Compositions and methods for targeted delivery of TGF [beta]

Abstract

The present disclosure provides a polypeptide complex comprising a target binding polypeptide that binds to a molecule on a target cell or a molecule in an extracellular matrix (ECM); and small latent complexes comprising in particular a dimer latent related polypeptide (LAP) or a fragment or derivative thereof, and a dimer mature transforming growth factor beta (TGF beta) family polypeptide or a fragment or derivative thereof. Polypeptides (e.g., fusion polypeptides), and related polynucleotides, vectors, cells, and pharmaceutical compositions are also provided. Also provided are methods of treating subjects using, for example, these polypeptide complexes and / or these fusion polypeptides or pharmaceutical compositions thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 479,679, filed on January 12, 2023, and U.S. Provisional Application No. 63 / 526,021, filed on July 11, 2023, the disclosures of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated 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. Technical Field

[0005] The present disclosure provides a polypeptide complex comprising a target binding polypeptide that binds to a molecule on a target cell or in an extracellular matrix (ECM); and a small latency complex (SLC) that specifically comprises a dimeric latency-associated polypeptide (LAP) or a fragment or derivative thereof, and a dimeric mature transforming growth factor beta (TGFβ) family polypeptide or a fragment or derivative thereof. Polypeptides (e.g., fusion polypeptides), and related polynucleotides, vectors, cells, and pharmaceutical compositions are also provided. Methods of treating a subject using, for example, these polypeptide complexes and / or these fusion polypeptides or their pharmaceutical compositions are also provided. Background Art

[0006] Transforming growth factor β (TGFβ, also referred to as TGFb, TGFB, TGFbeta, TGFBeta, etc.) consists 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 non-covalently associated with the mature domain, thereby inactivating the mature domain. The complex of LAP (e.g., LAP cleaved by furin) and the mature domain is referred to as a small latency complex (SLC). The SLC secreted in combination with environmental molecules is referred to as a large latency complex (LLC), which can be expressed on different cell types (e.g., endothelial cells, T cells, macrophages, and microglia) and incorporated into the extracellular matrix (ECM). LLC can be proteolytically or mechanically activated to release mature TGFβ, which can induce downstream TGFβ signaling. Reduced TGFβ signaling has been identified in various TGFβ dysregulation conditions, including inflammatory bowel disease (IBD), Marfan syndrome (MFS), autoimmune diseases, and other diseases associated with TGFβ loss-of-function mutations. Therefore, there is a need to target the delivery of TGFβ to specific cell types (which are activated only when TGFβ reaches the desired location) to benefit diseases and / or biological processes that are exacerbated by reduced TGFβ signaling. Summary of the Invention

[0007] As described above in the Background section, there is a significant need in the art to develop compositions and methods for targeted delivery of TGFβ to specific cell types, particularly in situations where TGFβ signaling is reduced, such as occurs in various TGFβ dysregulation disorders. The present application addresses these and other needs.

[0008] In one aspect, the present invention provides a polypeptide complex comprising:

[0009] a. a target binding polypeptide that binds to a molecule on a target cell or a molecule in the extracellular matrix (ECM); and

[0010] b. A small latent complex (SLC), comprising:

[0011] i. a dimer latency-associated polypeptide (LAP) or a fragment or derivative thereof; and

[0012] ii. a dimeric mature transforming growth factor beta (TGFβ) family polypeptide or a fragment or derivative thereof, wherein the mature TGFβ family polypeptide or a fragment or derivative thereof is inactivated due to interaction with the dimeric LAP or a fragment or derivative thereof.

[0013] In some embodiments, the LAP, or a fragment or derivative thereof, is covalently attached to the target binding polypeptide.

[0014] In some embodiments, the LAP, or a fragment or derivative thereof, is covalently attached to the target binding polypeptide via a linker.

[0015] 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).

[0016] 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).

[0017] In some embodiments, the linker comprises the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

[0018] In some embodiments, the linker consists of the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

[0019] In some embodiments, the linker comprises the sequence GSGESGGGSG (SEQ ID NO: 96).

[0020] In some embodiments, the linker consists of the sequence GSGESGGGSG (SEQ ID NO: 96).

[0021] In some embodiments, the target binding polypeptide binds both LAP or a fragment or derivative thereof, and a molecule on the target cell or a molecule in the ECM.

[0022] In some embodiments, the target binding polypeptide is an antibody or a fragment or derivative thereof.

[0023] In some embodiments of any of the aforementioned 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.

[0024] In some embodiments of any of the aforementioned polypeptide complexes, the mature TGF[beta] family polypeptide, or a fragment or derivative thereof, and the LAP, or a fragment or derivative thereof, are separated by a protease cleavage site.

[0025] In some embodiments, the protease cleavage site is a furin cleavage site.

[0026] In some embodiments, the furin cleavage site comprises the sequence RXXR (SEQ ID NO: 35).

[0027] In some embodiments, the furin cleavage site consists of the sequence RXXR (SEQ ID NO: 35).

[0028] 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).

[0029] In some embodiments of any of the aforementioned polypeptide complexes, the mature TGFβ family polypeptide, or fragment or derivative thereof, binds to the transforming growth factor β receptor (TGFβR) upon release of the mature TGFβ family polypeptide, or fragment or derivative thereof, from the SLC.

[0030] In some embodiments of any of the aforementioned polypeptide complexes, 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 released from the SLC.

[0031] In some embodiments of any of the aforementioned polypeptide complexes, the LAP or fragment or derivative thereof comprises an integrin binding motif.

[0032] In some embodiments, the integrin binding motif comprises the sequence RGD.

[0033] In some embodiments, the integrin is αvβ6 integrin or αvβ8 integrin.

[0034] In some embodiments of any of the aforementioned polypeptide complexes, the LAP or fragment or derivative thereof does not comprise an integrin binding motif.

[0035] 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.

[0036] In some embodiments, the antigen-binding polypeptide is an antibody or an antigen-binding fragment thereof.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain constant domain.

[0040] In some embodiments, the immunoglobulin heavy chain constant domain is an IgG1 domain.

[0041] In some embodiments, the immunoglobulin heavy chain constant domain is an IgG4 domain.

[0042] In some embodiments, the antigen binding polypeptide or antigen binding fragment thereof binds to CD63.

[0043] In some embodiments, the antigen binding polypeptide or antigen binding fragment thereof binds to the Extra Domain B of fibronectin (EDB-FN).

[0044] In some embodiments, the antigen binding polypeptide or antigen binding fragment thereof binds to epithelial cell adhesion molecule (Epcam).

[0045] In some embodiments, the antigen binding polypeptide or antigen binding fragment thereof binds to C-type lectin domain family 9 member A (Clec9a).

[0046] In some embodiments of any of the aforementioned polypeptide complexes, the target binding polypeptide is not internalized.

[0047] In some embodiments of any of the aforementioned polypeptide complexes, the mature TGFβ family polypeptide is a mature TGFβ polypeptide.

[0048] In some embodiments, the mature TGFβ polypeptide is a mature TGFβ1 polypeptide.

[0049] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:23.

[0050] In some embodiments, the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:23.

[0051] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:90.

[0052] In some embodiments, the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:90.

[0053] In some embodiments, the mature TGFβ polypeptide is a mature TGFβ2 polypeptide.

[0054] In some embodiments, the mature TGFβ2 polypeptide comprises the sequence of SEQ ID NO:27.

[0055] In some embodiments, the mature TGFβ2 polypeptide consists of the sequence of SEQ ID NO:27.

[0056] 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).

[0057] In some embodiments of any of the aforementioned polypeptide complexes, the LAP comprises the sequence of positions 30-274 of SEQ ID NO:82.

[0058] In some embodiments, the LAP consists of the sequence from positions 30-274 of SEQ ID NO:82.

[0059] In some embodiments of any of the aforementioned polypeptide complexes, the LAP comprises the sequence of positions 21-298 of SEQ ID NO:116.

[0060] In some embodiments, the LAP consists of the sequence from positions 21-298 of SEQ ID NO:116.

[0061] In some embodiments of any of the aforementioned polypeptide complexes, the LAP, or a fragment or derivative thereof, is heterologous to the mature TGF[beta] family polypeptide, or a fragment or derivative thereof.

[0062] In some embodiments of any of the aforementioned polypeptide complexes, the LAP, or a fragment or derivative thereof, comprises one or more mutations, wherein the one or more mutations allow for proteolytic activation of a mature TGFβ family polypeptide, or a fragment or derivative thereof.

[0063] In some embodiments of any of the aforementioned polypeptide complexes, the LAP, or a fragment or derivative thereof, comprises one or more mutations, wherein the one or more mutations allow for mechanical activation of a mature TGFβ family polypeptide, or a fragment or derivative thereof.

[0064] In some embodiments of any of the aforementioned polypeptide complexes, the LAP, or a fragment or derivative thereof, comprises one or more mutations, wherein the one or more mutations eliminate one or more protease cleavage sites within the LAP, or a fragment or derivative thereof.

[0065] In some embodiments of any of the aforementioned polypeptide complexes, LAP, or a fragment or derivative thereof, comprises one or more mutations, wherein the one or more mutations reduce binding of LAP, or a fragment or derivative thereof, to latency-associated binding protein (LTBP).

[0066] In some embodiments, the one or more mutations comprise a C33S mutation, wherein position 33 is related to the sequence of SEQ ID NO:82.

[0067] In some embodiments, the one or more mutations comprise a C24S mutation, wherein position 24 correlates to SEQ ID NO:116.

[0068] In some embodiments of any of the aforementioned polypeptide complexes, the LAP comprises the sequence of SEQ ID NO:31.

[0069] In some embodiments, the LAP consists of the sequence of SEQ ID NO:31.

[0070] In some embodiments of any of the aforementioned polypeptide complexes, the LAP comprises the sequence of SEQ ID NO:94.

[0071] In some embodiments, the LAP consists of the sequence of SEQ ID NO:94.

[0072] In some embodiments of any of the aforementioned 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 from the SLC in an active form.

[0073] In some embodiments, chemical dissociation comprises protease treatment, temperature treatment, acid treatment, or any combination thereof.

[0074] In some embodiments of any of the aforementioned 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 from the SLC in an active form.

[0075] In some embodiments, mechanical dissociation occurs due to interaction between LAP, or a fragment or derivative thereof, and the integrin polypeptide.

[0076] In another aspect, provided herein is a pharmaceutical composition comprising the polypeptide complex described herein.

[0077] In some embodiments, the pharmaceutical compositions described herein may further comprise a pharmaceutically acceptable carrier or diluent.

[0078] In another aspect, the present invention provides a fusion polypeptide comprising:

[0079] a. a target binding polypeptide that binds to a molecule on a target cell or a molecule in the extracellular matrix (ECM);

[0080] b. Latency-associated polypeptide (LAP) or a fragment or derivative thereof; and

[0081] c. Mature transforming growth factor beta (TGFβ) family polypeptides or fragments or derivatives thereof.

[0082] In some embodiments, the mature TGFβ family polypeptide, or fragment or derivative thereof, is inactivated due to interaction with LAP, or fragment or derivative thereof.

[0083] In some embodiments, the mature TGFβ family polypeptide, or fragment or derivative thereof, binds to the transforming growth factor beta receptor (TGFβR) upon release of the mature TGFβ family polypeptide, or fragment or derivative thereof, from the LAP, or fragment or derivative thereof.

[0084] In some embodiments of any of the aforementioned fusion polypeptides, 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 upon release of the mature TGFβ family polypeptide, or fragment or derivative thereof, from the LAP, or fragment or derivative thereof.

[0085] In some embodiments of any of the aforementioned fusion polypeptides, the fusion polypeptide comprises a linker.

[0086] In some embodiments, a linker is positioned between the target binding polypeptide and the LAP or a fragment or derivative thereof.

[0087] 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.

[0088] 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) a LAP or a fragment or derivative thereof, (iii) a linker, and (iv) a target binding polypeptide.

[0089] In some embodiments of any of the aforementioned 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).

[0090] 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).

[0091] In some embodiments, the linker comprises the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

[0092] In some embodiments, the linker consists of the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

[0093] In some embodiments, the linker comprises the sequence GSGESGGGSG (SEQ ID NO: 96).

[0094] In some embodiments, the linker consists of the sequence GSGESGGGSG (SEQ ID NO: 96).

[0095] In some embodiments of any of the aforementioned fusion polypeptides, the mature TGF[beta] family polypeptide, or a fragment or derivative thereof, and the LAP, or a fragment or derivative thereof, are separated by a protease cleavage site.

[0096] In some embodiments, the protease cleavage site is a furin cleavage site.

[0097] In some embodiments, the furin cleavage site comprises the sequence RXXR (SEQ ID NO: 35).

[0098] In some embodiments, the furin cleavage site consists of the sequence RXXR (SEQ ID NO: 35).

[0099] 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).

[0100] In some embodiments of any of the aforementioned fusion polypeptides, the fusion polypeptide further comprises a signal peptide.

[0101] In some embodiments, the signal peptide is an mROR signal peptide.

[0102] In some embodiments, the mROR signal peptide comprises the sequence MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 1).

[0103] In some embodiments, the mROR signal peptide consists of the sequence MHRPRRRGTRPPPLALLAALLLAARGADA (SEQ ID NO: 1).

[0104] In some embodiments of any of the aforementioned fusion polypeptides, the LAP or fragment or derivative thereof comprises an integrin binding motif.

[0105] In some embodiments, the integrin binding motif comprises the sequence RGD.

[0106] In some embodiments, the integrin is αvβ6 integrin or αvβ8 integrin.

[0107] In some embodiments of any of the aforementioned fusion polypeptides, the LAP, or fragment or derivative thereof, does not comprise an integrin binding motif.

[0108] In some embodiments of any of the aforementioned fusion polypeptides, the target binding polypeptide is an antigen binding polypeptide or an antigen binding fragment thereof.

[0109] In some embodiments, the antigen-binding polypeptide is an antibody or an antigen-binding fragment thereof.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain constant domain.

[0113] In some embodiments, the immunoglobulin heavy chain constant domain is an IgG1 domain.

[0114] In some embodiments, the immunoglobulin heavy chain constant domain is an IgG4 domain.

[0115] In some embodiments, the antigen binding polypeptide or antigen binding fragment thereof binds to CD63.

[0116] In some embodiments, the antigen binding polypeptide or antigen binding fragment thereof binds to the Extra Domain B of fibronectin (EDB-FN).

[0117] In some embodiments, the antigen binding polypeptide or antigen binding fragment thereof binds to epithelial cell adhesion molecule (Epcam).

[0118] In some embodiments, the antigen binding polypeptide or antigen binding fragment thereof binds to C-type lectin domain family 9 member A (Clec9a).

[0119] In some embodiments of any of the aforementioned fusion polypeptides, the target binding polypeptide is not internalized.

[0120] In some embodiments of any of the aforementioned fusion polypeptides, the mature TGFβ family polypeptide is a mature TGFβ polypeptide.

[0121] In some embodiments, the mature TGFβ polypeptide is a mature TGFβ1 polypeptide.

[0122] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:23.

[0123] In some embodiments, the mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO:23.

[0124] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:90.

[0125] In some embodiments, the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:90.

[0126] In some embodiments, the mature TGFβ polypeptide is a mature TGFβ2 polypeptide.

[0127] In some embodiments, the mature TGFβ2 polypeptide comprises the sequence of SEQ ID NO:27.

[0128] In some embodiments, the mature TGFβ2 polypeptide consists of the sequence of SEQ ID NO:27.

[0129] In some embodiments of any of the aforementioned 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).

[0130] In some embodiments of any of the aforementioned fusion polypeptides, the LAP comprises the sequence from positions 30-274 of SEQ ID NO:82.

[0131] In some embodiments, the LAP consists of the sequence from positions 30-274 of SEQ ID NO:82.

[0132] In some embodiments of any of the aforementioned fusion polypeptides, the LAP comprises the sequence from positions 21-298 of SEQ ID NO:116.

[0133] In some embodiments, the LAP consists of the sequence from positions 21-298 of SEQ ID NO:116.

[0134] In some embodiments of any of the aforementioned fusion polypeptides, the LAP, or a fragment or derivative thereof, is heterologous to the mature TGF[beta] family polypeptide, or a fragment or derivative thereof.

[0135] In some embodiments of any of the aforementioned fusion polypeptides, the LAP, or a fragment or derivative thereof, comprises one or more mutations, wherein the one or more mutations allow for proteolytic activation of a mature TGFβ family polypeptide, or a fragment or derivative thereof.

[0136] In some embodiments of any of the aforementioned fusion polypeptides, the LAP, or a fragment or derivative thereof, comprises one or more mutations, wherein the one or more mutations allow for mechanical activation of a mature TGFβ family polypeptide, or a fragment or derivative thereof.

[0137] In some embodiments of any of the aforementioned fusion polypeptides, the LAP, or a fragment or derivative thereof, comprises one or more mutations, wherein the one or more mutations eliminate one or more protease cleavage sites within the LAP, or a fragment or derivative thereof.

[0138] In some embodiments of any of the aforementioned fusion polypeptides, LAP, or a fragment or derivative thereof, comprises one or more mutations, wherein the one or more mutations reduce binding of LAP, or a fragment or derivative thereof, to latency-associated binding protein (LTBP).

[0139] In some embodiments, the one or more mutations comprise a C33S mutation, wherein position 33 is related to the sequence of SEQ ID NO:82.

[0140] In some embodiments, the one or more mutations comprise a C24S mutation, wherein position 24 correlates to SEQ ID NO:116.

[0141] In some embodiments, the LAP comprises the sequence of SEQ ID NO:31.

[0142] In some embodiments, the LAP consists of the sequence of SEQ ID NO:31.

[0143] In some embodiments, the LAP comprises the sequence of SEQ ID NO:94.

[0144] In some embodiments, the LAP consists of the sequence of SEQ ID NO:94.

[0145] In another aspect, provided herein is a polynucleotide encoding a fusion polypeptide described herein.

[0146] In another aspect, provided herein is a vector comprising a polynucleotide described herein.

[0147] In some embodiments, the sequence encoding the fusion polypeptide is operably linked to a promoter, wherein the promoter mediates expression of the fusion polypeptide.

[0148] In some embodiments, the vector is a viral vector.

[0149] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

[0150] 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.

[0151] In another aspect, provided herein is a method of preparing a polypeptide complex described herein, the method comprising incubating a cell comprising a polynucleotide described herein or a vector described herein under conditions that allow production of the polypeptide complex.

[0152] In some embodiments, the method further comprises collecting the cell culture medium and isolating the produced polypeptide complex by a method comprising affinity chromatography.

[0153] In some embodiments, affinity chromatography comprises a Protein A or Protein G column or beads.

[0154] In another aspect, provided herein is a method for treating a TGFβ disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polypeptide complex described herein, the pharmaceutical composition described herein, the polynucleotide described herein, or the vector described herein.

[0155] In some embodiments, the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered via injection.

[0156] In some embodiments, the injection is intravenous, intramuscular, subcutaneous, or intraperitoneal.

[0157] In some embodiments of any of the above methods for treating a TGF[beta] dysregulation disorder in a subject in need thereof, the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered via hydrodynamic delivery (HDD).

[0158] In some embodiments of any of the above methods for treating a TGF[beta] dysregulation disorder in a subject in need thereof, the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered to the liver of the subject.

[0159] In some embodiments of any of the above methods for treating a TGF[beta] dysregulation disorder in a subject in need thereof, the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered in conjunction with an additional therapeutic agent.

[0160] In some embodiments of any of the above methods for treating a TGF[beta] dysregulation disorder in a subject in need thereof, the TGF[beta] dysregulation disorder is inflammatory bowel disease (IBD).

[0161] In some embodiments of any of the above methods for treating a TGF[beta] dysregulation disorder in a subject in need thereof, the TGF[beta] dysregulation disorder is Marfan syndrome.

[0162] In some embodiments of any of the above methods for treating a TGF[beta] dysregulation disorder in a subject in need thereof, the TGF[beta] dysregulation disorder is an autoimmune disorder.

[0163] In some embodiments of any of the above methods for treating a TGF[beta] dysregulation disorder in a subject in need thereof, the TGF[beta] dysregulation disorder is a wound healing disorder.

[0164] In another aspect, provided herein is a method for promoting wound healing in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polypeptide complex described herein, the pharmaceutical composition described herein, the polynucleotide described herein, or the vector described herein.

[0165] In some embodiments, the polypeptide complex, pharmaceutical composition, polynucleotide, or vector is administered to a wound of a subject.

[0166] In some embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0167] Figure 1 Schematic representation of TGFβ1 in complex with latency-associated peptide (LAP).

[0168] Figure 2 showed that the TGFβ latent complex can be proteolytically or mechanically activated.

[0169] Figure 3A-Figure 3B Antibody-TGFβ small latency complex (SLC) fusion construct design is shown. Figure 3A Antibody-TGFβ SLC fusion construct design is shown. Also depicted are the large latent complex (LLC) in both the closed and open conformations. Figure 3B Figure 2 shows the processing and activation profile of the anti-CD63-TGFβ1 SLC fusion protein. Unprocessed anti-CD63-TGFβ1 SLC fusion (left) is an expression form that can be present in both, for example, cell lysates and conditioned medium (CM) and cannot be activated by integrins. Processed anti-CD63-TGFβ1 SLC fusion (right) is a targeted form that can be purified, present only in CM, and activated by integrins (e.g., integrin αvβ6). LAP, latency-associated peptide; SLC, small latency complex (LAP + mature domain).

[0170] Figures 4A-4C The construct design and amino acid sequence of the anti-hCD63-TGFβ construct are shown. Figure 4AThe amino acid sequence corresponding to anti-hCD63-TGFβ1.C33S chain 1 comprising mROR SP (signal peptide)+VH (heavy chain variable domain) anti-hCD63+hIgG4+linker+TGFβ1 [LAP.C33S+mature peptide] is shown. Figure 4B The amino acid sequence corresponding to anti-hCD63-TGFβ2.C33S chain 1 comprising mROR SP+VH anti-hCD63+hIgG4+linker+TGFβ2[LAP.C33S+mature peptide] is shown. Figure 4C The amino acid sequence corresponding to anti-hCD63-TGFβ.C33S chain 2 comprising mROR SP+VK (Kappa light chain variable domain) anti-hCD63+hKappa is shown.

[0171] Figure 5 Secretion of the anti-hCD63-TGFβ construct in the conditioned medium of ExpiCHO cells is shown.

[0172] Figure 6 Shown is the expression of anti-hCD63-TGFβ1 in FreedomCHO cells.

[0173] Figure 7 Shown is mature TGFβ1-induced Smad2 / 3 signaling in Hek293-CAGA reporter cells.

[0174] Figure 8 Shown are both heat- and acid-activation of Smad2 / 3 signaling induced by ExpiCHO conditioned medium (CM) expressing anti-hCD63-TGFβ1.

[0175] Figure 9 Anti-hCD63-TGFβ1 SLC is shown to be activated in CD63.Y235A (non-internalizing mutant) expressing cells co-cultured with integrin αvβ6 expressing cells.

[0176] Figure 10 The amino acid sequence corresponding to anti-hCD63-TGFβ1.C33S super-stealth chain 1 comprising mROR SP+VH anti-hCD63+hIgG4+linker+TGFβ1[LAP.C33S+mature peptide] is shown.

[0177] Figure 11 The amino acid sequence corresponding to anti-hCD63-TGFβ1.C33S super-stealth chain 2 comprising mROR SP+VK anti-hCD63+hKappa is shown.

[0178] Figure 12 Anti-hCD63-TGFβ1 secreted from FreedomCHO cells was shown to be fully processed.

[0179] Figure 13 Shown are the size exclusion chromatography (SEC) curve of the 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).

[0180] Figure 14 Anti-hCD63-TGFβ1 is shown to bind to cells expressing hCD63.Y235A, but not to hCD63 knockout (KO) Hek293 cells.

[0181] Figure 15 Shown are data on thermal activation of purified anti-CD63-TGFβ1 in NBL7 cells (American mink lung epithelial cells) carrying a Smad2 / 3 reporter vector.

[0182] Figure 16 Demonstrated that integrin αvβ6 antagonizes mechanical activation of CD63-TGFβ1 SLCs to induce Smad2 / 3 signaling.

[0183] Figure 17 Schematic representation 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 a latency-associated polypeptide (LAP) that allows for integrin-mediated activation of TGFβ1.

[0184] Figure 18 Shown is the purification of REGN14660-TGFβ1 by high performance liquid chromatography (HPLC).

[0185] Figure 19 REGN14660-TGFβ1 Western blot analysis is shown.

[0186] Figure 20 Shown is the purification of REGN14660-TGFβ2 by HPLC.

[0187] Figure 21 REGN14660-TGFβ2 Western blot analysis is shown.

[0188] Figure 22Shown is a Smad2 / 3 reporter gene assay showing the latency of the REGN14660-TGFβ1 fusion (HA = heat activation). The Smad2 / 3 reporter gene assay illustrates that REGN14660-TGFβ1 retains TGFβ in a latent form (box). TGFβ is released from LAP by heat activation (HA). Circles represent "free" active TGFβ present in the antibody preparation.

[0189] Figure 23 Shown is a Smad2 / 3 reporter gene assay showing the latency of the REGN14660-TGFβ2 fusion (HA = heat activation). The Smad2 / 3 reporter gene assay illustrates that REGN14660-TGFβ2 retains TGFβ in a latent form (box). TGFβ is released from LAP by heat activation (HA). Circles represent "free" active TGFβ present in the antibody preparation.

[0190] Figure 24 Illustrated is that REGN14660-TGFβ1 binds to the extra B domain of fibronectin (EDB-FN; extracellular matrix FN) with higher affinity (black line with filled circles) compared to plasma (soluble) FN (black line with open squares). TGFβ1 can be activated from SLC by αvβ6 integrin expressed by CHO (black line with filled circles) cells, but not by the parental cell line, Chinese Hamster Ovary (CHO) cells (black line with open triangles).

[0191] Figure 25 Images of aortas from wild-type (WT) and fibrillin 1 knockout (Fbn1KO) mice are shown. The upper panel shows an aneurysm in the ascending aorta of Fbn1KO mice. The lower panel shows immunostaining of WT and Fbn1KO aortic sections using the REGN14660 antibody and shows extra-domain B of fibronectin (EDB-FN) staining in the diseased area (ascending) of the Fbn1KO aorta.

[0192] Figures 26A-26B showed that EDB-FN detection can be correlated with the presence of abnormal microvessels (CD31 positive) formed within the media of Fbn1KO aortas ( Figure 26A EDB-FN was not detected in wild-type (WT) P15 aorta ( Figure 26B ).

[0193] Figure 27 Shown is a graph of the survival curve of fibrillin 1 knockout (Fbn1 KO) mice (MAID 9419).

[0194] Figure 28Schematic diagram depicting the function of fibrillin microfibrils. TGFβ, transforming growth factor β; BMP, bone morphogenetic protein; LTBP, latency-related binding protein; Magp1 / 2, microfibril-associated glycoproteins 1 and 2; ECM, extracellular matrix.

[0195] Figure 29 A model of aortic aneurysms in Marfan syndrome (MFS) is depicted, which is associated with reduced TGFβ signaling. Fewer fibrillin microfibrils in MFS lead to reduced incorporation of large latent complexes (LLCs) into the extracellular matrix (ECM), resulting in reduced TGFβ signaling and ultimately leading to aneurysms.

[0196] Figure 30 The use of fibronectin (FN) as a docking platform for delivering antibody-latent TGFβ fusions to restore local TGFβ in MFS is illustrated. Fibronectin is a matrix template for the deposition of fibrillin-1 microfibrils. Fibronectin and fibrillin coexist in all tissues in which they are co-expressed.

[0197] Figure 31 Illustrated plasma and cell fibronectin (FN) are different in three domains introduced by alternative splicing. Fibronectin is encoded by a single gene, but produces different forms of fibronectin by alternative splicing. Plasma FN is produced and secreted by hepatocytes in a soluble dimer form. Cell FN is expressed in a dimer or cross-linked polymer form by several mesenchymal cells, and it is deposited in the ECM in a protofibril form, which is necessary for fibrillin-1 and type I collagen (I type Col) to deposit in the ECM. Fibronectin participates in, for example, maintenance, development, and wound healing of cell adhesion, cell movement, cell shape. FNKO mice showed embryonic lethality at approximately day 8.5.

[0198] Figure 32 It is shown 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 produced by alternative splicing. The sequence of EDB in mice and humans is identical. Isoforms containing EDB are expressed during embryonic and postnatal development and in a variety of solid tumors (and can be a marker for newly formed blood vessels), but are barely detectable in normal adult tissues, except in some blood vessels in the endometrium and ovary during the proliferative phase.

[0199] Figure 33 An example of a single-dose pharmacokinetic (PK) and tissue distribution study design is shown.

[0200] Figure 34Depicted is the use of an enzyme-linked immunosorbent assay (ELISA) to determine whether the antibody-TGFβ SLC fusions disclosed herein remain latent in the circulation.

[0201] Figure 35 The antibody-TGFβ1 SLC fusions disclosed herein were shown to remain latent in the circulation.

[0202] Figure 36 The levels of Fc fusions of the antibodies disclosed herein - TGFβ1 SLC in circulation are exemplified.

[0203] Figure 37 Shown are the expressions of C-type lectin domain family 9 member A (Clec9a) and epithelial cell adhesion molecule (Epcam) in the ileum, colon, and heart (ArrayStudio).

[0204] Figure 38 Depicted is the use of tissue ELISA to quantify the biodistribution of the antibody-TGFβ SLC fusions disclosed herein.

[0205] Figure 39 Shown are the amounts of antibody-TGFβ1 SLC fusion delivered in the ileum and colon 2 hours (h) post-injection (top) and 18 hours (h) post-injection (bottom).

[0206] Figure 40 It was shown that anti-mEpcam-TGFβ1 SLC strongly induced Smad2 / 3 phosphorylation in the ileum.

[0207] Figure 41 Anti-mClec9a-TGFβ1 SLC was shown to induce Smad2 / 3 phosphorylation in the colon.

[0208] Figure 42 It was shown that the antibody-TGFβ1 SLC fusion does not induce Smad2 / 3 phosphorylation in the heart.

[0209] Figure 43 Anti-mEpcam-TGFβ1 SLC fusion was shown to be delivered to the colon.

[0210] Figure 44 Anti-mEpcam-TGFβ1 SLC fusion was shown to be delivered to the ileum.

[0211] Figure 45 It was shown that the anti-mEpcam-TGFβ1 SLC fusion induces phosphorylated (P)-Smad2 / 3 in the ileum.

[0212] Figure 46The amino acid sequence corresponding to anti-mEpcam-TGFβ1.C33S chain 1 comprising mROR SP+VH anti-mEpcam+mlgG1+linker+TGFβ1[LAP.C33S+mature peptide] is shown.

[0213] Figure 47 The amino acid sequence corresponding to anti-mEpcam-TGFβ1.C33S chain 2 comprising mROR SP+VK anti-mEpcam+mKappa is shown.

[0214] Figure 48 The amino acid sequence corresponding to anti-mClec9a-TGFβ1.C33S chain 1 comprising mROR SP+VH anti-mClec9a+mIgG1+linker+TGFβ1[LAP.C33S+mature peptide] is shown.

[0215] Figure 49 The amino acid sequence corresponding to anti-mClec9a-TGFβ1.C33S chain 2 comprising mROR SP+VK anti-mClec9a+mKappa is shown.

[0216] Figure 50 The amino acid sequence corresponding to mlgG1 isotype control antibody-TGFβ1.C33S chain 1 comprising mROR SP+VH mlgG1 isotype control antibody+mlgG1+linker+TGFβ1 [LAP.C33S+mature peptide] is shown.

[0217] Figure 51 The amino acid sequence corresponding to mIgG1 isotype control antibody-TGFβ1.C33S chain 2 comprising mROR SP+VK mIgG1 isotype control antibody+mKappa is shown. DETAILED DESCRIPTION

[0218] 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). In particular, the SLC comprises a dimeric latency-associated polypeptide (LAP) or a fragment or derivative thereof, and a dimeric mature transforming growth factor beta (TGFβ) family polypeptide or a fragment or derivative thereof. The mature TGFβ family polypeptide or fragment or derivative thereof can be inactivated due to interaction with the dimeric LAP. Upon activation (i.e., release from the SLC), the mature TGFβ family polypeptide or fragment or derivative thereof can induce TGFβ signaling, such as, but not limited to, Smad2 / 3 signaling. Fusion polypeptides are also provided, comprising 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 beta (TGFβ) family polypeptide or a fragment or derivative thereof. Also included are related polynucleotides (e.g., polynucleotides encoding the fusion polypeptides described herein), vectors, cells, and pharmaceutical compositions. Also disclosed is a method 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, pharmaceutical composition, polynucleotide and / or vector as described herein, such that, for example, a target binding polypeptide within the polypeptide complex binds to a molecule on a target cell. In certain aspects, the disclosure also provides a method for treating a TGFβ disorder (e.g., inflammatory bowel disease (IBD), Marfan syndrome, autoimmune disease and / or wound healing disorder) in a subject in need thereof. Specifically, the method includes administering to the subject a therapeutically effective amount of a polypeptide complex, pharmaceutical composition, polynucleotide or vector as described herein. Also included is a method for promoting wound healing in a subject, the method comprising administering to a subject in need thereof (e.g., a subject with a wound) a therapeutically effective amount of a polypeptide complex, pharmaceutical composition, polynucleotide or vector as described herein.

[0219] definition

[0220] Unless defined otherwise, 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.

[0221] Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. 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.

[0222] The term "about" or "approximately" includes within a statistically significant range of values. Such a range can be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5%. The permissible variation encompassed by the term "about" or "approximately" depends on the specific system under study and can be readily understood by those of ordinary skill in the art.

[0223] 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 into a host (animal or human) having an immune system (directly or when expressed in, for example, a DNA vaccine), is recognized by the host's immune system and is capable of eliciting an immune response.

[0224] The term "antigen-binding polypeptide" refers to an antigen-specific binding element, which can be any ligand or receptor fragment that binds to an antigen or polypeptide of interest or a fragment thereof. In some embodiments, the ligand and / or receptor fragment can be naturally derived. In some embodiments, the ligand and / or receptor fragment can be synthetic. Non-limiting examples of antigen-binding polypeptides include, for example, antibodies; polypeptides derived from antibodies, 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 can be artificially fused to signaling domains; and any ligand and / or receptor fragment that binds to an antigen of interest.

[0225] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.

[0226] The terms "antibody," "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 partially or wholly synthetically produced. The term includes monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), intrabodies, minibodies, diabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies directed against antigen-specific TCRs), as well as epitope-binding fragments of any of the above. The term "antibody" also refers to covalent double-chain antibodies, 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. The 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.

[0227] As used herein, the term "human antibody" is intended to include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies (mAbs) of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), for example, in complementary determining regions (CDRs), particularly in CDR3. However, as used herein, the term "human antibody" is not intended to include mAbs wherein the CDR sequences derived from the germline of another mammalian species (for example, mice) have been transplanted onto human framework (FR) sequences. The term is included in antibodies recombinantly produced in the cells of non-human mammals or non-human mammals. The term is not intended to include antibodies separated from human subjects or generated in human subjects.

[0228] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site called a paratope in the variable region of an antibody molecule. A single antigen may have more than one epitope. Therefore, different antibodies can bind to different regions on the antigen and may have different biological effects. The term "epitope" also refers to the site on the antigen to which B cells and / or T cells respond. It also refers to the region of the antigen that is bound by antibodies. An epitope can be defined as structural or functional. A functional epitope is typically a subset of a structural epitope and has those residues that directly contribute to the affinity of the interaction. An epitope can also be conformational, that is, composed of nonlinear amino acids. In certain embodiments, an epitope can include a determinant as a chemically active surface group of a molecule (such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group), and in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0229] The term "host cell" refers to any cell containing a heterologous nucleic acid. As a non-limiting example, the heterologous nucleic acid can be a vector. A host cell can be, for example, but not limited to, a cell from any organism that is used, manipulated, modified, selected, transformed, or grown to produce a substance, such as expression of an RNA or DNA sequence, gene, protein, or enzyme.

[0230] By "reduced" or "decreased" or "decreased" or "mitigated" or "attenuated" is meant any reduction in the level or activity of a gene / protein (e.g., encoded at a locus of interest). For example, a reduction in activity can include a reduction 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 reduction in level or activity compared to an appropriate control.

[0231] "Increase" means any increase in the level or activity of a gene / protein (e.g., encoded at a locus of interest). For example, an increase in activity can include an increase in the overall level or activity of a given protein, including, for example, an increase in activity level of 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120% or more compared to an appropriate control.

[0232] The terms "specific binding", "binding in a specific manner", "antigen specificity", etc., indicate that the molecules involved in the specific binding can form relatively stable complexes with each other under physiological conditions and cannot non-specifically form stable complexes with other molecules outside the specific binding pair. Specific binding can be measured by an equilibrium dissociation constant (K) in the low micromolar to picomolar range. D ) to characterize (i.e., smaller KD 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.

[0233] The terms "protein" and "polypeptide" are used interchangeably herein to encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins, and modified proteins, including but not limited to glycoproteins, as well as all other types of modified proteins (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."

[0234] Proteins are considered to have an "N-terminus" and a "C-terminus." The term "N-terminus" refers to the beginning of a protein's amino acid chain, which terminates in an amino acid with a free amine group (-NH2). The term "C-terminus" refers to the end of a protein's amino acid chain, which terminates in a free carboxyl group (-COOH).

[0235] The terms "nucleic acid," "polynucleotide," and "nucleotide," used interchangeably herein, encompass polymeric forms of nucleotides of any length, including ribonucleotides (RNA), deoxyribonucleotides (DNA), or analogs or modified forms thereof. These terms 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 derived nucleotide bases. A single-stranded nucleic acid can be the sense strand or the antisense strand.

[0236] Nucleic acids are considered to have a "5' end" and a "3' end" because mononucleotides are reacted to form oligonucleotides in such a way that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its adjacent mononucleotide pentose ring in one direction via a phosphodiester bond. If the 5' phosphate of an oligonucleotide is not linked to the 3' oxygen of a mononucleotide pentose ring, the end of the oligonucleotide is referred to as the "5' end". If the 3' oxygen of an oligonucleotide is not linked to the 5' phosphate of another mononucleotide pentose ring, the end of the oligonucleotide is referred to as the "3' end". A nucleic acid sequence may be considered to have a 5' end and a 3' end even if it is within a larger oligonucleotide. In a linear or circular DNA molecule, a discrete element is referred to as "upstream" or 5' of a "downstream" or 3' element.

[0237] When referring to a protein, the term "fragment" means a protein that is shorter or has fewer amino acids than the full-length protein. A fragment can be, for example, an N-terminal fragment (i.e., a portion of the C-terminus of a protein removed), a C-terminal fragment (i.e., a portion of the N-terminus of a protein removed), or an internal fragment. When referring to a nucleic acid, the term "fragment" means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, a 5' fragment (i.e., a portion of the 3' end of a nucleic acid removed), a 3' fragment (i.e., a portion of the 5' end of a protein removed), or an internal fragment.

[0238] The terms "derivative" and "variant" are used interchangeably herein and refer to an entity that has significant structural identity to a reference entity, but is structurally different from the reference entity in terms of the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, derivatives are also functionally different from their reference entities. Generally speaking, whether a particular entity is properly considered a "derivative" of a reference entity is based on the degree of structural identity between it and the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. By definition, a derivative is a unique entity that shares one or more such characteristic structural elements. To give just a few examples, a small molecule can have a characteristic core structural element (e.g., a macrocyclic core) and / or one or more characteristic side group moieties, such that the derivative of a small molecule is a derivative that shares a core structural element and a characteristic side group moiety but differs in other side group moieties and / or bond types (single bond vs. double bond, E vs. Z, etc.) present in the core. A derivative nucleic acid can have a characteristic sequence element consisting of a plurality of nucleotide residues that have a specified position relative to each other in a linear or three-dimensional space. In some embodiments, the nucleic acid sequence of the derivative may 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 higher identical to the full length of the reference sequence or its fragment. A derivative peptide or polypeptide may have a characteristic sequence element consisting of a plurality of amino acids that have a specified position relative to each other in linear or three-dimensional space and / or contribute to a specific biological function. Derivative peptides and polypeptides include peptides and polypeptides that differ from a reference peptide or polypeptide in amino acid sequence by insertion, deletion and / or substitution of one or more amino acids, but retain at least one biological activity of such a reference peptide or polypeptide (e.g., the ability to mediate viral cell infection, the ability to mediate membrane fusion, the ability to be bound by a specific antibody or to promote an immune response, etc.). In some non-limiting embodiments, the 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 sequence identity over the entire length of the reference peptide or polypeptide (or fragment thereof). Alternatively or in addition, the derivative peptide or polypeptide may differ from the reference peptide or polypeptide by one or more and / or one or more differences in the chemical moieties attached to the polypeptide backbone (e.g., in terms of glycosylation, phosphorylation, acetylation, myristoylation, palmitoylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).In some embodiments, the derived peptide or polypeptide lacks one or more of the biological activities of the reference polypeptide, or has a reduced or increased level of one or more biological activities compared to the reference polypeptide. Derivatives of specific peptides or polypeptides can be found in nature, or can be synthesized or recombinantly produced. As used herein, the terms "derivative" or "variant" also encompass various fusion proteins and conjugates, including fusions or conjugates with detection tags (e.g., HA tags, histidine tags, biotin, fusions with fluorescent or luminescent domains, etc.), dimerization / multimerization sequences, Fc, signaling sequences, etc.

[0239] "Sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences refers to the identical residues in the two sequences when aligned for maximum correspondence over a specified comparison window. When using percentage sequence identity with respect to a protein, non-identical residue positions are typically different due to conservative amino acid substitutions, in which amino acid residues are substituted by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), thereby not changing the functional properties of the molecule. When the conservative substitutions of a sequence are different, the percentage sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ due to such conservative substitutions are considered to have "sequence similarity" or "similarity." The means for making such adjustments are well known. Typically, this involves counting conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing percentage sequence identity. Thus, for example, when the score obtained for the identical amino acid is 1 and the score obtained for the non-conservative substitution is zero, the score obtained for the conservative substitution is between zero and 1. For example, the score for the conservative substitution is calculated, as implemented in the program PC / GENE.

[0240] "Percentage of sequence identity" includes the value (maximum number of fully matched residues) determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (not containing additions or deletions) to achieve optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue appears in the two sequences to obtain the number of matched positions, dividing the number of matched 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 indicated (e.g., where the shorter sequence comprises a linked heterologous sequence), the comparison window is the full length of the shorter of the two compared sequences.

[0241] The term "latency-associated peptide" or "LAP" may refer to a peptide prodomain that inhibits the binding of TGFβ to the transforming growth factor β receptor (TGFβR).

[0242] The term "integrin binding motif" or "integrin binding site" when used in conjunction with the latency associated peptides (LAPs) disclosed herein may refer to a region on the LAP that contains a site capable of being recognized by an integrin.

[0243] The term "small latency complex" or "SLC" may 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 may be non-covalently associated with LAP. The SLC may be linked to an additional protein (e.g., a target binding polypeptide).

[0244] The term "inactive" or "inactive form" or "latent" in the context of TGFβ can refer to a TGFβ family polypeptide or fragment or derivative thereof that is unable to bind to the transforming growth factor β receptor (TGFβR) and / or initiate TGFβ signaling (e.g., Smad2 / 3 signaling and / or ERK signaling, etc.). As a non-limiting example, a TGFβ family polypeptide or fragment or derivative thereof can be inactivated due to interaction with LAP or a fragment or derivative thereof.

[0245] The term "active" or "active form" in the context of TGFβ can refer to a form of a TGFβ family polypeptide or a fragment or derivative thereof that is capable of binding to the 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 the small latency complex (SLC).

[0246] The term "dimer" refers to any compound or molecule composed of two subunits. Depending on the nature of the subunits that form the dimeric compound or molecule, the dimeric compound or molecule (e.g., a protein dimer) can be considered a homodimer (e.g., formed by two identical proteins) or a heterodimer (e.g., formed by two different proteins). In some embodiments, the dimeric subunits can be attached via, for example, disulfide bonds. In some embodiments, the dimeric subunits can be non-covalently bound together.

[0247] 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 case of TGFβ, the process by which two TGFβ subunits are linked to form a single dimeric molecule). In some embodiments, the mature TGFβ polypeptide or fragment or derivative thereof may be inactivated, for example, by interaction with a latency-associated peptide (LAP) or a fragment or derivative thereof (e.g., dimeric LAP). In some embodiments, the dimeric mature TGFβ family polypeptide and the dimeric LAP may be complexed (i.e., associated) to form a small latency complex (SLC). In some embodiments, the mature TGFβ family polypeptide and the dimeric LAP may be associated via non-covalent interactions. In some embodiments, the mature TGFβ family polypeptide and the dimeric LAP may be covalently linked and may be 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 can remain non-covalently associated with a mature TGFβ family polypeptide or a fragment or derivative thereof. In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof can be released from SLC. In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof can induce Smad2 / 3 signaling in a target cell or in a cell adjacent to a target cell when released from 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 protein (BMP), and growth differentiation factor (GDF), TGF-β1, TGF-β2, and TGF-β3. In some embodiments, mature TGFβ family polypeptides may include, for example, TGFβ1, TGFβ2, TGFβ3, BMP4, and / or GDF11. In some embodiments, the mature TGFβ family polypeptide may comprise a latent BMP / TGF-β family ligand as described herein. In some embodiments, the mature TGFβ family polypeptide may be a mature TGFβ polypeptide. In some embodiments, the mature TGFβ polypeptide may be a mature TGFβ1 polypeptide. In some embodiments, the mature TGFβ polypeptide may be a mature TGFβ2 polypeptide. In some embodiments, the mature TGFβ polypeptide may be a mature growth differentiation factor 8 (GDF8) or a mature growth differentiation factor 11. In some embodiments, the mature TGFβ polypeptide may be a mature bone morphogenetic protein 4 (BMP4).

[0248] The term "operably linked" and the like refer to a juxtaposition in which the components are in a relationship that allows them to function in their intended manner. For example, a control sequence that is "operably linked" to a coding sequence is connected in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. An "operably linked" sequence includes both an expression control sequence that is adjacent to the gene of interest and an expression control sequence that acts in trans or at a distance to control the gene of interest (or sequence of interest). The term "expression control sequence" includes polynucleotide sequences that are necessary to affect the expression and processing of the coding sequence to which it is connected. "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 improve translation efficiency (i.e., Kozak consensus sequences); sequences that enhance polypeptide stability; and sequences that enhance polypeptide secretion, when desired. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence, while in eukaryotes, such control sequences typically include a promoter and a transcription termination sequence. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0249] The terms "vector", "expression vector" and "cloning vector" refer to any vector that can introduce a nucleotide sequence (e.g., an RNA sequence or a DNA sequence) encoding, for example, an exogenous gene into a cell (e.g., a host cell) to genetically modify the cell and promote the expression (e.g., transcription and translation) of the introduced nucleotide sequence. Non-limiting examples of vectors include synthetic RNA and DNA molecules, plasmids, viruses, bacteriophages, and the like. In some embodiments, the vector can be a viral vector, including but not limited to a baculovirus vector, a herpes virus vector, a lentivirus vector, a retrovirus vector, a vaccinia virus vector, an adeno-associated virus (AAV) vector, an adenovirus vector, and an alphavirus vector.

[0250] The term "isolated" refers to a homogenous population of molecules (such as polynucleotides or polypeptides) that have been substantially separated and / or purified from other components of a system (such as a recombinant cell) that produces the molecule, as well as proteins that have undergone at least one purification or separation step. "Isolated" refers to a molecule that is substantially free of other cellular material and / or chemicals, and encompasses molecules separated to a higher purity, such as molecules separated to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0251] The term "treating" or "treating" a state, disorder, disease, or condition includes: (1) preventing, delaying, or reducing the incidence and / or likelihood of the development of at least one clinical or subclinical symptom of the state, disorder, disease, or condition in a subject who may have or is susceptible to the state, disorder, disease, or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder, disease, or condition; or (2) inhibiting the state, disorder, disease, or condition, i.e., arresting, reducing, or delaying the development of the disease or its recurrence or at least one clinical or subclinical symptom thereof; or (3) alleviating the state, disorder, disease, or condition, i.e., causing regression of the state, disorder, disease, or condition or at least one of the clinical or subclinical symptoms of the state, disorder, disease, or condition. The benefit to the subject being treated is statistically significant or at least perceptible to the patient or physician.

[0252] The term "TGFβ dysregulation disorder" refers to a state, disorder, disease or condition associated with TGFβ dysregulation, including, for example, low TGFβ expression and / or expression of TGFβ variant forms. In some embodiments, TGFβ dysregulation disorders 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 dilatation and rupture (aortic aneurysm), autoimmune disorders, arthritis (e.g., rheumatoid arthritis or osteoarthritis), lupus (e.g., systemic lupus) and wound healing disorders.

[0253] "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 a preferred embodiment, the subject is a human.

[0254] The term "effective," as applied to dosage or amount, refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need thereof. Note that when a combination of active ingredients is administered, an effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. 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 specific drug or drugs employed, the mode of administration, and the like.

[0255] The phrase "pharmaceutically acceptable," as used in connection with the compositions described herein, refers to the molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce an adverse effect when administered to a mammal (e.g., a human). Preferably, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0256] The term "administering" or the like refers to and includes administering the composition to a subject or system (e.g., to a cell, organ, tissue, organism, or a collection of its associated components or components). It will be understood by those skilled in the art that route of administration can vary according to, for example, the subject or system to which the composition is administered, the properties of the composition, the purpose of administration, etc. For example, in certain embodiments, administration to an animal subject (e.g., to a human or rodent) can be bronchial (including by bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, and / or vitreous administration. In some embodiments, administration can involve intermittent administration. In some embodiments, administration can involve continuous administration (e.g., infusion) for at least a selected period of time.

[0257] In light of the disclosure herein, conventional molecular biology, microbiology, and recombinant DNA techniques can be employed within the skill of the art. Such techniques are fully explained in the literature. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (hereinafter "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover, ed. 1985); Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins, eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds., (1984)]; Animal Cell Culture [R. I. Freshney, ed., (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989; Cloning (1984); Ausubel, FM et al. (Editors) Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994.These techniques include site-directed mutagenesis as described in Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), U.S. Pat. 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 l4.3:74-75 (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 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.

[0258] Peptide complex

[0259] In certain aspects, the present disclosure provides a polypeptide complex comprising:

[0260] a) a target binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM); and

[0261] b) a small latency complex (SLC), said small latency complex comprising:

[0262] (i) a dimeric latency-associated polypeptide (LAP) or a fragment or derivative thereof; and

[0263] (ii) a dimeric mature transforming growth factor beta (TGFβ) family polypeptide or a fragment or derivative thereof,

[0264] The mature TGFβ family polypeptide or its fragment or derivative is inactivated due to interaction with the dimeric LAP.

[0265] In some embodiments, LAP or a fragment or derivative thereof is covalently attached to a target binding polypeptide. In some embodiments, LAP or a fragment or derivative thereof is covalently attached to a target binding polypeptide via a linker. In some embodiments, LAP or a fragment or derivative thereof is non-covalently bound to a target binding polypeptide. In some embodiments, the target binding polypeptide binds both LAP and a molecule on a target cell or a molecule in the ECM. In some embodiments, the target may include, for example, but not limited to, a molecule on a target cell or a molecule in the extracellular matrix (ECM).

[0266] In some embodiments, the mature TGF[beta] family polypeptide or fragment or derivative thereof and the dimeric LAP are associated via non-covalent interactions.

[0267] In some embodiments, the mature TGFβ family polypeptide, or fragment or derivative thereof, is covalently linked to the LAP and separated by a protease cleavage site, non-limiting examples of which 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 fragment or derivative thereof, binds to the transforming growth factor beta receptor (TGFβR) upon release of the mature TGFβ family polypeptide, or fragment or derivative thereof, from the SLC.

[0268] In some embodiments, the mature TGFβ family polypeptide, or 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, the mature TGFβ family polypeptide, or fragment or derivative thereof, induces Smad2 / 3 signaling in a target cell or a cell adjacent to a target cell upon release of the mature TGFβ family polypeptide from an SLC.

[0269] Small latency complex (SLC)

[0270] In certain aspects, the polypeptide complexes described herein may comprise a small latency complex (SLC) comprising a dimeric latency-associated peptide (LAP) or a fragment or derivative thereof; and a dimeric mature transforming growth factor beta (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 inactivated due to interaction with the dimeric LAP.

[0271] Without wishing to be bound by theory, the small latent complex (SLC) can comprise a complex of the LAP domain and the mature TGFβ domain. During secretion, a furin-like proprotein convertase can cut TGFβ at the protease cleavage site located at the junction between, for example, the mature domain and LAP. After furin cleavage, LAP can remain non-covalently associated with the mature TGFβ domain, thereby inactivating the mature TGFβ domain by blocking the binding of the mature TGFβ domain to the TGFβ signaling receptor. Therefore, if LAP remains associated with the mature TGFβ domain, the activity of the mature domain may be blocked. SLC can be secreted separately as a soluble molecule, but can be secreted when combined with, for example, a cell environment molecule that can be surface-bound. In some embodiments, environmental molecules can be covalently linked to 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). An SLC that is secreted in complex with, for example, an environmental molecule can be referred to as a large latency complex (LLC). The environmental molecules can each bind to the same epitope on LAP, such as via a disulfide bond (e.g., C33 in the LAP domain disclosed herein).

[0272] TGFβ protein family is by 33 gene encodings.Members of TGF-β family, including activin, anti-Müllerian hormone (AMH), bone morphogenetic protein (BMP), inhibin, Nodal and growth and differentiation factor (GDF), can participate in the specialization of for example front / back and dorsal / ventral axis, ectoderm, mesoderm and endoderm, and the specialization of the various features of left-right asymmetry and each organ.TGFβ protein family member TGFβ1, TGFβ2 and TGFβ3 play a key role in immune response, wound healing, development and tumor cell growth and inhibition.Especially, TGFβ1, TGFβ2 and TGFβ3 can participate in cellular process, such as but not limited to extracellular matrix (ECM) remodeling, cell migration, invasion, epithelial-mesenchymal transition (EMT) and growth inhibition and / or immunosuppression.

[0273] As a non-limiting example, transforming growth factor β1 (TGFβ1) can be a disulfide bonded homodimeric protein that is secreted. 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 the mature domain, there may be an 'RXXR' (SEQ ID NO: 35) furin processing site, and 'X' may 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 cut TGFβ1 at the junction between the mature domain and the LAP. After furin cleavage, LAP can remain non-covalently associated with the mature domain, thereby inactivating the mature domain by blocking its binding to signaling receptors. Therefore, if LAP remains associated with the mature domain, the activity of the mature domain may be blocked. LAP can be removed from the processed TGFβ complex by, for example, proteolytic and mechanical methods. Therefore, LAP removal can release active mature TGFβ, which can be able to induce downstream signaling, such as Smad2 / 3 signaling (see, e.g., Figure 1 and Figure 2 ).

[0274] In some embodiments, upon release of a mature TGFβ family polypeptide disclosed herein, or a fragment or derivative thereof, from an SLC disclosed herein, the mature TGFβ family polypeptide, or a fragment or derivative thereof, can bind to a transforming growth factor beta 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 to 7. Type II receptors include TGFβRII, activin RIIA, activin RIIB, BMPRII, and AMHRII. Type I and type II receptors are structurally related transmembrane glycoproteins that contain an extracellular N-terminal ligand binding domain with more than ten cysteine ​​residues, a transmembrane region, and a C-terminal serine / threonine kinase domain that can regulate dimer structure. Type I receptors have a highly conserved region, the GS domain, which is rich in glycine and serine residues in the juxtamembrane domain near the N-terminus of the kinase domain. In some embodiments, TGFβ signaling (e.g., Smad2 / 3 signaling) can be initiated by binding of a mature TGFβ family polypeptide to, for example, the TGFβ receptor type I (TGFβRI) and / or TGFβ receptor type II (TGFβRII) receptors on the cell membrane.

[0275] In some embodiments, upon release from the SLC disclosed herein, the mature TGFβ family polypeptides disclosed herein, or fragments or derivatives thereof, can induce TGFβ signaling, such as Smad2 / 3 signaling and / or ERK signaling, in cells (e.g., target cells or cells adjacent to target cells).

[0276] In some embodiments, the mature TGFβ family polypeptide or fragment or derivative thereof can be chemically dissociated from the LAP or fragment or derivative thereof and released from the SLC in an active form. Non-limiting examples of chemical dissociation include protease treatment, temperature treatment, acid treatment, or any combination thereof.

[0277] In some embodiments, the mature TGFβ family polypeptide, or fragment or derivative thereof, can be mechanically dissociated from the LAP, or fragment or derivative thereof, and released from the SLC in an active form. As a non-limiting example, mechanical dissociation can occur due to an interaction between the LAP, or fragment or derivative thereof, and an integrin polypeptide, such as via an integrin binding motif in the LAP. Non-limiting examples of integrins include αvβ6 integrin, αvβ8 integrin, and αvβ1 integrin.

[0278] In some embodiments, the small latency 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 latency 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 latency 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 latency complex (SLC) comprises the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the nucleotide sequence encoding the small latency complex (SLC) comprises the nucleotide sequence of SEQ ID NO: 22.

[0279] In some embodiments, the small latency 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 latency 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 latency 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 latency complex (SLC) comprises the amino acid sequence of SEQ ID NO: 25. In certain embodiments, the nucleotide sequence encoding the small latency complex (SLC) comprises the nucleotide sequence of SEQ ID NO: 26.

[0280] In some embodiments, the small latency 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 latency 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 latency 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 latency complex (SLC) comprises the amino acid sequence of SEQ ID NO: 92. In certain embodiments, the nucleotide sequence encoding the small latency complex (SLC) comprises the nucleotide sequence of SEQ ID NO: 93.

[0281] Latency-associated peptide (LAP)

[0282] In some embodiments, the polypeptide complex described herein may comprise a small latency complex (SLC) comprising a dimeric latency-associated polypeptide (LAP) or a fragment or derivative thereof.

[0283] In some embodiments, LAP or a fragment or derivative thereof can be attached to a target binding polypeptide disclosed herein. In some embodiments, LAP or a fragment or derivative thereof can be covalently attached to a target binding polypeptide. In some embodiments, LAP or a fragment or derivative thereof can be covalently attached to a target binding polypeptide via a linker. In some embodiments, a linker can be located between the target binding polypeptide and the LAP or a fragment or derivative thereof.

[0284] In some embodiments, the linker can be 1-10 amino acids long. In some embodiments, the linker can be 1-20 amino acids long. As non-limiting examples, 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 long. In some embodiments, the linker can be 1-30 amino acids long. In some embodiments, the linker can be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. 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 can be 1 amino acid to 20 amino acids long, 2 amino acids to 15 amino acids long, 3 amino acids to 12 amino acids long, including 4 amino acids to 10 amino acids long, 5 amino acids to 9 amino acids long, 6 amino acids to 8 amino acids long or 7 amino acids to 8 amino acids long, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker can be optimized so that the linker does not impose any restrictions on the conformation and / or interaction of the connected partner.

[0285] In some embodiments, joint is a flexible joint.Suitable joint can be easily selected, and can be any suitable different lengths, such as 1 amino acid (for example, Gly) to 20 amino acid, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids or 7 amino acids to 8 amino acids, and can be 1,2,3,4,5,6 or 7 amino acids.Exemplary flexible joints include glycine polymer (G) n, glycine-serine polymer (GS) n (wherein n is at least one integer (for example, 1-20) (SEQ ID NO:174)), glycine-alanine polymer, alanine-serine polymer and other flexible joints known in the art.

[0286] In some embodiments, the linker is a cleavable linker.In some embodiments, the linker is a non-cleavable linker.

[0287] Non-limiting examples of linkers that can be used include any one of SEQ ID NOs: 19, 46-79, and 96. In some embodiments, the linker comprises the amino acid sequence set forth in any one 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 NOs: 19, 46-79, or 96.

[0288] In some embodiments, the linker can comprise the sequence (GGGGS)n (SEQ ID NO: 46), wherein 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); GGGGSGGGGSGGGGS ((G4S)3; SEQ ID NO: 49); or GGGGSGGGGSGGGGSGGGGS ((G4S)4; SEQ ID NO: 50).

[0289] In some embodiments, the linker may comprise the sequence (GGGS)n (SEQ ID NO: 51), wherein 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 (G3S) 3 (SEQ ID NO: 19). In some embodiments, the linker may consist of the sequence GGGSGGGSGGGS (G3S) 3 (SEQ ID NO: 19).

[0290] 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.

[0291] 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).

[0292] 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.

[0293] In some embodiments, the LAP or a fragment or derivative thereof can be non-covalently bound to a target binding polypeptide. In some embodiments, the target binding polypeptide binds both the LAP and a molecule on the target cell or a molecule in the ECM. In some embodiments, the target binding polypeptide can comprise an antigen binding polypeptide. In some embodiments, the antigen binding polypeptide can comprise an antibody or a fragment or derivative thereof, such as an antigen binding fragment thereof.

[0294] In some embodiments, the dimeric LAP of the present disclosure can associate with a mature TGFβ family polypeptide disclosed herein, or a fragment or derivative thereof, for example, via non-covalent interactions. In some embodiments, the LAP and the mature TGFβ family polypeptide, or a fragment or derivative thereof, can be covalently linked. In some embodiments, the LAP and the mature TGFβ family polypeptide, or a fragment or derivative thereof, can be covalently linked and can be separated, for example, by a protease cleavage site.

[0295] In some embodiments, when 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.

[0296] In some embodiments, the protease cleavage site that can separate LAP and mature TGFβ family polypeptides can comprise a furin cleavage site. In some embodiments, the furin cleavage site can be located at the junction between LAP and mature TGFβ family polypeptides. In some embodiments, the furin cleavage site can 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).

[0297] Without wishing to be bound by theory, during secretion in the endoplasmic reticulum (ER) / Golgi apparatus, furin can cleave mature TGFβ family polypeptides from LAP, leaving LAP non-covalently 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 can be blocked, thereby inactivating the mature TGFβ family polypeptide. LAP can be removed from the mature TGFβ family polypeptide, for example, via proteolytic and / or mechanical methods. LAP removal can release active mature TGFβ, for example, to induce downstream Smad2 / 3 signaling. In proteolytic activation, proteases can degrade or cause conformational changes in the LAP domain to release mature TGFβ. In mechanical activation, integrins (e.g., αvβ6 integrin, αvβ8 integrin and / or αvβ1 integrin) can bind to the tripeptide Arg-Gly-Asp (arginine, glycine and aspartic acid) 'RGD' binding motif at the C-terminus of LAP. The integrin binding at the C-terminus of LAP and the environmental molecule association (by covalent bonding) of the cell surface binding, for example, at the N-terminus of LAP can generate a directional pulling force that can open LAP and release free mature TGFβ. Mature TGFβ can bind to, for example, TGFβR1 and TGFβR2 on the cell surface and induce signal transduction, such as, but not limited to, Smad2 / 3 signal transduction. The active form of mature TGFβ can induce Smad2 / 3 signal transduction. In some embodiments, the mature TGFβ described herein can be internalized in the cell. In some embodiments, when mature TGFβ is internalized in the cell, mature TGFβ can induce TGFβ signal transduction (e.g., Smad2 / 3 signal transduction and / or ERK signal transduction, etc.), such as in the endosome.

[0298] In some embodiments, a mature TGF[beta] family polypeptide disclosed herein, or a fragment or derivative thereof, may be inactive due to interaction (eg, association) with LAP, or a fragment or derivative thereof.

[0299] In some embodiments, activation of a mature TGFβ family polypeptide or a fragment or derivative thereof can involve the release of a mature TGFβ family polypeptide from the ECM, such as 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 participate in LAP proteolysis include matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), thrombospondin 1 (TSP-1), plasmin (PLN), and plasma kallikrein (PLK).

[0300] 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 an active form.

[0301] In some embodiments, LAP or a fragment or derivative thereof can 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).

[0302] 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 an arginine residue, a glycine residue, and an aspartic acid residue). In some embodiments, the integrin binding motif in LAP may be a region consisting of 1 to 30 amino acid residues that includes the RGD sequence. In some embodiments, the integrin binding motif in LAP may be a region consisting of 1 to 30 amino acid residues that does not include 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.

[0303] In some embodiments, the integrin binding motif in the LAP can be a region that can be 1-10 amino acids in length. In some embodiments, the integrin binding motif can be 1-20 amino acids long. As non-limiting examples, 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 long. In some embodiments, the integrin binding motif can be 1-30 amino acids long. In some embodiments, the integrin binding motif can be 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. In some embodiments, the integrin binding motif can be 1 amino acid to 20 amino acids long, 2 amino acids to 15 amino acids long, 3 amino acids to 12 amino acids long, including 4 amino acids to 10 amino acids long, 5 amino acids to 9 amino acids long, 6 amino acids to 8 amino acids long, or 7 amino acids to 8 amino acids long, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids long. In some embodiments, the integrin binding motif sequence can comprise an RGD sequence. In some embodiments, the RGD sequence may not comprise an integrin binding motif.

[0304] 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, during mechanical activation, the integrin can 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. The integrin binding at the C-terminus of LAP and the association of environmental molecules bound to the cell surface at the N-terminus of LAP (e.g., by covalent bonding) generate a directional pulling force that opens the LAP and releases free mature TGFβ. Mature TGFβ can bind to TGFβR1 and TGFβR2 on the cell surface and induce Smad2 / 3 signaling.

[0305] In some embodiments, the mature TGFβ described herein can bind to a TGFβ receptor at the surface of a cell as described herein, such as TGFβR1 and / or TGFβR2. In some embodiments, the ligand and receptor can remain at the cell surface, and the TGFβ ligand-receptor complex is not internalized. In some embodiments, when the TGFβ ligand-receptor complex is not internalized, TGFβ signaling (e.g., Smad2 / 3 signaling and / or ERK signaling, etc.) can be induced in the cell.

[0306] In some embodiments, mature TGFβ described herein can bind to TGFβ receptors on the cell surface, such as TGFβR1 and / or TGFβR2, thereby triggering internalization of the ligand and receptor. The TGFβ ligand-receptor complex can enter the endocytic system, such as via clathrin-mediated endocytosis (CME). In some embodiments, endocytosis of the ligand-receptor complex, for example, in early endosomes, can regulate (e.g., enhance) TGFβ signaling as described herein (e.g., Smad2 / 3 signaling and / or ERK signaling, etc.).

[0307] In some embodiments, the mature TGFβ disclosed herein can be internalized within 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 within a cell, the mature TGFβ can induce TGFβ signaling, such as within an endosome.

[0308] 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 embodiments, the RGD sequence comprising one or more mutations comprises the sequence RGE. In some embodiments, when the RGD sequence comprises a mutation, the mutant RGD sequence can inhibit or block mechanical activation of LAP. In some embodiments, when the mutant RGD sequence inhibits or blocks mechanical activation of LAP, activation of LAP can be limited to activation via chemical activation and / or proteolytic activation, for example, as described herein.

[0309] In some embodiments, the LAP, or fragment or derivative thereof, does not comprise an integrin binding motif.

[0310] 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.

[0311] 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, the 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.

[0312] 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.

[0313] In some embodiments, the LAP or a fragment or derivative thereof comprises the sequence of positions 30-274 of SEQ ID NO:82.

[0314] In some embodiments, the LAP comprises the amino acid sequence of positions 30-274 of the sequence 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 30-274 of the sequence of SEQ ID NO:82. In certain embodiments, the nucleotide sequence encoding LAP comprises a nucleotide sequence encoding the amino acid sequence of positions 30-274 of the sequence 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 30-274 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, 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 of positions 30-274 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.

[0315] In some embodiments, LAP or a fragment or derivative thereof comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence at positions 30-274 of the sequence of SEQ ID NO: 82. In some embodiments, LAP or a fragment or derivative thereof comprises the amino acid sequence at positions 30-274 of the sequence of SEQ ID NO: 82. In some embodiments, LAP consists of the amino acid sequence at positions 30-274 of the sequence of SEQ ID NO: 82.

[0316] 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, the 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.

[0317] 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.

[0318] In some embodiments, LAP or a fragment or derivative thereof comprises the amino acid sequence of positions 21-298 of the amino acid sequence of SEQ ID NO:116.

[0319] In some embodiments, the LAP comprises the amino acid sequence of positions 21-298 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 21-298 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 21-298 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 21-298 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 of positions 21-298 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.

[0320] In some embodiments, LAP or a fragment or derivative thereof comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence at positions 21-298 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 at positions 21-298 of the amino acid sequence of SEQ ID NO: 116. In some embodiments, LAP consists of the amino acid sequence at positions 21-298 of the amino acid sequence of SEQ ID NO: 116.

[0321] In some embodiments, a LAP of the present disclosure may comprise an amino acid sequence that has 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, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 9 ...0% or more, 90% or more, 9 0% or more, 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 to the parent sequence and any amino acid sequence that has an activity of a normally occurring (i.e., native) sequence or a parent sequence.

[0322] In some embodiments, the LAP, or a fragment or derivative thereof, may be heterologous to the mature TGF[beta] family polypeptide disclosed herein, or a fragment or derivative thereof.

[0323] In some embodiments, LAP or a fragment or derivative thereof comprises an amino acid mutation 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 at one or more positions. As non-limiting examples, LAP or a fragment or derivative thereof may comprise an amino acid substitution at 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, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 94, 95, 96, 97, 98, 99, or 100 or more positions.

[0324] In some embodiments, LAP or its fragment or derivative can comprise one or more amino acid replacements and / or insertions and / or deletions.Amino acid replacement means that amino acid residue is replaced by the replacement amino acid residue at the same position. The amino acid residue inserted can be inserted at any position, and can be inserted so that some or all of the amino acid residues inserted are next to each other, or can be inserted so that the amino acid residue without insertion is next to another amino acid residue inserted. One or more amino acids of the sequence of SEQ ID NO:29 can be replaced and / or inserted and / or deleted. One or more amino acids of the sequence of SEQ ID NO:82 can be replaced and / or inserted and / or deleted. One or more amino acids of the sequence of SEQ ID NO:118 can be replaced and / or inserted and / or deleted. One or more amino acids of the sequence of SEQ ID NO:116 can be replaced and / or inserted and / or deleted.

[0325] In some embodiments, the LAP or its fragment or derivative can comprise one or more amino acid substitutions and / or insertions and / or deletions at one or more positions in the amino acid sequence, e.g., compared to the amino acid sequence of a reference LAP. For example, the LAP can comprise a substitution of one or more amino acids in the amino acid sequence of the parent LAP with a similar or homologous amino acid or a dissimilar amino acid.

[0326] In certain embodiments, the amino acid mutations (e.g., substitutions) of a protein or portion thereof are mutations that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming a protein complex, 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) sequence or a 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 allow for 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, for example, an environmental molecule such as, but not limited to, latency-associated binding protein (LTBP).

[0327] In some embodiments, LAP or its fragment or derivative comprises one or more mutations that render LAP specific for proteolytic activation. In some embodiments, LAP or its fragment or derivative comprises one or more mutations that allow proteolytic activation of LAP. In some embodiments, LAP or its fragment or derivative comprises one or more mutations that allow mechanical activation of LAP. In some embodiments, LAP or its fragment or derivative comprises one or more mutations that promote mechanical activation of LAP. In some embodiments, LAP or its fragment or derivative comprises one or more mutations that inhibit or block mechanical activation of LAP. In some embodiments, LAP or its fragment or derivative comprises one or more mutations that eliminate one or more protease cleavage sites within LAP or its fragment or derivative. In some embodiments, LAP or its fragment or derivative comprises one or more mutations that reduce the binding of LAP to a potentially related binding protein (LTBP).

[0328] In various embodiments, LAP or its fragment or derivative as described herein may comprise one or more mutations as described herein. In some embodiments, one or more mutations may allow proteolytic activation of mature TGFβ family polypeptides or its fragment or derivative as described herein. In some embodiments, one or more mutations may allow mechanical activation of mature TGFβ family polypeptides or its fragment or derivative as described herein. In some embodiments, LAP or its fragment or derivative comprises one or more mutations that promote mechanical activation of LAP. In some embodiments, LAP or its fragment or derivative comprises one or more mutations that inhibit or block mechanical activation of LAP. In some embodiments, LAP or its fragment or derivative as described herein may be mutated, for example, to produce a self-activating form of TGF-β, i.e., TGFβ that does not require activation (such as by proteolysis or mechanical activation) to induce downstream signaling as described herein (e.g., Smad2 / 3 signaling and / or ERK signaling, etc.).

[0329] In some embodiments, LAP or a fragment or derivative thereof can comprise one or more mutations that can, for example, eliminate one or more protease cleavage sites within LAP or a fragment or derivative thereof. In some embodiments, one or more mutations can, for example, reduce the binding of LAP or a fragment or derivative thereof to latency-associated binding protein (LTBP).

[0330] In some embodiments, LAP or a fragment or derivative thereof can comprise one or more mutations that can, for example, introduce one or more protease cleavage sites into LAP or a fragment or derivative thereof, such as via insertion of a protease cleavage site described herein. In some embodiments, the insertion can comprise a PLGL insertion, such as one that can be used for MMP2 cleavage. In some embodiments, the insertion can comprise a PLGI insertion, such as one that can be used for MMP3, MMP7, and / or MMP8 cleavage.

[0331] In some embodiments, the LAPs disclosed herein may include 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.

[0332] In some embodiments, LAP or its fragment or derivative can comprise amino acid substitution at the position corresponding to, for example, amino acid residue C33 of LAP. In some embodiments, LAP or its fragment or derivative can comprise C33S mutation (cysteine ​​to serine mutation at position 33), and position 33 is relevant to the sequence of SEQ ID NO:82. In some embodiments, LAP or its fragment or derivative can comprise C33S mutation (cysteine ​​to serine mutation at position 33), and position 33 is relevant to the sequence of SEQ ID NO:116. In some embodiments, the LAP comprising C33S mutation can comprise the sequence of SEQ ID NO:31. In some embodiments, the LAP comprising C33S mutation can comprise the sequence of SEQ ID NO:80. In some embodiments, the LAP comprising C33S mutation can comprise the sequence of SEQ ID NO:88.

[0333] In some embodiments, LAP or a fragment or derivative thereof can comprise an amino acid substitution at a position corresponding to, for example, amino acid residue C24 of LAP. In some embodiments, LAP or a fragment or derivative thereof can comprise a C24S mutation (a cysteine ​​to serine mutation at position 24), and position 24 is associated with the sequence of SEQ ID NO: 116. In some embodiments, a LAP comprising a C24S mutation can comprise the sequence of SEQ ID NO: 94.

[0334] Since LAP can covalently link to environmental molecules bound to the cell surface at C33 or C24, the C33S mutation or the C24S mutation can prevent LAP from disulfide bonding to environmental molecules during secretion, which prevents the incorporation of environmental molecules into TGFβ SLC. There are three types of environmental molecules expressed on different cell types: (i) LTBP (latency-associated binding protein); (ii) GARP (LRRC32, glycoprotein-A repeat-dominant protein); and (iii) NRROS (LRRC33, leucine-rich repeat-containing protein 33). TGFβ complexed with LTBP1, LTBP3, and LTBP4 is stored in the extracellular matrix (ECM). TGFβ complexed with GARP is stored on the surface of endothelial cells and activated regulatory T cells. TGFβ complexed with LRRC33 is stored on macrophages and microglia.

[0335] In some embodiments, one or more mutations of the LAP disclosed herein can comprise a C33S mutation, and position 33 is associated with 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 embodiments, the LAP comprising the C33S mutation comprises the sequence of SEQ ID NO: 80. In some embodiments, the LAP comprising the C33S mutation comprises the sequence of SEQ ID NO: 88.

[0336] In some embodiments, one or more mutations of a LAP disclosed herein can comprise a C24S mutation, and position 24 is related to the sequence of SEQ ID NO: 116. In some embodiments, the LAP comprising the C24S mutation comprises the sequence of SEQ ID NO:94.

[0337] 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 a 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 comprising 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.

[0338] 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.

[0339] 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 a 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 comprising 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.

[0340] 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.

[0341] 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 a 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 comprising 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.

[0342] 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.

[0343] 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 a 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 comprising 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.

[0344] 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.

[0345] In some embodiments, one or more mutations of a LAP disclosed herein can comprise a C33S mutation, a C223S mutation, and / or a C225S mutation, and positions 33, 223, and / or 225 are related to the sequence of SEQ ID NO: 82. In some embodiments, a LAP comprising C33S, C223S, and C225S mutations comprises the sequence of SEQ ID NO: 80.

[0346] 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 a 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 comprising 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.

[0347] 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.

[0348] Transforming growth factor β (TGFβ) family peptides

[0349] In some embodiments, the polypeptide complexes described herein may comprise a transforming growth factor beta (TGFβ) family polypeptide or a fragment or derivative thereof. In some embodiments, the polypeptide complexes may comprise a dimer and / or a mature transforming growth factor beta (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 may be inactivated due to interaction with a LAP described herein. In some embodiments, the LAP is a dimeric LAP.

[0350] Non-limiting examples of TGFβ family members include activin, anti-Müllerian hormone (AMH), bone morphogenetic protein (BMP), inhibin, Nodal, growth and differentiation factor (GDF) and TGFβ isoforms (e.g., TGFβ1, TGFβ2 and TGFβ3). TGFβ isoforms (e.g., TGFβ1, TGFβ2 and TGFβ3) have each been identified in mammals and can share 70%-82% homology at the amino acid level. TGFβ isoforms can participate in various cellular processes, such as but not limited to extracellular matrix (ECM) remodeling, cell migration, invasion, epithelial-mesenchymal transition (EMT), growth inhibition and / or immunosuppression.

[0351] From the perspective of mechanism, TGFβ, like other members of the TGFβ family, can be synthesized as a precursor protein, which can form a homodimer that interacts with a latency-associated peptide (LAP) to form a small latency complex (SLC). Environmental molecules, such as latent TGF-β binding protein (LTBP), can be bound to LAP to form a larger complex, known as a large latency complex (LLC). The TGFβ gene encodes a preprotein sequence consisting of, for example, a signal peptide, a propeptide that ends with a protease cleavage site, and a mature TGFβ sequence. Furin can hydrolyze the protease cleavage site, thereby producing a homodimer derived from the TGFβ and propeptide of separation. Two homodimers remain non-covalently associated and can be secreted. This latent complex can maintain TGFβ in an inactivated form that cannot be bound to its receptor.

[0352] LLC can be activated by proteolytic or mechanical means. In proteolytic activation, proteases can degrade or cause conformational changes in the LAP domain to release mature TGFβ. Non-limiting examples of proteases that can participate in LAP proteolysis include matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), thrombospondin 1 (TSP-1), plasmin (PLN) and plasma kallikrein (PLK). In mechanical activation, integrins (αvβ6 integrin, αvβ8 integrin, αvβ1 integrin) bind to the 'RGD' binding motif at the C-terminus of LAP. The association of integrin binding at the C-terminus of LAP and the cell surface-bound environmental molecules at the N-terminus of LAP (by covalent bonding) generates a directional pulling force that opens the LAP, thereby releasing free mature TGFβ (see, e.g. Figure 3A , box). Mature TGFβ can then bind to the extracellular domains of, for example, TGFβR1 (TGFβ type I receptor) and / or TGFβR2 (TGFβ type II receptor) on the cell surface. Binding of TGFβR1 and / or TGFβR2 by mature TGFβ can bring the receptors into close proximity with each other, thereby placing the intracellular serine / threonine kinase domain of the TGFβ receptor in a conformation that can promote receptor phosphorylation and / or activation. However, in some cases, in the absence of a ligand (i.e., mature TGFβ), the TGFβRs may already be in close proximity with each other. In some embodiments, upon ligand binding, constitutively active TGFβR2 can phosphorylate TGFβR1, which, in turn, can phosphorylate intracellular Smad2 / 3 upon such activation. As a non-limiting example, the binding of active mature TGFβ can induce signal transduction via the Smad-dependent classical signal transduction pathway. In some embodiments, active mature TGFβ is capable of inducing Smad2 / 3 signaling (see, e.g., Figure 1 In the non-classical pathway, an activated TGFβ receptor complex (e.g., a complex comprising mature, active TGFβ and TGFβR1 and / or TGFβR2) can signal via other factors such as, but not limited to, nuclear factor-κB (NF-κB), 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).

[0353] In some embodiments, a mature TGFβ family polypeptide disclosed herein, or a fragment or derivative thereof, and a dimeric LAP disclosed herein can associate via non-covalent interactions. In some embodiments, a mature TGFβ family polypeptide, or a fragment or derivative thereof, can be inactivated due to the interaction of a mature TGFβ family polypeptide, or a fragment or derivative thereof, with LAP, or a fragment or derivative thereof.

[0354] In some embodiments, the TGFβ family polypeptides or fragments or derivatives thereof described herein may be self-activating TGFβ family polypeptides, i.e., TGFβ family polypeptides that do not require activation (such as 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 a self-activating form of TGFβ. The one or more mutations that generate a self-activating form of TGFβ may comprise any of the various mutations described herein.

[0355] In some embodiments, the LAP, or fragment or derivative thereof, is heterologous to the mature TGF[beta] family polypeptide, or fragment or derivative thereof.

[0356] In some embodiments, the mature TGFβ family polypeptide or fragment or derivative thereof and the LAP can be covalently linked and can be separated by a protease cleavage site. In some embodiments, the protease cleavage site can include, for example, but not limited to, a furin cleavage site comprising the sequence RXXR (SEQ ID NO: 35). In some embodiments, the protease cleavage site can 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).

[0357] In some embodiments, a mature TGFβ family polypeptide disclosed herein, or a fragment or derivative thereof, can bind to a transforming growth factor beta receptor (TGFβR) upon release of the mature TGFβ family polypeptide, or a fragment or derivative thereof, from an SLC disclosed herein. The transforming growth factor beta receptor (TGFβR) can comprise any of the various TGFβRs disclosed herein.

[0358] In some embodiments, the mature TGFβ family polypeptide, or fragment or derivative thereof, upon release from the SLC disclosed herein, induces signaling via the Smad-dependent canonical signaling pathway in a target cell or a cell adjacent to a target cell. In some embodiments, the mature TGFβ family polypeptide, or fragment or derivative thereof, upon release from the SLC disclosed herein, induces Smad2 / 3 signaling in a target cell or a cell adjacent to a target cell. In some embodiments, the mature TGFβ family polypeptide, or fragment or derivative thereof, upon release from the SLC disclosed herein, induces signaling via the non-canonical signaling pathway disclosed herein in a target cell or a cell adjacent to a target cell.

[0359] In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof induces signal transduction via a Smad-dependent classical signal transduction pathway in a target cell or a cell adjacent to a target cell when a mature TGFβ family polypeptide is activated. In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof induces Smad2 / 3 signal transduction in a target cell or a cell adjacent to a target cell when a mature TGFβ family polypeptide is activated. In some embodiments, a mature TGFβ family polypeptide or a fragment or derivative thereof induces signal transduction via a non-classical signal transduction pathway disclosed herein in a target cell or a cell adjacent to a target cell when a mature TGFβ family polypeptide is activated. Activation of a mature TGFβ family polypeptide can occur by any mechanism disclosed herein. Activation can occur, for example, via a mechanism involving proteolytic activation and / or mechanical activation. In some embodiments, activation can occur by chemical activation. In some embodiments, activation can occur by acid activation (e.g., a low pH that can be achieved with HCl, such as pH 3.0) and / or heat activation.

[0360] In some embodiments, the mature TGFβ family polypeptide or its fragment or derivative can be chemically dissociated from the LAP or its fragment or derivative and released from the SLC in an active form. In some embodiments, chemical dissociation includes, for example, but not limited to, protease treatment, temperature treatment, acid treatment, or any combination thereof.

[0361] In some embodiments, the mature TGFβ family polypeptide, or fragment or derivative thereof, is mechanically dissociated from the LAP, or fragment or derivative thereof, and released from the SLC in an active form. As a non-limiting example, mechanical release can occur due to an interaction between the LAP, or fragment or derivative thereof, and an integrin polypeptide, such as via an integrin binding motif in the LAP. Non-limiting examples of integrins include αvβ6 integrin, αvβ8 integrin, and αvβ1 integrin.

[0362] In some embodiments, the mature TGFβ family polypeptide may be a mature TGFβ polypeptide.

[0363] In some embodiments, the mature TGFβ polypeptide may be a mature TGFβ1 polypeptide.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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.

[0368] In some embodiments, the mature TGFβ polypeptide can be a mature TGFβ2 polypeptide.

[0369] 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.

[0370] 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.

[0371] Other non-limiting exemplary mature TGFβ family polypeptides useful in the practice of the present disclosure include mature growth differentiation factor 8 (GDF8), mature growth differentiation factor 11 (GDF11) polypeptides, and mature bone morphogenetic protein 4 (BMP4).

[0372] In some embodiments, the mature TGFβ family polypeptide may be a mature growth differentiation factor 8 (GDF8) polypeptide. In some embodiments, the mature TGFβ family polypeptide may be a mature growth differentiation factor 11 (GDF11) polypeptide. In some embodiments, the mature TGFβ family polypeptide may be a mature bone morphogenetic protein 4 (BMP4).

[0373] target binding polypeptide

[0374] In some embodiments, the polypeptide complexes described herein can comprise a target binding polypeptide. In some embodiments, the target binding polypeptide can bind to a molecule on a target cell. In some embodiments, the target binding polypeptide binds 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 is capable of internalization.

[0375] In some embodiments, the polypeptide complex disclosed herein may comprise a small latent complex (SLC) disclosed herein, comprising a dimeric LAP disclosed herein or a fragment or derivative thereof that can be attached to a target binding polypeptide. In some embodiments, the LAP or a fragment or derivative thereof can be covalently attached to the target binding polypeptide. In some embodiments, the LAP or a fragment or derivative thereof can be covalently attached to the target binding polypeptide via a linker disclosed herein. In some embodiments, the linker can 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 can be non-covalently bound to the target binding polypeptide. In some embodiments, the target binding polypeptide binds to both the target of the LAP and the target binding polypeptide, such as a molecule on a target cell and / or a molecule in the extracellular matrix (ECM).

[0376] In some embodiments, the target binding polypeptide is an antigen binding polypeptide or an antigen binding fragment thereof.

[0377] In some embodiments, the antigen-binding polypeptide binds to at least one antigen on the target cell. In some embodiments, the antigen-binding portion binds to two or more antigens on the target cell. In some embodiments, two or more antigens can associate with the same target cell. In some embodiments, two or more antigens can associate 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 include an immune cell, such as, but not limited to, a T cell (e.g., activated regulatory T cells, CD40+T cells, CD90+T cells), a natural killer (NK) cell, a macrophage, or a mast cell. In some embodiments, the target cell may be a microglia. 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 microglia. 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).

[0378] In some embodiments, antigens on T cells (e.g., CD4 or CD90) can be targeted by the antigen-binding polypeptides described herein, and polypeptide complexes comprising such antigen-binding polypeptides described herein or pharmaceutical compositions thereof can, for example, promote regulatory T cell (Treg) differentiation and / or maintain immune tolerance, for example in inflammatory bowel disease (IBD).

[0379] In some embodiments, antigens (e.g., mEpcam or mOlfm4) on epithelial cells (such as intestinal epithelial cells) can be targeted by the antigen-binding polypeptides described herein, and polypeptide complexes comprising such antigen-binding polypeptides described herein or pharmaceutical compositions thereof can, for example, enhance epithelial barrier integrity, for example in IBD.

[0380] In some embodiments, antigens on dendritic cells (e.g., mClec9a) can be targeted by an antigen-binding polypeptide described herein, and polypeptide complexes comprising such an antigen-binding polypeptide described herein, or a pharmaceutical composition thereof, can, for example, reduce antigen presentation and / or inhibit goblet cell differentiation, for example, in IBD.

[0381] In some embodiments, antigens on pancreatic beta cells (e.g., HLA-A2:INS) can be targeted by an antigen-binding polypeptide described herein, and polypeptide complexes comprising such an antigen-binding polypeptide described herein, or a pharmaceutical composition thereof, can, for example, inhibit autoreactive immune cells, such as in type 1 diabetes (T1D).

[0382] In some embodiments, the antigen-binding polypeptide is bound to at least one antigen of the extracellular matrix (ECM). In some embodiments, the antigen-binding moiety is bound to two or more antigens of the ECM. In some embodiments, two or more antigens are associated with the same ECM. In some embodiments, two or more antigens are associated with different ECMs. Examples of antigens associated with ECM can be associated with various ECM molecules, such as, but not limited to, collagen (e.g., type X collagen, also referred to 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.

[0383] In some embodiments, antibodies to the ECM (e.g., fibronectin) can be targeted by the antigen-binding polypeptides described herein, and polypeptide complexes comprising such antigen-binding polypeptides described herein, or pharmaceutical compositions thereof, can repopulate newly developed areas of the matrix, such as in aortic aneurysms in Marfan syndrome (MFS).

[0384] In some embodiments, antigens of the ECM (e.g., type X collagen) can be targeted by the antigen-binding polypeptides described herein, and polypeptide complexes comprising such antigen-binding polypeptides described herein, or pharmaceutical compositions thereof, can, for example, reduce inflammation in joints, such as in rheumatoid arthritis (RA).

[0385] In some embodiments, the antigen-binding polypeptide is bound to at least one antigen associated with a TGFβ disorder. In some embodiments, the antigen-binding polypeptide is bound to two or more antigens associated with a TGFβ disorder. In some embodiments, two or more antigens associated with a TGFβ disorder are associated with the same TGFβ disorder. In some embodiments, two or more antigens associated with a TGFβ disorder are associated with different antigens associated with a TGFβ disorder. Non-limiting examples of TGFβ 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), and wound healing disorders.

[0386] Examples of antigens that can 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, the antigen targeted by the antigen-binding polypeptides or antigen-binding fragments thereof may comprise HLA-A2:INS. In some embodiments, the antigen targeted by the antigen-binding polypeptides or antigen-binding fragments thereof may comprise fibronectin. In some embodiments, the antigen targeted by the antigen-binding polypeptides or antigen-binding fragments thereof may comprise collagen-10.

[0387] 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 participates in various biological processes, such as, but not limited to, cell migration, cell adhesion, thrombosis and hemostasis, as well as wound healing, and participates in the establishment and maintenance of normal cell morphology (i.e., cell shape), development, and oncogenic transformation. The structural diversity of plasma and cellular fibronectin (FN) results from alternative splicing of three domains of the primary FN transcript, which can produce 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 dimer. Cellular FN is expressed by several mesenchymal cells as a dimer or cross-linked multimer and is deposited in the ECM as fibrils. Cellular FN is required for the deposition of fibrillin-1 and type I Col 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.

[0388] FN isoforms containing an 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, for example, SEQ ID NO: 84) that is part of a fibronectin isoform produced by alternative splicing. The sequence of EDB in mice and humans is identical. Isoforms containing EDB are expressed during embryonic and postnatal development and in a variety of solid tumors (and can be a marker for newly formed blood vessels), but are barely detectable in normal adult tissues, except in some blood vessels in the endometrium and ovaries during the proliferative phase. In particular, FN isoforms containing EDB have been shown to play a role in, for example, protein stability, proliferation, angiogenesis, inflammation, opsonization (phagocytosis), and cell attachment. In addition, EDB-FN can increase the proteolytic sensitivity of FN, suggesting that EDB can increase the turnover rate of ECM. The presence of EDB can also upregulate the expression of vascular endothelial growth factor (VEGF) and can be associated with enhanced angiogenesis and endothelial proliferation.

[0389] Microfibrils within the ECM are composed of fibrillin polymers that can associate with elastin, other glycoproteins, and growth factors, and FN is required for microfibril formation. Fibrillin microfibrils impart strength to tissues (see, e.g., Figure 28 ), and dysregulation of microfibril assembly may be associated with disease states. Mutations in the fibrillin glycoproteins encoded by FBN1, FBN2, and FBN3 can be associated with various connective tissue disorders, most notably FBN1 causing Marfan syndrome (MFS) (see, e.g., Figures 28-30 ). Mutations in the FBN1 gene that can, for example, cause MFS can 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 out of cells. Such mutations can result in a severe reduction in the amount of fibrillin-1 available for microfibril formation. Aortic root dilatation / dissection is one of the cardinal features of MFS (see, e.g., Figure 29-30 ).

[0390] In some embodiments, the antigen binding polypeptide or antigen binding fragment thereof may target an antigen that may be associated with MFS.

[0391] In some embodiments, the antigen is associated with an autoimmune disease or condition. Antigens associated with an autoimmune disease or condition can be derived from, for example, cell receptors and / or cells that produce "self" directed antibodies. In some embodiments, the antigen is associated with an autoimmune disease or condition, 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 ( syndrome), sarcoidosis, systemic lupus erythematosus, type 1 diabetes mellitus, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, myasthenia gravis, ankylosing spondylitis, scleroderma, polymyositis, or dermatomyositis.

[0392] Non-limiting examples of autoimmune antigens include platelet antigens, islet cell antigens, myelin protein antigens, rheumatoid factor, anti-citrullinated proteins, glucose-6-phosphate isomerase, receptors such as lipocortin 1, neutrophil nuclear proteins such as lactoferrin and 25 kD-35 kD nuclear protein, Sm antigens (e.g., in snRNPs), granule proteins such as bactericidal permeability increasing protein (BPI), elastase, fibrin, vimentin, filaggrin, fibrinogen, collagen I and II peptides, plasminogen, α-enolase, translation initiation factor 4G1, perinuclear factors, keratins, Sa (cytoskeletal vimentin), citrullinated proteins and peptides such as CCP-1, CCP-2 (cyclic citrullinated peptides), circulating serum proteins such as RF (IgG, IgM), components of articular cartilage such as collagen II, IX and XI, nuclear components such as RA33 / hnRNP A2), ferritin, stress proteins (such as HSP-65, HSP-70, HSP-90, BiP), inflammatory / immune factors (such as B7-H1, IL-1α and IL-8), enzymes (such as α-enolase), calpastatin, dipeptidyl peptidase, eukaryotic translation elongation factor 1α1 aldolase-A, osteopontin, cathepsin G, myeloperoxidase, proteinase 3 antigen, rheumatoid factor, histones, nucleic acids (such as RNA, dsDNA, ssDNA and ribosomes), ribosomal P protein, myelin proteins, cardiolipin, vimentin, Sm antigens (including, for example, SmD's and SmB' / B), U1 RNP, A2 / B1 hnRNP, Ro (SSA) and La (SSB) antigens.

[0393] In some embodiments, the antigen is an endogenous molecule of the subject.In some embodiments, the antigen is targeted by an immune response in an autoimmune disease disclosed herein.

[0394] In some embodiments, the antigen is associated with a disease associated with a TGFβ loss-of-function mutation.A non-limiting example of a disease associated with a TGFβ loss-of-function mutation is aortic aneurysm.

[0395] 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.

[0396] In some embodiments, the antigen binding polypeptide may be an antibody or an antigen binding fragment thereof.

[0397] Without wishing to be bound by theory, the antibodies disclosed herein may comprise, for example, immunoglobulin molecules (i.e., "complete 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), 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 amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FR of the antibody (or its antigen-binding fragment) may be identical to a human germline sequence, 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 comprise, for example, one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences.

[0398] Antibodies useful in the practice of the present disclosure may be full length (e.g., IgG1 or IgG4 antibodies) or may comprise only an antigen binding portion (e.g., Fab, F(ab')2, or scFv fragments), and may be modified to affect functionality, such as increasing persistence in the host or eliminating residual effector function. In certain embodiments, the antibodies may be bispecific.

[0399] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include any naturally occurring, enzymatically obtainable, 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, fragments containing CDRs, 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, for example, complete antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and (optional) constant domains. Such DNA is known and / or easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and chemically manipulated or manipulated using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains into a suitable configuration, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0400] 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 region of an antibody (e.g., isolated complementary determining regions (CDRs), such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules (such as 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 shark variable IgNAR domains) are also encompassed within the term "antigen-binding fragment" as used herein.

[0401] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain can be of any size or amino acid composition and typically comprises at least one CDR that is adjacent to or in frame with one or more framework sequences. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH domain and the VL domain can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain a VH-VH, VH-VL, or VL-VL dimer. Alternatively, an antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0402] In certain embodiments, the antigen-binding fragment of an antibody may contain 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 within the 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 connected to each other, or may be connected by a complete or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which form a flexible or semi-flexible linker between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragments of the antibodies of the present disclosure may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above that are non-covalently associated with each other and / or with one or more monomeric VH or VL domains (e.g., via disulfide bonds).

[0403] As with complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Using conventional techniques available in the art, any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use with antigen-binding fragments of the antibodies of the present disclosure.

[0404] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise a heavy chain variable region (HCVR). In some embodiments, the antibodies or antigen-binding fragments thereof comprise a light chain variable region (LCVR). In some embodiments, the antibodies or antigen-binding fragments thereof comprise 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) may be an IgG1 domain. In some embodiments, the immunoglobulin heavy chain constant domain (CH) may be an IgG4 domain. In some embodiments, the antibodies or antigen-binding fragments thereof comprise an immunoglobulin light chain constant domain (CL).

[0405] In some embodiments, the antibody or its antigen-binding fragment of the present disclosure may include a heavy chain constant region, which includes one or more amino acid changes in the hinge region. In some embodiments, the amino acid changes in the hinge region can reduce the combination with Fc γ receptors. Examples of such modifications are disclosed in US2018 / 0282411, the content of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the antigen-binding polypeptide includes modifications made in amino acid positions 233-236 (by EU numbering) by replacing naturally occurring residues and / or deletions with glycine. In some embodiments, each of the amino acid positions 233-236 numbered by EU is occupied or unoccupied by G, such as GGG-(233-236), GG--(233-236), G---(233-236) or ----(233-236), wherein "-" represents an unoccupied position. In some embodiments, the heavy chain constant region comprising modification is a human IgG1 isotype. In some embodiments, the heavy chain constant region comprising the modification is a human IgG4 isotype. In some embodiments, the heavy chain constant region comprising the modification is a hybrid in which the domains are of different isotypes, such as a hybrid of IgG1 and IgG4 isotypes, in which one or more domains (e.g., CHI, CH2, or CH3 domains) and / or the hinge region are of one isotype, while the remaining domains are of different isotypes.

[0406] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise an anti-CD63 antibody or fragment or derivative thereof.

[0407] 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.

[0408] 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.

[0409] In some embodiments, the anti-CD63 antibody CHIgG4 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 CHIgG4 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.

[0410] In some embodiments, the anti-CD63 antibody CHIgG4 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 CHIgG4 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.

[0411] In some embodiments, the anti-CD63 antibody CHIgG4 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 CHIgG4 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.

[0412] 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.

[0413] 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.

[0414] 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.

[0415] 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.

[0416] 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.

[0417] 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.

[0418] 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.

[0419] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise anti-EDB-FN antibodies or fragments or derivatives thereof.

[0420] 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, the nucleotide sequence encoding the 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, the anti-EDB-FN antibody HCVR comprises the amino acid sequence of SEQ ID NO: 100. In certain embodiments, the nucleotide sequence encoding the anti-EDB-FN antibody HCVR comprises the nucleotide sequence of SEQ ID NO: 101.

[0421] 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.

[0422] In some embodiments, the anti-EDB-FN antibody CHIgG4 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 CH IgG4 domain of an anti-EDB-FN antibody 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.

[0423] In some embodiments, the anti-EDB-FN antibody CHIgG4 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 CH IgG4 domain of an anti-EDB-FN antibody 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.

[0424] In some embodiments, the anti-EDB-FN antibody CHIgG4 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 CH IgG4 domain of an anti-EDB-FN antibody 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.

[0425] 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.

[0426] 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.

[0427] 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.

[0428] 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.

[0429] 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.

[0430] 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.

[0431] 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.

[0432] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise anti-Epcam antibodies or fragments or derivatives thereof.

[0433] 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.

[0434] 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, the nucleotide sequence encoding the 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, the anti-Epcam antibody LCVR comprises the amino acid sequence of SEQ ID NO: 129. In certain embodiments, the nucleotide sequence encoding the anti-Epcam antibody LCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 129.

[0435] 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.

[0436] 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 anti-Epcam CL comprises a nucleotide sequence encoding 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 anti-Epcam antibody CL comprises the nucleotide sequence of SEQ ID NO: 133.

[0437] 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.

[0438] 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.

[0439] 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.

[0440] 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.

[0441] 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.

[0442] 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.

[0443] In some embodiments, the antibodies or antigen-binding fragments thereof disclosed herein comprise anti-mClec9a antibodies or fragments or derivatives thereof.

[0444] 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 mClec9a antibody HCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 138.

[0445] 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 mClec9a antibody LCVR comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 142.

[0446] 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.

[0447] 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 a nucleotide sequence encoding 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.

[0448] 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.

[0449] 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.

[0450] 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.

[0451] 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.

[0452] 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.

[0453] 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.

[0454] Fusion peptide

[0455] In certain aspects, the present disclosure provides a fusion polypeptide comprising:

[0456] a) a target binding polypeptide that binds to a molecule on a target cell or in the extracellular matrix (ECM);

[0457] b) latency-associated polypeptide (LAP) or a fragment or derivative thereof; and

[0458] c) Mature transforming growth factor beta (TGFβ) family polypeptides or fragments or derivatives thereof.

[0459] In some embodiments, a mature TGFβ family polypeptide, or a fragment or derivative thereof, can be inactivated due to its interaction with LAP, or a fragment or derivative thereof.

[0460] In some embodiments, a mature TGFβ family polypeptide, or a fragment or derivative thereof, can bind to a transforming growth factor beta receptor (TGFβR) when the mature TGFβ family polypeptide, or a fragment or derivative thereof, is dissociated from or released from LAP, or a fragment or derivative thereof. In some embodiments, a mature TGFβ family polypeptide, or a fragment or derivative thereof, can induce Smad2 / 3 signaling in a target cell or a cell adjacent to a target cell when the mature TGFβ family polypeptide, or a fragment or derivative thereof, is dissociated from or released from LAP, or a fragment or derivative thereof.

[0461] In some embodiments, the fusion polypeptide may comprise any of the various linkers described herein. By way of example and not limitation, 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 as 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.

[0462] In some embodiments, the linker can comprise the sequence (GGGGS)n (SEQ ID NO: 46), wherein 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); GGGGSGGGGSGGGGS ((G4S)3; SEQ ID NO: 49); or GGGGSGGGGSGGGGSGGGGS ((G4S)4; SEQ ID NO: 50).

[0463] In some embodiments, the linker may comprise the sequence (GGGS)n (SEQ ID NO: 51), wherein 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).

[0464] 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).

[0465] In some embodiments, a linker may be positioned between the target binding polypeptide and the LAP or a fragment or derivative thereof.

[0466] 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 described herein, or a fragment or derivative thereof, and (iv) a mature TGFβ family polypeptide described herein, or a fragment or derivative thereof.

[0467] 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 described herein, or a fragment or derivative thereof, (iii) a linker described herein, and (iv) a target binding polypeptide described herein.

[0468] In some embodiments, mature TGFβ family polypeptide or its fragment or derivative and LAP or its fragment or derivative can be separated by a protease cleavage site. The protease cleavage site can be any one of the various protease cleavage sites of the present disclosure, such as but not limited to the furin cleavage site. In some embodiments, the furin cleavage site can comprise the sequence RXXR (SEQ ID NO: 35). In some embodiments, the protease cleavage site can be the 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).

[0469] In various embodiments, the fusion polypeptide of the present disclosure may further include a signal peptide, for example, at the N-terminus of the fusion polypeptide. Without wishing to be bound by theory, the signal peptide may include a leader sequence at the amino terminus (N-terminus) of a nascent polypeptide (e.g., a fusion polypeptide described herein) that directs the nascent protein to the endoplasmic reticulum and / or subsequent surface expression or secretion in a co-translational or post-translational manner. Any of the various signal peptides known in the art, or fragments, derivatives, or combinations thereof, may be used in the practice of the present disclosure, for example, any of those described in signalpeptide.com / index.php?m=listspdb_mammalia, incorporated by reference for all intended purposes.

[0470] In some embodiments, the signal peptide may comprise an mROR signal peptide. In some embodiments, the signal peptide is an mROR signal peptide.

[0471] 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.

[0472] 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).

[0473] In some embodiments, a mature TGFβ family polypeptide, or a fragment or derivative thereof, induces signal transduction via a Smad-dependent classical signal transduction pathway in a target cell or a cell adjacent to a target cell upon activation of a mature TGFβ family polypeptide. In some embodiments, a mature TGFβ family polypeptide, or a fragment or derivative thereof, induces Smad2 / 3 signal transduction in a target cell or a cell adjacent to a target cell upon activation of a mature TGFβ family polypeptide. In some embodiments, a mature TGFβ family polypeptide, or a fragment or derivative thereof, induces signal transduction via a non-classical signal transduction pathway disclosed herein in a target cell or a cell adjacent to a target cell upon activation of a mature TGFβ family polypeptide. Activation of a mature TGFβ family polypeptide can occur by any mechanism disclosed herein.

[0474] In some embodiments, the TGFβ family polypeptides or fragments or derivatives thereof described herein may be self-activating TGFβ family polypeptides, i.e., TGFβ family polypeptides that do not require activation (such as by proteolysis or mechanical activation) to induce TGFβ signaling (e.g., Smad2 / 3 signaling and / or ERK kinase signaling, etc.). In some embodiments, the TGFβ family polypeptides or fragments or derivatives thereof may comprise one or more mutations to produce a self-activating form of TGF-β. The one or more mutations that produce a self-activating form of TGF-β may comprise any of the various mutations described herein. As a non-limiting example, the TGF-β family polypeptides described herein, such as TGF-β1 polypeptides, may comprise one or more cysteine ​​residues in the pro-region (e.g., LAP) of the TGF-β precursor, which have been substituted with one or more serine residues in the serine (S) residue. In certain embodiments, the TGF-β family polypeptide may comprise a C223S mutation, and position 223 is related to the sequence of SEQ ID NO: 82. In certain embodiments, the TGF-β family polypeptide may comprise a C225S mutation, and position 225 is related to the sequence of SEQ ID NO: 82. In certain embodiments, the TGF-β family polypeptide may comprise a C223S and / or C225S mutation, thereby rendering the TGF-β family polypeptide described herein self-activating.

[0475] 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.

[0476] In some embodiments, LAP or a fragment or derivative thereof can interact with an environmental molecule disclosed herein. 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).

[0477] In some embodiments, the fusion polypeptide disclosed herein may comprise a target binding polypeptide comprising an antigen-binding polypeptide or antigen-binding fragment thereof disclosed herein. The antigen that can be targeted by the antigen-binding polypeptide or antigen-binding fragment disclosed herein can be any of the various antigens disclosed herein, such as, 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.

[0478] In some embodiments, the target binding polypeptide is not internalized.

[0479] In some embodiments, the target binding peptide is capable of internalization.

[0480] 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 IgG1 domains and IgG4 domains.

[0481] In some embodiments, a mature TGFβ family polypeptide may be a mature TGFβ polypeptide disclosed herein. In some embodiments, a mature TGFβ family polypeptide may be a fragment of a mature TGFβ polypeptide disclosed herein or a derivative thereof. In some embodiments, a mature TGFβ polypeptide may be a fragment of a mature TGFβ1 polypeptide disclosed herein or a derivative thereof. In some embodiments, a mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO: 23. In some embodiments, a mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO: 23. In some embodiments, a mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO: 90. In some embodiments, a mature TGFβ1 polypeptide consists of the sequence of SEQ ID NO: 90. In some embodiments, a mature TGFβ2 polypeptide may be a fragment of a mature TGFβ2 polypeptide disclosed herein or a derivative thereof. In some embodiments, a mature TGFβ2 polypeptide comprises the sequence of SEQ ID NO: 27. In some embodiments, a mature TGFβ2 polypeptide consists of the sequence of SEQ ID NO: 27.

[0482] 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).

[0483] In some embodiments, LAP or its fragment or derivative can be any of the various LAP or its fragment or derivative described herein. In some embodiments, LAP can include the sequence of SEQ ID NO:29. In some embodiments, LAP can consist of the sequence of SEQ ID NO:29. In some embodiments, LAP can include the sequence of positions 30-274 of the sequence of SEQ ID NO:82. In some embodiments, LAP can consist of the sequence of positions 30-274 of the sequence of SEQ ID NO:82. In some embodiments, LAP can include the sequence of SEQ ID NO:118. In some embodiments, LAP can consist of the sequence of SEQ ID NO:118. In some embodiments, LAP can include the sequence of positions 21-298 of the sequence of SEQ ID NO:116. In some embodiments, LAP can consist of the sequence of positions 21-298 of the sequence of SEQ ID NO:116. In some embodiments, LAP can include the sequence of SEQ ID NO:31. In some embodiments, LAP can consist of the sequence of SEQ ID NO:31. In some embodiments, the LAP may 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 or a fragment or derivative thereof may be heterologous to a mature TGFβ family polypeptide or a fragment or derivative thereof.

[0484] In some embodiments, LAP or a fragment or derivative thereof may comprise one or more mutations. The one or more mutations in LAP may comprise any of the various mutations described herein, such as a C24S mutation, with position 24 associated with SEQ ID NO: 116; a C33S mutation, with position 33 associated with the sequence of SEQ ID NO: 82; a C223S mutation, with position 223 associated with the sequence of SEQ ID NO: 82; and a C225S mutation, with position 225 associated with 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 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 its fragment or derivative comprises one or more mutations that eliminate one or more protease cleavage sites within LAP or its fragment or derivative. In some embodiments, LAP or its fragment or derivative comprises one or more mutations that reduce the binding of LAP to a potentially related binding protein (LTBP).

[0485] In various embodiments, the LAPs described herein, or fragments or derivatives thereof, may comprise one or more mutations described herein. In some embodiments, the one or more mutations may allow for proteolytic activation of a mature TGFβ family polypeptide described herein, or fragments or derivatives thereof. In some embodiments, the one or more mutations may allow for mechanical activation of a mature TGFβ family polypeptide described herein, or fragments or derivatives thereof.

[0486] In various embodiments, LAP or a fragment or derivative thereof comprises one or more mutations that render the TGFβ family polypeptides described herein self-activating. Self-activating forms of TGFβ family polypeptides do not require activation (such as by proteolysis or mechanical activation) to induce TGFβ signaling (e.g., Smad2 / 3 signaling and / or ERK signaling, etc.).

[0487] In some embodiments, LAP or a fragment or derivative thereof can comprise one or more mutations that can, for example, eliminate one or more protease cleavage sites within LAP or a fragment or derivative thereof. In some embodiments, one or more mutations can, for example, reduce the binding of LAP or a fragment or derivative thereof to latency-associated binding protein (LTBP).

[0488] In some embodiments, LAP or a fragment or derivative thereof can comprise one or more mutations that can, for example, introduce one or more protease cleavage sites into LAP or a fragment or derivative thereof, such as via insertion of a protease cleavage site described herein. In some embodiments, the insertion can comprise a PLGL insertion, such as one that can be used for MMP2 cleavage. In some embodiments, the insertion can comprise a PLGI insertion, such as one that can be used for MMP3, MMP7, and / or MMP8 cleavage.

[0489] In some embodiments, LAP or a fragment or derivative thereof comprises an amino acid sequence having at least 80% sequence identity to the sequence at positions 30-274 of the sequence of SEQ ID NO: 82. In some embodiments, LAP or a fragment or derivative thereof comprises the sequence at positions 30-274 of the sequence of SEQ ID NO: 82. In some embodiments, LAP consists of the sequence at positions 30-274 of the sequence of SEQ ID NO: 82.

[0490] 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.

[0491] In some embodiments, LAP or a fragment or derivative thereof comprises an amino acid sequence having at least 80% sequence identity to the sequence of positions 21-298 of the sequence of SEQ ID NO: 116. In some embodiments, LAP or a fragment or derivative thereof comprises the sequence of positions 21-298 of the sequence of SEQ ID NO: 116. In some embodiments, LAP consists of the sequence of positions 21-298 of the sequence of SEQ ID NO: 116.

[0492] 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.

[0493] 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.

[0494] 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.

[0495] 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.

[0496] 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.

[0497] Non-limiting examples of fusion polypeptide sequences

[0498] 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.

[0499] 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.

[0500] 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.

[0501] 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.

[0502] 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.

[0503] 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.

[0504] 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.

[0505] 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.

[0506] 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.

[0507] 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.

[0508] 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.

[0509] 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.

[0510] Polynucleotides and vectors

[0511] 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.

[0512] In certain embodiments, the polynucleotide encoding the polypeptide disclosed herein may comprise one or more regulatory elements. Regulatory elements may be capable of regulating the expression of the polypeptide. Non-limiting examples of regulatory elements are promoters, initiation sites, polyadenylation (polyA) tails, IRES elements, enhancers, response elements, and termination signals.

[0513] In some embodiments, the polynucleotide sequence encoding a polypeptide as described herein (for example, a fusion polypeptide) can be operably connected to a promoter for expression. In some embodiments, when the sequence encoding a polypeptide as described herein is operably connected to a promoter, the promoter can mediate the expression of the polypeptide." promoter " is a regulatory region of DNA, which generally comprises a TATA box capable of guiding RNA polymerase II to initiate RNA synthesis at the appropriate transcription start site of a specific polynucleotide sequence. The promoter may also include other regions that affect transcription initiation rate. As used herein, the term "promoter" encompasses enhancers. Promoter sequences disclosed herein regulate the transcription of operably connected polynucleotides. Promoters can be active in one or more cell types in cell types disclosed herein (for example, eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, single-cell embryos, differentiated cells, or a combination thereof). Promoters may be, for example, constitutively active promoters, conditional promoters, inducible promoters, time-limited promoters (for example, promoters regulated by development) or spatially limited promoters (for example, cell-specific or tissue-specific promoters).

[0514] Examples of constitutive promoters include, but are not limited to, cytomegalovirus (CMV) promoter, EF1a, SV40, PGK1 (human or mouse), Ubc, human beta actin, CAG, Ac5, polyhedrin, TEF1, GDS, CaMV35S, Ubi, H1, and U6 promoters.

[0515] In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a CMV / EF1 hybrid promoter.

[0516] Inducible promoters can include, for example, chemically regulated promoters and physically regulated promoters. Chemically regulated promoters include, for example, alcohol regulated promoters (for example, alcohol dehydrogenase (alcA) gene promoter), tetracycline regulated promoters (for example, tetracycline responsive promoter, tetracycline operator sequence (tetO), tet-On promoter or tet-Off promoter), steroid regulated promoters (for example, rat glucocorticoid receptor, estrogen receptor promoter or ecdysone receptor promoter) or metal regulated promoters (for example, metalloprotein promoter). Physically regulated promoters include, for example, temperature regulated promoters (for example, heat shock promoters, such as promoters derived from Hsp70 and Hsp90) and light regulated promoters (for example, light induced promoters or light repressible promoters). Other inducible promoters include lac, sp6 and T7 promoters.

[0517] 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).

[0518] Developmentally regulated promoters include, for example, promoters that are active only during embryonic development or only in adult cells.

[0519] Other non-limiting examples of promoters that can be used in the nucleic acid molecules of the present disclosure include the CB7 / CAG promoter and associated upstream regulatory sequences, the EF-1α promoter, the mU1a promoter, the UB6 promoter, the chicken β-actin (CBA) promoter, and 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 the hypoxia-inducible promoter or the rapamycin-inducible promoter), or combinations thereof.

[0520] In some embodiments, the nucleic acid molecules of the present disclosure may comprise one promoter.In some embodiments, the nucleic acid molecules of the present disclosure may comprise more than one (eg, 2, 3, 4, or more) promoters.

[0521] In another aspect, the present disclosure provides a vector comprising any of the above-mentioned polynucleotides. Such vectors may comprise a polynucleotide encoding a polypeptide disclosed above. The vector may be a viral vector or a non-viral vector.

[0522] In some embodiments, the vector can be a viral vector. Non-limiting examples of viral vectors include adenovirus, adeno-associated virus (AAV, such as AAV8, AAV9, AAVrh10, AAVS3), lentivirus, helper virus-dependent adenovirus, herpes simplex virus, poxvirus, hemagglutinin virus of Japan (HVJ), alphavirus (e.g., Semliki forest virus (SFV), Sindbis virus (SIN)), vaccinia virus, baculovirus vectors and retroviral vectors (e.g., murine leukemia virus (MLV), human immunodeficiency virus (HIV)).

[0523] In some embodiments, the viral vectors described herein are recombinant viral vectors. In some embodiments, the viral vectors described herein are altered so that they are replication-defective in humans. In some embodiments, the viral vector is a hybrid vector, such as an AAV vector placed in a "helpless" 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 a VSV-G protein from vesicular stomatitis virus (VSV).

[0524] 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 synthesizing capsid proteins) (rep and cap proteins can be provided in trans by packaging cells). A variety of AAV serotypes have been identified. In some embodiments, the AAV-based vectors described herein comprise capsid components from one or more of: 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.Anc80L In some embodiments, the AAV-based vectors provided herein comprise components from one or more AAV serotypes. 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, neurons).

[0525] 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: long terminal repeats, primer binding sites, polypurine regions, att sites, and encapsidation sites.

[0526] In some embodiments, the viral vectors described herein are HIV-based viral vectors. In some embodiments, the HIV-based vectors described herein comprise at least two polynucleotides, wherein the gag and pol genes are from the HIV genome, and the env gene is from another virus.

[0527] 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 so that they do not contain one or more immediate early (IE) genes, thereby rendering them non-cytotoxic.

[0528] In some embodiments, the viral vectors provided herein are MLV-based viral vectors.In some embodiments, the MLV-based vectors provided herein comprise up to 8 kb of heterologous DNA in place of viral genes.

[0529] In some embodiments, the viral vectors provided herein are alphavirus-based viral vectors. In some embodiments, the alphavirus vectors provided herein are recombinant, replication-deficient alphaviruses. In some embodiments, the alphavirus replicons in the alphavirus vectors provided herein are targeted to specific cell types by displaying functional heterologous ligands on the surface of their virions.

[0530] In some embodiments, the vector can 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.

[0531] Cells and production methods

[0532] In one aspect, the present disclosure provides a cell comprising a polynucleotide and / or a recombinant vector as described herein, such as a host cell. In some embodiments, the polynucleotide can encode a fusion polypeptide as described herein, for example. In some embodiments, the vector can comprise a polynucleotide as described herein. The term "host cell" refers to any cell comprising a heterologous nucleic acid. As a non-limiting example, a heterologous nucleic acid can be a vector disclosed herein. A host cell (for example, but not limited to) can be a cell from any organism that is used, manipulated, modified, selected, transformed, or grown for producing a substance by a cell, for example, by expressing an RNA or DNA sequence, gene, protein, or enzyme by a cell. Suitable hosts can be determined.

[0533] 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 to replicate several types of vectors. Bacterial cells, including, 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 mammals, insects 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.

[0534] Packaging cells that can be used to produce the polynucleotides and / or recombinant vectors described herein include, for example, animal cells that are permissive for vectors (e.g., viral vectors), or cells modified to be permissive for vectors; or packaging cell constructs, for example, using transforming agents such as calcium phosphate. Non-limiting examples of packaging cell lines that can be used 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, human sarcoma cell line HT-1080 (CCL-121), lymphoblastoid cell line Raji (CCL-86), glioblastoma-astrocytoma epithelioid 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, CAP-T cells, etc.

[0535] Other non-limiting examples of packaging cells and / or systems that can be used in the production methods described herein include, for example, L929 cells, the FLY virus packaging cell system outlined in Cosset et al. (1995) J Virol 69, 7430-7436, NSO (murine 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).

[0536] Other packaging cells and systems, packaging techniques and vectors for packaging nucleic acid genomes into vectors can include method steps, including, for example, constructing a structural protein expression cassette comprising a plasmid for inducible expression of a vector for viral structural proteins, and amplifying by polymerase chain reaction (PCR) or by incorporating any other elements using synthetic oligonucleotides. As non-limiting examples, for the selection, screening and / or characterization of packaging cell lines, cells transfected with expression cassette constructs can be selected using, for example, G418 or hygromycin. The drug-resistant cell foci merged can be cloned by limiting dilution, and the packaging activity of individual clones can be screened, for example, by using, for example, lipofection or electroporation vector transfection. Those clones with the highest activity levels can be amplified for further use. Northern and Western blot analysis can be performed on the vector-specific or structural protein-specific RNA and protein expressed in packaging cells. The titer of the vector particles without replication ability in the packaging cell line culture supernatant can be determined, for example, by infecting naive monolayer cells, X-gal staining with serial dilutions and counting the total number of dyed cells per well at an appropriate dilution. Vector titers can be specified as infectious units (IU) / ml. Contaminating replication-competent virus can be detected in culture supernatants by standard plaque assays (plaque forming units or PFU / ml) and by serial undiluted passage in naive cells. Packaging methods include the use of packaging cells that permanently express viral components or by transient transfection of cells with plasmids.

[0537] In some embodiments, the present disclosure provides a cell (e.g., a FreedomCHO-S cell or an ExpiCHO cell) comprising a polypeptide complex or fusion polypeptide disclosed herein. In a related aspect, the present disclosure provides a cell comprising a polynucleotide disclosed herein. In another related aspect, the present disclosure provides a cell comprising a vector disclosed herein. By way of example and not limitation, any of the above cells may comprise a polypeptide complex, fusion polypeptide, polynucleotide, and / or vector disclosed herein. In some embodiments, the cell may be a FreedomCHO-S cell.

[0538] In some embodiments, the present disclosure provides a method for preparing a polypeptide complex or fusion polypeptide disclosed herein. The method may include incubating a cell comprising a polynucleotide disclosed herein and / or a vector disclosed herein under conditions that allow production of the polypeptide complex or fusion polypeptide.

[0539] Isolation or purification of a polypeptide complex or fusion polypeptide disclosed herein from, for example, a virus or viral extract can include, but is not limited to, techniques and / or method steps comprising any of the following: freeze / thaw cycles, 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 a crowding agent), cross-flow filtration, affinity purification, nanoscale flow cytometry, CsCl density gradients, iodixanol gradient centrifugation, chromatography (e.g., column chromatography, including the use of various resins such as, but not limited to, ion exchange, anion exchange and cation exchange, affinity (including antibody affinity), chromate focusing, desalting and buffer exchange, hydrophobic interaction, immunoprecipitation, multimodal, mixed modal, reverse phase and size exclusion, heparinized support matrix chromatography, DEAE Sepharose Fast Flow (FF), POROS 50 D, EMD DEAE(M), Macro-Prep DEAE Support, DEAE Ceramic 20 and Toyopearl DEAE-650M, and / or analytical chromatography using various media, such as ceramic hydroxyapatite, ceramic fluorapatite, ceramic hydroxyfluorapatite, cellulose sulfate media, including, for example, analytical ion exchange high performance liquid chromatography (HPLC), reverse phase HPLC, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis), or combinations thereof.

[0540] In some embodiments, the method of preparing a polypeptide complex or fusion polypeptide can include collecting cell culture medium and isolating the produced polypeptide complex or fusion polypeptide by a method including affinity chromatography. In some embodiments, the affinity chromatography can include, for example, a protein A column or beads or a protein G column or beads.

[0541] Pharmaceutical composition

[0542] In another aspect, the present disclosure provides a composition (e.g., a pharmaceutical composition) comprising a polypeptide complex, fusion polypeptide, polynucleotide, vector and / or cell disclosed herein, and, for example, a pharmaceutically acceptable carrier and / or diluent. The pharmaceutical composition of the present disclosure can be in any suitable form, depending on the desired method of administration to a subject.

[0543] The pharmaceutical composition may comprise a polypeptide complex or a molecule based on the polypeptide complex and / or a fusion polypeptide or a molecule based on the fusion polypeptide of the present disclosure in free form or in the form of a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of the disclosed polypeptide complex or fusion polypeptide, wherein the polypeptide or its complex is modified by preparing an acid salt or basic salt of a reagent. For example, an acid salt is prepared from a free base (typically wherein the neutral form of the drug has a neutral -NH2 group) and involves reacting with a suitable acid. Suitable acids for preparing acid salts include 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, and the like, as well as inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. 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.

[0544] The compositions of the present disclosure can comprise a plurality of polypeptide complexes and / or fusion polypeptides, for example, 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 as described herein. In some embodiments, the compositions of the present disclosure can comprise 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.

[0545] In some embodiments, the polypeptide complexes or polypeptide complex-based molecules and / or fusion polypeptides or fusion polypeptide-based molecules described herein can be administered at a concentration of about 1 μg / mL to 50 mg / mL, such as 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. , 0.7 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.

[0546] The pharmaceutical composition may be adapted for administration by any appropriate route, such as parenteral (including intraperitoneal, subcutaneous, intramuscular or intravenous), enteral (including oral or rectal), inhalation or intranasal.

[0547] Such compositions can be prepared, for example, by admixing the active ingredient with the carrier or excipient under sterile conditions.

[0548] Furthermore, disclosed herein are pharmaceutical dosage forms comprising polypeptide complexes or polypeptide complex-based molecules and / or fusion polypeptides or fusion polypeptide-based molecules, or conjugates comprising polypeptide complexes or polypeptide complex-based molecules and / or fusion polypeptides or fusion polypeptide-based molecules, nucleic acid molecules, vectors, cells, or target binding polypeptides of the present disclosure.

[0549] Pharmaceutical compositions based on polypeptide complexes or polypeptide complex-based molecules and / or fusion polypeptides or fusion polypeptide-based molecules, or conjugates comprising 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 disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. Polypeptide complexes or polypeptide complex-based molecules and / or fusion polypeptides or fusion polypeptide-based molecules, or conjugates comprising 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 by, for example, injection, inhalation or isolation (through the mouth or nose) or by oral, buccal, parenteral or rectal administration, or by direct administration to an organ or tissue.

[0550] The pharmaceutical composition can be formulated for a variety of modes of administration, including systemic, topical, or local 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 the purpose of injection, the pharmaceutical composition can be formulated in a liquid solution, preferably in a physiologically compatible buffer (such as Hank's solution or Ringer's solution). In addition, the pharmaceutical composition can be formulated in a solid form and redissolved or suspended immediately before use. Lyophilized forms of the pharmaceutical composition are also suitable.

[0551] In some embodiments, the pharmaceutical composition of the present disclosure can be lyophilized. As a non-limiting example, the lyophilized material obtained can be reconstituted into an aqueous composition by adding an aqueous solvent. In some embodiments, the aqueous composition can be directly administered to a patient parenterally. Therefore, in another embodiment of the present disclosure, the pharmaceutical composition can be an aqueous pharmaceutical composition, which can be obtained by reconstitution of the lyophilized material with an aqueous solvent.

[0552] In some embodiments, the pharmaceutical compositions disclosed herein may comprise a lyophilized formulation. As non-limiting examples, 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 As another non-limiting example, a lyophilized formulation may comprise a polypeptide complex or polypeptide complex-based molecule and / or fusion polypeptide or fusion polypeptide-based molecule disclosed herein, mannitol, and poloxamer 188. In some embodiments, a pharmaceutical composition may comprise a lyophilized formulation containing a reconstituted liquid composition.

[0553] In some embodiments, the pharmaceutical composition of the present disclosure can provide a formulation with enhanced solubility and / or moistening of a lyophilized product compared to previously known compositions. As a non-limiting example, an appropriate excipient composition can be used to achieve enhanced solubility and / or moistening of a lyophilized product. In this way, a pharmaceutical composition of the present disclosure comprising a polypeptide complex or a molecule based on a polypeptide complex and / or a fusion polypeptide or a molecule based on a fusion polypeptide or its variant can be developed to show desired storage stability at (e.g., -20°C, +5°C or +25°C) and can be easily redissolved so that the lyophilized product can be completely dissolved from a few seconds to two or more minutes using a buffer or other excipients with or without an ultrasonic homogenizer. In addition, the composition can be easily provided to a patient in need of treatment via any appropriate 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.

[0554] For oral administration, the pharmaceutical composition can take the form of, for example, tablets or capsules, which are prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can also be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be present as dry products for reconstitution with water or other suitable vehicles before use. Such liquid preparations can be prepared in a conventional manner with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); Emulsifiers (e.g., lecithin or gum arabic); Non-aqueous vehicles (e.g., ationd oil, oily esters, ethanol or fractionated vegetable oils); and preservatives (e.g., methylparaben or propylparaben or sorbic acid). The preparations may also contain buffer salts, flavorings, coloring agents and sweeteners as appropriate.

[0555] The pharmaceutical composition can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable formulations can be in unit dosage form, for example, in ampoules or in multidose containers with the optional addition of preservatives. The pharmaceutical composi...

Claims

1. A polypeptide complex, comprising: a. a target binding polypeptide that binds to a molecule on a target cell or a molecule in the extracellular matrix (ECM); and b. A small latent complex (SLC), comprising: i. a dimer 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, wherein the mature TGFβ family polypeptide or a fragment or derivative thereof is inactivated due to interaction with the dimeric LAP or a fragment or derivative thereof.

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

3. The polypeptide complex according to claim 2, wherein the LAP or the fragment or derivative thereof is covalently attached to the target binding polypeptide via a linker. The polypeptide complex according to 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). The polypeptide complex according to 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). The polypeptide complex according to claim 3 , wherein the linker comprises the sequence GGGSGGGSGGGS (SEQ ID NO: 19). The polypeptide complex according to claim 6 , wherein the linker consists of the sequence GGGSGGGSGGGS (SEQ ID NO: 19). The polypeptide complex according to claim 3 , wherein the linker comprises the sequence GSGESGGGSG (SEQ ID NO: 96). The polypeptide complex according to claim 8 , wherein the linker consists of the sequence GSGESGGGSG (SEQ ID NO: 96).

10. The polypeptide complex of claim 7 or claim 9, wherein the target binding polypeptide binds to both the LAP or the fragment or derivative thereof and the molecule on the target cell or the molecule in the ECM. The polypeptide complex according to claim 10 , wherein the target binding polypeptide is an antibody or a fragment or derivative thereof. 12 . The polypeptide complex according to claim 1 , wherein the mature TGFβ family polypeptide or the fragment or derivative thereof and the LAP or the fragment or derivative thereof are associated via non-covalent interactions. 13 . The polypeptide complex according to claim 1 , wherein the mature TGFβ family polypeptide or the fragment or derivative thereof and the LAP or the fragment or derivative thereof are separated by a protease cleavage site. The polypeptide complex according to claim 13 , wherein the protease cleavage site is a furin cleavage site. The polypeptide complex according to claim 14 , wherein the furin cleavage site comprises the sequence RXXR (SEQ ID NO: 35). The polypeptide complex according to claim 15 , wherein the furin cleavage site consists of the sequence RXXR (SEQ ID NO: 35). The polypeptide complex according to 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. The polypeptide complex according to any one of claims 1 to 17, wherein the mature TGFβ family polypeptide or the fragment or derivative thereof binds to the transforming growth factor β receptor (TGFβR) when the mature TGFβ family polypeptide or the fragment or derivative thereof is released from the SLC.

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

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

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

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

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. The polypeptide complex of claim 24 , wherein the antigen-binding polypeptide is an antibody or an antigen-binding fragment thereof. The polypeptide complex of claim 25 , wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region.

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

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

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

32. The polypeptide complex according to any one of claims 25 to 30, wherein the antigen-binding polypeptide or the antigen-binding fragment thereof binds to the Extra Domain B of Fibronectin (EDB-FN).

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

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

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. The polypeptide complex according to claim 36 , wherein the mature TGFβ polypeptide is a mature TGFβ1 polypeptide. The polypeptide complex according to claim 37 , wherein the mature TGFβ1 polypeptide comprises the sequence of SEQ ID NO:

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

23.

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

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

90.

42. The polypeptide complex of claim 36, wherein the mature TGFβ polypeptide is a mature TGFβ2 polypeptide. The polypeptide complex according to claim 42 , wherein the mature TGFβ2 polypeptide comprises the sequence of SEQ ID NO:

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

27.

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

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

82.

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

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

116.

50. The polypeptide complex according to 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. The polypeptide complex according to any one of claims 1 to 50, 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.

52. The polypeptide complex according to any one of claims 1 to 51, wherein 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.

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

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, wherein the one or more mutations reduce the binding of the LAP or a fragment or derivative thereof to latency-associated binding protein (LTBP).

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

82.

56. The polypeptide complex of claim 54, wherein the one or more mutations comprise a C24S mutation, wherein position 24 corresponds to SEQ ID NO:

116.

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

94.

61. The polypeptide complex according to any one of claims 1 to 60, wherein the mature TGFβ family polypeptide or fragment or derivative thereof is chemically dissociated from the LAP or fragment or derivative thereof and released from the SLC in an active form. The polypeptide complex according to claim 61 , wherein the chemical dissociation comprises protease treatment, temperature treatment, acid treatment, or any combination thereof.

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

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

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

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

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

68. The fusion polypeptide of claim 67, wherein the mature TGFβ family polypeptide, or fragment or derivative thereof, is inactivated due to interaction with the LAP, or fragment or derivative thereof.

69. The fusion polypeptide of claim 67 or claim 68, wherein the mature TGFβ family polypeptide, or fragment or derivative thereof, binds to the transforming growth factor β receptor (TGFβR) upon release of the mature TGFβ family polypeptide, or fragment or derivative thereof, from the LAP, or fragment or derivative thereof.

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 upon release of the mature TGFβ family polypeptide, or fragment or derivative thereof from the LAP, or fragment or derivative thereof.

71. The fusion polypeptide of any one of claims 67 to 70, wherein the fusion polypeptide comprises a linker.

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

73. The fusion polypeptide of claim 72, wherein the fusion polypeptide comprises, 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. The fusion polypeptide of claim 72, wherein the fusion polypeptide comprises, 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. 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. 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. The fusion polypeptide of claim 75, wherein the linker comprises the sequence GGGSGGGSGGGS (SEQ ID NO: 19).

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

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

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

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

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

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

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

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

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

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

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

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 fragment or derivative thereof does not comprise an integrin binding motif.

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

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

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

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

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. The fusion polypeptide of any one of claims 94 to 100, wherein the antigen binding polypeptide of an antigen binding fragment thereof binds to Extra Domain B of Fibronectin (EDB-FN).

103. 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. 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. 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. 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. 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. 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 of any one of claims 67 to 114, wherein the LAP comprises the sequence of positions 30-274 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 SEQ ID NO:

82.

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

116.

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

121. The fusion polypeptide of any one of claims 67 to 120, wherein the LAP, or a 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 a fragment or derivative thereof.

122. The fusion polypeptide of any one of claims 67 to 121, wherein the LAP, or a 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 a fragment or derivative thereof.

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

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, wherein the one or more mutations 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 comprise a C33S mutation, wherein position 33 is related to the sequence of SEQ ID NO:

82.

126. The fusion polypeptide of claim 124, wherein the one or more mutations comprise a C24S mutation, wherein position 24 correlates to SEQ ID NO:

116.

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

31.

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. 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 according to claim 131.

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

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

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

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

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

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

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

140. 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 the polypeptide complex of any one of claims 1 to 64, the pharmaceutical composition of claim 65 or claim 66, the polynucleotide of claim 131, or the vector of any one of claims 132 to 135.

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

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

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

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

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

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

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

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

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

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

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

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

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