL QRIHQGLIFY EKLLGSDIFTGEEPSLLPDSPVGQLHASLLG LSQLLQPEGH HWETQQIPSL SPSQPWQRLL LRFKILRSLQAFVAVAARVFAHGAATLSP, (SEQ ID NO: 63).

[0240] In one embodiment, the wild-type (wt.) IL-12B peptide has the sequence: MCHQQLVISW FSLVFLASPLVAIWELKKDV YVVELDWYPD APGEMVVLTC DTPEEDGITW TLDQSSEVLG SGKTLTIQVK EFGDAGQYTCHKGGEVLSHS LLLLHKKEDG IWSTDILKDQ KEPKNKTFLR CEAKNYSGRF TCWWLTTIST DLTFSVKSSRGSSDPQGVTC GAATLSAERV RGDNKEYEYS VECQEDSACP AAEESLPIEV MVDAVHKLKY ENYTSSFFIRDIIKPDPPKN LQLKPLKNSR QVEVSWEYPD TWSTPHSYFS LTFCVQVQGK SKREKKDRVF TDKTSATVICRKNASISVRA QDRYYSSSWS EWASVPCS, (SEQ ID NO:64), UniProtKB - P29460 protein, where aa1 to 22 are signal peptides and 23 to 328 are mature peptides.

[0241] Mature IL-12B polypeptides may have the aa sequence: IWELKKDV YVVELDWYPD APGEMVVLTC DTPEEDGITWTLDQSSEVLG SGKTLTIQVK EFGDAGQYTC HKGGEVLSHS LLLLHKKEDG IWSTDILKDQ KEPKNKTFLRCEAKNYSGRF TCWWLTTIST DLTFSVKSSR GSSDPQGVTC GAATLSAERV RGDNKEYEYS VECQEDSACPAAESLPIEV MVDAVHKLKY ENYTSSFFIR DIIKPDPPKN LQLKPLKNSR QVEVSWEYPD TWSTPHSYFSLTFCVQVQGK SKREKKDRVF TDKTSATVIC RKNASISVRA QDRYYSSSWS EWASVPCS, (SEQ ID NO: 65).

[0242] IL-23 signals via a dimer cell surface receptor that includes the IL-23 receptor polypeptide (IL-23R) and a subunit called 12RB1 or 12Rβ1, which is shared with the IL-12 receptor.

[0243] The IL-23R isoform 1 precursor protein sequence can be: MNQVTIQWDA VIALYILFSW CHGGITNINCSGHIWVEPAT IFKMGMNISI YCQAAIKNCQ PRKLHFYKNG IKERFQITRI NKTTARLWYK NFLEPHASMYCTAECPKHFQ ETLICGKDIS SGYPPDIPDE VTCVIYEYSG NMTCTWNAGK LTYIDTKYVV HVKSLETEEEQQYLTSSYIN ISTDSLQGGK KYLVWVQAAN ALGMEESKQL QIHLDDIVIP SAAVISRAET INATVPKTIIYWDSQTTIEK VSCEMRYKAT TNQTWNVKEF DTNFTYVQQS EFYLEPNIKY VFQVRCQETG KRYWQPWSSLFFHKTPETVP QVTSKAFQHD TWNSGLTVAS ISTGHLTSDN RGDIGLLLGM IVFAVMLSIL SLIGIFNRSFRTGIKRRILL LIPKWLYEDI PNMKNSNVVK MLQENSELMN NNSSEQVLYV DPMITEIKEI FIPEHKPTDYKKENTGPLET RDYPQNSLFD NTTVVYIPDL NTGYKPQISN FLPEGSHLSN NNEITSLTLK PPVDSLDSGNNPRLQKHPNF AFSVSSVNSL SNTIFLGELS LILNQGECSS PDIQNSVEEE TTMLLENDSP SETIPEQTLLPDEFVSCLGI VNEELPSINT YFPQNILESH FNRISLLEK, (SEQ ID NO:66) NCBI reference sequence NP_653302.2, in which aas 1-23 form the signal sequence, aas 24-629 are the mature polypeptide, aas 356 to 376 are transmembrane domains, and aas 24-355 is an extracellular domain.

[0244] The 12RB1 isoform 1 precursor protein aa sequence can be: MEPLVTWVVP LLFLFLLSRQ GAACRTSECCFQDPPYPDAD SGSASGPRDL RCYRISSDRY ECSWQYEGPT AGVSHFLRCC LSSGRCCYFA AGSATRLQFSDQAGVSVLYT VTLWVESWAR NQTEKSPEVT LQLYNSVKYE PPLGDIKVSK LAGQLRMEWE TPDNQVGAEVQFRHRTPSSP WKLGDCGPQD DDTESCLCPL EMNVAQEFQL RRRQLGSQGS SWSKWSSPVC VPPENPPQPQVRFSVEQLGQ DGRRRLTLKE QPTQLELPEG CQGLAPGTEV TYRLQLHMLS CPCKAKATRT LHLGKMPYLSGAAYNVAVIS SNQFGPGLNQ TWHIPADTHT EPVALNISVG TNGTTMYWPA RAQSMTYCIE WQPVGQDGGLATCSLTAPQD PDPAGMATYS WSRESGAMGQ EKCYYITIFA SAHPEKLTLW STVLSTYHFG GNASAAGTPHHVSVKNHSLD SVSVDWAPSL LSTCPGVLKE YVVRCRDEDS KQVSEHPVQP TETQVTLSGL RAGVAYTVQVRADTAWLRGV WSQPQRFSIE VQVSDWLIFF ASLGSFLSIL LVGVLGYLGL NRAARHLCPP LPTPCASSAIEFPGGKETWQ WINPVDFQEE ASLQEALVVE MSWDKGERTE PLEKTELPEG APELALDTEL SLEDGDRCKAKM,(SEQ ID NO:67) NCBI reference sequence NP_005526.1, where aas 1-23 form the signal sequence, aas 24-662 is the mature polypeptide, aas 546 to 570 are transmembrane domains, and aas 24-545 is an extracellular domain.

[0245] In some cases, variant IL-23 peptides (e.g., variants containing SEQ ID NO: 63 and / or 65) exhibit reduced binding affinity to mature IL-23 receptor sequences (e.g., IL-23 receptors containing all or part of the peptides listed in SEQ ID NO: 66 and 67, such as their extracellular domains) compared to the binding affinity of IL-23 peptides containing the amino acid sequences listed in SEQ ID NO: 63 or 65. For example, in some cases, the binding affinity of a variant of the IL-23 peptide (e.g., a variant comprising SEQ ID NO: 63 and / or 65) to the IL-23 receptor (e.g., a peptide comprising all or part of the peptides listed in SEQ ID NO: 66 and 67, such as their extracellular domains) is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% lower than the binding affinity of the IL-23 peptide comprising the amino acid sequence listed in SEQ ID NO: 63 or 65.

[0246] In some cases, the variant IL-23 peptide (e.g., variants comprising SEQ ID NO:63 and / or 65) has a binding affinity of 1 nM to 1 mM for the IL-23 receptor (e.g., comprising all or part of the peptides listed in SEQ ID NO:66 and 67, such as their extracellular domains). As another example, in some cases, variant IL-23 peptides (e.g., variants comprising SEQ ID NO: 63 and / or 65) have binding affinity for mature IL-23 receptors (e.g., peptides comprising all or part of the peptides listed in SEQ ID NO: 66 and 67, such as their extracellular domains) 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 1 μM, about 50 μM to about 1 μM, about 50 μM to about 1 μM, about 1 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 ... μM to approximately 75 μM, or approximately 75 μM to approximately 100 μM.

[0247] In some cases, the variant IL-23 polypeptide (e.g., variants comprising SEQ ID NO: 63 and / or 65) has a single aa substitution compared to the IL-23 polypeptide sequence listed in SEQ ID NO: 63 and / or 65. In some cases, the variant IL-23 polypeptide (e.g., variants comprising SEQ ID NO: 63 and / or 65) has 2 to 10 aa substitutions compared to the IL-23 polypeptide sequence listed in SEQ ID NO: 63 and / or 65. In some cases, the variant IL-23 polypeptide has 2 aa substitutions compared to the IL-23 polypeptide sequence listed in SEQ ID NO: 63 and / or 65. In some cases, the variant IL-23 polypeptide has 3 or 4 aa substitutions compared to the IL-23 polypeptide sequence listed in SEQ ID NO: 63 and / or 65. In some cases, the variant IL-23 polypeptide has 5 or 6 amino acid substitutions compared to the IL-23 polypeptide sequence listed in SEQ ID NO: 63 and / or 65. In some cases, the variant IL-23 polypeptide has 7 or 8 amino acid substitutions compared to the IL-23 polypeptide sequence listed in SEQ ID NO: 63 and / or 65. In some cases, the variant IL-23 polypeptide has 9 or 10 amino acid substitutions compared to the IL-23 polypeptide sequence listed in SEQ ID NO: 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% sequence identity with at least 50 consecutive 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 consecutive aa) of ...80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% aa sequence identity with SEQ ID NO:63 and / or 65 (and which have at least one aa substitution, deletion, or insertion).

[0249] 14 Fas ligands (FasL) and their variants In some cases, the MOD present in the masked TGF-β construct or complex of this disclosure is the Fas ligand (FasL). FasL is a homomeric type II transmembrane protein of the tumor necrosis factor (TNF) family. FasL signals through the trimerization of the Fas receptor in target cells, forming a death-inducing complex that leads to apoptosis in the target cells. Soluble FasL is produced by the cleavage of membrane-bound FasL at conserved sites by matrix metalloproteinase-7 (MMP-7).

[0250] In one implementation, the wt. Homo sapiens FasL protein has the sequence... MQQPFNYPYP QIYWVDSSAS SPWAPPGTVL PCPTSVPRRP GQRRPPPPPPPPPLPPPPPPPPLPPLPLPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG MFQLFHLQKELAELRESTSQMHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVLLSGVKYKKGGLVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMMSYCTTGQMWARSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L, (SEQ ID NO:143), NCBI reference sequence NP_000630.1, UniProtKB - P48023, wherein residues 1-80 are cytoplasmic, 810102 is a transmembrane domain and aas 103-281 is an extracellular domain.

[0251] Suitable FasL peptides contain all or part of the extracellular domain of FasL: QLFHLQKE LAELRESTSQMHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYFVYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVNVSELSLVNFE ESQTFFGLYK L. (SEQ ID NO144).

[0252] Fas receptors can have sequence MLGIWTLLPL VLTSVARLSS KSVNAQVTDI NSKGLELRKT VTTVETQNLE GLHHDGQFCHKPCPPGERKA RDCTVNGDEP DCVPCQEGKE YTDKAHFSSK CRRCRLCDEG HGLEVEINCTRTQNTKCRCKPNFFCNSTVC EHCDPCTKCE HGIIKECTLT SNTKCKEEGS RSNLGWLCLLLLPIPLIVWV KRKEVQKTCRKHRKENQGSH ESPTLNPETV AINLSDVDLS KYITTIAGVMTLSQVKGFVR KNGVNEAKID EIKNDNVQDTAEQKVQLLRN WHQLHGKKEA YDTLIKDLKKANLCTLAEKI QTIILKDITS DSENSNFRNE IQSLV, (SEQID NO:145) NCBI reference sequence: NP_000034.1, UniProtKB - P25445, where aas 26-173 form the extracellular domain, aas 174-190 form the transmembrane domain, and 191-335 is the cytoplasmic domain. The extracellular domain can be used to determine the binding affinity with FasL.

[0253] In some cases, variant FasL peptides (e.g., variants containing SEQ ID NO: 144) exhibit reduced binding affinity to mature Fas receptor sequences (e.g., FasL receptors containing all or part of the polypeptide listed in SEQ ID NO: 145, such as its extracellular domain) compared to the binding affinity of FasL peptides containing the amino acid sequence listed in SEQ ID NO: 144. For example, in some cases, the binding affinity of variant FasL peptides (e.g., variants containing SEQ ID NO: 144) to Fas receptors (e.g., those containing all or part of the polypeptide listed in SEQ ID NO: 145, such as its extracellular domain) is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more than 95% lower than the binding affinity of FasL peptides containing the amino acid sequence listed in SEQ ID NO: 144.

[0254] In some cases, the variant FasL peptide (e.g., the variant comprising SEQ ID NO:144) has a binding affinity of 1 nM to 1 mM for the Fas receptor (e.g., comprising all or part of the peptide listed in SEQ ID NO:145, such as its extracellular domain). As another example, in some cases, the binding affinity of the variant FasL peptide (e.g., comprising the variant of SEQ ID NO:144) to the mature Fas receptor (e.g., comprising all or part of the peptide listed in SEQ ID NO:145, such as its extracellular domain) is 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 1 μM, about 50 μM to about 1 μM, about 50 μM to about 1 μM, about 1 μM to about 20 μM, about 20 μM to about 30 μM, about 30 μM to about 50 ... μM to approximately 75 μM, or approximately 75 μM to approximately 100 μM.

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

[0256] Suitable variant 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% sequence identity with at least 50 consecutive 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, or at least 180 consecutive aa) of at least 50 consecutive aa (e.g., having at least one aa substitution, deletion, or insertion).

[0257] E. Stent Scaffold peptides, in particular, act as structural elements, providing a framework for the organization of masked TGF-β constructs or other components of the complex (see, for example, Figure 1Structure A, with an IgFc scaffold. When the peptide sequence masking the TGF-β peptide is trans-positioned with the TGF-β peptide (on different peptides in the complex), the scaffold sequence forming interspecies and non-interspecies duplexes (or higher-order structures) can maintain the masking peptide sequence associated with the TGF-β peptide, even when the complex is in an open form and the TGF-β peptide sequence is available to interact with other molecules (without direct contact with the masking sequence). Depending on the nature of the scaffold, it can also act as a tissue element, providing higher-order structures in terms of protein folding and dimerization or multimerization (e.g., homodimerization or heterodimerization accomplished by dimerizing sequences). The scaffold can also contribute to serum stability, especially when it is a constant region of the immunoglobulin heavy chain (e.g., IgFc). Suitable scaffold peptides are peptides with extended half-lives in some cases. In some cases, a suitable scaffold peptide 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 greater than 100-fold compared to a control masked TGF-β construct or complex having an Ig Fc peptide sequence replaced by a linker (e.g., a GGGS aa repeat sequence of equal sequence length). As an example, in some cases, an Ig Fc peptide sequence (e.g., including interspecies Ig sequences, such as mortar and pestle sequence pairs) 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 peptide sequence replaced by a linker (e.g., a mortar and pestle sequence of equal sequence length). 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 the Ig Fc peptide is used in a masked TGF-β construct, the Ig Fc may contain a mutation that prevents the spontaneous formation of a dimer of the masked TGF-β construct (see, for example, Tianlei Ying et al., J. Biol. Chem, 287(23), pp. 19399-19408 (June 1, 2012)), and may additionally include mutations that substantially reduce or eliminate the ability of the Ig peptide to induce cell lysis, for example, through complement-dependent cytotoxicity (CDC) and / or antibody-dependent cytotoxicity (ADCC) (e.g., the LALA mutation discussed below).

[0258] When the scaffold polypeptide sequence of a masked TGF-β construct contains one or more sequences that allow the scaffold to interact (specifically bind) with other scaffold molecules, the masked TGF-β construct can form a homodimeric complex (see, for example, Figure 1 Structure B) or heterodimeric complexes (see, for example, Figure 1 (structural CF). The masked TGF-β construct may also contain one or more aa sequences that allow the scaffold to interact (specifically bind) with other scaffold molecules to form higher-order structures. Sequences that form higher-order multimeric structures allow the formation of higher-order masked TGF-β constructs or complexes (e.g., trimers, tetramers, pentamers, etc.). For example, scaffold peptides containing an IgM Fc region (e.g., SEQ ID NO:83) allow the formation of pentamers (especially when the j-chain sequence is also expressed, e.g., SEQ ID NO:84) or hexamer-masked TGF-β constructs or complexes. Petr people, Med Hypotheses 77(6):959-61 (2011). Masked TGF-β construction systems that exist in the form of homodimers, heterodimers, trimers, tetramers, pentamers, etc. are called masked TGF-β complexes.

[0259] When it is necessary to combine 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 contain an interspecies dimerizing sequence that tends to form a heterodimer with its corresponding interspecies binding (dimerizing) sequence. In some cases, the interspecies binding sequence may form a homodimer, but preferentially dimers (binds more strongly) with its corresponding interspecies binding sequence. Therefore, when an interspecies dimerizing sequence and its corresponding interspecies binding sequence are incorporated into a pair of polypeptides, specific heterodimers may (preferably) form. For example, when an interspecies dimerizing sequence and its counterpart are incorporated into a pair of polypeptides to selectively form heterodimers, an equimolar mixture of peptides of greater than 60%, 70%, 80%, 90%, 95%, 98%, or 99% participates in heterodimer formation, and the remainder of the peptide exists as a monomer or homodimer.

[0260] Multiple amino acid (AA) sequences that bind specifically to each other or themselves with sufficient affinity can be used as dimerizing sequences in masked TGF-β constructs or complexes (see, for example, U.S. Patent Publication No. 2003 / 0138440). The sequences can have a relatively compact size (e.g., less than about 300, 250, 225, 200, 175, 150, 125, 100, 75, or 50 AA sequences). Dimerizing / multimerizing sequences include, but are not limited to: immunoglobulin heavy chain constant region (IgFc) polypeptide sequences (sequences containing the CH2-CH3 regions of immunoglobulins; see, for example, Figure 2A-2H(and SEQ ID NO: 68 to 83); Fc mortar sequences (e.g., SEQ ID NO: 77 and 78); polypeptides of the collagen lectin family (e.g., ACRP30 or ACRP30-like proteins) containing a collagen domain consisting of the collagen repeat sequence Gly-Xaa-Yaa; a coiled-coil domain; a leucine-zipper domain; a Fos / Jun binding pair; an Ig heavy chain region 1 (CH1) and a light chain constant region CL sequence (CH1 / CL pairs, such as CH1 sequences paired with κ or λ Ig light chain constant region sequences).

[0261] In some embodiments, the scaffold polypeptide sequence comprises an immunoglobulin heavy chain constant region (CH2-CH3) polypeptide sequence, which acts as a dimerizing or multiplying sequence (see, for example, Figure 2A-2H (and SEQ ID NO: 68 to 83). In embodiments, the Ig peptide does not substantially induce cell lysis, for example, through activation of complement-dependent cytotoxicity (CDC) and / or antibody-dependent cytotoxicity (ADCC), and therefore may include mutations that substantially reduce or eliminate the ability of the Ig peptide to induce cell lysis. In some cases, the Fc sequence is related to... Figures 2A to 2H The described Fc region aa 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. Such immunoglobulin sequences can covalently link polypeptides of the masked TGF-β complex together by forming one or two interchain disulfide bonds. As discussed below, additional disulfide bonds can be introduced to stabilize the dimer, especially when using a pair of interspecies Ig sequences such as a mortar and pestle polypeptide pair.

[0262] In one embodiment, the scaffold polypeptide sequence of the masked TGF-β complex contains [a sequence related to...]. Figure 2A The described IgAFc sequence (SEQ ID NO: 68) comprises at least 150 consecutive aas (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 consecutive aas) or all aas 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. In one embodiment, the scaffold polypeptide sequence comprises a sequence that is identical to... Figure 2BThe described IgD Fc sequence (SEQ ID NO: 69) comprises at least 150 consecutive aas (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 consecutive aas) or all aas 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. In one embodiment, the scaffold polypeptide sequence comprises a sequence that is identical to... Figure 2C The described IgE Fc sequence (SEQ ID NO: 70) comprises at least 125 consecutive amino acids (at least 150, at least 175, or at least 200 consecutive amino acids) or all amino acids 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% amino acid sequence identity. In one embodiment, the scaffold polypeptide sequence comprises a sequence similar to wt. IgG Fc polypeptide sequences, such as... Figure 2D The described IgG1Fc sequence (SEQ ID NO: 71-78) comprises at least 125 consecutive amino acids (at least 150, at least 175, or at least 200 consecutive amino acids) or all amino acids 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% amino acid sequence identity. In one embodiment, the scaffold polypeptide sequence comprises a sequence that is identical to... Figure 2E The described IgG2Fc polypeptide sequence (SEQ ID NO:79) comprises at least 125 consecutive aas (at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, or at least 300) or all aas having sequence identity of 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. In one embodiment, the scaffold polypeptide sequence comprises a sequence that is identical to... Figure 2F The described IgG3 Fc sequence (SEQ ID NO: 80) comprises at least 125 consecutive aas (at least 150, at least 175, at least 200, or at least 225) or all aas having sequence identity of 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. In one embodiment, the scaffold polypeptide sequence comprises a sequence that is identical to... Figure 2GThe described IgG4 Fc sequence (SEQ ID NO: 81 or 82) comprises at least 125 consecutive aas (at least 150, at least 175, at least 200, at least 225, or at least 250) or all aas having sequence identity of 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. In one embodiment, the scaffold polypeptide sequence comprises a sequence that is identical to... Figure 2H A sequence comprising at least 125 consecutive aas (at least 150, at least 175, at least 200, at least 225, or at least 250) or all aas 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 of the described IgM Fc polypeptide sequence (e.g., immunoglobulin scaffold polypeptide sequence). Figures 2A to 2H The aforementioned peptides of the masked TGF-β complex can be covalently linked together by forming one or two interchain disulfide bonds between cysteine ​​residues adjacent to their hinge regions.

[0263] In some cases, the dimerized sequence of the scaffold peptide present in the masked TGF-β construct or complex is similar to... Figure 2D The described human IgG1 Fc polypeptide has at least about 70% (e.g., at least about 80%, 90%, 95%, 98%, 99%, or 100%) aa sequence identity and contains aa amino acids substituted with alanine (N297A, or as shown in the original text). Figure 2D The substitution of N297 with N77 (numbered in SEQ ID NO:74). In some cases, the dimerized sequence of the scaffold peptide present in the masked TGF-β construct or complex contains Figure 2D The described aa sequence (human IgG1 Fc) is replaced by N297 with aa substituted for asparagine. Figure 2D Except for N77 of the described aa sequence. The substitution at N297 results in the removal of carbohydrate modification and, compared to the wt. protein, the resulting antibody sequence has reduced binding of complement component 1q (“C1q”), and therefore, reduced complement-dependent cytotoxicity.

[0264] In some cases, the dimerized sequence of the scaffold peptide present in the masked TGF-β construct or complex contains Figure 2D The described aa sequence (human IgG1 Fc) is replaced by L234 of aa except for leucine. Figure 2DExcept for L14 of the described aa sequence. L234 and other aas in the lower hinge region of IgG (e.g., aas 234-LLGGPS-239, which corresponds to aas 14-19 in SEQ ID NO:71) are involved in binding to the Fcλ receptor (FcλR), and therefore, mutations at said positions reduce binding to the receptor (relative to wt. protein). In some cases, the dimerized sequence of the scaffold polypeptide present in the masked TGF-β construct or complex contains Figure 2D The described aa sequence (human IgG1 Fc) is replaced by L235 of aa except for leucine. Figure 2D Except for L15 of the described aa sequence. In some cases, the dimerized sequence of the scaffold peptide present in the masked TGF-β construct or complex contains Figure 2D The described aa sequence (e.g., wt. human IgG1 sequence) has L234A and L235A (“LALA”) substitutions (corresponding to Figure 2D The positions of positions 14 and 15 of the described wt.aa sequence; see, for example, SEQ ID NO:75).

[0265] In some cases, the dimerized sequence of the scaffold peptide present in the masked TGF-β construct or complex contains Figure 2D The described aa sequence (human IgG1 Fc) has a P331 substitution for aa except proline. Figure 2D The described aa sequence (P111); in some cases, the substitution is a P331S substitution. Substitution at P331, like substitution at N297, leads to reduced protein binding to C1q relative to wt., thus decreasing complement-dependent cytotoxicity. For example, substitutions with alanine at D270, K322, and / or P329 (corresponding to...) Figure 2D D50, K122, and P119 of SEQ ID NO:71 can be used alone or as a substitute for P331 to reduce binding to C1q. In some cases, the dimerized sequence of the scaffold peptide present in the masked TGF-β construct or complex is an IgG1 Fc peptide containing L234A and / or L235A substitutions (replaced with Ala). Figure 2D The described aa sequence contains leucine residues at L14 and / or L15. In some cases, the dimerized sequence of the scaffold peptide present in the masked TGF-β construct or complex contains... Figure 2D The described aa sequence (wt. human IgG1 Fc) is represented by aas (excluding leucine) at L234 and / or L235. Figure 2D The substitution at L14 and / or L15 of the described aa sequence and the P331 of aa other than proline ( Figure 2D Except for the substitution of the described aa sequence (P111). In some cases, the dimerized sequence of the scaffold peptide present in the masked TGF-β construct or complex contains Figure 2D The "triple mutant" aa sequence (SEQ ID NO:73) described in (human IgG1 Fc) contains L234F, L235E, and P331S substitutions (corresponding to...) Figure 2D The aa positions in the described aa sequence are 14, 15, and 111.

[0266] When there is asymmetric pairing between two peptides in a TGF-β complex that needs to be masked, the dimerized sequence of the scaffold peptide present in the masked TGF-β construct or complex contains, is substantially composed of, or consists of interspecies binding sequences. Interspecies binding sequences favor the formation of heterodimers with their homologous peptide sequences (i.e., interspecies sequences and their corresponding interspecies sequences), especially those based on immunoglobulin Fc sequence variants. Such interspecies peptide sequences include the sequences mortar, HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107, which have or lack (KiH) Ss-stabilized disulfide bonds. An interspecies binding pair contains a T366Y and Y407T mutant pair at the CH3 domain interface of IgG1 or corresponding residues of other immunoglobulins. See Ridgway et al., Protein Engineering 9:7, 617-621 (1996) (substitutions are represented by the EU numbering scheme of Kabat et al. (1991)). A second type of interspecific binding pair involves the formation of a club via T366W substitution and the formation of a mortar via triple substitutions of T366S, L368A, and Y407V on complementary Fc sequences. See Xu et al. mAbs 7:1, 231-242 (2015). Another interspecific binding pair has a first Fc polypeptide containing substitutions of Y349C, T366S, L368A, and Y407V, and a second Fc polypeptide containing substitutions of S354C and T366W (disulfide bonds can be formed between Y349C and S354C). Brinkmann and Konthermann, mAbs 9:2, 182–212 (2015). Fc polypeptide sequences, with or without mortar modification, can be stabilized by forming disulfide bonds between Fc polypeptides (e.g., hinge region disulfide bonds). Several inter-peptide binding sequences are summarized in Table 1 and cross-referenced as follows: Figure 2D The numbers of the aa positions appearing in the listed wt. IgG1 sequence (SEQ ID NO: 71) are shown in brackets “{}”.

[0267] Table 1. Interspecific sequences and their homologous interspecific sequences Table 1 is from Ha et al., Frontiers in Immunol This is a modified version of .7:1-16 (2016).

[0268] * aa forms a stable disulfide bond.

[0269] In addition to the interspecific sequence pairs in Table 1, the interspecific “SEED” sequence has 45 residues derived from IgA in the IgG1 CH3 domain of the interspecific sequence and 57 residues derived from IgG1 in the IgA CH3 domain of the corresponding interspecific sequence. See Ha et al. Frontiers in Immunol .7:1-16 (2016).

[0270] In one embodiment, the scaffold sequence found in the masked TGF-β construct or complex comprises an interspecific binding sequence selected from the group consisting of: mortar (KiH); mortar with stable disulfide (KiHs-s); HA-TF; ZW-1; 7.8.60; DD-KK; EW-RVT; EW-RVTs-s; A107; or a SEED sequence.

[0271] In one embodiment, the masked TGF-β complex comprises: a first polypeptide containing an IgG1 scaffold with a T146W KiH sequence substitution and a second polypeptide containing an IgG1 scaffold with T146W, L148A and Y187V KiH sequence substitutions, wherein the scaffold contains a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity with at least 170, at least 180, at least 190, at least 200, at least 210, at least 220 or all 227 consecutive aas of IgG1 of SEQ ID NO:71. One or two scaffold aa sequences may optionally include substitutions at one or more of the following locations when using Kabat numbering: 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). Those that replace 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.

[0272] In one embodiment, the masked TGF-β complex comprises: a first polypeptide containing an IgG1 scaffold with a T146W KiH sequence substitution and a second polypeptide containing an IgG1 scaffold with T146S, L148A, and Y187V KiH sequence substitutions, wherein the scaffold comprises a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 consecutive aas of IgG1 of SEQ ID NO:71; wherein none, one, or both of the scaffold aa sequences contain L14 and L15 substitutions (e.g., L234A and L235A "LALA" in Kabat numbering), and / or N77 substitutions to remove effector function by blocking interaction with Fcγ receptors (N297, e.g., N297A or N297G in Kabat numbering). See, for example Figure 2D SEQ ID NO: 77 and 78, In one embodiment, the first and second peptides of the masked TGF-β complex contain T146W and S134C KiHs-s substitutions in the first scaffold sequence and T146S, L148A, Y187V, and Y129CKiHs-s substitutions in the second scaffold sequence, wherein the scaffold contains a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 consecutive aas of IgG1 of SEQ ID NO:71; wherein none, one, or both of the scaffold aa sequences contain L14 and L15 substitutions (e.g., L234A and L235A “LALA” in Kabat numbering), and / or N77 substitutions to remove effector function by blocking interaction with the Fcγ receptor (N297, e.g., N297A or N297G in Kabat numbering).

[0273] In one embodiment, the first and second peptides of the masked TGF-β complex contain S144H and F185A HA-TF substitutions in the first scaffold sequence and Y129T and T174F HA-TF substitutions in the second scaffold sequence, wherein the scaffold contains a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 consecutive aas of IgG1 of SEQ ID NO:71; wherein none, one, or both of the scaffold aa sequences contain L14 and L15 substitutions (e.g., L234A and L235A “LALA” in Kabat numbering), and / or N77 substitutions to remove effector function by blocking interaction with the Fcγ receptor (N297, e.g., N297A or N297G in Kabat numbering).

[0274] In one embodiment, the first and second peptides of the masked TGF-β complex contain T130V, L131Y, F185A, and Y187V ZW1 substitutions in the first scaffold sequence, and T130V, T146L, K172L, and T174W ZW1 substitutions in the second scaffold sequence, wherein the scaffold comprises the same as SEQ ID. NO:71 is a sequence of at least 170, at least 180, at least 190, at least 200, at least 210, at least 220 or all 227 consecutive aas having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity; wherein none, one or both of the scaffold aa sequences contain L14 and L15 substitutions (e.g., L234A and L235A “LALA” in Kabat numbering), and / or N77 substitutions to remove effector function by blocking interaction with Fcγ receptors (N297, e.g., N297A or N297G in Kabat numbering).

[0275] In one embodiment, the first and second peptides of the masked TGF-β complex contain K140D, D179M, and Y187A 7.8.60 substitutions in the first scaffold sequence, and E125R, Q127R, T146V, and K189V 7.8.60 substitutions in the second scaffold sequence, wherein the scaffold contains the same as SEQ ID. NO:71 is a sequence of at least 170, at least 180, at least 190, at least 200, at least 210, at least 220 or all 227 consecutive aas having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity; wherein none, one or both of the scaffold aa sequences contain L14 and L15 substitutions (e.g., L234A and L235A “LALA” in Kabat numbering), and / or N77 substitutions to remove effector function by blocking interaction with Fcγ receptors (N297, e.g., N297A or N297G in Kabat numbering).

[0276] In one embodiment, the first and second β peptides of the masked TGF-β complex contain K189D and K172D DD-KK substitutions in the first scaffold sequence and D179K and E136K DD-KK substitutions in the second scaffold sequence, wherein the scaffold contains a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 consecutive aas of IgG1 of SEQ ID NO:71; wherein none, one, or both of the scaffold aa sequences contain L14 and L15 substitutions (e.g., L234A and L235A "LALA" in Kabat numbering), and / or N77 substitutions to remove effector function by blocking interaction with the Fcγ receptor (N297, e.g., N297A or N297G in Kabat numbering). In one embodiment, the first and second peptides of the masked TGF-β complex contain K140E and K189W EW-RVT substitutions in the first scaffold sequence and Q127R, D179V, and F185T EW-RVT substitutions in the second scaffold sequence, wherein the scaffold contains a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 consecutive aas of IgG1 of SEQ ID NO:71; wherein none, one, or both of the scaffold aa sequences contain L14 and L15 substitutions (e.g., L234A and L235A "LALA" in Kabat numbering), and / or N77 substitutions to remove effector function by blocking interaction with the Fcγ receptor (N297, e.g., N297A or N297G in Kabat numbering).

[0277] In one embodiment, the first and second peptides of the masked TGF-β complex contain K140E, K189W, and Y129C EW-RVTs-s substitutions in the first scaffold sequence, and Q127R, D179V, F185T, and S134C EW-RVTs-s substitutions in the second scaffold sequence, wherein the scaffold comprises the same as SEQ ID. NO:71 is a sequence of at least 170, at least 180, at least 190, at least 200, at least 210, at least 220 or all 227 consecutive aas having at least 80%, 90%, 95%, 98%, 99% or 100% sequence identity; wherein none, one or both of the scaffold aa sequences contain L14 and L15 substitutions (e.g., L234A and L235A “LALA” in Kabat numbering), and / or N77 substitutions to remove effector function by blocking interaction with Fcγ receptors (N297, e.g., N297A or N297G in Kabat numbering).

[0278] In one embodiment, the first and second peptides of the masked TGF-β complex contain K150E and K189W A107 substitutions in the first scaffold sequence and E137N, D179V, and F185TA107 substitutions in the second scaffold sequence, wherein the scaffold contains a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity with at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 consecutive aas of IgG1 of SEQ ID NO:71; wherein none, one, or both of the scaffold aa sequences contain L14 and L15 substitutions (e.g., L234A and L235A “LALA” in Kabat numbering), and / or N77 substitutions to remove effector function by blocking interaction with Fcγ receptors (N297, e.g., N297A or N297G in Kabat numbering).

[0279] As an alternative to using the immunoglobulin heavy chain constant region as a scaffold, the immunoglobulin light chain constant region can pair with the heavy chain CH1 sequence as a dimerizing sequence, which forms a scaffold polypeptide sequence or a portion thereof. In one embodiment, the first and second polypeptides of the masked TGF-β complex contain 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 scaffold comprises sequences having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with at least 70, at least 80, at least 90, at least 100, or at least 110 consecutive aas of SEQ ID NO: 85 and / or 86, respectively. See also Figure 2J and 2K The CH1 and Igκ sequences can be modified to increase their affinity for each other, and thus improve the stability of any heterodimer formed using them as dimerizing sequences. Among the substitutions that improve the stability of the CH1-Igκ heterodimer is the substitution of the MD13 combination, as identified by Chen et al. MAbs , 8(4):761-774(2016). In MD13, two substitutions were introduced into each of the CH1 and Igκ sequences. The CH1 sequence was modified to contain S64E and S66V substitutions ( Figure 2J The Igκ sequence is modified to include S70E and S72V in SEQ ID NO:85. Figure 2K (S68L and T70S in SEQ ID NO:86 shown).

[0280] In another embodiment, the first and second peptides of the masked TGF-β complex contain an Ig CH1 domain (e.g., the peptide of SEQ ID NO: 85) in the first scaffold sequence and an Ig λ chain constant region sequence (SEQ ID NO: 87) in the second scaffold sequence, wherein the scaffold comprises sequences having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with at least 70, 80, 90, 100, or 110 consecutive aas of SEQ ID NO: 85 and / or 87, respectively. See also Figure 2J and 2K .

[0281] In some cases, the scaffold polypeptide sequences of the first and second peptides of the masked TGF-β complex each contain a leucine zipper polypeptide as a dimerizing sequence. The leucine zipper polypeptides bind to each other to form a dimer (e.g., a homodimer). Non-limiting examples of leucine-zipper peptides include peptides such as any one of the following aa sequences: RMKQIED-KIEEILS-KIYHIENEIARIKKLIGER (SEQ ID NO: 88); LSSIEK-KQEEQTS-WLIWISNELTL-IRNELAQS (SEQ ID NO: 89); LSSIEKKLE-EITSQLIQIS-NELTLIRNELAQ (SEQ ID NO: 90); LSSIE-KKL-E--EITSQLIQIRNELTLIRNELAQ (SEQ ID NO: 91); LSSIEKKL-EEITSQLQQ-IRNELTLI-RNELAQ (SEQ ID NO: 92); LSSLEKKLEEL-TSQLIQLRNELT-LLRNELAQ (SEQ ID NO: 93); ISSLE-KKIEE-LTSQI-QQLRNEITLLRNEIAQ (SEQ ID NO: 94). NO:94). In some cases, leucine zipper peptides contain the following aa sequence: LEIEAAFLERENT-ALET-RVAE-LRR-VRLR-NRV-SYRT-RYGPLGGGK (SEQ ID NO:95). Additional leucine-zipper peptides are known in the art, any of which are suitable for use as a scaffold or for incorporation into a scaffold as a dimerizing sequence.

[0282] In some cases, the scaffold peptide sequences of the first and second peptides 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 peptides include peptides such as any of the following aa sequences: LKSVENRLAVVENQLKT-VIEE-LK-TVKDLLSN (SEQ ID NO: 96); LARIE-EKLKTIKAQLSEIASTLNMIREQLAQ (SEQ ID NO: 97); VSRLE-EKVKT-LKSQV-TELAS-TVSLL-REQVAQ (SEQ ID NO: 98); IQSEKKIEDI-SSLI-G-QIQSEITLIRNEIAQ (SEQ ID NO: 99); LMSLE-KKLEE-LTQTLMQLQNELSMLKNELAQ (SEQ ID NO: 100).

[0283] In some cases, the scaffold polypeptide sequences that allow dimerization (co-dimerization) 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: human FasL polypeptide VDLEGSTSNGRQ-CAGIRL (SEQ ID NO:101); EDDVTTTEELAPALVPPPKGTCAGWMA (SEQ ID NO:102); and GHDQE-TTTQG-PGVLL-PLPKGACTGQMA (SEQ ID NO:103).

[0284] Peptides suitable as polymerizing (oligomerizing) sequences allow the formation of masked TGF-β complexes larger than dimers (e.g., trimers, tetramers, pentamers, hexamers, etc.), including but not limited to the formation of hexamer or pentamer IgM constant regions (see, for example, Figure 2H (especially in relation to, for example) Figure 2I (When a mature J-chain peptide combination lacking a signal sequence is provided). A collagen domain forming a trimer may also be employed. The collagen domain may contain (Gly-Xaa-Xaa)n, where Xaa is any amino acid, or / and where n is an integer (e.g., 10 to 40); where Xaa and Yaa are independently any amino acid and n is an integer from 10 to 40. In the Gly-Xaa-Yaa sequence, Xaa and Yaa are typically present in greater than 25%, 50%, 75%, 80%, 90%, or 95% of Gly-Xaa-Yaa, or each occurrence of Gly-Xaa-Yaa as proline and hydroxyproline, respectively. In some cases, the collagen domain contains the sequence (Gly-Xaa-Pro)n, where n is an integer (e.g., 10 to 40). Collagen oligopeptides may contain the following aa sequence: VTAFSNMDDMLQKAHL-VIEGTFIYLRDSTEFFIRVRDGW-KKLQLGE-LIPIP-ADSPP-PP-AL-SSNP (SEQ ID NO:104).

[0285] F. TGF-β polypeptide As shown above, the masked TGF-β construct or complex contains at least one TGF-β polypeptide (e.g., one or more independently selected TGF-β polypeptides). The amino acid sequence of the TGF-β polypeptide is known in the art. In some cases, the TGF-β polypeptide present in the masked TGF-β construct or complex is a TGF-β1 polypeptide. In some cases, the TGF-β polypeptide present in the masked TGF-β construct or complex is a TGF-β2 polypeptide. In some cases, the TGF-β polypeptide present in the masked TGF-β construct or complex is a TGF-β3 polypeptide.

[0286] While TGF-β1, TGF-β2, or TGF-β3 peptide sequences can be incorporated into masked TGF-β constructs or complexes, several factors can influence the choice of a specific TGF-β peptide and the particular sequence and aa substitution to be employed. For example, TGF-β1 and TGF-β3 undergo amino acid sequence “clipping” when expressed in many mammalian cell systems (e.g., CHO cells). Additionally, dimerized TGF-β (e.g., TGF-β2) has a higher affinity for TβR3 (a β-glycan receptor) than for the TβR2 receptor, which can lead to off-target binding and loss of the bioactively masked protein to a larger in vivo pool of non-signaling TβR3 molecules. To minimize off-target binding to TβR3 with high affinity, it may be necessary to substitute residues that generate the dimerized TGF-β molecule, which are bound by disulfide bonds. Thus, cysteine ​​77 (C77) can be substituted with an amino acid other than cysteine ​​(e.g., serine forming a C77S substitution).

[0287] A suitable TGF-β polypeptide may have a length of about 70 aas to about 125 aas; for example, a suitable TGF-β polypeptide may have a length of about 70 aas to about 80 aas, about 80 aas to about 90 aas, about 90 aas to about 100 aas, about 100 aas to about 105 aas, about 105 aas to about 110 aas, about 110 aas to about 112 aas, about 113 aas to about 120 aas, or about 120 aas to about 125 aas. A suitable TGF-β polypeptide may contain an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with at least 80, at least 90%, at least 100, or at least 110 consecutive aas of the mature form of human TGF-β1 polypeptide, human TGF-β2 polypeptide, or human TGF-β3 polypeptide.

[0288] 1 TGF-β1 peptide Suitable TGF-β1 peptides may contain an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 100, at least 110, or at least 112 amino acid sequences with at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% amino acid sequence identity with the following TGF-β1 amino acid sequences: AL DTNYCFSSTE KNCCVRQLYI DF R KDLGWKWIHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA SAAP C CVPQA LEPLPIVYY V G R KPKVEQLSNMIVRSCKCS (SEQ ID NO:105, 112 aas length); wherein the TGF-β1 polypeptide has a length of approximately 112 aas. The TGF-β1 precursor protein is... Figure 3 Provided as SEQ ID NO:106. Amino acids R25, C77, V92, and R94 are in bold and italics, see [link to documentation]. Figure 4 .

[0289] In some cases, suitable TGF-β1 peptides contain a C77S substitution. Therefore, in some cases, suitable TGF-β1 peptides contain an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with at least 70, 80, 90, 100, 110, or 112 aas of the following TGF-β1 amino acid sequences: AL DTNYCFSSTE KNCCVRQLYI DF R KDLGWKW IHEPKGYHANFCLGPCPYIW SLDTQYSKVL ALYNQHNPGA SAAP S CVPQA LEPLPIVYY V G R KPKVEQLS NMIVRSCKCS (SEQ ID NO:107), where amino acid 77 is Ser. Positions 25, 77, 92, and 94 are in bold and italics.

[0290] 2 TGF-β2 peptide Suitable TGF-β2 peptides may contain an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 100, at least 110, or at least 112 amino acid sequences with at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% amino acid sequence identity with the following TGF-β2 amino acid sequence: ALDAAYCFRNVQDNCCLRPLYIDF K RDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASPCCCVSQDLEPLTILYY I G K TPKIEQLSNMIVKSCKCS (SEQ ID NO:108), wherein the TGF-β2 polypeptide has a length of approximately 112 aas. The TGF-β2 precursor protein in... Figure 3 Provided as SEQ ID NO:109. Residues Lys 25, Ile 92 and / or Lys 94 are in bold and italics.

[0291] In some cases, suitable TGF-β2 peptides contain a C77S substitution. Therefore, in some cases, suitable TGF-β2 peptides contain 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% amino acid sequence identity with at least 70, 80, 90, 100, 110, or 112 aas of the following TGF-β2 amino acid sequence: ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPSCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS (SEQ ID NO110), wherein amino acid 77 is Ser.

[0292] 3 TGF-β3 peptide Suitable TGF-β3 peptides may contain an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 100, at least 110, or at least 112 amino acid sequences with at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% amino acid sequence identity with the following TGF-β3 amino acid sequences: ALDTNYCFRN LEENCCVRPL YIDF R QDLGWKWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPCCVP QDLEPLTILY YV G R TPKVEQLSNMVVKSCK CS (SEQ ID NO:111), wherein the TGF-β3 polypeptide has a length of approximately 112 aas. The TGF-β3 isoform 1 precursor protein is present in... Figure 3 Provided as SEQ ID NO:112. Positions 25, 92 and 94 are in bold and italics.

[0293] In some cases, suitable TGF-β3 peptides contain a C77S substitution. In some cases, suitable TGF-β3 peptides contain an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 100, at least 110, or at least 112 amino acid sequences of the following TGF-β3 amino acid sequence: ALDTNYCFRN LEENCCVRPL YIDF R QDLGW KWVHEPKGYY ANFCSGPCPYLRSADTTHST VLGLYNTLNP EASASPSCVP QDLEPLTILY Y V G R TPKVEQ LSNMVVKSCK CS (SEQ ID NO: 113), wherein amino acid 77 is Ser. Positions 25, 92 and 94 are in bold and italics.

[0294] 4. Additional TGF-β peptide sequence variations In addition to sequence variations that alter the dimerization of the TGF-β molecule (e.g., cysteine ​​77 substitution, such as C77S), TGF-β1-3 peptides with sequence variations affecting affinity and other properties can also be incorporated into masked TGF-β constructs or complexes. When a masked TGF-β construct or complex contains a TGF-β variant with a reduced affinity for the masking peptide (e.g., a TβR peptide, such as a TβRII peptide), those components are more readily dissociated, making the masked TGF-β peptide more readily available to cellular TβR proteins. Because TβRII proteins are typically the first peptide to interact with TGF-β in heterogeneous TβR signaling complexes, interaction with TβRII effectively controls TGF-β entry 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.

[0295] This disclosure includes and provides masked TGF-β constructs and complexes comprising (relative to otherwise identical masked TGF-β constructs or complexes without sequence variations) variant masked TβR (e.g., TβRII) polypeptide sequences and / or variant TGF-β polypeptides with altered (e.g., reduced) affinities to each other. The affinity between the TGF-β polypeptide and the TβR (e.g., TβRII) polypeptide can be determined using the method described above for MOD and its co-MOD (BLI).

[0296] a. Additional TGF-β2 sequence variants This disclosure includes and provides masked TGF-β2 constructs and complexes comprising a masked TβR (e.g., TβRII) polypeptide sequence and a wt. or variant TGF-β2 polypeptide; wherein the variant polypeptide (as opposed to the otherwise identical wt. TGF-β polypeptide sequence without sequence variation) has a reduced affinity for the masked TβR.

[0297] This disclosure provides a masked TGF-β construct or complex comprising a masked TβRII receptor sequence and a variant TGF-β2 polypeptide having greater than 85% (e.g., greater than 90%, 95%, 98%, or 99%) sequence identity to at least 100 consecutive amino acids of SEQ ID NO. 108, and comprising a substitution that reduces the affinity of the variant TGF-β2 polypeptide for the TβRII receptor sequence.

[0298] In some cases, the masked TGF-β construct or complex comprises a masked TβRII peptide and a variant TGF-β peptide (e.g., TGF-β2) containing substitutions at one or more, two or more, or all three of Lys 25, Ile 92, and / or Lys 94 (see SEQ ID NO:108 for the position of the residues, and see [link to SEQ ID NO:108] for the corresponding residues in TGF-β1 and TGF-β3). Figure 4 Those aa residues have been shown to affect the affinity of TGF-β2 for TβRII peptides (see Crescenzo et al., J. Mol. Biol.355: 47–62 (2006)). The masked TGF-β polypeptide optionally includes one or more independently selected MODs such as IL-2 or variants thereof. In one case, the 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 includes one or more independently selected MODs (e.g., one or more IL-2 MOD polypeptides or variants thereof with reduced affinity). The masked TGF-β construct or complex having a masked TβRII polypeptide may include 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 includes one or more independently selected MODs (e.g., one or more IL-2 MOD polypeptides or variants thereof with reduced affinity). A masked TGF-β construct or complex having a masked TβRII peptide may contain a TGF-β2 peptide having an aa other than Lys or Arg at position 94 of SEQ ID NO:108; and optionally contains one or more independently selected MODs (e.g., one or more IL-2 MOD peptides or their reduced-affinity variants). A masked TGF-β construct or complex having a masked TβRII peptide may contain a substituted TGF-β2 peptide at one or more, two or more, or all three of Lys 25, Ile 92, and / or Lys 94, and further contain one or more independently selected MODs. A masked TGF-β construct or complex having a masked TβRII peptide may contain a substituted TGF-β2 peptide at one or more, two or more, or all three of Lys 25, Ile 92, and / or Lys 94, and further contain one or more independently selected IL-2 MODs or their reduced-affinity variants.

[0299] b. Additional TGF-β1 and TGF-β3 sequence variants In some cases, the masked TGF-β construct or complex comprises a masked TβRII peptide and a variant TGF-β1 or TGF-β3 peptide containing substitutions at one or more, two or more, or all three aa positions corresponding to Lys 25, Ile 92, and / or Lys 94 in TGF-β2 SEQ ID NO:108. In TGF-β1 or TGF-β3, the aa corresponding to Lys 25 is Arg 25, Ile 92 is Val 92, and Lys 94 is Arg 94, each of which is a conserved substitution. For TGF-β1, see, for example, SEQ ID NO:106 and 107, and for TGF-β3, see SEQ ID NO:112 and 113.

[0300] As shown above, the masked TGF-β construct or complex optionally includes one or more independently selected MODs such as IL-2 or variants thereof. In one case, the masked TGF-β construct or complex having a masked TβRII peptide includes a TGF-β1 or β3 peptide having 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 peptides or variants thereof with reduced affinity). In another case, the masked TGF-β construct or complex having a masked TβRII peptide includes a TGF-β1 or β3 peptide having an aa other than Val or Ile (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 peptides or variants thereof with reduced affinity). In another case, the masked TGF-β construct or complex having a masked TβRII peptide comprises a TGF-β2 peptide having an amino acid other than Arg or Lys; and optionally comprises one or more independently selected MODs (e.g., one or more IL-2 MOD peptides or their reduced-affinity variants). In one particular case, the masked TGF-β construct or complex having a masked TβRII peptide comprises a substituted TGF-β1 or β3 peptide at one or more, two or more, or all three of Arg 25, Val 92, and / or Arg 94, and further comprises one or more independently selected MODs. In another particular case, the masked TGF-β construct or complex having a masked TβRII peptide comprises a substituted TGF-β1 or β3 peptide at one or more, two or more, or all three of Arg 25, Val 92, and / or Arg 94, and further comprises one or more independently selected IL-2 MODs or their reduced-affinity variants.

[0301] G. TGF-β receptor peptide and other peptides that bind to and mask TGF-β In any of the above-mentioned TGF-β peptides or peptide complexes, the peptide that binds to and masks the TGF-β peptide (“masking peptide”) may take various forms, including fragments of TβRI, TβRII, TβRIII and anti-TGF-β antibodies or fragments thereof (e.g., Fab, single-chain antibodies, etc.).

[0302] 1 TGF-β receptor polypeptide Masking of TGF-β in the masked TGF-β construct and complex can be accomplished by utilizing a TGF-β receptor fragment (e.g., an extracellular domain sequence of TβRI, TβRII, or TβRIII) containing a polypeptide sequence sufficient to bind a TGF-β polypeptide (e.g., TGF-β1, TGF-β2, or TGF-β3). In one embodiment, the masking sequence comprises all or part of the extracellular domain of TβRI, TβRII, or TβRIII.

[0303] a. TGF-β receptor I (TβRI) In one embodiment, the peptide sequence of TGF-β in the masked TGF-β construct or complex may be derived from TβRI (e.g., isoform 1 SEQ ID NO:114) and may contain all or a portion of the TβRI extracellular domain (aas 34-126). In some cases, a suitable TβRI peptide for masking TGF-β contains an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 100, or 103 aas sequence identity with the following TβRI extracellular domain aa sequence: LQCFCHL CTKDNFTCVTDGLCFVSVTE TTDKVIHNSM CIAEIDLIPR DRPFVCAPSS KTGSVTTTYC CNQDHCNKIE LPTTVKSSPGLGPVEL (SEQ ID NO:115).

[0304] b. TGF-β receptor II (TβRII) In the implementation, the peptide sequence of TGF-β in the masked TGF-β construct or complex may be derived from TβRII (e.g., isoform A SEQ ID NO: 116) and may contain all or part of the extracellular domain sequence (aas 24 to 177) of TβRII. In one embodiment, a suitable TβRII isoform A polypeptide for masking TGF-β may comprise an amino acid sequence having 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 154 aa sequences of the extracellular domain of the following TβRII isoform A: IPPHVQK SDVEMEAQKDEIICPSCNRT AHPLRHINND MIVTDNNGAV KFPQLCKFCD VRFSTCDNQK SCMSNCSITS ICEKPQEVCVAVWRKNDENI TLETVCHDPK LPYHDFILED AASPKCIMKE KKKPGETFFM CSCSS D ECND NIIFSEE (SEQ ID NO: 117). The position of the aspartic acid residue corresponding to D118 in isoform B is bold, underlined, and italicized.

[0305] In one embodiment, the peptide sequence of TGF-β in the masked TGF-β construct or complex may be derived from TβRII isoform B (SEQ ID NO: 118) and may contain all or part of the TβRII extracellular domain sequence (aas 24 to 166). In an example, a suitable TβRII isoform B peptide for masking TGF-β comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 100, or 103 aas sequence identity with the TβRII isoform B extracellular domain aa sequence: IPPHVQKSVN NDMIVTDNNG AVKFPQLCK F C D VRFSTCDN QKSCMSNCSI T S IC E KPQEV CVAVWRKNDENITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETF FMCSCSS DEC NDNIIFSEEYNTSNPDLLLVIFQ (SEQ ID NO:119). As discussed below, any one or more of F30, D32, S52, E55, or D118 (italic and bold) may be substituted at those positions with an amino acid other than naturally occurring aa (e.g., alanine). The masking TGF-β polypeptide sequence may comprise the SEQ ID NO:119 polypeptide with a D118A or D118R substitution. The masking TGF-β sequence may comprise the SEQ ID NO:119 peptide with a D118A or D118R substitution and one or more of F30A, D32N, S52L, and / or E55A substitutions.

[0306] While the extracellular domain of TβRII can be used as a masking peptide, regions of this protein contain charged and hydrophobic blocks that can generate unfavorable pIs and be toxic to cells expressing the peptide. Furthermore, combining the TβRII extracellular domain with an active TGF-β peptide can generate a complex that can bind to cell-surface TβRI and lead to activation of the signaling receptor (e.g., signal transduction via the Smad pathway). Modifying the TβRII extracellular domain sequence used for masking TGF-β by removing or altering the sequence involved in TβRI association can prevent unintentional stimulation of the cell by the masked TGF-β, except through its own cell-surface heterodimeric TβRI / TβRII complex. Modification of TβRII can also alter (e.g., reduce) the affinity of TβRII for TGF-β (e.g., TGF-β3), thereby allowing control over TGF-β demasking and its availability as a signaling molecule. TGF-β constructs or complexes containing masked TβR (e.g., TβRII) peptides with the highest affinity for TGF-β (e.g., TGF-β3) most tightly mask the TGF-β sequence and require higher doses to achieve the same effect. In contrast, aa substitutions in TβRII that reduce affinity demask the TGF-β peptide and are bioeffective at lower doses. See, for example, Example 3.

[0307] Therefore, when it is necessary to block / limit signal transduction by the masked TGF-β peptide and / or modify (e.g., reduce) the affinity of the masked TβRII peptide for TGF-β via TβRI, numerous modifications to TβRII can be incorporated into the TβRII peptide sequence. Manufacturable modifications include the aforementioned deletions (e.g., Δ14, Δ25) of the N-terminal 25 amino acids of length 1 to 25 aa and / or substitutions of one or more of L27, F30, D32, S49, 150, T51, S52, I53, E55, V77, D118, and / or E119. Certain modifications that reduce the association between TβRI and TβRII and decrease affinity for TGF-β include any one or more of the following based on SEQ ID NO: 119: L27A, F30A, D32A, D32N, S49A, I50A, T51A, S52A, S52L, I53A, E55A, V77A, D118A, D118R, E119A, and / or E119Q. See, for example, J. Groppe et al. Mol Cell 29, 157- 168 , (2008) and De Crescenzo et al. JMB 355, 47-62 (2006). For the effects of those substitutions on the TGF-β3-TβRII and TβRI-TβRII complexes, see Figure X. Modifications to TβRII include N-terminal Δ25 deletion and / or substitution at F24 (e.g., F24A substitution), which substantially or completely block signaling through the typical SMAD signaling pathway. In one aspect, the aspartic acid at position 118 (D118) of the mature TβRII B isoform (SEQ ID NO: 119) is replaced by an amino acid other than Asp or Glu, such as Ala, resulting in a "D118A" substitution, or by Arg, resulting in a D118R substitution. The Asp residue corresponding to D118 is indicated in SEQ ID NO. 117-123 (in... Figure 5B (In bold and underlined). N-terminal deletions of 1 to 25 aa lengths (e.g., Δ25 deletions) and / or substitutions at F24 (e.g., F24A substitutions) may be combined with D118 substitutions (e.g., D118A or D118R). N-terminal deletions of 1 to 25 aa lengths (e.g., Δ25 deletions) and / or substitutions at F24 (e.g., F24A substitutions) may also be combined with substitutions of any of L27, F30, D32, S49, 150, T51, S52, I53, E55, V77, D118 and / or E119 (e.g., D118A) and any of the specific affinity-altering substitutions listed, particularly for those positions in SEQ ID NO: 119 above.

[0308] N-terminal deletion of the TβRII peptide can also result in loss of TβRI interaction and prevent masked TGF-β constructs and complexes containing the TβRII peptide from acting as constitutively active complexes that connect and activate TβRI signaling. A 14 aa deletion (Δ14) of the TβRII peptide substantially reduces protein-TβRI interaction, and a Δ25 aa deletion of TβRII appears to completely eliminate interaction with TβRI. N-terminal deletion also substantially alters the protein's pI, with the Δ14 TβRII extracellular domain mutant exhibiting a pI of approximately 4.5–5.0 (e.g., approximately 4.74). Therefore, TGF-β constructs or complexes may contain TβRII extracellular domain peptides (e.g., peptides of SEQ ID NO: 117 or 118) with N-terminal deletions such as 14 to 25 aas (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aas). Extracellular domain sequences (including those that restrict interaction with TβRI) that can be used to modify TGF-β peptides in masked TGF-β constructs or complexes are described in the following paragraphs.

[0309] In one embodiment, the sequence of TGF-β in the masked TGF-β construct or complex comprises at least 70, at least 80, at least 90, at least 100, or 103 aas sequences 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 with the TβRII isoform B extracellular domain sequence: IPPHVQKSVN NDMIVTDNNG AVKFPQLCK F C D VRFSTCDN QKSCMSNCSI T S IC E KPQEV CVAVWRKNDENITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETFFMCSCSS D EC NDNIIFSEE (SEQ ID NO:120). Any one or more of F30, D32, S52, E55, or D118 (italic and bold) may be substituted at those positions with an amino acid other than naturally occurring aa (e.g., alanine). In one embodiment, the masked TGF-β sequence comprises the SEQ ID NO:120 peptide with a D118A substitution. In one embodiment, the masked TGF-β sequence comprises the SEQ ID NO:120 polypeptide with a D118A substitution and one or more of F30A, D32N, S52L, and / or E55A substitutions.

[0310] Combinations of N-terminal deletions of TβRIIs, such as 14 to 25 aas (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aas), that block unintended cellular signaling due to the interaction of the masked TGF-β / TβRII complex with TβRIs, can be combined with substitutions of other TβRII extracellular domains (including those substitutions at any one or more of F30, D32, S52, E55, and / or D118). The combination of deletions and substitutions ensures that the masked TGF-β construct or complex does not induce cellular signaling, except for those binding to TβRI and TβRII receptors across the cell membrane.

[0311] In one embodiment, the TGF-β sequence in the masked TGF-β construct or complex comprises a sequence having at least 70, 80, 90, 100, or 103 aas sequence isoforms of the TβRII extracellular domain sequence, with at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% aa sequence identity: TDNNGAVKFPQLCK F C D VRFSTCDN QKSCMSNCSI T S IC E KPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFILEDAASPKCIM KEKKKPGETF FMCSCSS D EC NDNIIFSEE (SEQ ID NO:121) has aas 1-14 (Δ14) deletion. Any one or more of F30, D32, S52, E55, or D118 (italic and bold) may be substituted at those positions with an amino acid other than naturally occurring aa (e.g., alanine). In one embodiment, the masked TGF-β sequence comprises the SEQ ID NO:120 peptide with a D118A substitution. In one embodiment, the masked TGF-β sequence comprises the SEQ ID NO:121 polypeptide with a D118A substitution and one or more of F30A, D32N, S52L, and / or E55A substitutions.

[0312] In one embodiment, the sequence of 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 100%, or at least 103 aas sequence identity with the following sequence: QLCK FC D VRFSTCDNQKSCMSNCSI T S IC E KPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIM KEKKKPGETFFMCSCSS D EC NDNIIFSEE (SEQ ID NO:122) has an aas 1-25 (Δ25) deletion. Any one or more of F30, D32, S52, E55, or D118 (italic and bold) may be substituted at those positions with an amino acid other than naturally occurring aa (e.g., alanine). In one embodiment, the masking TGF-β sequence comprises the SEQ ID NO:122 polypeptide with a D118A substitution (in... Figure 5B (Shown as SEQ ID NO:123). In one embodiment, the TGF-β sequence in the masked TGF-β construct or complex comprises the peptide of SEQ ID NO:122 with one or more of the following substitutions: D118A and F30A, D32N, S52L and / or E55A. In one embodiment, the TGF-β sequence in the masked TGF-β construct or complex comprises (see...) Figure 5B The peptide SEQ ID NO:122 contains D118A and F30A substitutions. In one embodiment, the sequence of TGF-β in the masked TGF-β construct or complex includes (see...) Figure 5B The peptide with D118A and D32N substitutions, SEQ ID NO:122. In one embodiment, the sequence of TGF-β in the masked TGF-β construct or complex includes (see...) Figure 5B The peptide SEQ ID NO:122 contains D118A and S52L substitutions. In one embodiment, the sequence of TGF-β in the masked TGF-β construct or complex includes (see...) Figure 5B SEQ ID NO:122 peptide with D118A and E55A.

[0313] c. TGF-β receptor III (TβRIII) In one embodiment, the peptide sequence of TGF-β in the masked TGF-β construct or complex may be derived from TβRIII (e.g., isoform A SEQ ID NO: 124 and isoform B 125) and may contain all or a portion of the TβRIII extracellular domain (aas 27-787 for isoform A or 27-786 for isoform B). In some cases, a suitable TβRIII peptide for masking TGF-β contains an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 100, or 120 aas sequence identity with the extracellular domain sequence of TβRIII isoform A or isoform B (e.g., in...). Figure 5C (As provided in SEQ ID NO:124 or SEQ ID NO:125).

[0314] 2 Antibodies While TGF-β receptor peptides (e.g., extracellular domain sequences) can act as binding agents to and masking TGF-β peptides in the TGF-β construct or complex, other peptide sequences (protein sequences) that bind to the TGF-β sequence can also be used as masking peptides. Among suitable peptide or protein sequences that can be used to mask TGF-β are antibodies with affinity for TGF-β (e.g., antibodies specific to one or more of TGF-β1, TGF-β2, or TGF-β3) or fragments thereof, nanobodies with affinity for TGF-β peptides, and especially single-chain anti-TGF-β antibodies (e.g., any of which may be humanized). Some antibodies that bind to and neutralize TGF-β have been described, including scFV antibodies. See, for example, US 9,090,685. Throughout the embodiments and / or aspects of the invention described in this disclosure, the TβR (e.g., TβRII) sequence used to mask the TGF-β peptide can be replaced with a masking antibody sequence (e.g., scFV or nanobody) having affinity for the TGF-β peptide. For example, in which the TGF-β receptor sequence is used to mask the TGF-β peptide... Figure 1 In each of the masked TGF-β constructs or complexes, the receptor peptide may be replaced with a masking antibody peptide (e.g., scFV or nanobody) that has an affinity for the TGF-β peptide.

[0315] One potential advantage of using antibodies (e.g., single-chain antibodies) as masking peptides is their ability to limit them to isoforms of the TGF-β peptide to be masked. For example, a single-chain antibody sequence based on metimolumab (CAT192) against TGF-β1 (e.g., Lord et al., mAbs 10(3): 444-452 (2018)) can be used to mask the TGF-β isoform present in a TGF-β construct or complex. In another embodiment, a single-chain antibody sequence specific to TGF-β2 is used to mask the TGF-β isoform present in a TGF-β construct or complex. In yet another embodiment, a single-chain antibody sequence specific to TGF-β3 is used to mask the TGF-β isoform present in a TGF-β construct or complex. Single-chain antibodies can also be specific to combinations of TGF-β isoforms (e.g., extracellular domain sequences appearing in masked TGF-β constructs or complexes selected from the group consisting of: TGF-β1 and TGF-β2; TGF-β1 and TGF-β3; and TGF-β2 and TGF-β3). Single-chain antibodies can also be broadly specific to TGF-β1, TGF-β2, and TGF-β3 extracellular domain sequences appearing in masked TGF-β constructs or complexes, see, for example, WO2014 / 164709. Antibodies and single-chain antibodies with desired specificity and affinity for TGF-β isoforms can be prepared by a variety of methods, including modification (e.g., combination modification) of variable region sequences for screening hybridomas and / or antibodies with affinity for target TGF-β peptide sequences.

[0316] In one embodiment, the masked TGF-β construct or complex comprises a single-chain antibody to mask the TGF-β sequence (e.g., the TGF-β3 sequence). In one such embodiment, the single-chain amino acid sequence is specific for TGF-β3 listed in SEQ ID NO:111 (see SEQ ID NO:112) containing a C77S substitution.

[0317] H. Connector As shown above, a masked TGF-β construct or complex may include a linker peptide / peptide sequence between any two elements of the masked TGF-β construct or complex. Although the term "linker" is used, the same sequence described below as a linker may also be placed at the N-terminus and / or C-terminus of the peptide of the masked TGF-β construct or complex to, for example, prevent proteolytic degradation.

[0318] Suitable connectors (also called "spacers") are readily selectable and can be any of many 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 connector lengths 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. Suitable connector lengths can be 25 to 35 aa. Suitable connector lengths can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 aa. Suitable connector lengths can also be 35 to 45 aa. Suitable connector lengths can be 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 aa. Suitable connector lengths can be 45 to 50 aa. Suitable connector lengths can be 45, 46, 47, 48, 49, or 50 aa.

[0319] Exemplary connectors include those containing glycine, or polyglycine containing a sequence 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 (including, for example, (GS)). n (GSGGS) n (SEQ ID NO:126) and (GGGS) n (SEQ ID NO:127), where n is an integer of at least 1 (e.g., 1-10, 10-20, or 20-30); a glycine-alanine polymer or an alanine-serine polymer (e.g., having a length of 1-10, 10-20, or 20-30 aa); and other flexible connectors known in the art. Glycine and glycine-serine polymers can be used; both Gly and Serine are relatively unstructured and therefore can act as neutral linkers between components. Glycine polymers can be used; glycine is significantly more readily accessible into the phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem(11173-142(1992)). Exemplary adapters may comprise amino acid sequences, including but not limited to GGSG (SEQ ID NO:128), GGSGG (SEQ ID NO:129), GGSSG (SEQ ID NO:130), GGSGG (SEQ ID NO:131), GGGSG (SEQ ID NO:132), GSSSG (SEQ ID NO:133), etc. Exemplary adapters may comprise, for example, GGSG (SEQ ID NO:134) repeated 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some cases, the adapter comprises an amino acid sequence (GSSSS) repeated 2, 3, or 4 times (SEQ ID NO:135). In some cases, the adapter comprises an amino acid sequence (GSSSS) repeated four or five times (SEQ ID NO:135). Exemplary adapters may include, for example, (GGGGS) (SEQ ID NO:136) repeated 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some cases, the linker contains an amino acid sequence repeated once or twice (GGGGS) (SEQ ID NO:136). In some cases, the linker contains an amino acid sequence repeated three or four times (GGGGS) (SEQ ID NO:136). In some cases, the linker contains an amino acid sequence repeated five, six, or seven times (GGGGS) (SEQ ID NO:136). In some cases, the linker contains an amino acid sequence repeated eight, nine, or ten times (GGGGS) (SEQ ID NO:136).

[0320] In some cases, the linker polypeptide present in the first polypeptide of the masked TGF-β complex includes a cysteine ​​residue that can form a disulfide bond with a cysteine ​​residue present in the second polypeptide of the masked TGF-β construct or complex. In some cases, for example, a suitable linker contains the amino acid sequence G. C GASGGGGSGGGGS (SEQ ID NO:137).

[0321] 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 peptide binding to and masking the TGF-β polypeptide sequence can take various forms. The masking peptide can be an antibody, a binding fragment of an antibody, a single-chain antibody or its TGF-β-binding portion (e.g., scFv), or a nanobody; any of these can be humanized. The masking peptide can also be a TGF-β receptor fragment (e.g., an extracellular domain sequence of TβRI, TβRII, or TβRIII) containing a polypeptide sequence sufficient to bind a TGF-β polypeptide (e.g., TGF-β1, TGF-β2, or TGF-β3).

[0322] In any of the masked TGF-β constructs and complexes described above, the TGF-β polypeptide sequence used may 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 in the masked TGF-β constructs and complexes; only a portion of TGF-β is needed that interacts with cell surface TβRIIs and allows the masked TGF-β complex to recruit TβRIs and thereby initiate signal transduction (e.g., signal transduction via Smad and non-Smad pathways).

[0323] While the immunomodulatory peptide (MOD) is not required for the delivery of masked TGF-β or its ability to activate cells with TβRI and TβRII as described above, the presence of the MOD can substantially affect the outcome of TGF-β cell activation. Therefore, incorporating a MOD into any of the masked TGF-β constructs and complexes described above can be used to drive various outcomes, including therapeutic outcomes, resulting 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, IL-2, IL-4, IL-6, IL-7, IL-21, IL-23, and variants of any one thereof, including those variants that have a reduced affinity for their co-MODs.

[0324] While MOD may need to be incorporated into masked TGF-β constructs and complexes, their presence is not essential in all cases, especially 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 factor). For example, when it is necessary to support the development of conventional CD8+ T cells (or the survival of low-affinity CD8+ T cells) by promoting the expression of interleukin-7 receptors (e.g., IL-7Ra) in thymocytes, a masked TGF-β construct and complex without MOD peptides (“MOD-free”) may be used. Similarly, when it is necessary to promote the development of T-cell populations induced by strong agonist ligands, a masked TGF-β construct and complex containing or without MOD may be used to support the survival of thymus-derived Tregs (tTregs), invariant natural killer T cells (iNKTs), and CD8αα+ T-cell precursors.

[0325] The following are non-limiting examples of masked TGF-β constructs and complexes.

[0326] 1. Masked TGF-β construct In the case of a masked TGF-β construct, all components (e.g., TGF-β, scaffold, masking polypeptide such as the TβRII sequence, and optionally one or more MODs) are parts of a single polypeptide chain (see, for example, Figure 1 (Structure A). In this embodiment, the scaffold peptide does not form dimers or higher-order structures with other scaffold peptides, and therefore the masked TGF-β construct does not exhibit homodimers, heterodimers, or higher-order multimers (trimers, etc.).

[0327] exist Figure 1 In the case of a masked TGF-β construct, the structure A polypeptide may comprise, from the N-terminus to the C-terminus: optionally one or more MODs; a scaffold polypeptide (without an interspecies binding sequence); a polypeptide that binds to and masks the TGF-β polypeptide; and the TGF-β polypeptide sequence. Such masked TGF-β constructs include the following constructs, wherein: (i) The polypeptide from the N-terminus to the C-terminus comprises: optionally one or more independently selected wt. or reduced-affinity variants of MOD; a scaffold polypeptide (without interspecies binding sequence); a TβR polypeptide that binds to and masks the TGF-β polypeptide; and the TGF-β polypeptide sequence; (ii) The polypeptide comprises, from the N-terminus to the C-terminus: optionally one or more independently selected wt. or reduced-affinity variants of MOD; a scaffold polypeptide (without an interspecies binding sequence); a TβRII polypeptide that binds to and masks the TGF-β polypeptide; and a TGF-β polypeptide sequence; (iii) The polypeptide from the N-terminus to the C-terminus comprises: one or more independently selected wt. or reduced-affinity variants of MOD; a scaffold polypeptide (without interspecies binding sequence); a TβR polypeptide that binds to and masks the TGF-β polypeptide; and the TGF-β polypeptide sequence; (iv) The polypeptide from the N-terminus to the C-terminus comprises: one or more independently selected wt. or reduced-affinity variants of IL-2 MOD; a scaffold polypeptide (without interspecies binding sequence); a TβR polypeptide that binds to and masks the TGF-β polypeptide; and the TGF-β polypeptide sequence; (v) The polypeptide from the N-terminus to the C-terminus comprises: one or more independently selected wt. or reduced-affinity variants of MOD; a scaffold polypeptide (without interspecies binding sequence); a TβR polypeptide that binds to and masks the TGF-β3 polypeptide; and the TGF-β3 polypeptide sequence; (vi) The polypeptide from the N-terminus to the C-terminus comprises: one or more independently selected wt. or reduced-affinity variants of MOD; a scaffold polypeptide (without an interspecies binding sequence); a TβRII polypeptide that binds to and masks the TGF-β3 polypeptide; and the TGF-β3 polypeptide sequence; and (vii) The polypeptide from the N-terminus to the C-terminus comprises: one or more independently selected wt. or reduced-affinity variants of IL-2 MOD; a scaffold polypeptide (without an interspecies binding sequence); a TβRII polypeptide that binds to and masks the TGF-β3 polypeptide; and the TGF-β3 polypeptide sequence.

[0328] In any of the masked TGF-β constructs described herein, C77 of the TGF-β polypeptide sequence is substituted to prevent dimerization (e.g., C77S substitution), and the TGF-β polypeptide may further (e.g., at one, two, or all three of aas 25, 92, and / or 94) include variations that reduce its affinity for the masked TβR polypeptide, as well as modifications in the MOD and TβR polypeptide sequences. Exemplary TβR polypeptide sequences that can be incorporated into the masked TGF-β construct include Δ14 or Δ25 TβRII polypeptides optionally having D118A or D118R substitutions to weaken TβRI linkage. MOD variants and their polypeptide sequences are described, and additional modifications to TβRI, TβRII, and TβRIII are as described above.

[0329] In one embodiment, the masked TGF-β construct has the sequence listed in SEQ ID NO:146 (see [link to SEQ ID NO:146]). Figure 7A In one embodiment, the masked TGF-β construct has the sequence listed in SEQ ID NO:147 (see [link to SEQ ID NO:147]). Figure 7BIn one embodiment, the masked TGF-β construct has the sequence listed in SEQ ID NO:157 (see [link to SEQ ID NO:157]). Figure 7G In one embodiment, the masked TGF-β construct has the sequence listed in SEQ ID NO:158 (see [link to SEQ ID NO:158]). Figure 7H In one embodiment, the masked TGF-β construct has the sequence listed in SEQ ID NO:159 (see [link to SEQ ID NO:159]). Figure 7I ).

[0330] 2. Masked TGF-β complex The masked TGF-β complex comprises at least two polypeptides, a first polypeptide and a second polypeptide, each containing a scaffold polypeptide associated with another scaffold polypeptide, thereby binding the first polypeptide and the second polypeptide together to form a complex. Therefore, the TGF-β polypeptide complex forms homodimer, heterodimer, or higher-order multimer structures. (i) In the first case, the masked TGF-β complex comprises at least one TGF-β polypeptide sequence, at least one polypeptide bound to and masking one or more TGF-β polypeptide sequences (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 homodimers (e.g., symmetrical dimers), as in Figure 1 In structure B. In such homodimers, the Ig Fc peptide may allow spontaneous disulfide bond formation between Ig Fc peptides in the scaffold of each construct and may include mutations (e.g., the LALA mutation discussed herein) that substantially reduce or eliminate the ability of the Ig peptide to induce cell lysis, for example, through complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC).

[0331] (ii) In the second case, the masked TGF-β complex contains (a) A first polypeptide comprising at least one TGF-β polypeptide sequence, at least one polypeptide (e.g., a masking sequence for each TGF-β polypeptide sequence) that binds to and masks one or more TGF-β polypeptide sequences, and optionally one or more immunomodulatory polypeptides (MODs) assembled on a scaffold structure comprising an interspecies dimerization sequence, and (b) A second polypeptide comprising at least one TGF-β polypeptide sequence, at least one polypeptide that binds to and masks at least one TGF-β polypeptide, and optionally one or more immunomodulatory polypeptides (MODs) assembled on a scaffold structure comprising an interspecies dimerization sequence of the first polypeptide. The first and second polypeptides form a heterodimer through the interaction of interspecies dimerization sequences, such as in... Figure 1 In structure C.

[0332] (iii) In the third case, the masked TGF-β complex contains (a) A first polypeptide comprising at least one TGF-β polypeptide sequence, at least one polypeptide (e.g., a masking sequence for each TGF-β polypeptide sequence) 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 structure comprising an interspecies dimerizing sequence, and (b) A second polypeptide comprising a scaffold structure containing a counterpart of the interspecies dimerization sequence of the first polypeptide and optionally one or more immunomodulatory polypeptides (MODs). The first and second polypeptides form a heterodimer through the interaction of interspecies dimerization sequences, such as in... Figure 1 In structure F, and (iv) In the fourth case, the masked TGF-β complex contains (a) A first polypeptide comprising at least one TGF-β polypeptide sequence and optionally, one or more immunomodulatory polypeptides (MODs) assembled on a scaffold structure comprising an interspecies dimerization sequence, 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 structure comprising an interspecies dimer sequence of the first polypeptide. The first and second polypeptides form a heterodimer through the interaction of interspecies dimerization sequences, such as in... Figure 1 In structures D and E.

[0333] In some cases, the masked TGF-β complex ( Figure 1 The sequence constituting the TGF-β polypeptide (first polypeptide), comprising structures B, C, and F, may include, from the N-terminus to the C-terminus: optionally one or more MODs; a scaffold polypeptide (with or without an interspecies binding sequence); a polypeptide that binds to and masks the TGF-β polypeptide; and the TGF-β polypeptide sequence. (Does not contain...) Figure 1 The polypeptide (second polypeptide) of the TGF-β sequence in structure F contains a scaffold polypeptide with an interspecies binding sequence and optionally includes MOD at the N-terminus, C-terminus, or both the N-terminus and C-terminus.

[0334] In some cases, Figure 1 The masked TGF-β complex in structures D and E, the polypeptide containing the TGF-β polypeptide sequence (the first polypeptide), may comprise from the N-terminus to the C-terminus: one or more optional MODs; a scaffold polypeptide (having an interspecies binding sequence); and the TGF-β polypeptide sequence. Figure 1The polypeptides (second polypeptides) in structures D and E that do not contain the TGF-β sequence may comprise, from the N-terminus to the C-terminus: optionally one or more MODs, a scaffold polypeptide having an interspecies binding sequence, and a polypeptide that binds to and masks the TGF-β polypeptide. Although not in Figure 1 The text states that, however, the first polypeptide containing the TGF-β polypeptide sequence may not contain one or more MODs, and the second polypeptide containing the masking sequence may contain one or more MODs.

[0335] The above-described cases of masked TGF-β complexes include those complexes in which the first polypeptide comprises, from the N-terminus to the C-terminus, the following: (i) Optionally one or more MODs; a scaffold polypeptide (having an interspecies binding sequence); and a TGF-β polypeptide sequence; (ii) Optionally, one or more independently selected wt. or reduced-affinity variants of MOD; scaffold peptides (with interspecies binding sequences); and TGF-β peptide sequences; (iii) One or more independently selected wt. or reduced-affinity variants of MOD; scaffold peptides (with interspecies binding sequences); and TGF-β1 or β2 peptide sequences; (iv) One or more independently selected wt. or reduced-affinity variants of IL-2 MOD; scaffold peptides (without interspecies binding sequences); and TGF-β peptide sequences; (v) One or more independently selected wt. or reduced-affinity variants of MOD; scaffold peptides (with interspecies binding sequences); and TGF-β3 peptide sequences; (vi) One or more independently selected wt. or reduced-affinity variants of MOD; scaffold peptides (with interspecies binding sequences); and TGF-β3 peptide sequences; or (vii) One or more independently selected wt. or reduced-affinity variants of IL-2 MOD; a scaffold polypeptide (having an interspecies binding sequence); and a TGF-β3 polypeptide sequence. In each case, the second polypeptide comprises, from the N-terminus to the C-terminus: a scaffold polypeptide containing the interspecies binding (dimerizing sequence) counterpart 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. Figure 1 In the case of a masked TGF-β complex in structure F, a TβR (e.g., TβRII) polypeptide may be located between an N-terminal MOD (if present) and a scaffold of the first polypeptide, and the second polypeptide contains an interspecies binding (dimerization sequence) counterpart of the first polypeptide, with one or more independently selected wt. or affinity-reduced variant MODs (e.g. wt. or variant IL-2 MOD) attached to the counterpart at the N-terminus or C-terminus.

[0336] In any of the masked TGF-β complexes described herein, C77 of the TGF-β polypeptide sequence may be substituted to prevent dimerization (e.g., C77S substitution), and the TGF-β polypeptide may further (e.g., at one, two, or all three of aas 25, 92, and / or 94) include variations that reduce its affinity for the masked TβR polypeptide, as well as modifications in the MOD and TβR polypeptide sequences. Exemplary TβR polypeptide sequences that can be incorporated into the masked TGF-β construct include Δ14 or Δ25 TβRII polypeptides optionally having D118A substitution. MOD variants and their polypeptide sequences are described, and additional modifications to TβRI, TβRII, and TβRIII are as described above.

[0337] In one embodiment, the masked TGF-β complex comprises a polypeptide having the sequences listed in SEQ ID NO: 148 and 149 (see [link to SEQ ID NO: 148 and 149]). Figure 7C In one embodiment, the masked TGF-β complex comprises a polypeptide having the sequences listed in SEQ ID NO: 150 and 151 (see [link to SEQ ID NO: 150 and 151]). Figure 7D In one embodiment, the masked TGF-β complex comprises a polypeptide having the sequences listed in SEQ ID NO:152 and 153 (see...). Figure 7E In one embodiment, the masked TGF-β complex comprises a polypeptide having the sequences listed in SEQ ID NO: 155 and 156 (see [link to SEQ ID NO: 155]). Figure 7F In one embodiment, the masked TGF-β complex comprises a polypeptide having the sequences listed in SEQ ID NO:148 and 160 (see [link to SEQ ID NO:148]). Figure 7J ).

[0338] J. Nucleic Acid This disclosure provides a nucleic acid comprising a nucleotide sequence encoding a masked TGF-β construct and a complex. In some cases, the nucleic acid is a recombinant expression vector; therefore, this disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding a masked TGF-β construct or complex. In some cases, the nucleic acid is a recombinant expression vector; therefore, this disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding multiple masked TGF-β constructs and complexes. The following discussion of nucleic acids relates to nucleic acids encoding the masked TGF-β constructs and complexes of this disclosure.

[0339] Nucleic acids encoding single-chain antigen-presenting peptides As described above, the masked TGF-β construct comprises a single polypeptide chain. Therefore, this disclosure provides a nucleic acid comprising a nucleotide sequence encoding a single-stranded masked TGF-β construct. The nucleic acid comprising the nucleotide sequence encoding a single-stranded masked TGF-β construct can be operatively linked to a transcriptional control element, such as a promoter.

[0340] Nucleic acid encoding the masked TGF-β complex As shown above, in some cases, the masked TGF-β complex comprises at least two separate polypeptide chains (a first polypeptide chain and a second polypeptide chain). This disclosure provides nucleic acids comprising a nucleotide sequence encoding a masked TGF-β complex. In some cases, the individual polypeptide chains of the masked TGF-β complex are encoded in separate nucleic acids. In some cases, all polypeptide chains of the masked TGF-β construct or complex are encoded in a single nucleic acid. In some cases, the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide of the 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 cases, a single nucleic acid comprises nucleotide sequences encoding both the first and second polypeptides of the masked TGF-β complex, which can be operatively linked to a single promoter or two independently selected promoters and under their transcriptional control.

[0341] Individual nucleic acids encoding individual polypeptide chains of the masked TGF-β construct or complex As shown above, in some cases, the individual polypeptide chains of the masked TGF-β complex are encoded by separate nucleic acids. In some cases, the nucleotide sequences encoding the individual polypeptide chains of the masked TGF-β complex are operatively linked to transcriptional control elements, such as promoters that function in eukaryotic cells, wherein the promoters can be constitutive or inducible promoters.

[0342] For example, this 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 wherein the second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of a masked TGF-β complex. In some cases, the nucleotide sequences encoding the first and second polypeptides are operatively linked to a transcriptional control element. In some cases, the transcriptional control element is a promoter that functions in a eukaryotic cell. In some cases, the nucleic acid is present in a separate expression vector.

[0343] In some cases, the nucleotide sequences encoding the first and second polypeptides are operatively linked to transcriptional control elements. In some cases, the transcriptional control elements are promoters that function in eukaryotic cells. In some cases, the nucleic acids are present in separate expression vectors.

[0344] Nucleic acids encoding two or more polypeptides present in the masked TGF-β complex This disclosure provides a nucleic acid comprising nucleotide sequences encoding at least a first polypeptide and a second polypeptide of a masked TGF-β complex. In some cases, when the masked TGF-β complex comprises a first polypeptide, a second polypeptide, and a third polypeptide, the nucleic acid comprises nucleotide sequences encoding the first polypeptide, the second polypeptide, and the third polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of the masked TGF-β complex encode a proteolytically cleavable site or linker between the encoded first polypeptide and the second polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of the masked TGF-β complex include nucleotides encoding an internal ribosome entry site (IRES) between the encoded first polypeptide and the second polypeptide. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide of the masked TGF-β complex include sequences encoding a ribosome jumping signal (or cis-acting hydrolase element, CHYSEL) between the nucleotide sequences encoding the first polypeptide and the second polypeptide.

[0345] In some cases, the first nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) contains the nucleotide sequence encoding a first polypeptide chain of the masked TGF-β complex; and the second nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) contains the nucleotide sequence encoding a second polypeptide chain of the masked TGF-β complex. In some cases, the nucleotide sequences encoding the first polypeptide and the second polypeptide are each operatively linked to independently selected transcriptional control elements, such as promoters that function in eukaryotic cells, wherein the promoter can be a constitutive promoter or an inducible promoter.

[0346] Recombinant expression vector This disclosure provides recombinant expression vectors containing nucleic acids. In some cases, the recombinant expression vector is a non-viral vector. In other cases, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus construct (see, for example, U.S. Patent No. 7,078,387), a recombinant adenovirus construct, a recombinant lentivirus construct, a recombinant retrovirus construct, a non-integrating viral vector, etc.

[0347] Suitable expression vectors include, but are not limited to, viral vectors (e.g., based on viral vectors such as vaccinia virus; poliovirus; adenovirus (see, for example, Li et al.)). Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, for example, 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 4:683690, 1997, Rolling et al. Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir (1989) 63:3822-3828; Mendelson et al., Virol (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, for example, Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); retroviral vectors (e.g., murine leukosis virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); etc. A large number of suitable expression vectors are known to those skilled in the art, and many expression vectors are commercially available.

[0348] Depending on the host / vector system used, any of a number of suitable transcriptional and translational control elements can be used in the expression vector, including constitutive and inducible promoters, transcriptional enhancer elements, transcription terminators, etc. (see, for example, Bitter et al. (1987)). Methods in Enzymology , 153:516-544).

[0349] In some cases, the nucleotide sequence of a polypeptide encoding a masked TGF-β construct and complex is operatively linked to control elements, such as transcriptional control elements, like promoters. Transcriptional control elements can function in eukaryotic cells, such as mammalian cells, or prokaryotic cells, such as bacterial or archaea cells. In some cases, the nucleotide sequence encoding DNA-targeting RNA and / or site-modified polypeptides is operatively linked to multiple control elements that allow the expression of the nucleotide sequence encoding DNA-targeting RNA and / or site-modified polypeptides in both prokaryotic and eukaryotic cells.

[0350] Non-limiting examples of suitable eukaryotic promoters (promoters that function in eukaryotic cells) include promoters derived 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. The selection of suitable vectors and promoters is entirely within the skill of a person skilled in the art. Expression vectors may also contain ribosome-binding sites for translation initiation and transcription terminators. Expression vectors may also include suitable sequences for amplifying expression.

[0351] Preparation of genetically modified host cells expressing masked TGF-β constructs and complexes, and purification of masked TGF-β constructs and complexes. This disclosure provides a genetically modified host cell, wherein the host cell is genetically modified with one or more nucleic acids encoding a masked TGF-β construct or complex.

[0352] Suitable host cells include eukaryotic cells, such as yeast cells, insect cells, and mammalian cells. In some cases, the host cell is a mammalian cell line. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, and rodent (e.g., mouse, rat) cell lines. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) CCL-2), CHO cells (e.g., ATCC CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC CCL10), PC12 cells (ATCC CRL1721), COS cells, COS-7 cells (ATCC CRL1651), RAT1 cells, mouse L cells (ATCC CCLI.3), human embryonic kidney (HEK) cells (ATCC CRL1573), HLHepG2 cells, etc.

[0353] Genetically modified host cells can be used to generate masked TGF-β constructs or complexes. For example, genetically modified host cells can be used to generate masked TGF-β complexes, or single-stranded masked TGF-β constructs, by introducing an expression vector containing a nucleotide sequence encoding a polypeptide, such as the expression vector described above, into the host cell, thereby generating genetically modified host cells. The host cell can constitutively express the masked TGF-β construct or complex, or express it in response to exposure to an inducer, wherein the promoter driving the expression is inducible (e.g., the CMV promoter and the anti-tetracycline operon induced by exposure to tetracycline).

[0354] The masked TGF-β construct or complex is obtained from cells, or from cell culture medium if the peptide is targeted to the secretion pathway by incorporating a signal sequence. The protein can be purified by any means known in the art, including one or more of the following: precipitation (e.g., 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. When the masked TGF-β construct or complex contains an immunoglobulin peptide sequence (e.g., as a scaffold), protein A or protein G can be used for affinity purification of the masked TGF-β construct or complex. In the absence of an immunoglobulin peptide, the complex can be affinity purified using an antibody against a peptide present in the masked TGF-β construct or complex; or alternatively, by incorporating an affinity tag such as a myc epitope (CEQKLISEEDL SEQID NO:154), a "HIS" tag (for divalent metal ion resin binding), or a "FLAG" tag. Purification and / or concentration steps that can be combined with any of the foregoing methods employ size-limited semipermeable membranes (e.g., dialysis membranes or pressure-sensitive elements) that can be used to remove contaminants of substantially different molecular weights and / or concentrate purified proteins.

[0355] In one embodiment, a masked TGF-β construct or complex is expressed from a nucleic acid sequence introduced into mammalian cells (e.g., CHO cells) and targeting the secretion pathway to induce 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 instance of this embodiment, the masked TGF-β construct or complex comprises an immunoglobulin scaffold (e.g., an IgG polypeptide sequence), and purification is performed by affinity chromatography (e.g., protein A or G), alone or in combination with size-based separation (size-based chromatography).

[0356] K. Composition This disclosure provides compositions comprising a masked TGF-β construct or complex, including pharmaceutical compositions. This disclosure also provides compositions comprising a nucleic acid or recombinant expression vector, including pharmaceutical compositions.

[0357] 1. A composition comprising a masked TGF-β construct or complex In addition to the masked TGF-β construct or complex, the compositions disclosed herein may also contain one or more of the following: salts, such as NaCl, MgCl2, KCl, MgSO4, etc.; buffers, such as Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; solubilizers; detergents, such as nonionic detergents, such as Tween-20, etc.; protease inhibitors; glycerol; etc.

[0358] Compositions may contain pharmaceutically acceptable excipients, many of which are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been well described in a variety of publications, including, for example, “Remington: The Science and Practice of Pharmacy,” 19th edition (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) HC Ansel et al., eds., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., 3rd edition, Amer. Pharmaceutical Assoc.

[0359] The pharmaceutical composition may comprise: i) a masked TGF-β construct or complex; and ii) a pharmaceutically acceptable excipient. In some cases, the pharmaceutical compositions of the present invention will be suitable for administration to a subject, for example, being sterile. For example, in some embodiments, the pharmaceutical compositions of the present invention are suitable for administration to human subjects, for example, wherein the composition is sterile and substantially free of detectable pyrogens and / or other toxins, or wherein such detectable pyrogens and / or other toxins are present at levels within acceptable limits set by an appropriate regulatory agency such as the U.S. Food and Drug Administration (USF&DA).

[0360] The protein composition may contain other components such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonates, etc. The composition may contain pharmaceutically acceptable excipients close to those required for physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, hydrochloride, sulfate, solvates (e.g., mixed ionic salts, water, organic matter), hydrates (e.g., water), etc.

[0361] For example, the composition may include aqueous solutions, powders, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc. The composition may be formulated according to the various routes of administration described below.

[0362] When the formulation is administered directly to tissues using a masked TGF-β construct or complex (e.g., introduced into a cell culture system) or as an injectable (e.g., subcutaneously, intraperitoneally, intramuscularly, intralymphaticly, and / or intravenously), the formulation may be provided in an off-the-shelf dosage form or in a non-aqueous form (e.g., a reparable, preservation-stable powder) or an aqueous form (e.g., a liquid composed of pharmaceutically acceptable carriers and excipients). Formulations containing proteins may also be provided in a form that extends the serum half-life of the protein of the invention after administration. For example, the protein may be provided in a liposome formulation, prepared as a colloid, or by other conventional techniques for extending serum half-life. Various methods can be used to prepare liposomes, such as those described in Szoka et al. 1980. Ann. Rev. Biophys. Bioeng As described in 9:467, U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028. The formulation may also be provided in a controlled-release or slow-release form.

[0363] In some cases, the composition comprises: a) a masked TGF-β construct or complex; and b) a saline solution (e.g., 0.9% NaCl). In some cases, the composition is sterile. In some cases, the composition is suitable for administration to a human subject, for example, wherein the composition is sterile and substantially free of detectable pyrogens and / or other toxins, or wherein such detectable pyrogens and / or other toxins are present in amounts within acceptable limits. Therefore, this disclosure provides a composition comprising: a) a masked TGF-β construct or complex; and b) a saline solution (e.g., 0.9% NaCl), wherein the composition is sterile and substantially free of detectable pyrogens and / or other toxins, or wherein such detectable pyrogens and / or other toxins are present in amounts within acceptable limits.

[0364] Other examples of formulations suitable for parenteral administration include isotonic sterile injectable solutions, antioxidants, bacteriostatic agents, and solutes, suspending agents, solubilizers, thickeners, stabilizers, and preservatives that make the formulation isotonic with the blood of the intended recipient. For example, the pharmaceutical compositions of the present invention may be contained in containers, such as sterile containers, like syringes. Formulations may be provided in single-dose or multi-dose sealed containers (such as ampoules and vials) and may be stored under lyophilized (freeze-dried) conditions requiring only the addition of a sterile liquid excipient, such as water for injection, just before use. Temporary injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0365] The concentration of the masked TGF-β construct or complex in the formulation can vary widely, from less than about 0.1% by weight (typically or at least about 2%) to as much as 20% to 50% or more (e.g., 0.1% to 1%, 1% to 5%, 5% to 10%, 10% to 20%, or 20% to 50% by weight) and is typically selected based on the specific administration modality chosen and the patient’s needs, primarily based on fluid volume, viscosity, and patient-based factors.

[0366] This disclosure provides a container comprising a composition (e.g., a liquid composition). The container may be, for example, a syringe, an ampoule, etc. In some cases, the container is sterile. In some cases, both the container and the composition are sterile.

[0367] 2. Compositions containing nucleic acids or recombinant expression vectors This disclosure provides compositions comprising the nucleic acids or recombinant expression vectors of this disclosure, such as pharmaceutical compositions. A variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been well described in numerous publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., 3rd edition, Amer. Pharmaceutical Assoc.

[0368] The compositions disclosed herein may comprise: a) one or more nucleic acids or one or more recombinant expression vectors comprising a nucleotide sequence encoding a masked TGF-β construct or complex; and b) one or more of the following: buffers, surfactants, antioxidants, hydrophilic polymers, dextrins, chelating agents, suspending agents, solubilizers, thickeners, stabilizers, antibacterial agents, wetting agents, and preservatives. Suitable buffers include, but are not limited to, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid) TAPSO, (N-tris(hydroxymethyl)-2-aminoethanesulfonic acid (TES), N-tris(hydroxymethyl)-methyl-glycine (Tricine), tris(hydroxymethyl)-aminomethane (Tris), etc.). Suitable salts include, for example, NaCl, MgCl2, KCl, MgSO4, etc.

[0369] Pharmaceutical formulations may contain about 0.001% to about 99% (w / w) (e.g., 0.001-0.1, 0.1-1.0, 1.0-10, 10-20, 20-40, 40-80, or 80-100% w / w) of a nucleic acid or recombinant expression vector. In the following description of the formulations, “subject nucleic acid or recombinant expression vector” should be understood to include nucleic acid or recombinant expression vectors. For example, in some cases, the formulations of the present invention contain nucleic acid or recombinant expression vectors.

[0370] The nucleic acid or recombinant expression vectors of the present invention can be blended with other compounds or mixtures of compounds, encapsulated with other compounds or mixtures of compounds, conjugated with other compounds or mixtures of compounds, or otherwise associated with other compounds or mixtures of compounds; such compounds may include, for example, liposomes or receptor-targeting molecules. The nucleic acid or recombinant expression vectors of the present invention can be combined in formulations with one or more components that aid in uptake, distribution, and / or absorption.

[0371] The nucleic acid or recombinant expression vector compositions of the present invention can be formulated into any of a number of possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The nucleic acid or recombinant expression vector compositions of the present invention can also be formulated into suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0372] Formulations containing the nucleic acid or recombinant expression vector of the present invention can be liposome formulations. As used herein, the term "liposome" means a vesicle composed of one or more spherical bilayers of amphiphilic lipids. Liposomes are monolayered or multilayered vesicles having a membrane formed of a lipophilic material and an aqueous lumen containing the composition to be delivered. Positively charged liposomes are positively charged liposomes that can interact with negatively charged DNA molecules to form stable complexes. pH-sensitive or negatively charged liposomes are considered to retain DNA rather than complex with it. Both cationic and non-cationic liposomes can be used to deliver the nucleic acid or recombinant expression vector of the present invention.

[0373] Liposomes also include “sterically stable” liposomes, as used herein; the term refers to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in an increased cycle life relative to liposomes lacking such specialized lipids. Examples of sterically stable liposomes are those in which a portion of the vesicle-forming lipids comprises one or more glycolipids or is partially derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG). Liposomes and their uses are further described in U.S. Patent No. 6,287,860, which is incorporated herein by reference in its entirety.

[0374] Formulations and compositions may also include surfactants. The use of surfactants in pharmaceutical products, formulations, and emulsions is well known in the art. Surfactants and their uses are further described in U.S. Patent No. 6,287,860.

[0375] In one embodiment, various permeation enhancers are included to achieve efficient delivery of nucleic acids. In addition to facilitating the diffusion of non-lipophilic drugs across the cell membrane, permeation enhancers also enhance the permeability of lipophilic drugs. Permeation enhancers can be classified into one of five major categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Permeation enhancers and their uses are further described in U.S. Patent No. 6,287,860, which is incorporated herein by reference in its entirety.

[0376] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in aqueous or non-aqueous media, capsules, gel capsules, small capsules, tablets, or microtablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersants, or binders may be required. Suitable oral formulations comprise those in which the antisense nucleic acid of the present invention is administered together with one or more of a penetration enhancer, surfactant, and chelating agent. Suitable surfactants include, but are not limited to, fatty acids and / or their esters or salts, cholic acids and / or their salts. Suitable cholic acids / salts and fatty acids and their uses are further described in U.S. Patent No. 6,287,860. Permeation enhancers, such as combinations of fatty acid / salts with cholic acid / salt combinations, are also suitable. An exemplary suitable combination is a sodium salt of lauric acid, decanoic acid, and UDCA. Other penetration enhancers include, but are not limited to, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-hexadecyl ether. Suitable penetration enhancers also include propylene glycol, dimethyl sulfoxide, triethanolamine, N,N-dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and its derivatives, tetrahydrofurfuryl alcohol, and AZONE™.

[0377] L. Preparations Suitable formulations are as described above, wherein the composition has a pharmaceutically acceptable level (e.g., the composition comprises pharmaceutically acceptable excipients and active molecules). In some cases, suitable formulations comprise: a) a masked TGF-β construct or complex; and b) a pharmaceutically acceptable excipient. In some cases, suitable formulations comprise: a) a nucleic acid comprising a nucleotide sequence encoding a masked TGF-β construct or complex; and b) a pharmaceutically acceptable excipient; in some cases, the nucleic acid is mRNA. In some cases, suitable formulations comprise: a) a first nucleic acid comprising a nucleotide sequence encoding a first polypeptide of a masked TGF-β construct or complex; b) a second nucleic acid comprising a nucleotide sequence encoding a second polypeptide of a masked TGF-β construct or complex; and c) a pharmaceutically acceptable excipient. In some cases, suitable formulations comprise: a) a recombinant expression vector comprising a nucleotide sequence encoding a masked TGF-β construct or complex; and b) a pharmaceutically acceptable excipient. In some cases, a suitable formulation comprises: a) a first recombinant expression vector containing a nucleotide sequence encoding a first polypeptide of a masked TGF-β construct or complex; b) a second recombinant expression vector containing a nucleotide sequence encoding a second polypeptide of a masked TGF-β construct or complex; and c) a pharmaceutically acceptable excipient.

[0378] Suitable pharmaceutically acceptable excipients are described above.

[0379] M. Method Masked TGF-β constructs or complexes are suitable for modulating T cell activity. Therefore, this disclosure provides a method for modulating T cell activity, the method generally involving contacting target T cells with a masked TGF-β construct or complex.

[0380] 1. Methods for regulating the activity of immune cells, including generating, stimulating or inhibiting specific types of immune cells.

[0381] This disclosure provides a method for selectively modulating the activity of cells expressing TβRI and TβRII, the method comprising contacting cells (e.g., T cells, B cells, and innate cells, including natural killer (NK) cells, macrophages, dendritic cells, and granulocytes) with a masked TGF-β construct or complex, wherein contact of T cells with the masked TGF-β construct or complex selectively modulates the activity of epitope-specific T cells. In some cases, the contact occurs in vitro. In some cases, the contact occurs in vivo. The activity of cells subjected to the masked TGF-β construct or complex (e.g., signaling via canonical pathways, atypical pathways, and / or downstream gene expression) can be assessed relative to a treatment group (e.g., a cell subject) not exposed to TGF-β or the masked TGF-β construct or complex.

[0382] This disclosure provides a method for reducing the number and / or activity of T cells or B cells (e.g., pathogen-reactive T cells and / or pathogen-reactive B cells); said method comprising administering (e.g., to a subject in need) one or more masked TGF-β constructs or complexes. In some cases, said method increases the number and / or activity of regulatory T cells (Tregs), resulting in a reduction in the number and / or activity of T cells or B cells (e.g., one or more autoreactive T cells and / or one or more autoreactive B cells), wherein the reduction in the number and / or activity of T cells or B cells subjected to one or more masked TGF-β constructs or complexes is assessed relative to a treatment group (e.g., a cellular subject) not exposed to TGF-β or one or more masked TGF-β constructs or complexes.

[0383] Administration of one or more masked TGF-β constructs or complexes optionally containing one or more independently selected wild-type or variant MODs can directly or indirectly affect various cell populations. For example, administration of a masked TGF-β construct or complex optionally containing one or more wild-type or variant IL-2 MODs can directly stimulate the development and / or survival of FoxP3+ Treg cells (in vivo or in vitro). In addition to any direct effects of the TGF-β / IL-2 complex on various immune cells, the resulting Treg cells can suppress immune responses by, for example, blocking the induction of T cell activation and / or the effector phase of T cell responses, inhibiting B cell activation and / or inhibiting the differentiation and / or proliferation of natural killer cells.

[0384] a. Treg (i) tTreg, pTreg, iTreg and TGF-β constructs or complexes containing IL-2 This disclosure provides a method to promote thymus-derived Tregs (tTregs) and / or peripheral Tregs (pTregs) (Tregs are CD4+ cells that can suppress autoreactive T cells and B cells). + FoxP3 + and CD25 +Methods for the development (e.g., expansion) and / or survival of cells; said methods include administering (e.g., to one or more subjects in need) one or more masked TGF-β constructs or complexes (e.g., in tissue cultures, blood, or specific tissue locations such as wounds), or contacting CD4+ T cells (e.g., naive CD4+ T cells) with them. The one or more masked TGF-β constructs or complexes administered or contacted in said methods may contain one or more (e.g., one, two, or three) independently selected IL-2 MOD polypeptide sequences and / or variant IL-2 MOD polypeptide sequences. Application or contact can be combined with the administration of vitamin D (e.g., vitamin D3 or its analogues), retinoic acid (e.g., all-trans retinoic acid), and / or an inhibitor of the mammalian target of rapamycin (mTOR) (e.g., rapamycin or its functional analogues, such as sirolimus, everolimus, or temsirolimus) to cells or with contact with said substance. Therefore, this disclosure provides a method for promoting the development and / or survival of induced regulatory T cells (iTregs), said cells being FoxpP3+, FoxP3+ thymus-derived Tregs (tTregs), and / or FoxP3+ peripheral Tregs (pTregs), said method comprising, optionally in the presence of vitamin D or its analogues, retinoic acid (e.g., all-trans retinoic acid) or its analogues, and / or rapamycin or its analogues, administering (e.g., to a subject in need) one or more masked TGF-β constructs or complexes containing one or more IL-2 MOD polypeptide sequences and / or variant IL-2 MOD polypeptide sequences, or contacting CD4+ T cells (e.g., naive CD4+ T cells) therewith. The effects of application or treatment with one or more masked TGF-β constructs or complexes can be assessed relative to baseline values ​​(e.g., cell number before treatment) or relative to treatment groups (e.g., cells or subjects) that are matched to the test group (e.g., identical in other respects) but not exposed to TGF-β or one or more masked TGF-β constructs or complexes.

[0385] This disclosure provides a method for increasing the induction / proliferation of Tregs, maintaining Tregs, and / or sustaining their function, the method comprising contacting T cells (e.g., CD4+ T cells in vivo or in vitro) with one or more masked TGF-β constructs or complexes comprising one or more (e.g., one, two, or three) independently selected IL-2 MOD peptide sequences and / or variant IL-2 MOD peptide sequences. The contact increases the induction / proliferation of Tregs, maintains Tregs, and / or sustains their function relative to a baseline value determined prior to contact or relative to a control group of otherwise identical cells not contacted with one or more masked TGF-β constructs or complexes. This disclosure includes and provides a masked TGF-β construct or complex comprising one or more (e.g., one, two, or three) independently selected IL-2 MOD peptide sequences and / or variant IL-2 MOD peptide sequences for use in the method. In one embodiment, the masked TGF-β construct or complex comprising one or more (e.g., one, two, or three) independently selected IL-2 MOD peptide sequences and / or variant IL-2 MOD peptide sequences has… Figure 1 The structural organization described by structures A, B, or C. In one embodiment, a masked TGF-β construct or complex comprising one or more (e.g., one, two, or three) independently selected IL-2 MOD peptide sequences and / or variant IL-2 MOD peptide sequences has Figure 1 The structural organization described by structure D or E. In one embodiment, a masked TGF-β construct or complex comprising one or more (e.g., one, two, or three) independently selected IL-2 MOD peptide sequences and / or variant IL-2 MOD peptide sequences has Figure 1 Structure F describes the structural organization.

[0386] This disclosure provides a method for increasing the induction / proliferation of Tregs, maintaining Tregs, and / or sustaining their function, the method comprising contacting T cells (e.g., CD4+ T cells in vivo or in vitro) with one or more masked TGF-β constructs or complexes comprising one or more (e.g., one, two, or three) independently selected PD-L1 or PD-L2 MOD peptide sequences and / or variant PD-L1 or PD-L2 MOD peptide sequences. The contact increases the induction / proliferation of Tregs, maintains Tregs, and / or sustains their function relative to a baseline value determined prior to contact or relative to a control group of otherwise identical cells not contacted with one or more masked TGF-β constructs or complexes. This disclosure includes and provides a masked TGF-β construct or complex comprising one or more (e.g., one, two, or three) independently selected PD-L1 or PD-L2 MOD peptide sequences and / or variant PD-L1 or PD-L2 MOD peptide sequences for use in the method. In one embodiment, a masked TGF-β construct or complex comprising one or more (e.g., one, two, or three) independently selected PD-L1 or PD-L2 MOD peptide sequences and / or variant PD-L1 or PD-L2 MOD peptide sequences has Figure 1 The structural organization described by structures A, B, or C. In one embodiment, a masked TGF-β construct or complex comprising one or more (e.g., one, two, or three) independently selected PD-L1 MOD peptide sequences and / or variant PD-L1 or PD-L2 MOD peptide sequences has Figure 1 The structural organization described in structure D or E. In one embodiment, a masked TGF-β construct or complex comprising one or more (e.g., one, two, or three) independently selected PD-L1 or PD-L2 MOD peptide sequences and / or variant PD-L1 or PD-L2 MOD peptide sequences has Figure 1 Structure F describes the structural organization. A masked TGF-β construct or complex containing one or more (e.g., one, two, or three) independently selected PD-L1 or PD-L2 MOD polypeptide sequences and / or variant PD...

Claims

1. A TGF-β complex comprising a first polypeptide and a second polypeptide as heterodimers, wherein: (i) The first polypeptide contains a) Scaffold polypeptide sequences containing interspecies dimerization sequences. b) A masking polypeptide sequence comprising a TGF-β receptor polypeptide sequence comprising an extracellular domain fragment of a type II TGF-β receptor (TβRII), the extracellular domain fragment having at least 95% sequence identity with QLCKFCDVRFSTCDN QKSCMSNCSI TSICEKPQEV CVAVWRKNDE NITLETVCHD PKLPYHDFIL EDAASPKCIMKEKKKPGETF FMCSCSSDEC NDNIIFSEE (SEQ ID NO:122). c) Optionally, one or more independently selected immunomodulatory polypeptide sequences, and d) Optionally, one or more independently selected linker polypeptide sequences are inserted between one or more sequences of the first polypeptide; and (ii) The second polypeptide comprises a) A scaffold polypeptide sequence comprising the corresponding interspecific dimer sequence of the interspecific dimer sequence in the first polypeptide. b) A TGF-β3 polypeptide sequence comprising a substitution of cysteine ​​77 (C77) and optionally substitutions at amino acids arginine 25 (R25), valine 92 (V92), and / or arginine 94 (R94). c) Optionally, one or more independently selected immunomodulatory polypeptide sequences, and d) Optionally, one or more independently selected linker polypeptide sequences inserted between one or more sequences of the second polypeptide; Complexes containing these elements are collectively referred to as "masked TGF-β complexes". The masking polypeptide sequence binds to the TGF-β3 polypeptide sequence; and The interspecific dimerization sequence and the corresponding interspecific dimerization sequence interact with each other in the heterodimer.

2. The masked TGF-β complex as described in claim 1, wherein: The first polypeptide comprises, from the N-terminus to the C-terminus, a) One or more independently selected immunomodulatory sequences, the scaffold polypeptide sequence comprising the interspecies dimerization sequence, and the masking polypeptide sequence; or b) The scaffold polypeptide sequence comprising the interspecies dimerization sequence and the masking polypeptide sequence; and The second polypeptide comprises, from its N-terminus to its C-terminus, one or more independently selected immunomodulatory sequences, the scaffold polypeptide sequence comprising the corresponding interspecies dimerization sequence, and the TGF-β3 polypeptide sequence, and One or more independently selected adapter polypeptide sequences may be inserted between one or more sequences of the first polypeptide and / or the second polypeptide.

3. The masked TGF-β complex of any one of claims 1 to 2, wherein the one or more scaffold polypeptide sequences are variant Ig Fc polypeptide sequences, and wherein the variant Ig Fc polypeptide sequence contains a mutation that substantially reduces or eliminates the ability of the variant Ig Fc polypeptide to induce cell lysis.

4. The masked TGF-β complex of claim 3, wherein each scaffold polypeptide comprises an IgG1 sequence having at least 80% amino acid sequence identity with SEQ ID NO:71, and optionally includes substitutions at one or more of L14 and L15.

5. The masked TGF-β complex of any one of claims 2 to 4, wherein the one or more independently selected immunomodulatory polypeptide sequences are wild-type or variant immunomodulatory polypeptide sequences selected from the group consisting of: PD-L1, FAS-L, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, and IL-23.

6. The masked TGF-β complex according to any one of claims 2 to 5, wherein at least one of the immunomodulatory polypeptide sequences is a variant IL-2 immunomodulatory polypeptide sequence having at least 95% amino acid sequence identity with SEQ ID NO:

9.

7. The masked TGF-β complex of claim 6, wherein the variant IL-2 immunomodulatory polypeptide sequence comprises substitutions at any one, any two, or all three of N88, F42, and / or H16.

8. The masked TGF-β complex of any one of claims 1 to 7, wherein the TGF-β3 polypeptide sequence comprises cysteine ​​77 replaced by serine (C77).

9. The masked TGF-β complex of any one of claims 1 to 7, wherein the TβRII polypeptide sequence comprises any one, any two, any three, any four, or all five substitutions of F30, D32, S52, E55, and / or D118.

10. The masked TGF-β complex of claim 9, wherein the TβRII polypeptide sequence comprises a D118A or D118R substitution.

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