TGF-β polypeptide

Abstract

To provide Transforming Growth Factor Beta (TGF-β) polypeptide constructs and complexes that find use in therapeutic treatment of diseases including autoimmune diseases, nucleic acids that encode the constructs and the complexes, and methods for preparing the constructs and complexes in cell-based expression systems.SOLUTION: A "masked TGF-β construct" includes: (i) a scaffold polypeptide sequence; (ii) a TGF-β polypeptide sequence; (iii) a masking polypeptide sequence optionally including a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; (iv) optionally, one or more independently selected MOD polypeptide sequences; and (v) optionally, one or more independently selected linker polypeptide sequences, as a first polypeptide, wherein the masking polypeptide sequence and the TGF-β polypeptide sequence bond with each other.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 925,227, filed October 23, 2019. No. 60 / 699,999, filed on Dec. 1, 2003, which application is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Sequence Listing This application contains an electronically submitted sequence listing, which may be easily accessed through paper copies and computer-readable media. The file, entitled "123640-8012WO00_seqlist.txt," serves as both a CRF and a It consists of a file, created on October 21, 2020, and is 341,966 bytes in size. The entire contents of which are incorporated herein by reference.

[0003] I. Introduction A. TGF-β and its effects Transforming growth factor beta (TGF-β) is a serogroup of three mammalian (human) isoforms: TGF-β1, TG It is a cytokine belonging to the transforming growth factor superfamily, which includes F-β2 and TGF-β3. TGF-β consists of the TGF-β sequence that homodimerizes as the active form of TGF-β, and an additional promoter. TGF-β is synthesized as a precursor molecule containing a peptide region. In other cell types, two other polypeptides—latent TGF-β binding protein (LTBP) and latent TGF-β binding protein (LTBP)—are expressed. It is secreted as a latent complex combined with latent TGF-β associated peptide (LAP). The complex is located in the extracellular matrix (ECM), for example, by thrombospondin-1 (which is linked to plasmin). on the cell surface by CD36 via ATP (which can be activated by ATP) or by latent transforming growth factors Retained by binding to LTBP1-4 .

[0004] The biological function of TGF-β is tightly regulated in response to ECM perturbation. TGF-β is found after activation of TGF-β. TGF-β activates various cell- or tissue-specific pathways, or Although it can be activated by pathways observed in multiple cell or tissue types, such activation The complete mechanism behind the activation pathway is not fully known. Including, but not limited to, proteases, integrins, pH, and reactive oxygen species (ROS) Upon activation, the cell / tissue-bound latent TGF-β complex functions and responds to environmental disturbances. In response to and sensing of TGF-β, the cells release active TGF-β in a spatial and / or temporal manner. The released TGF-β acts to promote or inhibit cell proliferation depending on the circumstances of its release. It also recruits stem / progenitor cells to participate in tissue regeneration / remodeling processes. TGF-β ligand expression, bioavailability, activation, receptor function, or translocation Abnormalities in post-transcriptional modifications can interfere with normal function and lead to excessive precursor mobilization (e.g., osteoarthritis or in Kamuraczi-Engelmann disease), or transdifferentiation of resident cells into undesired lineages (e.g. Many of these pathways are involved in the regulation of cellular functions, such as epithelial-to-mesenchymal transition during cancer metastasis or tissue / organ fibrosis. This can result in pathological consequences associated with the disease. Xu et al., Bone Research, 6 (Article No. .2)(2018).

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

[0006] Plasmin and several matrix metalloproteinases (MMPs) act on extracellular matrix Proteolytic degradation of TGF-β promotes tumor invasion and tissue remodeling. is activated by the action of such proteases, which release the latent complex from the matrix. This can be activated by LAP, followed by proteolysis of LAP and release of TGF-β to its receptor. Both matrix metalloproteinase-9 and -2 can cleave latent TGF-β. It is known.

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

[0008] Thrombospondin-1 (TSP-1), a glycoprotein present in the plasma of healthy individuals, TSP-1 is known to increase in response to wounding. It directly interacts with the latent TGF-β complex. It activates latent TGF-β by forming a phospholipase C (PcC) complex and preventing its binding to mature TGF-β. Thrombospondin-mediated activation is thought to be a factor in wound healing (e.g., skin wounds). It is thought to be involved in wound healing.

[0009] b. Activation by α(V)-containing integrins Integrins, particularly β6-, αV-, and β8-containing integrins, mediate the expression of latent TGF-β (e.g., TG It is understood that activation of the latent TGF-β1 complex contributes to its activation. by inducing conformational changes to release active TGF-β1 or by integrating It is thought to occur through a phospho-protease-dependent mechanism, particularly in epithelial cells. In this study, the conformational change leading to TGF-β1 activation without proteolysis was observed in LAP-β1 or The arginine-glycine-aspartic acid cell adhesion motif (RGD motif) present in LAP-β3 It is understood that this occurs when integrins bind to the RGD motif. LAP is recognized by most αV-containing integrins, such as αVβ6 integrin. activates TGF-β1 by binding to the RGD motif present in LAP-β1 and LAP-β3. In addition, integrin-protease-dependent activation of TGF-β can by forming a link between the latent TGF-β complex and MMPs such as MMP-2 and MMP-9. This can result in activation of TGF-β by proteolysis of the latent TGF-β complex. This can be done.

[0010] 2. TGF-β signal transduction and action Activated TGF-β plays an important role in cell differentiation and T cell regulation. , Cold Spring Harbor Perspect.Biol.2017;9:a022236 and citations therein. GF-β stimulates thymus-derived Tregs (tTregs), invariant natural killer T (iNKT), and CD8α + T cell precursors in the thymus by supporting the survival of several T cell lineages. Promotes development and thereby T cell development induced by potent agonist ligands TGF-β promotes the expression of interleukin (IL)-7Rα in thymocytes. TGF-β also supports conventional CD8+ T cells by inhibiting TGF-β expression. Regulating IL-7Rα expression on thymocytes by promoting IL-7-dependent survival of T cells and T cell receptor (TCR)-driven autoreactive or high-affinity T cell activation It regulates peripheral T cell homeostasis by inhibiting the differentiation of CD8+ T cells. In this context, TGF-β inhibits the formation of cytotoxic T lymphocytes (CTLs) and promotes the formation of short-lived effector cells (S While promoting the apoptosis of LECs, it also promotes differentiation of CD103-expressing tissue-resident memory (TRM) cells. TGF-β inhibits the differentiation of T helper 1 and 2 (Th1 and Th2) cells, but promotes the differentiation of other TGF-β, acting with IL-2, promotes the development of various T cells. It promotes the production of Th-17 cells together with IL-6, and promotes the production of Th-9 cells together with IL-4. promotes the production of T follicular helper (Tfh) cells, along with IL-21 and / or IL-23 .

[0011] In addition to its effects on T cells, it also affects B lymphocytes or "B cells," monocytes, and macrophages. A variety of other cells, including B cells, are regulated by TGF-β. It has an inhibitory effect on the immune system (see Li et al., Annual Review of Immunology. 24(1):99-146(2006) and and Roes et al., PNAS USA, 100(12):7241-7246(2003)), inhibiting B cell proliferation and inhibiting immature or induces apoptosis of resting B cells (Arsura et al., Immunity 5(1)31:-40:19 96) At least part of the effect of TGF-β on B cells is mediated by IL-1, TNF-α, and defensins. This may be due to the induction of IKBa, an inhibitor of NF-κB that regulates the production of cytokines, including β-glucan. See, for example, Cold Spring Harbor Perspect.Biol.2017;9:a022236 and citations therein. stomach.

[0012] In addition to its effects on B cells, TGF-β stimulates resting monocytes and activates macrophages. TGF-β inhibits macrophage proliferation stimulated by Toll-like receptor ("TLR") ligands. TGF-β stimulation is mediated by TLR ligands or other Stimulates the production of several pro-inflammatory cytokines by myeloid cells in the absence of cytokines TGF-β induces peripheral blood monocytes and macrophages into tissues and enhances the adhesive properties of monocytes. TGF-β has been shown to induce chemotaxis and enhance the adhesive properties of mast cells. See, for example, Cold Spring Harbor Perspect.Biol.2017;9:a022236 and See the quote.

[0013] It is understood that when TGF-β is activated, it acts through signaling receptors on the cell surface. Signal transduction occurs when activated TGF-β ligands bind to transforming growth factors on the cell surface. This interaction is initiated when the transforming factor binds to the transforming factor beta receptor II (TβRII). This can result in the recruitment of TGF-β receptor I ("TβRI"). TβRII binds TGF-β1 alone. TβRI can bind to ligands only in cooperation with TβRII. TβRI is activated by phosphorylation of TβRII, and the R-Smad proteins (Smad2 and Smad3) are activated. It also signals through the classical signaling pathway via recruitment and phosphorylation of mad3. These Smads then bind to a co-Smad (Smad4), and the combined complex Smith et al., Clin. Cancer Res.; 18(17):4514-21 (201 2) TGF-β is also activated by ligand-occupied receptors. MAP kinase pathway, Rho-like GTPase signaling pathway, and phosphatidylinositol-3- Signaling through non-canonical (non-Smad) pathways, including various branches such as the kinase / AKT pathway Signaling through non-canonical pathways can enhance, attenuate, or disrupt downstream cellular responses. or may be regulated in other ways. See Zhang Ye, Cell Res. 19(1):128-39 (2009). In contrast to TβRI and TβRII, transforming growth factor beta receptor III (TβRIII) receptor The receptor or "betaglycan" is not involved in TGF-β signaling, but rather is a lysing agent of TGF-β. It functions as a server.

[0014] Disruption of activators, abnormal levels of activated TGF-β, and / or altered TGF-β signaling This may result in uncontrolled TGF-β signaling levels, which may lead to several Indeed, TGF-β plays a key role in the development of inflammation, autoimmune diseases, fibrosis, and other diseases. It has been shown to have effects on a variety of conditions, including cancer and cataracts.

[0015] TGF-β is a factor involved in the induction of tolerance and is crucial for maintaining hemostasis in the immune system. TGF-β plays an important role in the regulation of immune responses, thereby influencing autoimmune responses. One mechanism by which this effect can be exerted is by T cells (e.g., CD4 + , CD8 + , CD4 - CD8 + and CD4 - CD8 - cells) into regulatory T cells or "T-Reg" cells (e.g., in the presence of IL-2) See, e.g., Bettini and Vagnali, Ann. NYAc. See, Ad. Sci., 1183:1-12 (2010). T-Reg cells are essential for maintaining immune tolerance. See Josefowicz et al., Annu Rev Immunol, 30:531-564. (2012). The role of TGF-β in inducing tolerance to antigens, including self-antigens, is well documented in the context of arthritis (rheumatoid arthritis or "R" arthritis). A"), type 1 diabetes ("T1D"), multiple sclerosis ("MS"), and systemic lupus erythematosus ("SLE"). TGF-β is an important factor in preventing the onset of diseases such as encephalopathy. Major functions include the regulation of autoimmune diseases and related inflammatory processes. This is especially true in the intestine, where it is a major contributor to conditions such as inflammatory bowel disease or "IBD." It is believed to suppress cytokine production by macrophages and mucosal inflammation. See, Cold Spring Harbor Perspect. Biol. 2017;9:a022236. RA is Like IBD, RA is an autoimmune disease with an inflammatory component directed at the joints. TGF-β is a cytokine that plays a key role in the development of rheumatoid arthritis. Schramm et al., Arthritis Res. Ther. 6:R114-R119 (2004) and Sanjab et al., Cold Spring Harbor Perspect. Biol. 2017;9:a022236), and See the references cited therein.

[0016] In the literature, it has been reported that the protein is trapped and its action is effectively neutralized. A number of approaches have been reported to modulate TGF-β action at the protein level. For example, methelimumab (CAT192) targets TGF-β1, and multiple isoforms of TGF-β are targeted. Monoclonal antibodies such as fresolimumab bind and capture TGF-β in vivo, Furthermore, it has been developed to bind tightly to TGF-β, capture it, and neutralize it. Therefore, receptor traps that capture and neutralize them have also been developed (e.g., Swagrtra et al. Mol Cancer Ther;11(7):1477-87(2012) and U.S. Patent Application Publication No. 2018 / 0327477 (see issue).

[0017] Designed to bind and capture TGF-β and function as an antagonist of TGF-β action Unlike the molecules described above, the masked TGF-β complexes described herein are not mutually exclusive. activating TGF-β polypeptides (e.g., TGF-β polypeptide sequences) to reversibly mask the TGF-β polypeptide sequence. TGF-β signaling pathway agonists) and masking polypeptides (e.g., TGF- The masked TGF-β complex comprises TGF-β and / or a masking β receptor fragment. The polypeptides may contain sequence mutations that reduce their affinity for each other and inhibit the activity of TGF-β. can contribute to masking by its binding via heteromeric cell surface receptors signal transduction (e.g., binding to TβRII followed by TβRI cleavage of heteromeric receptors) Heteromeric TβRI-TβRII polypeptide complexes with high affinity for TGF-β. Once formed, the complex can effectively compete with the masking polypeptide. Sequence variations in the β and / or its masking polypeptide may also be present in the masked It is also possible to avoid undesirable interactions between TGF-β polypeptides and other molecules. Such sequence mutations include the N-terminal portion of TβRII, which reduces binding to TβRI. deletions and / or TGF-β sequence mutations (e.g., C77S substitutions) that prevent its dimerization, , which relieves the target binding restriction to the reservoir of non-signaling TβRIII molecules. In addition to the above, masked TGF-β constructs and complexes have been shown to have in vitro effects and therapeutic potential. Substantially affect the outcome of TGF-β binding to target cells, including in vivo effects such as therapeutic effects. The polypeptide may comprise additional wild-type (wt) and / or mutant immunomodulatory polypeptide sequences (MOD).

[0018] II. Overview The present disclosure provides constructs in which TGF-β is masked by another polypeptide ("masking"). "Bound TGF-β constructs" (see, for example, structure A in Figure 1, which have a single polypeptide chain) and complexes in which TGF-β is masked by another polypeptide ("TGF-β polypeptide"). Preparation of a "peptide complex" (e.g., see structures B-F in Figure 1) containing two polypeptide chains These include constructs and complexes that may contain additional elements, and are described herein. In the present study, these are collectively referred to as "masked TGF-β constructs and complexes." F-β constructs and complexes are constructed around a scaffold polypeptide (e.g., an immunoglobulin Fc region). A masking polypeptide sequence ("masking polypeptide sequence") that binds to TGF-β is constructed. ", "masking polypeptide" or "masking sequence"). The TGF-β constructs and complexes also include one or more polypeptides, such as wild-type or mutant IL-2 polypeptide sequences. The immunomodulatory polypeptide may comprise any of the above independently selected immunomodulatory polypeptide sequences.

[0019] Masked non-targeted TGF-β activity is not as pronounced as that observed with unmasked TGF-β. Since masked TGF-β constructs and complexes have no adverse effects on cells, It can be expressed in a number of mammalian cell types.

[0020] The masked TGF-β construct comprises, as a first polypeptide: (i) a scaffold polypeptide sequence; and (ii) a TGF-β polypeptide sequence; and (iii) a mass spectrometry assay, optionally comprising a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; a KING polypeptide sequence; (iv) optionally, one or more independently selected MOD polypeptide sequences; (v) optionally, one or more independently selected linker polypeptide sequences; Constructs containing these elements are referred to herein as "masked TGF-β constructs." Collectively, the masking polypeptide sequence and the TGF-β polypeptide sequence bind to each other. The masked TGF-β construct may be, for example, (from the N-terminus to the C-terminus): (i) a scaffold polypeptide sequence, a masking polypeptide sequence, and a TGF-β polypeptide sequence; column, or (ii) a first MOD polypeptide sequence, a scaffold polypeptide sequence, and 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 polypeptide sequence, optionally one or more additional MOD polypeptide sequences, a scaffold polypeptide sequence, a masking polypeptide sequence; and a TGF-β polypeptide sequence, in that order; The masked TGF-β construct optionally comprises one or more independently selected linker molecules. The peptide sequence may include:

[0021] The scaffold polypeptide of the masked TGF-β construct described above induces homodimer formation. The dimerization sequences may include interspecific or non-interspecific dimerization sequences that cause In this case, the scaffold polypeptide sequences optionally have one or more covalent attachments to each other. do.

[0022] The scaffold polypeptides of the masked TGF-β constructs described above also undergo mutually specific dimerization. sequence, and further, the second sequence (described above) may be linked to the first sequence via a complementary mutually specific dimerization sequence. 1 polypeptide to form the masked TGF-β complex heterodimer. The second polypeptide may comprise a second polypeptide having one of the following structures: (i) a scaffold polypeptide sequence containing a complementary mutually specific dimerization sequence; (ii) one or two (or (or more) independently selected MOD sequences and a pair containing a mutually specific dimerization sequence. (iii) a scaffold polypeptide sequence comprising a complementary mutually specific dimerization sequence; and one independently selected MOD sequence; or (iv) one or two (or more) independently selected MOD sequences. a scaffold polypeptide sequence comprising a MOD sequence selected by the method of the present invention and a complementary mutually specific dimerization sequence; wherein the first and / or second polypeptides may optionally comprise one or more independently selected The second polypeptide thus comprises a linker polypeptide sequence from the N-terminus to the C-terminus. and one of the following structures: (i) a scaffold polypeptide comprising a complementary mutually specific dimerization sequence; (ii) one or two (or more) independently selected MOD sequences and their respective counterparts. a scaffold polypeptide sequence comprising a specific dimerization sequence; and (iii) a complementary mutually specific dimerization sequence. and one or two (or more) independently selected MOs. or (iv) one or two (or more) independently selected MOD sequences and a counterpart a scaffold polypeptide sequence comprising a side-specific dimerization sequence, The two polypeptides may optionally contain one or more independently selected linker polypeptide sequences. Alternatively, the masked TGF-β complex heterodimer may comprise, in order from N-terminus to C-terminus: (i) a scaffold polypeptide sequence containing a complementary mutually specific dimerization sequence and a masking polypeptide sequence; (ii) a first MOD polypeptide sequence and a TGF-β polypeptide sequence; a scaffold polypeptide sequence containing a specific dimerization sequence, a masking polypeptide sequence, and a TG an F-beta polypeptide sequence, or (iii) a first independently selected MOD polypeptide sequence; a second independently selected MOD polypeptide sequence and a complementary mutually specific dimerization sequence. a scaffold polypeptide sequence, a masking polypeptide sequence, and a TGF-β polypeptide sequence; It may include.

[0023] The masked TGF-β complex also forms a heterodimer of the masked TGF-β complex. and may comprise a first polypeptide and a second polypeptide, (i) the first polypeptide is (a) a scaffold polypeptide sequence comprising a mutually specific dimerization sequence; (b) a mask optionally containing a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; a coding polypeptide sequence; (c) optionally, one or more independently selected MOD polypeptide sequences; (d) optionally, one or more independently selected linker polypeptide sequences; (ii) the second polypeptide is (a) a mutually specific dimerization sequence that is a counterpart of the mutually specific dimerization sequence of the first polypeptide; a scaffold polypeptide sequence comprising a sequence of (b) a TGF-β polypeptide sequence; and (c) optionally, one or more independently selected MOD polypeptide sequences; (d) optionally, one or more independently selected linker polypeptide sequences; The complex containing these elements is collectively called the "masking TGF-β complex." The TGF-β polypeptide sequence and the TGF-β polypeptide sequence are provided on different polypeptide chains. , mutually coupled, The mutually specific binding sequence and the counterpart mutually specific binding sequence are mutually linked in the heterodimer. It acts, The first and / or second polypeptides of the masked TGF-β are optionally The polypeptide comprises one or more independently selected linker polypeptide sequences.

[0024] The TGF-β polypeptide sequence can be derived from any of the TGF-β isoforms, and the TGF-β sequence The masking sequence may include substitutions that limit the ability of the anti-TGF-β antibody to dimerize. The TGF-β receptor (TβR) ectodomain sequence may be a TGF-β receptor (TβR) ectodomain sequence. When used to mask β sequences, they are To avoid unintended signal transduction, the nucleotide sequence required for interaction with TGF-β was The protein may be modified (by deletion of all or part of the ectodomain that is not present).

[0025] The present disclosure also provides methods for producing masked TGF-β constructs and complexes, as well as various for effects on various cell types and for various diseases / disorders, including autoimmune and inflammatory diseases The methods of treatment described herein include: The masked TGF-β constructs and complexes may be coupled to other molecules, including but not limited to immunoglobulins. Regulatory substances (e.g., interleukins, cytokines, chemokines, etc.), antibodies and antibody fragments fragments (e.g., scFv, nanobodies, etc.), small molecule therapeutics (e.g., vitamin D or retinoic acid ), and combinations thereof to achieve a desired laboratory or therapeutic result. The present invention may include co-administration with other drugs that may be beneficial to the patient. [Brief explanation of the drawings]

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

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

[0028] [Figure 2I] The amino acid sequence of human J chain (SEQ ID NO: 84) is shown, including the underlined signal peptides ass1 to ass22.

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

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

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

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

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

[0034] [Figure 5B] 1 shows the sequence of TGF-β receptor type 2 (TβRII), its ectodomain, and a fragment of the ectodomain. In isoform B, the positions shown in bold and underlined are aas F30, D32, S52, E55, and D118 of the mature polypeptide, any of which may be substituted with an aa other than the naturally occurring aa.

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

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

[0037] [Figure 7A]

[0033] Figure 1 shows the aa sequence of a representative masked TGF-β (construct number: 3470) SEQ ID NO: 146, having the overall structure of Structure A in Figure 1. The polypeptide comprises, from N- to C-terminus, wild-type human IL-2 (hIL2), three repeats of G4S linkers, a human mono IgGFc with an LALA substitution, three repeats of G4S linkers, a human TβRII (hTβRII) Δ25 sequence with a D118A substitution, five repeats of G4S linkers, and a human TGF-β3 (hTGF-β3) sequence with a C77S substitution.

[0038] [Figure 7B]

[0039] Figure 1 shows the aa sequence of a representative masked TGF-β (construct number: 3334) SEQ ID NO: 147, which has the overall structure of Structure B in Figure 1. The polypeptide that forms a homodimer contains, from N- to C-terminus, hIL2 with H16T and F42A substitutions, three G4S repeats, human IgG1Fc with LALA substitutions, two repeats of G5S and G4S linkers, hTβRIIΔ25, D118A, five G4S repeats, and an hTGF-β3 sequence.

[0039] [Figure 7C]

[0039] Figure 1 shows the aa sequences of representative masked TGF-β constructs having the overall structure of Structure D in Figure 1, including first and second polypeptides. The first polypeptide construct number: 3618 (SEQ ID NO: 148) comprises, from N- to C-terminus, wild-type hIL-2, three repeats of the G4S linker, human IgG1Fc knob-in-hole (KiH) polypeptide chain A with a LALA substitution, five repeats of the G4S linker sequence, and an hTGF-β3 sequence with a C77S substitution. The second polypeptide (construct number: 3619) SEQ ID NO: 149 comprises, from N- to C-terminus, wild-type hIL-2, three repeats of the G4S linker, human IgG1Fc KiH polypeptide chain B with a LALA substitution, two repeats of the G5S linker and the G4S linker, and an hTβRIIΔ25, D118A sequence.

[0040] [Figure 7D]

[0039] Figure 1 shows the aa sequences of a representative masked TGF-β construct having the overall structure of Structure E in Figure 1, including first and second polypeptides. The first polypeptide (construct number: 3618) SEQ ID NO: 150 described above comprises, from N- to C-terminus, wild-type hIL-2, three repeats of a G4S linker, a human IgG1Fc knob-in-hole (KiH) polypeptide chain A with an LALA substitution, five repeats of a G4S linker sequence, and an hTGF-β3 sequence with a C77S substitution. The second polypeptide (construct number: 3855) SEQ ID NO: 151 comprises, from N- to C-terminus, a human IgG1Fc KiH polypeptide chain B with LALA, T366S, L368A, and Y407V substitutions, three repeats of a G4S linker, and an hTβRIIΔ25, D118A sequence.

[0041] [Figure 7E]

[0039] Figure 1 shows the aa sequences of representative masked TGF-β constructs having the overall structure of Structure F in Figure 1, including first and second polypeptides. The first polypeptide (Construct No.: 3891) SEQ ID NO: 152 comprises, from N- to C-terminus, hIL-2 with H16A, F42A, three repeats of a G4S linker sequence, a human IgG1Fc knob-in-hole (KiH) polypeptide chain A with a LALA and T366W substitution, and hTGF-β3 with two repeats of a G5S and G4S linker, hTβRIIΔ25, D118A, five repeats of a G4S linker, and a C77S substitution. The second polypeptide (Construct No.: 3664) SEQ ID NO: 153 comprises, from N- to C-terminus, a human IgG1Fc KiH polypeptide chain B with a LALA substitution.

[0042] [Figure 7F]

[0039] Figure 1 shows the aa sequences of representative masked TGF-β constructs having the overall structure of Structure F in Figure 1, including first and second polypeptides. The first polypeptide (construct number: 3715) SEQ ID NO: 155 comprises, from N- to C-terminus, human IgG1FcKiH polypeptide chain A with a LALA substitution, three repeats of a G4S linker, and a human wtIL2 sequence. The second polypeptide (construct number: 3714) SEQ ID NO: 156 comprises, from N- to C-terminus, human IgG1FcKiH polypeptide chain B with a LALA substitution, three repeats of a G4S linker, five repeats of the hTβRIIΔ25 (with D32N and D118A substitutions) G4S linker sequence, and an hTGF-β3 sequence with a C77S substitution.

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

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

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

[0046] [Figure 7I]

[0039] Figure 1 shows the aa sequence of SEQ ID NO: 159 of a representative masked TGF-β construct (construct number: 3468), having the overall structure of Structure A in Figure 1. The polypeptide comprises, from N- to C-terminus, hIL2 with H16T and F42A substitutions, three repeats of G4S linkers, a human mono IgGFc with a LALA substitution, a human TβRII (hTβRII) Δ25 sequence with three repeats of G4S linkers, D32N and D118A substitutions, five repeats of G4S linkers, and a human TGF-β3 (hTGF-β3) sequence with a C77S substitution.

[0047] [Figure 7J]

[0039] Figure 1 shows the aa sequences of representative masked TGF-β constructs having the overall structure of Structure D in Figure 1, including first and second polypeptides. The first polypeptide (Construct No. 3618) SEQ ID NO: 148 comprises, from N- to C-terminus, wild-type hIL-2, three repeats of the G4S linker, human IgG1Fc knob-in-hole (KiH) polypeptide chain A with a LALA substitution, five repeats of the G4S linker sequence, and an hTGF-β3 sequence with a C77S substitution. The second polypeptide (Construct No. 3621) SEQ ID NO: 160 comprises, from N- to C-terminus, wild-type hIL-2, three repeats of the G4S linker, human IgG1Fc KiH polypeptide chain B with a LALA substitution, three repeats of the G4S linker, and an hTβRIIΔ25 sequence with D32N and D118A substitutions.

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

[0049] [Figure 9] The effect of various aa substitutions in the mature TβRII polypeptide sequence (see Figure 5B) on the affinity for TβRII and TGF-β3 or TGF-β1 is shown (table, top left). Figure 9 also shows the effect of these three substitutions, E55A, D32N, and S52L, on the binding interaction between a TβRII-masked TGF-β construct and an immobilized TβRII-Fc receptor construct. The receptor construct is shown in a capture assay format with TGF-β3 in the top right of the figure, where mono-Fc is an Ig scaffold that does not form reciprocal specific binding to other scaffolds, and anti-IL-2 is an antibody against IL-2 labeled with biotin (B). Strep and HRP are streptavidin and horseradish peroxidase used for detection. The effect on binding was detected in the capture as binding curves generated for each of the four masked constructs (bottom). See Example 3.

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

[0051] [Figure 11]

[0039] Figure 1 shows the results of an experiment using PSM-4033-4039 to induce Foxp3+ iTregs from human peripheral naive CD4+ T cells. See Example 4.

[0052] [Figure 12]

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

[0053] [Figure 13A]

[0039] Figure 1 shows the results of an experiment using PSM-4033-4039 to induce Foxp3+ iTreg expression from human peripheral CD4+ T cells, including naive and memory CD4+ T cells. See Example 4. [Figure 13B]

[0039] Figure 1 shows the results of an experiment using PSM-4033-4039 to induce Foxp3+ iTreg expression from human peripheral CD4+ T cells, including naive and memory CD4+ T cells. See Example 4.

[0054] [Figure 14A]

[0039] Figure 1 shows the results of an experiment using PSM-4033-4039 to induce Foxp3+ iTregs from CD4+ T cells activated by an allogeneic lymphocyte response. See Example 4. [Figure 14B]

[0039] Figure 1 shows the results of an experiment using PSM-4033-4039 to induce Foxp3+ iTregs from CD4+ T cells activated by an allogeneic lymphocyte response. See Example 4.

[0055] [Figure 15]

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

[0056] IV. Detailed Description A.Definition As used herein, an amino acid (single unless the context allows for a plural) abbreviated as "aa" for the number form and "aas" for the plural) is a translational Naturally occurring proteinaceous substances that are incorporated into polypeptides and proteins during translation Unless otherwise specified, L (Leu, leucine), A (Ala, alanine), G ( Gly (glycine), S (Ser, serine), V (Val, valine), F (Phe, phenylalanine), Y (Tyr , tyrosine), H (His, histidine), R (Arg, arginine), N (Asn, asparagine), E (Glu , glutamic acid), D (Asp, asparagine), C (Cys, cysteine), Q (Gln, glutamine), I (Ile, isoleucine), M (Met, methionine), P (Pro, proline), T (Thr, threonine), K The amino acids are represented by α (Lys, lysine) and β (Trp, tryptophan). Hydroxyproline and selenosides are amino acids found in several proteins present in the body. This also includes stains.

[0057] As used herein, the terms "polypeptide," "polypeptide sequence," and "target" are used interchangeably. "Proteins" are synonyms and refer to the bond between their C-1 carboxyl group and their α-amine. It refers to a sequence of aas linked by peptide bonds to form the backbone of a polypeptide. Thus, the MOD polypeptide sequence, the scaffold polypeptide sequence, the TGF-β polypeptide sequence, and / or a masking polypeptide sequence (e.g., a TGF-β receptor polypeptide sequence or an anti-TG Each polypeptide (e.g., the first polypeptide) containing any one or more of the F-beta polypeptide sequences Polypeptides are those polypeptides that are organized as a single continuous backbone polypeptide chain. Such a polypeptide (e.g., a first polypeptide) may comprise any one or more of the following sequences: tides) are bound by covalent bonds (e.g., disulfide bonds between the side chains of cysteine ​​residues) Furthermore, as used herein, the term "polypeptide" refers to a polypeptide that is linked to another polypeptide. "Protein," "polypeptide," "polypeptide sequence," and "protein" are used to describe the structure of a protein that maintains a desired activity. Insofar as possible, modifications (generally of a conservative nature as known to those skilled in the art) to the native sequence, These modifications include, for example, deletions, additions, and substitutions. or due to accidental events such as mutations in the host producing the protein. It may be an error due to PCR amplification or other recombinant DNA techniques.

[0058] The embodiments, claims, or aspects may be directed to a particular species of mammal (e.g., human or non-human). When targeting a subject, the nucleic acid and polypeptide sequences are limited to sequences derived from those subjects. Unless otherwise specified, proteins (e.g., TGF-β, TβR, immunoglobulins, and MOD) is a human (Homo sapiens) sequence.

[0059] As used herein, "masked" refers to the ability of a molecule (e.g., a masked The polypeptide to be masked or the protein to be masked is preferably a masking molecule (e.g., a polypeptide to be masked or a protein to be masked). polypeptide, protein, or protein fragment) or to a masking molecule This means that the target molecule has the same affinity for the masked molecule. The masked molecule is then used to block the activity of other proteins (e.g., cell surface receptors) that have the same activity. The availability is limited.

[0060] As used herein, the term masked TGF-β construct refers to a TGF-β (e.g., For example, TGF-β1, TGF-β2, or TGF-β) polypeptide sequences and those that bind to TGF-β polypeptides. a masking polypeptide that interacts with or otherwise interacts with a TGF-β polypeptide; Unless otherwise specified, the term "masked" refers to a single polypeptide containing both the sequence and the The TGF-β construct comprises a scaffold polypeptide sequence, and optionally one or more independently selected Contains an immunomodulatory (MOD) polypeptide sequence.

[0061] As used herein, the term masked TGF-β complex refers to a complex comprising two or more Polypeptides (typically arranged as homodimers or heterodimers, but may also be in higher order forms) two polypeptides designated as the first and second polypeptides, which may also be multimers of A masked TGF-β complex refers to a TGF-β (e.g., TGF-β1, TGF-β2, or TGF -β) polypeptide sequence and a TGF-β polypeptide that binds to or otherwise inhibits TGF-β a masking polypeptide sequence that interacts with the polypeptide and, through it, the TGF-β complex; and a scaffold polypeptide comprising a dimerization or multimerization sequence to which the polypeptides of Any one or more TGF-β complex polypeptides may optionally be one or more independently selected The MOD polypeptide sequence is

[0062] The term "masked TGF-β construct or complex" refers to a masked TGF-β This abbreviation is used for construct or masked TGF-β complex. The present invention may be used in "linked TGF-β constructs or complexes."

[0063] The term "masked TGF-β constructs and complexes" refers to "masked TGF-β constructs and complexes." It is an abbreviation for both "masked TGF-β complex" and "masked TGF-β complex."

[0064] As used herein, dimerization and multimerization sequences refer to dimers (e.g., heterodimers). or homodimers), or multimers (homo- or multimers of three, four, five or more polypeptide sequences) allowing the association of polypeptide sequences (e.g., separate polypeptides) as heteromultimers Dimerization and multimerization sequences are polypeptide sequences that associate sequences in a non-covalent manner. In some circumstances, it may be possible to form covalent conjugates (e.g., polypeptides). (disulfide bond formation between amino acids).

[0065] The mutually specific binding sequences allow for asymmetric pairing of the polypeptides (heterodimerization). Mutual specific binding sequences are dimerization sequences that form homodimers (as opposed to homodimers). Their counterpart mutually specific binding sequence(s), i.e., their cognate Favors heterodimer formation with binding partners. Key-in-hole (or key-into-hole) A pair of Fc polypeptides is an example of a mutually specific binding sequence and its counterpart mutually specific binding sequence. Represents.

[0066] As used herein, Nanobodies® or nanobodies refer to whole antibodies as well as to antibodies. , consisting of a single monomeric antibody variable domain capable of selectively binding to a specific antigen It means an antibody fragment.

[0067] Wild-type (abbreviated as "wt") with respect to an aa sequence means that, as understood from the context, it is It is unchanged compared to the sequence found in nature (without any substitutions, deletions, a naturally occurring aa sequence or a contiguous portion of a naturally occurring aa sequence (without any insertions or fragments) Certain naturally occurring sequences may be designated as wt. sequences for reference. .

[0068] As used herein, "T cells" refers to, for example, helper T cells (CD4+ cells), cells All CD3-expressing cells, such as cytotoxic T cells (CD8+ cells), regulatory T cells (Treg), and NK-T cells, These include types of immune cells.

[0069] The term "binding," as used herein, refers to a non-covalent interaction between two molecules. For example, this refers to a non-covalent interaction between a MOD and its co-MOD. For example, between two molecules due to electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions. This refers to direct association, and includes interactions such as salt bridges and water bridges. Non-covalent interactions are typically Always, 10 -6 Under M, 10 -7 Under M, 10-8 Under M, 10 -9 Under M, 10 -10 Under M, 10 -11 Under M, 10 -12 M not yet Full, 10 -13 Under M, 10 -14 Less than M or 10 -15 A dissociation constant (K D ) is characterized by "specific binding." "Synchronization" generally refers to at least about 10 -7 M or more, e.g., 5 x 10 -7 M, 10 -8 M, 5 x 10 -8 M, 10 -9 "Non-specific binding" generally refers to binding with an affinity of about 10 M or greater. -7 Less than M Binding affinity (e.g., 10 -6 M, 10 -5 M, 10 -4 M affinity) (e.g., ligand binding) As used herein, the term "ligand binding" refers to the binding of a ligand to a component other than its designated binding site or receptor. When used in a context, "covalent binding" or "covalent bond" is , means that one or more covalent chemical bonds are formed between two different molecules.

[0070] "Affinity" refers to the strength of non-covalent binding; a high binding affinity has a low K D It correlates with As used herein, the term "affinity" refers to the affinity of two agents (e.g., an antibody and an antigen). It means the equilibrium constant in reverse binding, and also the dissociation constant (K D )

[0071] As used herein, the term "immunomodulating polypeptide" or MOD refers to a polypeptide that is expressed on T cells. specifically binds to its cognate co-immunomodulatory polypeptide ("co-MOD"), thereby transmitting a signal a polypeptide on an antigen-presenting cell (APC) (e.g., a dendritic cell, a B cell, etc.), or Examples of polypeptides on APCs include interleukins such as IL-2 and the like. Binding of a fragment of IL-2 (MOD) to the cell surface IL-2 receptor (co-MOD) provides a signal to the cell. MODs include, but are not limited to, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, and IL-21. and IL-23, PD-L1, and Fas ligand (FAS-L). MODs are, among others, expressed on T cells. Antibodies or antibodies that specifically bind to the co-MOD molecules present and mediate co-MOD signaling. As discussed herein, MODs also encompass antibody sequences (e.g., nanobodies). , a mutation of wt. MOD, such as a mutant MOD that has reduced binding affinity for co-MOD. Such a decrease in affinity can take several forms. For example, A heterologous IL-2 MOD has affinity for one or more of the α chain, β chain, and / or γ chain of the IL-2R. As discussed herein, mutant IL-2 MOD containing mutations at positions 16 and 42 may IL-2R α-chain and exhibits reduced affinity for the β-chain of IL-2R. In addition, MODs with two or more co-MODs (e.g., CD80 is associated with both CD28 and CTLA-4) can be used. In the case of a mutant MOD, the mutant MOD has a reduced affinity for one co-MOD and the other For purposes of this disclosure, TGF- β (eg, TGF-β1, TGF-β2, or TGF-β3), and fragments thereof, are not considered MODs.

[0072] Unless otherwise indicated, the term "substantially" includes "completely" and "largely but completely." For example, the term "antibody that does not substantially bind to the α chain of IL-2R" is intended to encompass both "antibody that does not substantially bind to the α chain of IL-2R" and "antibody that does not substantially bind to the α chain of IL-2R." The mutant IL-2 MODs not shown either do not bind to the α chain of IL-2R at all or only to the α chain of IL-2R. It is an IL-2 mutant MOD that does not bind to IL-2.

[0073] As used herein, the term "in vivo" refers to a gene that is produced within the body of, for example, an autoimmune patient. This refers to any process or procedure that involves

[0074] As used herein, "in vitro" includes procedures that may be referred to as ex vivo. refers to any process or procedure that occurs outside the body.

[0075] As used herein to describe the arrangement of MOD polypeptide sequences, "tandem" refers to " means that two or more MODs are adjacent to each other and at most separated by a linker. isolated (e.g., without an intervening scaffold, masking polypeptide, or TGF-β sequence) and This means that it is located on the polypeptide.

[0076] As used herein, the term "ectodomain" refers to a domain that extends into the extracellular space and transmembrane. The part of the membrane protein that anchors it in the cell membrane (the domain) does not contain a sufficient portion of the transmembrane domain. Main).

[0077] As used herein, "sequence identity" refers to the difference between two polynucleotide sequences or is a measure of aa or nucleotide identity between polypeptide sequences of a protein or A polynucleotide sequence is a specific percentage of another polynucleotide or polypeptide. The term "sequence identity" refers to the difference between two sequences when compared by alignment. This means that the percentage of bases or amino acids are identical and in the same relative positions. Sequence identity can be measured using a number of different methods. There are a variety of convenient methods and sites available on the World Wide Web to measure using a computer program (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.) , sequences can be aligned using the ncbi.nlm.nili.gov / BLAST and ebi.ac.uk / Too ls / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / See, e.g., Altschul et al., (1990), J. Mol. Biol. 215:403-10. Unless otherwise stated, sequence identities are based on the NCBI BLAST algorithm released on April 1, 2019. The algorithm, version BLAST+ 2.9.0 (BLASTP 2.9.0+ for proteins and B for nucleic acids) This is determined using alignments performed in LASTN 2.9.0+).

[0078] As used herein, "recombinant" means that a particular nucleic acid (DNA or RNA) is not present in a natural system. Constructs having structural coding or non-coding sequences that can be distinguished from the endogenous nucleic acid they contain. The resulting cloning step, restriction step, polymerase chain reaction (PCR) step and / or or the product of various combinations of ligation steps. The DNA sequence encoding the polypeptide is amplified from a cDNA fragment or a series of synthetic oligonucleotides. Assembled into recombinant transcripts contained within cells or cell-free transcription and translation systems. The nucleic acid may be expressed from a peptide, polynucleotide, or protein. Recombinant as used with respect to qualities means that they are prepared by expression from recombinant nucleic acid. Indicates that.

[0079] As used herein, the term "about" when used in connection with a quantity means that the amount stated is For example, "about 100" means an amount of 90 to 110. When used in the context of a range, "about" when used in reference to the lower quantity of the range, "About" when used in reference to the upper amount of a range means including an amount 10% below the lower amount. For example, about 100 to about 1000 is within the range. This means that the range is from 90 to 1100.

[0080] As used herein, the terms "treatment," "treating," and the like, broadly refer to the administration of a desired drug. The term "treatment" refers to obtaining a therapeutic effect and / or physiological effect that is effective against a disease or its symptoms. It may be prophylactic in the sense of completely or partially preventing a disease and / or and / or the partial or complete cure of the adverse effects resulting from the disease. As used herein, "treatment" refers to any treatment of a disease or condition in a mammal. (a) Treatment of a patient who is predisposed to, but has not yet been diagnosed with, the disease or condition; (b) preventing the onset of a disease or condition in a subject who has not yet developed the disease or condition; (c) to prevent the progression of the disease; and / or before, during, or after the onset of a disease or injury, i.e., causing the disease to regress. A therapeutic agent (e.g., a masked TGF-β construct or complex) may then be administered. Treatment of ongoing illnesses (which treatment stabilizes or suppresses undesirable clinical symptoms in patients) Such treatment is advantageous in that it is initiated before complete loss of function of the affected tissue occurs. Therapeutic treatment is administered during the symptomatic stage of the disease, and in some instances, after the symptomatic stage of the disease. It will be carried out.

[0081] The terms "individual," "subject," "host," and "patient" are used interchangeably herein. Mammal means any mammalian subject for whom diagnosis, treatment or therapy is desired. Examples include humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, rats, goats, sheep, horses, pigs, etc.), dogs Dogs (e.g., dogs), felines (e.g., cats), etc. (e.g., humans, bovines, dogs, cats, rodents) These include mammals such as cattle, rodents, goats, monkeys, sheep, cattle, horses, lappines, and pigs. It can be obtained.

[0082] As used in this specification and the accompanying embodiments and claims, the singular forms "a," "an," and "an" are used interchangeably. It should be noted that "the" and "the" include plural referents unless the context clearly dictates otherwise. Therefore, for example, a reference to "a Treg" encompasses a plurality of such Tregs, and "TGF-β The term "polypeptide" refers to one or more TGF-β polypeptides and their equivalents known to those skilled in the art. Furthermore, the claims may also include references to any optional element. Note that it can be made to be either inclusive or exclusive. That is, the declaration must be made in accordance with the scope of the claims (e.g., using terms such as "solely" and "only"). The use (inclusion) of such elements in the scope of the claims or their exclusion from the scope of the claims , either as the basis for the former, or as a "no" that excludes any particular element. The invention is intended to serve as a basis for a "definitive" limitation.

[0083] Certain aspects of the present invention are described in the context of individual aspects or embodiments for clarity. The features may also be combined in a single aspect or embodiment comprising the invention as defined in the dependent claims. Conversely, for the sake of brevity, Various features of the invention that are described in the context of a single embodiment may also be described separately. or in any suitable subcombination. Combinations are expressly encompassed by the present invention and each and every combination is treated individually. and are disclosed herein as if expressly disclosed. and all subcombinations of these elements are also expressly encompassed by the present invention. and each and every such subcombination is individually and expressly defined herein. The invention is disclosed herein as if it were disclosed.

[0084] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to any benefit whatsoever from any such publication by virtue of prior invention. Nothing in this document should be construed as an admission that you do not have any prior rights. Dates of publication shown may differ from the actual publication dates, which may need to be independently confirmed. There is a match.

[0085] B. Description 1. Introduction The TGF-β superfamily includes endogenous proteins with growth inhibitory functions. Increased expression of TGF-β or defects in cellular mechanisms that inhibit the action of TGF-β may, among other things, lead to the development of TG The immunosuppressive effect of TGF-β is correlated with the malignancy of many cancers. Dysregulation of TGF-β is also involved in autoimmune diseases. Therefore, it has been the target of numerous studies, and both TGF-β and its signaling pathways play important roles in inflammatory processes. It is considered a therapeutic target for the treatment of a variety of diseases, including encephalopathy and autoimmune disorders.

[0086] The ability to effectively produce and deliver TGF-β as a therapeutic agent depends on the toxicity of the molecule and the receptors for TGF-β. Due to the complexity of the system, large-scale production of TGF-β in mammalian cell expression systems is complicated. The cytotoxicity of TGF-β is limited by the toxicity of the protein to most mammalian cells. The cells in question are the most robust and commonly used for commercial protein production. Biomolecular phenotypes, such as Chinese hamster ovary (or "CHO") cells, are one of the cells that can be used to The use of TGF-β as a therapeutic agent is also being investigated. This is due to the high pI of TGF-β1, which has a pI of approximately 8.59 (as opposed to the pI of TGF-β3, which is approximately 6.1). The complexity of the drug is its stability / solubility under non-acidic conditions (such acidic conditions are generally In addition, the abundance of high-quality parenteral proteins present in mammalian systems is limited. The affinity of TβRIII receptors (e.g., about 5 nM for TGF-β2) is related to the affinity of TβRIII for TGF-β. It represents a significant pharmacodynamic sink compared to TGF-β-based biological activity (around 1-2 μM). Similarly, it recruits TGF-β to the TβRII / TβRI complex, limiting the access of targeted agents to target tissues. The role of TβRIII in targeting TGF-β may result in significant off-target delivery of TGF-β, resulting in unintended and unwanted effects. Such off-target delivery can result in undesirable and even toxic effects. This can lead to aberrant activation of TGF-β signaling, especially when TGF-β signaling is under feedforward control. When present, this can result in further production of active TGF-β, thereby resulting in further unintended See, e.g., Jiang et al., Redox Biol. 2:267-272. (2014).

[0087] The effective use of TGF-β as a therapeutic agent requires the administration of targeted drugs to cells to achieve the results of TGF-β stimulation. This is further complicated by the need to provide additional stimuli. As mentioned above, cytokines , for example, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21 and IL-23, PD-L1 and In particular, Fas ligand (FAS-L) can have a significant effect on the action of TGF-β. Therefore, the delivery of TGF-β and the ability to deliver additional stimuli in the form of cytokines etc. Advantageous for achieving a therapeutic or cell-mediated (e.g., in vitro or in vivo) effect. could be.

[0088] As discussed in more detail below, the present disclosure provides a method for the production of TβRI and TβRII proteins. The use of TGF-β polypeptides capable of interacting with cells and stimulating signal transduction is Advantageously, the TGF-β polypeptide is supported on a scaffold (e.g., an immunoglobulin Fc polypeptide). a TGF-β polypeptide sequence (e.g., one or two polypeptides, such as a tide) constructed around the TGF-β polypeptide sequence (e.g., a polypeptide that binds to and masks the TβRII sequence, which acts as a masking sequence Fusion proteins (single polypeptide chains) or fusion protein complexes (two or more polypeptide chains) also containing It is part of a masked TGF-β construct or complex, which is a polypeptide chain. An example of such a fusion protein or fusion protein complex is shown in Figure 1. When a TGF-β polypeptide interacts with, for example, the TβRIII of a cell, it binds to TGF-β and produces a mass The portion of the fusion protein that binds to TβRIII competes with TβRIII, allowing TGF-β to enter the TβRIII reservoir. In addition, a masking polypeptide for the TGF-β polypeptide is used to prevent it from being trapped. The affinity of can be deliberately altered using aa substitutions, as shown in Figure 9. Controlling the overall potency of the masking polypeptide against its target receptor (e.g., TβRII) TGF-β polypeptides bind to TβRIII primarily as disulfide-linked dimers. Therefore, its interaction with the receptor, which leads to sequestration, inhibits dimerization, as discussed below. or by including aa substitutions that limit dimer stability (e.g., C77S). The interaction between TGF-β fusion proteins or fusion protein complexes and TβRII can be attenuated. The interaction displaces the masking sequence and forms a cell surface-bound TGF-β / TβRII complex. The subsequent recruitment of TβRI to form heterodimeric TGF-β receptors is mediated by Maskin. High-affinity complexes (e.g., picopolypeptide sequences) that bind tightly to TGF-β Therefore, the masked TGF-β still , binds to the heterodimeric TβRI / TβRII receptor complex and mediates the classical and non-classical Smad protein pathways. It can signal through the mitochondrial Jun kinase pathway and the p38 signaling pathway. In effect, the masking polypeptide delivers TGF-β into cells and binds it to the cell surface TβRII component. The TβRI protein is then recruited to target the TGF-β polypeptide to the appropriate location. effectively maintains the formation of functional and active signaling complexes.

[0089] TGF-β binding proteins (e.g., TGF-β binding proteins containing all or part of their TGF-β binding ectodomains) TGF-β, masked by the TGF-β receptor fragment, is released into the body via unwanted off-target pathways. In addition to delivering the TGF-β in a form that avoids interactions, masked TGF-β constructs or complexes may also be used. The body produces immunomodulatory substances ("M") that can affect the outcome of TGF-β action on target cells. The TGF-β and TGF-β polypeptides may also contain one or more polypeptides that function as TGF-β and TGF-β receptors (e.g., TGF-β receptors ... and TGF-β receptors). The ability to deliver both immunomodulators together results in the action of TGF-β-activating signals. Not only does this allow for the same effect on target cells compared to the administration of an immunomodulator alone, This reduces the amount of immunomodulator required to produce the desired effect on the same target cells. It is obtained by having two polypeptide sequences that have affinity for the receptor (A This is the result of increased affinity (due to enhanced vidity). Masking that carries more than one MOD and a TGF-β construct or complex against both a TGF-β polypeptide and one or more MODs. Enhanced avidity between receptor-bearing target cells (MOD interactions with their receptors) (as a result of the increase in the free energy of binding, ΔG) is due to the masked TGF-β structure. Even when the construct or complex is present in less than saturating amounts, target cells that have both types of receptors are still viable. For example, structure A of FIG. 1 having IL-2 MOD or Masked TGF-β constructs such as those in FIG. 7G react with equivalent (equimolar) amounts of IL-2 MOD polypeptide. Even in the presence of IL-2, the TGF-β expression was significantly reduced compared to an otherwise identical masked TGF-β construct lacking IL-2 MOD. and is a more potent inducer of iTReg differentiation of naive CD4+ cells. For example, when similar doses and cell types are present, the results are similar for the IL-2 MOD-containing cells shown in Figure 7G. Constructs such as Figure 1 Structure A show increased TGF-β and IL-2 receptor activity compared to constructs lacking IL-2 MOD. The antibody selectively binds to cells that have both the

[0090] The action of the masked TGF-β construct or complex is not dependent on the action of the individual polypeptide sequences. It can be further specified by the incorporation of altering modifications. Incorporating aa substitutions that alter the dimerization ability of TGF-β (e.g., C77S substitution in TGF-β3) The monomeric form of TGF-β shows little affinity for TβRIII, but the TGF-β Incorporation of mutations that restrict the ability of TβRIII to dimerize relieves the restriction on target binding to TβRIII. This allows the complex to be drawn into the TGF-β "reservoir" and stimulates target cells. This can limit the ability of the

[0091] A TGF-β polypeptide comprising a masking polypeptide sequence and an immunomodulatory polypeptide sequence. Modifications (e.g., substitutions, deletions, insertions, etc.) to polypeptide sequences other than the peptide sequence may also be made. It can be done.

[0092] In addition to modifying the TGF-β polypeptide sequence, polypeptides that bind and mask TGF-β may also be used. Modifications to the polypeptide can be made to mask the TGF-β. The affinity of the peptide and the breathing rate of the masking polypeptide and TGF-β polypeptide TGF-β polypeptide sequences can be used to regulate the activity of TGF-β by altering the breathing rate (on and off rates). The availability of two different TGF-β polypeptides / masking Polypeptide complexes may have the same binding association constant (ratio of k to k), but with higher k and Complexes with koff and koc are expressed in a cell according to rate constants, particularly the koff rate. Figure 9 shows that TGF-β1 and TGF-β2 are more effectively available for binding to surface TβRII. A list of some aa substitutions in TβRII that alter the affinity for TGF-β3 is provided below. None or one of three substitutions, each with a different effect on TβRII binding to β3 1 shows a graph depicting the affinity of an identical TβRII-masked TGF-β construct, except that it has vinegar.

[0093] Various polypeptides are available to produce both single chain antibody sequences (e.g., humanized single chain antibody sequences). Although it is possible to mask TGF-β containing TGF-β, it is possible to mask the minimal TGF-β that binds to TGF-β. β receptor sequences (e.g., the ectodomains of TβRI, TβRII, or TβRIII) may be used. The ectodomain of TGF-β III can be used as a masking polypeptide for dimeric TGF-β. Its high affinity for TGF-β allows the binding of the TGF-β polypeptide sequence to TβRII to an antagonist. Nevertheless, the TβRIII masking sequence may be involved in the TGF-β polypeptide. This is effectively replaced by cell surface TβRI / TβRII complexes that have a higher affinity for TβRI. can be obtained, thereby allowing activation of their cell surface receptors.

[0094] The ectodomain of TβRII can be used as a masking polypeptide. Deletion of terminal amino acids (e.g., delta 14 or 25) in the presence or absence of the D118A substitution , suitable for masking TGF-β with a calculated pI of about 4.5 to about 5.0 (e.g., about 4.7 to 4.85). Proteins (or polypeptides) can be produced. TGF-β1 polypeptide (high pI value) and a TβRII masking polypeptide (including, for example, one having an N-terminal deletion). The combination can potentially neutralize the TGF-β1 polypeptide charge. The calculated pI of TGFβ1 (Ta25, D118A) / TGFβ1 is approximately 6.23, while TGFβ1 has a pI of approximately 8.59. Specifically, complexes of Delta14 or Delta25 TβRII and TGF-β3 (with or without D118A and / or C77S substitutions) The TβRII (regardless of the TβRII) has a calculated pI of about 4.9 to about 5.3 (pI of about 5.06 to 5.17). By combining the ectodomain with an active TGF-β polypeptide, it interacts with TβRI on the cell surface. This results in a complex that can interact with TGF-β, thereby affecting TGF-β signaling. can be used as an agonist, partial agonist, antagonist, or partial antagonist. Although this aa substitution limits the interaction with TβRI, and limiting or blocking the ability of the masked complex to participate in signal transduction activation. Therefore, as discussed below, N-terminal deletions ( For example, deletion of 14 to 25 aa, Δ14 to Δ25) or substitution (for example, D118A, D118R, etc. at D118) Incorporation of TβRII ectodomain sequences with any non-aspartic acid aa substitution TGF-β was masked using TβRII ectoprotein, which leaves the N-terminus of TβRII intact. Domain-masked TGF-β polypeptides can prevent cell stimulation. Therefore, the masked TGF-β complex contains TGF-β with an N-terminal deletion mutant of TβRII. This includes those masked by variants, and unmasking TGF-β (masking pane) The peptides bind to TβRII and TβRI in the target cells. It acts by combining with TβRI to form the active heterodimeric TβRI / TβRII signaling complex. It is possible.

[0095] Masking of TGF-β is a promising strategy for mammalian cells (e.g., C cells) without reducing cell viability. This allows for high levels of expression in HO cells, particularly in the presence of masked TGF-β. The Tβ polypeptide blocks binding to TβRI by N-terminal aa deletions, substitutions, and / or other mutations. This is the case when blocking TβRIII interactions (e.g., by blocking dimerization) Thus, the problems associated with cell expression can be further reduced.

[0096] C. Masked TGF-β constructs and masked TGF-β complexes The present disclosure provides masked TGF-β constructs (e.g., FIG. 1, having a single polypeptide chain). (see structure A) and the masked TGF-β complex (e.g., Figure 1, two polypeptide chains The preparation of masked TGF-β constructs and The masked and unmasked TGF-β complexes contain at least one TGF-β as their component. a polypeptide sequence and at least one polypeptide sequence that binds to and masks the TGF-β polypeptide(s); One polypeptide and, optionally, one or more (e.g., one, two, or three) immunomodulatory polypeptides. The masked TGF-β receptor (TGF-β) is a soluble form of TGF-β receptor agonist (TGF-β), which is a soluble form of TGF-β receptor agonist (TGF-β). The TGF-β construct and the masked TGF-β complex may be a portion of a membrane-bound protein (e.g., TGF- β receptor), but unless otherwise noted, they do not include a membrane anchor domain (e.g., expression The majority of the expressed protein is anchored to the cell membrane (e.g., of the expressed CHO cells). does not contain any part of the transmembrane domain.

[0097] Non-limiting examples of TGF-β constructs and complexes are described below, including those in the form shown in FIG. do.

[0098] MOD, scaffold, linker, TGF-β polypeptide and TGF-β masking polypeptide (e.g., , single chain antibody or TGF-β receptor ectodomain) and the components of the complex are described in the following sections, respectively.

[0099] D. Immunomodulatory Polypeptide Sequences ("MODs") 1. Masked TGF-β Constructs and Incorporation of MOD into Complexes As discussed above, immunomodulatory polypeptides (MODs) are polypeptides that act as masked TGF-β polypeptides. Delivery of peptides or other molecules that activate cells via the TβRI and TβRII heterodimeric receptors. Although not required for TGF-β activity, MOD substantially influences the outcome of TGF-β receptor activation. Thus, wild-type (wt.) or mutant MOD (e.g., "co-MOD," "coimmune Regulatory polypeptides" or cognate costimulatory receptors or their subtypes, Incorporating a compound that exhibits reduced affinity, increased affinity, or selectivity for a particular receptor It may be desirable to include TGF-β, which is also an immunomodulatory polypeptide, as described herein. As such, it is a central element in the masked TGF-β constructs and complexes described herein. As used herein, the term "MOD(s)" does not include TGF-β or its polypeptides. .

[0100] Masked TGF-β constructs and complexes (e.g., homodimeric or heterodimeric complexes) Suitable MODs for inclusion in either the PD-L1 or PD-L2 receptor agonist (e.g., PD-L1, FAS-L, IL-1, IL-2, IL-4, IL-6, These include, but are not limited to, IL-7, IL-10, IL-15, IL-21 and IL-23.

[0101] In some instances, the MOD may be a PD-L1, FAS-L, IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21 and IL-23 polypeptides, or fragments of any thereof, independently selected from the group consisting of: The masked TGF-β construct and the MOD polypeptide sequence (or sequences) incorporated into the complex are The MOD polypeptide fragments (possibly several fragments) may be expressed as either the secreted portion of the MOD polypeptide or the full length if it is membrane-anchored. It may include the extracellular portion of the mature MOD protein. Thus, for example, masking The MOD polypeptide sequence within the resulting TGF-β construct or complex may, in some instances, be The signal peptide, transmembrane domain, and / or intracellular domain typically found in native MODs One or more (e.g., each) of the options can be excluded.

[0102] In some instances, suitable for inclusion in the masked TGF-β constructs and complexes of the present disclosure Suitable MOD polypeptide sequences include all or part of the amino acid sequence of a naturally occurring MOD (e.g., , extracellular portion). In other examples, the masked TGF-β constructs and complexes of the present disclosure Suitable MODs for inclusion in the method include those that have at least one amino acid sequence that is at least as strong as that of a naturally occurring MOD. At least one amino acid insertion, substitution, and / or deletion (e.g., one , two, or three independently selected) mutant MODs.

[0103] In some instances, the mutant MOD may be a counterpart of the native MOD (e.g., a mutant MOD) to the co-MOD. The binding affinity to the co-MOD is lower than that of the MOD that does not contain the amino acid substitution(s) present in the co-MOD. As the affinity decreases, the affinity for those co-MODs also decreases. The use of a MOD allows the TGF-β polypeptide to have more influence on the binding interaction. or to make it predominant. The binding affinity of the TGF-β polypeptide is that of MOD. If the TGF-β receptor signal is higher than 100 kJ / s, the masked TGF-β construct or complex is activated to activate the TGF-β receptor system. Cells with TGF-βR1 and TGF-βR2 that form high-affinity TGF-β binding heterodimers 2) can associate with TGF-β receptors, even in the presence of sufficient co-MOD. This can restrict target binding to cells that lack or have little of the Qualitatively, the affinity (e.g., ΔG of binding) of a TGF-β polypeptide for its cellular receptor is If the affinity (e.g., ΔG of binding) of MODs for their co-MODs is greater than This influences the binding and specificity of the masked TGF-β construct or complex.

[0104] In one embodiment, any one of the masked TGF-β constructs and associated complexes The above MODs were either wild-type or the following mutant forms: PD-L1, FAS-L, IL-1, IL-2, IL-4, IL-6, IL-7, IL-1 0, IL-15, IL-21, IL-23, and combinations thereof. .

[0105] In some instances, such as when it is desirable to stimulate the production of Treg cells, masking The TGF-β construct and at least one MOD polypeptide present in the complex (e.g., one , two or three independently selected MODs) are IL-2 polypeptides or IL-2 variant polypeptides. The sequence variation in IL-2 results in the IL-2 polypeptide and the masked TGF-β of the construct or complex to target cells having different combinations of IL-2R receptor subunits. The IL-2 receptors are the common IL-2Rγ and two additional IL-2Rγ receptors. It is composed of IL-2Rα and / or IL-2Rβ subunits, and is a trimeric (IL-2Rα)2-IL-2Rγ, (IL-2R β)2-IL-2Rγ, or high affinity (Kd approx. 10 picomolar) IL-2Rα-IL-2Rβ-IL-2Rγ receptors. The α chain (CD25) is unique to IL-2, whereas the β chain (CD122) is shared with the IL-15 receptor and mediates signal transduction. The gamma chain (CD132), which is important for signaling, can cooperate with other cytokine receptor chains. Substitutions at H16A or H16T) or F42 (e.g., F42A or F42T) enhance the IL-2Rβ subunit This can bias binding in favor of receptors with , memory T cells and NK cells that present the β-γ receptor ((IL-2Rβ)2-IL-2Rγ), or high-affinity To activated T cells and Treg cells that present α-β-γ (IL-2Rα-IL-2Rβ-IL-2Rγ) receptors In contrast, substitution at N88 (e.g., N88R) reduces binding to IL-2Rβ. This can bias binding in favor of receptors with the IL-2Rα subunit, thus Therefore, the substitution at N88 is α-γ ((IL-2Rα)2-IL-2Rγ) and α-β-γ (IL-2Rα-IL-2Rβ-IL-2Rγ ) receptor-bearing cells, while simultaneously inhibiting the binding and Avoid activation. See, e.g., Skrombolas and Frelinger, Expert Rev Clin Immunol ., 10(2):207-217 (2014). IL-2 polypeptides with substituted MOD (aa substitutions) When used in the context of binding a co-MOD or a cell that displays a co-MOD, The presence of the substitution results in the same c as the substituted MOD compared to the interaction between the wt. MOD and the co-MOD. This means changing the degree of interaction with o-MOD, e.g., a lower side effect profile. N88, which has a morphine-like structure (e.g., is safer) and is better tolerated in human subjects. IL-2 sequences with substitutions at H16 and / or F42 (e.g., N88R) are also suitable for use with the above-mentioned substitutions at H16 and / or F42. may be included in

[0106] When it is desirable to stimulate the production of iTreg cells (CD4+FoxP3+ cells) (e.g., effector actively suppress T cells and / or promote peripheral tolerance to inhibit immune-mediated tissue damage In some instances, such as when it is desired to induce TGF-β expression, a masked TGF-β construct may be used. At least one MOD polypeptide (e.g., one, two, or three) present in the product or complex Independently selected MODs (i.e., independently selected wild-type or mutant PD-L1 MOD polypeptides) are See, for example, Francisco et al., J. Exp. Med., 206(13):3015-3029 (2009). In addition to the wt. or mutant PD-L1 sequence, the masked TGF-β construct or complex , can comprise one or more independently selected wt. or mutant IL-2 polypeptides. Sequence variations in IL-2 allow target cells with different combinations of IL-2R receptor subunits to differentiate. Selected to bias binding of IL-2 polypeptide and masked TGF-β construct to As discussed above, IL-2 mutants can bind to receptors having the IL-2Rβ subunit. H16 (e.g., H16A or H16T) and / or F42 (e.g., , F42A, or F42T); and / or a decrease in binding to IL-2Rβ is more pronounced in human subjects. IL-2 containing a substitution at N88 (e.g., N88R), which is well tolerated. In this study, the masked TGF-β construct / TGF-β complex contained both the H16T and F42A substitutions, or the H Contains both 16A and F42 substitutions, either of which pair is combined with an N88 substitution (e.g., N88R). It can be combined.

[0107] In some instances, such as when it is desirable to stimulate the production of Th17 cells, At least one MOD polypeptide (e.g., one, two, or three MOD polypeptides) present in the TGF-β construct and complex One or three independently selected MODs are selected from the group consisting of IL-6 polypeptides and IL-6 variant polypeptides. It's Chido.

[0108] In some instances, such as when it is desirable to stimulate the production of Th9 cells, At least one MOD polypeptide (e.g., one, two, or three MOD polypeptides) present in the TGF-β construct and complex One or three independently selected MODs are selected from the group consisting of IL-4 polypeptides and IL-4 variant polypeptides. For example, see Elyaman et al., Immunity., 36(4):623-634, Immunity. (2012) Please refer to.

[0109] Enhancement of IL-7-dependent survival of low-affinity T cells by regulating thymocyte IL-7Ra expression In some instances, such as when it is desirable to have a masked TGF-β construct or complex, at least one MOD polypeptide (e.g., one, two, or three independently selected polypeptides) present in the The IL-7 polypeptide (MOD) is an IL-7 polypeptide or an IL-7 mutant polypeptide.

[0110] In some instances, such as when it is desirable to stimulate the production of T follicular helper (Tfh) cells, Masked TGF-β constructs and the small amount of TGF-β present in the complex At least one MOD polypeptide (e.g., one, two, or three independently selected MODs) , IL-21 or IL-23 polypeptide, or a mutant IL-21 or IL-23 polypeptide. do.

[0111] In some instances, such as when it is desirable to induce tolerance, at least one MOD polynucleotide may be used. Peptides (e.g., one, two, or three independently selected MODs) are masked Within the TGF-β construct and / or variants of the Fas Ligand (FasL) polypeptide.

[0112] Inhibits type II innate lymphoid cells (ILC2 cells) (e.g., suppresses asthma and allergic inflammation) In some instances, a masked TGF-β construct may be desirable, e.g., to suppress TGF-β expression. At least one MOD polypeptide (e.g., one, two, or three) present in the product or complex The two independently selected MODs are an IL-10 polypeptide or a mutant IL-10 polypeptide. For example, see Rajas et al., J Allergy Clin Immunol, 139(5):1468(2017), and Ogasaw See Ara et al., J Allergy Clin Immunol, 141(3):1147-1151 (2018). ILC2 Inhibition of cells is achieved by type 2 cytokines in vivo (in tissues or body fluids) or in vitro (in culture medium). The IL-10 polypeptide can be assessed by a decrease in the production of the cytokines IL-5 and IL-13. IL-10M1 molecule, as described in Phinson et al., J. Biol. Chem. 275:13552-13557 (2000) or variants thereof, both of which are discussed below. The most active form is the intertwined IL-10 peptide pair, which binds two IL-10 molecules and four IL-10Ra receptors. In contrast to wild-type IL-10, which is formed in a complex with receptor chains, IL-10M1 is a soluble IL-1 It forms a 1:1 complex with Ra, with a dissociation constant of 30 nm, and is biocompatible in cell proliferation assays. It is biologically active (ibid.).

[0113] 2. Low-affinity mods and variant mods A preferred MOD that exhibits low affinity for its co-MOD has a 1 amino acid sequence relative to the wild-type MOD sequence. For example, in some cases, the mutations can be masked. The mutant MOD polypeptide present in the TGF-β construct complex has the amino acid sequence , 1aa, 2aa, 3aa, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, or As another example, in some instances, a masked TGF-β construct or complex may be used. Mutant MOD polypeptides present in the body are identical in amino acid sequence to the corresponding wild-type MOD polypeptides. Compared to the column, it differs by 11aa, 12aa, 13aa, 14aa, 15aa, 16aa, 17aa, 18aa, 19aa, or 20aa For example, in some instances, the TGF-β construct or complex may be present in a masked form. A mutant MOD polypeptide may have one, two, three or more amino acids, or more amino acids, as compared to a corresponding reference (e.g., wild-type) MOD sequence. In some instances, the masked TGF- The mutant MOD present in the β construct or complex is identical to the corresponding reference (e.g., wild-type) MOD sequence. In some instances, the masked TGF-β construct or multiple Mutant MODs present within a conjugate have two distinct sequences, one of which is a nucleotide sequence, compared to the corresponding reference (e.g., wild-type) MOD sequence. In some examples, the masked TGF-β The mutant MOD present in the construct or complex is compared to the corresponding reference (e.g., wild-type) MOD sequence. In some instances, the masked The mutant MOD present in a selected TGF-β construct or complex is expressed as a mutant MOD relative to the corresponding reference (e.g., wild-type) MOD. In some instances, the masked TGF- The mutant MOD present in the β construct or complex is identical to the corresponding reference (e.g., wild-type) MOD sequence. In some instances, the masked TGF-β construct contains 6 or 7 aa substitutions. or mutant MOD present in the complex, compared to the corresponding reference (e.g., wild-type) MOD sequence. In some instances, the masked TGF-β construct or complex comprises a substitution of 8 or 9 aa. Mutant MODs present in the body have 10 or more amino acid sequences that differ in length from the corresponding reference (e.g., wild-type) MOD sequence. contains 11 aa substitutions (eg, 10 or fewer aa substitutions).

[0114] In some instances, the mutant MOD present in the masked TGF-β construct or complex , which contain 11 or 12 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD sequence. In some instances, mutant MOD present in a masked TGF-β construct or complex may Contains 13 or 14 aa substitutions compared to the corresponding reference (e.g., wild-type) MOD sequence. In this example, the mutant MOD present in the masked TGF-β construct or complex is Some examples include 15 or 16 aa substitutions compared to a reference (e.g., wild-type) MOD sequence. In this study, the mutant MOD present in the masked TGF-β construct or complex was expressed in a manner similar to that of the corresponding reference MOD. It contains 17 or 18 aa substitutions compared to the reference (e.g., wild-type) MOD sequence. , the mutant MOD present in the masked TGF-β construct or complex is shown in the corresponding reference (e.g. For example, it contains 19 or 20 aa substitutions compared to the wild-type MOD sequence.

[0115] As discussed above, suitable for inclusion in a masked TGF-β construct or complex Mutant MODs exhibit increased affinity for their cognate co-MODs compared to the corresponding wild-type MODs. It may exhibit low affinity for co-MOD.

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

[0117] 3. Measurement of Binding Affinity The binding affinity between a MOD (e.g., a MOD polypeptide) and its cognate co-MOD can be determined by measuring the binding affinity of purified MOs. D and purified homologous co-MOD were measured by biolayer interferometry (BLI). A masked TGF-β construct or complex containing MOD and its cognate co-MOD can be used. The binding affinity between purified masked TGF-β constructs or complexes and MOD The same co-MOD can be used to measure the BLI. The BLI method is 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 Dun See Can (2014) J. Vis. Exp. 18:e51383. Between MOD and its cognate co-MOD, or between a masked TGF-β construct or complex with a MOD and its cognate co-MOD; The specific and relative binding affinities described in this disclosure can be measured using the following procedure. This can be done.

[0118] Binding affinity between a masked TGF-β construct or complex containing MOD and the MOD's cognate co-MOD To measure compatibility, an Octet RED96 (Pal ForteBio) instrument or similar instrument was used, as described below. A control masked TGF-β construct can be used to perform the BLI assay. a construct or complex (e.g., a masked TGF-β construct or complex containing wild-type MOD) Immobilized on an insoluble support ("biosensor"). The β construct or complex is the "target." Immobilization involves immobilizing the capture antibody on an insoluble support. The capture antibody can be used to capture the masked TGF-β construct or complex. For example, if the masked TGF-β construct or complex includes an IgFc scaffold, In this case, immobilization is performed by immobilizing an anti-IgFc (e.g., anti-human IgGFc) antibody on an insoluble support. The immobilized anti-IgFc antibody may be used to bind to a masked TGF-β construct or The co-MOD binds to the complex and immobilizes it. The masked TGF-β construct or complex is applied and the device's response is recorded. , 25 mM HEPES (pH 6.8), 5% poly(ethylene glycol) 6000, 50 mM KCl, 0.1% bovine serum Fixation is performed in a liquid medium containing albumin and 0.02% Tween 20 non-ionic surfactant. Binding of co-MOD to the modified masked TGF-β construct or complex is carried out at 30°C.

[0119] Use an antibody (e.g., a monoclonal antibody) as a positive control for binding and binding affinity The antibodies can be based on the specific structure of the masked TGF-β construct or complex. For example, TGF-β, TGF-β receptor, scaffold or The monoclonal antibody (mAb) against the MOD polypeptide sequence is Any substance that does not dissociate the TGF-β construct or complex from the support (biosensor) is suitable. The antibody can be used as a control for the efficacy of anti-MHC class I or class II monoclonal antibodies. A standard curve can be generated using serial dilutions. BLI involves two steps: (i) an immobilized polypeptide (the "target"), and (ii) an internal reference layer. The interference pattern of white light reflected from two surfaces is analyzed. A change in the number of molecules ("analytes"; e.g., co-MOD; anti-HLA antibodies) results in a shift in the interference pattern. This shift in the interference pattern can be measured in real time. Two kinetically related values ​​that describe the affinity of a target / analyte interaction are the association constant (k a ) and dissociation constant (k d ) The ratio of these two values ​​(k d / a ) is the affinity constant K D Give.

[0120] As described above, a MOD (e.g., IL-2 or an IL-2 variant) and its cognate co-MOD (e.g., IL-2R) Measurement of the binding affinity between the Similar to that described above for the masked TGF-β construct or complex. The experiment was carried out using an Octet RED 96 (Pal ForteBio) instrument or a similar instrument, as described below. MOD (e.g., a component of a masked TGF-β construct or complex) can be used. a mutant IL-2 polypeptide of the disclosure), and a control MOD (a control MOD is a wild-type MOD, e.g., , including wild-type IL-2) are separately immobilized on an insoluble support ("biosensor"). The OD is the "target." Immobilization is achieved by immobilizing the capture antibody on an insoluble support. For example, the target can be a target of interest that has been tagged with an immunoaffinity tag (e.g., When the antibody is fused to a soluble antibody (e.g., FLAG, human IgGFc), immobilization can be achieved by attaching the appropriate antibody to an immunoglobulin on an insoluble support. This can be achieved by immobilizing an immunoaffinity tag (e.g., anti-human IgGFc), and the immobilized The antibody binds to and immobilizes the MOD (which comprises an IgFc polypeptide). Several different concentrations of MOD are applied to the immobilized MOD and the response of the device is recorded. , co-MOD (or polypeptide) (e.g., IL-2 receptor heterotrimer, i.e., monomeric subunits) (for units + heterodimer subcomplexes, or complete heterotrimers) biosensors immobilized on a 100-well plate, and MOD was applied at several different concentrations to the immobilized co-MOD(s), The response of the device is recorded. The assay is performed in 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 surfactant Binding of co-MOD to immobilized MOD is performed at 30°C. The molecules are reflected from two surfaces: (i) the immobilized polypeptide (the "target"), and (ii) an internal reference layer. The interference pattern of the white light incident on the biosensor is analyzed. A change in the number of co-modulators (e.g., co-MODs) causes a shift in the interference pattern, This shift in the ΔΨ can be measured in real time. Two kinetically related values ​​that describe affinity are the association constant (k a ) and dissociation constant (k d ) These The ratio of the two values ​​of (k d / a ) is the affinity constant K D The co-MOD of wild-type MOD (e.g., IL-2) (e.g., its cognate binding partner or receptor, in the case of IL-2, IL-2R) and the co-MOD (e.g., IL-2 variants as disclosed herein) of the mutant MOD (e.g., IL-2 variants as disclosed herein). For example, in the case of IL-2 variants, the binding affinity to the IL-2R The relative binding affinity of mutant co-MODs to co-MODs compared with wild-type co-MODs was measured. That is, the binding affinity of a mutant MOD to its co-MOD can be compared with that of the same cognate co-MOD. It can be determined whether the binding affinity of the MOD to the target protein is reduced compared to that of wild-type MOD, and If so, what is the percentage reduction in binding affinity from that of wild-type co-MOD? It is possible.

[0121] The BLI assay is performed in multiwell plates. Define the port layout, define the assay steps, and connect the biosensor to the Octet Data Acq Assign in the installation software. The biosensor assembly is hydrated. The assembled biosensor assembly and assay plate were equilibrated on the Octet instrument for 10 minutes. Once the data is acquired, it is uploaded to the Octet Data Analysis software. The data is loaded after reference subtraction, y-axis alignment, step-to-step correction, and Savitzky-Golay filtering method is specified and processed in the processing window. The data is used to identify the analytical steps (association and dissociation) and to determine the curve fit in the analytical window. Analysis by selecting the fitting model (1:1), fitting method (global), and target window (in seconds) The quality of the fit is evaluated by the K D Values ​​are within a 3-fold range If KD The error value should be within one order of magnitude of the affinity constant value. No. R 2 The value should be greater than 0.95. See, for example, Abdiche et al. (2008) J. Anal. Bi See Ochem.377:209.

[0122] In some instances, (i) a control masked TGF-β construct versus the allogeneic co-MOD or The results of the complex (control masked TGF-β construct or complex containing wild-type MOD) (ii) the masked T containing the mutant form of wild-type MOD against the cognate co-MOD the ratio of the binding affinity of the GF-β construct or complex, as measured by BLI (as described above) , at least 1.5:1, at least 2:1, at least 5:1, at least 10:1, at least 1 5:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1, at least At least 500:1, at least 10 2 :1, at least 5 × 10 2 :1, at least 10 3 :1, at least Also 5×10 3 :1, at least 10 4 :1, at least 10 5 :1, or at least 10 6 :1. In some examples, (i) a control masked TGF-β construct or complex versus the allogeneic co-MOD. The binding parent of the complex (control masked TGF-β construct or complex contains wild-type MOD) (ii) a masked TGF- containing mutant form of wild-type MOD against the homologous co-MOD β construct or complex binding affinity, as measured by BLI, is 1.5:1-10 6 :1 , for example, 1.5:1 to 10:1, 10:1 to 50:1, 50:1 to 10 2 :1, 10 2 :1~10 3 :1, 10 3 :1~10 4 :1, 10 4 :1~10 5 :1 or 10 5 :1~10 6 :1 range.

[0123] In some instances, the mutant MOD present in the masked TGF-β construct or complex , and have a binding affinity of 1 nM to 100 nM or 100 nM to 100 μM to their cognate co-MOD. In some instances, the mutant MOD present in the masked TGF-β construct or complex is Relative to the same co-MOD, approximately 100nM to approximately 200nM, approximately 200nM to approximately 300nM, approximately 300nM to approximately 400nM, approximately 400nM M ~ about 500nM, about 500nM - about 600nM, about 600nM - about 700nM, about 700nM - about 800nM, about 800nM - 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 a binding affinity of 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 instances, the mutation present in the masked TGF-β construct or complex The MODs were approximately 1 nM to 5 nM, approximately 5 nM to 10 nM, approximately 10 nM to 50 nM, and approximately 50 nM relative to the same co-MOD. It has a binding affinity of about 100 nM.

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

[0125] The wild-type human PD-L1 polypeptide may comprise the following amino acid sequence: [ka] In the sequence, aas 1 to 18 form the signal sequence, and aas 19 to 127 form the Ig-like V-type or IgV domain. The residues 133 to 225 form an Ig-like C2 domain.

[0126] The wild-type human PD-L1 ectodomain may comprise the following amino acid sequence: [ka] In the sequence, aas 1 to 109 form an Ig-like V-type or "IgV" domain, and aas 115 to 207 form an Ig-like C2-type domain. Form an in.

[0127] A suitable wild-type PD-L1 IgV domain for use as a MOD is aa18, aas IgV aas of SEQ ID NO: 1. 19-127, and a carboxyl-terminal stabilizing sequence, e.g., SEQ ID NO: 138 [ka] The amino acids may include the last seven amino acids (bold and italicized) of A carboxyl stabilizing sequence is provided at about aa 122 containing a histidine (e.g., shown as aa 117 in SEQ ID NO: 138). In the case of a nucleotide sequence containing approximately 5 histidine residues C-terminal to Tyr (Y), the histidine is located at aas82 and and 83 can form stable electrostatic bonds with the backbone amides (SEQ ID NO: 138 (Q107 and L1 of SEQ ID NO: 1) 06) shown in bold and italics). Alternatively, aas82 or 83) (Q107 and L106 of SEQ ID NO: 1) one of the aa residues 121, 122, or 123 (corresponding to aa positions 139 to 141 of SEQ ID NO: 1) One of these can be substituted to form a stable disulfide bond.

[0128] The wild-type PD-1 polypeptide may comprise the following amino acid sequence: [ka] .

[0129] In some instances, a mutant PD-L1 polypeptide (e.g., SEQ ID NO: 2 or the IgV domain of PD-1) is used. (variant of) a PD-L1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) compared to the binding affinity of For example, in some instances, mutant PD-L1 polypeptides exhibit low binding affinity to the PD-L1 polypeptide. The peptide is a PD-L1 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. at least 10% lower, at least 20% lower, or at least 30% lower than the binding affinity of At least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, At least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3) with binding affinity )

[0130] In some instances, a mutant PD-L1 polypeptide (e.g., SEQ ID NO: 2 or its IgV domain) is used. mutant type) to PD-1 (for example, mutant type of SEQ ID NO: 3) at 1 nM to 1 mM (for example, 1 nM to 10 nM, Binding affinity of 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 another example, in some instances, a mutant PD-L1 polypeptide (e.g., SEQ ID NO: 2) can be used to express PD1 (e.g., a PD1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3). ) about 100nM to about 200nM, about 200nM to about 300nM, about 300nM to about 400nM, about 400nM to about 500nM M, about 500nM to about 600nM, about 600nM to about 700nM, about 700nM to about 800nM, about 800nM to about 900nM, about 900 nM~Approx. 1μM, Approx. 1μM~Approx. 5μM, Approx. 5μM~Approx. 10μM, Approx. 10μM~Approx. 20μM, Approx. 20μM~Approx. 30μM , having a binding affinity of about 30 μM to about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM. .

[0131] For the PD-L1 ectodomain sequences used as MOD, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: aas19 to 127 (IgV domain) of SEQ ID NO: 1 and at least 85 of any one of SEQ ID NO: 138 consecutive aas (e.g., at least 90, at least 95, at least 100, or at least to a sequence with greater than 90% (more than 95%, 98%, or 99%) sequence identity with the Many aa substitutions can be made, including (a) substitutions for disulfide bonds. (b) substitution pairs for salt bridge formation: D103C and G33C, or V104 and S34C. Q107D and K62R, or Q107D and S80R, and / or (c) Pi stacking The PD-L1 MOD sequence includes the sequence M36Y or M36F (based on SEQ ID NO: 1). At least 85 consecutive aas (e.g., at least 90, at least 95, at least a sequence having at least 100, or at least 105 consecutive aas, and at least one disulfide The PD-L1 MOD sequence may include aaas1 of SEQ ID NO: 1. At least 85 consecutive aas (e.g., at least 90, at least sequences having at least 95, at least 100, or at least 105 consecutive aas, and The PD-L1 MOD sequence may also contain one or more disulfide, salt bridge, or Pi stacking substitutions. s at least 85 contiguous aas (e.g., at least 90, at least 95, at least a sequence having at least 100, or at least 105 consecutive aas), and at least one It may contain disulfides, salt bridges, or Pi stacking substitutions.

[0132] In some examples, the mutant PD-L1 polypeptide is SEQ ID NO: 1, SEQ ID NO: 2, or PD-L1 IgV In some instances, the PD-L1 domain has one aa substitution compared to the PD-L1 amino acid sequence shown in The variant PD-L1 polypeptide may be a variant PD-L1 polypeptide as set forth in SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1. In some instances, the variant has 2-10 aa substitutions compared to the PD-L1 amino acid sequence of interest. The PD-L1 polypeptide may be a PD-L1 polypeptide as shown in SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1. In some instances, the mutant PD-L1 polypeptide has two aa substitutions compared to the amino acid sequence. The PD-L1 domain is compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1. In some instances, the mutant PD-L1 polypeptide has three or four aa substitutions in comparison to: Compared to the PD-L1 amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1 In some instances, the mutant PD-L1 polypeptide has SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 2, or the IgV domain of PD-L1. In some instances, the mutant PD-L1 polypeptide has the sequence SEQ ID NO: 1, the sequence SEQ ID NO: 2, the sequence SEQ ID NO: 3, the sequence SEQ ID NO: 4, the sequence SEQ ID NO: 5, the sequence SEQ ID NO: 6, the sequence SEQ ID NO: 7, the sequence SEQ ID NO: 8, the sequence SEQ ID NO: 9, the sequence SEQ ID NO: 10, the sequence SEQ ID NO: 11, the sequence 9 or 10 amino acids compared to the PD-L1 amino acid sequence shown in column 2 or the IgV domain of PD-L1 It has aa substitutions of:

[0133] A preferred mutant PD-L1 polypeptide sequence comprises at least 170 contiguous aa of SEQ ID NO:2 (e.g. For example, at least 80%, at least 180, 190, or 200 consecutive aa At least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the aa Polypeptides with sequence identity (e.g., with at least one aa insertion, deletion, or substitution) A preferred mutant PD-L1 IgV polypeptide sequence is a sequence from aas 1 to 10 of SEQ ID NO:2. At least 70 consecutive aas (e.g., at least 80, 90, 100, or 105 consecutive aas) of 9 At least 80%, at least 85%, at least 90%, at least 95% of the AAS %, at least 98%, or at least 99% aa sequence identity (e.g., at least 1 The present invention also includes polypeptide sequences having one or more aa insertions, deletions, or substitutions.

[0134] In some instances, the mutant PD-L1 polypeptide sequence has a sequence identity that is at least 90% (e.g., 90%) similar to SEQ ID NO:2. For example, polypeptides with at least 95%, 98%, or 99%, or 100% aa sequence identity. and the residue at position 8 is an aa other than D, in one such example, the residue is A. and in another example, R. The mutant PD-L1 polypeptide sequence may have at least 9 amino acids with respect to SEQ ID NO:2. 0% (e.g., at least 95%, 98%, or 99%), or 100% aa sequence identity and wherein the residue at position 36 is an aa other than I, and in one such example, the residue is A. and in another example D. The mutant PD-L1 polypeptide sequence may also be At least 90% (e.g., at least 95%, 98%, or 99%), or 100% aa sequence identity and the residue at position 54 is an aa other than E. In one example, the residue is A, and in another example, it is R.

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

[0136] The wild-type human IL-1α precursor polypeptide has the following amino acid sequence: [ka] UniProtKB-P01583, NCBI reference sequence NP_000566.3, but may contain naturally occurring variations It is possible that the patient has one or more of the following mutations: R85Q, A114S, N125D, D138N, and D176H. can.

[0137] The mature wild-type human IL-1α polypeptide has the following amino acid sequence: [ka] may include:

[0138] The wild-type human IL-1β precursor polypeptide has the following amino acid sequence: [ka] UniProtKB - P0158, may include NCBI reference sequence NP_000567.1.

[0139] The mature wild-type human IL-1β polypeptide has the following amino acid sequence: [ka] may include:

[0140] Both IL-1α and IL-1β bind to the IL-1 receptor, which has the following sequence: [ka] and may have NCBI reference sequence NP_000868.1, with aas 21 to 569 forming the mature polypeptide, The ectodomain of the IL-1α and IL-1β proteins can be used to measure their binding affinity to IL-1α and IL-1β. Cut.

[0141] In some instances, the variant IL-1α and IL-1β polypeptides are selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:7. The binding affinity of the IL-1 polypeptide shown in SEQ ID NO: 8 was compared to that of the IL-1 polypeptide containing the amino acid sequence shown. a low binding affinity to the IL-1 receptor or its ectodomain, comprising an amino acid sequence For example, in some instances, the variant IL-1α and IL-1β polypeptides may also include SEQ ID NO:5. or a binding partner of IL-1α and IL-1β polypeptides comprising the amino acid sequence shown in SEQ ID NO:7. At least 10% lower, at least 20% lower, at least 30% lower, at least at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower binding affinity It binds to the IL-1 receptor shown in SEQ ID NO:8.

[0142] In some instances, a mutant IL-1α or IL-1β polypeptide (e.g., SEQ ID NO: 5 or 7) is used. The mutant form) has a potency of 1 nM to 1 mM (e.g., , 1nM to 10nM, 10nM to 100nM, 100nM to 1μM, 1μM to 10μM, 10μM to 100μM, or 100μM to 1 It has a binding affinity of 100 mM. As another example, in some instances, a variant IL-1α or IL-1β polypeptide (e.g., SEQ ID NO: The IL-1 receptor having the sequence shown in SEQ ID NO: 8 has a nucleotide sequence of about 10 0nM ~ approx. 200nM, approx. 200nM ~ approx. 300nM, approx. 300nM ~ approx. 400nM, approx. 400nM ~ approx. 500nM, approx. 500nM ~ Approximately 600nM, approximately 600nM to approximately 700nM, approximately 700nM to approximately 800nM, approximately 800nM to approximately 900nM, approximately 900nM to approximately 1μM, Approximately 1μM to approximately 5μM, approximately 5μM to approximately 10μM, approximately 10μM to approximately 20μM, approximately 20μM to approximately 30μM, approximately 30μM to approximately It has a binding affinity of 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0143] In some examples, the mutant IL-1α or IL-1β polypeptide is SEQ ID NO: 5 or SEQ ID NO: 7 It has one aa substitution compared to the amino acid sequence of IL-1α or IL-1β shown in In one example, the mutant IL-1α or IL-1β polypeptide is shown in SEQ ID NO: 5 or SEQ ID NO: 7. The amino acid sequence of the corresponding IL-1α or IL-1β has 2 to 10 aa substitutions. In the present invention, the mutant IL-1α or IL-1β polypeptide is the IL-1α or IL-1β polypeptide shown in SEQ ID NO: 5 or SEQ ID NO: 2. In some instances, the variant has two aa substitutions compared to the amino acid sequence of IL-1α or IL-1β. The variant IL-1α or IL-1β polypeptide is the IL-1α or IL-1β polypeptide shown in SEQ ID NO: 5 or SEQ ID NO: 7. The amino acid sequence of IL-1β has three or four aa substitutions. The polypeptide of IL-1α or IL-1β is the IL-1α or IL-1β polypeptide shown in SEQ ID NO: 5 or SEQ ID NO: 7. In some instances, the variant has 5 or 6 aa substitutions compared to the amino acid sequence of -1β. The IL-1α or IL-1β polypeptide is the IL-1α or IL-1β polypeptide shown in SEQ ID NO: 5 or SEQ ID NO: 7. In some instances, the mutant IL-1 has 7 or 8 aa substitutions compared to the amino acid sequence of IL-1. The polypeptide of IL-1α or IL-1β is shown in SEQ ID NO: 5 or SEQ ID NO: 7. It has 9 or 10 aa substitutions compared to the amino acid sequence of

[0144] A suitable mutant IL-1α or IL-1β polypeptide sequence is set forth in SEQ ID NO:5 or SEQ ID NO:7 At least 80% of at least 100 consecutive aa of the amino acid sequence At least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the aa Polypeptide sequences with sequence identity (e.g., at least one aa substitution, deletion, or has an insertion).

[0145] 6. IL-2 and its variants As one non-limiting example, MOD or MOD-1 present in a masked TGF-β construct or complex. The mutant MOD is IL-2 or a mutant IL-2 polypeptide. In some instances, a masking The mutant MOD present in the TGF-β construct or complex is a mutant IL-2 polypeptide. Wild-type IL-2 binds to the IL-2 receptor (IL-2R). The wild-type IL-2 amino acid sequence is shown below. As follows: [ka] (aa 21 to 153 of UniProtP60568, SEQ ID NO: 9).

[0146] Wild-type IL-2 binds to the IL-2 receptor (IL-2R) on the surface of cells. Examples include the α chain (IL-2Rα; also called CD25), the β chain (IL-2Rβ; also called CD122), and Human IL-2 is a heterotrimeric polypeptide containing a γ chain (IL-2Rγ; also called CD132). The amino acid sequences of Ra, IL-2Rβ, and IL-2Rγ may be as follows:

[0147] Human IL-2Rα: [ka] .

[0148] Human IL-2Rβ: [ka] .

[0149] Human IL-2Rγ: [ka] .

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

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

[0152] In some instances, the variant IL-2 polypeptide (e.g., a variant of SEQ ID NO: 9) binds to the IL-2R (e.g., For example, it has a binding affinity of 100 nM to 100 μM to the sequences of SEQ ID NOs: 10 to 12. In some instances, the variant IL-2 polypeptide (e.g., a variant of SEQ ID NO: 9) binds to the IL-2R (e.g., For example, an IL-2R containing a polypeptide comprising the amino acid sequence shown in SEQ ID NOs: 10 to 12, Approximately 100nM to approximately 200nM, approximately 200nM to approximately 300nM, approximately 300nM to approximately 400nM, approximately 400nM to approximately 500nM, approximately 500nM ~about 600nM, about 600nM to about 700nM, about 700nM to about 800nM, about 800nM to about 900nM, about 900nM to about 1μM , about 1μM to about 5μM, about 5μM to about 10μM, about 10μM to about 20μM, about 20μM to about 30μM, about 30μM It has a binding affinity of about 50 μM, about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0153] In some examples, the variant IL-2 polypeptide has the IL-2 amino acid sequence set forth in SEQ ID NO:9. In some instances, the variant IL-2 polypeptide has one aa substitution compared to the sequence It has 2 to 10 aa substitutions compared to the IL-2 amino acid sequence shown in number 9. The mutant IL-2 polypeptide has two amino acid residues as compared to the IL-2 amino acid sequence shown in SEQ ID NO:9. In some examples, the mutant IL-2 polypeptide has an aa substitution of I as set forth in SEQ ID NO:9. In some instances, the mutant IL-2 polypeptide has three aa substitutions compared to the IL-2 amino acid sequence. The peptide has four aa substitutions compared to the IL-2 amino acid sequence shown in SEQ ID NO: 9. In some examples, the variant IL-2 polypeptide has a sequence similar to that of the IL-2 amino acid sequence set forth in SEQ ID NO:9. In some examples, the mutant IL-2 polypeptide has 5 aa substitutions compared to SEQ ID NO:9. In some instances, the mutation The IL-2 polypeptide has seven aa substitutions compared to the IL-2 amino acid sequence shown in SEQ ID NO:9. In some examples, the mutant IL-2 polypeptide has the IL-2 amino acid sequence shown in SEQ ID NO:9. In some instances, the mutant IL-2 polypeptide has eight aa substitutions compared to the amino acid sequence. has 9 aa substitutions compared to the IL-2 amino acid sequence shown in SEQ ID NO: 9. In examples, the variant IL-2 polypeptide has the following structure compared to the IL-2 amino acid sequence set forth in SEQ ID NO:9: , with 10 aa substitutions.

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

[0155] [ka] In the sequence, X is any amino acid other than Glu. In some examples, X is Ala.

[0156] [ka] In the sequence, X is any amino acid other than Asp. In some examples, X is Ala.

[0157] [ka] In the sequence, X (H16) is any amino acid other than His. In some examples, X is Ala (H16A). In some examples, X is Arg. In some examples, X is Asn. In some examples, In some instances, X is Asp. In some instances, X is Cys. In some instances, X is Glu. In some instances, X is Gln. In some instances, X is Gly. In some instances, X is Ile. In some instances, X is Lys. In some instances, X is Leu. In some instances, In some instances, X is Met. In some instances, X is Phe. In some instances, X is Pro. In some examples, X is Ser. In some examples, X is Thr(H16T). In some examples, X is is Tyr. In some instances, X is Trp. In some examples, X is Val.

[0158] [ka] In the sequence, X (F42) is any amino acid other than Phe. In some examples, X is Ala (F42A). In some instances, X is Thr(F42T).

[0159] [ka] In the sequence, X is any amino acid other than Tyr. In some examples, X is Ala.

[0160] [ka] In the sequence, X(N88) is any amino acid except Asn. In some examples, X is Ala. In some examples, X is Arg. [ka] In the sequence, X is any amino acid other than Gln. In some examples, X is Ala.

[0161] [ka] In the sequence, X1 (H16) is any amino acid other than His, and X2 (F42) is any amino acid other than Phe. In some instances, X1 is Ala. In some instances, X2 is Ala. In some examples, X1 is Ala and X2 is Ala (H16A, F42A). In some examples, X1 is Thr. and X2 is Ala (H16T, F42A). In some instances, X1 is Ala and X2 is Thr (H16A , F42T). In some instances, X1 is Thr and X2 is Thr (H16T, F42T).

[0162] [ka] N88R substitution, in which X1 (H16) is any amino acid other than His, and X2 (F42) is Phe In some examples, X1 is Ala. In some examples, X2 is A In some examples, X1 is Ala and X2 is Ala. In some examples, X1 is Thr. and X2 is Ala. In some examples, X1 is Ala and X2 is Thr. So, X1 is Thr and X2 is Thr.

[0163] [ka] In the sequence, X1 is any amino acid other than Asp, and X2 is any amino acid other than Phe. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X1 is Ala and X2 is Ala.

[0164] [ka] In the sequence, X1 is any amino acid other than Glu, and X2 is any amino acid other than Asp. , X3 is any amino acid other than Phe. In some examples, X1 is Ala. In some instances, X2 is Ala. In some instances, X3 is Ala. In some instances, X1 is Ala. X1 is Ala, and X2 is Ala. X3 is Ala.

[0165] [ka] In the sequence, X1 is any amino acid other than His, and X2 is any amino acid other than Asp. , X3 is any amino acid other than Phe. In some examples, X1 is Ala. In some instances, X2 is Ala. In some instances, X3 is Ala. In some instances, X1 is Ala. X2 is Ala and X3 is Ala.

[0166] [ka] In the sequence, X1 is any amino acid other than Asp, and X2 is any amino acid other than Phe. , X3 is any amino acid other than Gln. In some examples, X1 is Ala. In some instances, X2 is Ala. In some instances, X3 is Ala. In some instances, X1 is Ala. X2 is Ala and X3 is Ala.

[0167] [ka] In the sequence, X1 is any amino acid other than Asp, and X2 is any amino acid other than Phe. , X3 is any amino acid other than Tyr. In some examples, X1 is Ala. In some instances, X2 is Ala. In some instances, X3 is Ala. In some instances, X1 is Ala. X2 is Ala and X3 is Ala.

[0168] [ka] In the sequence, X1 is any amino acid other than His, and X2 is any amino acid other than Asp. , X3 is any amino acid other than Phe, and X4 is any amino acid other than Tyr. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Al. In some examples, X4 is Ala. In some examples, X1 is Ala and X2 is Ala. X3 is Ala and X4 is Ala.

[0169] [ka] In the sequence, X1 is any amino acid other than Asp, and X2 is any amino acid other than Phe. X3 is any amino acid other than Tyr, and X4 is any amino acid other than Gln. In some examples, X1 is Ala. In some examples, X2 is Ala. In some examples, X3 is Al. In some examples, X4 is Ala. In some examples, X1 is Ala and X2 is Ala. X3 is Ala and X4 is Ala.

[0170] [ka] In the sequence, X1 is any amino acid other than His, and X2 is any amino acid other than Asp. , X3 is any amino acid other than Phe, X4 is any amino acid other than Tyr, and X5 is , any amino acid other than Gln. In some examples, X1 is Ala. In some examples, X2 is Ala. In some instances, X3 is Ala. In some instances, X4 is Ala. In some examples, X5 is Ala. In some examples, X1 is Ala, X2 is Ala, and X3 is Ala. X1 is Ala, X2 is Ala, X3 is Ala, X4 is Ala, X5 is Ala, or any two, three, four, or five of these That is it.

[0171] [ka] In the sequence, X1 is any amino acid other than His, and X2 is any amino acid other than Phe. , X3 is any amino acid other than Gln. In some examples, X1 is Ala. In some instances, X2 is Ala. In some instances, X3 is Ala. In some instances, X1 is Ala. X2 is Ala, and X3 is Ala.

[0172] 7. IL-4 and its variants As one non-limiting example, MOD or MOD-1 present in a masked TGF-β construct or complex. A mutant MOD is IL-4 or a mutant IL-4 polypeptide. Wild-type IL-4 is composed of two isoforms: IL-4α and IL-4β, both of which bind to the membrane-bound IL-4 receptor (isoform 1) or It binds to its soluble counterpart (isoform 2).

[0173] The wild-type human IL-4 isoform 1 precursor polypeptide has the following amino acid sequence: [ka] It may include the NCBI reference sequence NP_000580.1.

[0174] The mature wild-type human IL-4 isoform 1 polypeptide has the following amino acid sequence: [ka] may include:

[0175] The wild-type human IL-4 isoform 2 precursor polypeptide has the following amino acid sequence: [ka] It may include the NCBI reference sequence NP_758858.1 (SEQ ID NO: 30).

[0176] The mature wild-type human IL-4 isoform 2 polypeptide has the following amino acid sequence: [ka] may include:

[0177] Both IL-4 isoform 1 and isoform 2 bind to the membrane-bound IL-4 receptor (IL-4R) and and / or its soluble isoform 2. Membrane-bound IL-4 has the following sequence: [ka] It may have the NCBI reference sequence NP_000409.1, in which aas 26 to 825 form the mature polypeptide and aas 233 to 2 56 forms the transmembrane region, and the ectodomain of the protein is used to bind IL-4 isoforms The binding affinity to 1 or 2 can be measured.

[0178] The following sequence [ka] The soluble isoform 2, which contains UniProtKB-P24394, also binds both IL-4 isoforms. It can be used to measure binding affinity.

[0179] In some instances, a mutant IL-4 isoform 1 or 2 polypeptide (e.g., SEQ ID NO: 29) or 31, variants thereof) is a mature IL-4 receptor agonist as shown in SEQ ID NO: 32 or the ectodomain thereof. 29 or SEQ ID NO: 33 for the soluble IL-4 receptor shown in SEQ ID NO: 33 31. It exhibits harmony. For example, in some instances, the mutant IL-4 isoform 1 or 2 polypeptide is SEQ ID NO: 32 (or the ectodomain thereof) or the mature IL-4 receptor shown in SEQ ID NO: 33, IL-4 isoform 1 or 2 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 31 At least 10% lower, at least 20% lower, or at least 30% lower than the binding affinity of the , at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower , at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower It binds with low binding affinity.

[0180] In some instances, a mutant IL-4 isoform 1 or 2 polypeptide (e.g., SEQ ID NO: 29) or 31, variants thereof) can be SEQ ID NO: 32 (or the exodomain thereof) or SEQ ID NO: 33 For the mature IL-4 receptor shown in 1 μM to 10 μM, 10 μM to 100 μM, or 100 μM to 1 mM). For example, in some instances, a mutant IL-4 isoform 1 or 2 polypeptide (e.g., a sequence The variants of SEQ ID NO: 29 or 31 are SEQ ID NO: 32 (or the ectodomain thereof) or SEQ ID NO: 33. For the mature IL-4 receptor shown in column 33, about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300nM~Approx. 400nM, Approx. 400nM~Approx. 500nM, Approx. 500nM~Approx. 600nM, Approx. 600nM~Approx. 700nM, Approx. 700nM~ Approximately 800nM, approximately 800nM to approximately 900nM, approximately 900nM to approximately 1μM, approximately 1μM to approximately 5μM, approximately 5μM to approximately 10μM, approximately 1 0 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 It has a binding affinity of about 100 μM.

[0181] In some instances, the variant IL-4 isoform 1 or 2 polypeptide is SEQ ID NO: 29 or SEQ ID NO: 30. Compared to the IL-4 isoform 1 or 2 amino acid sequence shown in column 31, one aa substitution is made. In some examples, the variant IL-4 isoform 1 or 2 polypeptide has the sequence set forth in SEQ ID NO:29. or 2 to 10 amino acids compared to the IL-4 isoform 1 or 2 amino acid sequence shown in SEQ ID NO: 31 In some examples, the variant IL-4 isoform 1 or 2 polypeptide has an aa substitution of: Compared to the IL-4 isoform 1 or 2 amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 31 In some instances, the mutant IL-4 isoform 1 or 2 polypeptide has two aa substitutions. The amino acid sequence of IL-4 isoform 1 or 2 shown in SEQ ID NO: 29 or SEQ ID NO: 31 is compared. In some instances, the mutant IL-4 polypeptide has three or four aa substitutions compared to the sequence Compared to the IL-4 isoform 1 or 2 amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 31, In some instances, the mutant IL-4 isoform 1 or 2 polypeptide has 5 or 6 aa substitutions. The peptide has the IL-4 isoform 1 or 2 amino acid sequence shown in SEQ ID NO: 29 or SEQ ID NO: 31. In some instances, the mutant IL-4 isoform has 7 or 8 aa substitutions compared to The IL-4 isoform 1 or 2 polypeptide is represented by SEQ ID NO: 29 or SEQ ID NO: 31. Compared to the amino acid sequence, there are 9 or 10 aa substitutions.

[0182] A suitable mutant IL-4 isoform 1 or 2 polypeptide sequence is SEQ ID NO: 29 or SEQ ID NO: 31 for at least 80 consecutive aa (e.g., at least 100, or 110 consecutive aa) and at least 80%, at least 85%, at least 90%, at least 95%, at least Polypeptide sequences having 98%, or at least 99% aa sequence identity (e.g., at least (also has one aa substitution, deletion or insertion).

[0183] 8. IL-6 and its variants As one non-limiting example, MOD or MOD-1 present in a masked TGF-β construct or complex. The mutant MOD is IL-6 or a mutant IL-6 polypeptide. Wild-type IL-6 is a β- and α-protein complex (gp13). 0) Binds to the IL-6 receptor, which is composed of IL-6 molecules and each subunit. They form a signaling hexamer of two trimers with the nucleotides α, β, and β. See, for example, Lacroix et al., J. See Biol.Chem.290(45)26943-953(2015).

[0184] The wild-type human IL-6 polypeptide has the following amino acid sequence: [ka] It may include the NCBI reference sequence NP_000591.1.

[0185] The mature wild-type human IL-6 polypeptide has the following amino acid sequence: [ka] may include:

[0186] IL-6 binds to the membrane-bound IL-6 receptor, which is composed of α and β subunits. The -6R α subunit has the sequence: [ka] NCBI reference sequence NP_000556.1, in which aas 26 to 825 form the mature polypeptide and aas 233 to 256 forms the transmembrane domain. After binding to IL-6, the IL-6α subunit translocates to the IL-6β subunit. It binds to

[0187] The human IL-6Rβ subunit has the following sequence: [ka] It may have UniProtKB-P40189, where aas 23 to 918 form the mature polypeptide and aas 620 to 641 form the membrane A through region is formed.

[0188] As an alternative to binding of IL-6 to the membrane-bound IL-6R α subunit, it binds to the IL-6R α subunit with the sequence: [ka] and capable of binding to a soluble form of the mature IL-6Rα subunit having the formula The peptide contains aas 20 to 468, UniProtKB - P08887.1. The soluble subunit is a membrane-bound IL-6 Can substitute for the Rα subunit and be used in binding affinity assays do.

[0189] In some examples, the variant IL-6 polypeptide (e.g., a variant of SEQ ID NO: 35) is 35, compared with the binding affinity of an IL-6 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 36 and 37, or SEQ ID NOs: 37 and 38. For example, in some instances, the mutant IL-6 polypeptide has the amino acid sequence set forth in SEQ ID NO:35. The binding affinity of the IL-6 polypeptide is at least 10% lower than that of the IL-6 polypeptide containing the IL-6 sequence. % lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60 % lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95 % lower or more than 95% lower binding affinity to SEQ ID NOs: 36 and 37, or SEQ ID NOs: 37 and 38 It binds to the mature IL-6 receptor shown in

[0190] In some examples, the variant IL-6 polypeptide (e.g., a variant of SEQ ID NO: 35) is 36 and 37, or the mature IL-6 receptor shown in SEQ ID NOs: 37 and 38, 1 nM to 1 mM (for example , 1nM to 10nM, 10nM to 100nM, 100nM to 1μM, 1μM to 10μM, 10μM to 100μM, or 100μ In another example, in some instances, the mutant IL-6 polypeptide has a binding affinity of 0.1 mM to 1 mM. The variants (e.g., variants of SEQ ID NO: 35) are shown in SEQ ID NOs: 36 and 37, or SEQ ID NOs: 37 and 38. For the mature IL-6 receptor, 100 nM to 100 μM (for example, 100 nM to 1 μM, 1 μM to 10 μM, or In some cases, the mutant IL-6 polypeptide has an affinity of 10 μM to 100 μM. The peptides (e.g., variants of SEQ ID NO: 35) are shown in SEQ ID NOs: 36 and 37 or SEQ ID NOs: 37 and 38. about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM against the mature IL-6 receptor , about 400nM to about 500nM, about 500nM to about 600nM, about 600nM to about 700nM, about 700nM to about 800nM, about 800n M ~ about 900nM, about 900nM - about 1μM, about 1μM - about 5μM, about 5μM - about 10μM, about 10μM - about 20μM, 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 It has affinity for

[0191] In some examples, the mutant IL-6 polypeptide has the IL-6 amino acid sequence set forth in SEQ ID NO:35. In some examples, the mutant IL-6 polypeptide has one aa substitution compared to the sequence Compared to the IL-6 amino acid sequence shown in No. 35, it has 2 to 10 aa substitutions. The mutant IL-6 polypeptide has the following structure compared to the IL-6 amino acid sequence shown in SEQ ID NO: 35: In some examples, the mutant IL-6 polypeptide has two aa substitutions. In some instances, the mutations The IL-6 polypeptide has 5 or 6 amino acids compared to the IL-6 amino acid sequence shown in SEQ ID NO: 35. In some examples, the mutant IL-6 polypeptide has an aa substitution of: In some instances, the mutant IL-6 has 7 or 8 aa substitutions compared to the IL-6 amino acid sequence. The IL-6 polypeptide has 9 or 8 aa changes compared to the IL-6 amino acid sequence shown in SEQ ID NO: 35. It has substitutions.

[0192] A preferred variant IL-6 polypeptide sequence is at least 80 consecutive aa of SEQ ID NO: 35 (e.g. For example, at least 80% of the sequences are identical for at least 100 or 110 consecutive aa. At least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the aa Polypeptide sequences with sequence identity (e.g., at least one aa substitution, deletion, or insertion) (including those with an entry).

[0193] 9. IL-7 and its variants As one non-limiting example, MOD or MOD-1 present in a masked TGF-β construct or complex. A mutant MOD is IL-7 or a mutant IL-7 polypeptide. Wild-type IL-7 is composed of four isoforms: They all have two subunits, the α (α) and common γ (common γ) chains. It binds to the membrane-bound IL-7 receptor.

[0194] The wild-type human IL-7 isoform 1 precursor polypeptide has the following amino acid sequence: [ka] UniProtKB-P13232, which may include NCBI reference sequence NP_000871.1.

[0195] The mature wild-type human IL-7 isoform 1 polypeptide has the following amino acid sequence: [ka] may include:

[0196] The wild-type human IL-7 isoform 2 precursor polypeptide has the following amino acid sequence: [ka] It may include the NCBI reference sequence NP_001186815.1.

[0197] The mature wild-type human IL-7 isoform 2 polypeptide has the following amino acid sequence: [ka] may include:

[0198] The wild-type human IL-7 isoform 3 precursor polypeptide has the following amino acid sequence: [ka] NCBI reference sequence: NP_001186816.1.

[0199] The mature wild-type human IL-7 isoform 3 polypeptide has the following amino acid sequence: [ka] may include:

[0200] The wild-type human IL-7 isoform 4 precursor polypeptide has the following amino acid sequence: [ka] It may include the NCBI reference sequence NP_001186817.1 (SEQ ID NO: 45).

[0201] The mature wild-type human IL-7 isoform 4 polypeptide has the following amino acid sequence: [ka] may include:

[0202] The IL-7 receptor alpha subunit has the following sequence: [ka] NCBI reference sequence NP_002176.2, wherein aas 21 to 459 form the mature polypeptide and aas 240 to 2 64 forms the transmembrane region. The receptor subunits of proteins (e.g., ectodomains ( Using all or part of aas 21 to 239), IL-7 isoforms can be synthesized in combination with the IL-7 receptor γ subunit. The binding affinity to the form can be measured.

[0203] The common gamma subunit (IL-7RG or IL-R7γ) has the following sequence: [ka] It may have the NCBI reference sequence NP_000197.1 (SEQ ID NO: 48), in which aas 23 to 369 form the mature polypeptide The aas 263 to 283 form the transmembrane domain. The whole or part of the target domain (aas23-262) was used to bind to IL-7 together with the α subunit. Binding affinity can be measured.

[0204] In some instances, a mutant IL-7 isoform 1, 2, 3, or 4 polypeptide (e.g., The polypeptide of SEQ ID NO: 40, 42, 44, or 46 is the polypeptide of SEQ ID NO: 40, 42, 44, or 46. the binding affinity of the mature IL-7 receptor compared to that of an IL-7 polypeptide comprising the amino acid sequence of IL-7 polypeptides comprising all or part of the sequences (e.g., SEQ ID NOs: 47 and 48) exhibit low binding affinity to receptors, e.g., their ectodomains. In some examples, the variant IL-7 isoform 1, 2, 3, or 4 polypeptide has the sequence set forth in SEQ ID NO: 40. IL-7 isoform 1, 2, 3, or 4 polynucleotides containing the amino acid sequence shown in Table 1, 42, 44, or 46. At least 10% lower, at least 20% lower, at least 30% lower than the binding affinity of the peptide % lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70 % lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% and a polypeptide having a binding affinity relatively low to the polypeptides set forth in SEQ ID NOs: 47 and 48. Binds to the IL-7 receptor.

[0205] In some instances, variants of IL-7 isoform 1, 2, 3, or 4 polypeptides (e.g., , variants of SEQ ID NOs: 40, 42, 44, or 46) are polypeptides shown in SEQ ID NOs: 47 and 48. 1 nM for the IL-7 receptor, including all or part of the IL-7 receptor ectodomain, e.g., In some instances, mutant IL-7 isoforms 1, 2, 3, or The polypeptides of SEQ ID NO: 4 (e.g., variants of SEQ ID NO: 40, 42, 44, or 46) are SEQ ID NO: 47 and and 48, including all or part of the polypeptide, e.g., the ectodomain thereof. For the mature IL-7 receptor, 100 nM to 100 μM (e.g., 100 nM to 1 μM, 1 μM to 10 μM, or For example, in some instances, the mutant IL-7 isoforms have a binding affinity of 10 μM to 100 μM. Polypeptides of groups 1, 2, 3, or 4 are all of the polypeptides set forth in SEQ ID NOs: 47 and 48. or a portion thereof, for example, about 100 nM to about 200 nM, about 200 nM to about 300 nM relative to the ectodomain thereof. 00nM, about 300nM to about 400nM, about 400nM to about 500nM, about 500nM to about 600nM, about 600nM to about 700nM, about 700nM to about 800nM, about 800nM to about 900nM, about 900nM to about 1μM, about 1μM to about 5μM, about 5μM to about 10 μM, approximately 10 μM ~ approximately 20 μM, approximately 20 μM ~ approximately 30 μM, approximately 30 μM ~ approximately 50 μM, approximately 50 μM ~ approximately 75 μM, Or, it has a binding affinity of about 75 μM to about 100 μM.

[0206] In some instances, the mutant IL-7 isoform 1, 2, 3, or 4 polypeptide is selected from the group consisting of SEQ ID NOs: 40, 42, 44, or 46. In some instances, mutant IL-7 isoforms 1, 2, 3, or 4 have one aa substitution. The polypeptide may be selected from the group consisting of IL-7 isoforms 1, 2, 3, 40, 42, 44, and 46. or 4 amino acid sequence, and have 2 to 10 aa substitutions. Isoform 1, 2, 3, or 4 polypeptides are set forth in SEQ ID NOs: 40, 42, 44, or 46. It has two aa substitutions compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequence. In some examples, the variant IL-7 isoform 1, 2, 3, or 4 polypeptide is SEQ ID NO: 40, 42, 44, or 46, compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequence shown in In some examples, the mutant IL-7 polypeptide has 3 or 4 aa substitutions. , 42, 44, or 46. In some examples, mutant IL-7 isoforms 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 4 polypeptide is IL-7 isoform 1, 2, or 3 as set forth in SEQ ID NO: 40, 42, 44, or 46. In some instances, the variants have 7 or 8 aa substitutions compared to the 1, 3, or 4 amino acid sequence. A variant IL-7 isoform 1, 2, 3, or 4 polypeptide is selected from the group consisting of SEQ ID NOs: 40, 42, 44, and 46. 9 or 10 aa compared to the IL-7 isoform 1, 2, 3, or 4 amino acid sequences shown It has substitutions.

[0207] A suitable mutant IL-7 isoform 1, 2, 3, or 4 polypeptide sequence is SEQ ID NO: 40: 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) At least 80%, at least 85%, at least 90%, at least 95%, polypeptide sequences having 98%, or at least 99% aa sequence identity with each other (e.g., at least (having at least one aa substitution, deletion, or insertion).

[0208] 10. IL-10 and its variants As one non-limiting example, MOD or MOD-1 present in a masked TGF-β construct or complex. The mutant MOD is described, for example, in Josephson et al., J. Biol. Chem. 275:13552-13557 (2000). Monomeric IL-10 variant with an insertion in the hinge region between the D and E helices Wild-type IL-10 is an isoform of IL-10 or a mutant IL-10 polypeptide, such as a variant. all of which contain both the alpha (α) IL-10RA and beta (β) IL-10RB subunits. This receptor binds to the membrane-bound IL-10 receptor, which has the structure It exists as a tetramer on the surface of various cells (NK cells, mast cells, and dendritic cells).

[0209] The wild-type human IL-10 isoform 1 precursor polypeptide has the following amino acid sequence: [ka] UniProtKB-P22301, which may contain the NCBI reference sequence NP_000563.1 and may have the sequence mutation H227L.

[0210] The mature wild-type human IL-10 polypeptide has the following amino acid sequence: [ka] may include:

[0211] The human IL-10 polypeptide has the following amino acid sequence: [ka] may include:

[0212] The IL-10 polypeptide contains an insertion in the hinge region between the D and E helices of the IL-10 polypeptide. (For example, a position adjacent to any of E48, N49, K50, or S51 of SEQ ID NO: 51, or a position adjacent to any of SEQ ID NO: 52, 49 or 50) thereby providing a single The monomeric IL-10 polypeptide is a polypeptide between N49 and K50 of SEQ ID NO: 51 (or SEQ ID NO: The insertion of 5-7 aa at the corresponding position in SEQ ID NO: 49 or 50 can be carried out. In an example, the 5 to 7 amino acids include Ala, Gly, and / or Ser. In one example, the 5 to 7 aas are selected from Gly and Ser. The insertion is a sequence of IL-10M1aa between N49 and K50 of SEQ ID NO: 51 (or the corresponding positions of SEQ ID NO: 49 or 50). Insert [ka] In one example, the IL-10 variant comprises the amino acid sequence between aa 49 and 50 of SEQ ID NO: 51. [ka] The fragment is composed of IL-10M1 (SEQ ID NO: 189) into which the fragment is inserted. See, for example, Josephson et al., J. Biol. Chem. .275:13552-13557(2000).

[0213] The IL-10 receptor alpha subunit has the following sequence: [ka] It may have the NCBI reference sequence NP_001549.2, in which aas 21 to 587 form the mature polypeptide and aas 236 to 2 56 forms the transmembrane region. All or part of the receptor subunit of the protein (e.g., The ectodomain (aas21-235) of the IL-10 receptor was used to express the IL-10 receptor β subunit in combination with the IL-10 receptor. The binding affinity to the isoform can be measured.

[0214] The IL-10 receptor beta subunit has the following sequence: [ka] It may have the NCBI reference sequence NP_000619.3 (SEQ ID NO: 53), wherein aas 20 to 325 form the mature polypeptide The aas 221 to 242 form the transmembrane domain. A portion of the protein (e.g., the ectodomain (aas 20-220)) is used to encode IL-10 together with the α subunit. The binding affinity of the antibody can be measured.

[0215] In some instances, a mutant IL-10 isoform polypeptide (e.g., SEQ ID NOs: 50, 51) is used. mutants, or those having a 5-7 aa insertion in the hinge between the D and E helices described above. Monomeric IL-10 variants of these sequences are IL-10 variants comprising the amino acid sequence shown in SEQ ID NO: 50 or 51. The binding affinity of the mature IL-10 receptor sequence (SEQ ID NO: 52 or 53) was compared with that of the mature IL-10 polypeptide. ILs comprising all or part of the polypeptides shown, such as the ectodomains thereof. For example, in some instances, the IL-10 polypeptide exhibits low binding affinity to the IL-10 receptor. Variants of the nucleotides (e.g., variants of SEQ ID NO: 50 or 51) are shown in SEQ ID NO: 52 or 53. IL-10 receptor polypeptides, including all or part of the polypeptide (e.g., the ectodomain thereof). and the whole or part of the polypeptide shown in SEQ ID NO: 52 or 53 (e.g., the exons thereof). Binding of an IL-10 isoform (e.g., of SEQ ID NO: 50 or 51) to a target domain (e.g., a target domain) At least 10% lower, at least 20% lower, at least 30% lower, or less than the affinity at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower To bond harmoniously.

[0216] In some instances, a mutant IL-10 polypeptide (e.g., a region of the hinge between the D and E helices) The variants of SEQ ID NO: 50 or 51, which may contain one of the above-mentioned insertions into the IL-10 receptor ( All or part of the polypeptides set forth in SEQ ID NOs: 52 and 53, e.g., their ectodomains 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). The mutant IL-10 polypeptide (e.g., the mutant of SEQ ID NO: 50 or 51) binds to the mature IL-10 receptor. Enzymes (e.g., all or part of the polypeptides set forth in SEQ ID NOs: 52 and 53, e.g., those ectodomain, containing), 100 nM to 100 μM (e.g., 100 nM to 1 μM, 1 μM to 10 μM) For example, in some instances, the mutant IL-10 polypeptide has a binding affinity of 100 μM to 100 μM. The polypeptide (e.g., a variant of SEQ ID NO: 50 or 51) is a polypeptide that binds to the mature IL-10 receptor (e.g., a variant of SEQ ID NO: 50 or 51). All or part of the polypeptides set forth in Nos. 52 and 53, e.g., the ectodomains thereof; With respect to the Approx. 500nM, Approx. 500nM ~ Approx. 600nM, Approx. 600nM ~ Approx. 700nM, Approx. 700nM ~ Approx. 800nM, Approx. 800nM ~ Approx. 900nM , about 900nM to about 1μM, about 1μM to about 5μM, about 5μM to about 10μM, about 10μM to about 20μM, about 20μM A binding affinity of 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. Has.

[0217] In some instances, the variant IL-10 polypeptide is an IL-10 amino acid sequence set forth in SEQ ID NO: 50 or 51. In some examples, the mutant IL-10 polypeptide has one aa substitution compared to the amino acid sequence. has 2 to 10 aa substitutions compared to the IL-10 amino acid sequence shown in SEQ ID NO: 50 or 51. In some examples, the mutant IL-10 polypeptide is an IL-10 polypeptide set forth in SEQ ID NO: 50 or 51. In some instances, the mutant IL-10 polypeptide has two aa substitutions compared to the amino acid sequence. The IL-10 amino acid sequence shown in SEQ ID NO: 50 or 51 has three or four aa substitutions. In some examples, the mutant IL-10 polypeptide has the I sequence shown in SEQ ID NO: 50 or 51. In some instances, the mutant IL-10 has 5 or 6 aa substitutions compared to the IL-10 amino acid sequence. The IL-10 polypeptide has 7 or 8 amino acids compared to the IL-10 amino acid sequence shown in SEQ ID NO: 50 or 51. has 8 aa substitutions. In some examples, the mutant IL-10 polypeptide has SEQ ID NO: 50 or 51. In the present specification, the mutant IL-10 polypeptide has the IL-10 polypeptide sequence shown in SEQ ID NO: 50 or 51. has 1-10 aa substitutions compared to , and 5-7 aa substitutions in the hinge region between the D and E helices. a substitution (e.g., K49 and N50 of SEQ ID NO: 50).

[0218] A preferred variant IL-10 polypeptide sequence is at least 50 contiguous sequences of SEQ ID NO: 50 or 51. aa (e.g., at least 60, at least 70, at least 80, at least 90, at least 1 00, at least 110, at least 120, at least 130, at least 140, at least 150, or at least 160 consecutive aas, at least 80%, at least 85%, at least have 90%, at least 95%, at least 98%, or at least 99% aa sequence identity with a polypeptide sequence (e.g., having at least one aa substitution, deletion, or insertion) include.

[0219] 11. IL-15 and its variants In some instances, the MOD present in the masked TGF-β construct or complex of the present disclosure is the IL-15 polypeptide. Alternative splicing events give rise to different precursor proteins. The sequence of the IL-15 polypeptide, including the two isoforms formed by cloning, is known in the art. It is known in the art. In one embodiment, the wt. IL-15 polypeptide has the following sequence: [ka] UniProtKB-P40933, NCBI reference NP_000576.1, aa 1-29 in IL-15 preprotein This is the signal peptide, and 30 to 48 is the propeptide.

[0220] For purposes of this disclosure, the mature IL-15 polypeptide, designated as isoform 1, is Form of reference: [ka] It can have:

[0221] IL-15 is structurally similar to IL-2, and has the same β and γ chains as the IL-2 receptor, but IL-15 is secreted via a cell surface trimeric receptor with distinct IL-15 receptor α (IL-15Rα) subunits. The amino acid sequence of the human IL-15Rα isoform 1 precursor protein is: [ka] NCBI reference NP_002180, where aas 1 to 30 form the signal sequence and aas peptide The sequences of IL-2Rβ and IL-2Rγ are shown in SEQ ID NOs: 11 and 12. and show.

[0222] In some instances, a mutant IL-15 isoform 1 polypeptide (e.g., a mutant of SEQ ID NO: 55) is used. The binding affinity of the IL-15 polypeptide (variant) is compared to that of the IL-15 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In comparison, the mature IL-15 receptor sequences (e.g., the polypeptides shown in SEQ ID NOS: 11, 12, and 56) low binding to all or part of the IL-15 receptor, including their ectodomains For example, in some instances, variants of IL-15 polypeptides (e.g., those with the sequence SEQ ID NO: 55) is a variant of an IL-15 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 55. At least 10% lower, at least 20% lower, at least 30% lower, or less than the affinity at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or more than 95% lower and the whole or part of the polypeptides set forth in SEQ ID NOs: 11, 12, and 56, e.g., It binds to the ectodomain-containing IL-15 receptor.

[0223] In some examples, the variant IL-15 polypeptide (e.g., a variant of SEQ ID NO: 55) is All or part of the polypeptides shown in Nos. 11, 12, and 56, e.g., their ectodomains. For IL-15 receptors containing IL-15, the concentration should be between 1 nM and 1 mM (e.g., 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1 For example, In some examples, the variant IL-15 polypeptide (e.g., a variant of SEQ ID NO: 55) is a variant of SEQ ID NO: 1 1, 12, and 56, including all or part of the polypeptides shown therein, e.g., their ectodomains. about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM against mature IL-15 receptor containing IL-15 receptor Approx. 400nM, Approx. 400nM ~ Approx. 500nM, Approx. 500nM ~ Approx. 600nM, Approx. 600nM ~ Approx. 700nM, Approx. 700nM ~ Approx. 800nM , about 800nM to about 900nM, about 900nM to about 1μM, about 1μM to about 5μM, about 5μM to about 10μM, about 10μM 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 10 μM It has a binding affinity of 0 μM.

[0224] In some examples, the variant IL-15 polypeptide (e.g., a variant of SEQ ID NO: 55) is 55. The mutant IL-15 polypeptide (e.g., the mutant of SEQ ID NO: 55) is a polypeptide of the invention shown in SEQ ID NO: 55. In some instances, variant I has 2 to 10 aa substitutions compared to the I-15 polypeptide sequence. The IL-15 polypeptide contains two aa substitutions compared to the IL-15 amino acid sequence shown in SEQ ID NO:55. In some examples, the mutant IL-15 polypeptide has the IL-15 amino acid sequence set forth in SEQ ID NO:55. In some instances, the mutant IL-15 polypeptide has three or four aa substitutions compared to the amino acid sequence. The polypeptide has 5 or 6 aa substitutions compared to the IL-15 amino acid sequence shown in SEQ ID NO: 55. In some examples, the mutant IL-15 polypeptide has the IL-15 polypeptide set forth in SEQ ID NO:55. In some instances, the mutant IL-15 has 7 or 8 aa substitutions compared to the amino acid sequence. The polypeptide has 9 or 10 aa changes compared to the IL-15 amino acid sequence shown in SEQ ID NO: 55. It has substitutions.

[0225] A preferred variant IL-15 polypeptide sequence comprises at least 50 contiguous aa of SEQ ID NO: 55 (e.g. For example, at least 60, at least 70, at least 80, at least 90, at least 100, or For at least 110 consecutive aa), at least 80%, at least 85%, at least also have 90%, at least 95%, at least 98%, or at least 99% aa sequence identity, It includes polypeptide sequences having at least one aa substitution, deletion, or insertion.

[0226] 12. IL-21 and its variants In some instances, the MOD present in the masked TGF-β construct or complex of the present disclosure is the IL-21 polypeptide. Alternative splicing events give rise to different precursor proteins. The sequence of the IL-21 polypeptide, including the two isoforms formed by cloning, is known in the art. It is known in the art.

[0227] In one embodiment, the wild-type (wt.) IL-21 isoform 1 polypeptide has the following sequence: [ka] UniProtKB - Q9HBE4, with NCBI reference NP_068575.1, with aa 1-29 of the IL-21 protein signal It is a peptide.

[0228] The mature IL-21 isoform 1 polypeptide has the following aa sequence: [ka] may have:

[0229] In one embodiment, the wild-type (wt.) IL-21 isoform 2 polypeptide has the following sequence: [ka] NP_001193935.1, and aa 1 to 29 of the IL-21 protein are the signal peptide.

[0230] The mature IL-21 isoform 2 polypeptide has the following aa sequence: [ka] may have:

[0231] IL-21 has the same gamma chain as the IL-2 receptor, but a different IL-21R receptor subunit. It signals through a dimeric cell surface receptor, the human IL-21R isoform 1 precursor. The aa sequence of the protein is as follows: [ka] The NCBI reference sequence may be NP_068570.1, where aas 1 to 19 form the signal sequence and aas 20 to 538 form the complement. It is a mature polypeptide, with aas 233-253 being the transmembrane domain and aas 20-232 being the ectodomain. It's in.

[0232] In some examples, the variant IL-21 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 58 or 60. The binding affinity of the mature IL-21 receptor sequence (e.g., For example, all or part of the polypeptides set forth in SEQ ID NOs: 12 and 61, e.g., their ectodomains. For example, in some instances, Variants of IL-21 polypeptides comprising SEQ ID NO: 58 or 60 are those shown in SEQ ID NO: 58 or 60. a binding affinity that is at least 10% lower than that of a wt. IL-21 polypeptide containing the amino acid sequence At least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least At least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 12 and 61 with at least 95% lower or more than 95% lower binding affinity. The peptides may be used to bind to the IL-21 receptor, including all or part of the peptide, for example, the ectodomain thereof. do.

[0233] In some instances, the variant IL-21 polypeptide (e.g., a variant of SEQ ID NO: 58 or 60) is L-21 receptor (e.g., all or part of the polypeptides set forth in SEQ ID NOs: 12 and 61, e.g., , including their ectodomains) at 1 nM to 1 mM (e.g., 1 nM to 10 nM, 10 nM to 100 nM, The antibody has a binding affinity of 100 nM to 1 μM, 1 μM to 10 μM, 10 μM to 100 μM, or 100 μM to 1 mM. In some instances, the variant IL-21 polypeptide (e.g., a variant of SEQ ID NO: 58 or 60) is The whole or a part of the mature IL-21 receptor (for example, the whole polypeptide shown in SEQ ID NOs: 12 and 61) or a portion thereof, for example, including the ectodomain thereof) at 100 nM to 100 μM (for example , 100 nM to 1 μM, 1 μM to 10 μM, or 10 μM to 100 μM). In some examples, the variant IL-21 polypeptide (e.g., variants of SEQ ID NO: 58 or 60) is an IL-21 receptor. All or part of the polypeptide (e.g., all or part of the polypeptides set forth in SEQ ID NOS: 12 and 61) , for example, including their ectodomains), about 100 nM to about 200 nM, about 200 nM to about 30 0nM, about 300nM to about 400nM, about 400nM to about 500nM, about 500nM to about 600nM, about 600nM to about 700nM, about 7 00nM to about 800nM, about 800nM to about 900nM, about 900nM to about 1μM, about 1μM to about 5μM, about 5μM to about 10μ M, 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 It has a binding affinity of about 75 μM to about 100 μM.

[0234] In some examples, the variant IL-21 polypeptide (e.g., a variant of SEQ ID NO: 58 or 60) is It has one aa substitution compared to the IL-21 polypeptide sequence shown in SEQ ID NO: 58 or 60. In some instances, the variant IL-21 polypeptide (e.g., a variant of SEQ ID NO: 58 or 60) is IL-21 polypeptide sequence shown in sequence number 58 or 60, which has 2 to 10 aa substitutions. In some examples, the mutant IL-21 polypeptide is an IL-21 amino acid sequence set forth in SEQ ID NO: 58 or 60. In some instances, the mutant IL-21 polypeptide has two aa substitutions compared to the amino acid sequence. has 3 or 4 aa substitutions compared to the IL-21 amino acid sequence shown in SEQ ID NO: 58 or 60. In some examples, the variant IL-21 polypeptide has the IL-21 sequence set forth in SEQ ID NO: 58 or 60. In some instances, the mutant IL-2 has 5 or 6 aa substitutions compared to the 21 amino acid sequence. 1 polypeptide has 7 or 8 amino acids compared to the IL-21 amino acid sequence shown in SEQ ID NO: 58 or 60. In some examples, the mutant IL-21 polypeptide has aa substitutions. The IL-21 amino acid sequence has 9 or 10 aa substitutions compared to the IL-21 amino acid sequence shown in

[0235] A preferred IL-21 polypeptide sequence is at least 50 contiguous aa of SEQ ID NO: 58 or 60 (e.g., For example, at least 60, at least 70, at least 80, at least 90, at least 100, or For at least 110 consecutive aa), at least 80%, at least 85%, at least also have 90%, at least 95%, at least 98%, or at least 99% aa sequence identity, It includes polypeptide sequences having at least one aa substitution, deletion, or insertion.

[0236] 13. IL-23 and its variants In some instances, the MOD present in the masked TGF-β construct or complex of the present disclosure is an IL-23 polypeptide. IL-23 is composed of the IL-23A (IL-23p19) subunit and the IL-12B (IL- It is a heterodimeric cytokine composed of a 12p40 subunit (shared with IL-12). do.

[0237] In one embodiment, the wild-type (wt.) IL-23A polypeptide has the following sequence: [ka] The NCBI reference sequence is NP_057668.1, and aa 1 to 19 of the protein is the signal peptide, and aa 20 to 189 is the mature peptide.

[0238] The mature IL-23A polypeptide has the following aa sequence: [ka] may have:

[0239] In one embodiment, the wild-type (wt) IL-12B polypeptide has the following sequence: [ka] It has Unprot-PKKB-29460, and aa 1 to 22 of the protein is a signal peptide, and aa 23 to 328 is This is the mature peptide.

[0240] The mature IL-12B polypeptide has the following aa sequence: [ka] may have:

[0241] IL-23 is composed of the IL-23 receptor polypeptide (IL-23R) and a subunit common to the IL-12 receptor (IL-12R). RB1 or 12Rβ1) and signaling via a dimeric cell surface receptor composed of To communicate.

[0242] The IL-23R isoform 1 precursor protein sequence is as follows: [ka] The NCBI reference sequence may be NP_653302.2, where aas 1 to 23 form the signal sequence and aas 24 to 629 form the complement. It is a mature polypeptide, with aas 356-376 being the transmembrane domain and aas 24-355 being the ectodomain. It's in.

[0243] The 12RB1 isoform 1 precursor protein aa sequence is: [ka] (SEQ ID NO: 67) NCBI Reference Sequence NP_005526.1, wherein aas 1 to 23 form the signal sequence and aas Aas 24 to 662 is the mature polypeptide, aas 546 to 570 is the transmembrane domain, and aas 24 to 545 is It is an ectodomain.

[0244] In some instances, a mutant IL-23 (e.g., comprising a variant of SEQ ID NO: 63 and / or 65) polypeptide is The peptide is a binding parent of an IL-23 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 63 or 65. The mature IL-23 receptor sequence (e.g., the polypeptides shown in SEQ ID NOs: 66 and 67) is compared to the homologous sequence. low for all or part of the IL-23 receptor, including their ectodomains For example, in some instances, variants of IL-23 polypeptides (e.g., sequences thereof) may be used. (including variants of SEQ ID NO: 63 and / or 65) contains the amino acid sequence shown in SEQ ID NO: 63 or 65. at least 10% lower, at least 20% lower than the binding affinity of the IL-23 polypeptide, At least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, At least 70% lower, at least 80% lower, at least 90% lower, at least 95% lower, or with greater than 95% lower binding affinity (e.g., the polypeptides set forth in SEQ ID NOs: 66 and 67). The IL-23 receptor binds to the IL-23 receptor (including all or part of the domain, for example, the ectodomain thereof).

[0245] In some instances, a variant IL-23 polypeptide (e.g., a variant of SEQ ID NO: 63 and / or 65) is used. IL-23 receptor (e.g., all or part of the polypeptides set forth in SEQ ID NOs: 66 and 67) 1 nM to 1 mM (e.g., 1 nM to 10 nM) for a portion, e.g., including their ectodomains , 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 example, in some instances, a mutant IL-23 polypeptide (e.g., SEQ ID NO: 63) and / or variants of 65) may be expressed as mature IL-23 receptors (e.g., as shown in SEQ ID NOS: 66 and 67). about 1 for all or a portion of a polypeptide (including, for example, the ectodomain thereof) 00nM to about 200nM, about 200nM to about 300nM, about 300nM to about 400nM, about 400nM to about 500nM, about 500nM to about 600nM, about 600nM to about 700nM, about 700nM to about 800nM, about 800nM to about 900nM, about 900nM to about 1μM, about 1μM to about 5μM, about 5μM to about 10μM, about 10μM to about 20μM, about 20μM to about 30μM, about 30μM to about 50 The antibody has a binding affinity of about 50 μM to about 75 μM, or about 75 μM to about 100 μM.

[0246] In some instances, a variant IL-23 polypeptide (e.g., a variant of SEQ ID NO: 63 and / or 65) is used. ) has one amino acid sequence similar to that of the IL-23 polypeptide sequence shown in SEQ ID NO: 63 and / or 65. In some examples, a mutant IL-23 polypeptide (e.g., SEQ ID NO: 63 and / or SEQ ID NO: 64) has aa substitution. or 65) can be compared with the IL-23 polypeptide sequence shown in SEQ ID NO: 63 and / or 65. In some instances, the mutant IL-23 polypeptide has 2 to 10 aa substitutions in comparison to the sequence Compared to the IL-23 amino acid sequence shown in columns 63 and / or 65, it has two aa substitutions. In some examples, the mutant IL-23 polypeptide is an IL-23 polypeptide set forth in SEQ ID NOs: 63 and / or 65. In some instances, the mutant IL-23 has three or four aa substitutions compared to the amino acid sequence. The polypeptide has 5 amino acids different from the IL-23 amino acid sequence shown in SEQ ID NO: 63 and / or 65. or 6 aa substitutions. In some examples, the mutant IL-23 polypeptide has SEQ ID NO: 63 or and / or 7 or 8 aa substitutions compared to the IL-23 amino acid sequence shown in SEQ ID NO: 65. In some examples, the mutant IL-23 polypeptide is an IL-23 amino acid sequence shown in SEQ ID NO: 63 and / or 65. It has 9 or 10 aa substitutions compared to the amino acid sequence.

[0247] Preferred variant IL-23 polypeptide sequences include at least 50 sequences of SEQ ID NO: 63 and / or 65. the following 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 1 80, 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 aas) At least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or have at least 99% aa sequence identity and at least one aa substitution, deletion, or insertion The polypeptide sequence includes:

[0248] 14. Fas Ligand (FasL) and its Mutants In some instances, the MOD present in the masked TGF-β construct or complex of the present disclosure is the Fas ligand (FasL). FasL is a homomer of the tumor necrosis factor (TNF) family. FasL is a type II transmembrane protein. It activates Fas receptors by trimerization within target cells. signal, which forms a cell death-inducing complex that leads to apoptosis of the target cell. Soluble FasL is produced by matrix metalloproteinase-7 (MMP-7)-mediated cleavage of membrane-bound FasL. It results from cleavage at a conserved site.

[0249] In one embodiment, the wt. Homo sapiens FasL protein has the following sequence: [ka] (SEQ ID NO: 143), NCBI reference sequence NP_000630.1, UniProtKB-P48023, wherein residue 1 ~80 is present in the cytoplasm, 81-102 are transmembrane domains, and aas103-281 are present extracellularly (It is an ectodomain).

[0250] A suitable FasL polypeptide is FasL: [ka] It contains all or part of the ectodomain of

[0251] The Fas receptor has the following sequence: [ka] (SEQ ID NO: 145), NCBI reference sequence NP_000034.1, UniProtKB-P25445, in which aas26 ~173 forms the ectodomain (extracellular domain), and aas174-190 forms the transmembrane domain The cytoplasmic domain is formed by segments 191-335. The ectodomain is used to bind to FasL. Compatibility can be measured.

[0252] In some instances, the mutant FasL polypeptide (e.g., comprising a variant of SEQ ID NO: 144) comprises: compared to the binding affinity of a FasL polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 144, The mature Fas receptor sequence (all or part of the polypeptide shown in SEQ ID NO: 145, for example, It exhibits low binding affinity to FasL receptors, including the ectodomain of In some instances, the mutant FasL polypeptide (e.g., comprising a variant of SEQ ID NO: 144) is The binding affinity of the FasL polypeptide containing the amino acid sequence shown in SEQ ID NO: 144 is less than that of the FasL polypeptide containing the amino acid sequence shown in SEQ ID NO: 144. at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least at least 90% lower, at least 95% lower, or more than 95% lower binding affinity to the Fas receptor (sequence All or part of the polypeptide shown in sequence number 145, such as the ectodomain thereof, (including

[0253] In some instances, the mutant FasL polypeptide (e.g., comprising the variant of SEQ ID NO: 144) is as receptor (all or part of the polypeptide shown in SEQ ID NO: 145, for example, the ectodomain thereof) 1 nM to 1 mM (e.g., 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1 μM) , 1 μM to 10 μM, 10 μM to 100 μM, or 100 μM to 1 mM). In one example, the mutant FasL polypeptide (e.g., comprising the mutant of SEQ ID NO: 144) binds to the Fas receptor (All or part of the polypeptide shown in SEQ ID NO: 145, for example, the ectodomain thereof) , including) about 100 nM to about 200 nM, about 200 nM to about 300 nM, about 300 nM to about 400 nM, about 400 nM ~about 500nM, about 500nM to about 600nM, about 600nM to about 700nM, about 700nM to about 800nM, about 800nM to about 900n M, about 900nM to about 1μM, about 1μM to about 5μM, about 5μM to about 10μM, about 10μM to about 20μM, about 20μM to A binding affinity of 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. Has.

[0254] In some instances, the mutant FasL polypeptide (e.g., comprising a variant of SEQ ID NO: 144) comprises: It has one aa substitution compared to the FasL polypeptide sequence shown in SEQ ID NO: 144. In one example, a mutant FasL polypeptide (including, for example, a variant of SEQ ID NO: 144) may be selected from the group consisting of SEQ ID NO: 1 44. In the present specification, the mutant FasL polypeptide is compared to the FasL polypeptide sequence shown in SEQ ID NO: 144. In some examples, the mutant FasL polypeptide has two aa substitutions. Some examples include: In the present specification, the mutant FasL polypeptide is compared to the FasL polypeptide sequence shown in SEQ ID NO: 144. In some instances, the mutant FasL polypeptide has the sequence It has 7 or 8 aa substitutions compared to the FasL polypeptide sequence shown in number 144. In some examples, the mutant FasL polypeptide is the FasL polypeptide sequence set forth in SEQ ID NO:144. Compared to the sequence, there are 9 or 10 aa substitutions.

[0255] A preferred mutant FasL polypeptide sequence is at least 50 consecutive aa of SEQ ID NO: 144 (e.g. For example, 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 For consecutive aa), at least 80%, at least 85%, at least 90%, at least 9 Polypeptide sequences with 5%, at least 98%, or at least 99% aa sequence identity (e.g., For example, having at least one aa substitution, deletion, or insertion).

[0256] E. Scaffolding The scaffold polypeptide may, inter alia, comprise a masked TGF-β construct or other component of a complex. Acts as a structural element that provides a framework within which elements are organized (e.g. (See Figure 1, structure A with IgFc scaffold). The peptide sequence and the TGF-β polypeptide are located in trans (on different polypeptides of the complex). When the two are in the same position, the mutually specific and non-mutually specific molecules form a duplex (or higher-order structure). The scaffold sequence is such that the complex is in the open form and the TGF-β polypeptide sequence is in the open form (masking sequence). Even during periods when the molecules are able to interact with other molecules (not in direct contact with the matrix), The binding polypeptide sequence can be maintained in association with the TGF-β polypeptide. Depending on their nature, the field can induce protein folding and dimerization or multimerization (e.g. For example, in terms of homodimerization or heterodimerization achieved through a dimerization sequence, They can also function as organizational elements that provide higher-order structure. It also has a stable antibody in serum, particularly when it is an immunoglobulin heavy chain constant region (e.g., IgFc). Suitable scaffold polypeptides may, in some instances, contribute to increased half-life. In some instances, a suitable scaffold polypeptide is a different, non-immune polypeptide. Control masked TGF-β construct with a scaffold polypeptide having a globulin sequence The in vivo half-life ( For example, serum half-life) by at least about 10%, at least about 15%, at least about 25%, At least about 50%, at least about 2 times, at least about 2 times, at least about 5 times, 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 extension For example, in some instances, an IgFc polypeptide sequence (e.g., a knob-in-hole sequence) may be used. A linker (e.g., GGGSaa repeats of equal sequence length) is inserted between the two sequences (including mutually specific Ig sequences such as a pair of sequences). Control masked TGF-β with the IgFc polypeptide sequence replaced by the stability and / or potency of the masked TGF-β construct or complex compared to the normal TGF-β construct or complex; or prolonging the in vivo half-life (e.g., serum half-life). Prolonging the in vivo half-life may be achieved 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 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, The IgFc polypeptide may be at least about 50-fold, at least about 100-fold, or more than 100-fold. When used with masked TGF-β constructs, IgFc binds to the dimer of the masked TGF-β construct. These may include mutations that prevent the spontaneous formation of the body (see, e.g., Tianlei Ying et al., JB Chem., 287(23), pp. 19399-19408 (June 1, 2012)). Inducing cell lysis via complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC) Mutations that substantially reduce or eliminate the ability of an Ig polypeptide to bind to a target protein (e.g., the LAL mutations discussed below) A mutation).

[0257] The scaffold polypeptide sequence of the masked TGF-β construct is such that the scaffold interacts with other scaffold molecules. If the target gene contains one or more sequences that allow it to interact (specifically bind) with the target gene, it is considered to be masked. The TGF-β constructs may be homodimeric complexes (see, e.g., Figure 1, structure B) or heterodimeric complexes. (See, for example, Figure 1, structures C-F.) Masked TGF-β structures The scaffold also interacts (specifically binds) with other scaffold molecules to form higher-order structures. The sequence that forms a higher order multimeric structure may contain one or more aa sequences that allow for the formation of a higher order multimeric structure. Higher order masked TGF-β constructs or complexes (e.g., trimers, tetramers, pentamers, etc.) For example, a scaffold polypeptide containing an IgM Fc region (e.g., SEQ ID NO: 83) can be formed. The peptide may be pentameric (especially if the J chain sequence of, for example, SEQ ID NO: 84, is also expressed) or hexameric. This allows for the formation of masked TGF-β constructs or complexes. See Hypotheses. 77(6):959-61 (2011). Homo- and hetero-dimers, trimers, Masked TGF-β constructs in the form of tetramers, pentamers, etc., are useful for the production of masked TGF-β. It is called a complex.

[0258] The masked TGF-β is then coupled to one or more other polypeptide sequences (one or more specific MODs or MOs). If it is desired to combine two or more copies of the scaffold polypeptide sequence with one another, such as a copy of a corresponding one of the scaffold polypeptide sequences, the scaffold polypeptide sequence may be Mutual specific dimers tend to form heterodimers with the corresponding mutual specific binding (dimerization) sequences. The mutually specific binding sequences may, in some instances, form homodimers. Although it can form a complex, it preferentially dimerizes (binds more strongly) with its counterpart's mutually specific binding sequence. Therefore, a particular heterodimer consists of a mutually specific dimerization sequence and its counterpart. It can be formed when heterologous binding sequences join to form a pair of polypeptides. For example, a mutually specific dimerization sequence and its counterpart mutually specific binding sequence may When they combine to form a pair of polypeptides, selectively forming heterodimers, the peptide More than 60%, 70%, 80%, 90%, 95%, 98% or 99% of an equimolar mixture of The remaining peptides exist as monomers or homodimers.

[0259] Various aa sequences that bind specifically to each other or to themselves with sufficient affinity are masking These sequences can be used as dimerization sequences in TGF-β constructs or complexes (see, e.g., US Pat. No. 6,139,163). See Patent Application Publication No. 2003 / 0138440). The array has a relatively compact size (e.g. , about 300, 250, 225, 200, 175, 150, 125, 100, 75, or less than 50 aa, etc. The merization / multimerization sequence may be an immunoglobulin heavy chain constant region (IgFc) polypeptide sequence (immunoglobulin heavy chain constant region (IgFc) polypeptide sequence). a sequence comprising the CH2 to CH3 regions of an immunoglobulin; see, for example, Figures 2A to 2H and SEQ ID NOs: 68 to 83) , Fc knob-in-hole sequences (e.g., SEQ ID NOs: 77 and 78), collagen repeats Gly-Xaa-Yaa to Collagen domain-containing collectin family polypeptides (e.g., ACRP30 or is ACRP30-like protein), coiled-coil domain, leucine zipper domain, Fos / Jun Binding pair, Ig heavy chain region 1 (CH1) and light chain constant region CL sequence (e.g., kappa or lambda Ig light chain constant region sequence) Examples of suitable nucleotide sequences include, but are not limited to, a CH1 / CL pair, such as a CH1 sequence paired with a CH1 sequence.

[0260] In some embodiments, the scaffold polypeptide sequence functions as a dimerization or multimerization sequence. The polypeptide sequence of the immunoglobulin heavy chain constant region (CH2-CH3) capable of carrying out the antibody synthesis (e.g., Figures 2A-2H) and SEQ ID NOS: 68-83). In embodiments, the Ig polypeptide is, for example, a complement-dependent Substantially induces cell lysis via activation of cellular cytotoxicity (CDC) and / or antibody-dependent cellular cytotoxicity (ADCC). If the antibody would not induce cytolysis by Ig polypeptides, the ability to induce cytolysis by Ig polypeptides would be substantially reduced. In some instances, the Fc sequence may contain mutations that reduce or eliminate the Fc domains shown in Figures 2A-2H. At least about 70%, at least about 80%, or at least about 90% of the aa sequence of the Fc region , at least about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity Such immunoglobulin sequences have one or two interchain disulfide bonds. By this, the polypeptides of the masked TGF-β complex can be covalently bound. As discussed below, pairs such as knob-in-hole polypeptide pairs are particularly suitable. When mutually specific Ig sequences are used, additional disulfides are added to stabilize the dimer. A bond can be introduced.

[0261] In one embodiment, the scaffold polypeptide sequence of the masked TGF-β complex is shown in FIG. 2A (sequence At least 150 consecutive aas (at least 175, at least 180) of the IgA Fc sequence shown in column 68 at least 200, at least 225, at least 250, at least 275, at least 300, at least At least 325, or at least 350 consecutive aas), or for all aas, at least about 70 %, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least In one embodiment, the scaffold polypeptide comprises a sequence with at least about 99%, or 100%, aa sequence identity. The peptide sequence is a sequence consisting of at least 150 consecutive aas ( At least 175, at least 200, at least 225, at least 250, at least 275, at least at least 300, at least 325, or at least 350 consecutive aas), or for all aas At least about 70%, at least about 80%, at least about 90%, at least about 95%, In one embodiment, the sequence comprises a sequence with at least about 98%, at least about 99%, or 100% aa sequence identity. In the present specification, the scaffold polypeptide sequence is at least 12 of the IgE Fc sequence shown in Figure 2C (SEQ ID NO: 70). 5 consecutive aas (at least 150, at least 175, at least 200 consecutive aas), or For all AAS, 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 the IgG1 Fc sequence shown in Figure 2D ( At least 125 consecutive aas (at least 125 consecutive aas) of a wt. IgG Fc polypeptide sequence, such as SEQ ID NOs: 71 to 78. at least 150, at least 175, or at least 200 consecutive aas), or for all aas At least about 70%, at least about 80%, at least about 90%, at least about 95%, These sequences include sequences having at least about 98%, at least about 99%, or 100% aa sequence identity. In an embodiment, the scaffold polypeptide sequence is the IgG2 Fc polypeptide shown in Figure 2E (SEQ ID NO: 79). At least 125 consecutive aas (at least 150, at least 175, at least 2 00, at least 225, at least 250, at least 275, or at least 300), or all At least about 70%, at least about 80%, at least about 90%, at least Sequences with about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity In one embodiment, the scaffold polypeptide sequence comprises the IgG3 F At least 125 consecutive aas of the c sequence (at least 150, at least 175, at least 200 , or at least 225), or at least about 70%, at least about 80% of all aas %, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or In one embodiment, the scaffold polypeptide sequence comprises a sequence having 100% aa sequence identity. At least 125 consecutive aas (at least 125 consecutive aas) of the IgG4 Fc sequence shown in Figure 2G (SEQ ID NO: 81 or 82) at least 150, at least 175, at least 200, at least 225, or at least 250; or For all AAS, at least about 70%, at least about 80%, at least about 90%, have about 95%, at least about 98%, at least about 99%, or 100% aa sequence identity In one embodiment, the scaffold polypeptide sequence comprises the Ig sequence shown in Figure 2H (SEQ ID NO: 83). M Fc polypeptide sequence of at least 125 consecutive aas (at least 150, at least 175 , at least 200, at least 225, or at least 250), or at least at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least The immunoglobulins include sequences having about 98%, at least about 99%, or 100% aa sequence identity. The above-mentioned polypeptides of the masked TGF-β complex containing the phosphoscaffold polypeptide sequence ( For example, those shown in Figures 2A-2H have one or more cysteines between adjacent cysteines in their hinge regions. They can be covalently linked by the formation of two interchain disulfide bonds.

[0262] In some instances, the scaffold polypeptide present in the masked TGF-β construct or complex The dimerization sequence of the nucleotide sequence is at least about 700 nm for the human IgG1 Fc polypeptide shown in FIG. 2D. 0% (e.g., at least about 80%, 90%, 95%, 98%, 99%, or 100%) aa sequence identity and a substitution of N297 with alanine (FIG. 2D, N297A substitution as numbered in SEQ ID NO: 74, or In some instances, the masked TGF-β construct or complex may contain a N77 (N77) substitution. The dimerization sequence of the scaffold polypeptide includes the aa sequence shown in FIG. 2D (human IgG1Fc), but except for the substitution of N297 with an aa other than asparagine (N77 in the aa sequence shown in Figure 2D). Substitution at 7 results in the removal of the carbohydrate moiety modification, thereby reducing the This results in antibody sequences with reduced binding to complement component 1q ("C1q"), thereby inhibiting complement-dependent cell proliferation. This results in a reduction in cell damage.

[0263] In some instances, the scaffold polypeptide present within the masked TGF-β construct or complex may be The dimerization sequence of the peptide comprises the aa sequence shown in Figure 2D (human IgG1Fc), except for L234, Except for substitutions with aa other than syn (L14 in the aa sequence shown in Figure 2D), the lower hinge region of IgG (e.g. For example, L234 and other aas in aas234-LLGGPS-239, which corresponds to aas 14 to 19 of SEQ ID NO: 71, involved in binding to the Fc lambda receptor (FcλR), and therefore mutations at that position may In some instances, masked TGF-β receptors are reduced. The dimerization sequence of the scaffold polypeptide present in the construct or complex is the aa sequence shown in Figure 2D. (human IgG1 Fc), except for the substitution of L235 with an aa other than leucine (aa shown in Figure 2D). In some instances, the masked TGF-β construct or complex may contain a nucleotide sequence other than L15. The dimerization sequence of the scaffold polypeptide containing the L234A and L235A ("LALA") substitutions (shown in Figure 2D) positions corresponding to positions 14 and 15 of the wt. aa sequence; see, e.g., SEQ ID NO: 75). The aa sequences include those shown in FIG. 2D (e.g., wt. human IgG1 sequences).

[0264] In some instances, the scaffold polypeptide present within the masked TGF-β construct or complex may be The dimerization sequence of the peptide is determined by substitution of an aa other than proline at P331 (P111 in the aa sequence shown in Figure 2D). 2D (human IgG1 Fc), including the substitution P331S The substitution at P331, like the substitution at N297, increases the binding of C1q to the protein compared to the wt. protein. Reduced binding and therefore reduced complement-dependent cytotoxicity. D270, K322, and / or P32 9 (corresponding to D50, K122, and P119 of SEQ ID NO: 71 in Figure 2D) with, for example, alanine may be used alone or in place of the P331 substitution to reduce binding to C1q. In some instances, the masked TGF-β construct or scaffold polypeptide present in the complex The dimerization sequence may contain L234A and / or L235A substitutions (L14 and / or L15 of the aa sequence shown in Figure 2D). In some instances, the IgG1 Fc polypeptide contains a substitution of leucine with Ala. The dimerization sequence of the scaffold polypeptide present in the masked TGF-β construct or complex is 2D (wt. human IgG1Fc), except for L234 and / or L235 (shown in FIG. 2D). substitution of aa other than leucine at P331 (Figure 2) In some cases, substitution of aa other than proline at P111 of the aa sequence shown in D is omitted. The dimerization sequence of the scaffold polypeptide present in the masked TGF-β construct or complex The columns represent the L234F, L235E, and P331S substitutions (at aa positions 14, 15, and 111 of the aa sequence shown in Figure 2D). 2D (human IgG1Fc) comprising the "triple mutant" aa sequence (SEQ ID NO: 73) shown in FIG. 2D (human IgG1Fc) .

[0265] When asymmetric pairing between the two polypeptides of the masked TGF-β complex is desired , a dimerization sequence of a scaffold polypeptide present in a masked TGF-β construct or complex. comprises, consists essentially of, or consists of a mutually specific binding sequence. Heterologous binding sequences are those that are homologous to their polypeptide sequences (i.e., mutually specific sequences and their Mutually specific sequences (companion sequences), especially those based on variants of immunoglobulin Fc sequences, Such mutually specific polypeptide sequences include stabilizing disulfides. Knob-in-hole (KiH) without disulfide bonds or (KiH) with stabilizing disulfide bonds s, HA-TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, and A107 sequences. The mutually specific binding pair is the T366Y and Y407T mutants on the CH3 domain interface of IgG1. or corresponding residues in other immunoglobulins. Ring 9:7, 617-621 (1996) (substitutions are indicated according to the EU numbering scheme of Kabat et al. (1991) The second mutually specific binding pair is formed by the T366W substitution to form a knob, and with the formation of a hole by the triple substitutions T366S, L368A, and Y407V on the complementary Fc sequence. Xu et al. See mAbs 7:1, 231-242 (2015). Another mutually specific binding pair is Y349C, T366 a first Fc polypeptide having S, L368A, and Y407V substitutions and a second Fc polypeptide having S354C, and T366W substitutions; and a second Fc polypeptide comprising the amino acid sequence Y349C and S354C (a disulfide bond is formed between Y349C and S354C). (See Brinkmann and Konthermann, mAbs 9:2, 182-212 (2015). Fc poly The peptides, with or without knob-in-hole modifications, are designed to mimic the disulfide bonds between Fc polypeptides. It can be stabilized by the formation of bonds (e.g., hinge region disulfide bonds). Some interspecific inter-polypeptide binding sequences are summarized in Table 1 and shown in Figure 2D in brackets "{}". Shown are the aa positions shown with a cross reference to the numbering shown in the wt. IgG1 sequence (SEQ ID NO: 71). [Table 1]

[0266] In addition to the mutually specific pairs of sequences in Table 1, mutually specific "seed" (SEED) sequences are 45 residues from IgA in the IgG1 CH3 domain of the target sequence, and the interspecific sequence of the counterpart The IgA CH3 has 57 residues derived from IgG1. Ha et al., Frontiers in Immuno See 1.7:1-16 (2016).

[0267] In one embodiment, the scaffold sequence found in a masked TGF-β construct or complex is Columns indicate knob-in-hole (KiH), knob-in-hole with stabilizing disulfide (KiHs-s), HA- TF, ZW-1, 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, A107, or a seed sequence. The binding sequence includes a mutually specific binding sequence or its counterpart mutually specific binding sequence, which is selected from the binding sequence of the target molecule.

[0268] In one embodiment, the masked TGF-β complex comprises an IgG1 having a T146W KiH sequence substitution. a first polypeptide comprising a scaffold and an IgG1 scaffold having T146W, L148A, and Y187V KiH sequence substitutions; and a second polypeptide comprising a scaffold, the scaffold comprising at least 170 of an IgG1 of SEQ ID NO: 71; At least 180, at least 190, at least 200, at least 210, at least 220, or 2 At least 80%, 90%, 95%, 98%, 99%, or 100% for all 27 consecutive aas One or both scaffold aa sequences may optionally be numbered according to the Kabat numbering system. 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., P 395V), F405 (e.g., F405R), Y407 (e.g., Y407A), and K409 (e.g., K409Y). These substitutions include one or more substitutions at L14 and L15 (e.g., in the IgG1 sequence of SEQ ID NO: 71). , L14A / L15A "LALA" or L14F / L15E), N77 (e.g., N77A), P111 (e.g., P111S), L131 ( For example, L131K), T146 (for example, T146S), P175 (for example, P175V), F185 (for example, F185R), Y 187 (e.g., Y187A), and K189 (e.g., K189Y).

[0269] In one embodiment, the masked TGF-β complex comprises an IgG1 having a T146W KiH sequence substitution. a first polypeptide comprising a scaffold and an IgG1 scaffold having T146S, L148A, and Y187VKiH sequence substitutions; and a second polypeptide comprising a scaffold, the scaffold comprising at least 170 of an IgG1 of SEQ ID NO: 71; At least 180, at least 190, at least 200, at least 210, at least 220, or 2 At least 80%, 90%, 95%, 98%, 99%, or 100% for all 27 consecutive aas wherein none, one or both of the scaffold aa sequences have a sequence identity of Fc L14 and L2, which eliminate effector function by blocking interaction with the γ receptor. 15 substitutions (e.g., L234A and L235A "LALA" in Kabat numbering), and / or N77 substitutions (N2 97, e.g., N297A or N297G in the Kabat numbering). For example, SEQ ID NO: 77 in Figure 2D See also 78.

[0270] In one embodiment, the first and second polypeptides of the masked TGF-β complex are T146W and S134C KiHs-s substitutions in the first scaffold sequence and T146S, L148A, Y187V and Y 129C KiHs-s substitutions, and the scaffold comprises at least 170 of the IgG1 of SEQ ID NO: 71, at least 180, at least 190, at least 200, at least 210, at least 220, or all 227 At least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity for consecutive aas wherein 0, 1 or both of the scaffold aa sequences have a sequence having a sequence similar to that of an Fcγ receptor. L14 and L15 substitutions (e.g., For example, L234A and L235A "LALA" in Kabat numbering), and / or N77 substitutions (N297, e.g., For example, N297A or N297G in the Kabat numbering.

[0271] In one embodiment, the first and second polypeptides of the masked TGF-β complex are S144H and F185A HA-TF substitutions in one scaffold sequence and Y129T and T174F HA-TF substitutions in the second scaffold sequence and the scaffold comprises at least 170, at least 180, at least 190 of an IgG1 of SEQ ID NO: 71. For all 190, at least 200, at least 210, at least 220, or 227 consecutive aas and sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity. wherein none, one or both of the scaffold aa sequences block the interaction with the Fcγ receptor. L14 and L15 substitutions (e.g., Kabat numbering) that eliminate effector function by disrupting "LALA" for L234A and L235A in N297A or N297G in

[0272] In one embodiment, the first and second polypeptides of the masked TGF-β complex are T130V, L131Y, F185A, and Y187V ZW1 substitutions in the first scaffold sequence and T130V, T146 and ZW1 substitutions of at least 170 of the IgG1 of SEQ ID NO: 71. , at least 180, at least 190, at least 200, at least 210, at least 220, or is at least 80%, 90%, 95%, 98%, 99%, or 10% for all 227 consecutive aas. 0% sequence identity, wherein 0, 1, or both of the scaffold aa sequences are , which eliminates effector function by blocking interaction with Fcγ receptors, L14 and L15 substitutions (e.g., L234A and L235A "LALA" in Kabat numbering), and / or N77 substitutions Substitutions (N297, e.g., N297A or N297G in the Kabat numbering) are included.

[0273] In one embodiment, the first and second polypeptides of the masked TGF-β complex are K140D, D179M, and Y187A substitutions in the first scaffold sequence and E125R, Q127R, T 146V, and K189V 7.8.60 substitutions, and the scaffold comprises at least 170 of the IgG1 of SEQ ID NO: 71. , at least 180, at least 190, at least 200, at least 210, at least 220, or is at least 80%, 90%, 95%, 98%, 99%, or 10% for all 227 consecutive aas. 0% sequence identity, wherein 0, 1, or both of the scaffold aa sequences are , which eliminates effector function by blocking interaction with Fcγ receptors, L14 and L15 substitutions (e.g., L234A and L235A "LALA" in Kabat numbering), and / or N77 substitutions Substitutions (N297, e.g., N297A or N297G in the Kabat numbering) are included.

[0274] In one embodiment, the first and second β polypeptides of the masked TGF-β complex are K189D and K172D DD-KK substitutions in one scaffold sequence and D179K and E136KD D-KK substitutions in the second scaffold sequence and the scaffold comprises at least 170, at least 180, at least 190 of an IgG1 of SEQ ID NO: 71. For all 190, at least 200, at least 210, at least 220, or 227 consecutive aas and sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity. wherein none, one or both of the scaffold aa sequences block the interaction with the Fcγ receptor. L14 and L15 substitutions (e.g., Kabat numbering) that eliminate effector function by disrupting "LALA" for L234A and L235A in N297A or N297G in

[0275] In one embodiment, the first and second polypeptides of the masked TGF-β complex are K140E and K189W EW-RVT substitutions in the first scaffold sequence and Q127R, D179V, and F185T in the second scaffold sequence EW-RVT substitutions, and the scaffold comprises at least 170, at least 180, At least 190, at least 200, at least 210, at least 220, or all 227 consecutive At least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to the corresponding aas. wherein zero, one or both of the scaffold aa sequences have an interaction with an Fcγ receptor. L14 and L15 substitutions (e.g., , L234A and L235A "LALA" in Kabat numbering), and / or N77 substitutions (N297, e.g., K Includes N297A or N297G in the abat numbering.

[0276] In one embodiment, the first and second polypeptides of the masked TGF-β complex are K140E, K189W, and Y129C EW-RVTs-s substitutions in the first scaffold sequence and Q127R, D179V in the second scaffold sequence , F185T, and S134C EW-RVTs-s substitutions, and the scaffold comprises at least one of the IgG1 of SEQ ID NO: 71. At least 170, at least 180, at least 190, at least 200, at least 210, at least 220 or at least 80%, 90%, 95%, 98%, 99%, or more for all 227 consecutive aas comprises sequences with 100% sequence identity, where none, one or both of the scaffold aa sequences The former eliminates effector function by blocking interaction with Fcγ receptors. L14 and L15 substitutions (e.g., L234A and L235A "LALA" in Kabat numbering), and / or N7 Contains 7 substitutions (N297, e.g., N297A or N297G in Kabat numbering).

[0277] In one embodiment, the first and second polypeptides of the masked TGF-β complex are K150E and K189W A107 substitutions in the first scaffold sequence and E137N, D179V, and F185T A1 in the second scaffold sequence. 07 substitutions, the scaffold comprising at least 170, at least 180, at least 190 of the IgG1 of SEQ ID NO: 71. At least 190, at least 200, at least 210, at least 220, or all 227 consecutive a has at least 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to as wherein zero, one or both of the scaffold aa sequences interact with an Fcγ receptor. L14 and L15 substitutions (e.g., Kab) that eliminate effector function by blocking its use. at numbering), and / or N77 substitutions (N297, e.g., Kabat Includes N297A or N297G in the numbering.

[0278] As an alternative to using immunoglobulin heavy chain constant regions as scaffolds, immunoglobulin light chain constant regions have been used. The constant region is used to form a scaffold polypeptide sequence or a part of a scaffold polypeptide sequence. In one embodiment, the heavy chain CH1 sequence is a dimerization sequence. The first and second polypeptides of the scaffolded TGF-β complex comprise, in the first scaffold sequence, a gCH1 domain (e.g., the polypeptide of SEQ ID NO: 85), and in the second scaffold sequence, an Ig The scaffolds comprise the kappa chain constant region sequence (SEQ ID NO: 86), SEQ ID NO: 85 and / or SEQ ID NO: 86, respectively. At least 70, at least 80, at least 90, at least 100, or at least 110 repetitions of At least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the sequence of the following aas See Figures 2J and 2K. The CH1 and Igκ sequences are identical to each other. and thus, any desired cleavage sites formed using them as dimerization sequences can be enhanced. The stability of the CH1-Igκ heterodimer can be increased by modifying the nucleotide sequence of ... Substitutions that increase the quality include the MD13 combination in Chen et al., MAbs, 8(4):761-774 (2016). MD13 has two sequences each for CH1 and Igκ. Two substitutions were introduced: S64E and S66V substitutions (S70E of SEQ ID NO: 85 shown in Figure 2J). and S72V). The Igκ sequence is modified to contain S69L and T71S substitutions (sequence shown in Figure 2K). The nucleotide sequence is modified to include S68L and T70S (SEQ ID NO: 86).

[0279] In another embodiment, the first and second polypeptides of the masked TGF-β complex are In one scaffold sequence, the IgCH1 domain (e.g., the polypeptide of SEQ ID NO: 85) is included, and in the second and the scaffold comprises an Ig λ chain constant region sequence (SEQ ID NO: 87), each of which is At least 70, at least 80, at least 90, at least 100 of SEQ ID NO: 85 and / or 87; or at least 80%, 85%, 90%, 95%, 98% for at least 110 consecutive aas , 99%, or 100% sequence identity. See Figures 2J and 2K.

[0280] In some instances, the scaffold polymer of the first and second polypeptides of the masked TGF-β complex is The polypeptide sequences each contain a leucine zipper polypeptide as a dimerization sequence. The syn-zipper polypeptides associate with each other to form dimers (eg, homodimers). Non-limiting examples of leucine zipper polypeptides include, for example, those having the following aa sequences: Any one of the peptides: [ka] In some examples, the leucine zipper polypeptide has the following aa sequence: [ka] Includes. Additional leucine zipper polypeptides are known in the art, among these are Both are suitable for use as scaffolds or for incorporation into scaffolds as dimerization sequences.

[0281] In some instances, the scaffold polymer of the first and second polypeptides of the masked TGF-β complex is Each of the polypeptide sequences is a coiled-coil peptide that forms a dimer (e.g., a homodimer). Non-limiting examples of coiled-coil polypeptides include, for example, those having the following aa sequences: Any one of the peptides can be mentioned: [ka] .

[0282] In some instances, a dimer of the first and second polypeptides of the masked TGF-β complex Each scaffold polypeptide sequence allows for homodimerization by forming disulfide bonds. The polypeptide sequence includes at least one cysteine ​​residue capable of An example of a polypeptide sequence that can be used is the human FasL polypeptide: [ka] Examples include:

[0283] Masked TGF-β complexes larger than dimers (e.g., trimers, tetramers, pentamers) Peptides suitable as multimerization (oligomerization) sequences that allow the formation of mers, hexamers, etc. The nucleotides may form, but are not limited to, hexamers or pentamers (particularly, the nucleotide sequences shown in Figure 2I). When combined with a mature J chain peptide lacking a signal sequence, the IgM constant region (see, e.g., Figure 2H) Collagen domains that form trimers can also be used. The collagen domain may comprise (Gly-Xaa-Xaa)n, where Xaa is any aa, or n is is an integer (e.g., 10 to 40), Xaa and Yaa are independently any aa, and n is 10 to 40. In the Gly-Xaa-Yaa sequence, Xaa and Yaa are integers. 25% and 50% of the occurrences of Gly-Xaa-Yaa are , 75%, 80%, 90% or more than 95%, or at each occurrence of Gly-Xaa-Yaa, These are often proline and hydroxyproline, respectively. The main chain contains the sequence (Gly-Xaa-Pro)n, where n is an integer (e.g., 10 to 40). The ligomerization peptide may comprise the following aa sequence: [ka] .

[0284] F. TGF-β polypeptide As described above, the masked TGF-β construct or complex comprises at least one TGF-β polypeptide (e.g., one or more independently selected TGF-β polypeptides). The amino acid sequence of a β polypeptide is known in the art. The TGF-β polypeptide present in the bound TGF-β construct or complex is a TGF-β1 polypeptide. In some instances, the peptide is present within a masked TGF-β construct or complex. The TGF-β polypeptide is a TGF-β2 polypeptide. The TGF-β polypeptide present in the TGF-β construct or complex is a TGF-β3 polypeptide. be.

[0285] The TGF-β1, TGF-β2, or TGF-β3 polypeptide sequence may be a masked TGF-β construct. or can be incorporated into a complex, various factors will determine the choice of the particular TGF-β polypeptide, as well as The specific sequences and aa substitutions used may influence the choice of TGF-β1 and TGF-β2. β3, when expressed in many mammalian cell lines (e.g., CHO cells), In addition, dimerized TGF-β (e.g., TGF-β2) undergoes "clipping" of the amino acid sequence. It has a higher affinity for the βR3 (betaglycan receptor) than for the TβR2 receptor, This results in off-target binding and large in vivo activity of non-signaling TβR3 molecules. This may result in the loss of biologically active masked protein to the TβR3 pool. High affinity to the target protein, linked by disulfide bonds to minimize off-target binding It may be desirable to substitute residues that result in combined dimeric TGF-β molecules. Thus, cysteine ​​77 (C77) can be substituted with an amino acid other than cysteine ​​(e.g., a cysteine ​​residue forming a C77S substitution). may be substituted by phosphorus).

[0286] Suitable TGF-β polypeptides can have from about 70 aas to about 125 aas, for example, suitable Suitable TGF-β polypeptides include those having 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 100aas to about 105aas, about 105aas to about 110aas, about 110aas to about 112aas, about 113aas to about 120aas, Alternatively, the TGF-β polypeptide may have a length of about 120 aas to about 125 aas. The composition of human TGF-β1 polypeptide, human TGF-β2 polypeptide, or human TGF-β3 polypeptide At least 80, at least 90, at least 100, or at least 110 consecutive ripe For aas, 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 may comprise an amino acid sequence with 100% aa sequence identity.

[0287] 1. TGF-β1 polypeptide A suitable TGF-β1 polypeptide has the TGF-β1 amino acid sequence [ka] (SEQ ID NO: 105, length of 112 aas) For at least 100, at least 110, or at least 112 aas, at least 60%, at least At least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least The TGF-β1 may contain an amino acid sequence having 99% or 100% aa sequence identity. The polypeptide has a length of approximately 112 aas. TGF-β1 preproprotein is shown in SEQ ID NO: 106 The amino acids R25, C77, V92 and R94 are shown in bold and italics (see Figure 4).

[0288] In some instances, a suitable TGF-β1 polypeptide comprises a C77S substitution. Thus, in some instances, a suitable TGF-β1 polypeptide may have the following TGF-β1 amino acid sequence: Column: [ka] at least 70, at least 80, at least 90, at least 100, at least 110, or 11 For 2 aas, 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 The amino acid sequence is as follows: Show it with your body.

[0289] 2. TGF-β2 polypeptide A suitable TGF-β2 polypeptide has the following TGF-β2 amino acid sequence: [ka] at least 70, at least 80, at least 90, at least 100, at least 110, or 11 For 2 aas, 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 The TGF-β2 polypeptide may comprise an amino acid sequence having a length of about 112 aas. The TGF-β2 preproprotein is shown in FIG. 3 as SEQ ID NO: 109. Residues Lys25, Ile92, and / or Lys94 are shown in bold and italics.

[0290] In some instances, a suitable TGF-β2 polypeptide comprises a C77S substitution. Thus, in some instances, a suitable TGF-β2 polypeptide may have the following TGF-β2 amino acid sequence: Column: [ka] at least 70, at least 80, at least 90, at least 100, at least 110, or 11 For 2 aas, 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 The amino acid sequence can include the amino acid sequence shown below, wherein amino acid 77 is Ser.

[0291] 3. TGF-β3 Polypeptide A suitable TGF-β3 polypeptide has the following TGF-β3 amino acid sequence: [ka] at least 70, at least 80, at least 90, at least 100, at least 110, or 11 For 2 aas, 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 The TGF-β3 polypeptide may comprise an amino acid sequence having a length of about 112 aas. The TGF-β3 isoform 1 preproprotein is shown in FIG. 3 as SEQ ID NO: 112. 25, 92, and 94 are shown in bold and italic.

[0292] In some instances, a suitable TGF-β3 polypeptide comprises a C77S substitution. A suitable TGF-β3 polypeptide has the following TGF-β3 amino acid sequence: [ka] at least 70, at least 80, at least 90, at least 100, at least 110, or 11 For 2 aas, 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 The amino acid sequence is as follows: Shown in.

[0293] 4. Additional TGF-β Polypeptide Sequence Variations Sequence mutations that alter the dimerization of TGF-β molecules (e.g., cysteine ​​77 substitutions such as C77S) Additionally, TGF-β1-3 polypeptides with sequence variations that affect affinity and other properties are also available. , can be incorporated into a masked TGF-β construct or complex. The construct or complex may comprise a masking polypeptide (e.g., a TβR polypeptide, such as a TβRII polypeptide). When TGF-β mutants with low affinity for the polypeptide are included, these components more readily dissociated and masked TGF-β polypeptides to cellular TβR proteins TβRII proteins are generally heteromeric TβR complexes that interact with TGF-β. Since it is the first peptide in the signal transduction complex, its interaction with TβRII regulates the activation signal of TGF-β. This effectively regulates the participation of TGF-β in the signal transduction complex. Mutations controlling compatibility masked TGF-β constructs and active signaling of complexes. Effectively control participation in the complex.

[0294] The present disclosure is not intended to limit the scope of the present invention to the use of other identical masked TGF-β constructs (or constructs) that do not have the sequence mutation(s). mutant matrices that have altered (e.g., decreased) affinity for each other (compared to the complex); a masked TβR (e.g., TβRII) polypeptide sequence and / or a mutant TGF-β polypeptide The present invention encompasses and provides masked TGF-β constructs and complexes containing TGF-β. The affinity between a polypeptide and a TβR (e.g., TβRII) polypeptide is determined by the MOD and its co-MO D can be measured using the (BLI) method described above.

[0295] a. Additional TGF-β2 sequence mutations The present disclosure provides a method for the preparation of a masked TβR (e.g., TβRII) polypeptide sequence and a wt. TGF-β2 polypeptide. and a masked TGF-β2 construct comprising either a nucleotide or a mutant TGF-β2 polypeptide. The present invention encompasses and provides compounds and conjugates, wherein the mutant polypeptides are (compared to an otherwise identical wt. TGF-β polypeptide sequence without any mutations) It has an affinity for

[0296] The present disclosure provides a method for the preparation of a masked TβRII receptor sequence and at least 100 contiguous sequences of SEQ ID NO: 108. have greater than 85% (e.g., greater than 90%, greater than 95%, greater than 98%, or greater than 99%) sequence identity to the aa A masked TGF-β construct comprising a mutant TGF-β2 polypeptide comprising an amino acid sequence the amino acid sequence is a sequence of the mutant TGF-β against the TβRII receptor sequence; 2 polypeptide.

[0297] In some instances, the masked TGF-β construct or complex comprises a masked TβRII polynucleotide. peptide and one or more, two or more, or all three of Lys25, Ile92, and / or Lys94. and mutant TGF-β (e.g., TGF-β2) polypeptides containing substitutions (see, for residue positions, See Figure 4 for SEQ ID NO: 108 and corresponding residues in TGF-β1 and TGF-β3 These aa residues affect the affinity of TGF-β2 for the TGF-β2 polypeptide. (See also Crescenzo et al., J. Mol. Biol. 355:47-62 (2006)). The masked TGF-β polypeptide may optionally be one or more polypeptides, such as IL-2 or a variant thereof. In one example, the masked TGF-β construct or complex comprises an independently selected MOD. The present invention relates to a masked TβRII polypeptide and a polypeptide having an aa other than Lys or Arg at position 25 of SEQ ID NO: 108. and optionally, one or more independently selected MODs (e.g., 1 The present invention relates to a method for treating IL-2 MOD polypeptides, including the steps of: The masked TGF-β construct or complex having the polypeptide has I at position 92 of SEQ ID NO: 108. TGF-β2 polynucleotides having an aa other than Ile or Val (or an aa other than Ile, Val or Leu at position 92) peptide, and optionally one or more independently selected MODs (e.g., one or more IL-2 M OD polypeptide or an affinity-reduced variant thereof). A masked TGF-β construct or complex having Lys or Arg at position 94 of SEQ ID NO: 108. and optionally, one or more independently selected TGF-β2 polypeptides having aa other than MOD (e.g., one or more IL-2 MOD polypeptides or affinity-reduced variants thereof). The masked TGF-β construct or complex having a masked TβRII polypeptide is selected from the group consisting of Lys25, I TGF-β2 polypeptides containing one or more, two or more, or all three substitutions of le92, and / or Lys94. The TβR masking peptide may further comprise one or more independently selected MODs. The masked TGF-β construct or complex having the II polypeptide is and / or Lys94. and further comprising one or more independently selected IL-2 MODs or affinity-reduced variants thereof. It can be seen.

[0298] b. Additional TGF-β1 and TGF-β3 sequence mutations In some instances, the masked TGF-β construct or complex comprises a masked TβRII polynucleotide. peptide and one corresponding to Lys25, Ile92, and / or Lys94 in SEQ ID NO: 108 of TGF-β2. Mutant TGF-β1 or TGF-β3 polypeptides containing substitutions at one, two or more, or all three aa positions. In TGF-β1 or TGF-β3, the aa corresponding to Lys25 is Arg25, and the aa corresponding to Ile92 is The corresponding aa is Val92, and the corresponding aa to Lys94 is Arg94, each of which is a conservative substitution. For example, SEQ ID NOs: 106 and 107 for TGF-β1 and SEQ ID NO: 108 for TGF-β3. See 112 and 113.

[0299] As noted above, the masked TGF-β construct or complex may optionally be IL-2 or a variant thereof. In one example, the masking TβRII polynucleotide comprises one or more independently selected MODs, such as variants. The masked TGF-β construct or complex having a peptide at position 25 other than Arg or Lys. aa, and optionally one or more independently selected TGF-β1 or TGF-β3 polypeptides having The IL-2 MOD polypeptides include one or more IL-2 MOD polypeptides or affinity-reduced variants thereof. In an example, a masked TGF-β construct or complex having a masked TβRII polypeptide. The nucleotide sequence is a T nucleotide sequence having an aa other than Val or Ile at position 92 (or an aa other than Ile, Val, or Leu at position 92). Transforming Growth Factor-beta 1 (TGF-β1) or transforming growth factor-beta 3 (TGF-β3) polypeptide, and optionally one or more independently selected MODs (e.g., In another example, the IL-2 MOD polypeptide may comprise one or more IL-2 MOD polypeptides or affinity-reduced variants thereof. The masked TGF-β construct or complex having a masked TβRII polypeptide may be selected from the group consisting of Arg or a TGF-β2 polypeptide having an aa other than Lys, and optionally one or more independently selected The present invention also includes a MOD (eg, one or more IL-2 MOD polypeptides or affinity-reduced variants thereof) that is administered intracellularly. In one particular example, a masked TGF-β construct having a masked TβRII polypeptide is The construct or complex may comprise one or more, two or more, or three of Arg25, Val92, and / or Arg94. and one or more independently selected TGF-β1 or TGF-β3 polypeptides containing all of the substitutions. In another specific example, the masking TβRII polypeptide further comprises a MOD comprising a masking TβRII polypeptide. The TGF-β construct or complex may contain one or more of Arg25, Val92, and / or Arg94. , two or more, or all three substitutions, and one or more or an affinity-reduced variant thereof.

[0300] G. TGF-β Receptor Polypeptides and Other Polypeptides that Bind and Mask TGF-β In any of the above-described TGF-β polypeptides or polypeptide complexes, the TGF-β polypeptide The polypeptide that binds to and masks the TβRI peptide (the "masking polypeptide") is , TβRII, TβRIII, and anti-TGF-β antibodies or fragments thereof (e.g., Fab, single-chain antibodies, etc.). Which can take a variety of forms.

[0301] 1. TGF-β receptor polypeptide The masking of TGF-β in masked TGF-β constructs and complexes is due to the TGF-β polyclonal antibody. A polypeptide sufficient to bind to a peptide (e.g., TGF-β1, TGF-β2, or TGF-β3). a TGF-β receptor fragment (e.g., a TβRI, TβRII, or TβRIII ectodomain sequence) comprising the sequence In one embodiment, the masking sequence is TβRI, T It contains all or part of the βRII or TβRIII ectodomain.

[0302] TGF-β receptor I (TβRI) In one embodiment, the masking agent for TGF-β within the masked TGF-β construct or complex is a The polypeptide sequence may be derived from TβRI (e.g., SEQ ID NO: 114 for isoform 1). In some instances, the TGF-βRI domain may comprise all or part of the TβRI ectodomain (aas 34-126). A suitable TβRI polypeptide for masking TβRI ectodomain aa array: [ka] for at least 70, at least 80, at least 90, at least 100, or 103 aas of At least 60%, at least 70%, at least 80%, at least 90%, at least 95% , an amino acid sequence having at least 98%, at least 99%, or 100% aa sequence identity. include.

[0303] b. TGF-β receptor II (TβRII) In an embodiment, the TGF-β in the masked TGF-β construct or complex is masked. The polypeptide sequence may be derived from TβRII (e.g., SEQ ID NO: 116 of isoform A). In one embodiment, the TβRII ectodomain sequence (aas 24 to 177) may be included in its entirety or in part. Suitable TβRII isoform A polypeptides for masking TGF-β are listed below. aa sequence of βRII isoform A ectodomain: [ka] At least 70, at least 80, at least 90, at least 100, at least 110, or less At least 120, at least 130, at least 140, at least 150, or at least 154 aas At least 60%, at least 70%, at least 80%, at least 90%, at least Amino acids with at least 95%, at least 98%, at least 99%, or 100% aa sequence identity The position of the aspartic acid residue corresponding to D118 in the B isoform may be Indicated in bold, underlined, and italic.

[0304] In one embodiment, the masking agent for TGF-β in the masked TGF-β construct or complex is a The polypeptide sequence can be derived from TβRII isoform B (SEQ ID NO: 118) and can be a TβRII ectodomain. In one embodiment, TGF-β is masked by a nucleotide sequence encoding a TGF-β domain (aas 24 to 166). Suitable TβRII isoform B polypeptides for use in cloning include those described below. Form B ectodomain aa sequence: [ka] for at least 70, at least 80, at least 90, at least 100, or 103 aas of 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 As discussed below, F30, D32, S52, E55, or D118 (italic and bold) Any one or more of the amino acids other than the naturally occurring aa at those positions (e.g., alanine) may be present. The polypeptide sequence that masks TGF-β may be replaced by D118A or D118 The TGF-β masking sequence may comprise the polypeptide of SEQ ID NO: 119 with an R substitution. D118A or D118R substitution and one or more of F30A, D32N, S52L and / or E55A substitutions The peptide may comprise the peptide of SEQ ID NO: 119.

[0305] The ectodomain of TβRII can be used as a masking polypeptide, but the protein The region of the protein has charged and hydrophobic patches, and these patches are unfavorable pI nd In addition, TβRII may be toxic to cells expressing the polypeptide. Combining the ectodomain with an active TGF-β polypeptide allows for association with cell surface TβRI. These can be combined to activate their signaling receptors (e.g., via the Smad pathway). This complex can induce signal transduction. The TβRII ectodomain used to mask TGF-β is removed or modified. By modifying the main sequence, the masked TGF-β was able to bind to the cell surface. To avoid unintended intracellular stimulation other than via the heterodimeric TβRI / TβRII complex. Furthermore, modification of TβRII can increase the affinity of TβRII for TGF-β (e.g., TGF-β3). can alter (e.g., decrease) the activity of TGF-β, thereby unmasking it. This allows for the regulation of TGF-β and increases the availability of TGF-β as a signaling molecule. a mask containing a TβR (e.g., TβRII) peptide with the highest affinity for TGF-β3; The masked TGF-β construct or complex most tightly masks the TGF-β sequence and has the same effect. In contrast, higher doses are required to achieve the desired affinity for TβRII. The aa substitutions in unmask the TGF-β polypeptide and are biologically effective at low doses. See, for example, Example 3.

[0306] Thus, signaling by masked TGF-β polypeptides via TβRI Blocking / limiting and / or altering the affinity of the masked TβRII polypeptide for TGF-β ( Many modifications to TβRII can be made to the TβRII polypeptide sequence if desired (e.g., reduction of TβRII activity). Possible modifications include the deletion of the N-terminal 25 amino acids of the above length of 1 to 25 aa. Loss (e.g., Δ14, Δ25) and / or L27, F30, D32, S49, 150, T51, S52, I53, E55, V77 The association of TβRI with TβRII may be mediated by substitution at one or more of D118, D119, and / or E119. Some specific modifications that result in reduced binding and reduced affinity for TGF-β include: Based on column number 119, L27A, F30A, D32A, D32N, S49A, I50A, T51A, S52A, S52L, I53A, E 55A, V77A, D118A, D118R, E119A, and / or E119Q. For example, see J. Groppe et al., MolCell 29, 157-168, (2008) and De Crescenzo et al., See JMB355, 47-62 (2006). These substitutions are useful for TGF-β3-TβRII and TβRI-TβRII complexes. See Figure X for the effect on synthesis. Modifications of TβRII, including substitutions (e.g., F24A), promote transcription through the canonical SMAD signaling pathway. In one embodiment, the mature TβRIIB isoform (sequence number 1001001) is used to substantially or completely block the signal. Aspartic acid at position 118 (D118) of column 119 can be substituted with an amino acid other than Asp or Glu, such as Ala. D118 is substituted with Arg to give a "D118A" substitution, or D118R to Arg. The corresponding Asp residues are shown in SEQ ID NOs: 117-123 (bold and underlined in Figure 5B). N-terminal deletion of 25 aa (e.g., deletion of Δ25) and / or substitution at F24 (e.g., substitution of F24A) It can be combined with a 118 substitution (e.g., D118A or D118R). It can also be combined with an N-terminal deletion ( For example, deletion of Δ25) and / or substitution at F24 (for example, substitution of F24A) Substitution with any of the following substitutions: 9, 150, T51, S52, I53, E55, V77, D118, and / or E119 (e.g., D118A), and in particular those positions listed in SEQ ID NO: 119 above. Specific substitutions may be combined with any of the above to modify affinity.

[0307] Deletions of the N-terminus of the TβRII polypeptide can also result in loss of interaction with TβRI, Masked TGF-β constructs and complexes containing βRII polypeptides inhibit TβRI signaling. This prevents the TβRII polypeptide from acting as a constitutively active complex that participates in and activates the TβRII polypeptide. A 14-aa deletion (Δ14) of the peptide substantially reduced the interaction of the protein with TβRI, resulting in TβRII. The Δ25aa deletion of βRI completely blocks the interaction with TβRI. , substantially altering the pI of the protein, with the Δ14 TβRII ectodomain mutant changing it to approximately 4.5–5. 0 (e.g., about 4.74). Thus, the TGF-β construct or complex has a pI of 14 to 25 aas (e.g., and N-terminal deletions such as 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 aa. and a TβRII ectodomain polypeptide (e.g., the polypeptide of SEQ ID NO: 117 or 118). The TGF-β polypeptide in the masked TGF-β construct or complex may be masked. Modified ectodomains, including those that limit interaction with TβRI, can be utilized to The sequences are listed in the paragraphs below.

[0308] In one embodiment, the masking agent for TGF-β in the masked TGF-β construct or complex is a The sequence is the TβRII isoform B ectodomain sequence: [ka] for at least 70, at least 80, at least 90, at least 100, or 103 aas of 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. Any one or more of 30, D32, S52, E55, or D118 (italic and bold) may be substituted with an amino acid other than the naturally occurring aa (e.g., alanine). The TGF-β masking sequence includes the peptide of SEQ ID NO: 120 with a D118A substitution. In an embodiment, the TGF-β masking sequence comprises a D118A substitution and F30A, D32N, S52L, and / or and one or more of the E55A substitutions.

[0309] N-terminal deletions of TβRII, e.g., 14–25 aas (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 2 2, 23, 24, or 25 aa) to produce masked TGF-β / Tβ that interact with TβRI. Blocks unintended intracellular signaling caused by the βRII complex, and inhibits F30, D32, S52, and E55 and / or other TβRII ectodomain substitutions at any one or more positions in D118. This may be in combination with a βRII ectodomain substitution. The combination of deletions and substitutions allows the masked TGF-β construct or complex to remain membrane-bound in cells. It is confirmed that the compound does not induce cell signaling other than that mediated by TβRI and TβRII receptors. To do so.

[0310] In one embodiment, the TGF-β in the masked TGF-β construct or complex is The sequence is the TβRII isoform B ectodomain sequence: [ka] (In this sequence, aas1 to 14 are deleted (Δ14)) For at least 90, at least 100, or 103 aas, at least 60%, at least 70%, or at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or contains sequences with 100% aa sequence identity. F30, D32, S52, E55, or D118 (italic and bold) Any one or more of the amino acids (aa, b, c) may be an amino acid other than the naturally occurring aa at those positions (e.g., In one embodiment, the TGF-β masking sequence is In one embodiment, the TGF-β is masked by a peptide of SEQ ID NO: 120 having a D118A substitution. The matching sequence contains a D118A substitution and one or more of the following substitutions: F30A, D32N, S52L, and / or E55A. The polypeptide of SEQ ID NO: 121 has the formula:

[0311] In one embodiment, the masking agent for TGF-β in the masked TGF-β construct or complex is a The sequence is as follows: [ka] (In this sequence, aas1 to 25 are deleted (Δ25)) For at least 90, at least 100, or 103 aas, at least 60%, at least 70%, or at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or contains sequences with 100% aa sequence identity. F30, D32, S52, E55, or D118 (italic and bold) Any one or more of the amino acids (aa, b, c) may be an amino acid other than the naturally occurring aa at those positions (e.g., In one embodiment, the TGF-β masking sequence is , comprising the polypeptide of SEQ ID NO: 122 with a D118A substitution (shown as SEQ ID NO: 123 in Figure 5B) In one embodiment, the masking of TGF-β in the masked TGF-β construct or complex is carried out by The sequence has a D118A substitution and one or more of the following substitutions: F30A, D32N, S52L, and / or E55A. In one embodiment, the masked TGF-β construct comprises the peptide of SEQ ID NO: 122. or the sequence masking TGF-β in the complex is SEQ ID NO: 122 with D118A and F30A substitutions (See Figure 5B). In one embodiment, the masked TGF-β construct comprises a peptide of The sequence masking TGF-β in the product or complex is SEQ ID NO: 1 with D118A and D32N substitutions. 22 (see FIG. 5B). In one embodiment, the masked TGF-β structure The sequence masking TGF-β in the construct or complex is SEQ ID NO: 1 with D118A and S52L substitutions. No. 122 (see Figure 5B). In one embodiment, the masked TGF- The sequence masking TGF-β in the β construct or complex is SEQ ID NO: 1, which has D118A and E55A. No. 122 (see Figure 5B).

[0312] c. TGF-β receptor III (TβRIII) In one embodiment, the masking agent for TGF-β in the masked TGF-β construct or complex is a The polypeptide sequence can be derived from TβRIII (e.g., SEQ ID NO: 124 for isoform A and isoform B, SEQ ID NO: 125), all or part of the TβRIII ectodomain (A isoform In some instances, the TGF-β may contain aaas 27-787 of the A isoform or 27-786 of the B isoform. Suitable TβRIII polypeptides for masking include the TβRIIIA isoform or the B isoform. The ectodomain sequence of the form (e.g., SEQ ID NO: 124 or SEQ ID NO: 125, as provided in Figure 5C) At least 70, at least 80, at least 90, at least 100, or 120 a as at least 60%, at least 70%, at least 80%, at least 90%, at least Amino acids having at least 95%, at least 98%, at least 99%, or 100% aa sequence identity. Contains the amino acid sequence.

[0313] 2. Antibodies The TGF-β receptor polypeptide (e.g., the ectodomain sequence) is a masked TGF-β It may also function to bind to and mask the TGF-β polypeptide in the construct or complex. However, other polypeptide sequences (protein sequences) that bind to the TGF-β sequence may also be used to mask the TGF-β sequence. It can be used as a masking polypeptide to mask TGF-β. Suitable polypeptide or protein sequences that can be used include those that have affinity for TGF-β. antibodies specific for one or more of TGF-β1, TGF-β2, or TGF-β3, or The present invention relates to nanobodies, fragments thereof, nanobodies with affinity for TGF-β polypeptides, and in particular single and α-, β- and β-receptor antibodies (e.g., any of which may be humanized). Several neutralizing antibodies, including scFV antibodies, have been reported. See, for example, U.S. Pat. No. 9,090,688. In view of the embodiments and / or aspects of the invention described in this disclosure, The TβR (e.g., TβRII) sequence used to mask the TGF-β polypeptide is TG A masking antibody sequence (e.g., scFV or nanobodies) with affinity for F-beta polypeptides. For example, the TGF-β receptor sequence may be replaced with a masking sequence of the TGF-β polypeptide. In each of the masked TGF-β constructs or complexes of FIG. 1 used for The receptor polypeptide is a masking antibody having affinity for a TGF-β polypeptide. It may be replaced by a polypeptide (e.g., an scFV or a nanobody).

[0314] One potential advantage of using an antibody (e.g., a single-chain antibody) as a masking polypeptide is One is to limit it to the isoform(s) of TGF-β polypeptide that is / are masked. For example, the ability to bind to TGF-β1 using a single-chain antibody sequence based on methimumab (CAT192) (e.g. For example, Lord et al., mAbs 10(3):444-452 (2018)) were used to detect TGF-β isoforms. , which can be masked when present in a TGF-β construct or complex. In an embodiment, a single chain antibody sequence specific for TGF-β2 is used to identify TGF-β isoforms. , when present in a TGF-β construct or complex. , using a single-chain antibody sequence specific for TGF-β3, TGF-β isoforms were identified, which are TGF- The single chain antibody also masks the TGF-β antigen when present in a TGF-β construct or complex. Isoform combinations (e.g., the following: TGF-β1 and TGF-β2, TGF-β1 and TGF-β3, and a masked TGF-β construct selected from the group consisting of TGF-β2 and TGF-β3; The ectodomain sequence present in the complex can be specific for the ectodomain sequence. The single chain antibody also binds to TGF-β1, TGF-β2, and the like present in the masked TGF-β construct or complex. , and may be approximately specific for the TGF-β3 ectodomain sequence. See, for example, US Pat. No. 5,049,014 / 164709. The antibody and single chain antibody have affinity for the target TGF-β polypeptide sequence. Hybridomas and / or modifications (e.g., combinatorial modifications) to the variable region sequences of The antibodies can be prepared by a variety of methods, including screening for the following:

[0315] In one embodiment, the masked TGF-β construct or complex comprises a TGF-β sequence (e.g., T In one such embodiment, the single chain antibody is a single chain antibody for masking the GF-β3 sequence. The amino acid sequence corresponds to TGF-β3 as set forth in SEQ ID NO: 111, which contains a C77S substitution (see SEQ ID NO: 112). It is specific.

[0316] H. Linker As described above, the masked TGF-β construct or complex may be a masked TGF-β A linker peptide / polypeptide inserted between any two elements of the construct or complex Although the term "linker" is used, it is understood that the term "linker" is the same as the linker described below. TGF-β constructs in which the nucleotide sequence is masked, e.g., as protection against proteolysis. They may also be located at the N-terminus and / or C-terminus of the polypeptide of the compound or complex.

[0317] Suitable linkers (also called "spacers") can be easily selected and include 1aa to 2 5aa, 3aa to 20aa, 2aa to 15aa, 3aa to 12aa, 4aa to 10aa, 5aa to 9aa, 6aa to 8aa, or 7aa to 8a Suitable linkers may be any of a number of suitable lengths, such as 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 25aa Suitable linkers may be 25-35 aa in length. Suitable linkers are: It may be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 aa in length. can also be 35-45 aa in length. Suitable linkers include 35, 36, 37, 38, 39, 40, 41, It can be 42, 43, 44, or 45 aa in length. Suitable linkers can be 45-50 aa in length. Suitable linkers may be 45, 46, 47, 48, 49, or 50 aa in length.

[0318] Exemplary linkers include those containing glycine or those containing about 2 to about 50 (e.g., 2 to 4, 4 to 7) , 7 to 10, 10 to 20, 20 to 35, or 35 to 50) consecutive glycine residues , glycine-serine polymers (e.g., [ka] and the like, where n is an integer of at least 1 (e.g., 1 to 10, 10 to 20, or 20 to 30), alanine-serine polymer, or alanine-serine polymer (e.g., 1-10, 10-20, or 20 30 aa in length), as well as other flexible linkers known in the art. Glycine polymers or glycine-serine polymers can be used, In this case, both Gly and Ser are relatively unstructured and act as neutral tethers between the components. Glycine polymers can be used, and glycine can be substituted with alanine. The phi-psi space is significantly more restricted than that of residues with long side chains. (See Scheraga, Rev. Computational Chem. 11173-142 (1992)). An explicit linker is [ka] Exemplary linkers may include amino acid sequences including, but not limited to, For example, it may be repeated 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. [ka] In some examples, the linker may be repeated two, three, or four times. Amino acid sequence [ka] In some examples, the linker comprises an amino acid sequence repeated four or five times: [ka] Exemplary linkers include, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of can be [ka] In some examples, the linker may include an amino acid sequence that is repeated only once or twice. [ka] In some examples, the linker comprises an amino acid sequence repeated three or four times: [ka] In some examples, the linker comprises an amino acid sequence repeated 5, 6, or 7 times: [ka] In some examples, the linker comprises an amino acid sequence repeated 8, 9, or 10 times: [ka] Includes:

[0319] In some instances, the phospholipid present in the first polypeptide of the masked TGF-β complex is The car polypeptide is linked to the second polypeptide of the masked TGF-β construct or complex. Contains a cysteine ​​residue that can form a disulfide bond with an existing cysteine ​​residue. In some instances, for example, a suitable linker may have the amino acid sequence [ka] Includes:

[0320] I. Exemplary Masked TGF-β Constructs and Complexes As discussed above, the masked TGF-β constructs and complexes described in the present disclosure In either case, a masking polynucleotide that binds to and masks the TGF-β polypeptide sequence is used. The peptide can take various forms. Masking peptides can be used to mask the binding of antibodies, antibodies, fragments, single chain antibodies (e.g., scFv) or portions thereof, or nanobodies that bind to TGF-β. The masking polypeptide may also be a TGF-β polypeptide, any of which may be humanized. a polypeptide sufficient to bind to a polypeptide (e.g., TGF-β1, TGF-β2, or TGF-β3) a TGF-β receptor fragment containing the ectodomain of TβRI, TβRII, or TβRIII array).

[0321] In any of the masked TGF-β constructs and complexes described above, the TGF-β used The β polypeptide sequence may be based on TGF-β1, TGF-β2, or TGF-β3. The TGF-β polypeptide comprises a TGF-β3 sequence. In addition, the full-length mature TGF-β protein sequence is not essential for interaction with cell surface TβRII. The masked TGF-β complex, together with cell surface TβRII, recruits TβRI, thereby to initiate signal transduction (e.g., signaling through Smad and non-Smad pathways). Only the portion of TGF-β that is necessary is essential.

[0322] Delivery of masked TGF-β, or cells with TβRI and TβRII, as described above Its ability to activate does not require immunomodulatory polypeptides (MODs), but the presence of MODs This can substantially affect the outcome of TGF-β cell activation. Incorporation of MOD into any of the TGF-β constructs and complexes described herein can be used in conjunction with the masking techniques described herein. To obtain various effects, including therapeutic effects, from the use of engineered TGF-β constructs and complexes. In one embodiment, the masked TGF-β construct or complex can be used MODs present in the PD-L1, Fas-L, IL-2, IL-4, IL-6, IL-7, IL-21, IL-23, and their related A group consisting of any of these mutant forms, including those with reduced affinity for co-MOD. is selected from.

[0323] It may be desirable to incorporate MOD into masked TGF-β constructs and complexes. However, its presence is not essential in all cases, especially in the case of masked TGF-β constructs. and the complex is a cytokine (e.g., one or more independently selected interleukins, and other materials, including steroids, anti-inflammatory drugs ... When administered together, it is not necessary. For example, interleukin-7 receptor (e.g., IL-7 Ra) in thymocytes, thereby inhibiting conventional CD8+ T cells (or low-affinity CD8+ T cells). If it is desired to support the development of 8+ T cell survival, do not include MOD polypeptide. Masked ("MOD-free") TGF-β constructs and complexes may be used. In some cases, it may be desirable to promote the development of T cell populations induced by specific agonist ligands. In this case, thymus-derived Tregs (tTregs), invariant natural killer T (iNKT), and CD8αα T cells were Masked TGF-β with or without MOD was used to support the survival of cellular precursors. Constructs and complexes may be used.

[0324] Below are non-limiting examples of masked TGF-β constructs and complexes.

[0325] 1. Masked TGF-β Construct For masked TGF-β constructs, components (e.g., TGF-β, scaffold, TβRII sequence) and optionally one or more MODs) are all contained in a single polypeptide. In such embodiments, the scaffold is part of a chain (see, for example, Figure 1, structure A). The polypeptide does not form dimers or higher order structures with other scaffold polypeptides, but Thus, the masked TGF-β construct may be a homodimer, heterodimer, or higher order multimer. It is not in the form of a dimeric structure (such as a trimer).

[0326] For the masked TGF-β construct of FIG. 1, in structure A, the polypeptide is N-terminal to C Optionally, one or more MODs and a scaffold polypeptide (without mutually specific binding sequences) to the terminus. a polypeptide that binds to and masks a TGF-β polypeptide; and a polypeptide that binds to and masks a TGF-β polypeptide. and a sequence. Such masked TGF-β constructs include: Can be: (i) from the N-terminus to the C-terminus, optionally containing one or more independently selected wild-type or affinity-reducing variants; A heterologous MOD, a scaffold polypeptide (without a mutually specific binding sequence), and a TGF-β polypeptide. a polypeptide comprising a TβR polypeptide that binds to and masks a TGF-β polypeptide sequence; Petite; (ii) from the N-terminus to the C-terminus, optionally containing one or more independently selected wild-type or affinity-reducing variants; A heterologous MOD, a scaffold polypeptide (without a mutually specific binding sequence), and a TGF-β polypeptide. a TβRII polypeptide that binds to and masks a TGF-β polypeptide sequence; peptide; (iii) one or more independently selected wild-type or affinity-reduced mutant M Binding to OD, a scaffold polypeptide (not containing a mutually specific binding sequence), and a TGF-β polypeptide a polypeptide comprising a TβR polypeptide that masks the TGF-β polypeptide sequence; ; (iv) one or more independently selected wild-type or affinity-reduced mutant ILs, from the N-terminus to the C-terminus; -2 MOD, a scaffold polypeptide (not containing a mutually specific binding sequence), and a TGF-β polypeptide. a polypeptide comprising a masking TβR polypeptide and a TGF-β polypeptide sequence; (v) one or more independently selected wild-type or affinity-reduced mutant MODs, from the N-terminus to the C-terminus; and a scaffold polypeptide (not including a mutually specific binding sequence) that binds to a TGF-β3 polypeptide. a polypeptide comprising a TβR polypeptide that masks the TGF-β3 polypeptide sequence; Do; (vi) one or more independently selected wild-type or affinity-reduced mutant MOs, from the N-terminus to the C-terminus; D, a scaffold polypeptide (not including a mutually specific binding sequence), and a TGF-β3 polypeptide. a polypeptide comprising a TβRII polypeptide that masks the TGF-β3 polypeptide sequence; Chid; and (vii) one or more independently selected wild-type or affinity-reduced variants I, from the N-terminus to the C-terminus; L-2 MOD, a scaffold polypeptide (not including a mutually specific binding sequence), and a TGF-β3 polypeptide a TβRII polypeptide that binds to and masks TGF-β3, and a TGF-β3 polypeptide sequence. Lipeptide.

[0327] In any of the examples of masked TGF-β constructs described herein, the TGF-β polypeptide C77 of the peptide sequence can be substituted to prevent dimerization (e.g., C77S substitution), and the TGF-β polypeptide can be The polypeptide, along with modifications of the MOD and TβR polypeptide sequences, is a masking TβR polypeptide. Mutations to reduce affinity for the nucleotide (e.g., one of aas 25, 92, and / or 94) The masked TGF-β construct may further comprise one, two, or all three of the following: Exemplary TβR polypeptide sequences that can be used include those that attenuate TβRI engagement. and Δ14 or Δ25 TβRII polypeptides, optionally with a D118A or D118R substitution for The MOD variants are described with their polypeptide sequences, and the TβRI, TβRII, and Additional modifications of TβRIII are described above.

[0328] In one embodiment, the masked TGF-β construct has the sequence shown in SEQ ID NO: 146. (See Figure 7A.) ​​In one embodiment, the masked TGF-β construct is SEQ ID NO: 147. (See Figure 7B.) In one embodiment, the masked TGF-β construct has the sequence shown. , having the sequence shown in SEQ ID NO: 157 (see Figure 7G). The TGF-β construct has the sequence shown in SEQ ID NO: 158 (see Figure 7H). The masked TGF-β construct has the sequence shown in SEQ ID NO: 159 (see Figure 7I) .

[0329] 2. Masked TGF-β complex The masked TGF-β complex comprises at least two polypeptides: a first and a second polypeptide. and two polypeptides, each polypeptide associated with a separate scaffold polypeptide, forming a first and a second polypeptide. It comprises a scaffold polypeptide that holds together a second polypeptide into a complex. GF-β polypeptide complexes may form homodimeric, heterodimeric, or higher order multimeric structures. Form. (i) In the first example, the masked TGF-β complex dimerizes into a homodimer, as shown in structure B in Figure 1. Assembled on a scaffold structure capable of forming mono-dimers (e.g., symmetric dimers), A compound comprising at least one TGF-β polypeptide sequence and a mass that binds to one or more TGF-β polypeptides. At least one polypeptide to be masked (e.g., a masking sequence for each TGF-β polypeptide) and, optionally, one or more immunomodulatory polypeptides (MODs). In the body, the IgFc polypeptides are spaced apart between the IgFc polypeptides within the scaffold of each construct. It can allow spontaneous formation of disulfide bonds, e.g., complement-dependent cytotoxicity. The ability of Ig polypeptides to induce cell lysis via (CDC) and antibody-dependent cellular cytotoxicity (ADCC) The polypeptide may include a mutation that substantially reduces or eliminates the cleavage of the nucleus (e.g., the LALA mutation discussed herein). (ii) In a second example, the masked TGF-β complex is (a) At least one TGF-α complex assembled on a scaffold containing a mutually specific dimerization sequence. β polypeptide sequence and at least one sequence that binds and masks one or more TGF-β polypeptides. and optionally a second polypeptide (e.g., a masking sequence for each TGF-β polypeptide sequence). a first polypeptide comprising one or more immunomodulatory polypeptides (MODs); (b) on a scaffold structure comprising a sequence that is a counterpart of the mutually specific dimerization sequence of the first polypeptide; at least one TGF-β polypeptide sequence and at least one TGF-β at least one polypeptide that binds to and masks the polypeptide, and optionally one or more and a second polypeptide comprising an immunomodulatory polypeptide (MOD) of wherein the first and second polypeptides have mutually specific dimerization sequences, such as structure C in FIG. They form heterodimers through interactions. (iii) In a third example, the masked TGF-β complex is (a) At least one TGF-α complex assembled on a scaffold containing a mutually specific dimerization sequence. a TGF-β polypeptide sequence and a nucleotide sequence that binds and masks at least one TGF-β polypeptide; at least one polypeptide (e.g., a masking sequence for each TGF-β polypeptide sequence) and optionally one or more immunomodulatory polypeptides (MODs), and to (b) a sequence that is a counterpart of the mutually specific dimerization sequence of the first polypeptide, and optionally one a second polypeptide comprising a scaffold comprising the above immunomodulatory polypeptide (MOD); wherein the first and second polypeptides have mutually specific dimerization sequences, such as structure F in FIG. They form heterodimers through interactions. (iv) In a fourth example, the masked TGF-β complex is (a) At least one TGF-α complex assembled on a scaffold containing a mutually specific dimerization sequence. a first polypeptide sequence comprising a β polypeptide sequence and, optionally, one or more immunomodulatory polypeptides (MODs); Polypeptides, and (b) on a scaffold structure comprising a sequence that is a counterpart of the mutually specific dimerization sequence of the first polypeptide; a small molecule that binds and masks at least one TGF-β polypeptide, at least one polypeptide and, optionally, one or more immunomodulatory polypeptides (MODs) a second polypeptide, wherein the first and second polypeptides form a specific interacting dimer, such as structures D and E in FIG. They form heterodimers via a covalent sequence.

[0330] In some instances, in masked TGF-β complexes (Fig. 1, structures B, C, and F), TGF- The sequence comprising the β polypeptide (first polypeptide) may optionally comprise, from the N-terminus to the C-terminus, one or more MODs, a scaffold polypeptide (with or without mutually specific binding sequences), and a TGF a polypeptide that binds to and masks a TGF-β polypeptide; and a TGF-β polypeptide sequence. The TGF-β sequence-free polypeptide (second polypeptide) of Figure 1, Structure F, may comprise: a scaffold polypeptide having mutually specific binding sequences, optionally at the N-terminus, C-terminus, or N-terminus; It contains MODs at both the terminal and C-terminus.

[0331] In some instances, in the masked TGF-β complexes of structures D and E in FIG. 1, TGF-β The polypeptide-containing polypeptide (first polypeptide) is a polypeptide having, from the N-terminus to the C-terminus, one or more Any of the above MODs, a scaffold polypeptide (having a mutually specific binding sequence), and a TGF-β polypeptide The polypeptides of structures D and E in FIG. 1 that do not contain the TGF-β sequence (second polypeptide) optionally linked from the N-terminus to the C-terminus to one or more MODs and a scaffold polypeptide having a sequence and a polypeptide that binds to and masks the TGF-β polypeptide; Although not illustrated in FIG. 1, it may also include a TGF-β polypeptide sequence. The first polypeptide may not contain one or more MODs, and the second polypeptide may contain a masking sequence. The polypeptide may contain one or more MODs.

[0332] An example of the masked TGF-β complex described above is a complex in which the first polypeptide is N-terminally Towards the C-terminus, these include: (i) optionally, one or more MODs, a scaffold polypeptide (having mutually specific binding sequences), and a TGF- β polypeptide sequence; (ii) optionally, one or more independently selected wt. or affinity-reduced mutant MODs (mutually specific) a scaffold polypeptide having a binding sequence) and a TGF-β polypeptide sequence; (iii) one or more independently selected wt. or affinity-reduced mutant MODs and (mutually specific binding sequences) a scaffold polypeptide having a sequence) and a TGF-β1 or 2 polypeptide sequence; (iv) one or more independently selected wt. or affinity-reduced mutant IL-2 MODs (mutually specific binding); a scaffold polypeptide (not including any fusion sequences) and a TGF-β polypeptide sequence; (v) one or more independently selected wild-type or affinity-reduced mutant MODs and (mutually specific binding sequences a scaffold polypeptide having a TGF-β3 polypeptide sequence; (vi) one or more independently selected wild-type or affinity-reduced mutant MODs and (mutually specific binding sequences a scaffold polypeptide having a TGF-β3 polypeptide sequence; (vii) one or more independently selected wt. or affinity-reduced mutant IL-2 MODs (mutually specific binding); a scaffold polypeptide (having a nucleotide sequence identical to that of the TGF-β3 polypeptide) and a TGF-β3 polypeptide sequence. In each instance, the second polypeptide has, from the N-terminus to the C-terminus, the mutually specific structure of the first polypeptide. A scaffold polypeptide containing a heterologous binding (dimerization sequence) partner, followed by a first polypeptide. TβR (e.g., TβRII) polypeptides that bind to and mask the TGF-β polypeptide of the peptide In the case of a masked TGF-β complex, such as that shown in Figure 1, structure F, the TβR (e.g., For example, a TβRII polypeptide may comprise a nucleotide sequence comprising an N-terminal MOD (if present) and a scaffold for a first polypeptide. The second polypeptide may be positioned between the first polypeptide and the second polypeptide may be positioned between the first polypeptide and the second polypeptide. sequence), wherein at the N-terminus or C-terminus, there is one or more independently selected Either wt. or reduced affinity mutant MOD (eg, wt. or mutant IL-2 MOD) may bind.

[0333] In any of the examples of masked TGF-β complexes described herein, the TGF-β polypeptide C77 of the peptide sequence can be substituted to prevent dimerization (e.g., C77S substitution), and the TGF-β polypeptide can be The polypeptide, along with modifications of the MOD and TβR polypeptide sequences, is a masking TβR polypeptide. Mutations to reduce affinity for the nucleotide (e.g., one of aas 25, 92, and / or 94) The masked TGF-β construct may further comprise one, two, or all three of the following: Exemplary TβR polypeptide sequences that may be used include Δ14 or Δ2, optionally with a D118A substitution. 5 TβRII polypeptide. For MOD variants, the polypeptide sequence is and additional modifications of TβRI, TβRII, and TβRIII are described above. do.

[0334] In one embodiment, the masked TGF-β complex has the sequences shown in SEQ ID NOs: 148 and 149. In one embodiment, the masked TGF-β comprises a polypeptide having a sequence similar to that of the TGF-β polypeptide (see FIG. 7C). The complex comprises polypeptides having the sequences set forth in SEQ ID NOs: 150 and 151 (see Figure 7D). In one embodiment, the masked TGF-β complex has the sequences shown in SEQ ID NOs: 152 and 153. In one embodiment, the masked TGF-β comprises a polypeptide having a sequence similar to that of the TGF-β polypeptide (see FIG. 7E). The complex comprises polypeptides having the sequences set forth in SEQ ID NOs: 155 and 156 (see Figure 7F). In one embodiment, the masked TGF-β complex has the sequences shown in SEQ ID NOs: 148 and 160. The polypeptides contain sequences (see Figure 7J).

[0335] J. Nucleic acids The present disclosure provides nucleotide sequences encoding masked TGF-β constructs or complexes. In some instances, the nucleic acid is a recombinant expression vector, and thus, The present disclosure includes nucleotide sequences encoding masked TGF-β constructs or complexes. In some examples, the nucleic acid is a recombinant expression vector, Thus, the present disclosure provides nucleosides encoding masked TGF-β constructs and complexes. In the discussion of nucleic acids below, the mask sequences of the present disclosure are provided. The present invention deals with nucleic acids encoding encoded TGF-β constructs and complexes.

[0336] Nucleic Acids Encoding Single-Chain Antigen-Presenting Polypeptides As mentioned above, the masked TGF-β construct comprises a single polypeptide chain. Thus, the present disclosure provides a nucleotide sequence encoding a single-chain masked TGF-β construct. The present invention provides a nucleic acid comprising a nucleotide sequence encoding a single-stranded masked TGF-β construct. The nucleic acid containing the gene sequence is operably linked to a transcriptional control element(s), e.g., a promoter. They can be linked together.

[0337] Nucleic acid(s) encoding the masked TGF-β complex As noted above, in some instances, the masked TGF-β complex comprises at least two The present disclosure provides a method for preparing a polypeptide comprising: The present invention provides a nucleic acid comprising a nucleotide sequence encoding a masked TGF-β complex. In some instances, the individual polypeptide chains of the masked TGF-β complex are represented by individual nucleic acid fragments. In some instances, the entire masked TGF-β construct or complex is encoded within The polypeptide chains are encoded within a single nucleic acid. In some instances, the first nucleic acid is a mass a nucleotide sequence encoding a first polypeptide of a linked TGF-β complex, The second nucleic acid comprises a nucleotide sequence encoding a second polypeptide of the masked TGF-β complex. In some instances, the single nucleic acid comprises the first sequence of the masked TGF-β complex. and a nucleotide sequence encoding a second polypeptide, which are operably linked under the transcriptional control of a single promoter or two independently selected promoters. It could be.

[0338] Individual nuclei encoding the individual polypeptide chains of the masked TGF-β construct or complex acid As noted above, in some instances, the individual polypeptides of the masked TGF-β complex The chains are encoded within individual nucleic acids. In some instances, the masked TGF-β complex The nucleotide sequences encoding the individual polypeptide chains are controlled by transcriptional control elements, e.g. operably linked to a promoter, e.g., a promoter functional in a eukaryotic cell, The promoter may be a constitutive promoter or an inducible promoter.

[0339] For example, the present disclosure provides a first nucleic acid and a second nucleic acid, wherein the first nucleic acid is a masked nucleic acid. a nucleotide sequence encoding a first polypeptide of a TGF-β complex, and a second nucleic acid a nucleotide sequence encoding the second polypeptide of the masked TGF-β complex; In some instances, a nucleotide sequence encoding a first polypeptide and a nucleotide sequence encoding a second polypeptide are included. The nucleotide sequence encoding the peptide is operably linked to a transcriptional control element. In some instances, the transcriptional control element is a promoter that is functional in eukaryotic cells. In some instances, the nucleic acids are present in individual expression vectors.

[0340] In some instances, a nucleotide sequence encoding a first polypeptide and a nucleotide sequence encoding a second polypeptide are The nucleotide sequence encoding the peptide is operably linked to a transcriptional control element. In this example, the transcriptional control element is a promoter that is functional in eukaryotic cells. In some instances, the nucleic acids are present in individual expression vectors.

[0341] Nucleic acids encoding two or more polypeptides present in a masked TGF-β complex The present disclosure provides a first polypeptide and a second polypeptide of a masked TGF-β complex. In some instances, a nucleic acid is provided that comprises a nucleotide sequence encoding at least The bound TGF-β complex comprises a first, second and third polypeptide, and the nucleic acid is In some instances, the vector comprises a nucleotide sequence encoding a first polypeptide, a second polypeptide, and a third polypeptide. Nucleotides encoding the first and second polypeptides of a masked TGF-β complex The nucleotide sequence is located between the encoded first and second polypeptides. In some instances, the nucleic acid sequence encodes a proteolytic cleavable site or linker. Encoding the first and second polypeptides of a masked TGF-β complex. The nucleotide sequence is located between the encoded first and second polypeptides. In some instances, the nucleic acid sequence includes nucleotides encoding an internal ribosome entry site (IRES). , encoding a first polypeptide and a second polypeptide of a masked TGF-β complex. The nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide are A ribosomal skipping signal is placed between the nucleotide sequence encoding the peptide. It contains a sequence encoding nal (or cis-acting hydrolase element, CHYSEL).

[0342] In some instances, the first nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) , a nucleotide sequence encoding the first polypeptide chain of the masked TGF-β complex and the second nucleic acid (e.g., a recombinant expression vector, mRNA, viral RNA, etc.) comprises a masking The nucleotide sequence encoding the second polypeptide chain of the TGF-β complex is In some examples, the nucleotide sequence encoding the first polypeptide and the second polypeptide are Each encoding second nucleotide sequence comprises an independently selected transcriptional control element, e.g., For example, a promoter, e.g., a promoter that is functional in a eukaryotic cell, can be used. The promoter may be a constitutive promoter or an inducible promoter.

[0343] Recombinant Expression Vectors The disclosure provides a recombinant expression vector comprising the nucleic acid. In some examples, the recombinant expression vector In some instances, the recombinant expression vector is a viral vector. viral constructs, such as recombinant adeno-associated viral constructs (see, e.g., U.S. Pat. No. 7,078,387); (see, e.g., ref. 1), recombinant adenoviral constructs, recombinant lentiviral constructs, recombinant recombinant retroviral constructs, non-integral viral vectors, and the like.

[0344] Suitable expression vectors include viral vectors (e.g., vaccinia virus, poly oviruses, adenoviruses (e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92: 7700 7704, 1995, Sakamoto et al., H Gene Ther 5:1088 1097, 1999, International Publication No. WO94 / See WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984, and WO95 / 00655. adeno-associated virus (e.g., Ali et al., Hum Gene Ther 9:81-86, 1998; Fil annery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis S ci 38:2857 2863,1997, Jomary et al., Gene Ther 4:683 690,1997, Rolling et al. Hum Gene Ther 10:641-648, 1999; Ali et al., Hum Mol Genet 5:591-594, 1996; Srivastava, International Publication No. 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 (199 3) 90:10613-10617), SV40, herpes simplex virus, human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 7 3:7812 7816, 1999) based on viral vectors, retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus), as well as Rous sarcoma virus, Bayer sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, bone marrow virus Vectors derived from retroviruses such as myeloproliferative sarcoma virus and mammary tumor virus Many suitable expression vectors are known to those of skill in the art. and many of them are commercially available.

[0345] Depending on the host / vector system utilized, constitutive and inducible promoters, transcription enhancers, Many suitable transcriptional and translational control elements, including promoter elements, transcription terminators, etc. Any of the following can be used in the expression vector (see, e.g., Bitter et al., (1987) Methods in Enzymology, 153:516-544).

[0346] In some instances, the polypeptides encoding the masked TGF-β constructs and complexes are The nucleotide sequence may be a transcription control element, such as a promoter. The transcriptional control element is operably linked to a transcription factor in eukaryotic cells (e.g., mammalian cells) or It can be functional in either prokaryotic cells (e.g., bacterial cells or archaeal cells). In some instances, nucleotides encoding DNA-targeting RNA and / or site-specific modifying polypeptides are The tide sequences are useful for DNA-targeting RNA and / or site-specific modification in both prokaryotic and eukaryotic cells. A plurality of control elements that allow expression of a nucleotide sequence encoding a polypeptide. Functionally linked.

[0347] Non-limiting examples of suitable eukaryotic promoters (promoters functional in eukaryotic cells) include: Cytomegalovirus (CMV) immediate early stage, herpes simplex virus (HSV) thymidine kinase , early and late SV40, retrovirus-derived long terminal repeats (LTRs), and mouse metallothionein For selection of an appropriate vector and promoter, see Expression vectors also contain ribosome binding sites for translation initiation. Expression vectors may also contain a transcriptional site and a transcription terminator. The nucleic acid sequence may include an appropriate sequence for achieving this.

[0348] Preparation and cloning of genetically modified host cells expressing masked TGF-β constructs and complexes Purification of skinned TGF-β constructs and complexes The present disclosure provides a genetically engineered host cell, the host cell containing a masked TGF-β construct. It is genetically engineered with one or more nucleic acid(s) encoding the construct or complex.

[0349] Suitable host cells include eukaryotic cells such as yeast cells, insect cells, and mammalian cells. In some instances, the host cells are cells of a mammalian cell line. Animal cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) Suitable mammalian cell lines include HeLa cells (e.g., American HeLa cells). and Type Culture Collection (ATCC) number CCL-2), CHO cells (e.g., ATCC number CRL9618, CCL 61, CRL9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH 3T3 cells (e.g., A TCC number CRL-1658), Huh-7 cells, BHK cells (e.g. ATCC number CCL10), PC12 cells (ATCC number CR L1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI. 3), human embryonic kidney (HEK) cells (ATCC number CRL1573), and HLHepG2 cells. Not limited to.

[0350] Genetically engineered host cells are used to produce masked TGF-β constructs or complexes. For example, the recombinant host cell can be a The expression vector(s) containing the nucleotide sequence as described above are introduced into a host cell, Therefore, by generating genetically modified host cells, the masked TGF-β complex, Alternatively, it can be used to generate single chain masked TGF-β constructs. Constitutive expression of the masked TGF-β construct or complex in the chief cells may also be achieved. or a promoter driving expression (e.g., a CMV promoter and a promoter for tetracycline to an inducible inducer using a tetracycline resistance operon) It may also be possible to express it in response to exposure to

[0351] The masked TGF-β construct or complex may be obtained from a cell or may be a polypeptide (complex). When a protein (possibly several) is designed to transit the secretory pathway by incorporating a signal sequence, The protein is obtained from the cell culture medium. precipitation (with ethanol), isoelectric focusing, and one or more types of chromatography, The purified product may be purified by any means known in the art, including one or more of the following: Chromatographic methods include size-based chromatographic separation (e.g., size exclusion or gel permeation), hydrophobic interaction chromatography, ion exchange chromatography These include, but are not limited to, masking, and affinity chromatography. The resulting TGF-β construct or complex may comprise an immunoglobulin polypeptide (e.g., as a scaffold). If the sequence contains a masked TGF-β construct, use Protein A or Protein G. The substance or complex can be affinity purified. The complex recognizes the polypeptide present in the masked TGF-β construct or complex. Alternatively, antibodies recognizing myc epitopes may be used. [ka] affinity tags such as "HIS" tag (for divalent metal ion resin binding), or "FLAG" tag In combination with any of the above methods, The resulting purification and / or concentration step removes contaminants having substantially different molecular weights. size-restricted proteins that can be used to isolate and / or concentrate purified proteins A certain semi-permeable membrane (eg, a dialysis membrane or a pressure cell) is used.

[0352] In one embodiment, the masked TGF-β construct or complex is expressed in a mammalian cell (e.g., The protein is expressed from a nucleic acid sequence introduced into a CHO cell, targeted to the secretory pathway, and released from the cell. The masked TGF-β construct or The complexes can be separated using affinity chromatography alone or by size-based separation (e.g. In combination with cell culture In a particular example of such an embodiment, the masked TGF-β construct or The complex comprises an immunoglobulin scaffold (e.g., an IgG polypeptide sequence), and the purification comprises: Affinity chromatography (e.g., Protein A or G) alone or size-based separation This is performed in combination with size-based chromatography.

[0353] K. Composition The present disclosure provides compositions (including pharmaceutical compositions) comprising masked TGF-β constructs or complexes. The present disclosure also provides compositions (including pharmaceutical compositions) comprising the nucleic acid or recombinant expression vector. (including)

[0354] 1. Compositions Comprising Masked TGF-β Constructs or Complexes The compositions of the present disclosure may contain, in addition to the masked TGF-β construct or complex, a salt, e.g., NaCl, MgCl, KCl, MgSO, etc.; buffers, e.g., Tris buffer, N-(2-hydroxyethyl ) piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (ME S), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propane Sulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TA solubilizing agents; surfactants, such as nonionic surfactants such as Tween-20; protease inhibitors; glycerol, and the like.

[0355] The composition may contain pharmaceutically acceptable excipients, the types of which are known in the art. Pharmaceutically acceptable excipients are well known in the art and need not be discussed at length here. For example, "Remington: The Science and Practice of Pharmacy," 19th Edition (1995) or the 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., 7th ed. Lippincott, Williams, & Wilkins and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., 3rd ed., Amer. Pharma It has been well described in various publications, including the European Association of Neuropathology.

[0356] The pharmaceutical composition comprises (i) a masked TGF-β construct or complex; and (ii) a pharmaceutically acceptable In some instances, the pharmaceutical composition may be suitable for administration to a subject. For example, in some embodiments, the pharmaceutical composition comprises: Suitable for administration to human subjects, e.g., the composition is sterile and detectably pyrogen-free. and / or other toxins or are substantially free of such detectable pyrogens and / or Other toxins may be regulated by applicable regulatory agencies, e.g., the U.S. Food and Drug Administration. Present at levels within acceptable limits.

[0357] The protein composition may contain other ingredients such as pharmaceutical grade mannitol, lactose, , starch, magnesium stearate, sodium saccharin, talcum, cellulose , glucose, sucrose, magnesium, carbonate, etc. The composition may contain pharmaceutically acceptable adjuvants necessary to approximate physiological conditions, e.g., pH adjusting agents. agents, pH buffers, toxicity adjusters, etc., such as sodium acetate, sodium chloride, potassium chloride Salts such as ammonium, calcium chloride, sodium lactate, hydrochloride, sulfate, solvates (e.g., mixed ions) The compound may contain inorganic salts, water, organic substances, hydrates (e.g., water), etc.

[0358] For example, the composition may be in the form of an aqueous liquid, a powder, a granule, a tablet, a pill, a suppository, a capsule, or the like. The compositions may be administered by various routes, as described below. It may be formulated accordingly.

[0359] A masked TGF-β construct or complex is utilized (e.g., introduced into a cell culture system). or as an injection directly into tissues (e.g., subcutaneous, intraperitoneal, intramuscular, intralymphatic, and When administered intravenously (intravenously and / or intravenously), the formulation may be in a ready-to-use form or in a non-aqueous form (e.g. The pharmaceutical composition may be in a form such as a reconstituted shelf-stable powder) or in an aqueous form (e.g., a pharmaceutically acceptable The protein-containing formulation may be provided as a liquid or other formulation comprising a carrier and excipients. It may also be provided in a form that extends the serum half-life of the target protein after administration. For example, the protein may be provided as a liposomal formulation, which is prepared into a colloid, or can be provided using other conventional techniques for extending serum half-life. Oka et al., 1980 Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871 and 4,501, Various methods for preparing liposomes are described in US Pat. Nos. 728, 4,837,028 and 4,837,028. The formulations may also be provided in controlled or sustained release forms.

[0360] In some instances, the composition comprises (a) a masked TGF-β construct or complex; and (b) a physiologically In some instances, the composition is sterile. In some instances, the composition is suitable for administration to a human subject, e.g., the composition is sterile and and is substantially free of detectable pyrogens and / or other toxins or Possible pyrogens and / or other toxins are present in amounts within acceptable limits. The present disclosure provides a method for treating a TGF-β infection comprising administering to a subject a subject a) a masked TGF-β construct or complex and (b) a saline solution (e.g., 0.9 mL). % NaCl), wherein the composition is sterile and has no detectable pyrogens or and / or other toxins, or are substantially free of such detectable pyrogens and and / or other toxins are present in acceptable amounts.

[0361] Other examples of formulations suitable for parenteral administration include isotonic sterile injection solutions, antioxidants, Bacteriostatic agents, solutes that make the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizing agents, thickening agents For example, the pharmaceutical composition may be packaged in a container, e.g., The formulation may be provided in a sterile container such as a syringe. The pharmaceutical composition may be provided in an ampule or vial, for example, and may be dispensed immediately before use. in a freeze-dried (lyophilized) state that requires only the addition of a sterile liquid excipient (e.g., water) Extemporaneous injection solutions and extemporaneous injection suspensions may be prepared in the form of sterile powders, granules, and It may also be prepared from tablets.

[0362] The concentration of the masked TGF-β construct or complex in the formulation is less than about 0.1% by weight (usually about 2% by weight, or at least about 2% by weight, to 20% to 50% by weight or more (e.g., 0.1% to 1%, 1 % to 5%, 5% to 10%, 10% to 20%, or 20% to 50% by weight), and usually, mainly, the liquid volume, Based on viscosity and patient-based factors according to the particular method of administration selected and patient needs. The selection is based on

[0363] The present disclosure provides a container containing a composition (e.g., a liquid composition). The container can be, for example, a syringe. In some instances, the container may be a sterile container, an ampoule, or the like. In this case, both the container and the composition are sterile.

[0364] 2. Compositions Comprising Nucleic Acids or Recombinant Expression Vectors The present disclosure also provides compositions, e.g., pharmaceutical compositions, comprising the nucleic acids or recombinant expression vectors of the present disclosure. A wide variety of pharmaceutically acceptable excipients are well known in the art, and Pharmaceutically acceptable excipients need not be discussed in detail in the specification. For example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy" 20th edition, Lippincott,Williams,& Wilkins;Pharmaceutical Dosage Forms and Drug Delivery Sy stems (1999) HC Cansel et al., 7th ed. Lippincott, Williams, & Wilkins; and Handbook of P Harmful Excipients (2000) AH Kibbe et al., 3rd ed., American Pharmaceutical Assoc. This is well explained in various publications.

[0365] The compositions of the disclosure include (a) a nucleic acid encoding a masked TGF-β construct or complex; (b) one or more nucleic acids or one or more recombinant expression vectors containing a nucleotide sequence; and Surfactants, antioxidants, hydrophilic polymers, dextrins, chelating agents, suspending agents, solubilizing agents and one or more of a surfactant, a thickener, a stabilizer, a bacteriostat, a humectant, and a preservative. Suitable buffering agents include, for example, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonyl. carboxylic 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) Phenylic Acid) (PIPES), Sodium Bicarbonate, 3-(N-Tris(hydroxymethyl)-methyl-amino)-2 -Hydroxy-propanesulfonic acid (TAPSO), N-tris(hydroxymethyl)methyl-2-amino Ethanesulfonic acid (TES), N-tris(hydroxymethyl)methyl-glycine (tricine), tris(hydroxymethyl)methyl-glycine (hydroxymethyl)-aminomethane (tris), and the like. Suitable salts include, for example, NaCl, MgCl2, KCl, MgSO4, and the like.

[0366] The pharmaceutical preparation may contain from about 0.001% to about 99% (weight / weight) (e.g., 0.001 to 0.1, 0.1 to 1.0, 1.0 to 10 , 10-20, 20-40, 40-80, or 80-100% (weight / weight) of the nucleic acid or recombinant expression vector. In the following description of the formulation, "the nucleic acid or recombinant expression vector" refers to a nucleic acid or recombinant expression vector. It is understood that the term "antibody" includes an acid or a recombinant expression vector. For example, in some instances, The formulation comprises a nucleic acid or a recombinant expression vector.

[0367] The nucleic acid or recombinant expression vector may be mixed with other compounds or mixtures of compounds. Rather, they may be encapsulated therein, conjugated to them or otherwise attached thereto. Such compounds may include, for example, liposomes or receptor-targeting molecules. The nucleic acid or recombinant expression vector may contain one or more molecules that aid in uptake, distribution, and / or absorption. The above ingredients may be combined in the formulation.

[0368] The nucleic acid or recombinant expression vector composition may be formulated into various forms, including, but not limited to, tablets, capsules, or the like. Many formulations are available, including syrups, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The nucleic acid or recombinant expression vector composition may be formulated into any of the following possible dosage forms: They may also be formulated as suspensions in aqueous, non-aqueous or mixed media. The suspending agent may further contain substances that increase the viscosity of the suspending agent, such as: For example, sodium carboxymethylcellulose, sorbitol and / or dextran The suspension may also contain stabilizers.

[0369] The formulation containing the nucleic acid or recombinant expression vector may be a liposomal formulation. As used herein, the term "liposome" refers to a polymeric material composed of a spherical bilayer or a plurality of spherical bilayers. Liposomes are vesicles composed of amphiphilic lipids placed in a lipophilic space. A unilamellar membrane having a membrane formed of a substance and an aqueous interior containing the composition to be delivered. or multilamellar vesicles. Cationic liposomes interact with negatively charged DNA molecules. These are positively charged liposomes that can interact to form stable complexes. Sensitive or negatively charged liposomes do not complex with DNA but rather take up DNA. Both cationic and non-cationic liposomes are It can be used to deliver a nucleic acid or a recombinant expression vector.

[0370] Liposomes also include "sterically stabilized" liposomes, as used herein. In this case, the term refers to a liposome containing one or more specialized lipids, and Incorporating lipids into liposomes improves their performance compared to liposomes lacking these specialized lipids. Examples of sterically stabilized liposomes include liposomal vesicle formation. A portion of the lipid moiety may contain one or more glycolipids or polyethylene glycol (PEG) moieties. and sterically stabilized liposomes derivatized with one or more hydrophilic polymers such as Thesomes and their uses are further described in U.S. Pat. No. 6,287,860. , the entirety of which is incorporated herein by reference.

[0371] The formulations and compositions may also include surfactants. The use of surfactants in the preparation of emulsions is well known in the art. and its uses are further described in US Pat. No. 6,287,860.

[0372] In one embodiment, various penetration enhancers are included to achieve efficient delivery of the nucleic acid. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the penetration of lipophilic drugs. Penetration enhancers fall into five broad categories: surfactants, lipids, and Belonging to one of the following groups: fatty acids, bile salts, chelating agents, and non-chelating non-surfactants Penetration enhancers and their uses are described in U.S. Patent No. 6,287,860. and is further described in US Pat. No. 6,223,999, the entire contents of which are incorporated herein by reference.

[0373] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticles, and the like. Microparticles, suspensions or solutions in water or aqueous media, capsules, gel capsules, Sachets, tablets or mini-tablets, thickeners, flavourings, diluents, emulsifiers, Dispersing aids or binders may be desirable. Suitable oral formulations include one or more osmotic Oral formulations include those in which the antisense nucleic acids are administered in conjunction with enhancing surfactants and chelating agents. Suitable surfactants include fatty acids and / or esters or salts thereof, bile acids and / or Suitable bile acids / salts and fatty acids and their salts include, but are not limited to, Uses are further described in U.S. Patent No. 6,287,860. Combinations are also suitable, for example, fatty acids / salts in combination with bile acids / salts. The combination is that of the sodium salts of lauric acid, capric acid, and UDCA. Examples of penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene Suitable penetration enhancers include, but are not limited to, ethylene-20-cetyl ether. Also, propylene glycol, dimethyl sulfoxide, triethanolamine, N,N- Dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and its derivatives, tetrahydrofuran Also included are dihydrofurfuryl alcohol and Azone™.

[0374] L. Preparation Suitable formulations are described above, but the compositions are pharmaceutically acceptable. The composition is of a suitable composition (e.g., the composition comprises a pharmaceutically acceptable excipient(s) and an active molecule). In some instances, a suitable formulation comprises (a) a masked TGF-β construct or complex; and (b) a drug In some instances, suitable formulations include: (a) a masked (b) a nucleic acid comprising a nucleotide sequence encoding a TGF-β construct or complex; and an acceptable excipient, and in some instances, the nucleic acid is mRNA. A preferred formulation comprises (a) a first polypeptide of a masked TGF-β construct or complex; and (b) a first nucleic acid comprising a nucleotide sequence encoding a masked TGF-β construct or complex. (c) a second nucleic acid comprising a nucleotide sequence encoding a second polypeptide of the present invention; and acceptable excipients. In some instances, suitable formulations include: (a) a masked TGF-β (b) a recombinant expression vector comprising a nucleotide sequence encoding a β-β construct or complex; and In some instances, suitable formulations include: (a) a masked a nucleotide sequence encoding the first polypeptide of the TGF-β construct or complex (b) a first recombinant expression vector and (c) a second polypeptide of the masked TGF-β construct or complex. (c) a second recombinant expression vector comprising a nucleotide sequence encoding the peptide; Suitable pharmaceutically acceptable excipients are described above. It is.

[0375] M. Method Masked TGF-β constructs or complexes are useful for modulating T cell activity Thus, the present disclosure provides a method for modulating T cell activation, which method broadly comprises: This involves contacting the target T cells with a masked TGF-β construct or complex.

[0376] 1. Methods for modulating immune cell activity, including the generation, stimulation, or inhibition of specific immune cell types The present disclosure provides methods for selectively modulating the activity of cells expressing TβRI and TβRII. The method includes the step of detecting cells (e.g., T cells, B cells, and natural killer (NK) cells, macrophages, and the like) in a mammalian cell line. phage, dendritic cells, and innate cells including granulocytes) with a masked TGF-β construct. or complex, wherein the T cell is contacted with a masked TGF-β construct or complex. By contacting the epitope with the antigen, the activity of epitope-specific T cells is selectively modulated. In some instances, the contacting occurs in vitro. In some instances, the contacting occurs in vivo. The cellular activity (e.g., classical pathway, non-classical pathway) that the bound TGF-β construct or complex acts on The classical pathway, and / or downstream gene expression) signaling is mediated by TGF-β or maskin. compared to a treatment group (e.g., cells, controls) that was not exposed to the TGF-β construct or complex. can be evaluated as such.

[0377] The present disclosure provides methods for detecting T cells or B cells (e.g., pathogenic autoreactive T cells and / or pathogenic autoreactive T cells). and / or a method for reducing the number and / or activity of inflammatory B cells (immune B cells), the method comprising one or more masking and administering the TGF-β construct or complex (e.g., to a subject in need thereof). In some instances, the method increases the number and / or activity of regulatory T cells (Tregs), T cells or the number of B cells (e.g., one or more autoreactive T cells and / or one or more autoreactive B cells) and / or reduce the activity of one or more masked TGF-β constructs or The reduction in the number and / or activity of T cells or B cells affected by the combination is due to the incorporation of TGF-β or one or more mass spectrometry inhibitors. a treatment group (e.g., cells, subjects) that was not exposed to the induced TGF-β construct or complex; are evaluated comparatively.

[0378] One or more (e.g., one, two or more, or three or more) independently selected wild-type or mutant Administration of one or more masked TGF-β constructs or complexes, optionally including a heterologous MOD, can be used to treat various The present invention may affect various cell populations directly or indirectly. For example, the present invention may optionally include one or more wild-type Administration of a masked TGF-β construct or complex containing a normal or mutant IL-2 MOD induces FoxP The development and / or survival of 3+ Treg cells can be directly stimulated (in vivo or in vitro). In addition to any direct effects of the TGF-β / IL-2 complex on various immune cells, the resulting T Reg cells, for example, block the induction of T cell activation and / or the effector stage of the T cell response. suppressing B cell activation and / or inhibiting natural killer cell differentiation and / or proliferation By doing so, the immune response can be suppressed.

[0379] a.Treg (i) TGF-β constructs or complexes containing tTregs, pTregs, iTregs, and IL-2 The present disclosure provides a method for the treatment of thymus-derived Tregs (tTregs) and / or peripheral Tregs (pTregs) (Tregs are autoreactive T cells). and CD4, which can suppress B cells + , FoxP3 + , and CD25 + Cellular development (e.g. The present invention provides a method for promoting the growth and / or survival of a cell in a mammalian cell line, the method comprising the step of: administering the induced TGF-β construct or complex (e.g., to a subject in need thereof); or CD4+ T cells (e.g., naive CD4+ T cells) and (e.g., in tissue culture, blood, or wound The method of administering or contacting includes contacting the subject with a substance (e.g., at a specific tissue location such as a wound). The one or more masked TGF-β constructs or complexes may be one or more (e.g., one, two, three, or four) one, two, or three) independently selected IL-2 MOD polypeptide sequences and / or variant IL-2 The administration or contact may include a MOD polypeptide sequence. and analogs thereof), retinoic acid (e.g., all-trans retinoic acid), and / or rapamycin rapamycin or its functional analogues, e.g., sirolimus, everolimus In conjunction with co-administration or contact with cells of an inhibitor of the mammalian target of receptor (mTOR) (e.g., temsirolimus or temsirolimus). Thus, the present disclosure provides methods for the treatment of FoxpP3+, FoxP3+ thymus-derived Tregs (tTregs) and / or F Promotes the development and / or survival of induced regulatory T cells (iTregs), which are oxP3+ peripheral Tregs (pTregs). The present invention provides a method for promoting the growth of erythrocytes in vitro, the method comprising the step of: For example, all-trans retinoic acid) or analogs thereof, and / or rapamycin or its analogs. In the presence of an analog, one or more IL-2 MOD polypeptide sequences and / or variant IL-2 MOD polypeptides One or more masked TGF-β constructs or complexes containing the peptide sequence are to a subject in need thereof), or to administer CD4+ T cells (e.g., naive CD4+ T cells) Injection with one or more masked TGF-β constructs or complexes The effect of a dose or treatment may be measured relative to a baseline value (e.g., cell count before treatment) or relative to a baseline value, e.g., cell count before treatment. not been exposed to TGF-β or one or more masked TGF-β constructs or complexes The effect is assessed in comparison to a treatment group (e.g., cells or controls), such as an otherwise identical test group. It is possible.

[0380] The present disclosure provides methods for increasing the induction / expansion of Tregs, maintaining Tregs, and / or maintaining their function. The present invention provides a method for isolating T cells (e.g., CD4+ T cells in vivo or in vitro) independently. one or more (e.g., one, two, or three) independently selected IL-2 MOD polypeptide sequences and and / or one or more masked TGF-β constructs comprising a variant IL-2 MOD polypeptide sequence. or a complex, wherein the contacting comprises contacting the complex with one or more masked TGF-β constructs. than the baseline value measured before or after contact with the substance or complex. Increased Treg induction / expansion, Treg maintenance, and Treg proliferation compared to otherwise identical control cells. The present disclosure provides one or more (e.g., , one, two or three) independently selected IL-2 MOD polypeptide sequences and / or mutations. and a masked TGF-β construct or complex comprising a IL-2 MOD polypeptide sequence. In one embodiment, the masked TGF-β construct or complex comprises one or more (e.g., (e.g., one, two, or three) independently selected IL-2 MOD polypeptide sequences and / or IL-2 MOD polypeptide sequences having the structural organization depicted in Structures A, B, or C of FIG. In one embodiment, the masked TGF-β construct or complex comprises one or more (e.g., one, two, or three) independently selected IL-2 MOD polypeptide sequences and / or variants I It has the structural organization depicted in Structure D or E of Figure 1, including the L-2 MOD polypeptide sequence. In embodiments, the masked TGF-β construct or complex comprises one or more (e.g., one, two, or more) or three) independently selected IL-2 MOD polypeptide sequences and / or variant IL-2 MODs. It has the structural organization depicted in Structure F of Figure 1, including the polypeptide sequence.

[0381] The present disclosure provides methods for increasing the induction / expansion of Tregs, maintaining Tregs, and / or maintaining their function. The present invention provides a method for isolating T cells (e.g., CD4+ T cells in vivo or in vitro) from a single cell. or more (e.g., one, two, three) independently selected PD-L1 or PD-L2 MOD polypeptide sequences. and / or one or more masked polypeptide sequences comprising a variant PD-L1 or PD-L2 MOD polypeptide sequence. The contacting comprises contacting the TGF-β construct or complex with one or more masked TGF-β constructs or complexes. than a baseline value measured prior to contact with the TGF-β construct or complex, or The cells showed increased Treg induction / proliferation compared to the otherwise identical control group of cells that had not been contacted. The present disclosure provides a method for the preparation of a medicament for use in the method, the method comprising: one or more (e.g., one, two, or three) independently selected PD-L1 or PD-L2 MOD polynucleotides. masking peptide sequences and / or variant PD-L1 or PD-L2 MOD polypeptide sequences In one embodiment, the present invention provides a masked TGF-β construct or complex. The TGF-β construct or complex may comprise one or more (e.g., one, two, or three) independently selected PD-L1 or PD-L2 MOD polypeptide sequences and / or variant PD-L1 or PD-L2 MOD polypeptides In one embodiment, the structural organization is as set forth in Structures A, B, or C of Figure 1, including a methylated ... The masked TGF-β construct or complex may be one or more independently selected (e.g., one or more , ...

Claims

1. As the first polypeptide, (i) a scaffold polypeptide sequence; and (ii) a TGF-β polypeptide sequence; and (iii) a mass spectrometry assay, optionally comprising a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; a KING polypeptide sequence; (iv) optionally, one or more independently selected MOD polypeptide sequences; (v) optionally, one or more independently selected linker polypeptide sequences. And, Constructs containing these elements are collectively referred to as "masked TGF-β constructs" the masking polypeptide sequence and the TGF-β polypeptide sequence bind to each other; Described structure.

2. 2. The masked TGF-β construct of claim 1, wherein the first polypeptide comprises: From the N-terminus to the C-terminus, (i) the scaffold polypeptide sequence, the masking polypeptide sequence, and the TGF-β polypeptide sequence; Lipid sequence, (ii) a first MOD polypeptide sequence, the scaffold polypeptide sequence, the masking polypeptide sequence, a TGF-β polypeptide sequence, and the TGF-β polypeptide sequence; or (iii) a first independently selected MOD polypeptide sequence, a second independently selected MOD polypeptide sequence, the scaffold polypeptide sequence, the masking polypeptide sequence, and a TGF-β polypeptide sequence as defined above, The masked TGF-β construct optionally contains one or more independently selected linkers. - the masked TGF-β construct comprising a polypeptide sequence.

3. The scaffold polypeptide comprises a dimerization sequence, and optionally, the scaffold polypeptide 3. The masked TGF-β construct of claim 1 or claim 2, comprising a reciprocal dimerization sequence. thing.

4. dimerizing the first polypeptide to form a masked TGF-β complex heterodimer; and further comprising a second polypeptide that forms the second polypeptide is (i) a scaffold polypeptide sequence; and (ii) a TGF-β polypeptide sequence; and (iii) a mass spectrometry assay, optionally comprising a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; a KING polypeptide sequence; (iv) optionally, one or more independently selected MOD polypeptide sequences; (v) optionally, one or more independently selected linker polypeptide sequences. Item 4. The masked TGF-β construct according to any one of items 1 to 3.

5. the first polypeptide comprises a scaffold polypeptide comprising mutually specific dimerization sequences; The second polypeptide is the counterpart of the mutually specific binding sequence of the first polypeptide. a scaffold polypeptide sequence comprising a mutually specific dimerization sequence, The mutually specific binding sequence and the counterpart mutually specific binding sequence are mutually specific in the heterodimer.

5. The masked TGF-β construct of claim 4, wherein the TGF-β construct interacts with the TGF-β receptor.

6. A TGF-β complex comprising a first polypeptide and a second polypeptide as a heterodimer. There was, (i) the first polypeptide is (a) a scaffold polypeptide sequence comprising a mutually specific dimerization sequence; (b) a mask optionally containing a TGF-β receptor polypeptide sequence or an anti-TGF-β polypeptide sequence; a coding polypeptide sequence; (c) optionally, one or more independently selected MOD polypeptide sequences; (d) optionally, one or more independently selected linker polypeptide sequences; (ii) the second polypeptide is (a) a mutually specific dimerization sequence of the first polypeptide, which is a counterpart of the mutually specific dimerization sequence of the first polypeptide; a scaffold polypeptide sequence comprising the sequence (b) a TGF-β polypeptide sequence; and (c) optionally, one or more independently selected MOD polypeptide sequences; (d) optionally, one or more independently selected linker polypeptide sequences; The complex containing these elements is collectively called the "masked TGF-β complex." the masking polypeptide sequence and the TGF-β polypeptide sequence bind to each other; The mutually specific binding sequence and the counterpart mutually specific binding sequence bind to each other in the heterodimer. interacts (e.g., covalently or non-covalently) with The first polypeptide and / or the second polypeptide of the masked TGF-β are any of and one or more independently selected linker polypeptide sequences. 。

7. the first polypeptide comprising, from N-terminus to C-terminus: (a) one or two (or more) independently selected MOD sequences and a scaffold polypeptide sequence comprising a merization sequence, and the masking polypeptide sequence. or (b) a scaffold polypeptide sequence comprising a mutually specific dimerization sequence and a masking polypeptide sequence; a polypeptide sequence (e.g., a TGF-β receptor polypeptide sequence), the second polypeptide comprises, from the N-terminus to the C-terminus, one or two (or more) independent A scaffold polypeptide sequence comprising a MOD sequence selected by the above method and a complementary mutually specific dimerization sequence. and the TGF-β polypeptide sequence. β complex heterodimer.

8. the scaffold polypeptide sequence(s) consist of an IgFc polypeptide sequence and variants thereof; and optionally, said IgFc polypeptide sequence is selected from the group consisting of complement component 1q of said polypeptide. (C1q) and / or Fc lambda receptor (FcλR), and / or of peptides, e.g., via complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Any of claims 1 to 12, comprising a mutation that substantially reduces or eliminates the ability to induce cell lysis. A masked TGF-β construct or complex according to any one of claims 1 to 14.

9. The one or more independently selected MOD polypeptide sequences may be 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, IL-23 MOD polypeptide sequences and any of these.

14. The mask according to any one of claims 1 to 13, selected from the group consisting of: A ligated TGF-β construct or complex.

10. At least one of the MOD polypeptide sequences is a wt. IL-2 MOD polypeptide sequence. There is, or (i) at least 80, 90, 100, 110, 120, 130, or 133 consecutive aas of SEQ ID NO: 9 At least 80%, at least 85%, at least 90%, at least 95%, at least or have 98% or at least 99% aa sequence identity; (ii) at least 80, 90, 100, 110, 120, 130, or 133 consecutive sequences of SEQ ID NOs: 13-27 For AASs, at least 80%, at least 85%, at least 90%, at least 95%, have at least 98% or at least 99% aa sequence identity or 100% sequence identity 10. The masking agent of claim 1, wherein the masking agent is a mutant IL-2 MOD polypeptide sequence. A ligated TGF-β construct or complex.

11. The masking polypeptide sequence is a TGF-β receptor (「TβR」) polypeptide sequence and comprises an ectodomain fragment of type I (TβRI), type II (TβRII) or type III (TβRIII) of TβR. The masked TGF-β construct or complex according to any one of claims 1 to 10.

12. The complex is formed from a combination of polypeptide construct 4033 (SEQ ID NO: 191) and polypeptide construct 4039 (SEQ ID NO: 192). The masked TGF-β construct or complex according to claim 7.

13. One or more nucleic acids encoding the masked TGF-β complex according to any one of claims 1 to 12.

14. A method for inducing Treg cells in a mammalian subject or treating a disease or condition in the subject, comprising administering to the subject one or more masked TGF-β constructs or complexes according to any one of claims 1 to 12, or the nucleic acid according to claim 13. The method.

15. The method is for the treatment of autoimmune symptoms or diseases selected from the group consisting of Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune encephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune infertility, autoimmune thrombocytopenic purpura, bullous pemphigoid, celiac disease, Crohn's disease, Goodpasture syndrome, glomerulonephritis, Graves' disease, Hashimoto's thyroiditis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis (MG), pemphigus, pernicious anemia, polymyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus (SLE), type 1 diabetes (T1D), celiac disease, vasculitis and vitiligo. The method according to claim 14. ​

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