Compositions and methods for targeted delivery of TGFβ

Abstract

The present disclosure provides polypeptide complexes comprising a target-binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM) and a small latent complex, particularly a small latent complex comprising a dimeric latency-associated polypeptide (LAP) or a fragment or derivative thereof, and a dimeric mature transforming growth factor β (TGFβ) family polypeptide or a fragment or derivative thereof. Additionally, polypeptides (e.g., fusion polypeptides), as well as related polynucleotides, vectors, cells, and pharmaceutical compositions, are provided. Also provided are methods for treating a subject, e.g., using the polypeptide complexes and / or fusion polypeptides, or pharmaceutical compositions thereof.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 479,679, filed January 12, 2023, and U.S. Provisional Patent Application No. 63 / 526,021, filed July 11, 2023, the disclosures of both of which are incorporated herein by reference in their entireties.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy, created on January 8, 2024, is named 250298_000604_SL.xml and is 204,303 bytes in size.

[0003] FIELD OF THE INVENTION The present disclosure provides a polypeptide complex comprising a target-binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM) and a small latent complex (SLC), particularly the small latent complex (SLC) comprising a dimeric latency-associated polypeptide (LAP) or a fragment or derivative thereof, and a dimeric mature transforming growth factor β (TGFβ) family polypeptide or a fragment or derivative thereof. Additionally, polypeptides (e.g., fusion polypeptides), as well as related polynucleotides, vectors, cells, and pharmaceutical compositions, are provided. Also provided are methods for treating a subject, e.g., using the polypeptide complex and / or fusion polypeptide, or pharmaceutical compositions thereof. [Background technology]

[0004] Transforming growth factor beta (TGFβ, TGFb, TGFB, TGFbeta, TGFBeta, etc.) is composed of a signal peptide, a latency-associated peptide (LAP), and a mature TGFβ domain. After furin cleavage at the furin cleavage site at the junction between LAP and the mature domain during secretion, LAP remains noncovalently associated with the mature domain, thereby inactivating it. The complex of LAP (e.g., furin-cleaved LAP) and the mature domain is called the small latency complex (SLC). SLC secreted in complex with environmental molecules is called the large latency complex (LLC), which is expressed on different cell types (e.g., endothelial cells, T cells, macrophages, and microglia) and can be incorporated into the extracellular matrix (ECM). The LLC can be proteolytically or mechanically activated to release mature TGFβ, which can induce downstream TGFβ signaling. Decreased TGFβ signaling has been identified in a variety of TGFβ dysregulated disorders, including inflammatory bowel disease (IBD), Marfan syndrome (MFS), autoimmune diseases, and other diseases associated with loss-of-function mutations in TGFβ. Therefore, targeted delivery of TGFβ to specific cell types, where it is activated only when it reaches the desired location, is required to benefit diseases and / or biological processes exacerbated by decreased TGFβ signaling. Summary of the Invention [Means for solving the problem]

[0005] As identified in the Background section above, there is a great need in the art for the development of compositions and methods for the targeted delivery of TGFβ to specific cell types, particularly in the context of reduced TGFβ signaling such as occurs in various TGFβ dysregulation disorders. The present application addresses these and other needs.

[0006] In one aspect, there is provided a polypeptide complex comprising: a. a target-binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM); b. Small Latent Complex (SLC), i. dimeric latency associated polypeptide (LAP) or a fragment or derivative thereof; and ii. A dimeric mature transforming growth factor beta (TGFβ) family polypeptide or a fragment or derivative thereof, which is inactive as a result of its interaction with dimeric LAP or a fragment or derivative thereof. Small latent complex (SLC) containing Provided herein is a polypeptide complex comprising:

[0007] In some embodiments, the LAP, or a fragment or derivative thereof, is covalently attached to the target binding polypeptide.

[0008] In some embodiments, the LAP, or a fragment or derivative thereof, is covalently attached to the target binding polypeptide via a linker.

[0009] In some embodiments, the linker comprises the sequence (GGGGS)n (SEQ ID NO:46), (GGGS)n (SEQ ID NO:51), or GSGESGGGSG (SEQ ID NO:96).

[0010] In some embodiments, the linker consists of the sequence (GGGGS)n (SEQ ID NO:46), (GGGS)n (SEQ ID NO:51), or GSGESGGGSG (SEQ ID NO:96).

[0011] In some embodiments, the linker comprises the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

[0012] In some embodiments, the linker consists of the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

[0013] In some embodiments, the linker comprises the sequence GSGESGGGSG (SEQ ID NO: 96).

[0014] In some embodiments, the linker consists of the sequence GSGESGGGSG (SEQ ID NO: 96).

[0015] In some embodiments, the target-binding polypeptide binds to both LAP or a fragment or derivative thereof and to a molecule on a target cell or in the ECM.

[0016] In some embodiments, the target-binding polypeptide is an antibody or a fragment or derivative thereof.

[0017] In some embodiments of any of the above-described polypeptide complexes, the mature TGFβ family polypeptide, or a fragment or derivative thereof, and the LAP, or a fragment or derivative thereof, are associated via non-covalent interactions.

[0018] In some embodiments of any of the above-described polypeptide complexes, the mature TGFβ family polypeptide, or a fragment or derivative thereof, and the LAP, or a fragment or derivative thereof, are separated by a protease cleavage site.

[0019] In some embodiments, the protease cleavage site is a furin cleavage site.

[0020] In some embodiments, the furin cleavage site comprises the sequence RXXR (SEQ ID NO: 35).

[0021] In some embodiments, the furin cleavage site consists of the sequence RXXR (SEQ ID NO: 35).

[0022] In some embodiments, RXXR (SEQ ID NO: 35) is RHRR (SEQ ID NO: 85), RRKR (SEQ ID NO: 86), or RKKR (SEQ ID NO: 87).

[0023] In some embodiments of any of the above-described polypeptide complexes, the mature TGFβ family polypeptide or fragment or derivative thereof binds to a transforming growth factor β receptor (TGFβR) upon release of the mature TGFβ family polypeptide or fragment or derivative thereof from the SLC.

[0024] In some embodiments of any of the above-described polypeptide complexes, the mature TGFβ family polypeptide or fragment or derivative thereof, upon release from the SLC, induces Smad2 / 3 signaling in the target cell or in a cell adjacent to the target cell.

[0025] In some embodiments of any of the above-described polypeptide complexes, the LAP, or a fragment or derivative thereof, comprises an integrin-binding motif.

[0026] In some embodiments, the integrin binding motif comprises the sequence RGD.

[0027] In some embodiments, the integrin is αvβ6 integrin or αvβ8 integrin.

[0028] In some embodiments of any of the above-described polypeptide complexes, the LAP or fragment or derivative thereof does not comprise an integrin-binding motif.

[0029] In some embodiments of any of the aforementioned polypeptide complexes, the target-binding polypeptide is an antigen-binding polypeptide or an antigen-binding fragment thereof.

[0030] In some embodiments, the antigen-binding polypeptide is an antibody or an antigen-binding fragment thereof.

[0031] In some aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region.

[0032] In some aspects, the antibody or antigen-binding fragment thereof comprises a light chain variable region.

[0033] In some aspects, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain constant domain.

[0034] In some embodiments, the immunoglobulin heavy chain constant domain is an IgG1 domain.

[0035] In some embodiments, the immunoglobulin heavy chain constant domain is an IgG4 domain.

[0036] In some embodiments, the antigen-binding polypeptide or antigen-binding fragment thereof binds to CD63.

[0037] In some embodiments, the antigen-binding polypeptide or antigen-binding fragment thereof binds to the extra domain B of fibronectin (EDB-FN).

[0038] In some embodiments, the antigen-binding polypeptide or antigen-binding fragment thereof binds to epithelial cell adhesion molecule (Epcam).

[0039] In some embodiments, the antigen-binding polypeptide or antigen-binding fragment thereof binds to C-type lectin domain family 9 member A (Clec9a).

[0040] In some embodiments of any of the above-described polypeptide complexes, the target-binding polypeptide is not internalized.

[0041] In some embodiments of any of the aforementioned polypeptide complexes, the mature TGFβ family polypeptide is a mature TGFβ polypeptide.

[0042] In some embodiments, the mature TGFβ polypeptide is a mature TGFβ1 polypeptide.

[0043] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:23.

[0044] In some embodiments, the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:23.

[0045] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:90.

[0046] In some embodiments, the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:90.

[0047] In some embodiments, the mature TGFβ polypeptide is a mature TGFβ2 polypeptide.

[0048] In some embodiments, the mature TGFβ2 polypeptide comprises the sequence of SEQ ID NO:27.

[0049] In some embodiments, the mature TGFβ2 polypeptide consists of the sequence of SEQ ID NO:27.

[0050] In some embodiments, the mature TGFβ family polypeptide is a mature growth differentiation factor 8 (GDF8), a mature growth differentiation factor 11 (GDF11) polypeptide, or a mature bone morphogenetic protein 4 (BMP4).

[0051] In some embodiments of any of the aforementioned polypeptide complexes, the LAP comprises the sequence from positions 30 to 274 of the sequence of SEQ ID NO:82.

[0052] In some embodiments, the LAP consists of the sequence from positions 30 to 274 of SEQ ID NO:82.

[0053] In some embodiments of any of the aforementioned polypeptide complexes, the LAP comprises the sequence from positions 21 to 298 of the sequence of SEQ ID NO:116.

[0054] In some embodiments, the LAP consists of the sequence from positions 21 to 298 of SEQ ID NO:116.

[0055] In some embodiments of any of the aforementioned polypeptide complexes, the LAP or fragment or derivative thereof is heterologous to the mature TGFβ family polypeptide or fragment or derivative thereof.

[0056] In some embodiments of any of the aforementioned polypeptide complexes, the LAP or fragment or derivative thereof comprises one or more mutations, wherein the one or more mutations allow for proteolytic activation of the mature TGFβ family polypeptide or fragment or derivative thereof.

[0057] In some embodiments of any of the aforementioned polypeptide complexes, the LAP or fragment or derivative thereof comprises one or more mutations, wherein the one or more mutations allow for mechanical activation of the mature TGFβ family polypeptide or fragment or derivative thereof.

[0058] In some embodiments of any of the above-described polypeptide complexes, the LAP or fragment or derivative thereof comprises one or more mutations that eliminate one or more protease cleavage sites within the LAP or fragment or derivative thereof.

[0059] In some embodiments of any of the above-described polypeptide complexes, the LAP or fragment or derivative thereof comprises one or more mutations that reduce binding of the LAP or fragment or derivative thereof to latency-associated binding protein (LTBP).

[0060] In some embodiments, the one or more mutations comprise a C33S mutation, position 33 relative to the sequence of SEQ ID NO:82.

[0061] In some embodiments, the one or more mutations comprise a C24S mutation, where position 24 is relative to SEQ ID NO:116.

[0062] In some embodiments of any of the aforementioned polypeptide complexes, the LAP comprises the sequence of SEQ ID NO:31.

[0063] In some embodiments, the LAP consists of the sequence of SEQ ID NO:31.

[0064] In some embodiments of any of the aforementioned polypeptide complexes, the LAP comprises the sequence of SEQ ID NO:94.

[0065] In some embodiments, the LAP consists of the sequence of SEQ ID NO:94.

[0066] In some embodiments of any of the above-described polypeptide complexes, the mature TGFβ family polypeptide or fragment or derivative thereof is chemically dissociated from the LAP or fragment or derivative thereof and released in an active form from the SLC.

[0067] In some embodiments, chemical dissociation comprises protease treatment, temperature treatment, acid treatment, or any combination thereof.

[0068] In some embodiments of any of the above-described polypeptide complexes, the mature TGFβ family polypeptide, or fragment or derivative thereof, is mechanically dissociated from the LAP, or fragment or derivative thereof, and released in an active form from the SLC.

[0069] In some embodiments, the mechanical dissociation occurs as a result of an interaction between LAP, or a fragment or derivative thereof, and an integrin polypeptide.

[0070] In another aspect, provided herein is a pharmaceutical composition comprising a polypeptide complex described herein.

[0071] In some embodiments, the pharmaceutical compositions described herein may further comprise a pharmaceutically acceptable carrier or diluent.

[0072] In another aspect, a fusion polypeptide comprising: a. a target-binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM); b. Latency-associated polypeptide (LAP) or a fragment or derivative thereof; c. Mature transforming growth factor β (TGFβ) family polypeptide or a fragment or derivative thereof Provided herein is a fusion polypeptide comprising:

[0073] In some embodiments, the mature TGFβ family polypeptide or fragment or derivative thereof is inactive as a result of interaction with LAP or a fragment or derivative thereof.

[0074] In some embodiments, the mature TGFβ family polypeptide or fragment or derivative thereof binds to a transforming growth factor β receptor (TGFβR) once the mature TGFβ family polypeptide or fragment or derivative thereof is released from the LAP or fragment or derivative thereof.

[0075] In some embodiments of any of the above-described fusion polypeptides, the mature TGFβ family polypeptide or fragment or derivative thereof induces Smad2 / 3 signaling in the target cell or in a cell adjacent to the target cell when the mature TGFβ family polypeptide or fragment or derivative thereof is released from the LAP or fragment or derivative thereof.

[0076] In some embodiments of any of the above-described fusion polypeptides, the fusion polypeptide comprises a linker.

[0077] In some embodiments, a linker is positioned between the target binding polypeptide and the LAP or fragment or derivative thereof.

[0078] In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, (i) a target-binding polypeptide, (ii) a linker, (iii) LAP or a fragment or derivative thereof, and (iv) a mature TGFβ family polypeptide or a fragment or derivative thereof.

[0079] In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus: (i) a mature TGFβ family polypeptide or a fragment or derivative thereof; (ii) LAP or a fragment or derivative thereof; (iii) a linker; and (iv) a target-binding polypeptide.

[0080] In some embodiments of any of the above-described fusion polypeptides, the linker comprises the sequence (GGGGS)n (SEQ ID NO:46), (GGGS)n (SEQ ID NO:51), or GSGESGGGSG (SEQ ID NO:96).

[0081] In some embodiments, the linker consists of the sequence (GGGGS)n (SEQ ID NO:46), (GGGS)n (SEQ ID NO:51), or GSGESGGGSG (SEQ ID NO:96).

[0082] In some embodiments, the linker comprises the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

[0083] In some embodiments, the linker consists of the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

[0084] In some embodiments, the linker comprises the sequence GSGESGGGSG (SEQ ID NO: 96).

[0085] In some embodiments, the linker consists of the sequence GSGESGGGSG (SEQ ID NO: 96).

[0086] In some embodiments of any of the above-described fusion polypeptides, the mature TGFβ family polypeptide, or a fragment or derivative thereof, and the LAP, or a fragment or derivative thereof, are separated by a protease cleavage site.

[0087] In some embodiments, the protease cleavage site is a furin cleavage site.

[0088] In some embodiments, the furin cleavage site comprises the sequence RXXR (SEQ ID NO: 35).

[0089] In some embodiments, the furin cleavage site consists of the sequence RXXR (SEQ ID NO: 35).

[0090] In some embodiments, RXXR (SEQ ID NO: 35) is RHRR (SEQ ID NO: 85), RRKR (SEQ ID NO: 86), or RKKR (SEQ ID NO: 87).

[0091] In some embodiments of any of the above-described fusion polypeptides, the fusion polypeptide further comprises a signal peptide.

[0092] In some embodiments, the signal peptide is an mROR signal peptide.

[0093] In some embodiments, the mROR signal peptide comprises the sequence MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 1).

[0094] In some embodiments, the mROR signal peptide consists of the sequence MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 1).

[0095] In some embodiments of any of the above-described fusion polypeptides, the LAP, or a fragment or derivative thereof, comprises an integrin-binding motif.

[0096] In some embodiments, the integrin binding motif comprises the sequence RGD.

[0097] In some embodiments, the integrin is αvβ6 integrin or αvβ8 integrin.

[0098] In some embodiments of any of the above-described fusion polypeptides, LAP or a fragment or derivative thereof does not comprise an integrin-binding motif.

[0099] In some embodiments of any of the above-described fusion polypeptides, the target-binding polypeptide is an antigen-binding polypeptide or an antigen-binding fragment thereof.

[0100] In some embodiments, the antigen-binding polypeptide is an antibody or an antigen-binding fragment thereof.

[0101] In some aspects, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region.

[0102] In some aspects, the antibody or antigen-binding fragment thereof comprises a light chain variable region.

[0103] In some aspects, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain constant domain.

[0104] In some embodiments, the immunoglobulin heavy chain constant domain is an IgG1 domain.

[0105] In some embodiments, the immunoglobulin heavy chain constant domain is an IgG4 domain.

[0106] In some embodiments, the antigen-binding polypeptide or antigen-binding fragment thereof binds to CD63.

[0107] In some embodiments, the antigen-binding polypeptide or antigen-binding fragment thereof binds to the extra domain B of fibronectin (EDB-FN).

[0108] In some embodiments, the antigen-binding polypeptide or antigen-binding fragment thereof binds to epithelial cell adhesion molecule (Epcam).

[0109] In some embodiments, the antigen-binding polypeptide or antigen-binding fragment thereof binds to C-type lectin domain family 9 member A (Clec9a).

[0110] In some embodiments of any of the above-described fusion polypeptides, the target-binding polypeptide is not internalized.

[0111] In some embodiments of any of the above-described fusion polypeptides, the mature TGFβ family polypeptide is a mature TGFβ polypeptide.

[0112] In some embodiments, the mature TGFβ polypeptide is a mature TGFβ1 polypeptide.

[0113] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:23.

[0114] In some embodiments, the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:23.

[0115] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:90.

[0116] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:90.

[0117] In some embodiments, the mature TGFβ polypeptide is a mature TGFβ2 polypeptide.

[0118] In some embodiments, the mature TGFβ2 polypeptide comprises the sequence of SEQ ID NO:27.

[0119] In some embodiments, the mature TGFβ2 polypeptide consists of the sequence of SEQ ID NO:27.

[0120] In some embodiments of any of the above-described fusion polypeptides, the mature TGFβ family polypeptide is a mature growth differentiation factor 8 (GDF8), a mature growth differentiation factor 11 (GDF11) polypeptide, or a mature bone morphogenetic protein 4 (BMP4).

[0121] In some embodiments of any of the above-described fusion polypeptides, the LAP comprises the sequence from positions 30 to 274 of the sequence of SEQ ID NO:82.

[0122] In some embodiments, the LAP consists of the sequence from positions 30 to 274 of SEQ ID NO:82.

[0123] In some embodiments of any of the above-described fusion polypeptides, the LAP comprises the sequence from positions 21 to 298 of the sequence of SEQ ID NO:116.

[0124] In some embodiments, the LAP consists of the sequence from positions 21 to 298 of SEQ ID NO:116.

[0125] In some embodiments of any of the above-described fusion polypeptides, the LAP or fragment or derivative thereof is heterologous to the mature TGFβ family polypeptide or fragment or derivative thereof.

[0126] In some embodiments of any of the above-described fusion polypeptides, the LAP or fragment or derivative thereof comprises one or more mutations, wherein the one or more mutations allow for proteolytic activation of the mature TGFβ family polypeptide or fragment or derivative thereof.

[0127] In some embodiments of any of the above-described fusion polypeptides, the LAP or fragment or derivative thereof comprises one or more mutations, wherein the one or more mutations allow for mechanical activation of the mature TGFβ family polypeptide or fragment or derivative thereof.

[0128] In some embodiments of any of the above-described fusion polypeptides, the LAP or fragment or derivative thereof comprises one or more mutations that eliminate one or more protease cleavage sites within the LAP or fragment or derivative thereof.

[0129] In some embodiments of any of the above-described fusion polypeptides, the LAP or fragment or derivative thereof comprises one or more mutations that reduce binding of the LAP or fragment or derivative thereof to latency-associated binding protein (LTBP).

[0130] In some embodiments, the one or more mutations comprise a C33S mutation, position 33 relative to the sequence of SEQ ID NO:82.

[0131] In some embodiments, the one or more mutations comprise a C24S mutation, where position 24 is relative to SEQ ID NO:116.

[0132] In some embodiments, the LAP comprises the sequence of SEQ ID NO:31.

[0133] In some embodiments, the LAP consists of the sequence of SEQ ID NO:31.

[0134] In some embodiments, the LAP comprises the sequence of SEQ ID NO:94.

[0135] In some embodiments, the LAP consists of the sequence of SEQ ID NO:94.

[0136] In another aspect, provided herein are polynucleotides encoding the fusion polypeptides described herein.

[0137] In another aspect, provided herein is a vector comprising a polynucleotide described herein.

[0138] In some embodiments, the sequence encoding the fusion polypeptide is operably linked to a promoter, and the promoter mediates expression of the fusion polypeptide.

[0139] In some embodiments, the vector is a viral vector.

[0140] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0141] In another aspect, provided herein is a cell comprising a polypeptide complex described herein, a fusion polypeptide described herein, a polynucleotide described herein, or a vector described herein.

[0142] In another aspect, provided herein is a method of making a polypeptide complex described herein, comprising incubating a cell comprising a polynucleotide described herein or a vector described herein under conditions that allow for the production of the polypeptide complex.

[0143] In some embodiments, the method further comprises collecting the cell culture medium and isolating the produced polypeptide complex by a process comprising affinity chromatography.

[0144] In some embodiments, the affinity chromatography comprises a Protein A or Protein G column or beads.

[0145] In another aspect, provided herein is a method for treating a TGFβ dysregulation disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide complex described herein, a pharmaceutical composition described herein, a polynucleotide described herein, or a vector described herein.

[0146] In some embodiments, the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered by injection.

[0147] In some embodiments, the injection is intravenous, intramuscular, subcutaneous, or intraperitoneal.

[0148] In some embodiments of any of the aforementioned methods for treating a TGFβ dysregulation disorder in a subject in need thereof, the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered by hydrodynamic delivery (HDD).

[0149] In some embodiments of any of the aforementioned methods for treating a TGFβ dysregulation disorder in a subject in need thereof, the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered to the liver of the subject.

[0150] In some embodiments of any of the above-described methods for treating a TGFβ dysregulation disorder in a subject in need thereof, the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered in combination with an additional therapeutic agent.

[0151] In some embodiments of any of the aforementioned methods for treating a TGFβ dysregulation disorder in a subject in need thereof, the TGFβ dysregulation disorder is inflammatory bowel disease (IBD).

[0152] In some embodiments of any of the aforementioned methods for treating a TGFβ dysregulation disorder in a subject in need thereof, the TGFβ dysregulation disorder is Marfan syndrome.

[0153] In some embodiments of any of the aforementioned methods for treating a TGFβ dysregulation disorder in a subject in need thereof, the TGFβ dysregulation disorder is an autoimmune disorder.

[0154] In some embodiments of any of the aforementioned methods for treating a TGFβ dysregulation disorder in a subject in need thereof, the TGFβ dysregulation disorder is a wound healing disorder.

[0155] In another aspect, provided herein is a method for promoting wound healing in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide complex described herein, a pharmaceutical composition described herein, a polynucleotide described herein, or a vector described herein.

[0156] In some embodiments, the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered to a wound in a subject.

[0157] In some embodiments, the subject is a human. [Brief explanation of the drawings]

[0158] [Figure 1] FIG. 1 is a schematic diagram of TGFβ1 in complex with latency-associated peptide (LAP).

[0159] [Figure 2] We show that the TGFβ latent complex can be proteolytically or mechanically activated.

[0160] [Figure 3A] Figure 3A shows the antibody-TGFβ small latent complex (SLC) fusion construct design. Figure 3B shows the antibody-TGFβ SLC fusion construct design. The large latent complex (LLC) in both closed and open conformations is also shown. LAP, latency-associated peptide; SLC, small latent complex (LAP + mature domain). [Figure 3B]Figure 3B shows the antibody-TGFβ small latent complex (SLC) fusion construct design. Figure 3B shows the processing and activation profile of the anti-CD63-TGFβ1 SLC fusion protein. The unprocessed anti-CD63-TGFβ1 SLC fusion (left) is an expression form that can be present in both cell lysates and conditioned medium (CM), for example, and cannot be activated by integrins. The processed anti-CD63-TGFβ1 SLC fusion (right) is a purified, targeted form that is present only in CM and can be activated by integrins (e.g., integrin αvβ6). LAP, latency-associated peptide; SLC, small latent complex (LAP + mature domain).

[0161] [Figure 4A] Figure 4A shows the construct design and amino acid sequence of the anti-hCD63-TGFβ construct. Figure 4A shows the amino acid sequence corresponding to anti-hCD63-TGFβ1.C33S chain 1, which contains mROR SP (signal peptide) + VH (heavy chain variable domain) anti-hCD63 + hIgG4 + linker + TGFβ1 [LAP.C33S + mature peptide]. [Figure 4B] Figure 4B shows the construct design and amino acid sequence of the anti-hCD63-TGFβ construct. Figure 4B shows the amino acid sequence corresponding to anti-hCD63-TGFβ2.C33S chain 1, which contains mROR SP + VH anti-hCD63 + hIgG4 + linker + TGFβ2 [LAP.C33S + mature peptide]. [Figure 4C] Figure 4C shows the construct design and amino acid sequence of the anti-hCD63-TGFβ construct. Figure 4C shows the amino acid sequence corresponding to anti-hCD63-TGFβ.C33S chain 2, which contains mROR SP + VK (K light chain variable domain) anti-hCD63 + hKappa.

[0162] [Figure 5] Figure 1 shows the secretion of anti-hCD63-TGFβ constructs in the conditioned medium of ExpiCHO cells.

[0163] [Figure 6] 1 shows the expression of anti-hCD63-TGFβ1 in Freedom CHO cells.

[0164] [Figure 7] Figure 1 shows that mature TGFβ1 induced Smad2 / 3 signaling in Hek293-CAGA reporter cells.

[0165] [Figure 8] Figure 1 shows that both heat and acid activation of anti-hCD63-TGFβ1-expressing ExpiCHO conditioned medium (CM) induced Smad2 / 3 signaling.

[0166] [Figure 9] Figure 1 shows that anti-hCD63-TGFβ1 SLC was activated in CD63.Y235A (non-internalizing mutant) expressing cells co-cultured with integrin αvβ6 expressing cells.

[0167] [Figure 10] The amino acid sequence corresponding to anti-hCD63-TGFβ1.C33S uber stealth chain 1 containing mROR SP + VH anti-hCD63 + hIgG4 + linker + TGFβ1 [LAP.C33S + mature peptide] is shown.

[0168] [Figure 11] The amino acid sequence corresponding to anti-hCD63-TGFβ1.C33S uber stealth chain 2 containing mROR SP+VK anti-hCD63+hKappa is shown.

[0169] [Figure 12] 1 shows that anti-hCD63-TGFβ1 secreted from Freedom CHO cells is completely processed.

[0170] [Figure 13] Shown is the size exclusion chromatography (SEC) profile of purified anti-hCD63-TGFβ1 fusion protein (upper panel) and the accompanying SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of the eluate (lower panel).

[0171] [Figure 14] Anti-hCD63-TGFβ1 bound to hCD63.Y235A-expressing cells but not to hCD63 knockout (KO) Hek293 cells.

[0172] [Figure 15] 1 shows heat activation data of purified anti-CD63-TGFβ1 in NBL7 cells (American mink lung epithelial cells) harboring a Smad2 / 3 reporter vector.

[0173] [Figure 16] We show that mechanical activation of anti-CD63-TGFβ1 SLCs by integrin αvβ6 induces Smad2 / 3 signaling.

[0174] [Figure 17] Schematic diagrams of the REGN14660-TGFβ1 and REGN14660-TGFβ2 fusions described herein are shown. The REGN14660-TGFβ1 fusion contains the tripeptide Arg-Gly-Asp (arginine, glycine, and aspartic acid) "RGD" binding domain in latency-associated polypeptide (LAP), which enables integrin-mediated activation of TGFβ1.

[0175] [Figure 18] 1 shows REGN14660-TGFβ1 purification by high performance liquid chromatography (HPLC).

[0176] [Figure 19] REGN14660-TGFβ1 Western blot analysis is shown.

[0177] [Figure 20] 1 shows REGN14660-TGFβ2 purification by HPLC.

[0178] [Figure 21] REGN14660-TGFβ2 Western blot analysis is shown.

[0179] [Figure 22] Figure 1 shows a Smad2 / 3 reporter assay demonstrating the latency of the REGN14660-TGFβ1 fusion (HA = heat activated). The Smad2 / 3 reporter assay demonstrates that REGN14660-TGFβ1 retains TGFβ in a latent form (squares). TGFβ is released from the LAP upon heat activation (HA). Circles indicate "free" active TGFβ present in the antibody preparation.

[0180] [Figure 23] Figure 1 shows a Smad2 / 3 reporter assay demonstrating the latency of the REGN14660-TGFβ2 fusion (HA = heat activated). The Smad2 / 3 reporter assay demonstrates that REGN14660-TGFβ2 retains TGFβ in a latent form (squares). TGFβ is released from the LAP upon heat activation (HA). Circles indicate "free" active TGFβ present in the antibody preparation.

[0181] [Figure 24] REGN14660-TGFβ1 binds to fibronectin extra domain B (EDB-FN; extracellular matrix FN) with higher affinity (black line with black circles) than plasma (soluble) FN (black line with white squares). TGFβ1 could be activated from SLC by αvβ6 integrin expressed by CHO cells (black line with black circles), but not from the parental cell line, Chinese hamster ovary CHO cells (black line with white triangles).

[0182] [Figure 25] Images of aortas from wild-type (WT) and fibrillin-1 knockout (Fbn1KO) mice are shown. The top panel shows an aneurysm in the ascending aorta of an Fbn1KO mouse. The bottom panel shows immunostaining of WT and Fbn1KO aortic sections using the REGN14660 antibody, demonstrating fibronectin extra domain B (EDB-FN) staining in the diseased (ascending) region of the Fbn1KO aorta.

[0183] [Figure 26A] We show that EDB-FN detection can be associated with the presence of abnormal microvessels (CD31 positive) formed within the medial layer of Fbn1KO aortas (Fig. 26A). EDB-FN was not detected in wild-type (WT) P15 aortas (Fig. 26B). [Figure 26B] We show that EDB-FN detection can be associated with the presence of abnormal microvessels (CD31 positive) formed within the medial layer of Fbn1KO aortas (Fig. 26A). EDB-FN was not detected in wild-type (WT) P15 aortas (Fig. 26B).

[0184] [Figure 27] 1 shows a survival curve plot of fibrillin 1 knockout (Fbn1KO) mice (MAID 9419).

[0185] [Figure 28] Schematic diagram of the microfibrillar function of fibrillin. TGFβ, transforming growth factor β; BMP, bone morphogenetic protein; LTBP, latency-associated binding protein; Magp1 / 2, microfibril-associated glycoproteins 1 and 2; ECM, extracellular matrix.

[0186] [Figure 29] This shows a model of aortic aneurysm in Marfan syndrome (MFS) associated with reduced TGFβ signaling. Fewer fibrillin microfibrils in MFS reduces the incorporation of large latent complexes (LLCs) into the extracellular matrix (ECM), leading to reduced TGFβ signaling and ultimately to aneurysms.

[0187] [Figure 30]We demonstrate the use of fibronectin (FN) as a docking platform to deliver antibody-cryptic TGFβ fusions for local TGFβ restoration in MFS. Fibronectin is a matrix template for the deposition of fibrillin-1 microfibers. Fibronectin and fibrillin coexist in all tissues where they are coexpressed.

[0188] [Figure 31] Plasma and cellular fibronectin (FN) differ in three domains introduced by alternative splicing. Fibronectin is encoded by a single gene, but different forms of fibronectin arise through alternative splicing. Plasma FN is produced and secreted by hepatocytes in a soluble dimeric form. Cellular FN is expressed by some mesenchymal cells as a dimeric or cross-linked multimeric form and deposited as fibrils in the ECM, which is required for fibrillin-1 and type I collagen (Col I) deposition in the ECM. Fibronectin is involved in, for example, cell adhesion, cell motility, maintenance of cell shape, development, and wound healing. FN KO mice exhibit embryonic lethality at approximately day 8.5.

[0189] [Figure 32] demonstrates that fibronectin isoforms containing the EDB domain (cellular FN) are expressed in growing and remodeling tissues. EDB is a small domain of 91 amino acids (SEQ ID NO: 84) that is part of a fibronectin isoform formed by alternative splicing. The sequence of EDB is identical in mice and humans. EDB-containing isoforms are expressed during embryonic and postnatal development and in various solid tumors (and may be a marker for newly forming blood vessels), but are virtually undetectable in normal adult tissues, except for some blood vessels in the endometrium and ovaries during the proliferative phase.

[0190] [Figure 33] An example of a single-dose pharmacokinetic (PK) and tissue distribution study design is shown.

[0191] [Figure 34] 1 shows the use of an enzyme-linked immunosorbent assay (ELISA) to determine whether the antibody-TGFβ SLC fusions disclosed herein remained latent in the circulation.

[0192] [Figure 35] Figure 1 shows that the antibody-TGFβ1 SLC fusions disclosed herein remained latent in the circulation.

[0193] [Figure 36] 1 shows the levels of Fc fusions of antibodies disclosed herein-TGFβ1 SLC in circulation.

[0194] [Figure 37] Expression of C-type lectin domain family 9 member A (Clec9a) and epithelial cell adhesion molecule (Epcam) in the ileum, colon, and heart is shown (ArrayStudio).

[0195] [Figure 38] 1 shows the use of tissue ELISA to quantify the biodistribution of antibody-TGFβ SLC fusions disclosed herein.

[0196] [Figure 39] The delivered amount of antibody-TGFβ1 SLC fusion in the ileum and colon 2 hours post-injection (top) and 18 hours post-injection (bottom) is shown.

[0197] [Figure 40] 1 shows that anti-mEpcam-TGFβ1 SLC strongly induced Smad2 / 3 phosphorylation in the ileum.

[0198] [Figure 41] 1 shows that anti-mClec9a-TGFβ1 SLC induced Smad2 / 3 phosphorylation in the colon.

[0199] [Figure 42] 1 shows that antibody-TGFβ1 SLC fusions did not induce Smad2 / 3 phosphorylation in the heart.

[0200] [Figure 43] 1 shows that the anti-mEpcam-TGFβ1 SLC fusion was delivered to the colon.

[0201] [Figure 44] 1 shows that the anti-mEpcam-TGFβ1 SLC fusion was delivered to the ileum.

[0202] [Figure 45] 1 shows that anti-mEpcam-TGFβ1 SLC fusion induced phosphorylated (P)-Smad2 / 3 in the ileum.

[0203] [Figure 46] The amino acid sequence corresponding to anti-mEpcam-TGFβ1.C33S, chain 1, containing mROR SP+VH anti-mEpcam+mIgG1+linker+TGFβ1[LAP.C33S+mature peptide] is shown.

[0204] [Figure 47] The amino acid sequence corresponding to anti-mEpcam-TGFβ1.C33S, chain 2, containing mROR SP+VK anti-mEpcam+mKappa is shown.

[0205] [Figure 48] The amino acid sequence corresponding to anti-mClec9a-TGFβ1.C33S, chain 1, contains mROR SP+VH anti-mClec9a+mIgG1+linker+TGFβ1[LAP.C33S+mature peptide].

[0206] [Figure 49] The amino acid sequence corresponding to anti-mClec9a-TGFβ1.C33S, chain 2, containing mROR SP+VK anti-mClec9a+mKappa is shown.

[0207] [Figure 50] The amino acid sequence corresponding to mIgG1 isotype control antibody-TGFβ1.C33S, chain 1 includes mROR SP+VH mIgG1 isotype control antibody+mIgG1+linker+TGFβ1[LAP.C33S+mature peptide] is shown.

[0208] [Figure 51] The amino acid sequence corresponding to mROR SP + VK mIgG1 isotype control antibody + mIgG1 isotype control antibody with mKappa - TGFβ1.C33S, chain 2 is shown. DETAILED DESCRIPTION OF THE INVENTION

[0209] The present application provides, inter alia, compositions and methods relating to polypeptide complexes comprising a target-binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM) and a small latency complex (SLC). The SLC, in particular, comprises a dimeric latency-associated polypeptide (LAP) or a fragment or derivative thereof and a dimeric mature transforming growth factor β (TGFβ) family polypeptide or a fragment or derivative thereof. The mature TGFβ family polypeptide or a fragment or derivative thereof can be inactive as a result of interaction with the dimeric LAP. Upon activation, i.e., release from the SLC, the mature TGFβ family polypeptide or a fragment or derivative thereof can induce TGFβ signaling, for example, but not limited to, Smad2 / 3 signaling. Additionally, fusion polypeptides are provided that comprise a target-binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM), a latency-associated polypeptide (LAP) or a fragment or derivative thereof, and a mature transforming growth factor β (TGFβ) family polypeptide or a fragment or derivative thereof. Related polynucleotides, such as polynucleotides, vectors, cells, and pharmaceutical compositions encoding the fusion polypeptides described herein, are also included. Also disclosed are methods for delivering a mature TGFβ family polypeptide or a fragment or derivative thereof to a target cell in a subject in need thereof. Specifically, the method includes administering a polypeptide complex(es), pharmaceutical composition(s), polynucleotide(s), and / or vector(s) described herein, e.g., such that a target-binding polypeptide in the polypeptide complex binds to a molecule on the target cell. In certain aspects, the present disclosure also provides methods for treating a TGFβ dysregulated disorder (e.g., inflammatory bowel disease (IBD), Marfan syndrome, an autoimmune disease, and / or a wound healing disorder) in a subject in need of such treatment. Specifically, the method includes administering to the subject a therapeutically effective amount of a polypeptide complex(es), pharmaceutical composition(s), polynucleotide(s), or vector(s) described herein.Also included are methods for promoting wound healing in a subject, comprising administering to a subject in need thereof (e.g., a subject having a wound) a therapeutically effective amount of a polypeptide complex(es), pharmaceutical composition(s), polynucleotide(s), or vector(s) described herein. definition

[0210] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0211] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those skilled in the art upon reading this disclosure.

[0212] The term "about" or "approximately" includes within a statistically significant range of values. Such a range can be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The allowable variation encompassed by the term "about" or "approximately" depends on the particular system being studied and can be readily appreciated by one of ordinary skill in the art.

[0213] The term "antigen" refers to any agent (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portion thereof, or combination thereof) that, when introduced (either directly or when expressed, e.g., in a DNA vaccine) into a host, animal, or human having an immune system, can be recognized by the host's immune system and elicit an immune response.

[0214] The term "antigen-binding polypeptide" refers to an antigen-specific binding element, which may be any ligand or receptor fragment that binds to an antigen of interest or a polypeptide or fragment thereof. In some embodiments, the ligand and / or receptor fragment may be naturally derived. In some embodiments, the ligand and / or receptor fragment may be synthetic. Non-limiting examples of antigen-binding polypeptides include, for example, antibodies; antibody-derived polypeptides, such as Fab, Fab', F(ab')2, single-chain variable fragments (scFv), and Fv fragments; polypeptides derived from T cell receptors (TCRs), such as TCR variable domains; secreted factors (e.g., growth factors, cytokines) that may be artificially fused to signaling domains; and any ligand and / or receptor fragment that binds to an antigen of interest.

[0215] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0216] The terms "antibody," "antibodies," "immunoglobulin," and the like refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced. The term includes monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, diabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against an antigen-specific TCR), as well as epitope-binding fragments of any of the above. The terms "antibody" and "antibodies" also refer to covalently linked diabodies, such as those disclosed in U.S. Patent Application Publication No. 2007 / 0004909, which is incorporated herein by reference in its entirety, and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication No. 2009 / 0060910, which is incorporated herein by reference in its entirety. Antibodies useful in the present disclosure include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0217] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies (mAbs) of the present disclosure may include amino acid residues, for example, in the complementarity-determining regions (CDRs), particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework (FR) sequences. This term includes antibodies recombinantly produced in a non-human mammal or in the cells of a non-human mammal. This term is not intended to include antibodies isolated from or produced in a human subject.

[0218] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen can have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0219] The term "host cell" refers to any cell that contains heterologous nucleic acid. As a non-limiting example, the heterologous nucleic acid may be a vector. A host cell may be, for example, but not limited to, a cell from any organism that is used, engineered, modified, selected, transformed, or grown to produce a substance by the cell, for example, for expression of an RNA or DNA sequence, gene, protein, or enzyme by the cell.

[0220] By "decreased" or "reduced" or "lowered" or "lessened" or "attenuated" is intended any decrease in the level or activity of a gene / protein (e.g., encoded at a locus of interest). For example, decreased activity can include a decrease in the overall level or activity of a given protein, including, for example, a 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120% or more decreased level or activity when compared to an appropriate control.

[0221] By "increased" is intended any increase in the level or activity of a gene / protein (e.g., encoded at a locus of interest). For example, increased activity can include an increase in the overall level or activity of a given protein, including, for example, an increased level of activity of 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120% or more, as compared to an appropriate control.

[0222] Terms such as "specifically bind," "bind in a specific manner," and "antigen-specific" indicate that the molecules involved in specific binding are capable of forming relatively stable complexes with each other under physiological conditions and are unable to form nonspecifically stable complexes with other molecules outside the specific binding pair. Specific binding is characterized by an equilibrium dissociation constant (K) in the low micromolar to picomolar range. D ) (i.e., smaller K D(A higher specificity indicates tighter binding.) High specificity can be in the low nanomolar range, and very high specificity is in the picomolar range. Methods for determining whether two molecules specifically bind to each other are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0223] The terms "protein" and "polypeptide," used interchangeably herein, encompass all types of naturally occurring and synthetic proteins, including protein fragments of any length, fusion proteins, and modified proteins, including, but not limited to, glycoproteins, as well as any other type of modified protein (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.). Small polypeptides of less than 100 amino acids, preferably less than 50 amino acids, may be referred to as "peptides."

[0224] Proteins are said to have an "N-terminus" and a "C-terminus." The term "N-terminus" refers to the beginning of the amino acid chain of a protein, terminated by an amino acid having a free amine group (-NH). The term "C-terminus" refers to the end of the amino acid chain of a protein, terminated by a free carboxyl group (-COOH).

[0225] The terms "nucleic acid," "polynucleotide," and "nucleotide," used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides (RNA), deoxyribonucleotides (DNA), or analogs or modified versions thereof. These include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases. A single-stranded nucleic acid can be a sense strand or an antisense strand.

[0226] Nucleic acids are said to have a "5' end" and a "3' end" because mononucleotides react to create oligonucleotides in a manner in which the 5' phosphate of one mononucleotide pentose ring is attached in one direction to the 3' oxygen of its neighbor via a phosphodiester linkage. An end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to the 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if it is internal to a larger oligonucleotide, can also be said to have 5' and 3' ends. In either a linear or circular DNA molecule, distinct elements are referred to as being "upstream" or "downstream" 5' or 3' elements.

[0227] The term "fragment," when referring to a protein, means a protein that is shorter or has fewer amino acids than the full-length protein. A fragment may be, for example, an N-terminal fragment (i.e., removal of a portion of the C-terminus of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminus of the protein), or an internal fragment. The term "fragment," when referring to a nucleic acid, means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment may be, for example, a 5' fragment (i.e., removal of a portion of the 3' end of the nucleic acid), a 3' fragment (i.e., removal of a portion of the 5' end of the protein), or an internal fragment.

[0228] The terms "derivative" and "variant" are used interchangeably herein to refer to an entity that has significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, a derivative also differs functionally from its reference entity. Generally, whether a particular entity is properly considered a "derivative" of a reference entity is based on its degree of structural identity with the reference entity. As will be understood by those skilled in the art, any biological or chemical reference entity possesses certain characteristic structural elements. By definition, a derivative is a distinct entity that shares one or more such characteristic structural elements. To give just a few examples, a small molecule may have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic pendant moieties; thus, derivatives of this small molecule share the core structural element and characteristic pendant moieties but differ in other pendant moieties and / or the type of bond present within the core (single bond vs. double bond, E vs. Z, etc.). Derivative nucleic acids may have characteristic sequence elements composed of multiple nucleotide residues with designated positions relative to each other in linear or three-dimensional space. In some embodiments, a derivative nucleic acid sequence can be 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical over the entire length of a reference sequence or fragment thereof. Derivative peptides or polypeptides can have designated positions relative to each other in linear or three-dimensional space and / or characteristic sequence elements composed of multiple amino acids that contribute to a particular biological function. Derivative peptides and polypeptides include peptides and polypeptides that differ in amino acid sequence from a reference peptide or polypeptide by the insertion, deletion, and / or substitution of one or more amino acids, but retain at least one biological activity of such reference peptide or polypeptide (e.g., the ability to mediate cell infection by a virus, the ability to mediate membrane fusion, the ability to be bound by a specific antibody, or the ability to stimulate an immune response, etc.).In some non-limiting embodiments, a derivative peptide or polypeptide exhibits at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more full-length sequence identity with a reference peptide or polypeptide (or fragment thereof). Alternatively, or additionally, a derivative peptide or polypeptide may differ from a reference peptide or polypeptide as a result of one or more chemical moieties attached to the polypeptide backbone (e.g., glycosylation, phosphorylation, acetylation, myristoylation, palmitoylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.) and / or one or more variations therein. In some embodiments, a derivative peptide or polypeptide lacks one or more biological activities of a reference polypeptide, or has a reduced or increased level of one or more biological activities compared to the reference polypeptide. Derivatives of a particular peptide or polypeptide can be found in nature or can be synthetically or recombinantly produced. As used herein, the term "derivative" or "variant" also encompasses various fusion proteins and conjugates, including fusions or conjugates with detection tags (e.g., HA tags, histidine tags, biotin, fluorescent or luminescent domains, etc.), dimerization / multimerization sequences, Fc, signaling sequences, etc.

[0229] "Sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues of the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When using percentage sequence identity with respect to proteins, non-identical residue positions often differ by conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and thus does not alter the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score of 0 to 1. Scoring of conservative substitutions is calculated, for example, as carried out in the program PC / GENE.

[0230] "Percentage of sequence identity" includes values ​​determined by comparing two optimally aligned sequences (maximum number of perfectly matched residues) over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) when compared to a reference sequence (no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes concatenated non-homologous sequences), the comparison window is the entire length of the shorter of the two sequences being compared.

[0231] The term "latency-associated peptide" or "LAP" can refer to a peptide prodomain that prevents binding of TGFβ to the transforming growth factor β receptor (TGFβR).

[0232] The terms "integrin-binding motif" or "integrin-binding site," when used in connection with a latency-associated peptide (LAP) disclosed herein, may refer to a region on the LAP that contains a site that can be recognized by an integrin.

[0233] The term "small latent complex" or "SLC" can refer to a complex formed by a TGFβ family polypeptide, or a fragment or derivative thereof, and a latency-associated peptide (LAP), or a fragment or derivative thereof. In some embodiments, TGFβ can be non-covalently associated with LAP. The SLC can be linked to an additional protein, for example, a target-binding polypeptide.

[0234] The terms "inactive" or "inactive form" or "latent" in the context of TGFβ may refer to a TGFβ family polypeptide, or a fragment or derivative thereof, that is unable to bind to a transforming growth factor β receptor (TGFβR) and / or initiate TGFβ signaling (such as, for example, Smad2 / 3 signaling and / or ERK signaling). As a non-limiting example, a TGFβ family polypeptide, or a fragment or derivative thereof, may be inactive as a result of interaction with LAP, or a fragment or derivative thereof.

[0235] The term "active" or "active form" in the context of TGFβ may refer to a form of a TGFβ family polypeptide, or a fragment or derivative thereof, that is capable of binding to a transforming growth factor β receptor (TGFβR) and / or inducing TGFβ signaling (e.g., Smad2 / 3 signaling and / or ERK signaling, etc.), for example, upon release from a small latent complex (SLC).

[0236] The term "dimer" refers to any compound or molecule composed of two subunits. A dimeric compound or molecule, e.g., a protein dimer, may be considered a homodimer (e.g., formed by two identical proteins) or a heterodimer (e.g., formed by two different proteins) depending on the nature of the subunits that form the dimeric compound or molecule. In some embodiments, the dimeric subunits may be attached, for example, via disulfide bonds. In some embodiments, the dimeric subunits may be non-covalently bound to each other.

[0237] The term "mature TGFβ family polypeptide" refers to any TGFβ family polypeptide, or fragment or derivative thereof, that has undergone dimerization, i.e., in the context of TGFβ, the process by which two TGFβ subunits combine to form a single dimeric molecule. In some embodiments, a mature TGFβ polypeptide, or fragment or derivative thereof, may be inactive, for example, as a result of interaction with a latency-associated peptide (LAP) or a fragment or derivative thereof, such as a dimeric LAP. In some embodiments, a dimeric mature TGFβ family polypeptide and a dimeric LAP may be in a complex (i.e., associated) and form a small latent complex (SLC). In some embodiments, a mature TGFβ family polypeptide and a dimeric LAP may be associated via non-covalent interactions. In some embodiments, a mature TGFβ family polypeptide and a dimeric LAP may be covalently linked and separated, for example, by a protease cleavage site (such as, but not limited to, a furin cleavage site). Other non-limiting examples of protease cleavage sites include PC1 / 3, PC2, PC4, PC5 / 6, PACE4, PC7, SKI-1 / S1P, and PCSK9 cleavage sites. In some embodiments, after protease cleavage, LAP may remain non-covalently associated with the mature TGFβ family polypeptide or a fragment or derivative thereof. In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof may be released from the SLC. In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof may induce Smad2 / 3 signaling in target cells or cells adjacent to the target cells upon release from the SLC. Non-limiting examples of mature TGFβ family polypeptides include mature growth differentiation factor 8 (GDF8), mature growth differentiation factor 11 (GDF11), and mature bone morphogenetic protein 4 (BMP4) polypeptides. Members of the TGFβ family include, for example, nodal, activin, inhibin, bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs), TGF-β1, TGF-β2 and TGF-β3.In some embodiments, a mature TGFβ family polypeptide can include, for example, TGFβ1, TGFβ2, TGFβ3, BMP4, and / or GDF11. In some embodiments, a mature TGFβ family polypeptide can include a potential BMP / TGF-β family ligand described herein. In some embodiments, a mature TGFβ family polypeptide can be a mature TGFβ polypeptide. In some embodiments, a mature TGFβ polypeptide can be a mature TGFβ1 polypeptide. In some embodiments, a mature TGFβ polypeptide can be a mature TGFβ2 polypeptide. In some embodiments, a mature TGFβ polypeptide can be a mature growth differentiation factor 8 (GDF8) or mature growth differentiation factor 11. In some embodiments, a mature TGFβ polypeptide can be a mature bone morphogenetic protein 4 (BMP4).

[0238] Terms such as "operably linked" refer to a juxtaposition in which the described components are in a relationship permitting them to function in their intended manner. For example, a control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Operably linked" sequences include both expression control sequences contiguous with a gene of interest and expression control sequences that act in trans or remotely to control the gene of interest (or sequence of interest). The term "expression control sequence" includes polynucleotide sequences necessary to affect the expression and processing of coding sequences to which they are ligated. "Expression control sequences" include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance polypeptide stability; and, if desired, sequences that enhance polypeptide secretion. The nature of such control sequences will vary depending on the host organism. For example, in prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences, while in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0239] The terms "vector," "expression vector," and "cloning vector" refer to any vehicle capable of introducing a nucleotide sequence, e.g., an RNA or DNA sequence, encoding, e.g., a foreign gene, into a cell (e.g., a host cell) to genetically modify the cell and promote expression (e.g., transcription and translation) of the introduced nucleotide sequence. Non-limiting examples of vectors include synthetic RNA and DNA molecules, plasmids, viruses, phages, and the like. In some embodiments, the vector may be a viral vector, including, but not limited to, a baculovirus vector, a herpesvirus vector, a lentivirus vector, a retrovirus vector, a vaccinia virus vector, an adeno-associated virus (AAV) vector, an adenovirus vector, and an alphavirus vector.

[0240] The term "isolated" refers to a homogenous population of molecules (e.g., polynucleotides or polypeptides) that have been substantially separated and / or purified from other components of the system in which they are produced, e.g., recombinant cells, and proteins that have been subjected to at least one purification or isolation step. "Isolated" refers to molecules that are substantially free of other cellular material and / or chemicals and includes molecules isolated to greater degrees of purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0241] The term "treating" a condition, disorder, disease, or condition or "treatment" thereof includes (1) preventing, delaying, or reducing the occurrence and / or likelihood of the appearance of at least one clinical or subclinical symptom of the condition, disorder, disease, or condition in a subject who is suffering from or may be susceptible to the condition, disorder, disease, or condition but who has not yet experienced or displayed clinical or subclinical symptoms of the condition, disorder, disease, or condition; or (2) inhibiting the condition, disorder, disease, or condition, i.e., arresting, reducing, or delaying the onset or recurrence of the disease, or at least one clinical or subclinical symptom thereof; or (3) palliating the condition, disorder, disease, or condition, i.e., causing regression of the condition, disorder, disease, or condition, or at least one clinical or subclinical symptom of the condition, disorder, disease, or condition. The benefit to a treated subject will be statistically significant or at least perceptible to the patient or physician.

[0242] The term "TGFβ dysregulation disorder" refers to a condition, disorder, disease, or condition associated with dysregulation of TGFβ, including, for example, low TGFβ expression and / or expression of variants of TGFβ. In some embodiments, a TGFβ dysregulation disorder can be treated by targeted delivery of TGFβ. Non-limiting examples of TGFβ dysregulation disorders include type 1 diabetes (T1D), inflammatory bowel disease (IBD), colitis, Marfan syndrome (MFS), aortic dilation and rupture (aortic aneurysm), autoimmune disorders, arthritis (e.g., rheumatoid arthritis or osteoarthritis), lupus (e.g., systemic lupus erythematosus), and wound healing disorders.

[0243] "Individual" or "subject" or "animal" refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats). In some embodiments, the subject is a human.

[0244] The term "effective" as applied to a dose or amount refers to the amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs used, the mode of administration, etc.

[0245] The phrase "pharmaceutically acceptable," when used in connection with the compositions described herein, refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to a mammal (e.g., a human). Preferably, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in mammals, more particularly humans.

[0246] Terms such as "administration" refer to and include administration of a composition to a subject or system (e.g., to a cell, organ, tissue, organism, or related component or set of components thereof). One of skill in the art will understand that the route of administration can vary depending, for example, on the subject or system to which the composition is being administered, the nature of the composition, the purpose of the administration, etc. For example, in certain embodiments, administration to an animal subject (e.g., a human or rodent) may be bronchial (including by bronchial instillation), oral, enteral, intradermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intracerebroventricular, transmucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, intravaginal, and / or intravitreal. In some embodiments, administration may include intermittent dosing. In some embodiments, administration may include continuous dosing (e.g., perfusion) for at least a selected period of time.

[0247] In light of the disclosure herein, one skilled in the art can employ conventional molecular biology, microbiology, and recombinant DNA techniques, which are explained fully in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (herein referred to as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (DNGlover ed.1985); Oligonucleotide Synthesis (MJGait ed.1984);Nucleic Acid Hybridization[BDHames & SJHiggins eds.(1985)];Transcription And Translation[BDHames & SJHiggins,eds.(1984)];Animal Cell Culture[RIFreshney,ed.(1986)];Immobilized Cells And Enzymes[IRL Press,(1986)];B.Perbal,A Practical Guide To Molecular Cloning(1984);Ausubel,FMet al.(eds.).Current See Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994.These techniques are described in Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), U.S. Patent No. 5,071,743; Fukuoka et al., Biochem. Biophys. Res. Commun. 263:357-360 (1999); Kim and Maas, BioTech. 28:196-198 (2000); Parikh and Guengerich, BioTech. 24:4 28-431 (1998); Ray and Nickoloff, BioTech. 13:342-346 (1992); Wang et al., BioTech. 19:556-559 (1995); Wang and Malcolm, BioTech. 26:680-682 (1999); Xu and Gong, BioTech. 26:639-641 (1999), U.S. Patent Nos. 5,789,166 and 5,932,419; Hogrefe, Strategies 14.3:74-75 (2001); U.S. Patent Nos. 5,702,931, 5,780,270 and 6,242,222; Angag and Schutz, Biotech. 30:486-488 (2001); Wang and Wilkinson, Biotech. 29:976-978 (2000); Kang et al., Biotech. 20:44-46 (1996); Ogel and McPherson, Protein Engineer. 5:467-468 (1992); Kirsch and This includes site-directed mutagenesis, as described in Joly, Nucl. Acids. Res. 26:1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178:3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28:197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22:541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57:229-237, and Pons et al., Meth. Molec. Biol. 67:209-218. Polypeptide Complex

[0248] In certain aspects, the present disclosure provides a polypeptide complex comprising: a) a target-binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM); b) Small Latent Complexes (SLCs), (i) a dimeric latency-associated polypeptide (LAP) or a fragment or derivative thereof; and (ii) a small latent complex (SLC) comprising a dimeric mature transforming growth factor β (TGFβ) family polypeptide or a fragment or derivative thereof; The present invention provides a polypeptide complex comprising: The mature TGFβ family polypeptide, or a fragment or derivative thereof, is inactive as a result of interaction with dimeric LAP.

[0249] In some embodiments, the LAP or a fragment or derivative thereof is covalently attached to the target-binding polypeptide. In some embodiments, the LAP or a fragment or derivative thereof is covalently attached to the target-binding polypeptide via a linker. In some embodiments, the LAP or a fragment or derivative thereof is non-covalently bound to the target-binding polypeptide. In some embodiments, the target-binding polypeptide binds both the LAP and a molecule on a target cell or in the ECM. In some embodiments, the target can include, for example, but not limited to, a molecule on a target cell or a molecule in the extracellular matrix (ECM).

[0250] In some embodiments, the mature TGFβ family polypeptide or fragment or derivative thereof and the dimeric LAP associate via non-covalent interactions.

[0251] In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof and the LAP are covalently linked and separated by a protease cleavage site. Non-limiting examples of protease cleavage sites include furin, PC1 / 3, PC2, PC4, PC5 / 6, PACE4, PC7, SKI-1 / S1P, and PCSK9 cleavage sites. In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof binds to a transforming growth factor β receptor (TGFβR) once the mature TGFβ family polypeptide or a fragment or derivative thereof is released from the SLC.

[0252] In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof induces Smad2 / 3 signaling in the target cell or a cell adjacent to the target cell upon activation of the mature TGFβ family polypeptide, hi some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof induces Smad2 / 3 signaling in the target cell or a cell adjacent to the target cell upon release of the mature TGFβ family polypeptide from the SLC. small latent complex (SLC)

[0253] In certain aspects, the polypeptide complexes described herein may comprise a small latent complex (SLC) comprising a dimeric latency-associated peptide (LAP) or a fragment or derivative thereof; and a dimeric mature transforming growth factor β (TGFβ) family polypeptide or a fragment or derivative thereof. In one aspect, the mature TGFβ family polypeptide or a fragment or derivative thereof may be inactive as a result of interaction with the dimeric LAP.

[0254] Without wishing to be bound by theory, the small latent complex (SLC) may comprise a complex of the LAP domain and the mature TGFβ domain. During secretion, a furin-like proprotein convertase may cleave TGFβ, for example, at a protease cleavage site located at the junction between the mature domain and LAP. After furin cleavage, LAP may remain noncovalently associated with the mature TGFβ domain, thereby inactivating the mature TGFβ domain by blocking its binding to the TGFβ signaling receptor. Thus, when LAP remains associated with the mature TGFβ domain, the activity of the mature domain may be blocked. SLC may be secreted alone as a soluble molecule, or secreted while tethered to a cellular environmental molecule, which may be, for example, surface-bound. In some embodiments, the environmental molecule may be covalently linked to the SLC. Non-limiting examples of environmental molecules include latency-associated binding protein (LTBP), glycoprotein A repeat-dominant protein (GARP), leucine-rich repeat-containing protein 32 (LRRC32), and leucine-rich repeat-containing protein 33 (LRRC33 / NRROS). For example, an SLC secreted in a complex with an environmental molecule(s) may be referred to as a large latent complex (LLC). The environmental molecules may each bind to the same epitope on LAP, such as via a disulfide bond (e.g., C33 in the LAP domain disclosed herein).

[0255] The TGF-β protein family is encoded by 33 genes. Members of the TGF-β family, including activin, anti-Müllerian hormone (AMH), bone morphogenetic protein (BMP), inhibin, nodal, and growth and differentiation factors (GDFs), may be involved in, for example, the anterior / posterior and dorsal / ventral axes, the specification of ectoderm, mesoderm, and endoderm, as well as left-right asymmetry and various characteristics of individual organs. TGF-β protein family members TGFβ1, TGFβ2, and TGFβ3 play important roles in immune response, wound healing, development, and tumor cell growth and inhibition. In particular, TGFβ1, TGFβ2, and TGFβ3 may be involved in cellular processes such as, but not limited to, extracellular matrix (ECM) remodeling, cell migration, invasion, epithelial-mesenchymal transition (EMT), and growth inhibition and / or immunosuppression.

[0256] As a non-limiting example, transforming growth factor beta 1 (TGFβ1) can be a secreted disulfide-linked homodimeric protein. Full-length TGFβ1 can include a signal peptide (amino acids 1-29), a latency-associated peptide (LAP or prodomain; amino acids 30-274), and a mature domain (amino acids 279-390). At the junction between the LAP and mature domain, an "RXXR" (SEQ ID NO: 35) furin processing site can be present, where "X" can be any amino acid. Non-limiting examples of "RXXR" (SEQ ID NO: 35) furin processing sites include, for example, RHRR (SEQ ID NO: 85), RRKR (SEQ ID NO: 86), and RKKR (SEQ ID NO: 87). During secretion, a furin-like proprotein convertase can cleave TGFβ1 at the junction between the mature domain and LAP. After furin cleavage, LAP may remain non-covalently associated with the mature domain, thereby inactivating the mature domain by blocking its binding to a signaling receptor(s). Thus, when LAP remains associated with the mature domain, the activity of the mature domain may be blocked. LAP may be removed from the processed TGFβ complex, for example, by proteolytic and mechanical methods. Thus, LAP removal can release active mature TGFβ, which may be capable of inducing downstream signaling, such as Smad2 / 3 signaling (see, for example, Figures 1 and 2).

[0257] In some embodiments, upon release of a mature TGFβ family polypeptide or a fragment or derivative thereof disclosed herein from an SLC disclosed herein, the mature TGFβ family polypeptide or a fragment or derivative thereof can bind to a transforming growth factor β receptor (TGFβR). Without wishing to be bound by theory, seven TGFβ superfamily type I receptors and five type II receptors have been identified in mammals. The type I receptor family includes activin-like kinases (ALK) 1-7. Type II receptors include TGFβRII, activin RIIA, activin RIIB, BMPRII, and AMHRII. Type I and type II receptors are structurally related transmembrane glycoproteins containing an extracellular N-terminal ligand-binding domain with more than 10 cysteine ​​residues that may regulate dimeric structure, a transmembrane region, and a C-terminal serine / threonine kinase domain. Type I receptors have a highly conserved GS domain, a region rich in glycine and serine residues, in the juxtamembrane domain adjacent to the N-terminus of the kinase domain. In some embodiments, TGFβ signaling (e.g., Smad2 / 3 signaling) can be initiated by the binding of a mature TGFβ family polypeptide to, for example, TGFβ receptor type I (TGFβRI) and / or TGFβ receptor type II (TGFβRII) receptors on the cell membrane.

[0258] In some embodiments, upon release from the SLC disclosed herein, the mature TGFβ family polypeptides disclosed herein, or fragments or derivatives thereof, may induce TGFβ signaling, such as Smad2 / 3 signaling and / or ERK signaling, in a cell, e.g., a target cell or a cell adjacent to a target cell.

[0259] In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof can be chemically dissociated from the LAP or fragment or derivative thereof and released in an active form from the SLC. Non-limiting examples of chemical dissociation include protease treatment, temperature treatment, acid treatment, or any combination thereof.

[0260] In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof can be mechanically dissociated from the LAP or a fragment or derivative thereof and released in an active form from the SLC. As a non-limiting example, mechanical dissociation can occur as a result of interaction between the LAP or a fragment or derivative thereof and an integrin polypeptide, e.g., via an integrin-binding motif in the LAP. Non-limiting examples of integrins include αβ, αβ, and αβ.

[0261] In some embodiments, the small latent complex (SLC) comprises the amino acid sequence of SEQ ID NO:21, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:21. In certain embodiments, the nucleotide sequence encoding the small latent complex (SLC) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:21, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:21. In certain embodiments, the nucleotide sequence encoding the small latent complex (SLC) comprises the nucleotide sequence of SEQ ID NO: 22, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 22. In certain embodiments, the small latent complex (SLC) comprises the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the nucleotide sequence encoding the small latent complex (SLC) comprises the nucleotide sequence of SEQ ID NO: 22.

[0262] In some embodiments, the small latent complex (SLC) comprises the amino acid sequence of SEQ ID NO:25, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:25. In certain embodiments, the nucleotide sequence encoding the small latent complex (SLC) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:25, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:25. In certain embodiments, the nucleotide sequence encoding the small latent complex (SLC) comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 26. In certain embodiments, the small latent complex (SLC) comprises the amino acid sequence of SEQ ID NO: 25. In certain embodiments, the nucleotide sequence encoding the small latent complex (SLC) comprises the nucleotide sequence of SEQ ID NO: 26.

[0263] In some embodiments, the small latent complex (SLC) comprises the amino acid sequence of SEQ ID NO:92, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:92. In certain embodiments, the nucleotide sequence encoding the small latent complex (SLC) comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:92, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:92. In certain embodiments, the nucleotide sequence encoding the small latent complex (SLC) comprises the nucleotide sequence of SEQ ID NO:93, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:93. In certain embodiments, the small latent complex (SLC) comprises the amino acid sequence of SEQ ID NO:92. In certain embodiments, the nucleotide sequence encoding the small latent complex (SLC) comprises the nucleotide sequence of SEQ ID NO:93. Latency-associated peptide (LAP)

[0264] In some embodiments, a polypeptide complex described herein can comprise a small latent complex (SLC) comprising a dimeric latency-associated polypeptide (LAP) or a fragment or derivative thereof.

[0265] In some embodiments, the LAP or a fragment or derivative thereof may be attached to a target-binding polypeptide disclosed herein. In some embodiments, the LAP or a fragment or derivative thereof may be covalently attached to the target-binding polypeptide. In some embodiments, the LAP or a fragment or derivative thereof may be covalently attached to the target-binding polypeptide via a linker. In some embodiments, the linker may be located between the target-binding polypeptide and the LAP or a fragment or derivative thereof.

[0266] In some embodiments, the linker can be 1 to 10 amino acids in length. In some embodiments, the linker can be 1 to 20 amino acids in length. By way of non-limiting example, the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the linker can be 1 to 30 amino acids in length. In some embodiments, the linker can be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In some embodiments, the linker can be a flexible linker. In some embodiments, the linker can be a rigid linker. In some embodiments, the linker may be 1 to 20 amino acids in length, 2 to 15 amino acids in length, 3 to 12 amino acids in length (including 4 to 10 amino acids in length, 5 to 9 amino acids in length, 6 to 8 amino acids in length, or 7 to 8 amino acids in length), and may be 1, 2, 3, 4, 5, 6, or 7 amino acids in length. In some embodiments, the linker is 12 amino acids in length. In some embodiments, the linker may be optimized so that it does not impose any constraints on the conformation and / or interactions of the linked partners.

[0267] In some embodiments, the linker is a flexible linker. Suitable linkers can be readily selected and can be of any of a variety of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids (including from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids), and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (GS)n (where n is an integer of at least 1 (e.g., 1 to 20)) (SEQ ID NO: 174), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.

[0268] In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0269] Non-limiting examples of linkers that can be used include any of SEQ ID NOs: 19, 46-79, and 96. In some embodiments, the linker comprises an amino acid sequence set forth in any of SEQ ID NOs: 19, 46-79, or 96, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NO: 19, 46-79, or 96.

[0270] In some embodiments, the linker can comprise the sequence (GGGGS)n (SEQ ID NO:46), where n=1-10, or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker can consist of the sequence (GGGGS)n (SEQ ID NO:46). As non-limiting examples, the linker can comprise the sequence GGGGS (SEQ ID NO:47); GGGGSGGGGS((G4S)2; SEQ ID NO:48); GGGSGGGGSGGGGS((G4S)3; SEQ ID NO:49); or GGGGSGGGGSGGGGSGGGGS((G4S)4; SEQ ID NO:50).

[0271] In some embodiments, the linker can comprise the sequence (GGGS)n (SEQ ID NO:51), where n=1-10, or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker can consist of the sequence (GGGS)n (SEQ ID NO:51). In some embodiments, the linker can comprise the sequence GGGSGGGSGGGS(G3S)3 (SEQ ID NO:19). In some embodiments, the linker can consist of the sequence GGGSGGGSGGGS(G3S)3 (SEQ ID NO:19).

[0272] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 19. In certain embodiments, the nucleotide sequence encoding the linker comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:19, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:19. In certain embodiments, the nucleotide sequence encoding the linker comprises the nucleotide sequence of SEQ ID NO:20, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:20. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:19. In certain embodiments, the nucleotide sequence encoding the linker comprises the nucleotide sequence of SEQ ID NO:20.

[0273] In some embodiments, the linker can comprise the sequence GSGESGGGSG (SEQ ID NO: 96). In some embodiments, the linker can consist of the sequence GSGESGGGSG (SEQ ID NO: 96).

[0274] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:96, or a variant thereof having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% sequence identity to SEQ ID NO:96. In certain embodiments, the nucleotide sequence encoding the linker comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:96, or a variant thereof having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% sequence identity to SEQ ID NO:96. In certain embodiments, the nucleotide sequence encoding the linker comprises the nucleotide sequence of SEQ ID NO:97, or a nucleotide sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% sequence identity to SEQ ID NO:97. In certain embodiments, the linker comprises the amino acid sequence of SEQ ID NO:96. In certain embodiments, the nucleotide sequence encoding the linker comprises the nucleotide sequence of SEQ ID NO:97.

[0275] In some embodiments, the LAP or a fragment or derivative thereof may be non-covalently bound to the target-binding polypeptide. In some embodiments, the target-binding polypeptide binds to both the LAP and a molecule on the target cell or in the ECM. In some embodiments, the target-binding polypeptide may comprise an antigen-binding polypeptide. In some embodiments, the antigen-binding polypeptide may comprise an antibody or a fragment or derivative thereof, such as an antigen-binding fragment thereof.

[0276] In some embodiments, a dimeric LAP of the present disclosure may associate with a mature TGFβ family polypeptide or a fragment or derivative thereof disclosed herein, for example, via a non-covalent interaction. In some embodiments, the LAP and the mature TGFβ family polypeptide or a fragment or derivative thereof may be covalently linked. In some embodiments, the LAP and the mature TGFβ family polypeptide or a fragment or derivative thereof may be covalently linked and separated, for example, by a protease cleavage site.

[0277] In some embodiments, when a LAP or a fragment or derivative thereof can be attached (e.g., covalently attached) or bound (e.g., non-covalently bound) to a target-binding polypeptide, the target-binding polypeptide can bind to both the LAP and the target.

[0278] In some embodiments, the protease cleavage site that can separate LAP and the mature TGFβ family polypeptide may comprise a furin cleavage site. In some embodiments, the furin cleavage site may be located at the junction between LAP and the mature TGFβ family polypeptide. In some embodiments, the furin cleavage site may comprise the sequence RXXR (SEQ ID NO: 35). In some embodiments, the protease cleavage site is a furin cleavage site. In some embodiments, the furin cleavage site comprises the sequence RXXR (SEQ ID NO: 35). In some embodiments, the furin cleavage site comprises the sequence RHRR (SEQ ID NO: 85). In some embodiments, the furin cleavage site comprises the sequence RRKR (SEQ ID NO: 86). In some embodiments, the furin cleavage site comprises the sequence RKKR (SEQ ID NO: 87).

[0279] Without wishing to be bound by theory, during secretion in the endoplasmic reticulum (ER) / Golgi, furin may cleave mature TGFβ family polypeptides from LAP, leaving LAP noncovalently associated with the mature TGFβ family polypeptide. If LAP remains associated with the mature TGFβ family polypeptide, the activity of the mature TGFβ family polypeptide may be blocked, thereby rendering the mature TGFβ family polypeptide inactive. LAP may be removed from the mature TGFβ family polypeptide, for example, via proteolytic and / or mechanical methods. LAP removal may release active mature TGFβ to induce, for example, downstream Smad2 / 3 signaling. During proteolytic activation, proteases may degrade the LAP domain or cause a conformational change in the LAP domain, releasing mature TGFβ. Upon mechanical activation, integrins (e.g., αvβ6 integrin, αvβ8 integrin, and / or αvβ1 integrin) can bind to the tripeptide Arg-Gly-Asp (arginine, glycine, and aspartate) "RGD" binding motif at the C-terminus of LAP. Integrin binding at the C-terminus of LAP and cell surface-bound environmental molecule association (via covalent bonds), for example, at the N-terminus of LAP, can generate a directional tensile force that can open the LAP and release free mature TGFβ. Mature TGFβ can bind to, for example, TGFβR1 and TGFβR2 on the cell surface and induce signaling, including, but not limited to, Smad2 / 3 signaling. The active form of mature TGFβ can induce Smad2 / 3 signaling. In some embodiments, the mature TGFβ described herein can be internalized within cells. In some embodiments, when mature TGFβ is internalized within a cell, it can induce TGFβ signaling (e.g., Smad2 / 3 signaling and / or ERK signaling), such as within an endosome.

[0280] In some embodiments, as a result of interacting with (eg, associating with) LAP, or a fragment or derivative thereof, a mature TGFβ family polypeptide disclosed herein, or a fragment or derivative thereof, may be inactive.

[0281] In some embodiments, activation of a mature TGFβ family polypeptide or a fragment or derivative thereof can involve release of the mature TGFβ family polypeptide from the ECM, e.g., release of LLC from the ECM, followed by further proteolysis of LAP by any of a variety of proteases to release active TGFβ. Non-limiting examples of proteases that can be involved in the proteolysis of LAP include matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), thrombospondin 1 (TSP-1), plasmin (PLN), and plasma kallikrein (PLK).

[0282] In some embodiments, upon activation, a mature TGFβ family polypeptide disclosed herein, or a fragment or derivative thereof, can bind to a TGFβR disclosed herein. In some embodiments, upon activation, a mature TGFβ family polypeptide disclosed herein, or a fragment or derivative thereof, can induce Smad2 / 3 signaling in a cell (e.g., a target cell or a cell adjacent to a target cell). Upon activation, a mature TGFβ family polypeptide can be considered to be in an active form.

[0283] In some embodiments, LAP or a fragment or derivative thereof may interact with latency-associated binding protein (LTBP), glycoprotein A repeat-dominant protein (GARP), leucine-rich repeat-containing protein 32 (LRRC32), or leucine-rich repeat-containing protein 33 (LRRC33 / NRROS).

[0284] In some embodiments, LAP or a fragment or derivative thereof comprises an integrin-binding motif. In some embodiments, the integrin-binding motif may comprise an RGD sequence (i.e., a sequence consisting of arginine, glycine, and aspartic acid residues). In some embodiments, the integrin-binding motif in LAP may be a region consisting of 1 to 30 amino acid residues including the RGD sequence. In some embodiments, the integrin-binding motif in LAP may be a region consisting of 1 to 30 amino acid residues excluding the RGD sequence. In some embodiments, the integrin-binding motif comprises the sequence RGD. In some embodiments, the integrin is αvβ6 integrin. In some embodiments, the integrin is αvβ8 integrin.

[0285] In some embodiments, the integrin binding motif in the LAP can be a region that can be 1 to 10 amino acids in length. In some embodiments, the integrin binding motif can be 1 to 20 amino acids in length. By way of non-limiting example, the integrin binding motif can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the integrin binding motif can be 1 to 30 amino acids in length. In some embodiments, the integrin binding motif can be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In some embodiments, the integrin-binding motif may be 1 to 20 amino acids in length, 2 to 15 amino acids in length, 3 to 12 amino acids in length (including 4 to 10 amino acids in length, 5 to 9 amino acids in length, 6 to 8 amino acids in length, or 7 to 8 amino acids in length), or may be 1, 2, 3, 4, 5, 6, or 7 amino acids in length. In some embodiments, the integrin-binding motif sequence may comprise an RGD sequence. In some embodiments, the RGD sequence may not comprise an integrin-binding motif.

[0286] In some embodiments, the integrin-binding motif comprises an RGD sequence. In some embodiments, the integrin-binding motif consists of an RGD sequence. Without wishing to be bound by theory, upon mechanical activation, an integrin may bind to the integrin-binding motif at the C-terminus of LAP. Non-limiting examples of integrins include αvβ6 integrin, αvβ8 integrin, and αvβ1 integrin. In some embodiments, the integrin is αvβ6 integrin. In some embodiments, the integrin is αvβ8 integrin. Integrin binding at the C-terminus of LAP and cell surface-bound environmental molecule association (e.g., via covalent bonds) at the N-terminus of LAP generates a directional tensile force that opens the LAP and releases free mature TGFβ. Mature TGFβ may bind to TGFβR1 and TGFβR2 on the cell surface and induce Smad2 / 3 signaling.

[0287] In some embodiments, a mature TGFβ described herein can bind to a TGFβ receptor, e.g., TGFβR1 and / or TGFβR2, on the surface of a cell described herein. In some embodiments, the ligand and receptor can remain on the cell surface and the TGFβ ligand-receptor complex is not internalized. In some embodiments, if the TGFβ ligand-receptor complex is not internalized, TGFβ signaling (e.g., Smad2 / 3 signaling and / or ERK signaling) can be induced within the cell.

[0288] In some embodiments, a mature TGFβ described herein can bind to a TGFβ receptor, e.g., TGFβR1 and / or TGFβR2, on the cell surface, thereby triggering internalization of the ligand and receptor. The TGFβ ligand-receptor complex can enter the endocytic system, e.g., via clathrin-mediated endocytosis (CME). In some embodiments, endocytosis of the ligand-receptor complex, e.g., in an early endosome, can modulate (e.g., enhance) TGFβ signaling (e.g., Smad2 / 3 signaling and / or ERK signaling) as described herein.

[0289] In some embodiments, the mature TGFβ disclosed herein can be internalized into a cell described herein (e.g., a target cell or a cell adjacent to a target cell). In some embodiments, when the mature TGFβ is internalized into a cell, the mature TGFβ can induce TGFβ signaling, such as in an endosome.

[0290] In some embodiments, the integrin binding motif comprises an RGD sequence comprising one or more mutations. In some embodiments, the integrin binding motif consists of an RGD sequence comprising one or more mutations. In some aspects, the RGD sequence comprising one or more mutations comprises the sequence RGE. In some embodiments, when the RGD sequence comprises a mutation(s), the mutated RGD sequence may inhibit or block mechanical activation of LAP. In some embodiments, when the mutated RGD sequence inhibits or blocks mechanical activation of LAP, activation of LAP may be limited to activation by, for example, chemical activation and / or proteolytic activation as described herein.

[0291] In some embodiments, LAP or a fragment or derivative thereof does not comprise an integrin binding motif.

[0292] In some embodiments, the integrin binding motif comprises the amino acid sequence of SEQ ID NO: 33, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 33. In certain embodiments, the nucleotide sequence encoding the integrin-binding motif comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:33, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:33. In certain embodiments, the nucleotide sequence encoding the integrin-binding motif comprises the nucleotide sequence of SEQ ID NO: 34, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 34. In certain embodiments, the integrin-binding motif comprises the amino acid sequence of SEQ ID NO: 33. In certain embodiments, the nucleotide sequence encoding the integrin-binding motif comprises the nucleotide sequence of SEQ ID NO: 34.

[0293] In some embodiments, the LAP comprises the amino acid sequence of SEQ ID NO:29, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:29. In certain embodiments, the nucleotide sequence encoding LAP comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:29, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:29. In certain embodiments, the nucleotide sequence encoding LAP comprises the nucleotide sequence of SEQ ID NO: 30, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 30. In certain embodiments, LAP comprises the amino acid sequence of SEQ ID NO: 29. In certain embodiments, the nucleotide sequence encoding LAP comprises the nucleotide sequence of SEQ ID NO: 30.

[0294] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 29. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 29.

[0295] In some embodiments, LAP or a fragment or derivative thereof comprises the sequence from positions 30 to 274 of SEQ ID NO:82.

[0296] In some embodiments, the LAP comprises the amino acid sequence of positions 30 to 274 of SEQ ID NO: 82, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of positions 30 to 274 of SEQ ID NO: 82. In certain embodiments, the nucleotide sequence encoding LAP comprises a nucleotide sequence encoding the amino acid sequence of positions 30 to 274 of SEQ ID NO: 82, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of positions 30 to 274 of SEQ ID NO: 82. In certain embodiments, the nucleotide sequence encoding LAP comprises the nucleotide sequence of SEQ ID NO: 83, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 83. In certain embodiments, LAP comprises the amino acid sequence from positions 30 to 274 of the sequence of SEQ ID NO: 82. In certain embodiments, the nucleotide sequence encoding LAP comprises the nucleotide sequence of SEQ ID NO: 83.

[0297] In some embodiments, LAP or a fragment or derivative thereof comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of positions 30 to 274 of SEQ ID NO: 82. In some embodiments, LAP or a fragment or derivative thereof comprises the amino acid sequence of positions 30 to 274 of SEQ ID NO: 82. In some embodiments, LAP consists of the amino acid sequence of positions 30 to 274 of SEQ ID NO: 82.

[0298] In some embodiments, the LAP comprises the amino acid sequence of SEQ ID NO:118, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:118. In certain embodiments, the nucleotide sequence encoding LAP comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:118, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:118. In certain embodiments, the nucleotide sequence encoding LAP comprises the nucleotide sequence of SEQ ID NO: 119, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 119. In certain embodiments, LAP comprises the amino acid sequence of SEQ ID NO: 118. In certain embodiments, the nucleotide sequence encoding LAP comprises the nucleotide sequence of SEQ ID NO: 119.

[0299] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 118. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 118. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 118.

[0300] In some embodiments, LAP or a fragment or derivative thereof comprises the amino acid sequence of positions 21 to 298 of the amino acid sequence of SEQ ID NO:116.

[0301] In some embodiments, the LAP comprises the amino acid sequence of positions 21 to 298 of the amino acid sequence of SEQ ID NO: 116, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of positions 21 to 298 of the amino acid sequence of SEQ ID NO: 116. In certain embodiments, the nucleotide sequence encoding LAP comprises a nucleotide sequence encoding the amino acid sequence of positions 21 to 298 of the amino acid sequence of SEQ ID NO: 116, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of positions 21 to 298 of the amino acid sequence of SEQ ID NO: 116. In certain embodiments, the nucleotide sequence encoding LAP comprises the nucleotide sequence of SEQ ID NO: 117, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 117. In certain embodiments, LAP comprises the amino acid sequence from positions 21 to 298 of the amino acid sequence of SEQ ID NO: 116. In certain embodiments, the nucleotide sequence encoding LAP comprises the nucleotide sequence of SEQ ID NO: 117.

[0302] In some embodiments, LAP or a fragment or derivative thereof comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of positions 21 to 298 of the amino acid sequence of SEQ ID NO: 116. In some embodiments, LAP or a fragment or derivative thereof comprises the amino acid sequence of positions 21 to 298 of the amino acid sequence of SEQ ID NO: 116. In some embodiments, LAP consists of the amino acid sequence of positions 21 to 298 of the amino acid sequence of SEQ ID NO: 116.

[0303] In some embodiments, a LAP of the disclosure has an amino acid sequence that is at least about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more of its native or parental amino acid sequence. , 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identity and having the activity of the normally occurring (i.e., naturally occurring) or parent sequence.

[0304] In some embodiments, the LAP or fragment or derivative thereof may be heterologous to the mature TGFβ family polypeptide or fragment or derivative thereof disclosed herein.

[0305] In some embodiments, LAP or a fragment or derivative thereof comprises an amino acid mutation(s) at one or more positions. Non-limiting examples of amino acid mutations include amino acid substitutions and / or insertions and / or deletions. In some embodiments, LAP or a fragment or derivative thereof comprises an amino acid substitution(s) at one or more positions. Non-limiting examples of LAP or a fragment or derivative thereof include 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, The amino acid substitution(s) may be at positions 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 or more.

[0306] In some embodiments, LAP or a fragment or derivative thereof may contain one or more amino acid substitutions and / or insertions and / or deletions. An amino acid substitution means that an amino acid residue is replaced with an alternative amino acid residue at the same position. The inserted amino acid residues may be inserted at any position, and may be inserted so that some or all of the inserted amino acid residues are immediately adjacent to one another, or so that none of the inserted amino acid residues are immediately adjacent to another inserted amino acid residue. One or more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO:29. One or more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO:82. One or more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO:118. One or more amino acids may be substituted and / or inserted and / or deleted from the sequence of SEQ ID NO:116.

[0307] In some embodiments, a LAP or a fragment or derivative thereof may comprise, for example, one or more amino acid substitutions and / or insertions and / or deletions at one or more positions in the amino acid sequence compared to the amino acid sequence of a reference LAP, e.g., a LAP may comprise substitution(s) of one or more amino acids in the amino acid sequence of the parent LAP with similar or homologous amino acid(s) or dissimilar amino acid(s).

[0308] In certain embodiments, amino acid mutations (e.g., substitutions) to a protein or portion thereof (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, or (4) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in a normally occurring (i.e., native) or parent sequence. In some embodiments, the amino acid sequence of a LAP disclosed herein can be mutated, for example, to render the LAP specific for proteolytic activation. In some embodiments, the amino acid sequence of a LAP disclosed herein can be mutated, for example, to enable mechanical activation of the LAP. In some embodiments, the amino acid sequence of a LAP disclosed herein can be mutated, for example, to eliminate one or more protease cleavage sites within the LAP or a fragment or derivative thereof. In some embodiments, the amino acid sequence of a LAP disclosed herein can be mutated, for example, to reduce binding of the LAP to environmental molecules, such as, but not limited to, latency-associated binding protein (LTBP).

[0309] In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that render LAP specific for proteolytic activation. In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that allow proteolytic activation of LAP. In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that allow mechanical activation of LAP. In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that promote mechanical activation of LAP. In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that inhibit or block mechanical activation of LAP. In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that eliminate one or more protease cleavage sites within LAP or a fragment or derivative thereof. In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that reduce binding of LAP to latency-associated binding protein (LTBP).

[0310] In various embodiments, the LAP or fragment or derivative thereof described herein can comprise one or more mutations described herein. In some embodiments, the one or more mutations can enable proteolytic activation of a mature TGFβ family polypeptide described herein, or a fragment or derivative thereof. In some embodiments, the one or more mutations can enable mechanical activation of a mature TGFβ family polypeptide described herein, or a fragment or derivative thereof. In some embodiments, the LAP or fragment or derivative thereof comprises one or more mutations that promote mechanical activation of the LAP. In some embodiments, the LAP or fragment or derivative thereof comprises one or more mutations that inhibit or block mechanical activation of the LAP. In some embodiments, the LAP or fragment or derivative thereof described herein can be mutated to generate, for example, an autoactive form of TGFβ, i.e., a TGFβ that does not require activation (e.g., by proteolysis or mechanical activation) to induce downstream signaling (e.g., Smad2 / 3 signaling and / or ERK signaling) as described herein.

[0311] In some embodiments, LAP or a fragment or derivative thereof may include one or more mutations that may, for example, eliminate one or more protease cleavage sites within LAP or a fragment or derivative thereof, hi some embodiments, the one or more mutations may, for example, reduce binding of LAP or a fragment or derivative thereof to latency-associated binding protein (LTBP).

[0312] In some embodiments, LAP or a fragment or derivative thereof may include one or more mutations that may introduce one or more protease cleavage sites into LAP or a fragment or derivative thereof, for example, via insertion of a protease cleavage site as described herein. In some embodiments, the insertion may include a PLGL insertion, such as may be useful for MMP2 cleavage. In some embodiments, the insertion may include a PLGI insertion, such as may be used for MMP3, MMP7, and / or MMP8 cleavage.

[0313] In some embodiments, the LAPs disclosed herein may contain conservative modifications and / or substitutions. Conservative amino acid modifications and / or substitutions should not substantially alter the structural characteristics of the parent sequence. For example, amino acids belonging to one of the following groups represent conservative mutations: Group I: Ala, Pro, Gly, Gln, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Val, Ile, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu.

[0314] In some embodiments, LAP or a fragment or derivative thereof may comprise an amino acid substitution, for example, at a position corresponding to amino acid residue C33 of LAP. In some embodiments, LAP or a fragment or derivative thereof may comprise a C33S mutation (a cysteine ​​to serine mutation at position 33), where position 33 is relative to the sequence of SEQ ID NO: 82. In some embodiments, LAP or a fragment or derivative thereof may comprise a C33S mutation (a cysteine ​​to serine mutation at position 33), where position 33 is relative to the sequence of SEQ ID NO: 116. In some embodiments, a LAP comprising a C33S mutation may comprise the sequence of SEQ ID NO: 31. In some embodiments, a LAP comprising a C33S mutation may comprise the sequence of SEQ ID NO: 80. In some embodiments, a LAP comprising a C33S mutation may comprise the sequence of SEQ ID NO: 88.

[0315] In some embodiments, LAP or a fragment or derivative thereof may comprise an amino acid substitution, for example, at a position corresponding to amino acid residue C24 of LAP. In some embodiments, LAP or a fragment or derivative thereof may comprise a C24S mutation (a cysteine ​​to serine mutation at position 24), where position 24 is relative to the sequence of SEQ ID NO: 116. In some embodiments, a LAP comprising a C24S mutation may comprise the sequence of SEQ ID NO: 94.

[0316] Because LAP can be covalently linked to cell surface-bound environmental molecules at C33 or C24, the C33S or C24S mutation may prevent disulfide binding of LAP to the environmental molecule(s) during secretion, preventing their incorporation into TGFβ SLC. There are three classes of environmental molecules expressed on different cell types: (i) LTBPs (latency-associated binding proteins); (ii) GARPs (LRRC32, glycoprotein A repeat-dominant proteins); and (iii) NRROS (LRRC33, leucine-rich repeat-containing proteins 33). TGFβ complexed with LTBP1, LTBP3, and LTBP4 is stored in the extracellular matrix (ECM). TGFβ complexed with GARPs is stored on the surface of endothelium and activated regulatory T cells. TGFβ complexed with LRRC33 is stored on macrophages and microglia.

[0317] In some embodiments, one or more mutations of the LAP disclosed herein may comprise a C33S mutation, where position 33 is relative to the sequence of SEQ ID NO: 82. In some embodiments, the LAP comprising the C33S mutation comprises the sequence of SEQ ID NO: 31. In some aspects, the LAP comprising the C33S mutation comprises the sequence of SEQ ID NO: 80. In some aspects, the LAP comprising the C33S mutation comprises the sequence of SEQ ID NO: 88.

[0318] In some embodiments, one or more mutations of the LAP disclosed herein may comprise a C24S mutation, where position 24 is relative to the sequence of SEQ ID NO: 116. In some aspects, the LAP comprising the C24S mutation comprises the sequence of SEQ ID NO: 94.

[0319] In some embodiments, the LAP comprising one or more mutations disclosed herein comprises the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 31. In certain embodiments, the nucleotide sequence encoding the LAP comprising one or more mutations comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 31, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 31. In certain embodiments, the nucleotide sequence encoding the LAP having one or more mutations comprises the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 32. In certain embodiments, the LAP comprising one or more mutations comprises the amino acid sequence of SEQ ID NO: 31. In certain embodiments, the nucleotide sequence encoding the LAP comprises the nucleotide sequence of SEQ ID NO: 32.

[0320] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 31. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 31.

[0321] In some embodiments, the LAP comprising one or more mutations disclosed herein comprises the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 80. In certain embodiments, the nucleotide sequence encoding the LAP comprising one or more mutations comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:80, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:80. In certain embodiments, the nucleotide sequence encoding the LAP having one or more mutations comprises the nucleotide sequence of SEQ ID NO: 81, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 81. In certain embodiments, the LAP comprising one or more mutations comprises the amino acid sequence of SEQ ID NO: 80. In certain embodiments, the nucleotide sequence encoding the LAP comprises the nucleotide sequence of SEQ ID NO: 81.

[0322] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 80.

[0323] In some embodiments, the LAP comprising one or more mutations disclosed herein comprises the amino acid sequence of SEQ ID NO: 88, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 88. In certain embodiments, the nucleotide sequence encoding the LAP comprising one or more mutations comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:88, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:88. In certain embodiments, the nucleotide sequence encoding the LAP having one or more mutations comprises the nucleotide sequence of SEQ ID NO: 89, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 89. In certain embodiments, the LAP comprising one or more mutations comprises the amino acid sequence of SEQ ID NO: 88. In certain embodiments, the nucleotide sequence encoding the LAP comprises the nucleotide sequence of SEQ ID NO: 89.

[0324] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 88. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 88.

[0325] In some embodiments, the LAP comprising one or more mutations disclosed herein comprises the amino acid sequence of SEQ ID NO:94, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:94. In certain embodiments, the nucleotide sequence encoding the LAP comprising one or more mutations comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:94, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:94. In certain embodiments, the nucleotide sequence encoding the LAP having one or more mutations comprises the nucleotide sequence of SEQ ID NO: 95, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 95. In certain embodiments, the LAP comprising one or more mutations comprises the amino acid sequence of SEQ ID NO: 94. In certain embodiments, the nucleotide sequence encoding the LAP comprises the nucleotide sequence of SEQ ID NO: 95.

[0326] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 94. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 94. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 94.

[0327] In some embodiments, one or more mutations of a LAP disclosed herein may comprise a C33S mutation, a C223S mutation, and / or a C225S mutation, where positions 33, 223 and / or 225 are relative to the sequence of SEQ ID NO: 82. In some embodiments, a LAP comprising the C33S, C223S and C225S mutations comprises the sequence of SEQ ID NO: 80.

[0328] In some embodiments, the LAP comprising one or more mutations disclosed herein comprises the amino acid sequence of SEQ ID NO: 80, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 80. In certain embodiments, the nucleotide sequence encoding the LAP comprising one or more mutations comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:80, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:80. In certain embodiments, the nucleotide sequence encoding the LAP having one or more mutations comprises the nucleotide sequence of SEQ ID NO: 81, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 81. In certain embodiments, the LAP comprising one or more mutations comprises the amino acid sequence of SEQ ID NO: 80. In certain embodiments, the nucleotide sequence encoding the LAP comprises the nucleotide sequence of SEQ ID NO: 81.

[0329] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 80. Transforming growth factor β (TGFβ) family polypeptides

[0330] In some embodiments, a polypeptide complex described herein can comprise a transforming growth factor β (TGFβ) family polypeptide or a fragment or derivative thereof. In some embodiments, a polypeptide complex can comprise a dimeric and / or mature transforming growth factor β (TGFβ) family polypeptide or a fragment or derivative thereof. In some embodiments, a TGFβ family polypeptide (e.g., a mature dimeric TGFβ family polypeptide) or a fragment or derivative thereof can be inactive as a result of interaction with a LAP described herein. In some embodiments, the LAP is a dimeric LAP.

[0331] Non-limiting examples of members of the TGF-β family include activin, anti-Müllerian hormone (AMH), bone morphogenetic protein (BMP), inhibin, nodal, growth and differentiation factor (GDF), and TGF-β isoforms (e.g., TGF-β1, TGF-β2, and TGF-β3). TGF-β isoforms (e.g., TGF-β1, TGF-β2, and TGF-β3) have each been identified in mammals and may share 70-82% homology at the amino acid level. TGF-β isoforms may be involved in various cellular processes, including, but not limited to, extracellular matrix (ECM) remodeling, cell migration, invasion, epithelial-mesenchymal transition (EMT), growth inhibition, and / or immunosuppression.

[0332] From a mechanistic perspective, TGF-β, like other members of the TGF-β family, can be synthesized as a precursor protein, which can interact with latency-associated peptide (LAP) to form a homodimer, forming the small latent complex (SLC). Environmental molecules (e.g., latent TGF-beta binding protein (LTBP)) can bind to LAP to form a larger complex called the large latent complex (LLC). The TGF-β gene encodes a preproprotein sequence consisting of, for example, a signal peptide, a propeptide terminating in a protease cleavage site, and the mature TGF-β sequence. Furin can hydrolyze the protease cleavage site, thereby generating distinct TGF-β- and propeptide-derived homodimers. The two homodimers remain noncovalently associated and can be secreted. This latent complex can maintain TGF-β in an inactive form that cannot bind to its receptor.

[0333] LLC can be activated proteolytically or mechanically. During proteolytic activation, proteases can degrade the LAP domain or induce conformational changes in the LAP domain, releasing mature TGFβ. Non-limiting examples of proteases that may be involved in the proteolysis of LAP include matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), thrombospondin 1 (TSP-1), plasmin (PLN), and plasma kallikrein (PLK). During mechanical activation, integrins (αvβ6 integrin, αvβ8 integrin, αvβ1 integrin) bind to the "RGD" binding motif at the C-terminus of LAP. Integrin binding at the C-terminus of LAP and cell surface-bound environmental molecule association (via covalent bonds) at the N-terminus of LAP generate a directional tensile force that opens the LAP, thereby releasing free mature TGFβ (see, for example, the box in Figure 3A). Mature TGFβ can then bind to extracellular domain(s), such as TGFβR1 (TGFβ type I receptor) and / or TGFβR2 (TGFβ type II receptor), e.g., on the cell surface. Binding of TGFβR1 and / or TGFβR2 by mature TGFβ can bring the receptors into close proximity with each other, thereby positioning the intracellular serine / threonine kinase domains of the TGFβ receptors in a conformation that can facilitate phosphorylation and / or activation of the receptor(s). However, in some instances, TGFβRs may already be in close proximity with each other in the absence of ligand (i.e., mature TGFβ). In some embodiments, upon ligand binding, constitutively active TGFβR2 can phosphorylate TGFβR1, which can then phosphorylate intracellular Smad2 / 3 upon such activation. As a non-limiting example, binding of active mature TGFβ can induce signaling through a Smad-dependent canonical signaling pathway. In some embodiments, active mature TGFβ can induce Smad2 / 3 signaling (see, eg, FIG. 1).In non-canonical pathway(s), activated TGFβ receptor complexes, e.g., activated TGFβ complexes including mature active TGFβ complexes complexed with TGFβR1 and / or TGFβR2, can signal through other factors, such as, but not limited to, nuclear factor-kappa B (NF-kappaB), TRAF6, extracellular signal-regulated kinase (ERK), TGFβ-activated kinase 1 (TAK1, also known as MAP3K7), tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4), RHO, phosphoinositide 3-kinase (PI3K), p38 mitogen-activated protein kinase (p38 MAPK), AKT (also known as protein kinase B), and / or JUN N-terminal kinase (JNK).

[0334] In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof disclosed herein and a dimeric LAP disclosed herein may associate via a non-covalent interaction, hi some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof may be inactive as a result of the interaction of the mature TGFβ family polypeptide or a fragment or derivative thereof with the LAP or a fragment or derivative thereof.

[0335] In some embodiments, the TGFβ family polypeptides or fragments or derivatives thereof described herein may be autoactive TGFβ family polypeptides, i.e., TGFβ family polypeptides that do not require activation (e.g., by proteolysis or mechanical activation) to induce TGFβ signaling (e.g., Smad2 / 3 signaling and / or ERK signaling, etc.). In some embodiments, the TGFβ family polypeptides or fragments or derivatives thereof may comprise one or more mutations to generate an autoactivated form of TGFβ. The one or more mutations to generate an autoactivated form of TGFβ may comprise any of the various mutations described herein.

[0336] In some embodiments, the LAP or fragment or derivative thereof is heterologous to the mature TGFβ family polypeptide or fragment or derivative thereof.

[0337] In some embodiments, the mature TGFβ family polypeptide or fragment or derivative thereof and the LAP may be covalently linked and separated by a protease cleavage site. In some embodiments, the protease cleavage site may comprise a furin cleavage site, including, for example, but not limited to, the sequence RXXR (SEQ ID NO: 35). In some embodiments, the protease cleavage site may consist of the sequence RXXR (SEQ ID NO: 35). In some embodiments, the protease cleavage site is RHRR (SEQ ID NO: 85), RRKR (SEQ ID NO: 86), or RKKR (SEQ ID NO: 87).

[0338] In some embodiments, a mature TGFβ family polypeptide or fragment or derivative thereof disclosed herein, upon release of the mature TGFβ family polypeptide or fragment or derivative thereof from an SLC disclosed herein, can bind to a transforming growth factor β receptor (TGFβR), which can include any of the various TGFβRs disclosed herein.

[0339] In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof, upon release from an SLC disclosed herein, induces signaling in a target cell or a cell adjacent to the target cell via a Smad-dependent canonical signaling pathway. In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof, upon release from an SLC disclosed herein, induces Smad2 / 3 signaling in a target cell or a cell adjacent to the target cell. In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof, upon release from an SLC disclosed herein, induces signaling in a target cell or a cell adjacent to the target cell via a non-canonical signaling pathway disclosed herein.

[0340] In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof induces signal transduction in a target cell or a cell adjacent to a target cell via a Smad-dependent canonical signaling pathway upon activation of the mature TGFβ family polypeptide. In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof induces Smad2 / 3 signaling in a target cell or a cell adjacent to a target cell upon activation of the mature TGFβ family polypeptide. In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof induces signal transduction in a target cell or a cell adjacent to a target cell via a non-canonical signaling pathway disclosed herein upon activation of the mature TGFβ family polypeptide. Activation of a mature TGFβ family polypeptide can occur by any mechanism disclosed herein. Activation can occur via mechanisms including, for example, proteolytic activation and / or mechanical activation. In some embodiments, activation can occur by chemical activation. In some embodiments, activation can occur by low pH, such as pH 3.0, which can be achieved with acid, e.g., HCl, and / or thermal activation.

[0341] In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof can be chemically dissociated from the LAP or a fragment or derivative thereof and released in an active form from the SLC, hi some embodiments, chemical dissociation includes, for example, but is not limited to, protease treatment, temperature treatment, acid treatment, or any combination thereof.

[0342] In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof is mechanically dissociated from the LAP or a fragment or derivative thereof and released in an active form from the SLC. As a non-limiting example, mechanical release can occur as a result of interaction between the LAP or a fragment or derivative thereof and an integrin polypeptide, e.g., via an integrin-binding motif in the LAP. Non-limiting examples of integrins include αvβ6 integrin, αvβ8 integrin, and αvβ1 integrin.

[0343] In some embodiments, the mature TGFβ family polypeptide may be a mature TGFβ polypeptide.

[0344] In some embodiments, the mature TGFβ polypeptide may be a mature TGFβ1 polypeptide.

[0345] In some embodiments, the mature TGFβ1 polypeptide comprises the amino acid sequence of SEQ ID NO:23, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:23. In certain embodiments, the nucleotide sequence encoding the mature TGFβ1 polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:23, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:23. In certain embodiments, the nucleotide sequence encoding the mature TGFβ1 polypeptide comprises the nucleotide sequence of SEQ ID NO:24, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:24. In certain embodiments, the mature TGFβ1 polypeptide comprises the amino acid sequence of SEQ ID NO:23. In certain embodiments, the nucleotide sequence encoding the mature TGFβ1 polypeptide comprises the nucleotide sequence of SEQ ID NO:24.

[0346] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO: 23. In some embodiments, the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:23.

[0347] In some embodiments, the mature TGFβ1 polypeptide comprises the amino acid sequence of SEQ ID NO:90, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:90. In certain embodiments, the nucleotide sequence encoding the mature TGFβ1 polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:90, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:90. In certain embodiments, the nucleotide sequence encoding the mature TGFβ1 polypeptide comprises the nucleotide sequence of SEQ ID NO:91, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 91. In certain embodiments, the mature TGFβ1 polypeptide comprises the amino acid sequence of SEQ ID NO:90. In certain embodiments, the nucleotide sequence encoding the mature TGFβ1 polypeptide comprises the nucleotide sequence of SEQ ID NO:91.

[0348] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO: 90. In some embodiments, the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:90.

[0349] In some embodiments, the mature TGFβ polypeptide may be a mature TGFβ2 polypeptide.

[0350] In some embodiments, the mature TGFβ2 polypeptide comprises the amino acid sequence of SEQ ID NO:27, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:27. In certain embodiments, the nucleotide sequence encoding the mature TGFβ2 polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:27, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:27. In certain embodiments, the nucleotide sequence encoding the mature TGFβ2 polypeptide comprises the nucleotide sequence of SEQ ID NO:28, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:28. In certain embodiments, the mature TGFβ2 polypeptide comprises the amino acid sequence of SEQ ID NO:27. In certain embodiments, the nucleotide sequence encoding the mature TGFβ2 polypeptide comprises the nucleotide sequence of SEQ ID NO:28.

[0351] In some embodiments, the mature TGFβ2 polypeptide comprises the sequence of SEQ ID NO: 27. In some embodiments, the mature TGFβ2 polypeptide consists of the sequence of SEQ ID NO:27.

[0352] Other non-limiting examples of mature TGFβ family polypeptides that may be useful in practicing the present disclosure include mature growth differentiation factor 8 (GDF8), mature growth differentiation factor 11 (GDF11) polypeptide, and mature bone morphogenetic protein 4 (BMP4).

[0353] In some embodiments, the mature TGFβ family polypeptide can be a mature growth differentiation factor 8 (GDF8) polypeptide. In some embodiments, the mature TGFβ family polypeptide can be a mature growth differentiation factor 11 (GDF11) polypeptide. In some embodiments, the mature TGFβ family polypeptide can be a mature bone morphogenetic protein 4 (BMP4). Target-binding polypeptides

[0354] In some embodiments, the polypeptide complexes described herein may comprise a target-binding polypeptide. In some embodiments, the target-binding polypeptide may bind to a molecule on a target cell. In some embodiments, the target-binding polypeptide may bind to a molecule in the extracellular matrix (ECM). In some embodiments, the target-binding peptide is not internalized. In some embodiments, the target-binding peptide may be internalized.

[0355] In some embodiments, a polypeptide complex disclosed herein may comprise a small latent complex (SLC) disclosed herein comprising a dimeric LAP or a fragment or derivative thereof disclosed herein, which may be attached to a target-binding polypeptide. In some embodiments, the LAP or a fragment or derivative thereof may be covalently attached to the target-binding polypeptide. In some embodiments, the LAP or a fragment or derivative thereof may be covalently attached to the target-binding polypeptide via a linker disclosed herein. In some embodiments, the linker may be located between the target-binding polypeptide and the LAP or a fragment or derivative thereof disclosed herein. In some embodiments, the LAP or a fragment or derivative thereof may be non-covalently bound to the target-binding polypeptide. In some embodiments, the target-binding polypeptide binds to both the LAP and the target(s) of the target-binding polypeptide, e.g., a molecule on a target cell and / or a molecule in the extracellular matrix (ECM).

[0356] In some embodiments, the target-binding polypeptide is an antigen-binding polypeptide or an antigen-binding fragment thereof.

[0357] In some embodiments, the antigen-binding polypeptide binds to at least one antigen on a target cell. In some embodiments, the antigen-binding moiety binds to two or more antigens on a target cell. In some embodiments, two or more antigens may be associated with the same target cell. In some embodiments, two or more antigens may be associated with different target cells. Non-limiting examples of target cells include fibroblasts, chondroblasts, osteoblasts, myofibroblasts, plasma cells, adipocytes, and leukocytes. In some embodiments, the target cell may be an immune cell, such as, but not limited to, a T cell (e.g., activated regulatory T cell, CD40+ T cell, CD90+ T cell), natural killer (NK) cell, macrophage, or mast cell. In some embodiments, the target cell may be a microglial cell. In some embodiments, the target cell may be an endothelial cell. In some embodiments, the target cell may be an epithelial cell (e.g., an intestinal epithelial cell). In some embodiments, the target cell may be a microglial cell. In some embodiments, the target cell may be a dendritic cell. In some embodiments, the target cell may be a beta cell (e.g., a pancreatic beta cell).

[0358] In some embodiments, antigens on T cells (e.g., CD4 or CD90) may be targeted by antigen-binding polypeptides described herein, and polypeptide complexes comprising such antigen-binding polypeptides described herein or pharmaceutical compositions thereof may, for example, promote regulatory T cell (Treg) differentiation and / or maintain immune tolerance, such as in inflammatory bowel disease (IBD).

[0359] In some embodiments, antigens on epithelial cells, such as intestinal epithelial cells (e.g., mEpcam or mOlfm4), may be targeted by the antigen-binding polypeptides described herein, and polypeptide complexes comprising such antigen-binding polypeptides described herein or pharmaceutical compositions thereof may improve epithelial barrier integrity, for example, in IBD.

[0360] In some embodiments, antigens (e.g., mClec9a) on dendritic cells may be targeted by antigen-binding polypeptides described herein, and polypeptide complexes comprising such antigen-binding polypeptides described herein or pharmaceutical compositions thereof may, for example, reduce antigen presentation and / or inhibit goblet cell differentiation, e.g., in IBD.

[0361] In some embodiments, antigens on pancreatic beta cells (e.g., HLA-A2:INS) can be targeted, e.g., by antigen-binding polypeptides described herein, and polypeptide complexes comprising such antigen-binding polypeptides described herein or pharmaceutical compositions thereof can suppress autoreactive immune cells, e.g., in type 1 diabetes (T1D).

[0362] In some embodiments, the antigen-binding polypeptide binds to at least one antigen of the extracellular matrix (ECM). In some embodiments, the antigen-binding moiety binds to two or more antigens of the ECM. In some embodiments, the two or more antigens are associated with the same ECM. In some embodiments, the two or more antigens are associated with different ECMs. Examples of antigens associated with ECM may be associated with various ECM molecules, such as, but not limited to, collagen (e.g., type X collagen, also known as collagen X), fibrillar collagen, fibronectin, elastin, and / or laminin. In some embodiments, the antigen-binding polypeptide targets collagen. In some embodiments, the antigen-binding polypeptide targets fibronectin.

[0363] In some embodiments, antigens of the ECM (e.g., fibronectin) may be targeted by the antigen-binding polypeptides described herein, and polypeptide complexes comprising such antigen-binding polypeptides described herein, or pharmaceutical compositions thereof, may be relocated to, for example, de novo areas of the matrix, such as aortic aneurysms in Marfan syndrome (MFS).

[0364] In some embodiments, antigens of the ECM (e.g., type X collagen) may be targeted by the antigen-binding polypeptides described herein, and polypeptide complexes comprising such antigen-binding polypeptides described herein or pharmaceutical compositions thereof may reduce inflammation in joints, such as in rheumatoid arthritis (RA).

[0365] In some embodiments, the antigen-binding polypeptide binds to at least one antigen associated with a TGFβ dysregulation disorder. In some embodiments, the antigen-binding polypeptide binds to two or more antigens associated with a TGFβ dysregulation disorder. In some embodiments, the two or more antigens associated with a TGFβ dysregulation disorder are associated with the same TGFβ dysregulation disorder. In some embodiments, the two or more antigens associated with a TGFβ dysregulation disorder are associated with different antigens associated with a TGFβ dysregulation disorder. Non-limiting examples of TGFβ dysregulation disorders include type 1 diabetes, inflammatory bowel disease (IBD), Marfan syndrome (MFS), aortic aneurysms in MFS, autoimmune disorders, arthritis (e.g., rheumatoid arthritis (RA)), lupus (e.g., systemic lupus erythematosus), and wound healing disorders.

[0366] Examples of antigens that may be targeted by antigen-binding polypeptides or antigen-binding fragments thereof include, but are not limited to, PD-1, EDB-FN, CD63, CD4, CD90, Epcam, Madcam, a4b7, CCR9, Clec9a, Xcr1, and Olfm4. In some embodiments, an antigen targeted by an antigen-binding polypeptide or antigen-binding fragment thereof may comprise HLA-A2:INS. In some embodiments, an antigen targeted by an antigen-binding polypeptide or antigen-binding fragment thereof may comprise fibronectin. In some embodiments, an antigen targeted by an antigen-binding polypeptide or antigen-binding fragment thereof may comprise collagen-10.

[0367] In some embodiments, the antigen-binding polypeptide or antigen-binding fragment thereof may comprise fibronectin (FN) or a fragment or derivative thereof. Fibronectin (FN) is a multifunctional, high-molecular-weight glycoprotein component of both body fluids (e.g., plasma) and the extracellular matrix (ECM). FN is involved in various biological processes, including, but not limited to, cell migration, cell adhesion, thrombosis and hemostasis, and wound healing, as well as the establishment and maintenance of normal cell morphology (i.e., cell shape), development, and oncogenic transformation. Structural diversity in plasma and cellular fibronectin (FN) arises from alternative splicing of three domains of the primary FN transcript, which can generate at least 20 different isoforms that can be differentially expressed, for example, in tumors and normal tissues (see, e.g., Figure 31). Plasma FN is produced and secreted by hepatocytes as a soluble dimeric form. Cellular FN is expressed by some mesenchymal cells as a dimeric or cross-linked multimeric form and deposited as fibrils in the ECM. Cellular FN is required for the deposition of fibrillin-1 and Col type I in the ECM. FN can interact with many other ECM proteins, as well as small molecules, growth factors, glycosaminoglycans (GAGs), cell surface receptors, and other FN molecules.

[0368] FN isoforms containing the EDB domain (e.g., cellular FN) are expressed in growing and remodeling tissues (see, e.g., Figure 32). EDB is a small domain of 91 amino acids (see, e.g., SEQ ID NO: 84) that is part of a fibronectin isoform formed by alternative splicing. The sequence of EDB is identical between mice and humans. EDB-containing isoforms are expressed during embryonic and postnatal development and in various solid tumors (and may be a marker of newly forming blood vessels), but are virtually undetectable in normal adult tissues, except for some blood vessels in the endometrium and ovaries during the proliferative phase. EDB-containing FN isoforms have been shown to play roles, among other things, in protein stability, proliferation, angiogenesis, inflammation, opsonization (phagocytosis), and cell adhesion. Furthermore, EDB-FN can increase the proteolytic susceptibility of FN, suggesting that EDB may increase the rate of ECM turnover. The presence of EDB may also upregulate the expression of vascular endothelial growth factor (VEGF) and may be associated with enhanced angiogenesis and endothelial growth.

[0369] Microfibrils within the ECM are composed of fibrillin polymers that can associate with elastin, other glycoproteins, and growth factors, and FN is essential for microfibril formation. Fibrillin microfibrils provide strength to tissues (see, e.g., Figure 28), and dysregulation of microfibril assembly can contribute to disease states. Mutations in fibrillin glycoproteins encoded by FBN1, FBN2, and FBN3 may be associated with various connective tissue disorders, most notably FBN1, which causes Marfan syndrome (MFS) (see, e.g., Figures 28-30). For example, FBN1 gene mutations that can cause MFS may alter the structure or stability of fibrillin-1, reduce the amount of fibrillin-1 produced by cells, and / or impair the transport of fibrillin-1 from cells. Such mutations can result in a significant reduction in the amount of fibrillin-1 available to form microfibrils. Aortic root dilatation / dissection is one of the cardinal features of MFS (see, eg, Figures 29-30).

[0370] In some embodiments, the antigen-binding polypeptide or antigen-binding fragment thereof may target an antigen that may be associated with MFS.

[0371] In some embodiments, the antigen is associated with an autoimmune disease or disorder. Antigens associated with an autoimmune disease or disorder can be derived, for example, from cell receptors and / or cells that produce "self"-directed antibodies. In some embodiments, the antigen is associated with an autoimmune disease or disorder, such as autoimmune gastritis, vasculitis, Wegener's granulomatosis, Hashimoto's thyroiditis, psoriasis (Graves' disease), Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (Crohn's disease), ulcerative colitis, rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, systemic lupus erythematosus, type 1 diabetes, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, myasthenia gravis, ankylosing spondylitis, scleroderma, polymyositis, or dermatomyositis.

[0372] Non-limiting examples of autoimmune antigens include platelet antigens, islet cell antigens, myelin protein antigens, rheumatoid factors, anti-citrullinated proteins, glucose-6-phosphate isomerase, receptors such as lipocortin 1, neutrophil nuclear proteins such as lactoferrin and 25-35 kD nuclear protein, Sm antigens such as snRNPs, granule proteins such as bactericidal permeability-increasing protein (BPI), citrullinated proteins and peptides such as elastase, fibrin, vimentin, filaggrin, fibrinogen, collagen I and II peptides, plasminogen, alpha-enolase, translation initiation factor 4G1, perinuclear factor, keratin, Sa (cytoskeletal protein vimentin), CCP-1, CCP-2 (cyclic citrullinated peptides), circulating serum proteins such as RF (IgG, IgM), components of articular cartilage such as collagens II, IX, and XI, and RA33 / hnRNP. These include nuclear components such as A2, stress proteins such as ferritin, HSP-65, -70, -90, and BiP, inflammatory / immune factors such as B7-H1, IL-1 alpha, and IL-8, enzymes such as alpha-enolase, calpastatin, dipeptidyl peptidase, eukaryotic translation elongation factor 1 alpha 1 aldolase-A, osteopontin, cathepsin G, myeloperoxidase, proteinase 3 antigen, rheumatoid factor, histones, nucleic acids such as RNA, dsDNA, ssDNA, and ribonucleoparticles, ribosomal P proteins, myelin proteins, cardiolipin, vimentin, Sm antigens (including, for example, SmD' and SmB' / B), U1RNP, A2 / B1 hnRNP, Ro(SSA), and La(SSB) antigens.

[0373] In some embodiments, the antigen is an endogenous molecule of the subject, hi some embodiments, the antigen is targeted by the immune response in an autoimmune disease disclosed herein.

[0374] In some embodiments, the antigen is associated with a disease associated with a TGFβ loss-of-function mutation(s). A non-limiting example of a disease associated with a TGFβ loss-of-function mutation(s) is an aortic aneurysm.

[0375] In some embodiments, the target-binding polypeptide may comprise an antibody or a fragment or derivative thereof. In some embodiments, the antigen-binding polypeptide may comprise an antibody or an antigen-binding fragment thereof.

[0376] In some embodiments, the antigen-binding polypeptide can be an antibody or an antigen-binding fragment thereof.

[0377] Without wishing to be bound by theory, the antibodies disclosed herein may include, for example, immunoglobulin molecules (i.e., "intact antibody molecules") composed of four polypeptide chains, two immunoglobulin heavy (H) chains (HC) and two immunoglobulin light chains (LC), interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain may be composed of a heavy chain variable region ("HCVR" or "VH") and a heavy chain constant region (composed of domains CH1, CH2, and CH3). Each light chain may be composed of a light chain variable region ("LCVR" or "VL") and a light chain constant region (CL). The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL may be composed of three CDRs and four FRs, which may be arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. In some embodiments, the antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions, for example, in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences.

[0378] Antibodies that may be useful in practicing the present disclosure may be full-length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and may be modified to affect functionality, for example, to increase persistence in the host or to eliminate residual effector function. In certain embodiments, antibodies may be bispecific.

[0379] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, CDR-containing fragments, or isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0380] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0381] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be dimeric and may comprise a VH-VH, VH-VL, or VL-VL dimer. Alternatively, the antigen-binding fragment of an antibody may comprise a monomeric VH or VL domain.

[0382] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids and provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may comprise homodimers or heterodimers (or other multimers) of any of the above-listed variable and constant domain configurations non-covalently associated with each other and / or with one or more monomeric VH or VL domains (e.g., by disulfide bond(s)).

[0383] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of the antigen-binding fragments of antibodies of the present disclosure using routine techniques available in the art.

[0384] In some embodiments, an antibody or antigen-binding fragment thereof disclosed herein comprises a heavy chain variable region (HCVR). In some embodiments, an antibody or antigen-binding fragment thereof comprises a light chain variable region (LCVR). In some embodiments, an antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain constant domain (CH), such as, but not limited to, an IgG1 domain or an IgG4 domain. In some embodiments, the immunoglobulin heavy chain constant domain (CH) can be an IgG1 domain. In some embodiments, the immunoglobulin heavy chain constant domain (CH) can be an IgG4 domain. In some aspects, an antibody or antigen-binding fragment thereof comprises an immunoglobulin light chain constant domain (CL).

[0385] In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure may comprise a heavy chain constant region comprising one or more amino acid changes in the hinge region. In some embodiments, the amino acid change(s) in the hinge region may reduce binding to Fcγ receptors. Examples of such modifications are disclosed in US 2018 / 0282411, the contents of which are incorporated herein by reference in their entirety for all purposes. In some embodiments, the antigen-binding polypeptide comprises modifications within amino acid positions 233-236 (according to EU numbering) by substituting naturally occurring residues with glycine(s) and / or deletion(s). In some embodiments, each of the amino acids at EU numbering positions 233-236 is occupied by G or is unoccupied, e.g., GGG(233-236), GG--(233-236), G---(233-236), or ----(233-236), where "-" represents an unoccupied position. In some embodiments, the heavy chain constant region comprising the modification(s) is of the human IgG1 isotype. In some embodiments, the heavy chain constant region comprising the modification(s) is of the human IgG4 isotype. In some embodiments, the heavy chain constant region comprising the modification(s) is a hybrid in which the domains are of different isotypes, e.g., a hybrid of IgG1 and IgG4 isotypes in which one or more domains (e.g., the CHI, CH2, or CH3 domains) and / or hinge region are of one isotype and the remaining domains are of a different isotype.

[0386] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise anti-CD63 antibodies or fragments or derivatives thereof.

[0387] In some embodiments, the anti-CD63 antibody HCVR comprises the amino acid sequence of SEQ ID NO:3, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:3. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody HCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:3. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody HCVR comprises the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 4. In certain embodiments, the anti-CD63 antibody HCVR comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody HCVR comprises the nucleotide sequence of SEQ ID NO: 4.

[0388] In some embodiments, the anti-CD63 antibody LCVR comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 8. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody LCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 8. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody LCVR comprises the nucleotide sequence of SEQ ID NO: 9, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 9. In certain embodiments, the anti-CD63 antibody LCVR comprises the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody LCVR comprises the nucleotide sequence of SEQ ID NO: 9.

[0389] In some embodiments, the anti-CD63 antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO: 13, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 13. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody CH IgG4 domain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 13. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 14. In certain embodiments, the anti-CD63 antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO: 14.

[0390] In some embodiments, the anti-CD63 antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 15. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody CH IgG4 domain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 15. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 16. In certain embodiments, the anti-CD63 antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO: 16.

[0391] In some embodiments, the anti-CD63 antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO:98, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:98. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody CH IgG4 domain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:98, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:98. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO: 99, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 99. In certain embodiments, the anti-CD63 antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO: 98. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO:99.

[0392] In some embodiments, the anti-CD63 antibody CL comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 17. In certain embodiments, the nucleotide sequence encoding the anti-CD63 CL comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 17. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody CL comprises the nucleotide sequence of SEQ ID NO: 18, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 18. In certain embodiments, the anti-CD63 CL comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the nucleotide sequence encoding the anti-CD63 antibody CL comprises the nucleotide sequence of SEQ ID NO: 18.

[0393] In some embodiments, the anti-CD63 antibody HCDR1 comprises the amino acid sequence of SEQ ID NO:5, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:5.

[0394] In some embodiments, the anti-CD63 antibody HCDR2 comprises the amino acid sequence of SEQ ID NO:6, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:6.

[0395] In some embodiments, the anti-CD63 antibody HCDR3 comprises the amino acid sequence of SEQ ID NO:7, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:7.

[0396] In some embodiments, the anti-CD63 antibody LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 10.

[0397] In some embodiments, the anti-CD63 antibody LCDR2 comprises the amino acid sequence of SEQ ID NO:11, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:11.

[0398] In some embodiments, the anti-CD63 antibody LCDR3 comprises the amino acid sequence of SEQ ID NO: 12, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 12.

[0399] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise anti-EDB-FN antibodies or fragments or derivatives thereof.

[0400] In some embodiments, the anti-EDB-FN antibody HCVR comprises the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 100. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody HCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:3. In certain embodiments, a nucleotide sequence encoding an anti-EDB-FN antibody HCVR comprises the nucleotide sequence of SEQ ID NO: 101, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 101. In certain embodiments, an anti-EDB-FN antibody HCVR comprises the amino acid sequence of SEQ ID NO: 100. In certain embodiments, a nucleotide sequence encoding an anti-EDB-FN antibody HCVR comprises the nucleotide sequence of SEQ ID NO: 101.

[0401] In some embodiments, the anti-EDB-FN antibody LCVR comprises the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 105. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody LCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 105, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 105. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody LCVR comprises the nucleotide sequence of SEQ ID NO: 106, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 106. In certain embodiments, the anti-EDB-FN antibody LCVR comprises the amino acid sequence of SEQ ID NO: 105. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody LCVR comprises the nucleotide sequence of SEQ ID NO: 106.

[0402] In some embodiments, the anti-EDB-FN antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO: 13, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 13. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody CH IgG4 domain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 13. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 14. In certain embodiments, the anti-EDB-FN antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO: 14.

[0403] In some embodiments, the anti-EDB-FN antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 15. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody CH IgG4 domain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 15. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO: 16, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 16. In certain embodiments, the anti-EDB-FN antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO: 16.

[0404] In some embodiments, the anti-EDB-FN antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO:98, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:98. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody CH IgG4 domain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:98, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:98. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO: 99, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 99. In certain embodiments, the anti-EDB-FN antibody CH IgG4 domain comprises the amino acid sequence of SEQ ID NO: 98. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody CH IgG4 domain comprises the nucleotide sequence of SEQ ID NO:99.

[0405] In some embodiments, the anti-EDB-FN antibody CL comprises the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 17. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN CL comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 17. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody CL comprises the nucleotide sequence of SEQ ID NO: 18, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 18. In certain embodiments, the anti-EDB-FN CL comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody CL comprises the nucleotide sequence of SEQ ID NO: 18.

[0406] In some embodiments, the anti-EDB-FN antibody HCDR1 comprises the amino acid sequence of SEQ ID NO: 102, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 102.

[0407] In some embodiments, the anti-EDB-FN antibody HCDR2 comprises the amino acid sequence of SEQ ID NO: 103, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 103.

[0408] In some embodiments, the anti-EDB-FN antibody HCDR3 comprises the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 104.

[0409] In some embodiments, the anti-EDB-FN antibody LCDR1 comprises the amino acid sequence of SEQ ID NO: 107, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 107.

[0410] In some embodiments, the anti-EDB-FN antibody LCDR2 comprises the amino acid sequence of SEQ ID NO: 108, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 108.

[0411] In some embodiments, the anti-EDB-FN antibody LCDR3 comprises the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 109.

[0412] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise anti-Epcam antibodies or fragments or derivatives thereof.

[0413] In some embodiments, the anti-Epcam antibody HCVR comprises the amino acid sequence of SEQ ID NO: 124, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 124. In certain embodiments, the nucleotide sequence encoding the anti-Epcam antibody HCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 124, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 124. In certain embodiments, the anti-Epcam antibody HCVR comprises the amino acid sequence of SEQ ID NO: 124. In certain embodiments, the nucleotide sequence encoding the anti-Epcam antibody HCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 124.

[0414] In some embodiments, the anti-Epcam antibody LCVR comprises the amino acid sequence of SEQ ID NO: 129, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 129. In certain embodiments, a nucleotide sequence encoding an anti-Epcam antibody LCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 129, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 129. In certain embodiments, an anti-Epcam antibody LCVR comprises the amino acid sequence of SEQ ID NO: 129. In certain embodiments, a nucleotide sequence encoding an anti-Epcam antibody LCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 129.

[0415] In some embodiments, the anti-Epcam antibody CH IgG1 domain comprises the amino acid sequence of SEQ ID NO: 128, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 128. In certain embodiments, the nucleotide sequence encoding the anti-Epcam antibody CH IgG1 domain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 128, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 128. In certain embodiments, the anti-Epcam antibody CH IgG1 domain comprises the amino acid sequence of SEQ ID NO: 128. In certain embodiments, the nucleotide sequence encoding the anti-Epcam antibody CH IgG1 domain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 128.

[0416] In some embodiments, the anti-Epcam antibody CL comprises the amino acid sequence of SEQ ID NO: 133, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 133. In certain embodiments, the nucleotide sequence encoding the anti-Epcam CL comprises the amino acid sequence of SEQ ID NO: 133, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 133. In certain embodiments, the anti-Epcam CL comprises the amino acid sequence of SEQ ID NO: 133. In certain embodiments, the nucleotide sequence encoding the anti-Epcam antibody CL comprises the nucleotide sequence of SEQ ID NO: 133.

[0417] In some embodiments, the anti-Epcam antibody HCDR1 comprises the amino acid sequence of SEQ ID NO: 125, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 125.

[0418] In some embodiments, the anti-Epcam antibody HCDR2 comprises the amino acid sequence of SEQ ID NO: 126, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 126.

[0419] In some embodiments, the anti-Epcam antibody HCDR3 comprises the amino acid sequence of SEQ ID NO: 127, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 127.

[0420] In some embodiments, the anti-Epcam antibody LCDR1 comprises the amino acid sequence of SEQ ID NO: 130, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 130.

[0421] In some embodiments, the anti-Epcam antibody LCDR2 comprises the amino acid sequence of SEQ ID NO: 131, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 131.

[0422] In some embodiments, the anti-Epcam antibody LCDR3 comprises the amino acid sequence of SEQ ID NO: 132, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 132.

[0423] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise anti-mClec9a antibodies or fragments or derivatives thereof.

[0424] In some embodiments, the anti-mClec9a antibody HCVR comprises the amino acid sequence of SEQ ID NO: 138, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 138. In certain embodiments, the nucleotide sequence encoding the anti-mClec9a antibody HCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 138, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 138. In certain embodiments, the anti-mClec9a antibody HCVR comprises the amino acid sequence of SEQ ID NO: 138. In certain embodiments, the nucleotide sequence encoding the anti-mClec9a antibody HCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 138.

[0425] In some embodiments, the anti-mClec9a antibody LCVR comprises the amino acid sequence of SEQ ID NO: 142, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 142. In certain embodiments, the nucleotide sequence encoding the anti-mClec9a antibody LCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 142, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 142. In certain embodiments, the anti-mClec9a antibody LCVR comprises the amino acid sequence of SEQ ID NO: 142. In certain embodiments, the nucleotide sequence encoding the anti-mClec9a antibody LCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 142.

[0426] In some embodiments, the anti-mClec9a antibody CH IgG1 Domain comprises the amino acid sequence of SEQ ID NO: 128, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 128. In certain embodiments, the nucleotide sequence encoding the anti-mClec9a antibody CH IgG1 domain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 128, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 128. In certain embodiments, the anti-mClec9a antibody CH IgG1 domain comprises the amino acid sequence of SEQ ID NO: 128. In certain embodiments, the nucleotide sequence encoding the anti-mClec9a antibody CH IgG1 domain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 128.

[0427] In some embodiments, the anti-mClec9a antibody CL comprises the amino acid sequence of SEQ ID NO: 133, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 133. In certain embodiments, the nucleotide sequence encoding the anti-mClec9a CL comprises the amino acid sequence of SEQ ID NO: 133, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 133. In certain embodiments, the anti-mClec9a CL comprises the amino acid sequence of SEQ ID NO: 133. In certain embodiments, the nucleotide sequence encoding the anti-mClec9a antibody CL comprises the nucleotide sequence of SEQ ID NO: 133.

[0428] In some embodiments, the anti-mClec9a antibody HCDR1 comprises the amino acid sequence of SEQ ID NO: 139, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 139.

[0429] In some embodiments, the anti-mClec9a antibody HCDR2 comprises the amino acid sequence of SEQ ID NO: 140, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 140.

[0430] In some embodiments, the anti-mClec9a antibody HCDR3 comprises the amino acid sequence of SEQ ID NO: 141, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 141.

[0431] In some embodiments, the anti-mClec9a antibody LCDR1 comprises the amino acid sequence of SEQ ID NO: 143, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 143.

[0432] In some embodiments, the anti-mClec9a antibody LCDR2 comprises the amino acid sequence of SEQ ID NO: 144, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 144.

[0433] In some embodiments, the anti-mClec9a antibody LCDR3 comprises the amino acid sequence of SEQ ID NO: 145, or a variant thereof having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 145. Fusion Polypeptides

[0434] In certain aspects, the present disclosure provides a fusion polypeptide comprising: a) a target-binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM); b) a latency-associated polypeptide (LAP) or a fragment or derivative thereof; c) a mature transforming growth factor β (TGFβ) family polypeptide or a fragment or derivative thereof; The present invention provides a fusion polypeptide comprising:

[0435] In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof may be inactive as a result of its interaction with LAP or a fragment or derivative thereof.

[0436] In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof can bind to a transforming growth factor β receptor (TGFβR) when the mature TGFβ family polypeptide or a fragment or derivative thereof is dissociated or released from the LAP or a fragment or derivative thereof. In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof can induce Smad2 / 3 signaling in a target cell or a cell adjacent to the target cell when the mature TGFβ family polypeptide or a fragment or derivative thereof is dissociated or released from the LAP or a fragment or derivative thereof.

[0437] In some embodiments, the fusion polypeptide may include any of a variety of linker(s) described herein. By way of example, and not limitation, non-limiting examples of linkers that may be used include any of SEQ ID NOs: 19, 46-79, and / or 96. In some embodiments, the linker comprises an amino acid sequence set forth in any of SEQ ID NOs: 19, 46-79, and / or 96, or a variant thereof having at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, or at least 99% sequence identity to SEQ ID NOs: 19, 46-79, and / or 96.

[0438] In some embodiments, the linker can comprise the sequence (GGGGS)n (SEQ ID NO:46), where n=1-10, or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker can consist of the sequence (GGGGS)n (SEQ ID NO:46). As non-limiting examples, the linker can comprise the sequence GGGGS (SEQ ID NO:47); GGGGSGGGGS((G4S)2; SEQ ID NO:48); GGGSGGGGSGGGGS((G4S)3; SEQ ID NO:49); or GGGGSGGGGSGGGGSGGGGS((G4S)4; SEQ ID NO:50).

[0439] In some embodiments, the linker may comprise the sequence (GGGS)n (SEQ ID NO:51), where n=1-10, or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker may consist of the sequence (GGGS)n (SEQ ID NO:51). In some embodiments, the linker may comprise the sequence GGGSGGGSGGGS (SEQ ID NO:19). In some embodiments, the linker consists of the sequence GGGSGGGSGGGS (SEQ ID NO:19).

[0440] In some embodiments, the linker can comprise the sequence GSGESGGGSG (SEQ ID NO: 96). In some embodiments, the linker consists of the sequence GSGESGGGSG (SEQ ID NO: 96).

[0441] In some embodiments, a linker may be located between the target binding polypeptide and the LAP or fragment or derivative thereof.

[0442] In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, (i) a target-binding polypeptide described herein, (ii) a linker described herein, (iii) a LAP or a fragment or derivative thereof described herein, and (iv) a mature TGFβ family polypeptide or a fragment or derivative thereof described herein.

[0443] In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, (i) a mature TGFβ family polypeptide described herein or a fragment or derivative thereof, (ii) a LAP or a fragment or derivative thereof described herein, (iii) a linker described herein, and (iv) a target-binding polypeptide described herein.

[0444] In some embodiments, the mature TGFβ family polypeptide or a fragment or derivative thereof and LAP or a fragment or derivative thereof may be separated by a protease cleavage site. The protease cleavage site may be any of the various protease cleavage sites of the present disclosure, such as, but not limited to, a furin cleavage site. In some embodiments, the furin cleavage site may comprise the sequence RXXR (SEQ ID NO: 35). In some embodiments, the protease cleavage site may be a furin cleavage site. In some embodiments, the furin cleavage site comprises the sequence RXXR (SEQ ID NO: 35). In some embodiments, the furin cleavage site consists of the sequence RXXR (SEQ ID NO: 35). In some embodiments, the furin cleavage site is RHRR (SEQ ID NO: 85), RRKR (SEQ ID NO: 86), or RKKR (SEQ ID NO: 87).

[0445] In various embodiments, a fusion polypeptide of the present disclosure can further comprise a signal peptide, for example, at the N-terminus of the fusion polypeptide. Without wishing to be bound by theory, a signal peptide can comprise a leader sequence at the amino-terminus (N-terminus) of a nascent polypeptide, such as a fusion polypeptide described herein, which co- or post-translationally directs the nascent protein to the endoplasmic reticulum and / or subsequent surface expression or secretion. Any of a variety of signal peptides known in the art, or fragments, derivatives, or combinations thereof, can be used in this practice, such as, for example, any of those described at signalpeptide.com / index.php?m=listspdb_mammalia, which is incorporated by reference for all intended purposes.

[0446] In some embodiments, the signal peptide can include an mROR signal peptide. In some embodiments, the signal peptide is an mROR signal peptide.

[0447] In some embodiments, the mROR signal peptide comprises the amino acid sequence of SEQ ID NO:1, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:1. In certain embodiments, the nucleotide sequence encoding the mROR signal peptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:1. In certain embodiments, the nucleotide sequence encoding the mROR signal peptide comprises the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:2. In certain embodiments, the mROR signal peptide comprises the amino acid sequence of SEQ ID NO:1. In certain embodiments, the nucleotide sequence encoding the mROR signal peptide comprises the nucleotide sequence of SEQ ID NO:2.

[0448] In some embodiments, the mROR signal peptide comprises the sequence MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 1). In some embodiments, the mROR signal peptide consists of the sequence MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 1).

[0449] In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof induces signal transduction via a Smad-dependent canonical signaling pathway in a target cell or a cell adjacent to a target cell upon activation of the mature TGFβ family polypeptide. In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof induces Smad2 / 3 signaling in a target cell or a cell adjacent to a target cell upon activation of the mature TGFβ family polypeptide. In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof induces signal transduction via a non-canonical signaling pathway disclosed herein in a target cell or a cell adjacent to a target cell upon activation of the mature TGFβ family polypeptide. Activation of a mature TGFβ family polypeptide can occur by any of the mechanisms disclosed herein.

[0450] In some embodiments, the TGF-β family polypeptides or fragments or derivatives thereof described herein may be autoactive TGF-β family polypeptides, i.e., TGF-β family polypeptides that do not require activation (e.g., by proteolysis or mechanical activation) to induce TGF-β signaling (e.g., Smad2 / 3 signaling and / or ERK kinase signaling, etc.). In some embodiments, the TGF-β family polypeptides or fragments or derivatives thereof may comprise one or more mutations to generate an autoactivated form of TGF-β. The one or more mutations to generate an autoactivated form of TGF-β may include any of the various mutations described herein. As a non-limiting example, a TGF-β family polypeptide described herein, e.g., a TGF-β1 polypeptide, may comprise one or more cysteine ​​(C) residue(s) in the pro-region of the TGF-β precursor (e.g., LAP) substituted with one or more serine (S) residue(s). In certain embodiments, the TGF-β family polypeptide may comprise a C223S mutation, where position 223 is relative to the sequence of SEQ ID NO: 82. In certain embodiments, a TGF-β family polypeptide may comprise a C225S mutation, where position 225 is relative to the sequence of SEQ ID NO: 82. In certain embodiments, a TGF-β family polypeptide may comprise a C223S and / or C225S mutation, which may render the TGF-β family polypeptide described herein autoactivating.

[0451] In some embodiments, LAP or a fragment or derivative thereof may comprise an integrin-binding motif disclosed herein. As a non-limiting example, the integrin-binding motif may comprise the sequence RGD. In some embodiments, the integrin-binding motif may consist of the sequence RGD. In some embodiments, LAP or a fragment or derivative thereof does not comprise an integrin-binding motif. Non-limiting examples of integrins include αvβ6 integrin, αvβ8 integrin, and αvβ1 integrin.

[0452] In some embodiments, LAP or a fragment or derivative thereof may interact with environmental molecule(s) disclosed herein, non-limiting examples of which include latency-associated binding protein (LTBP), glycoprotein A repeat-dominant protein (GARP), leucine-rich repeat-containing protein 32 (LRRC32), and leucine-rich repeat-containing protein 33 (LRRC33 / NRROS).

[0453] In some embodiments, the fusion polypeptides disclosed herein may comprise a target-binding polypeptide comprising an antigen-binding polypeptide or antigen-binding fragment thereof disclosed herein. The antigen that may be targeted by the antigen-binding polypeptide or antigen-binding fragment thereof disclosed herein may be any of the various antigens disclosed herein, including, but not limited to, PD-1, EDB-FN, CD63, CD4, CD90, Epcam, Madcam, a4b7, CCR9, Clec9a, Xcr1, and Olfm4. In some embodiments, the antigen targeted by the antigen-binding polypeptide or antigen-binding fragment thereof may comprise HLA-A2:INS. In some embodiments, the antigen targeted by the antigen-binding polypeptide or antigen-binding fragment thereof may comprise fibronectin. In some embodiments, the antigen targeted by the antigen-binding polypeptide or antigen-binding fragment thereof may comprise collagen-10.

[0454] In some embodiments, the target-binding polypeptide is not internalized.

[0455] In some embodiments, the target-binding peptide is capable of being internalized.

[0456] In various embodiments, the antigen-binding polypeptide may comprise any of the various antibodies or antigen-binding fragments thereof described herein. The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region disclosed herein. The antibody or antigen-binding fragment thereof may comprise a light chain variable region disclosed herein. The antibody or antigen-binding fragment thereof may comprise an immunoglobulin heavy chain constant domain disclosed herein. Non-limiting examples of immunoglobulin heavy chain constant domains are the IgG1 domain and the IgG4 domain.

[0457] In some embodiments, the mature TGFβ family polypeptide can be a mature TGFβ polypeptide disclosed herein. In some embodiments, the mature TGFβ family polypeptide can be a fragment of a mature TGFβ polypeptide or a derivative thereof disclosed herein. In some embodiments, the mature TGFβ polypeptide can be a fragment of a mature TGFβ1 polypeptide or a derivative thereof disclosed herein. In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:23. In some embodiments, the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:23. In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:90. In some embodiments, the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:90. In some embodiments, the mature TGFβ polypeptide can be a fragment of a mature TGFβ2 polypeptide or a derivative thereof disclosed herein. In some embodiments, the mature TGFβ2 comprises the sequence of SEQ ID NO:27. In some embodiments, the mature TGFβ2 consists of the sequence of SEQ ID NO:27.

[0458] In some embodiments, the mature TGFβ family polypeptide can be a mature growth differentiation factor 8 (GDF8) or a mature growth differentiation factor 11 (GDF11) polypeptide. In some embodiments, the mature TGFβ family polypeptide can be a mature growth differentiation factor 8 (GDF8) or a mature bone morphogenetic protein 4 (BMP4).

[0459] In some embodiments, the LAP or fragment or derivative thereof can be any of the various LAPs or fragments or derivatives thereof described herein. In some embodiments, the LAP can comprise the sequence of SEQ ID NO: 29. In some embodiments, the LAP can consist of the sequence of SEQ ID NO: 29. In some embodiments, the LAP can comprise the sequence of positions 30-274 of the sequence of SEQ ID NO: 82. In some embodiments, the LAP can consist of the sequence of positions 30-274 of the sequence of SEQ ID NO: 82. In some embodiments, the LAP can comprise the sequence of SEQ ID NO: 118. In some embodiments, the LAP can comprise the sequence of SEQ ID NO: 118. In some embodiments, the LAP can comprise the sequence of positions 21-298 of the sequence of SEQ ID NO: 116. In some embodiments, the LAP can consist of the sequence of positions 21-298 of the sequence of SEQ ID NO: 116. In some embodiments, the LAP can comprise the sequence of SEQ ID NO: 31. In some embodiments, the LAP can comprise the sequence of SEQ ID NO: 31. In some embodiments, the LAP can comprise the sequence of SEQ ID NO: 80. In some embodiments, the LAP may consist of the sequence of SEQ ID NO: 80. In some embodiments, the LAP may comprise the sequence of SEQ ID NO: 88. In some embodiments, the LAP may consist of the sequence of SEQ ID NO: 88. In some embodiments, the LAP may comprise the sequence of SEQ ID NO: 94. In some embodiments, the LAP may consist of the sequence of SEQ ID NO: 94. In some embodiments, the LAP or a fragment or derivative thereof may be heterologous to the mature TGFβ family polypeptide or a fragment or derivative thereof.

[0460] In some embodiments, LAP or a fragment or derivative thereof may comprise one or more mutations. The one or more mutations in LAP may include any of the various mutations described herein, such as a C24S mutation (position 24 is relative to the sequence of SEQ ID NO: 116); a C33S mutation (position 33 is relative to the sequence of SEQ ID NO: 82); a C223S mutation (position 223 is relative to the sequence of SEQ ID NO: 82); or a C225S (position 225 is relative to the sequence of SEQ ID NO: 82). In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that render LAP specific for proteolytic activation. In some embodiments, a fragment of LAP or a derivative thereof comprises one or more mutations that enable proteolytic activation of LAP. In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that enable mechanical activation of LAP. In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that promote mechanical activation of LAP. In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that inhibit or block mechanical activation of LAP. In some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that eliminate one or more protease cleavage sites within LAP or a fragment or derivative thereof, hi some embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that reduce binding of LAP to latency-associated binding protein (LTBP).

[0461] In various embodiments, the LAP or fragment or derivative thereof described herein may comprise one or more mutations described herein. In some embodiments, the one or more mutations may enable proteolytic activation of the mature TGFβ family polypeptide or fragment or derivative thereof described herein. In some embodiments, the one or more mutations may enable mechanical activation of the mature TGFβ family polypeptide or fragment or derivative thereof described herein.

[0462] In various embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that autoactivate a TGFβ family polypeptide described herein, where the autoactivating form of a TGFβ family polypeptide does not require activation (e.g., by proteolytic or mechanical activation) to induce TGFβ signaling (e.g., Smad2 / 3 signaling and / or ERK signaling).

[0463] In some embodiments, LAP or a fragment or derivative thereof may include one or more mutations that may, for example, eliminate one or more protease cleavage sites within LAP or a fragment or derivative thereof, hi some embodiments, the one or more mutations may, for example, reduce binding of LAP or a fragment or derivative thereof to latency-associated binding protein (LTBP).

[0464] In some embodiments, LAP or a fragment or derivative thereof may include one or more mutations that may introduce one or more protease cleavage sites into LAP or a fragment or derivative thereof, for example, via insertion of a protease cleavage site as described herein. In some embodiments, the insertion may include a PLGL insertion, such as may be useful for MMP2 cleavage. In some embodiments, the insertion may include a PLGI insertion, such as may be useful for MMP3, MMP7, and / or MMP8 cleavage.

[0465] In some embodiments, LAP or a fragment or derivative thereof comprises an amino acid sequence having at least 80% sequence identity with the sequence of positions 30 to 274 of SEQ ID NO: 82. In some embodiments, LAP or a fragment or derivative thereof comprises the sequence of positions 30 to 274 of SEQ ID NO: 82. In some embodiments, LAP consists of the sequence of positions 30 to 274 of SEQ ID NO: 82.

[0466] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 29. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 29.

[0467] In some embodiments, LAP or a fragment or derivative thereof comprises an amino acid sequence having at least 80% sequence identity with the sequence of positions 21 to 298 of SEQ ID NO: 116. In some embodiments, LAP or a fragment or derivative thereof comprises the sequence of positions 21 to 298 of SEQ ID NO: 116. In some embodiments, LAP consists of the sequence of positions 21 to 298 of SEQ ID NO: 116.

[0468] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 118. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 118. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 118.

[0469] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 31. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 31.

[0470] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 80.

[0471] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 88. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 88.

[0472] In some embodiments, LAP, or a fragment or derivative thereof, comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 94. In some embodiments, LAP, or a fragment or derivative thereof, comprises the amino acid sequence of SEQ ID NO: 94. In some embodiments, LAP consists of the amino acid sequence of SEQ ID NO: 94. Non-limiting examples of fusion polypeptide sequences

[0473] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:36, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:36. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:36, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:36. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 37, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 37. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 36. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 37.

[0474] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:38, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:38. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:38, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:38. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 39, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 39. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 39.

[0475] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:40, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:40. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:40, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:40. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 41, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 41. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 40. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 41.

[0476] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:42, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:42. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:42, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:42. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 43, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 43. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 43.

[0477] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:44, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:44. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:44, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:44. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 45, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 45. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 44. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 45.

[0478] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:110, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:110. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:110, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:110. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 111, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 111. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 110. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 111.

[0479] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:112, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:112. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:112, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:112. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 113, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 113. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 112. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 113.

[0480] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:114, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:114. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:114, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:114. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 115, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 115. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 114. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 115.

[0481] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 120, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 120. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:120, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:120. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 121, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 121. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 120. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 121.

[0482] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 122, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 122. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:122, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:122. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 123, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 123. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 122. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 123.

[0483] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 134, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 134. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:134, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:134. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 135, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 135. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 134. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 135.

[0484] In various embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 136, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 136. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 136, or a variant thereof having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 136. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 137, or a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 137. In certain embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 136. In certain embodiments, the nucleotide sequence encoding the fusion polypeptide comprises the nucleotide sequence of SEQ ID NO: 137. Polynucleotides and Vectors

[0485] In certain aspects, the present disclosure provides polynucleotides encoding one or more of the above-mentioned polypeptides. In one aspect, the present disclosure provides polynucleotides encoding the polypeptide complexes disclosed herein. In one aspect, the present disclosure provides polynucleotides encoding the fusion polypeptides disclosed herein. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA.

[0486] In certain embodiments, a polynucleotide encoding a polypeptide disclosed herein may contain one or more regulatory elements. Regulatory elements can regulate the expression of the polypeptide. Non-limiting examples of regulatory elements include promoters, initiation sites, polyadenylation (polyA) tails, IRES elements, enhancers, response elements, and termination signals.

[0487] In some embodiments, a polynucleotide sequence encoding a polypeptide (e.g., a fusion polypeptide) described herein can be operably linked to a promoter for expression. In some embodiments, when a sequence encoding a polypeptide(s) described herein is operably linked to a promoter, the promoter can mediate expression of the polypeptide(s). A "promoter" is a regulatory region of DNA that typically contains a TATA box that can direct RNA polymerase II to begin RNA synthesis at the appropriate transcription start site for a particular polynucleotide sequence. A promoter may additionally contain other regions that affect the rate of transcription initiation. As used herein, the term "promoter" encompasses enhancers. The promoter sequences disclosed herein regulate transcription of an operably linked polynucleotide. A promoter can be active in one or more cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or a combination thereof). The promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter).

[0488] Examples of constitutive promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, EF1a, SV40, PGK1 (human or mouse), Ubc, human beta-actin, CAG, Ac5, polyhedrin, TEF1, GDS, CaMV35S, Ubi, H1, and U6 promoters.

[0489] In some embodiments, the promoter can be a CMV promoter. In some embodiments, the promoter can be a CMV / EF1 hybrid promoter.

[0490] Inducible promoters can include, for example, chemically regulated promoters and physically regulated promoters. Chemically regulated promoters include, for example, alcohol-regulated promoters (e.g., alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., tetracycline-responsive promoters, tetracycline operator sequence (tetO), tet-On promoters, or tet-Off promoters), steroid-regulated promoters (e.g., rat glucocorticoid receptor, estrogen receptor promoters, or ecdysone receptor promoters), or metal-regulated promoters (e.g., metalloprotein promoters). Physically regulated promoters include, for example, temperature-regulated promoters (e.g., heat shock promoters, such as Hsp70-derived promoters and Hsp90-derived promoters), and light-regulated promoters (e.g., light-inducible promoters or light-repressible promoters). Other inducible promoters include lac, sp6, and T7 promoters.

[0491] The tissue-specific promoter can be, for example, a neuron-specific promoter, a glial-specific promoter, a muscle cell-specific promoter, a cardiac cell-specific promoter, a kidney cell-specific promoter, a bone cell-specific promoter, an endothelial cell-specific promoter, or an immune cell-specific promoter (e.g., a B cell promoter or a T cell promoter).

[0492] Developmentally-regulated promoters include, for example, promoters that are active only during embryonic stages of development or only in adult cells.

[0493] Other non-limiting examples of promoters useful in the nucleic acid molecules of the present disclosure include the CB7 / CAG promoter and associated upstream regulatory sequences, the EF-1 alpha promoter, the mU1a promoter, the UB6 promoter, the chicken beta-actin (CBA) promoter, as well as liver-specific promoters such as the TBG (thyroxine-binding globulin) promoter, the APOA2 promoter, the SERPINA1 (hAAT) promoter, ApoE.hAAT, or muscle-specific promoters such as the human desmin promoter, the CK8 promoter, or the Pitx3 promoter, inducible promoters such as hypoxia-inducible promoters or rapamycin-inducible promoters, or combinations thereof.

[0494] In some embodiments, a nucleic acid molecule of the present disclosure can include one promoter. In some embodiments, a nucleic acid molecule of the present disclosure can include more than one promoter (e.g., two, three, four, or more).

[0495] In a further aspect, the present disclosure provides a vector comprising any of the above-described polynucleotides. Such a vector may comprise a polynucleotide encoding a polypeptide disclosed above. The vector may be a viral vector or a non-viral vector.

[0496] In some embodiments, the vector may be a viral vector. Non-limiting examples of viral vectors include adenovirus, adeno-associated virus (AAV, e.g., AAV8, AAV9, AAVrhlO, AAVS3), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, Sendai virus (HVJ), alphavirus (e.g., Semliki Forest virus (SFV), Sindbis virus (SIN)), vaccinia virus, baculovirus vectors, and retrovirus vectors (e.g., murine leukemia virus (MLV), human immunodeficiency virus (HIV)).

[0497] In some embodiments, the viral vectors described herein are recombinant viral vectors. In some embodiments, the viral vectors described herein are modified so that they are replication-deficient in humans. In some embodiments, the viral vector is a hybrid vector, such as an AAV vector placed in a "disabled" adenoviral vector. In some embodiments, the viral vector comprises a viral capsid from a first virus and a viral envelope protein from a second virus, such as the VSV-G protein from vesicular stomatitis virus (VSV).

[0498] In some embodiments, the viral vectors described herein are AAV-based viral vectors. In some embodiments, the AAV-based vectors described herein do not encode the AAV rep gene (required for replication) and / or the AAV cap gene (required for synthesis of capsid proteins) (rep and cap proteins can be provided in trans by the packaging cell). Multiple AAV serotypes have been identified. In some embodiments, the AAV-based vectors described herein include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAVS3, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.rh46, AAV.rh73, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP AAV-based vectors may comprise capsid components from one or more of AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other rAAV particles, or a combination of two or more thereof. In some embodiments, the AAV-based vectors provided herein comprise components from one or more serotypes of AAV. In some embodiments, the AAV-based vectors described herein comprise components from one or more serotypes of AAV that have tropism for a desired tissue (e.g., liver, muscle, heart, kidney, neuron).

[0499] In some embodiments, the viral vectors described herein are lentiviral-based viral vectors. In some embodiments, the lentiviral vectors described herein are derived from human lentiviruses. In some embodiments, the lentiviral vectors described herein are derived from non-human lentiviruses. In some embodiments, the lentiviral vectors described herein are packaged into lentiviral capsids. In some embodiments, the lentiviral vectors described herein comprise one or more of the following elements: a long terminal repeat sequence, a primer binding site, a polypurine tract, an att site, and an encapsidation site.

[0500] In some embodiments, the viral vectors described herein are HIV-based viral vectors, hi some embodiments, the HIV-based vectors described herein comprise at least two polynucleotides, the gag and pol genes from the HIV genome and the env gene from another virus.

[0501] In some embodiments, the viral vectors described herein are herpes simplex virus-based viral vectors. In some embodiments, the herpes simplex virus-based vectors described herein are modified to not contain one or more immediate early (IE) genes, rendering them non-cytotoxic.

[0502] In some embodiments, the viral vectors provided herein are MLV-based viral vectors. In some embodiments, the MLV-based vectors provided herein contain up to 8 kb of heterologous DNA in place of viral genes.

[0503] In some embodiments, the viral vectors provided herein are alphavirus-based viral vectors. In some embodiments, the alphavirus vectors provided herein are recombinant replication-defective alphaviruses. In some embodiments, the alphavirus replicons within the alphavirus vectors provided herein are targeted to specific cell types by displaying a functional heterologous ligand on the virion surface.

[0504] In some embodiments, the vector may be a non-viral vector. Non-limiting examples of non-viral vectors include plasmids (e.g., minicircle plasmids), Sleeping Beauty transposons, piggyBac transposons, or single-stranded or double-stranded DNA molecules used as templates for homology-directed repair (HDR)-based gene editing. Cells and production methods

[0505] In one aspect, the present disclosure provides cells, e.g., host cells, comprising a polynucleotide and / or recombinant vector described herein. In some embodiments, the polynucleotide can encode, for example, a fusion polypeptide described herein. In some embodiments, a vector can comprise a polynucleotide described herein. The term "host cell" refers to any cell that contains heterologous nucleic acid. As a non-limiting example, the heterologous nucleic acid can be a vector disclosed herein. A host cell can be, for example, but not limited to, a cell from any organism that can be used, engineered, modified, selected, transformed, or grown for the production of a substance by the cell, e.g., for the expression of an RNA or DNA sequence, gene, protein, or enzyme by the cell. An appropriate host can be determined.

[0506] In some embodiments, host cells can be selected based on the vector backbone. In some embodiments, cosmids or plasmids can be introduced into prokaryotic host cells for replication of some types of vectors. Bacterial cells can be used as host cells for vector replication and / or expression, or for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, mammalian, insect, and yeast. Non-limiting examples of mammalian eukaryotic host cells are PC12, NIH3T3, HeLa, COS, Jurkat, 293, CHO (Chinese Hamster Ovary), ExpiCHO-S, FreedomCHO-S, and Saos.

[0507] Packaging cells useful for producing the polynucleotides and / or recombinant vectors described herein include, for example, animal cells that are permissive for the vector, e.g., a viral vector, or cells modified to be permissive for the vector; or packaging cell constructs using, for example, a transforming agent such as calcium phosphate. Non-limiting examples of packaging cell lines useful in the production methods described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), HEK-293 cells containing the SV40 large T antigen (HEK-293T or 293T), HEK293T / 17 cells, the human sarcoma cell line HT-1080 (CCL-121), the lymphoblastoid cell line Raj i(CCL-86), glioblastoma-astrocytoma epithelial-like cell line U87-MG (HTB-14), T lymphoma cell line HuT78 (TIB-161), NIH / 3T3 cells, Chinese hamster ovary cells (CHO) (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC No. CCL-2), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, and CAP-T cells.

[0508] Further non-limiting examples of packaging cells and / or lines that may be useful in the production methods described herein include, for example, L929 cells, the FLY virus packaging cell line reviewed in Cosset et al (1995) J Virol 69, 7430-7436, NS0 (mouse myeloma) cells, human amniotic cells (e.g., CAP, CAP-T), yeast cells (including but not limited to, S. cerevisiae, Pichia pastoris), plant cells (including but not limited to, tobacco NT1, BY-2), insect cells (including but not limited to, SF9, S2, SF21, Tni (e.g., High 5)), or bacterial cells including but not limited to, E. coli.

[0509] Additional packaging cells and systems, packaging techniques, and vectors for packaging nucleic acid genomes into vectors may include method steps, including, for example, construction of a structural protein expression cassette containing a plasmid for vector-inducible expression of viral structural proteins, and incorporation of any additional elements by polymerase chain reaction (PCR) amplification or by using synthetic oligonucleotides. As a non-limiting example, for selection, screening, and / or characterization of packaging cell lines, cells transfected with the expression cassette construct may be selected, for example, with G418 or hygromycin. Pooled drug-resistant cell foci may be cloned by limiting dilution, and individual clones may be screened for packaging activity, for example, by transfection with the vector, using, for example, lipofection or electroporation. Those clones with the highest levels of activity may be expanded for further use. Northern blot and Western blot analysis of vector-specific or structural protein-specific RNA and proteins expressed in packaging cells may be performed. The titer of replication-incompetent vector particles in clarified packaging cell line culture supernatant can be determined, for example, by infecting naive monolayers with serial dilutions, X-gal staining, and counting the total number of stained cells per well at the appropriate dilution. Vector titer can be specified as infectious units (IU) / ml. Contaminating replication-competent virus in the culture supernatant can be detected by standard plaque assays (plaque-forming units or PFU / mL) and by serial undiluted passaging in naive cells. Packaging methods include using packaging cells that permanently express viral components or transiently transfecting cells with plasmids.

[0510] In some embodiments, the present disclosure provides cells (e.g., FreedomCHO-S cells or ExpiCHO cells) comprising a polypeptide complex or fusion polypeptide disclosed herein. In a related aspect, the present disclosure provides cells comprising a polynucleotide disclosed herein. In yet another related aspect, the present disclosure provides cells comprising a vector disclosed herein. By way of example and not limitation, any of the above-described cells can comprise a polypeptide complex(es), fusion polypeptide(s), polynucleotide(s), and / or vector(s) disclosed herein. In some embodiments, the cell can be a FreedomCHO-S cell.

[0511] In some embodiments, the present disclosure provides methods of making a polypeptide complex or fusion polypeptide disclosed herein, which may include incubating a cell comprising a polynucleotide disclosed herein and / or a vector disclosed herein under conditions that allow for the production of the polypeptide complex or fusion polypeptide.

[0512] Isolation or purification of a polypeptide complex or fusion polypeptide disclosed herein, for example, from a virus or viral extract, can involve any of the techniques and / or method steps including, but not limited to, freeze / thaw cycling, microfluidization, filtration, e.g., nanofiltration and cross-flow filtration, osmotic shock, nuclease, detergent and / or protease treatment, cell lysis and DNA digestion, clarification (including filtration and centrifugation), ultracentrifugation, precipitation, e.g., precipitation with crowding agents, cross-flow filtration, affinity purification, nanoscale flow cytometry, CsCl density gradients, iodixanol gradient centrifugation, chromatography, e.g., column chromatography, including, but not limited to, ion exchange, anion and cation exchange, affinity, including antibody affinity, chromate focusing, desalting and buffer exchange, hydrophobic interaction, immunoprecipitation, multimode, mixed-mode, reversed-phase, and size exclusion, heparinized support matrix chromatography, DEAE Sepharose Fast Flow (FF), POROS 50 D, Fractogel® EMD DEAE (M), Macro-Prep DEAE Support, DEAE Ceramic HyperD® 20, and Toyopearl DEAE-650M, and / or the use of various media, for example, the application of various resins such as ceramic hydroxyapatite, ceramic fluoroapatite, ceramic hydroxyfluoroapatite, Cerfin sulfate media analytical chromatography methods, or combinations thereof, including analytical ion-exchange high performance liquid chromatography (HPLC), reverse-phase HPLC, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

[0513] In some embodiments, methods of producing a polypeptide complex or fusion polypeptide can include collecting the cell culture medium and isolating the produced polypeptide complex or fusion polypeptide by a process comprising affinity chromatography, which in some embodiments can comprise, for example, a Protein A column or beads or a Protein G column or beads. Pharmaceutical Compositions

[0514] In a further aspect, the present disclosure provides compositions (e.g., pharmaceutical compositions) comprising the polypeptide complexes, fusion polypeptides, polynucleotides, vectors, and / or cells disclosed herein, e.g., together with a pharmaceutically acceptable carrier and / or diluent. Pharmaceutical compositions of the present disclosure can be in any suitable form, depending on the desired method of administration to a subject.

[0515] Pharmaceutical compositions can include the disclosed polypeptide complexes or polypeptide complex-based molecules, and / or fusion polypeptides or fusion polypeptide-based molecules, either in free form or in the form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt," as used herein, refers to a derivative of the disclosed polypeptide complex(es) or fusion polypeptide(s), in which the polypeptide(s) or their complexes are modified by making an acid or base salt of the drug. For example, an acid salt is prepared from the free base (typically, the neutral form of the drug has a neutral -NH group) by reaction with a suitable acid. Acids suitable for preparing acid salts include both organic acids, such as acetic acid, benzoic acid, citric acid, propionic acid, glycolic acid, trifluoroacetic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, maleic acid, succinic acid, fumaric acid, tartaric acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc., as well as inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Conversely, basic salts of acidic moieties that may be present on the polypeptide are prepared using pharmaceutically acceptable bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.

[0516] Compositions of the disclosure can include a plurality of polypeptide complexes and / or fusion polypeptides, e.g., 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 polypeptide complexes and / or fusion polypeptides described herein. In some embodiments, compositions of the disclosure can include 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, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 polypeptide complexes and / or fusion polypeptides, or pharmaceutically acceptable salts thereof.

[0517] In some embodiments, the polypeptide complexes or polypeptide complex-based molecules and / or fusion polypeptides or fusion polypeptide-based molecules described herein have a concentration of about 1 μg / mL to 50 mg / mL, e.g., about 0.1 mg / mL to 10 mg / mL, about 0.2 mg / mL to 5 mg / mL, about 0.5 mg / mL to 8 mg / mL, about 0.8 mg / mL to 12 mg / mL, about 1 mg / mL to 15 mg / mL, about 2 mg / mL to 20 mg / mL, or about 5 mg / mL to 25 mg / mL, or about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL The compound may be present in solution at a concentration of 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.25 mg / mL, 1.5 mg / mL, 1.75 mg / mL, 2 mg / mL, 2.25 mg / mL, 2.5 mg / mL, 2.75 mg / mL, 3 mg / mL, 3.25 mg / mL, 3.5 mg / mL, 3.75 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL or 20 mg / mL.

[0518] The pharmaceutical compositions may be adapted for administration by any suitable route, such as, for example, parenteral (including intraperitoneal, subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation, or intranasal routes.

[0519] Such compositions may be prepared, for example, by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

[0520] Further disclosed herein are pharmaceutical dosage forms comprising a polypeptide complex or polypeptide complex-based molecule and / or a fusion polypeptide or fusion polypeptide-based molecule of the present disclosure, or a conjugate comprising a polypeptide complex or polypeptide complex-based molecule and / or a fusion polypeptide or fusion polypeptide-based molecule, a nucleic acid molecule, a vector, a cell, or a target-binding polypeptide.

[0521] Pharmaceutical compositions based on the polypeptide complexes or polypeptide complex-based molecules and / or fusion polypeptides or fusion polypeptide-based molecules disclosed herein, or conjugates comprising the polypeptide complexes or polypeptide complex-based molecules and / or fusion polypeptides or fusion polypeptide-based molecules, nucleic acid molecules, vectors, cells, or target-binding moieties, can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. The polypeptide complexes or polypeptide complex-based molecules and / or fusion polypeptides or fusion polypeptide-based molecules, or conjugates comprising the polypeptide complexes or polypeptide complex-based molecules and / or fusion polypeptides or fusion polypeptide-based molecules, nucleic acid molecules, vectors, cells, or target-binding moieties can be formulated for administration, for example, by injection, inhalation, or sequestration (either through the mouth or nose), or by oral, buccal, parenteral, or rectal administration, or by direct administration to an organ or tissue.

[0522] Pharmaceutical compositions can be formulated for various modes of administration, including systemic, local, or topical administration. Techniques and formulations can be found, for example, in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection purposes, pharmaceutical compositions can be formulated into a liquid solution, preferably a physiologically compatible buffer, such as Hank's solution or Ringer's solution. Furthermore, pharmaceutical compositions can be formulated in solid form and redissolved or suspended immediately before use. Lyophilized forms of pharmaceutical compositions are also suitable.

[0523] In some embodiments, the pharmaceutical composition of the present disclosure may be lyophilized. As a non-limiting example, the resulting lyophilized product may be reconstituted into an aqueous composition by adding an aqueous solvent. In some embodiments, the aqueous composition may be directly administered parenterally to a patient. Thus, in a further embodiment of the present disclosure, the pharmaceutical composition may be an aqueous pharmaceutical composition obtainable by reconstituting the lyophilized product with an aqueous solvent.

[0524] In some embodiments, pharmaceutical compositions disclosed herein may comprise lyophilized formulations. As a non-limiting example, the lyophilized formulation may comprise a polypeptide complex or polypeptide complex-based molecule and / or a fusion polypeptide or fusion polypeptide-based molecule of the present disclosure, mannitol, and / or TWEEN 80®. As another non-limiting example, the lyophilized formulation may comprise a polypeptide complex or polypeptide complex-based molecule and / or a fusion polypeptide or fusion polypeptide-based molecule of the present disclosure, mannitol, and poloxamer 188. In some embodiments, pharmaceutical compositions may comprise lyophilized formulations comprising a reconstituted liquid composition.

[0525] In some embodiments, pharmaceutical compositions of the present disclosure may provide formulations with higher solubility and / or wettability than previously known compositions. As a non-limiting example, improved solubility and / or wettability of lyophilizates can be achieved using appropriate excipient compositions. In this manner, pharmaceutical compositions of the present disclosure, including polypeptide complexes or polypeptide complex-based molecules and / or fusion polypeptides or fusion polypeptide-based molecule variants thereof, can be developed to exhibit desired storage stability (e.g., at -20°C, +5°C, or +25°C) and can be easily resolubilized with buffers or other excipients, allowing complete dissolution of the lyophilizate in a matter of seconds up to 2 minutes or more, with or without the use of an ultrasonic homogenizer. Furthermore, the compositions can be easily provided to patients in need of treatment via any suitable delivery route disclosed herein, such as parenteral (including intraperitoneal, subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation, or intranasal routes. As a non-limiting example, the pH value of the resulting solution can be between pH 2.7 and pH 9.0.

[0526] For oral administration, the pharmaceutical compositions can take the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cel...

Claims

1. 1. A polypeptide complex comprising: a. a target-binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM); b. Small Latent Complex (SLC), i. dimeric latency associated polypeptide (LAP) or a fragment or derivative thereof; and ii. A dimeric mature transforming growth factor beta (TGFβ) family polypeptide or a fragment or derivative thereof, which is inactive as a result of its interaction with dimeric LAP or a fragment or derivative thereof. and small latent complexes (SLCs) containing A polypeptide complex comprising:

2. The polypeptide complex of claim 1 , wherein the LAP or a fragment or derivative thereof is covalently attached to the target-binding polypeptide.

3. 3. The polypeptide complex of claim 2, wherein the LAP or a fragment or derivative thereof is covalently attached to the target-binding polypeptide via a linker.

4. 4. The polypeptide complex of claim 3, wherein the linker comprises the sequence (GGGGS)n (SEQ ID NO: 46), (GGGS)n (SEQ ID NO: 51), or GSGESGGGSG (SEQ ID NO: 96).

5. 5. The polypeptide complex of claim 4, wherein the linker consists of the sequence (GGGGS)n (SEQ ID NO: 46), (GGGS)n (SEQ ID NO: 51), or GSGESGGGSG (SEQ ID NO: 96).

6. The polypeptide complex of claim 3 , wherein the linker comprises the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

7. The polypeptide complex of claim 6, wherein the linker consists of the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

8. The polypeptide complex of claim 3 , wherein the linker comprises the sequence GSGESGGGSG (SEQ ID NO: 96).

9. The polypeptide complex of claim 8, wherein the linker consists of the sequence GSGESGGGSG (SEQ ID NO: 96).

10. 10. The polypeptide complex of claim 7 or claim 9, wherein the target-binding polypeptide binds to both the LAP or a fragment or derivative thereof and a molecule on the target cell or in the ECM.

11. 11. The polypeptide complex of claim 10, wherein the target-binding polypeptide is an antibody or a fragment or derivative thereof.

12. The polypeptide complex according to any one of claims 1 to 11, wherein the mature TGFβ family polypeptide or a fragment or derivative thereof and the LAP or a fragment or derivative thereof are associated via a non-covalent interaction.

13. The polypeptide complex according to any one of claims 1 to 11, wherein the mature TGFβ family polypeptide or a fragment or derivative thereof and the LAP or a fragment or derivative thereof are separated by a protease cleavage site.

14. The polypeptide complex of claim 13, wherein the protease cleavage site is a furin cleavage site.

15. 15. The polypeptide complex of claim 14, wherein the furin cleavage site comprises the sequence RXXR (SEQ ID NO: 35).

16. 16. The polypeptide complex of claim 15, wherein the furin cleavage site consists of the sequence RXXR (SEQ ID NO: 35).

17. 17. The polypeptide complex of claim 16, wherein the RXXR (SEQ ID NO: 35) is RHRR (SEQ ID NO: 85), RRKR (SEQ ID NO: 86), or RKKR (SEQ ID NO: 87).

18. 18. The polypeptide complex of any one of claims 1 to 17, wherein the mature TGFβ family polypeptide or a fragment or derivative thereof binds to a transforming growth factor β receptor (TGFβR) upon release of the mature TGFβ family polypeptide or a fragment or derivative thereof from the SLC.

19. 19. The polypeptide complex of any one of claims 1 to 18, wherein the mature TGFβ family polypeptide or a fragment or derivative thereof, upon release from the SLC, induces Smad2 / 3 signaling in the target cell or a cell adjacent to the target cell.

20. The polypeptide complex of any one of claims 1 to 19, wherein the LAP or a fragment or derivative thereof comprises an integrin binding motif.

21. 21. The polypeptide complex of claim 20, wherein the integrin binding motif comprises the sequence RGD.

22. The polypeptide complex of claim 20, wherein the integrin is αvβ6 integrin or αvβ8 integrin.

23. The polypeptide complex of any one of claims 1 to 19, wherein the LAP or a fragment or derivative thereof does not contain an integrin-binding motif.

24. 24. The polypeptide complex of any one of claims 1 to 23, wherein the target-binding polypeptide is an antigen-binding polypeptide or an antigen-binding fragment thereof.

25. 25. The polypeptide complex of claim 24, wherein the antigen-binding polypeptide is an antibody or an antigen-binding fragment thereof.

26. 26. The polypeptide complex of claim 25, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region.

27. 27. The polypeptide complex of claim 25 or claim 26, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region.

28. The polypeptide complex of any one of claims 25 to 27, wherein the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain constant domain.

29. 29. The polypeptide complex of claim 28, wherein the immunoglobulin heavy chain constant domain is an IgG1 domain.

30. 29. The polypeptide complex of claim 28, wherein the immunoglobulin heavy chain constant domain is an IgG4 domain.

31. 31. The polypeptide complex of any one of claims 25 to 30, wherein the antigen-binding polypeptide or antigen-binding fragment thereof binds to CD63.

32. The polypeptide complex of any one of claims 25 to 30, wherein the antigen-binding polypeptide or antigen-binding fragment thereof binds to the extra domain B of fibronectin (EDB-FN).

33. 31. The polypeptide complex of any one of claims 25 to 30, wherein the antigen-binding polypeptide or antigen-binding fragment thereof binds to epithelial cell adhesion molecule (Epcam).

34. 31. The polypeptide complex of any one of claims 25 to 30, wherein the antigen-binding polypeptide or antigen-binding fragment thereof binds to C-type lectin domain family 9 member A (Clec9a).

35. 35. The polypeptide complex of any one of claims 1 to 34, wherein the target-binding polypeptide is not internalized.

36. The polypeptide complex according to any one of claims 1 to 35, wherein the mature TGFβ family polypeptide is a mature TGFβ polypeptide.

37. 37. The polypeptide complex of claim 36, wherein the mature TGFβ polypeptide is a mature TGFβ1 polypeptide.

38. 38. The polypeptide complex of claim 37, wherein the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:

23.

39. 39. The polypeptide complex of claim 38, wherein the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:

23.

40. 38. The polypeptide complex of claim 37, wherein the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:

90.

41. 41. The polypeptide complex of claim 40, wherein the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:

90.

42. 37. The polypeptide complex of claim 36, wherein the mature TGFβ polypeptide is a mature TGFβ2 polypeptide.

43. 43. The polypeptide complex of claim 42, wherein the mature TGFβ2 polypeptide comprises the sequence of SEQ ID NO:

27.

44. 44. The polypeptide complex of claim 43, wherein the mature TGFβ2 polypeptide consists of the sequence of SEQ ID NO:

27.

45. 36. The polypeptide complex of any one of claims 1 to 35, wherein the mature TGFβ family polypeptide is a mature growth differentiation factor 8 (GDF8), a mature growth differentiation factor 11 (GDF11) polypeptide, or a mature bone morphogenetic protein 4 (BMP4).

46. The polypeptide complex of any one of claims 1 to 44, wherein the LAP comprises the sequence from positions 30 to 274 of the sequence of SEQ ID NO:

82.

47. The polypeptide complex of claim 46, wherein the LAP consists of the sequence from positions 30 to 274 of SEQ ID NO:

82.

48. The polypeptide complex of any one of claims 1 to 44, wherein the LAP comprises the sequence from positions 21 to 298 of the sequence of SEQ ID NO:

116.

49. The polypeptide complex of claim 48, wherein the LAP consists of the sequence from positions 21 to 298 of the sequence of SEQ ID NO:

116.

50. 50. The polypeptide complex of any one of claims 1 to 49, wherein the LAP or fragment or derivative thereof is heterologous to the mature TGFβ family polypeptide or fragment or derivative thereof.

51. 51. The polypeptide complex of any one of claims 1 to 50, wherein the LAP or a fragment or derivative thereof comprises one or more mutations, wherein the one or more mutations allow proteolytic activation of the mature TGFβ family polypeptide or a fragment or derivative thereof.

52. 52. The polypeptide complex of any one of claims 1 to 51, wherein the LAP or a fragment or derivative thereof comprises one or more mutations, wherein the one or more mutations enable mechanical activation of the mature TGFβ family polypeptide or a fragment or derivative thereof.

53. 53. The polypeptide complex of any one of claims 1 to 52, wherein the LAP or a fragment or derivative thereof comprises one or more mutations that eliminate one or more protease cleavage sites within the LAP or a fragment or derivative thereof.

54. 54. The polypeptide complex of any one of claims 1 to 53, wherein the LAP or a fragment or derivative thereof comprises one or more mutations, which reduce binding of the LAP or a fragment or derivative thereof to latency-associated binding protein (LTBP).

55. 55. The polypeptide complex of claim 54, wherein the one or more mutations include a C33S mutation, position 33 relative to the sequence of SEQ ID NO:

82.

56. 55. The polypeptide complex of claim 54, wherein the one or more mutations include a C24S mutation, where position 24 is relative to SEQ ID NO:

116.

57. 56. The polypeptide complex of any one of claims 1 to 44 and 54 to 55, wherein the LAP comprises the sequence of SEQ ID NO:

31.

58. 58. The polypeptide complex of claim 57, wherein the LAP consists of the sequence of SEQ ID NO:

31.

59. The polypeptide complex of any one of claims 1 to 44, 54, and 56, wherein the LAP comprises the sequence of SEQ ID NO:

94.

60. 60. The polypeptide complex of claim 59, wherein the LAP consists of the sequence of SEQ ID NO:

94.

61. 61. The polypeptide complex of any one of claims 1 to 60, wherein the mature TGFβ family polypeptide or a fragment or derivative thereof is chemically dissociated from the LAP or a fragment or derivative thereof and released in an active form from the SLC.

62. 62. The polypeptide complex of claim 61, wherein the chemical dissociation comprises protease treatment, temperature treatment, acid treatment, or any combination thereof.

63. 61. The polypeptide complex of any one of claims 1 to 60, wherein the mature TGFβ family polypeptide or a fragment or derivative thereof is mechanically dissociated from the LAP or a fragment or derivative thereof and released in an active form from the SLC.

64. 64. The polypeptide complex of claim 63, wherein mechanical dissociation occurs as a result of an interaction between the LAP or a fragment or derivative thereof and an integrin polypeptide.

65. A pharmaceutical composition comprising a polypeptide complex according to any one of claims 1 to 64.

66. 66. The pharmaceutical composition of claim 65, further comprising a pharmaceutically acceptable carrier or diluent.

67. 1. A fusion polypeptide comprising: a. a target-binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM); b. Latency-associated polypeptide (LAP) or a fragment or derivative thereof; c. Mature transforming growth factor β (TGFβ) family polypeptide or a fragment or derivative thereof A fusion polypeptide comprising:

68. 68. The fusion polypeptide of claim 67, wherein the mature TGFβ family polypeptide or a fragment or derivative thereof is inactive as a result of interaction with the LAP or a fragment or derivative thereof.

69. 69. The fusion polypeptide of claim 67 or claim 68, wherein the mature TGFβ family polypeptide or a fragment or derivative thereof binds to a transforming growth factor beta receptor (TGFβR) when the mature TGFβ family polypeptide or a fragment or derivative thereof is released from the LAP or a fragment or derivative thereof.

70. 70. The fusion polypeptide of any one of claims 67 to 69, wherein the mature TGFβ family polypeptide or fragment or derivative thereof induces Smad2 / 3 signaling in the target cell or a cell adjacent to the target cell when the mature TGFβ family polypeptide or fragment or derivative thereof is released from the LAP or fragment or derivative thereof.

71. A fusion polypeptide according to any one of claims 67 to 70, comprising a linker.

72. 72. The fusion polypeptide of claim 71, wherein the linker is located between the target-binding polypeptide and the LAP or fragment or derivative thereof.

73. 73. The fusion polypeptide of claim 72, comprising, from N-terminus to C-terminus: (i) the target-binding polypeptide; (ii) the linker; (iii) the LAP or a fragment or derivative thereof; and (iv) the mature TGFβ family polypeptide or a fragment or derivative thereof.

74. 73. The fusion polypeptide of claim 72, comprising, from N-terminus to C-terminus: (i) the mature TGFβ family polypeptide or a fragment or derivative thereof; (ii) the LAP or a fragment or derivative thereof; (iii) the linker; and (iv) the target-binding polypeptide.

75. 75. The fusion polypeptide of any one of claims 71 to 74, wherein the linker comprises the sequence (GGGGS)n (SEQ ID NO: 46), (GGGS)n (SEQ ID NO: 51), or GSGESGGGSG (SEQ ID NO: 96).

76. 76. The fusion polypeptide of claim 75, wherein the linker consists of the sequence (GGGGS)n (SEQ ID NO: 46), (GGGS)n (SEQ ID NO: 51), or GSGESGGGSG (SEQ ID NO: 96).

77. 76. The fusion polypeptide of claim 75, wherein the linker comprises the sequence GGGSGGGGSGGGS (SEQ ID NO: 19).

78. 78. The fusion polypeptide of claim 77, wherein the linker consists of the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

79. 76. The fusion polypeptide complex of claim 75, wherein the linker comprises the sequence GSGESGGGSG (SEQ ID NO: 96).

80. 80. The fusion polypeptide complex of claim 79, wherein the linker consists of the sequence GSGESGGGSG (SEQ ID NO: 96).

81. 81. The fusion polypeptide of any one of claims 67 to 80, wherein the mature TGFβ family polypeptide or a fragment or derivative thereof and the LAP or a fragment or derivative thereof are separated by a protease cleavage site.

82. 82. The fusion polypeptide of claim 81, wherein the protease cleavage site is a furin cleavage site.

83. 83. The fusion polypeptide of claim 82, wherein the furin cleavage site comprises the sequence RXXR (SEQ ID NO: 35).

84. 84. The fusion polypeptide of claim 83, wherein the furin cleavage site consists of the sequence RXXR (SEQ ID NO: 35).

85. 85. The fusion polypeptide of claim 84, wherein the RXXR (SEQ ID NO: 35) is RHRR (SEQ ID NO: 85), RRKR (SEQ ID NO: 86), or RKKR (SEQ ID NO: 87).

86. The fusion polypeptide of any one of claims 67 to 84, further comprising a signal peptide.

87. 87. The fusion polypeptide of claim 86, wherein the signal peptide is an mROR signal peptide.

88. 88. The fusion polypeptide of claim 87, wherein the mROR signal peptide comprises the sequence MHRPRRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 1).

89. 89. The fusion polypeptide of claim 88, wherein the mROR signal peptide consists of the sequence MHRPRRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 1).

90. The fusion polypeptide of any one of claims 67 to 89, wherein the LAP or a fragment or derivative thereof comprises an integrin binding motif.

91. 91. The fusion polypeptide of claim 90, wherein the integrin binding motif comprises the sequence RGD.

92. The fusion polypeptide of claim 90, wherein the integrin is αvβ6 integrin or αvβ8 integrin.

93. The fusion polypeptide of any one of claims 67 to 89, wherein the LAP or a fragment or derivative thereof does not contain an integrin binding motif.

94. 94. The fusion polypeptide of any one of claims 67 to 93, wherein the target-binding polypeptide is an antigen-binding polypeptide or an antigen-binding fragment thereof.

95. 95. The fusion polypeptide of claim 94, wherein the antigen-binding polypeptide is an antibody or an antigen-binding fragment thereof.

96. 96. The fusion polypeptide of claim 95, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region.

97. 97. The fusion polypeptide of claim 95 or claim 96, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region.

98. 98. The fusion polypeptide of any one of claims 95 to 97, wherein the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain constant domain.

99. 99. The fusion polypeptide of claim 98, wherein the immunoglobulin heavy chain constant domain is an IgG1 domain.

100. 99. The fusion polypeptide of claim 98, wherein the immunoglobulin heavy chain constant domain is an IgG4 domain.

101. 101. The fusion polypeptide of any one of claims 94 to 100, wherein the antigen-binding polypeptide or antigen-binding fragment thereof binds to CD63.

102. 101. The fusion polypeptide of any one of claims 94 to 100, wherein the antigen-binding polypeptide or antigen-binding fragment thereof binds to the extra domain B of fibronectin (EDB-FN).

103. 101. The fusion polypeptide of any one of claims 94 to 100, wherein the antigen-binding polypeptide or antigen-binding fragment thereof binds to epithelial cell adhesion molecule (Epcam).

104. 101. The fusion polypeptide of any one of claims 94 to 100, wherein the antigen-binding polypeptide or antigen-binding fragment thereof binds to C-type lectin domain family 9 member A (Clec9a).

105. 105. The fusion polypeptide of any one of claims 67 to 104, wherein the target-binding polypeptide is not internalized.

106. The fusion polypeptide of any one of claims 67 to 105, wherein the mature TGFβ family polypeptide is a mature TGFβ polypeptide.

107. 107. The fusion polypeptide of claim 106, wherein the mature TGFβ polypeptide is a mature TGFβ1 polypeptide.

108. The fusion polypeptide of claim 107, wherein the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:

23.

109. The fusion polypeptide of claim 108, wherein the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:

23.

110. The fusion polypeptide of claim 107, wherein the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:

90.

111. The fusion polypeptide of claim 110, wherein the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:

90.

112. 107. The fusion polypeptide of claim 106, wherein the mature TGFβ polypeptide is a mature TGFβ2 polypeptide.

113. The fusion polypeptide of claim 112, wherein the mature TGFβ2 polypeptide comprises the sequence of SEQ ID NO:

27.

114. The fusion polypeptide of claim 113, wherein the mature TGFβ2 polypeptide consists of the sequence of SEQ ID NO:

27.

115. 106. The fusion polypeptide of any one of claims 67 to 105, wherein the mature TGFβ family polypeptide is a mature growth differentiation factor 8 (GDF8), a mature growth differentiation factor 11 (GDF11) polypeptide, or a mature bone morphogenetic protein 4 (BMP4).

116. The fusion polypeptide according to any one of claims 67 to 114, wherein the LAP comprises the sequence from positions 30 to 274 of the sequence of SEQ ID NO:

82.

117. The fusion polypeptide of claim 116, wherein the LAP consists of the sequence from positions 30 to 274 of the sequence of SEQ ID NO:

82.

118. The fusion polypeptide according to any one of claims 67 to 114, wherein the LAP comprises the sequence from positions 21 to 298 of the sequence of SEQ ID NO:

116.

119. The fusion polypeptide of claim 118, wherein the LAP consists of the sequence from positions 21 to 298 of the sequence of SEQ ID NO:

116.

120. The fusion polypeptide of any one of claims 67 to 119, wherein the LAP or fragment or derivative thereof is heterologous to the mature TGFβ family polypeptide or fragment or derivative thereof.

121. 121. The fusion polypeptide of any one of claims 67 to 120, wherein the LAP or fragment or derivative thereof comprises one or more mutations, wherein the one or more mutations allow proteolytic activation of the mature TGFβ family polypeptide or fragment or derivative thereof.

122. 122. The fusion polypeptide of any one of claims 67 to 121, wherein the LAP or fragment or derivative thereof comprises one or more mutations, wherein the one or more mutations allow mechanical activation of the mature TGFβ family polypeptide or fragment or derivative thereof.

123. 123. The fusion polypeptide of any one of claims 67 to 122, wherein the LAP or a fragment or derivative thereof comprises one or more mutations that eliminate one or more protease cleavage sites within the LAP or a fragment or derivative thereof.

124. 124. The fusion polypeptide of any one of claims 67 to 123, wherein the LAP or a fragment or derivative thereof comprises one or more mutations that reduce binding of the LAP or a fragment or derivative thereof to latency-associated binding protein (LTBP).

125. The fusion polypeptide of claim 124, wherein the one or more mutations include a C33S mutation, where position 33 is relative to the sequence of SEQ ID NO:

82.

126. 125. The fusion polypeptide of claim 124, wherein the one or more mutations include a C24S mutation, where position 24 is relative to SEQ ID NO:

116.

127. A fusion polypeptide according to any one of claims 67 to 114 and 124 to 125, wherein the LAP comprises the sequence of SEQ ID NO:

31.

128. 128. The fusion polypeptide of claim 127, wherein the LAP consists of the sequence of SEQ ID NO:

31.

129. The fusion polypeptide of any one of claims 67 to 114, 124 and 126, wherein the LAP comprises the sequence of SEQ ID NO:

94.

130. 130. The fusion polypeptide of claim 129, wherein the LAP consists of the sequence of SEQ ID NO:

94.

131. A polynucleotide encoding the fusion polypeptide of any one of claims 67 to 128.

132. A vector comprising the polynucleotide of claim 131.

133. 133. The vector of claim 132, wherein the sequence encoding the fusion polypeptide is operably linked to a promoter, and the promoter mediates expression of the fusion polypeptide.

134. 134. The vector of claim 132 or claim 133, which is a viral vector.

135. 135. The vector of claim 134, wherein the viral vector is an adeno-associated viral (AAV) vector.

136. A cell comprising a polypeptide complex according to any one of claims 1 to 64, a fusion polypeptide according to any one of claims 67 to 130, a polynucleotide according to claim 131, or a vector according to any one of claims 132 to 135.

137. 136. A method of making a polypeptide complex of any one of claims 1 to 64, comprising incubating said cell comprising the polynucleotide of claim 131 or the vector of any one of claims 132 to 135 under conditions that allow the production of said polypeptide complex.

138. 138. The method of claim 137, further comprising collecting the cell culture medium and isolating the produced polypeptide complex by a process comprising affinity chromatography.

139. 139. The method of claim 138, wherein the affinity chromatography comprises a Protein A or Protein G column or beads.

140. 131. A method for treating a TGFβ dysregulation disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide complex of any one of claims 1 to 64, a pharmaceutical composition of claim 65 or claim 66, a polynucleotide of claim 131, or a vector of any one of claims 132 to 135.

141. 141. The method of claim 140, wherein the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered by injection.

142. 142. The method of claim 141, wherein the injection is intravenous, intramuscular, subcutaneous, or intraperitoneal.

143. 143. The method of any one of claims 140 to 142, wherein the polypeptide complex, pharmaceutical composition, polynucleotide or vector is administered by hydrodynamic delivery (HDD).

144. 144. The method of any one of claims 140 to 143, wherein the polypeptide complex, pharmaceutical composition, polynucleotide or vector is administered to the liver of the subject.

145. 145. The method of any one of claims 140 to 144, wherein said polypeptide complex, pharmaceutical composition, polynucleotide or vector is administered in combination with a further therapeutic agent.

146. 146. The method of any one of claims 140 to 145, wherein the TGFβ dysregulated disorder is inflammatory bowel disease (IBD).

147. 146. The method of any one of claims 140 to 145, wherein the TGFβ dysregulation disorder is Marfan syndrome.

148. 146. The method of any one of claims 140 to 145, wherein the TGFβ dysregulation disorder is an autoimmune disorder.

149. 146. The method of any one of claims 140 to 145, wherein the TGFβ dysregulation disorder is a wound healing disorder.

150. 135. A method for promoting wound healing in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide complex of any one of claims 1 to 64, a pharmaceutical composition of claim 65 or claim 66, a polynucleotide of claim 131, or a vector of any one of claims 132 to 135.

151. 151. The method of claim 150, wherein the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered to a wound of the subject.

152. 152. The method of any one of claims 140 to 151, wherein the subject is a human.

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