TGFβ1-binding molecules, GARP-TGFβ1-binding molecules and their pharmaceutical uses

Abstract

The present disclosure relates to TGFβ1-binding molecules, GARP-TGFβ1-binding molecules, and pharmaceutical uses thereof. Specifically, the present disclosure relates to TGFβ1-binding molecules, GARP-TGFβ1-binding molecules, pharmaceutical compositions comprising them, methods for preparing them, and pharmaceutical uses thereof. In another aspect, the present disclosure relates to methods for treating and / or preventing diseases (e.g., tumors or cancers) using the TGFβ1-binding molecules, GARP-TGFβ1-binding molecules of the present disclosure, and pharmaceutical compositions comprising them.

Description

[Technical Field]

[0001] This disclosure claims priority to Chinese Patent Application No. 202310027965.9 filed on January 9, 2023, and Chinese Patent Application No. 202310037692.6 filed on January 9, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the biomedicine field, and in particular to TGFβ1 binding molecules, pharmaceutical compositions comprising TGFβ1 binding molecules, as well as pharmaceutical uses, methods of preparation, and methods of preventing or treating diseases associated with the TGFβ1 signaling pathway. [Background technology]

[0003] Transforming growth factor β (TGFβ) is a class of multi-function growth factors that includes TGFβ1, TGFβ2, and TGFβ3. They regulate many important physiological processes, such as cell growth, differentiation, proliferation, apoptosis, and matrix production. Research has shown that TGFβ can inhibit tumor growth in the early stages of tumorigenesis but promote it in the later stages of tumor progression (Liu S, Ren J, Ten Dijke P. Signal Transduct Target Ther. 2021 Jan 8;6(1):8.). One important mechanism by which TGFβ promotes aggressive tumor growth is by inhibiting immune cells in the tumor microenvironment. Here, TGFβ1, instead of TGFβ2 and TGFβ3, is a key factor promoting aggressive tumor growth (Constance J Martin, et al. Sci Transl Med. 2020 Mar 25;12(536):eaay8456.). TGFβ1, produced by tumor cells, regulatory T cells (Treg), and inhibitory phagocytes, can directly promote the activity of Treg cells and inhibit effector T cells and antigen-presenting cells, thereby achieving an immunosuppressive effect.

[0004] Currently, anti-PD-1 (Programmed Cell Death-1) antibodies, tumor immune checkpoint inhibitors, have shown great success in tumor immunotherapy, but the overall patient response rate is only about 20%. Combining anti-TGF-β1 antibodies with anti-PD-1 antibodies significantly overcomes tumor immune evasion and enhances the efficacy of anti-PD-1 antibodies in tumor models such as EMT-6, MBT-2, Cloudman S91, CT26, and MC38. However, pan-TGF-β antibodies targeting mature TGF-β1 exhibit dose-dependent in vivo toxicity in experimental studies. Targeting the pro / latent TGF-β1 complex inhibits TGF-β1 production from its source, which has better efficacy in animal models and significantly reduced toxicity compared to targeting mature TGF-β1.

[0005] TGF-β1 production is tightly regulated through a multistep process. In the TGF-β1 precursor protein, the mature TGF-β1 domain at its C-terminus remains covalently or noncovalently bound to an N-terminal domain called latency-associated peptide (LAP). LAP prevents mature TGF-β1 from binding to its receptor, rendering it normally inactive. This TGF-β1 precursor protein can also form large latent complexes with GARP, LRRC33, LTBP1, and LTBP3. In the tumor microenvironment, GARP-TGF-β1 is primarily produced by Tregs and tumor cells, LRRC33-TGF-β1 is primarily produced by immune-inhibitory phagocytes, while LTBP1-TGF-β1 and LTBP3-TGF-β1 are primarily present in the tumor matrix. Currently, no commercial products for TGF-β1 precursor protein or TGF-β1 complexes are available worldwide. There remains a need in the art for agents that can bind with high affinity to the TGFβ1 precursor protein or the TGFβ1 complex and block its activation, and that can be used to treat TGFβ-related diseases such as tumors and fibrosis. Summary of the Invention

[0006] The present disclosure provides TGFβ1 binding molecules, GARP-TGFβ1 binding molecules, pharmaceutical compositions comprising said binding molecules, as well as pharmaceutical uses, methods of preparation, and methods of preventing or treating diseases associated with the TGFβ signaling pathway, in particular methods of preventing or treating cancer or tumors.

[0007] TGFβ1 binding molecule In a first aspect, the present disclosure provides a TGFβ1 binding molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL).

[0008] In some embodiments, the VH comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 11, and / or the VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 12, 21 to 35.

[0009] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some specific embodiments, the CDRs are defined according to the Kabat definition method.

[0010] In some embodiments, the CDRs may also be defined by other numbering systems, such as the IMGT, Chothia, AbM, or Contact numbering systems. Illustratively, the HCDR1, HCDR2, and HCDR3 in the VH of SEQ ID NO: 11 and the LCDR1, LCDR2, and LCDR3 in the VL of SEQ ID NO: 35 are provided below, as defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems.

[0011] [Table 1] [Table 2]

[0012] In some embodiments, the TGFβ1 binding molecule comprises the VH and VL shown below: The VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, and / or the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 38, 64 and 18.

[0013] wherein the amino acid sequence represented by SEQ ID NO: 38 is RASQX1ISX2YLN, wherein X1 is selected from S, A, F, G, I, P, Y, V, or K, and X2 is selected from S, D, E, P, or H; The amino acid sequence represented by SEQ ID NO: 64 is X3ASX4LX5S, where X3 is selected from A, T, S or M, X4 is selected from S, Y, A, E or G, and X5 is selected from Q, T, D or E.

[0014] In some embodiments, the amino acid sequence represented by SEQ ID NO: 38 is RASQX1ISX2YLN, where X1 is selected from F, Y, or A, and X2 is selected from D or P; and the amino acid sequence represented by SEQ ID NO: 64 is X3ASX4LX5S, where X3 is selected from A, T, or S, X4 is selected from S, Y, or E, and X5 is selected from Q, D, or E.

[0015] In some embodiments, the TGFβ1 binding molecule, wherein: The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 13. The amino acid sequence of the HCDR2 is shown in SEQ ID NO: 14. The amino acid sequence of the HCDR3 is shown in SEQ ID NO: 15. The amino acid sequence of the LCDR1 is represented by any one of SEQ ID NOs: 16, 36, 39, 41 to 42, 44, 46, 49, 52, 54, and 56 to 59; The amino acid sequence of the LCDR2 is represented by any one of SEQ ID NOs: 17, 37, 40, 43, 45, 47 to 48, 50 to 51, 53, and 55, and / or The amino acid sequence of the LCDR3 is shown in SEQ ID NO:18.

[0016] In some embodiments, the TGFβ1 binding molecule, wherein: a) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 59, 40, and 18; b) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 36 to 37, 18; c) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 39 to 40, 18; d) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 41, 40, and 18; e) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 42 to 43, 18; f) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 44 to 45, 18; g) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 46 to 47, 18; h) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 46, 48, and 18; j) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 49 to 50, 18; k) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 46, 51, and 18; l) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 52 to 53, 18; m) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 54 to 55, 18; n) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 56, 53, and 18; o) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 57, 45, and 18; p) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 58, 50 and 18, or q) The VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 16 to 18.

[0017] In some embodiments, the TGFβ1 binding molecule comprises a VH and a VL, wherein: Any one of the HCDRs contained in the VH shown has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any one of the HCDRs above, and / or any one of the LCDRs contained in the VL shown has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any one of the LCDRs above.

[0018] In some specific embodiments, the amino acid mutations in the HCDRs or LCDRs are conservative substitutions.

[0019] In some embodiments, the TGFβ1 binding molecules provided by the present disclosure comprise any one or a combination of more than one (eg, 2, 3, 4, 5, or 6) of the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3.

[0020] In some embodiments, the TGFβ1 binding molecule is an anti-TGFβ1 antibody or an antigen-binding fragment thereof.

[0021] In some embodiments, the anti-TGFβ1 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antigen-binding fragment of any one of the above.

[0022] In some specific embodiments, the anti-TGFβ1 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, or a human antibody or antigen-binding fragment thereof, hi some specific embodiments, the anti-TGFβ1 antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.

[0023] In some specific embodiments, the anti-TGFβ1 antibody or antigen-binding fragment thereof is modified by affinity maturation. The anti-TGFβ1 antibody or antigen-binding fragment thereof modified by affinity maturation comprises one or more mutations located in the VH and / or VL. Illustratively, the mutations located in the VH comprise mutations at any one or any combination of positions 99, 100, 53, 56, 58, 31, 33, 95, and 100. Illustratively, the mutations located in the VL comprise mutations at any one or any combination of positions 24, 28, 31, 32, 50, 53, 55, 92, 93, and 94. The positions of the mutations are numbered according to the Kabat numbering convention.

[0024] In some embodiments, the TGFβ1 binding molecule comprises a VH and a VL, wherein: The VH comprises the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least 80% sequence identity thereto; and / or The VL comprises an amino acid sequence shown in any one of SEQ ID NOs: 12, 21 to 35, or an amino acid sequence having at least 80% sequence identity thereto.

[0025] In some embodiments, the TGFβ1 binding molecule comprises a VH and a VL, wherein the designated VH has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to any one of the VHs, and / or the designated VL has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to any one of the VLs.

[0026] In some embodiments, the TGFβ1 binding molecules provided by the present disclosure comprise any one or a combination of any two of the above VHs and VLs.

[0027] In some embodiments, the TGFβ1-binding molecules provided by the present disclosure further comprise an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, IgG4, or a variant of any one of the above. In some embodiments, the immunoglobulin Fc region is derived from human IgG4 or a variant thereof. In some embodiments, the human IgG4 variant comprises a mutation that reduces or eliminates Fc effector function. Illustratively, the human IgG4 variant comprises the mutation S228P. The mutation sites are numbered according to the EU numbering convention.

[0028] In some embodiments, the TGFβ1 binding molecule further comprises a light chain constant region and / or a heavy chain constant region.

[0029] In some embodiments, the light chain constant region is derived from a κ light chain, a λ light chain, or a variant of any one of the above. In some embodiments, the light chain constant region is derived from a human κ light chain, a human λ light chain, or a variant of any one of the above.

[0030] In some specific embodiments, the light chain constant region is derived from a human kappa light chain or a variant thereof.

[0031] In some specific embodiments, the light chain constant region is derived from a mouse kappa light chain or a variant thereof.

[0032] In some specific embodiments, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity thereto.

[0033] In some embodiments, the heavy chain constant region is derived from IgG1, IgG2, IgG3, IgG4, or a variant of any one of the above. In some embodiments, the heavy chain constant region is derived from human IgG1, human IgG2, human IgG3, human IgG4, or a variant of any one of the above.

[0034] In some specific embodiments, the heavy chain constant region is derived from human IgG4 or a variant thereof.

[0035] In some specific embodiments, the human IgG4 variant comprises a mutation that reduces or eliminates Fc effector function. Illustratively, the human IgG4 variant comprises the mutation S228P. The mutation sites are numbered according to the EU numbering convention.

[0036] In some specific embodiments, the heavy chain constant region is derived from murine IgG2 or a variant thereof.

[0037] In some specific embodiments, the murine IgG2 mutant comprises mutations that reduce or eliminate ADCC effectors. Illustratively, the murine IgG2 mutant comprises at least one mutation shown below: L234A / L235E / G237A / D327Q / A330S / P331S. The mutation sites are numbered according to the EU numbering convention.

[0038] In some embodiments, the immunoglobulin Fc region is an Fc region that reduces or eliminates effector function, e.g., has a mutation that reduces or eliminates ADCC effector function. Illustratively, the mutation that reduces or eliminates ADCC effector function is: Contains at least one of L234A / L235E / G237A / D327Q / A330S / P331S (IgG2a), N297A or N297Q (IgG1), L234A / L235A (IgG1), V234A / G237A (IgG2), L235A / G237A / E318A (IgG4), H268Q / V309L / A330S / A331S (IgG2), C220S / C226S / C229S / P238S (IgG1), C226S / C229S / E233P / L234V / L235A (IgG1), L234F / L235E / P331S (IgG1), or S267E / L328F (IgG1). The positions of the mutations are numbered according to the EU numbering rules.

[0039] In some specific embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 20, or an amino acid sequence having at least 80% sequence identity thereto.

[0040] In some embodiments, the TGFβ1 binding molecule comprises a heavy chain and a light chain, wherein: the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 66, or an amino acid sequence having at least 80% sequence identity thereto; and / or The light chain comprises an amino acid sequence shown in any one of SEQ ID NOs: 65, 67 to 81, or an amino acid sequence having at least 80% sequence identity thereto.

[0041] In some embodiments, the TGFβ1 binding molecule comprises a heavy chain and a light chain, wherein: the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 82, or an amino acid sequence having at least 80% sequence identity thereto; and / or The light chain comprises the amino acid sequence set forth in SEQ ID NO: 83, or an amino acid sequence having at least 80% sequence identity thereto.

[0042] In some embodiments, the TGFβ1 binding molecule comprises a heavy chain and a light chain, wherein the designated heavy chain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to any one of the heavy chains, and / or the designated light chain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations compared to any one of the light chains.

[0043] In some specific embodiments, the amino acid mutations in the heavy and / or light chains are conservative substitutions.

[0044] In some embodiments, the TGFβ1 binding molecules of the present disclosure comprise any one or a combination of any two of the above heavy chains, light chains.

[0045] In the context of the present disclosure, "at least 80%" includes 80% or more, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and any numerical range therebetween.

[0046] In some embodiments, the TGFβ1 binding molecule specifically binds to a TGFβ1 precursor protein or a TGFβ1 complex.

[0047] In some embodiments, the TGFβ1 in the TGFβ1 complex is present as a TGFβ1 precursor protein.

[0048] In some embodiments, the TGFβ1 complex comprises: (2) LTBP1-TGFβ1 complex, (3) LTBP3-TGFβ1 complex, (4) LRRC33-TGFβ1 complex, (5) Contains any one of the GARP-TGFβ1 complexes.

[0049] As used in this disclosure, "TGFβ1" or "TGFβ1 protein" is understood broadly to include the proprotein form (also called pro-TGFβ1) or the latent form (latent TGFβ1) of transforming growth factor-β1 (TGFβ1) protein, or mature TGFβ1 after release.

[0050] In some embodiments, "TGFβ1" or "TGFβ1 protein" as used in this disclosure refers to "TGFβ1 precursor protein."

[0051] In some embodiments, the TGFβ1 precursor protein comprises (i) a mature TGFβ1 domain, and (ii) a latency-associated peptide (LAP).

[0052] Exemplary TGFβ1 precursor proteins are pro-TGFβ1 or latent TGFβ1. In pro-TGFβ1, the mature TGFβ1 domain is covalently linked to latency-associated peptides (LAPs), and in latent TGFβ1, the mature TGFβ1 domain is non-covalently linked to latency-associated peptides (LAPs).

[0053] In the present disclosure, "pro-TGFβ1" or "latent TGFβ1" are used interchangeably.

[0054] During the translation process, latent TGFβ1 (also called small latent complex (SLC)) is linked to a "presenting molecule" through disulfide bridges, thereby forming a large latent complex (LLC), for example, by forming a GARP-TGFβ1 complex with GARP. The TGFβ1 present in the GARP-TGFβ1 complex may be in its latent form (latent TGFβ1) or its precursor form (pro-TGFβ1).

[0055] In some embodiments, the TGFβ1 precursor protein or TGFβ1 complex further comprises a protein fragment, functional variant, etc. of any of the above proteins or protein complexes.

[0056] Exemplary "fragments" and "protein fragments" include, but are not limited to, growth factor domains, N-terminal prodomains, latency-associated peptides (LAPs), LAP-like domains, straight jacket regions, fastener regions, furin cleavage site regions, arm regions, fingers regions, latency loops, alpha1 helical regions, alpha2 helical regions, RGD sequence regions, trigger loop regions, extracellular domains, transmembrane domains, intracellular domains, and the like.

[0057] Illustratively, "variant" and "functional variant" refer to a protein or protein complex that contains one or more amino acid substitutions, deletions and / or additions and that has equivalent biological activity.

[0058] Illustratively, the mutant TGFβ1 precursor protein comprises an amino acid mutation at position 4, counting in whole numbers relative to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the mutant TGFβ1 precursor protein comprises a mutated C4S, and the site of the mutation is a position counted in whole numbers relative to the amino acid sequence set forth in SEQ ID NO: 6.

[0059] In some embodiments, the TGFβ1 complex is a complex formed between the TGFβ1 precursor protein and other types of proteins or their protein fragments or functional variants, such as LTBP1S, LTBP4, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4, etc.

[0060] In some embodiments, the TGFβ1 precursor protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 6-7, 9-10, or an amino acid sequence having at least 80% sequence identity thereto.

[0061] Exemplary, the TGFβ1 complex is a GARP-TGFβ1 complex comprising GARP and a TGFβ1 precursor protein, wherein: GARP comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 5, 8, 100, and 103, or an amino acid sequence having at least 80% sequence identity thereto; And / or, the TGFβ1 precursor protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 1, 3, 6-7, 9-10, or an amino acid sequence having at least 80% sequence identity thereto.

[0062] In some embodiments, the TGFβ1-binding molecule is an anti-TGFβ1 antibody or antigen-binding fragment thereof that specifically binds to an antigenic epitope of the TGFβ1 precursor protein or TGFβ1 complex. In some embodiments, the anti-TGFβ1 antibody or antigen-binding fragment thereof does not inhibit the binding of TGFβ1 to integrins. For example, in some embodiments, the anti-TGFβ1 antibody or antigen-binding fragment thereof does not mask the integrin-binding site of TGFβ1.

[0063] In some embodiments, the TGFβ1 binding molecule is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M or lower K D and binds to a TGFβ1 precursor protein or a TGFβ1 complex (e.g., an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, an LRRC33-TGFβ1 complex, a GARP-TGFβ1 complex, etc.). In some embodiments, the TGFβ1 complex comprises the TGFβ1 precursor protein.

[0064] In some embodiments, the TGFβ1 binding molecule does not bind or binds very weakly to TGFβ2 protein or TGFβ2 complex. Illustratively, the anti-TGFβ1 antibody or antigen-binding fragment thereof does not specifically bind or binds very weakly to at least one of mature TGFβ2, TGFβ2 precursor protein, GARP-TGFβ2 complex, and LTBP-TGFβ2 complex.

[0065] In some embodiments, the TGFβ1 binding molecule does not bind or binds very weakly to TGFβ3 protein or TGFβ3 complex. Illustratively, the anti-TGFβ1 antibody or antigen-binding fragment thereof does not specifically bind or binds very weakly to at least one of mature TGFβ3, TGFβ3 precursor protein, GARP-TGFβ3 complex, and LTBP-TGFβ3 complex.

[0066] In the present disclosure, "TGFβ2 protein" should be understood in a broad sense to cover mature TGFβ2 as well as inactive precursor protein forms such as pro-TGFβ2 and latent TGFβ2. "Pro-TGFβ2" and "latent TGFβ2" are used interchangeably.

[0067] In the present disclosure, "TGFβ3 protein" should be understood in a broad sense to cover mature TGFβ3 as well as inactive precursor protein forms such as pro-TGFβ3 and latent TGFβ3. "Pro-TGFβ3" and "latent TGFβ3" are used interchangeably.

[0068] In some embodiments, the TGFβ1-binding molecule of the present disclosure inhibits TGFβ1 activity. For example, the TGFβ1-binding molecule binds to a TGFβ1 precursor protein (pro / latent TGFβ1) or a TGFβ1 complex (e.g., a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, or an LRRC33-TGFβ1 complex) and selectively inhibits the activity of TGFβ1 (e.g., inhibits the activation of TGFβ1 and / or inhibits the release of mature TGFβ1 and / or inhibits TGFβ1 signaling). Furthermore, the TGFβ1-binding molecule does not selectively inhibit the activity of TGFβ2 and / or TGFβ3, e.g., selectively inhibits the activation of TGFβ1 but not the activation of TGFβ2 and / or TGFβ3. Here, TGFβ1 in the TGFβ1 complex exists in the form of a TGFβ1 precursor protein.

[0069] In some embodiments, the TGFβ1 binding molecules of the disclosure have improved safety (eg, reduced in vivo toxicity and / or adverse reactions).

[0070] In some embodiments, the TGFβ1 binding molecules of the present disclosure bind to regulatory T (T reg ) further have at least one property exhibited by inhibiting the immunosuppressive activity of cells, inhibiting tumor growth, inhibiting fibrosis.

[0071] In some embodiments, the TGFβ1 binding molecule is an anti-TGFβ1 antibody or an antigen-binding fragment thereof, further including Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibodies, domain antibodies, and multispecific antibodies (diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv), e.g., specifically scFv, Fv, Fab, or Fab' fragments.

[0072] In some embodiments, the present invention provides an anti-TGFβ1 antibody or antigen-binding fragment thereof that binds to the TGFβ1-binding molecule or competes for binding to the same epitope in the TGFβ1 precursor protein or TGFβ1 complex (e.g., human or mouse GARP-TGFβ1 complex, LTBP-TGFβ1 complex, LRRC33-TGFβ1 complex). In some embodiments, TGFβ1 in the TGFβ1 complex is present in the form of the TGFβ1 precursor protein.

[0073] In some embodiments, an anti-TGFβ1 antibody or antigen-binding fragment thereof is provided that blocks binding of the TGFβ1-binding molecule to a TGFβ1 precursor protein or a TGFβ1 complex (e.g., a human or mouse GARP-TGFβ1 complex, an LTBP-TGFβ1 complex, or an LRRC33-TGFβ1 complex). In some embodiments, TGFβ1 in a TGFβ1 complex exists in the form of a TGFβ1 precursor protein.

[0074] In some embodiments, an anti-TGFβ1 antibody or antigen-binding fragment thereof is provided, whose binding to a TGFβ1 protein or TGFβ1 complex (e.g., a human or mouse GARP-TGFβ1 complex, an LTBP-TGFβ1 complex, or an LRRC33-TGFβ1 complex) is blocked by the TGFβ1-binding molecule. In some embodiments, TGFβ1 in the TGFβ1 complex is present in the form of a TGFβ1 precursor protein.

[0075] GARP- TGFβ1 binding molecule In a second aspect, the disclosure provides a GARP-TGFβ1 binding molecule comprising at least one immunoglobulin single variable domain that binds to a TGFβ1 complex.

[0076] In some embodiments, the TGFβ1 complex is a GARP-TGFβ1 complex. In some embodiments, the TGFβ1 in the GARP-TGFβ1 complex is a TGFβ1 precursor protein. Illustratively, the TGFβ1 precursor protein is selected from pro-TGFβ1 or latent TGFβ1. In the present disclosure, "pro-TGFβ1" and "latent TGFβ1" are used interchangeably.

[0077] In some embodiments, the disclosure provides a GARP-TGFβ1 binding molecule comprising at least one immunoglobulin single variable domain that binds to a GARP-TGFβ1 complex.

[0078] In some embodiments, the immunoglobulin single variable domain comprises three complementarity determining regions CDR1, CDR2, CDR3, wherein: a) the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence set forth in SEQ ID NO: 84, b) the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence set forth in SEQ ID NO: 90, c) the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence set forth in SEQ ID NO: 91; or d) The immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in SEQ ID NO:92.

[0079] The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the CDRs are defined according to the Kabat numbering system. Exemplarily, CDR1, CDR2, and CDR3 defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system are provided below.

[0080] [Table 3]

[0081] In some embodiments, the immunoglobulin single variable domain comprises three complementarity determining regions CDR1, CDR2, CDR3, wherein: CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 85; CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 86, and / or CDR3 comprises the amino acid sequence shown in SEQ ID NO:87.

[0082] In some embodiments, the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 set forth in SEQ ID NOs: 85-87.

[0083] In some embodiments, the CDR1 comprised in the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 85, and / or the CDR2 comprised in said immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 86, and / or The CDR3 comprised in said immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO:87.

[0084] In some specific embodiments, the amino acid mutations in CDR1, CDR2, or CDR3 are conservative substitutions.

[0085] In some embodiments, the GARP-TGFβ1 binding molecule comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, etc.) immunoglobulin single variable domains described above. In some embodiments, the GARP-TGFβ1 binding molecule comprises two or more immunoglobulin single variable domains, and any two of the immunoglobulin single variable domains may be the same or different.

[0086] In some embodiments, the GARP-TGFβ1 binding molecule may comprise any one of the above complete immunoglobulin single variable domains, or may further comprise a functional portion of any one of the above immunoglobulin single variable domains or variants thereof, such as CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, FR1-CDR1-FR2-CDR2-FR3-CDR3-CDR3.

[0087] In some embodiments, the variant of the functional part of the immunoglobulin single variable domain may be a polypeptide that retains the serum albumin binding function of CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, or FR1-CDR1-FR2-CDR2-FR3-CDR3 and has at least 80%, at least 90%, sequence homology thereto, for example a polypeptide that retains the TGFβ1 complex binding function of CDR3 and has a certain sequence homology thereto, for example a polypeptide that has at least 80%, at least 90%, sequence homology thereto with any one of the above CDR3s.

[0088] In some embodiments, the immunoglobulin single variable domain is a VHH. Illustratively, the immunoglobulin single variable domain is a camelid VHH, a fully human VHH, or a humanized VHH.

[0089] In some specific embodiments, the immunoglobulin single variable domain is a humanized VHH.

[0090] In some embodiments, the human germline template of the humanized VHH is at least one selected from IGHV3-23 and IGJH4. In some specific embodiments, the humanized VHH comprises FR1, FR2, and FR3 derived from IGHV3-23, and the humanized VHH comprises FR4 derived from IGJH4.

[0091] In some embodiments, the humanized VHH comprises at least one back mutation as set forth below: 23T, 29Y, 30C, 37Y, 44E, 45R, 47F, 71Q, 74A, 75R, 78G, 81E, 93K, and 94T. In some specific embodiments, the framework regions of the humanized VHH comprise at least one of the above back mutations.

[0092] The sites of the backmutations are numbered according to the Kabat numbering convention.

[0093] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 84, 90 to 92, or an amino acid sequence having at least 80% sequence identity thereto.

[0094] In some embodiments, the amino acid sequence comprised in the immunoglobulin single variable domain has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid mutations compared to the sequence set forth in any one of SEQ ID NOs: 84, 90-92.

[0095] In some specific embodiments, the amino acid mutations in the immunoglobulin single variable domain are conservative substitutions.

[0096] In some embodiments, the GARP-TGFβ1 binding molecule further comprises an immunoglobulin Fc region. Illustratively, the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulin Fc region is derived from IgG2 or IgG4. In some specific embodiments, the immunoglobulin Fc region is derived from human IgG4.

[0097] In some embodiments, the immunoglobulin Fc region comprises a mutation that reduces or eliminates an Fc effector function. Illustratively, the immunoglobulin Fc region comprises the mutation S228P. The positions of the mutations are numbered according to the EU numbering convention.

[0098] In some specific embodiments, the immunoglobulin Fc region comprises the amino acid sequence set forth in SEQ ID NO: 88, or an amino acid sequence having at least 80% sequence identity thereto.

[0099] In some embodiments, the immunoglobulin Fc region is derived from mouse IgG2.

[0100] In some embodiments, the immunoglobulin Fc region is an Fc region that reduces or eliminates effector function, for example, having mutations that reduce or eliminate ADCC effector function. Exemplary mutations that reduce or eliminate ADCC effector function include L234A / L235E / G237A / D327Q / A330S / P331S (IgG2a), N297A or N297Q (IgG1), L234A / L235A (IgG1), V234A / G237A (IgG2), L235A / G237A / E318A (IgG4), H The mutations include at least one of 268Q / V309L / A330S / A331S (IgG2), C220S / C226S / C229S / P238S (IgG1), C226S / C229S / E233P / L234V / L235A (IgG1), L234F / L235E / P331S (IgG1), or S267E / L328F (IgG1). The positions of the mutations are numbered according to the EU numbering rules.

[0101] In some embodiments, the GARP-TGFβ1 binding molecule is an antibody or antigen-binding fragment thereof that binds to a GARP-TGFβ1 complex.

[0102] In some embodiments, the GARP-TGFβ1 binding molecule comprises an amino acid sequence set forth in any one of SEQ ID NOs: 89, 93-95, and 106, or an amino acid sequence having at least 80%, at least 90% sequence identity thereto.

[0103] In some embodiments, the GARP-TGFβ1 binding molecule comprises an amino acid sequence that has one or more (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid mutations compared to the amino acid sequence set forth in any one of SEQ ID NOs: 89, 93 to 95, and 106, and has functional activity of specifically binding to a GARP-TGFβ1 complex.

[0104] In some specific embodiments, the amino acid mutations in the GARP-TGFβ1 binding molecule are conservative substitutions.

[0105] In the context of the present disclosure, "at least 80%" includes 80% or more, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and any numerical range therebetween.

[0106] In some embodiments, the GARP-TGFβ1 binding molecule specifically binds to the GARP-TGFβ1 complex, thereby inhibiting the release of mature TGFβ1 from the GARP-TGFβ1 complex, and / or inhibiting TGFβ1 activity, and / or inhibiting TGFβ1 signaling.

[0107] In some embodiments, the GARP-TGFβ1 binding molecule does not bind to free, mature TGFβ1.

[0108] In some embodiments, the GARP-TGFβ1 binding molecule does not bind or binds very weakly to TGFβ2 protein or TGFβ2 complex, e.g., the TGFβ1 binding molecule does not bind or binds very weakly to mature TGFβ2, TGFβ2 precursor protein, GARP-TGFβ2 complex, LTBP-TGFβ2 complex, etc.

[0109] In some embodiments, the GARP-TGFβ1 binding molecule does not bind or binds very weakly to TGFβ3 protein or TGFβ3 complex, e.g., the GARP-TGFβ1 binding molecule does not bind or binds very weakly to mature TGFβ3, TGFβ3 precursor protein, GARP-TGFβ3 complex, LTBP-TGFβ3 complex, etc.

[0110] In the present disclosure, the term "TGFβ2 protein" should be understood in a broad sense to cover mature TGFβ2 and inactive precursor protein forms such as pro-TGFβ2 and latent TGFβ2. The term "TGFβ3 protein" should be understood in a broad sense to cover mature TGFβ3 and inactive precursor protein forms such as pro-TGFβ3 and latent TGFβ3.

[0111] In some embodiments, the GARP-TGFβ1 binding molecule can selectively inhibit TGFβ1 activity, but not TGFβ2 and / or TGFβ3 activity. For example, a GARP-TGFβ1 binding molecule selectively inhibits the activation of TGFβ1, but not TGFβ2 and / or TGFβ3. Thus, the GARP-TGFβ1 binding molecules of the present disclosure have improved safety (e.g., reduced in vivo toxicity and / or adverse reactions).

[0112] In some embodiments of the present disclosure, mature TGFβ1, mature TGFβ2, and / or mature TGFβ3 are present in a free state in cells.

[0113] In some embodiments, the GARP-TGFβ1 binding molecule specifically binds to a GARP-TGFβ1 complex, wherein the GARP-TGFβ1 complex is a) glycoprotein A repeat dominant sequence (GARP); b) the mature TGFβ1 domain, and c) Contains latency-associated peptide (LAP).

[0114] In some embodiments, the GARP comprises the amino acid sequence set forth in SEQ ID NO: 100 or 103, or an amino acid sequence having at least 90% sequence identity thereto, the mature TGFβ1 domain comprises the amino acid sequence set forth in SEQ ID NO: 101 or 104, or an amino acid sequence having at least 90% sequence identity thereto, and / or the LAP comprises the amino acid sequence set forth in SEQ ID NO: 102 or 105, or an amino acid sequence having at least 90% sequence identity thereto.

[0115] In some embodiments, the GARP-TGFβ1 binding molecule is 10 -7 M, 10 -8 M, 10 -9 M or lower K D and binds to the GARP-TGFβ1 complex or a fragment thereof.

[0116] In some embodiments, the GARP-TGFβ1 binding molecule has properties that exhibit inhibition of TGFβ1 activity, inhibition of the immunosuppressive activity of regulatory T cells, and / or inhibition of tumor growth.

[0117] In some embodiments, the GARP-TGFβ1 binding molecule is an anti-GARP-TGFβ1 antibody or an antigen-binding fragment thereof, and in some specific embodiments, a chimeric antibody, a camelid antibody, a humanized antibody, a fully human antibody, or an antigen-binding fragment thereof.

[0118] In some embodiments, the GARP-TGFβ1 binding molecule is an anti-GARP-TGFβ1 antibody or an antigen-binding fragment thereof, and in some specific embodiments, the anti-GARP-TGFβ1 antibody or antigen-binding fragment thereof includes, but is not limited to, Fab, Fv, sFv, Fab', F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv), for example, scFv, Fv, Fab, or Fab' fragments.

[0119] In some embodiments, anti-GARP-TGFβ1 antibodies or antigen-binding fragments thereof are provided that bind to the above-mentioned GARP-TGFβ1 binding molecules or compete for binding to the same epitope in the GARP-TGFβ1 complex (e.g., human or mouse GARP-TGFβ1 complex).

[0120] In some embodiments, an anti-GARP-TGFβ1 antibody or antigen-binding fragment thereof is provided that blocks binding of the GARP-TGFβ1 binding molecule to GARP-TGFβ1 (eg, human or mouse GARP-TGFβ1 complex).

[0121] In some embodiments, an anti-GARP-TGFβ1 antibody or antigen-binding fragment thereof is provided whose binding to a GARP-TGFβ1 complex (e.g., a human or mouse GARP-TGFβ1 complex) is blocked by the GARP-TGFβ1 binding molecule.

[0122] In some embodiments, the GARP-TGFβ1 complex comprises (i) GARP and (ii) a TGFβ1 precursor protein. Illustratively, in the GARP-TGFβ1 complex: GARP comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 5, 8, 100, and 103, or an amino acid sequence having at least 80% sequence identity thereto; And / or, the TGFβ1 precursor protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 1, 3, 6-7, 9-10, or an amino acid sequence having at least 80% sequence identity thereto.

[0123] Protein-binding molecules In some embodiments, the present disclosure provides TGFβ1 binding molecules.

[0124] In some embodiments, the TGFβ1 binding molecule specifically binds to a TGFβ1 precursor protein or a TGFβ1 complex. In some embodiments, the TGFβ1 complex comprises a TGFβ1 precursor protein.

[0125] As used in this disclosure, "TGFβ1" or "TGFβ1 protein" is understood broadly to include the proprotein form (also called pro-TGFβ1) or latent form (latent TGFβ1) of transforming growth factor-β1 (TGFβ1) protein, or mature TGFβ1 after release. In some embodiments, TGFβ1 or TGFβ1 protein refers to the TGFβ1 precursor protein.

[0126] In some embodiments, the TGFβ1 precursor protein comprises (i) a mature TGFβ1 domain, and (ii) a latency-associated peptide (LAP).

[0127] Exemplary TGFβ1 precursor proteins are pro-TGFβ1 or latent TGFβ1. In pro-TGFβ1, the mature TGFβ1 domain is covalently linked to latency-associated peptides (LAPs), and in latent TGFβ1, the mature TGFβ1 domain is non-covalently linked to latency-associated peptides (LAPs).

[0128] In the present disclosure, "pro-TGFβ1" or "latent TGFβ1" are used interchangeably.

[0129] Illustratively, the mature TGFβ1 domain comprises the amino acid sequence set forth in SEQ ID NO: 101 or 104, or an amino acid sequence having at least 80% sequence identity thereto.

[0130] Illustratively, the LAP comprises the amino acid sequence set forth in SEQ ID NO: 102 or 105, or an amino acid sequence having at least 80% sequence identity thereto.

[0131] Illustratively, the TGFβ1 precursor protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 6-7, 9-10, or an amino acid sequence having at least 80% sequence identity thereto.

[0132] In some embodiments, the TGFβ1 precursor protein or TGFβ1 complex further comprises a protein fragment, functional variant, etc. of any of the above proteins or protein complexes.

[0133] In some embodiments, the TGFβ1 precursor protein or TGFβ1 complex is derived from any vertebrate, including mammals such as primates (e.g., humans) and other species (e.g., mice, rats, guinea pigs, rabbits, dogs, pigs, sheep, etc.).

[0134] Exemplary "fragments" and "protein fragments" include, but are not limited to, growth factor domains, N-terminal prodomains, latency-associated peptides (LAPs), LAP-like domains, straight jacket regions, fastener regions, furin cleavage site regions, arm regions, fingers regions, latency loops, alpha1 helical regions, alpha2 helical regions, RGD sequence regions, trigger loop regions, extracellular domains, transmembrane domains, intracellular domains, and the like.

[0135] Illustratively, "variant" and "functional variant" refer to a protein or protein complex that contains one or more amino acid substitutions, deletions and / or additions and that has equivalent biological activity.

[0136] Illustratively, the mutant TGFβ1 precursor protein comprises an amino acid mutation at position 4, counting in whole numbers relative to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the mutant TGFβ1 precursor protein comprises a mutated C4S, and the site of the mutation is a position counted in whole numbers relative to the amino acid sequence set forth in SEQ ID NO: 6.

[0137] In some embodiments, the TGFβ1 precursor protein or TGFβ1 complex may or may not include a leader sequence. Illustratively, the leader sequence is a signal peptide sequence. In some embodiments, the TGFβ1 precursor protein or TGFβ1 complex may or may not include a tag sequence. Illustratively, the tag sequence is a His-tag, AVI-tag, myc-tag, fluorescent tag, or the like.

[0138] In some embodiments, the TGFβ1 complex comprises a TGFβ1 protein and any second protein such as those represented by glycoprotein A repeat dominant sequences (GARPs), latent TGF-β-binding proteins (LTBPs, e.g., LTBP1, LTBP1S, LTBP2, LTBP3, LTBP4), fibrillins (e.g., fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4), LRRC33 (Leucine-Rich Repeat-Containing Protein 33), or a variant or protein fragment of any one of the above.

[0139] Exemplary "protein fragments" or "fragments" include, but are not limited to, extracellular domains, transmembrane domains, or intracellular domains.

[0140] In some embodiments, the TGFβ1 complex includes, but is not limited to, a GARP-TGFβ1 complex, an LTBP-TGFβ1 complex (e.g., an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex), or an LRRC33-TGFβ1 complex. In some embodiments, in the TGFβ1 complex, TGFβ1 exists in the form of a TGFβ1 precursor protein.

[0141] Illustratively, the GARP-TGFβ1 complex is a) glycoprotein A repeat dominant sequence (GARP); b) the mature TGFβ1 domain, and c) Contains latency-associated peptide (LAP).

[0142] In some embodiments, the GARP comprises the amino acid sequence set forth in SEQ ID NO: 100 or 103 or an amino acid sequence having at least 80% sequence identity thereto, the TGFβ1 comprises the amino acid sequence set forth in SEQ ID NO: 101 or 104 or an amino acid sequence having at least 80% sequence identity thereto, and / or the LAP comprises the amino acid sequence set forth in SEQ ID NO: 102 or 105 or an amino acid sequence having at least 80% sequence identity thereto.

[0143] In some embodiments, the GARP-TGFβ1 complex is formed from GARP and TGFβ1 precursor protein.

[0144] In some specific embodiments, the GARP-TGFβ1 complex is formed from the extracellular domain of GARP and the TGFβ1 precursor protein.

[0145] In the present disclosure, "GARP" may be a naturally occurring wild-type GARP or a functional variant of GARP (e.g., containing one or more modifications, truncations, and / or mutations compared to wild-type GARP). In some embodiments, GARP may be full-length or a partial domain thereof (e.g., an extracellular domain, a transmembrane domain, or an intracellular domain). For example, naturally occurring means derived from any vertebrate, including mammals such as primates (e.g., humans) and other species (e.g., mice, rats, guinea pigs, rabbits, dogs, pigs, sheep, etc.).

[0146] In some embodiments, a GARP may or may not include a leader sequence. Illustratively, the leader sequence is a signal peptide sequence. In some embodiments, a GARP may include one or more tag sequences, such as a His-tag, an AVI-tag, a myc-tag, a fluorescent tag, or the like. In some embodiments, a GARP may not include a tag sequence.

[0147] Illustratively, the extracellular domain of GARP comprises the amino acid sequence set forth in SEQ ID NO: 100 or 103, or an amino acid sequence having at least 80% sequence identity thereto.

[0148] Illustratively, the GARP comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 5, 8, 100, 103, or an amino acid sequence having at least 80% sequence identity thereto.

[0149] In some embodiments, the protein-binding molecule that binds to the TGFβ1 precursor protein or the TGFβ1 complex is selected from antibodies or antigen-binding fragments thereof. Exemplary antibodies or antigen-binding fragments thereof include, but are not limited to, linear antibodies, single-chain antibodies (scFv), single-domain antibodies (sdAb), nanobodies, peptibodies, domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv), Fab, Fv, sFv, Fab', and F(ab')2.

[0150] In some embodiments, an antibody or antigen-binding fragment thereof specifically binds to an epitope of the TGFβ1 precursor protein or TGFβ1 complex. In some embodiments, when TGFβ1 is in the precursor protein form or in a complex with GARP, LTBP1, LTBP3, and / or LRRC33, the epitope can be used for binding to the antibody or antigen-binding fragment thereof. In some embodiments, the epitope is available due to a conformational change in TGFβ1 upon complexing with GARP, LTBP, and / or LRRC33. In some embodiments, the epitope in TGFβ1 to which the antibody or antigen-binding fragment thereof binds is unavailable when TGFβ1 is not complexed with GARP, LTBP, and / or LRRC33, or when TGFβ1 is mature TGFβ1. In some embodiments, the antibody or antigen-binding fragment thereof does not inhibit the binding of TGFβ1 to integrins. For example, in some embodiments, the antibody or antigen-binding fragment thereof does not mask the integrin-binding site of TGFβ1.

[0151] In some embodiments, the protein-binding molecule does not bind or binds very weakly to TGFβ2 protein or TGFβ2 complex. Illustratively, the protein-binding molecule does not specifically bind to at least one of mature TGFβ2, TGFβ2 precursor protein, GARP-TGFβ2 complex, LTBP-TGFβ2 complex, etc.

[0152] In some embodiments, the protein-binding molecule does not bind or binds very weakly to TGFβ3 protein or the TGFβ3 complex. Illustratively, the protein-binding molecule does not specifically bind to at least one of mature TGFβ3, TGFβ3 precursor protein, GARP-TGFβ3 complex, LTBP-TGFβ3 complex, etc.

[0153] In the present disclosure, "TGFβ2 protein" should be understood in a broad sense to cover mature TGFβ2 as well as inactive precursor protein forms such as pro-TGFβ2 and latent TGFβ2. "Pro-TGFβ2" and "latent TGFβ2" are used interchangeably.

[0154] In the present disclosure, "TGFβ3 protein" should be understood in a broad sense to cover mature TGFβ3 as well as inactive precursor protein forms such as pro-TGFβ3 and latent TGFβ3. "Pro-TGFβ3" and "latent TGFβ3" are used interchangeably.

[0155] In some embodiments, mature TGFβ1, mature TGFβ2, and / or mature TGFβ3 are present in a free state in cells.

[0156] In some embodiments, the protein-binding molecule can selectively inhibit the activity of TGFβ1 by specifically binding to a TGFβ1 precursor protein or a TGFβ1 complex (e.g., a GARP-TGFβ1 complex, an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, an LRRC33-TGFβ1 complex, etc.), e.g., by inhibiting TGFβ1 activation and / or inhibiting the release of mature TGFβ1 and / or inhibiting TGFβ1 signaling.

[0157] Polynucleotides The present disclosure provides a polynucleotide encoding any one of the above protein-binding molecules. In some embodiments, the present disclosure provides a polynucleotide encoding any one of the above TGFβ1-binding molecules, GARP-TGFβ1-binding molecules.

[0158] In some embodiments, the present disclosure provides a polynucleotide encoding a TGFβ1 binding molecule according to the first aspect of the disclosure. In some embodiments, the present disclosure provides a polynucleotide encoding a GARP-TGFβ1 binding molecule according to the second aspect of the disclosure.

[0159] In some embodiments, the polynucleotides of the present disclosure may be RNA, DNA, or cDNA.

[0160] In some embodiments, the polynucleotides of the present disclosure are isolated polynucleotides.

[0161] In some embodiments, a polynucleotide of the present disclosure may be in the form of, present in, and / or part of a vector, which may be a eukaryotic, prokaryotic, or viral vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may be, in particular, an expression vector, i.e., a vector capable of providing expression of a binding molecule (e.g., a TGFβ1-binding molecule) in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Such an expression vector typically contains at least one polynucleotide of the present disclosure operably linked to one or more appropriate expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the skill of the art. Regulatory elements and other elements useful or necessary for expression of a protein-binding molecule, TGFβ1-binding molecule, or GARP-TGFβ1-binding molecule of the present disclosure include, for example, promoters, enhancers, terminators, integrons, selectable markers, leader sequences, and reporter genes.

[0162] The polynucleotides of the present disclosure may be prepared or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA techniques) based on information about the amino acid sequences of the polypeptides of the present disclosure, and / or may be isolated from suitable natural sources.

[0163] In some embodiments, the polynucleotides and vectors of the present disclosure can be used to prepare TGFβ1-binding molecules, hi some embodiments, the polynucleotides and vectors of the present disclosure can be used to express TGFβ1-binding molecules in vitro or in vivo that bind to TGFβ1 precursor protein or TGFβ1 complexes for different purposes, such as detection, diagnosis, treatment, or regulation.

[0164] In some embodiments, the polynucleotides and vectors of the present disclosure can be used to prepare GARP-TGFβ1 binding molecules. In some embodiments, the polynucleotides and vectors of the present disclosure can be used to express GARP-TGFβ1 binding molecules in vitro or in vivo, which bind to GARP-TGFβ1 complexes for different purposes, such as detection, diagnosis, treatment, and regulation.

[0165] host cell The present disclosure provides host cells that express one or more protein-binding molecules of the present disclosure. In some embodiments, the host cells provided by the present disclosure comprise any one of the polynucleotides or vectors described above, or the host cells express any one of the TGFβ1-binding molecules or GARP-TGFβ1-binding molecules described above.

[0166] In some embodiments, the host cell expresses a TGFβ1 binding molecule according to the first aspect of the present disclosure. In some embodiments, the host cell expresses a GARP-TGFβ1 binding molecule according to the second aspect of the present disclosure.

[0167] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0168] Illustratively, bacterial cells include, for example, cells of Gram-negative strains (e.g., Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0169] Exemplary fungal cells include, for example, cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0170] Exemplary mammalian cells include, for example, monkey kidney CV1 cells (COS-7), human embryonic kidney cells (293 or 293T cells), baby mouse kidney cells (BHK), mouse Sertoli cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (HepG2), human glioma cells (LN229 cells), human cervical cancer cells (HeLa cells), human breast cancer cells (MCF-7), human prostate cancer cells (PC3), mouse breast cancer cells (EMT-6 cells), mouse mammary tumor cells (MMT060562), mouse colon cancer cells (CT26 cells), TRI cells (see, e.g., Mather et al., Annals of NY Acad. Sci 383, 44-68 (1982)), MRC5 cells and FS4 cells, Chinese hamster ovary (CHO) cells, myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0.

[0171] The present disclosure may use amphibian cells, insect cells, plant cells and any other cells in the art for expressing heterologous proteins.

[0172] The cells of the present disclosure are not capable of developing into complete plants or animals.

[0173] Production or preparation method The present disclosure provides methods for preparing the protein-binding molecules of the present disclosure.

[0174] In some embodiments, the present disclosure provides a method of preparing a TGFβ1 binding molecule according to the first aspect, comprising: a. forming a TGFβ1 complex containing a TGFβ1 precursor protein or the TGFβ1 precursor protein as an antigen protein; b. constructing a human antibody phage library; c. screening for a TGFβ1-binding molecule that specifically binds to the antigen protein using the phage library.

[0175] In some embodiments, the method for preparing a TGFβ1 binding molecule comprises: d. Further comprising the step of performing affinity maturation modifications on said TGFβ1 binding molecule.

[0176] The above preparation method is also a method for screening for a TGFβ1 binding molecule that specifically binds to a TGFβ1 precursor protein or a TGFβ1 complex containing the above TGFβ1 precursor protein.

[0177] In some embodiments, the present disclosure provides a method for preparing a GARP-TGFβ1 binding molecule according to the second aspect, comprising: a. forming a complex containing human GARP and human TGFβ1 precursor protein as an antigen protein; b. Immunizing an animal with the antigen protein to construct a phage library; c. screening for a GARP-TGFβ1 binding molecule comprising an immunoglobulin single variable domain that binds to a GARP-TGFβ1 complex using the phage library.

[0178] In some embodiments, the method for preparing a GARP-TGFβ1 binding molecule comprises: d. Further comprising the step of humanizing the GARP-TGFβ1 binding molecule.

[0179] In some specific embodiments, step b further comprises: A camel is immunized with the antigen protein, peripheral blood is collected from the immunized camel, nucleic acid is extracted, and a phage library is constructed using the nucleic acid.

[0180] The above preparation method is also a method for screening for a GARP-TGFβ1 binding molecule that specifically binds to the GARP-TGFβ1 complex.

[0181] In some other embodiments, the disclosure provides a method of preparing any one of the TGFβ1 binding molecules or GARP-TGFβ1 binding molecules described above, comprising: - culturing a host cell of the present disclosure under conditions that allow expression of a TGFβ1 binding molecule or a GARP-TGFβ1 binding molecule of the present disclosure; - recovering the target protein expressed in said host cells from the culture; - optionally further purifying and / or modifying the target protein of the present disclosure.

[0182] The TGFβ1 binding molecules or GARP-TGFβ1 binding molecules of the present disclosure may be produced intracellularly in cells such as those described above (e.g., in the cytoplasm, periplasm, or in inclusion bodies), then isolated from the host cells and, optionally, further purified, or may be produced extracellularly (e.g., in the medium in which the host cells are cultured), then isolated from the medium and, optionally, further purified.

[0183] Methods and reagents for recombinantly producing proteins or polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selectable markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, target protein isolation and purification techniques applicable to the production of binding molecules or antibodies of the present disclosure are known to those of skill in the art. Methods for producing and purifying antibodies are well known in the art and can be found, for example, in the Cold Spring Harbor Antibody Laboratory Techniques Manual (Chapters 5-8 and 15). Engineered antibodies of the present disclosure may be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains may be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can be used to stably transfect cells. Mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free medium in bioreactors to produce antibodies. The culture medium into which the antibody is secreted can be purified and collected by conventional techniques. The antibody can be concentrated by filtration using conventional methods. Soluble contaminants and polymers can be removed using conventional methods such as molecular sieving or ion exchange. The resulting product should be immediately frozen at, for example, -70°C or lyophilized.

[0184] However, the TGFβ1 binding molecules or GARP-TGFβ1 binding molecules of the disclosure can also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0185] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions comprising any one or a combination of the above-described protein-binding molecules or polynucleotides encoding the same. In some embodiments, the pharmaceutical composition comprises a prophylactically or therapeutically effective amount of any one of the above-described TGFβ1-binding molecules or GARP-TGFβ1-binding molecules, or a polynucleotide or vector encoding the TGFβ1-binding molecule or GARP-TGFβ1-binding molecule.

[0186] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable additives, diluents, buffers, or excipients.

[0187] In some embodiments, the pharmaceutical composition can be prepared in any dosage form known in the medical arts, with the choice of dosage form being determined by the anticipated mode of administration and therapeutic use.

[0188] In some embodiments, the pharmaceutical composition further comprises an immune checkpoint inhibitor. For example, the immune checkpoint inhibitor includes, but is not limited to, an inhibitor of one or more of PD-1, PD-L1, and PD-L2. In some embodiments, the pharmaceutical composition further comprises an anti-PD-1 antibody or antigen-binding fragment thereof.

[0189] The TGFβ1-binding molecule or GARP-TGFβ1-binding molecule of the present disclosure can inhibit TGFβ1 activity and inhibit the immunosuppressive activity of regulatory T cells. The combined use of a TGFβ1-binding molecule or GARP-TGFβ1-binding molecule with an anti-PD-1 antibody can eliminate the tumor immunosuppressive microenvironment. Examples of the present disclosure have demonstrated that the combined use of a TGFβ1-binding molecule or GARP-TGFβ1-binding molecule with an anti-PD-1 antibody exhibits significantly improved therapeutic effects in tumor treatment.

[0190] In some embodiments, a unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of the TGFβ1-binding molecule. In some specific embodiments, the amount of the TGFβ1-binding molecule contained in a unit dose of the pharmaceutical composition is 0.1 to 2000 mg, and in some specific embodiments, 1 to 1000 mg.

[0191] In some embodiments, a unit dose of the pharmaceutical composition can contain 0.01 to 99% by weight of the GARP-TGFβ1 binding molecule. In some other specific embodiments, a unit dose of the pharmaceutical composition contains 0.1 to 2000 mg of the GARP-TGFβ1 binding molecule, and in some specific embodiments, 1 to 1000 mg.

[0192] Combination administration The present disclosure provides use of any one of the protein binding molecules or its encoding polynucleotide in combination with an immune checkpoint inhibitor in the treatment of a disease associated with the TGFβ signaling pathway. Exemplary immune checkpoint inhibitors include, but are not limited to, inhibitors of one or more of PD-1, PD-L1, and PD-L2.

[0193] In some embodiments, there is provided a use of a TGFβ1 binding molecule in combination with an immune checkpoint inhibitor in the treatment of a disease associated with the TGFβ signaling pathway.

[0194] In some embodiments, there is provided a use of a TGFβ1 binding molecule in combination with an anti-PD-1 antibody or antigen-binding fragment thereof in the treatment of a disease associated with the TGFβ signaling pathway.

[0195] In some embodiments, there is provided the use of a GARP-TGFβ1 binding molecule in combination with an immune checkpoint inhibitor in the treatment of a disease associated with the TGFβ signaling pathway.

[0196] In some embodiments, there is provided a use of a GARP-TGFβ1 binding molecule in combination with an anti-PD-1 antibody or antigen-binding fragment thereof in the treatment of a disease associated with the TGFβ signaling pathway.

[0197] The term "disease associated with the TGFβ signaling pathway" refers to any disease, disorder, and / or condition associated with the expression, activity, and / or metabolism of a TGFβ family protein, or any disease, disorder, and / or condition that may benefit from modulation of the activity and / or levels of one or more TGFβ family proteins. Diseases associated with the TGFβ signaling pathway can include, but are not limited to, tumors or cancers.

[0198] In some embodiments, the disease associated with the TGFβ signaling pathway is cancer. The present disclosure provides for the use of a TGFβ1 binding molecule or a GARP-TGFβ1 binding molecule in combination with an anti-PD-1 antibody or antigen-binding fragment thereof in the treatment of cancer.

[0199] The TGFβ1 binding molecule or GARP-TGFβ1 binding molecule significantly improves the immune-inhibitory microenvironment of tumors, and after being combined with an anti-PD-1 antibody, exerts significantly improved therapeutic effects in tumor treatment.

[0200] In some embodiments, the cancer is selected from lung cancer, intestinal cancer, kidney cancer, bladder cancer, liver cancer, gastric cancer, breast cancer, colon cancer, cervical cancer, prostate cancer, and head and neck cancer.

[0201] Reagent kit (or kit) The present disclosure provides a reagent kit or kit comprising one or more containers, each independently containing a protein-binding molecule of the present disclosure (e.g., the TGFβ1-binding molecule or GARP-TGFβ1-binding molecule described above), or any one or combination thereof selected from the group consisting of a polynucleotide encoding the molecule, a vector encoding the molecule, and the like.

[0202] Disease prevention and treatment methods and pharmaceutical uses The present disclosure provides pharmaceutical uses of protein-binding molecules, or their encoding polynucleotides, vectors, pharmaceutical compositions, and methods for preventing, treating, or alleviating diseases or symptoms.

[0203] In some embodiments, the present disclosure provides (1) inhibiting TGFβ1 activity, such as inhibiting TGFβ1 activation, inhibiting release of mature TGFβ1 from the TGFβ1 complex, and / or inhibiting TGFβ1 signaling; (2) preparation of an agent for inhibiting TGFβ1 activity; (3) Inhibition of the immunosuppressive activity of regulatory T cells (4) Preparation of a drug for inhibiting the immunosuppressive activity of regulatory T cells; (5) Use of a TGFβ1-binding molecule, encoding polynucleotide, vector, or pharmaceutical composition in at least one of the prevention and treatment of a disease or condition associated with the TGFβ signaling pathway is provided.

[0204] In some embodiments, the present disclosure provides a method for preventing or treating a disease or condition associated with the TGFβ signaling pathway, comprising administering to a subject a prophylactically or therapeutically effective amount of a TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition.

[0205] In some embodiments, the disclosure provides a method of inhibiting TGFβ1 activity in vitro, the method comprising administering a TGFβ1 antibody or antigen-binding fragment thereof, encoding polynucleotide, vector, or pharmaceutical composition in vitro. In some specific embodiments, an inhibitory effective amount of a TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition is administered.

[0206] In some embodiments, the present disclosure provides a method of inhibiting TGFβ1 activity in vivo, comprising administering a TGFβ1-binding molecule, encoding polynucleotide, vector, or pharmaceutical composition to a subject. In some specific embodiments, an inhibitory effective amount of a TGFβ1-binding molecule, encoding polynucleotide, vector, or pharmaceutical composition is administered to a subject.

[0207] In some specific embodiments, the subject suffers from a disease or condition associated with the TGFβ signaling pathway.

[0208] The term "disease associated with the TGFβ signaling pathway" refers to any disease, disorder, and / or condition associated with the expression, activity, and / or metabolism of a TGFβ family protein, or any disease, disorder, and / or condition that may benefit from modulation of the activity and / or levels of one or more TGFβ family proteins. Diseases associated with the TGFβ signaling pathway can include, but are not limited to, diseases or conditions associated with fibrosis, tumors, or cancer.

[0209] In some embodiments, the disease associated with the TGFβ signaling pathway is cancer.

[0210] In some embodiments, the disease or condition associated with the TGFβ signaling pathway is fibrosis.

[0211] In some embodiments, a method for preventing or treating cancer comprises administering a prophylactically or therapeutically effective amount of the components shown in 1) and 2) below, i.e., 1) an anti-TGFβ1 antibody or an antigen-binding fragment thereof, or an encoding polynucleotide, vector, or pharmaceutical composition; 2) A method is provided in which the compound is administered in combination with an immune checkpoint inhibitor.

[0212] Illustratively, the PD-1 signaling pathway inhibitor includes, but is not limited to, an inhibitor of one or more of PD-1, PD-L1, and PD-L2. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0213] In some embodiments, the cancer is selected from lung cancer, intestinal cancer, kidney cancer, bladder cancer, liver cancer, stomach cancer, breast cancer, colon cancer, cervical cancer, prostate cancer, and head and neck cancer.

[0214] In some embodiments, the TGFβ1 binding molecules or pharmaceutical compositions of the disclosure can be administered by any suitable method known in the art, and administration can be systemic or local.

[0215] In some embodiments, the dosage regimen can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response), e.g., a single administration, multiple administrations over a period of time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0216] In some embodiments, the present disclosure provides (1) inhibiting TGFβ1 activity, such as inhibiting TGFβ1 activation, inhibiting release of mature TGFβ1 from the TGFβ1 complex, and / or inhibiting TGFβ1 signaling; (2) preparation of an agent for inhibiting TGFβ1 activity; (3) Inhibition of the immunosuppressive activity of regulatory T cells (4) Preparation of a drug for inhibiting the immunosuppressive activity of regulatory T cells; (5) Use of a GARP-TGFβ1 binding molecule, an encoding polynucleotide, a vector, or a pharmaceutical composition in at least one of the prevention and treatment of a disease or condition associated with the TGFβ signaling pathway is provided.

[0217] In some embodiments, the disclosure provides a method for preventing or treating a disease or condition associated with the TGFβ signaling pathway, the method comprising administering to a subject a prophylactically or therapeutically effective amount of a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition.

[0218] In some embodiments, the disclosure provides a method for preventing or treating a disease associated with the TGFβ signaling pathway, the method comprising administering to a subject a prophylactically or therapeutically effective amount of a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition, and a prophylactically or therapeutically effective amount of an immune checkpoint inhibitor.

[0219] Exemplary immune checkpoint inhibitors include, but are not limited to, inhibitors of one or more of PD-1, PD-L1, and PD-L2, hi some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody or antigen-binding fragment thereof.

[0220] In some embodiments, the disclosure provides methods for inhibiting TGFβ1 activity in vitro, the methods comprising administering a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition in vitro. In some specific embodiments, an inhibitory effective amount of a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition is administered.

[0221] In some embodiments, the disclosure provides methods for inhibiting TGFβ1 activation or inhibiting release of mature TGFβ1 from a GARP-TGFβ1 complex in vitro, the method comprising administering a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition in vitro. In some specific embodiments, an inhibitory-effective amount of a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition is administered.

[0222] In some embodiments, the disclosure provides methods for inhibiting TGFβ1 activity in vivo, the methods comprising administering a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition to a subject. In some specific embodiments, an inhibitory-effective amount of a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition is administered to a subject.

[0223] In some embodiments, the present disclosure provides methods for inhibiting TGFβ1 activation or inhibiting release of mature TGFβ1 from a GARP-TGFβ1 complex in vivo, the methods comprising administering a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition to a subject. In some specific embodiments, an inhibitory-effective amount of a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition is administered to the subject.

[0224] In some specific embodiments, the subject suffers from a disease or condition associated with the TGFβ signaling pathway.

[0225] In some embodiments, the disease associated with the TGFβ signaling pathway is cancer.

[0226] In some embodiments, the cancer is selected from lung cancer, intestinal cancer, kidney cancer, bladder cancer, liver cancer, stomach cancer, breast cancer, colon cancer, cervical cancer, prostate cancer, and head and neck cancer.

[0227] In some embodiments, a GARP-TGFβ1 binding molecule or pharmaceutical composition of the disclosure can be administered by any suitable method known in the art, and administration can be systemic or local.

[0228] In some embodiments, the dosage regimen can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response), e.g., a single administration, multiple administrations over a period of time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0229] definition In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this disclosure, all other technical and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art.

[0230] Unless the context clearly indicates otherwise, throughout the specification and claims, the words "comprises," "having," "including," and the like are to be understood to have an inclusive meaning, i.e., "including but not limited to," rather than an exclusive or exhaustive meaning.

[0231] "Optionally" or "optionally" means that the subsequently described event or circumstance may occur, but need not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0232] "About" or "approximately" refers to a numerical value that is within an acceptable error range for the specified value as determined by one of ordinary skill in the art, as determined by how the numerical portion is measured or determined (i.e., the limitations of the measurement system). For example, "about" may mean within or more than 1 standard deviation. Alternatively, "about" or "essentially including" may mean a variation within a range of at most 20%, e.g., between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%, and in this disclosure, whenever the term "about" appears before a number or numerical range, the specified number of embodiments is included. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" or "essentially including" should be assumed to be within an acceptable error range for the specified value.

[0233] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0234] The term "transforming growth factor-β" family comprises a class of structurally and functionally related polypeptide growth factor subfamilies that are involved in many different biological pathways. In addition to TGF-β, it also includes activins, inhibins, growth and differentiation factors (GDFs), bone morphogenetic proteins (BMPs), etc. TGF-β has three isoforms, TGF-β1, TGF-β2, and TGF-β3, and is widely expressed in almost all mammalian cell types.

[0235] Unlike other cytokines, TGF-β superfamily members are not secreted as active growth factors but instead as dimeric precursor proteins consisting of an N-terminal prodomain and a C-terminal growth factor domain. Pro-TGF-β1 undergoes furin cleavage to separate the homodimeric growth factor domain from its prodomain (also called latency-associated peptide (LAP)). However, the growth factor and LAP remain noncovalently bound, forming a latent complex that cannot bind to its receptor and trigger signaling. During translation, latent TGF-β1 (also called small latent complex (SLC)) links to a "presentation molecule" through disulfide bridges, thereby forming a large latent complex (LLC). These molecules enable the presence of pro-TGF-β1 in specific cellular or tissue contexts. Two cysteines proximal to the N-terminus of latent TGF-β1 link to appropriately positioned cysteines on the presentation molecule. The identity of the presentation molecule depends on the environment and the cell type producing latent TGF-β1. For example, fibroblasts secrete latent TGFβ1 linked to TGFβ-binding proteins (LTBPs), which then bind to proteins in the extracellular matrix (ECM) (i.e., fibronectin, fibulin-1) to tether latent TGFβ to the ECM (Robertson et al., Matrix Biol 47:44-53 (2015)). On the surface of activated regulatory T cells, latent TGFβ1 is covalently bound to the transmembrane protein GARP, and GARP, a protein closely related to LRRC33, has recently been identified as a presenting molecule for TGFβ1 on the surface of monocytes, macrophages, and microglia (Wang, R. et al., Mol Biol Cell, 2012.23(6):pp.1129-39 and TA Springer, Int. BMP Conference 2016).

[0236] Within the scope of the present disclosure, TGFβ1 protein should be understood in the broadest sense. This term encompasses naturally occurring forms of TGFβ1 in nature, naturally occurring mutants, as well as artificially expressed forms, functional mutants, etc. Unless otherwise specified in the context, TGFβ1 encompasses mature TGFβ1, TGFβ1 precursor protein, and a range of protein epitopes thereof. TGFβ1 precursor protein encompasses pro-TGFβ1, latent TGFβ1, and fragments thereof. TGFβ1 sequences can be obtained from GenBank, UniProt, etc. Examples include human pro-TGFβ1 (Uniprot: P01137) and mouse pro-TGFβ1 (Uniprot: P04202).

[0237] In the context of the present disclosure, "pro-TGFβ1" and "latent TGFβ1" are used interchangeably.

[0238] As used herein, the term "TGFβ1 complex" refers to a complex formed by disulfide cross-linking between TGFβ1 and a protein molecule, including, but not limited to, GARP, LRRC33, LTBP3, or LTBP1, and is also referred to as an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, an LRRC33-TGFβ1 complex, or a GARP-TGFβ1 complex. In the TGFβ1 complex, TGFβ1 exists in the form of a TGFβ1 precursor protein (e.g., pro / latent TGFβ1).

[0239] The term "functional variant" includes, but is not limited to, homologues, fragments, truncations, mutants, modifications, etc. of a wild-type protein, where the functional variant of the protein has an enhanced, reduced or maintained activity of the protein compared to the wild-type protein.

[0240] The term "binding molecule" encompasses any molecule capable of specifically binding to an antigen or antigenic epitope, for example, an antibody, an antigen-binding fragment thereof or a conjugate thereof, or a fusion protein as defined in this disclosure.

[0241] The term "antibody" is used in the broadest sense to refer to any antibody that exhibits the desired antigen-binding activity, and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments or antigen-binding portions). Antibodies may also refer to immunoglobulins, which have a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Immunoglobulins differ in the amino acid composition and sequence of the heavy chain constant regions, resulting in different antigenicities. Therefore, immunoglobulins can be divided into five types, or immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses based on differences in the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains and λ chains based on differences in the constant region. Each of the five types of Ig may have either κ chains or λ chains. In antibody heavy and light chains, the sequence of approximately 110 amino acids near the N-terminus is highly variable and forms the variable region (V region), while the remaining amino acid sequence near the C-terminus is relatively stable and forms the constant region (C region). The variable region contains three hypervariable regions (CDRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0242] The determination or definition of CDRs can be achieved by solving the structure of the antibody and / or the structure of the antibody-ligand complex, thereby accurately delineating the CDRs and identifying the residues that comprise the antibody's binding site. This can be accomplished by any one of a variety of techniques known to those of skill in the art, such as X-ray crystallography. Various analytical methods can be used to identify CDRs, including, but not limited to, the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions.

[0243] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but takes into account the location of certain structural loop regions (see, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses an integrated suite of computer programs from the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK, Oxford Molecular, Ltd.). The AbM numbering system models the tertiary structure of antibodies from the base sequence using a combination of knowledge databases and ab initio methods (see, e.g., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in Samudrala et al., 1999, PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In conformational definitions, CDR positions can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).Additionally, the definition of the boundaries of other CDRs may not strictly follow one of the above methods, but may be shortened or extended depending on predictions or experimental results that show that a particular residue or group of residues does not significantly affect antigen binding, while still overlapping with at least a portion of the Kabat CDRs. As used in this disclosure, CDR can refer to a CDR defined by any method (including a combination of methods) known in the art. The correspondence between each numbering system is well known to those skilled in the art and is illustratively shown in Table 4 below.

[0244] [Table 4-1] [Table 4-2]

[0245] A "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of the protein and can often be added, removed, or transferred to other proteins without loss of function of the other parts and / or domains of the protein.

[0246] An "immunoglobulin variable domain" refers to an immunoglobulin domain that is essentially composed of four "framework regions," referred to herein and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, wherein the framework regions are spaced apart by three "complementarity-determining regions" or "CDRs," referred to herein and hereinafter as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. The general structure or sequence of an immunoglobulin variable domain may therefore be depicted as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain contains the antigen-binding site and thus confers specificity for the antigen.

[0247] "Antibody framework (FR)" refers to the part of a variable domain that serves as a support for the antigen binding loops (CDRs) of that variable domain.

[0248] The term "immunoglobulin single variable domain" is typically used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain and comprises a VH, VHH or VL domain) that is capable of forming a functional antigen-binding site when it does not interact with other variable domains (e.g., in the absence of the necessary VH / VL interactions between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domains" are nanobodies (including VHH, camelized VH such as humanized VHH and / or camelized human VH), IgNARs, domains, (single domain) antibodies (e.g., dAbs) as or derived from a VH domain. TM ) and (single domain) antibodies (e.g., dAbs) as or derived from the VL domain. TM) Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are generally preferred. One specific example of an immunoglobulin single variable domain is a "VHH domain" (or abbreviated "VHH"), defined as follows:

[0249] "VHH domain" refers to a heavy chain single domain antibody, VHH, V H H domains, also known as VHH antibody fragments, VHH antibodies, or nanobodies, are variable domains of antigen-binding immunoglobulins called "heavy-chain antibodies" (i.e., "light-chain-depleted antibodies") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy-chain variable domain (referred to in the present disclosure as the "VH domain") and light-chain variable domain (referred to in the present disclosure as the "VL domain") present in conventional tetrapeptide chain antibodies. VHH domains specifically bind to an epitope without the need for another antigen-binding domain (this is the opposite of the VH or VL domains in conventional tetrapeptide chain antibodies, where the epitope is recognized by both the VL and VH domains). VHH domains are small, stable, and efficient antigen recognition units formed by a single immunoglobulin domain. HThe terms "H domain," "VHH antibody fragment," "VHH antibody," "Nanobody," and "Nanobody domain" may be used interchangeably. A "VHH domain" includes, but is not limited to, natural antibodies produced by camelids, antibodies produced by camelids and then humanized, or fully human antibodies screened by phage display technology. The total number of amino acid residues in a VHH domain is typically in the range of 110-120, and often between 112-115. However, it should be noted that both relatively short and relatively long sequences may also be suitable for the purposes described in this disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have previously been disclosed in the following publications: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, June 17, 2009; and WO94 / 04678.

[0250] As is known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions based on the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows). This generally means that the Kabat numbering may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or conventions include Chothia, IMGT, and AbM.

[0251] A "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting nonhuman CDR sequences onto a human antibody variable region framework. This antibody can overcome the strong immune response induced by chimeric antibodies containing a large amount of nonhuman protein components. To avoid reduced activity associated with reduced immunogenicity, activity can be maintained by performing minimal back mutations on the fully human antibody variable region. An example of "humanization" includes a VHH domain derived from a camelid, which can be "humanized" by replacing one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a normal human tetrapeptide chain structure antibody (also referred to as "sequence optimization" in the present disclosure, which may include, in addition to humanization, one or more mutations added to the sequence to confer improved properties of the VHH, such as removal of potential post-translational modification sites). A humanized VHH domain may contain one or more fully human framework region sequences. Furthermore, in order to avoid a decrease in activity associated with a decrease in immunogenicity, the activity can be maintained by performing minimal back mutations or reverse mutations on the framework sequences of the variable regions of the human antibody.

[0252] A "fully human antibody" or "fully human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Fully human antibodies according to the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). A "fully human antibody" does not include a "humanized antibody."

[0253] "Competition," when used in reference to competition between antigen-binding proteins for the same epitope (e.g., neutralizing antigen-binding protein or neutralizing antibody), refers to competition between antigen-binding proteins, as measured by the prevention or inhibition (e.g., reduction) of specific binding between a reference antigen-binding protein (e.g., a ligand or reference antibody) and a common antigen by the antigen-binding protein to be detected (e.g., an antibody or immunologically functional fragment thereof). A variety of competitive binding assays can be used to determine whether one antigen-binding protein competes with another, including, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahl et al., 1983, Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct label assays, solid-phase direct label sandwich assays (see, e.g., Harlow & Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct label RIA using an I-125 marker (see, e.g., Morel et al., 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), and the like. These assays include the direct biotin-avidin solid-phase EIA (see, e.g., Cheung et al., 1990, Virology 176:546-552), and the direct labeling RIA (see, e.g., Cheung et al., 1990, Scand. J. Immunol. 32:77-82). These assays typically involve the use of purified antigens (the antigens are on a solid surface or cell surface) that are capable of binding to an unlabeled detection antigen-binding protein and a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the antigen-binding protein to be measured. The antigen-binding protein to be measured is typically present in excess.Antigen-binding proteins identified by competitive assays (competing antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein and antigen-binding proteins that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antigen-binding protein so that the two epitopes spatially interfere with each other's binding. Typically, when a competing antigen-binding protein is present in excess, specific binding between the reference antigen-binding protein and a common antigen is inhibited (e.g., reduced) by at least 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, or 75% or more. Binding may also be inhibited by at least 80% to 85%, 85% to 90%, 90% to 95%, 95% to 97%, or 97% or more.

[0254] Conventional techniques known to those skilled in the art can be used to competitively screen antibodies for binding to the same epitope. For example, competition and cross-competition studies can be performed to obtain antibodies that bind to an antigen by competing with or cross-competing with each other. A high-throughput method for obtaining antibodies that bind to the same epitope by cross-competition is described in International Patent Publication WO 03 / 48731. Therefore, conventional techniques known to those skilled in the art can be used to obtain antibodies that compete with the antibody molecules of the present disclosure for binding to the same epitope on an antigen protein.

[0255] The term "glycoprotein-A repeat predominant (GARP)" refers to a protein with a single transmembrane structure. GARP is also called leucine-rich repeat containing 32 (LRRC32) and belongs to the leucine-rich repeat family. GARP is expressed on the cell surface of activated Tregs and can form a complex with TGF-β precursors (e.g., latent TGF-β). GARP sequences can be obtained from GenBank, UniProt, etc. Examples include human GARP (LRRC32, Uniprot:Q14392) and mouse GARP (LRRC32, Uniprot:G3XA59).

[0256] Within the scope of this disclosure, GARP should be understood in the broadest sense. The term encompasses naturally occurring forms of GARP in nature, naturally occurring variants, as well as artificially expressed forms, functional variants, etc. Unless otherwise indicated by the context, when referring to antigen-antibody interactions, GARP encompasses the complete protein, the extracellular domain, and its epitope coverage.

[0257] The terms "GARP-TGFβ1 complex," "GARP / TGFβ1 protein," and "GARP / TGFβ1" refer to a protein complex comprising a precursor protein of transforming growth factor-β1 (TGFβ1) protein and a glycoprotein-A repeat as the major protein (GARP). In some embodiments, the proprotein or latent form of the TGFβ1 protein can be referred to as a "pro / latent TGFβ1 protein." In some embodiments, a GARP-TGFβ1 complex comprises a GARP covalently bound to a TGFβ1 precursor protein (pro / latent TGFβ1 protein) by one or more disulfide bonds. In other embodiments, a GARP-TGFβ1 complex comprises a GARP non-covalently bound to a TGFβ1 precursor protein (pro / latent TGFβ1 protein). In some embodiments, the GARP-TGFβ1 complex is a naturally occurring complex, e.g., a GARP-TGFβ1 complex in a cell.

[0258] The term "LTBP" refers to latent transforming growth factor β binding proteins. LTBPs are important components of the extracellular matrix (ECM), and their primary function is to regulate fibrillin and transforming growth factor β (TGF-β). There are four known LTBPs in mammals, LTBP1-4, each of which has multiple splice variants (Robertson, IB et al., Matrix Biol, 2015. 47: pp. 44-53).

[0259] The terms "GARP-TGFβ1 complex," "GARP / TGFβ1 protein," and "GARP / TGFβ1" refer to a protein complex comprising the precursor or latent form of transforming growth factor-β1 (TGFβ1) protein and glycoprotein-A repeat as the major protein (GARP). In some embodiments, the proprotein or latent form of the TGFβ1 protein can be referred to as the "pro / latent TGFβ1 protein." In some embodiments, the GARP-TGFβ1 complex comprises GARP covalently bound to the TGFβ1 precursor protein (pro / latent TGFβ1 protein) by one or more disulfide bonds. In other embodiments, the GARP-TGFβ1 complex comprises GARP noncovalently bound to the TGFβ1 precursor protein (pro / latent TGFβ1 protein). In some embodiments, the GARP-TGFβ1 complex is a naturally occurring complex, e.g., a GARP-TGFβ1 complex in a cell.

[0260] The term "LTBP1-TGFβ1 complex" refers to a protein complex comprising a precursor protein of transforming growth factor β1 (TGFβ1) protein and a latent TGFβ-binding protein (e.g., LTBP1, LTBP3). In some embodiments, the LTBP-TGFβ1 complex comprises LTBP1 covalently bound to the TGFβ1 precursor protein by one or more disulfide bonds. In other embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 non-covalently bound to the TGFβ1 precursor protein. In some embodiments, the LTBP1-TGFβ1 complex is a naturally occurring complex, e.g., an LTBP1-TGFβ1 complex in a cell.

[0261] The term "antigen" refers to a molecule that is used to immunize an immunocompetent vertebrate to produce antibodies that recognize the antigen, or to screen an expression library (e.g., phage, yeast, or ribosome display libraries, among others). In the present disclosure, antigen is defined more broadly to include a target molecule that is specifically recognized by an antibody, and also includes portions or mimetics of molecules used in the immunization process to produce antibodies or in library screening to select antibodies. For example, antibodies of the present disclosure that bind to the human GARP-TGFβ1 complex, truncated mutants and other mutants of the human GARP-TGFβ1 complex, are all referred to as antigens.

[0262] The term "epitope" refers to the site on an antigen that binds to an immunoglobulin or antibody. Epitopes may be formed from contiguous amino acids or from juxtaposed non-contiguous amino acids formed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically contain at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody are well known in the art and include Western blotting and immunoprecipitation assays. Methods for determining the spatial conformation of epitopes include techniques known in the art and described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0263] "Specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. For example, when the human GARP-TGFβ1 complex or an epitope thereof is used as an analyte and an antibody is used as a ligand, and measurement is performed by surface plasmon resonance (SPR) technology in an instrument, the antibody binds to an epitope on a specific antigen at a specific binding site of about 10 -7 The equilibrium dissociation constant (K D) and its binding affinity to the predetermined antigen or its epitope is at least twice its binding affinity to a non-specific antigen other than the predetermined antigen (or its epitope) or a closely related antigen (e.g., BSA, etc.). The term "antibody that recognizes an antigen" may be used interchangeably with "antibody that specifically binds" in this disclosure.

[0264] "Binding affinity" or "affinity" is used in the present disclosure as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or a portion thereof and an antigen). The binding affinity between two molecules can be quantified by determining the dissociation constant (KD). KD can be determined, for example, by measuring the kinetics of complex formation and dissociation using surface plasmon resonance (SPR) (Biacore). The rate constants corresponding to the binding and dissociation of a monovalent complex are called the binding rate constant ka (or k) and the dissociation rate constant kd (or koff), respectively. K D is K D The dissociation constants ka and kd are related by the equation ka = kd / ka. The value of the dissociation constant can be determined directly by well-known methods and can also be calculated for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, the K can be calculated by double filtration nitrocellulose filter binding assays such as those disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432). D Other standard assays for assessing the binding ability of an antibody to a target antigen are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays mentioned elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can be determined by standard assays known in the art, such as surface plasmon resonance (SPR), e.g., Biacore. TM The K of each antibody / antigen complex may be evaluated by the KinExA system or KinExA. DBy comparing K values, it is possible to compare the binding affinities associated with interactions with different molecules, for example, to compare the binding affinities of different antibodies to a given antigen. Similarly, the specificity of an interaction can be determined by comparing the K values ​​of the interaction of interest (e.g., the specific interaction between an antibody and an antigen). D value and the K of a non-target interaction (e.g., a known control antibody that does not bind to the antigen). D It can be evaluated by determining and comparing values.

[0265] The terms "conservative substitution" and "conservative replacement" refer to the substitution of an amino acid residue with another amino acid residue having similar properties to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Furthermore, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it is clear to those skilled in the art that even when amino acid residues in the above-mentioned group showing similar properties are substituted, it does not show a specific change in properties.

[0266] The terms "homology," "identity," or "sequence identity" refer to the sequence similarity between two polynucleotide sequences or two polypeptides. When every position in two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, when each position in two DNA molecules is occupied by the same nucleotide, the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences, divided by the number of positions compared, multiplied by 100%. For example, if 6 out of 10 positions in two sequences are matched or homologous when the sequences are optimally aligned, the two sequences are 60% homologous. Typically, two sequences are compared when aligned to obtain the maximum percentage of homology.

[0267] The terms "nucleic acid molecule" and "polynucleotide" may be used interchangeably and refer to DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded DNA. A nucleic acid is "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of the coding sequence.

[0268] The term "host cell" includes each cell or cell culture that may be, or has been, a recipient of a vector for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, and progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected and / or transformed in vivo with a polynucleotide according to the present disclosure. "Cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include their progeny. It should also be understood that, due to deliberate or unintentional mutation, all progeny may not be precisely identical in DNA content. Mutant progeny that have the same function or biological activity as screened for from the originally transformed cell are included.

[0269] The terms "inhibition" and "blocking" may be used interchangeably and encompass both partial and complete inhibition / blocking. For example, an "inhibitory antibody" refers to an antibody that inhibits the release of a mature growth factor or reduces the activity of a growth factor. Inhibitory antibodies include antibodies that target any epitope that reduces the release or activity of a growth factor when bound to such an antibody. Such epitopes can be located in the prodomain of a TGFβ protein (e.g., TGFβ1), the growth factor, or other epitopes that reduce the activity of a growth factor when bound to the antibody. Inhibitory antibodies of the present invention include, but are not limited to, TGFβ1-inhibitory antibodies.

[0270] An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disorder. Effective amount further refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used in a subject can vary depending on factors such as the condition being treated, the subject's overall health, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or human subject.

[0271] The term "pharmaceutical composition" refers to a mixture of one or more active ingredients described herein or physiologically / medicinally acceptable salts or prodrugs thereof with other chemical components, and other components such as physiologically / medicinally acceptable carriers and excipients, to facilitate administration to the body and contribute to the absorption of the active ingredients to further exert their biological activity.

[0272] The term "pharmaceutically acceptable additive" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions such as oil-in-water emulsions, and various wetting agents. In some embodiments, the diluent used for aerosol or parenteral administration is phosphate-buffered saline (PBS) or normal (0.9%) saline. Compositions containing such vectors are prepared by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990, and R. Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0273] "Cancer," "cancerous," "proliferative condition," and "tumor" are not mutually exclusive when referred to in this disclosure.

[0274] The terms "giving," "administration," and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to the contact of an exogenous agent, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, e.g., therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contact of a reagent with a cell and contact of a reagent with a fluid, where the fluid contacts the cell. "Giving," "administration," and "treatment" also refer to ex vivo and in vitro treatment, e.g., of cells, with a reagent, diagnostic, binding composition, or through another cell. When applied to a human, veterinary, or research subject, they refer to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic uses.

[0275] The term "treatment" refers to administering a therapeutic agent, e.g., one comprising any one of the fusion proteins or insulin analogs disclosed herein, to a subject suffering from, at risk of, or prone to one or more diabetes- or hyperglycemia-related diseases or symptoms thereof, where the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is provided to the subject or population being treated in an amount that effectively alleviates one or more disease symptoms by preventing or delaying the onset of symptoms or complications, alleviating symptoms or complications, or eliminating the disease, condition, or disease to any clinically measurable extent. The amount of therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary depending on several factors, including the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Reduction of disease symptoms can be assessed by any clinical detection method commonly used by physicians or other professional healthcare providers to assess the severity or progression of the condition. Although embodiments of the present disclosure (e.g., therapeutic methods or products) may be ineffective in alleviating target disease symptoms in some subjects, they should alleviate target disease symptoms in a statistically significant number of subjects, as determined by any statistical testing method known in the art, such as, for example, Student's t-test, chi-square test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test. The patient to be treated is a mammal, and preferably a human.

[0276] The term "prevention" refers to reducing the risk or incidence of one or more conditions, symptoms, complications or diseases, or eliminating or alleviating the progression of one or more conditions, symptoms, complications or diseases.

[0277] The terms "subject" and "patient" refer to mammals, particularly primates, and especially humans. [Brief explanation of the drawings]

[0278] [Figure 1] Figure 1 shows the results of detecting the inhibition of activity of human or mouse TGFβ1 complexes (human proTGFβ1, GARP-TGFβ1 complex, LRRC33-TGFβ1 complex, and mouse GARP-TGFβ1 complex) by anti-TGFβ1 antibodies (H27, SL2-2, SL2-9, SL2-12, SL2-19, and SL2-22). In Figure 1, A is a graph showing the inhibitory effect of antibodies on human proTGFβ1 activation, B is a graph showing the inhibitory effect of antibodies on human GARP-TGFβ1 complex activation, C is a graph showing the inhibitory effect of antibodies on human LRRC33-TGFβ1 complex activation, and D is a graph showing the inhibitory effect of antibodies on mouse GARP-TGFβ1 complex activation. [Figure 2] This figure shows the results of an experiment to detect the binding of an anti-TGFβ1 antibody (SL2-22, Ab6) to HEK293E cells expressing human or mouse pro-TGFβ1, TGFβ2, TGFβ3, and TGFβ complexes (GARP-TGFβ1 complex, GARP-TGFβ2 complex, GARP-TGFβ3 complex). [Figure 3A] ~ [Figure 3C] This shows a graph of the results of tumor growth inhibition in a mouse EMT-6 model by the combination of an anti-TGFβ1 antibody and an anti-PD-1 antibody (RMP1-14-mIgG2a-FcS+SL2-22-mIgG2a-FcS). [Figure 3A] FIG. 1 is a graph showing tumor volume curves in mice. [Figure 3B] FIG. 1 is a graph showing mouse survival rate curves. [Figure 3C] FIG. 1 is a graph showing the body weight curve of mice. [Figure 4A] ~ [Figure 4C] This shows a graph of the results of tumor growth inhibition in a mouse CT26 model by the combination of an anti-TGFβ1 antibody and an anti-PD-1 antibody (RMP1-14-mIgG2a-FcS+SL2-22-mIgG2a-FcS). [Figure 4A] FIG. 1 is a graph showing tumor volume curves in mice. [Figure 4B] FIG. 1 is a graph showing mouse survival rate curves. [Figure 4C] FIG. 1 is a graph showing the body weight curve of mice. [Figure 5A] ~ [Figure 5E] FIG. 1 shows a graph showing the results of fibrosis inhibition in a mouse idiopathic fibrosis model by anti-TGFβ1 antibody (SL2-22-mIgG2a-FcS). [Figure 5A] 1 shows a weight change curve of a pulmonary fibrosis model mouse. [Figure 5B] 1 shows a survival curve of a pulmonary fibrosis model mouse. [Figure 5C] 1 shows lung weight and lung tissue hydroxyproline content in mice at the end of the pulmonary fibrosis model experiment. [Figure 5D] This is a lung section from a mouse at the end of a pulmonary fibrosis model experiment. [Figure 5E] Masson stained collagen area ratio. [Figure 6] 1 shows the detection of inhibition of GARP-TGFβ1 complex activation by anti-GARP-TGFβ1 single domain antibodies in an LN229 experiment. LN229 cells were transfected with human or mouse pro-TGFβ1 and human or mouse GARP expression plasmids, and the inhibition of human or mouse GARP-TGFβ1 complex activation by Abbv-151, C19, C19-3, C19-7, and C19-8 was detected using HepG2 CAGA12-luc luciferase reporter cells. [Figure 6A] FIG. 10 is a graph showing the inhibitory effect of antibodies on the activation of the human GARP-TGFβ1 complex. [Figure 6B] FIG. 10 is a graph showing the inhibitory effect of antibodies on the activation of mouse GARP-TGFβ1 complex. [Figure 7] This figure shows the results of a binding experiment of C19-8 to HEK293E cells expressing human or mouse GARP-TGFβ1 complexes. HEK293E cells were transfected with the plasmids in Table 3, and then C19-8 and Abbv-151 were added, followed by staining and detection, and the results were obtained. [Figure 8A] ~ [Figure 8C]This shows a graph of tumor growth inhibition in the mouse EMT-6 model by the combination of C19-8 and an anti-PD-1 antibody (RMP1-14-mIgG2a-FcS+C19-8-mIgG2a-FcS). [Figure 8A] FIG. 1 is a graph showing tumor volume curves in mice. [Figure 8B] FIG. 1 is a graph showing mouse survival rate curves. [Figure 8C] FIG. 1 is a graph showing the body weight curve of mice. [Figure 9A] ~ [Figure 9C] This shows a graph of tumor growth inhibition in a mouse CT26 model by the combination of C19-8 and an anti-PD-1 antibody (RMP1-14-mIgG2a-FcS+C19-8-mIgG2a-FcS). [Figure 9A] FIG. 1 is a graph showing tumor volume curves in mice. [Figure 9B] FIG. 1 is a graph showing mouse survival rate curves. [Figure 9C] FIG. 1 is a graph showing the body weight curve of mice. DETAILED DESCRIPTION OF THE INVENTION

[0279] Example The present disclosure will be further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure.

[0280] Experimental methods for which specific conditions are not specified in the examples or experimental examples of this disclosure generally follow conventional conditions or conditions recommended by the manufacturers of materials or products. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, and Modern Methods in Molecular Biology, Ausubel et al., Greene Publishing Company, Wiley Interscience, NY. Reagents for which specific sources are not specified are conventional, commercially available reagents.

[0281] Example 1. Preparation of antigen and detection protein Human pro-TGFβ1 (Uniprot: P01137), human GARP (LRRC32, Uniprot: Q14392), mouse pro-TGFβ1 (Uniprot: P04202), and mouse GARP (LRRC32, Uniprot: G3XA59) were used as templates to design the amino acid sequences of related proteins for screening and detection.

[0282] >Full length amino acid sequence of human pro-TGFβ1: [ka] >Mature human TGFβ1 (mature transforming growth factor beta-1): [ka] >Human LAP: [ka] >Full length amino acid sequence of human GARP: [ka] >Human GARP extracellular domain: [ka] >Mouse pro-TGFβ1 full-length amino acid sequence: [ka] >Mature mouse TGFβ1: [ka] > Mouse LAP: [ka] >Full-length mouse GARP amino acid sequence: [ka] >Mouse GARP extracellular domain: [ka] The human GARP / TGFβ1 complex for screening and detection was prepared as follows: human GARP-avi-his and human native TGFβ1 (shown in SEQ ID NOs: 5 and 6, respectively) were coexpressed in ExpiCHO cells (ThermoFisher, A29127) and purified to obtain the protein complex, the human TGFβ1 C4S protein of which is shown in SEQ ID NO: 7. The mouse GARP / TGFβ1 complex was prepared as follows: mouse GARP-avi-his and mouse native TGFβ1 (shown in SEQ ID NOs: 8 and 9, respectively) were coexpressed in ExpiCHO cells (ThermoFisher, A29127) and purified to obtain the protein complex, the mouse TGFβ1 C4S protein of which is shown in SEQ ID NO: 10.

[0283] Human GARP-avi-his amino acid sequence: [ka] >Human TGFβ1-Native amino acid sequence: [ka] >Human TGFβ1 C4S amino acid sequence: [ka] >Mouse GARP-avi-his amino acid sequence: [ka] >Mouse TGFβ1-Native amino acid sequence: [ka] >Mouse TGFβ1 C4S amino acid sequence: [ka] All of the above sequences can be obtained by expression, purification and isolation using methods commonly used in this field.

[0284] Example 2. Specific binding to human TGFβ1 complex Anti-TGFβ1 monoclonal antibody Screening Antibodies with high affinity for the human TGF-β1 complex were obtained by screening four human antibody phage libraries: 1) semi-synthetic human Fab library 1 (germline 3-23), 2) semi-synthetic human Fab library 2 (germline 1-69), 3) fully human naive Fab library, and 4) fully human scFV library, all of which were kindly provided by Shanghai Hengrui Pharmaceutical Co., Ltd. Ten micrograms of biotinylated human GARP-TGFβ1 complex (containing SEQ ID NOs: 5 and 6) protein was bound to 1 mg of Dynabeads M-280 streptavidin (Cat. No. 11206D, Invitrogen), and the mixture was incubated at room temperature for 0.5 hours. After washing three times with 1×PBS, 2% skim milk was added and the mixture was blocked at room temperature for 1 hour. At the same time, 2% skim milk and 1 mg of Dynabeads M-280 streptavidin were used to block and deplete the human antibody phage display library. The phage library was then added to the antigen-bound beads and incubated at room temperature for 1 hour. After 1x PBST (containing 0.05% Tween-20) was used to remove unbound phages, the cells were washed 10 times with a pH 7.4 solution and two more times with 1x PBS. Phages that specifically bound to human GARP / TGFβ1 were then eluted with 0.5 mL of trypsin (1 mg / mL), infected into logarithmic-phase E. coli TG1, and grown overnight on 2YT (containing 2% glucose) resistant plates. Phages were produced and purified from the plates and used for the next round of screening.

[0285] The same screening process was repeated two rounds. To obtain antibodies that cross-link with the mouse TGFβ1 complex, biotinylated mouse TGFβ1_C4S (SEQ ID NO: 10) was used as the screening antigen in the second round. Biotinylated human GARP-TGFβ1 complex (comprising SEQ ID NOs: 5 and 6) was used as the screening antigen in the third round. After three rounds of screening, positive clones were enriched.

[0286] From the screened and enriched clones, 6x92 monoclonals were selected and packaged into monoclonal phages for phage ELISA testing. ELISA plates (Cat. No. 9018, Corning) were coated with 2 μg / mL of human GARP-TGFβ1 complex (SEQ ID NOs: 5 and 6) and mouse TGFβ1_C4S (SEQ ID NO: 10) protein, respectively, and incubated overnight at 4°C. After washing three times with 1×PBST, the plates were blocked with 2% BSA at 37°C for 1 hour. After washing three times with PBST, phage supernatant diluted with blocking solution was added and incubated at room temperature for 1 hour. After washing six times with PBST, anti-M13 HRP (Cat. No. 11973-MM05T-H, Yiqiao Shenzhou) was added and incubated at room temperature for 1 hour. After washing three times with PBST, 100 μL of TMB chromogenic substrate was added. The reaction was stopped with 100 μL of 1 M sulfuric acid, and the absorbance at 450 nm was read using a SpectraMax M5 plate reader. Clones with OD450 readings three times higher than the negative control in ELISA binding assays were sequenced, and 43 specific antibody sequences were identified. SPR experiments showed that antibodies H5, H14, H16, H17, H19, H20, H21, H23, H24, H27, H28, H30, H32, H34, and H39 could bind to the TGFβ1 complex. However, cellular function experiments showed that only H27 had a relatively good inhibitory function and could effectively bind to both human and mouse GARP-TGFβ1 complexes and human and mouse TGFβ1_C4S (i.e., TGFβ1 precursor protein).

[0287] The H27 sequence here is shown below.

[0288] >H27 VH [ka] >H27 VL [ka] In the sequences of SEQ ID NOs: 11 and 12, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italics in the sequences indicate FR sequences, and the underlines indicate CDR1, CDR2, and CDR3 sequences, respectively. The numbering convention for the human antibodies provided by the present disclosure is Kabat, and Table 5 shows the CDR sequences.

[0289] [Table 5]

[0290] Example 3. Construction, expression and purification of intact monoclonal antibodies The sequence of H27 obtained by screening the human antibody phage library in Example 2 was used to construct a complete recombinant antibody: the light chain variable region (VL) of the antibody was cloned into a pTT5 expression vector containing the human kappa light chain constant region (SEQ ID NO: 19), and the heavy chain variable region (VH) of the antibody was cloned into a pTT5 expression vector containing the human IgG4-S228P (CH1-CH2-CH3) heavy chain constant region (SEQ ID NO: 20).

[0291] >Human kappa light chain constant region: [ka] >Human IgG4-S228P(CH1-CH2-CH3) heavy chain constant region: [ka] The H27 complete antibody sequence is as follows: >H27 light chain: [ka] >H27 heavy chain: [ka] The cloned light and heavy chain plasmids were paired and co-transfected into HEK293E cells (gift from Shanghai Hengrui Pharmaceutical Co., Ltd.) or ExpiCHO (Cat No. A29127, ThermoFisher) cells. Cell culture supernatants were collected on day 5 (37°C, HEK293E cells) or day 10–12 (32°C, expiCHO cells) post-transfection, centrifuged at 4000 rpm for 20 minutes, and filtered through a 0.45 μm filter. The first affinity purification step was then performed using a MabSelectSure LX column (GE Healthcare). The culture supernatant was loaded onto a PBS-equilibrated MabSelectSure LX column and washed with PBS. The target protein was eluted with 0.1 M glycine at pH 3.0, neutralized with 1 M MES (pH 6.0), and finally purified using a HiTrap SP HP ion column. Detection yielded target antibodies.

[0292] Example 4. Affinity measurement of TGFβ1 monoclonal antibody The affinity of the antibodies to human / mouse GARP / TGFβ1 and human / mouse TGFβ1_C4S was measured using Biacore T200 (GE Healthcare).

[0293] A fixed amount of antibody to be measured was affinity captured using a Protein A biosensor chip (Cat. No. #29127556, GE). A series of gradient dilutions of human or mouse GARP-TGFβ1 complex or TGFβ1_C4S antigen was then passed over the chip surface at a flow rate of 50 μL / min, with a dissociation time of 5 min. After each cycle, the chip was regenerated with glycine-HCl (Cat. No. BR-1003-54, GE), pH 1.5. The reaction buffer was HBS-EP+ buffer solution (Cat. No. BR-1006-69, GE), pH 7.4, diluted to 1x with distilled water.

[0294] The binding-dissociation curves were obtained by detecting the reaction signals in real time using Biacore T200, and the obtained data were fitted with the Langmuir 1:1 binding model using BIAevaluation version 4.1, GE software, to obtain affinity values, and the results are shown in Table 6.

[0295] [Table 6]

[0296] Example 5. Affinity maturation of TGFβ1 monoclonal antibody H27 Three-dimensional structural simulations were performed on the H27 antibody molecule. Based on the results of human germline gene mutation hotspots and three-dimensional structural simulations, important amino acid residues were selected in the framework and CDR regions, and four randomly mutated phage libraries were established (Table 7). Functional antibodies with improved affinity were screened using phage library display technology. The new amino acid residues obtained from the different libraries were combined and validated to obtain functional antibodies with improved affinity and function.

[0297] [Table 7]

[0298] Four random mutation phage libraries were screened, and biotinylated human GARP-TGFβ1 (including SEQ ID NOs: 5 and 6) was used as the screening antigen. After three to four rounds of screening, the resulting clones were sequenced. Based on the phage ELISA results, 62 antibody sequences were selected for clone construction. The antibody light chain variable region (VL) was cloned into a pTT5 expression vector containing the human kappa light chain constant region (SEQ ID NO: 19), and the antibody heavy chain variable region (VH) was cloned into a pTT5 expression vector containing the human hIgG4-S228P (CH1-CH2-CH3) heavy chain constant region (SEQ ID NO: 20).

[0299] The cloned light chain and heavy chain plasmids were paired and co-transfected into HEK293E cells. After 5 days, the cell culture supernatant was collected and centrifuged at 4000 rpm to remove cells. The supernatant was purified using MabSelect Sure, and affinity measurements were performed according to the method described in Example 4.

[0300] All of the antibodies with affinities five times or more higher than H27, as determined by SPR protein interaction, were from the second random mutation library, and the antibody heavy chains in this library were H27 heavy chains. The sequences of their light chain variable regions are shown in Table 8 (the underlined portions are CDR regions using the Kabat numbering convention). The full-length light chain sequences and CDR combinations are shown in Tables 9 and 10, respectively.

[0301] [Table 8-1] [Table 8-2] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 10-1] [Table 10-2]

[0302] H27, SL2-1, SL2-2, SL2-3, SL2-4, SL2-5, SL2-6, SL2-8, SL2-9, SL2-12, SL2-13, SL2-15, SL2-17, SL2-18, SL2-19, SL2-22 have the following general structure: LCDR1 is RASQX1ISX2YLN (SEQ ID NO: 38), wherein X1 is selected from S, A, F, G, I, P, Y, V, or K, and X2 is selected from S, D, E, P, or H; LCDR2 is X3ASX4LX5S (SEQ ID NO: 64), where X3 is selected from A, T, S, or M; X4 is selected from S, Y, A, E, or G; and X5 is selected from Q, T, D, or E.

[0303] SL2-2, SL2-9, SL2-12, SL2-19, and SL2-22 have the following general structure: LCDR1 is RASQX1ISX2YLN (SEQ ID NO: 38), where X1 is selected from F, Y, or A, and X2 is selected from D or P; LCDR2 is X3ASX4LX5S (SEQ ID NO: 64), where X3 is selected from A, T, or S; X4 is selected from S, Y, or E; and X5 is selected from Q, D, or E.

[0304] Example 6. Affinity measurement of TGFβ1 affinity-matured monoclonal antibodies The negative control in this example was HBS-EP, and the positive controls were Ab6 (Scholar Rock, WO2020014460Al) and Abbv-151 (Abbvie, US10793627B2). Ab6 binds to the GARP-TGFβ1 complex, LRRC33-TGFβ1 complex, LTBP3-TGFβ1 complex, and LTBP1-TGFβ1 complex, with the binding epitope located entirely on TGFβ1 (Martin et al., 2020). On the other hand, the binding epitope of Abbv-151 includes GARP and TGFβ1 in the GARP-TGFβ1 complex, and does not bind to GARP or TGFβ1 alone (Streel et al., 2020). The sequences of Ab6 and Abbv-151 are as follows: >Ab6 antibody heavy chain sequence: [ka] >Ab6 antibody light chain sequence: [ka] >Abbv-151 antibody heavy chain sequence: [ka] >Abbv-151 antibody light chain: [ka] Using Biacore T200 (GE Healthcare), the affinities of several affinity-matured monoclonal antibodies for human and mouse GARP-TGFβ1 complexes were measured according to the method of Example 4. The affinity measurement results are shown in Tables 11 and 12.

[0305] [Table 11] [Table 12]

[0306] Example 7. Inhibition of GARP-TGFβ1 complex function in vitro by TGFβ1 monoclonal antibodies 2×10 6 LN229 cells (Procell, CL-0578) were transferred to a T75 culture flask (Nunc) and transfected with 293- TM Human or mouse pro-TGFβ1 was transfected singly using the (Invitrogen) transfection reagent, or human or mouse pro-TGFβ1 and GARP or LRRC33 plasmids were co-transfected. After 24 h, cells were cultured at 1 × 10 4 The cells were transferred to a white opaque 96-well cell culture plate (PerkinElmer) at 1 / well. After 24 h, the medium was aspirated, and HepG2 CAGA12-luc luciferase reporter cells resuspended in DMEM + 0.5% BSA were added. Five-fold diluted H27, H27 affinity-matured antibody, and control antibody Ab6 were simultaneously added. After 20 h of co-culture, ONE-Glo TM Luciferase reagent was added, and after 5 minutes of incubation, chemiluminescence values ​​were read using a multifunction plate reader (SpectraMax M5). Luciferase activity in the solvent-treated group was normalized to 1, and relative activity was calculated as luciferase activity in the antibody-treated group divided by the solvent control group. Reaction models were plotted using Prism9 with nonlinear fitting and a three-parameter logarithmic inhibitor model.

[0307] As shown in Figures 1A and 1B, the affinity-matured SL2-2, SL2-9, SL2-12, SL2-19, and SL2-22 all had higher inhibitory activity against pro-TGFβ1 and TGFβ1 complexes than the parent H27. The inhibitory activity of SL2-2, SL2-9, SL2-12, SL2-19, and SL2-22 against human pro-TGFβ1, the GARP-TGFβ1 complex, and the mouse GARP-TGFβ1 complex was comparable to that of the positive control Ab6, but their inhibitory activity against the human LRRC33-TGFβ1 complex was superior to that of Ab6.

[0308] Example 8. Binding experiments of TGFβ1 monoclonal antibody SL2-22 to cells expressing different TGFβ complexes 1 × 10 HEK293E cells 6 5 mL of the solution was placed in a 50 mL culture tube and incubated with 293fectin TM The cells were transfected with the plasmids listed in Table 13 using the reagents. After 48 h, the cells were washed twice with 1x PBS and transfected with 1x10 5 Cells were placed in a 96-well round-bottom cell culture plate (Nunc) at 100 μL per well. 10 nM antibody or control antibody (100 μL, prepared in 1x PBS) was added and stained at room temperature for 1 hour. After washing three times with 1x PBS, 100 μL of FITC-labeled anti-human Fc antibody was added and stained at room temperature for 30 minutes. After staining, the cells were washed three times with 1x PBS. 7-AAD was added and incubated for 5 minutes. After washing three times with 1x PBS, cells were detected by flow cytometry. The number of FITC-positive cells among live cells was analyzed using Flowjo, and the percentage of FITC-positive cells out of the total number of live cells was calculated and plotted.

[0309] [Table 13-1] [Table 13-2]

[0310] As shown in Figure 2, the results showed that both SL2-22 and Ab6 bound to human and mouse TGFβ1 (corresponding to TGFβ1 and mTGFβ1, respectively, in Figure 2) and the GARP-TGFβ1 complex (corresponding to TGFβ1 + GARP and mTGFβ1 + mGARP, respectively, in Figure 2), but did not bind to TGFβ2 or the GARP-TGFβ2 complex (corresponding to TGFβ2 + GARP in Figure 2).The difference was that Ab6 also bound to some extent to human TGFβ3 and the GARP-TGFβ3 complex (corresponding to TGFβ3 + GARP in Figure 2), while SL2-22 specifically bound only to the TGFβ1 complex.

[0311] Example 9. Epitope competition studies of TGFβ1 antibodies The negative control in this example was HBS-EP, and the positive controls were Ab6 and Abbv-151.

[0312] Using Biacore T200 (GE Healthcare), competition between anti-human TGFβ1 antibody SL2-22 and a positive control antibody for the epitope of the human GARP-TGFβ1 complex was studied.

[0313] Using an amine coupling kit (Cat. No. BR-1000-50, GE) and a His capture reagent kit (Cat. No. 28-9950-56, GE), anti-His tag antibodies were coupled to a CM5 sensor chip (Cat. 29-1049-88, GE) according to the kit instructions. After the surface response value reached approximately 10,000 RU, the chip was blocked with ethanolamine and then prepared for use.

[0314] The epitope competition study steps (samples using duplex mode) are as follows (antibodies are paired in cycles of two): 1) Antigen capture: human GARP-TGFβ1 complex 2) First antibody: concentration 100 μg / mL, flow rate 50 μL / min, 60 seconds, HBS-EP buffer as control 3) Second antibody: concentration 100 μg / mL, flow rate 50 μL / min, 60 seconds, HBS-EP buffer as control 4) The sensorgrams were analyzed and it was found that SL2-22 partially competed with Ab6 for the binding epitope on the human GARP-TGFβ1 complex, but did not completely compete with Abbv151 for the binding epitope on the human GARP-TGFβ1 complex (Table 14).

[0315] [Table 14]

[0316] Example 10. Inhibition of tumor growth in the murine EMT-6 model by TGFβ1 monoclonal antibody SL2-22 To detect the tumor-inhibitory activity of SL2-22 in vivo, BALB / c mice (female, 6 weeks old, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were adapted for breeding for 1 week and then numbered and weighed on the day of the experiment. Logarithmically growing mouse breast cancer cells EMT-6 (Procell, CL-0573) were harvested, resuspended in PBS, and diluted to 5 × 10 6 The virus was inoculated subcutaneously into the right rib of BALB / c mice at a concentration of 0.1 mL / mouse at a concentration of 0.1 mL / mouse. Three days after inoculation, the average tumor volume was approximately 60 mm. 3 When the tumor volume reached 100 μg / mL, mice with an appropriate tumor volume were selected and randomly assigned to groups as shown in Table 15. mIgG2a-FcS was an mIgG2a Fc with ADCC effector-deleted L234A / L235E / G237A / D327Q / A330S / P331S as an isotype control, and RMP1-14-mIgG2a-FcS was a mIgG2a Fc with ADCC effector-deleted L234A / L235E / G237A / D327Q / A330S / P331S as an isotype control. RMP1-14-mIgG2a-FcS was a mIgG2a Fc with ADCC effector-deleted L234A / L235E / G237A / D327Q / A330S / P331S as an isotype control. SL2-22-mIgG2a-FcS is an antibody in which the variable region of SL2-22 is fused with an mIgG2a heavy chain constant region having L234A / L235E / G237A / D327Q / A330S / P331S / mouse kappa light chain constant region, from which the ADCC effector has been removed, and the sequences are as follows: >SL2-22-mIgG2a-FcS antibody heavy chain: [ka] >SL2-22-mIgG2a-FcS antibody light chain: [ka] The administration started on the day of grouping, and the same molar dose was administered twice a week for a total of seven times. The administration method was intraperitoneal injection. The body weight and tumor volume of the mice were measured twice a week, and the tumor volume was calculated using the formula TV = L 長 ×L 短 2 The tumor volume of each group was expressed as the mean ± standard deviation, and statistical analysis was performed using two-way ANOVA to calculate the tumor inhibition rate (%TGI), which was calculated using the formula: %TGI = [1 - (T - T0) / (C - C0)] × 100%.

[0317] As shown in Figures 3A and 3B and Table 15, the combination of SL2-22-mIgG2a-FcS and the anti-PD-1 antibody RMP1-14-mIgG2a-FcS significantly inhibited mouse EMT-6 tumor growth (p=0.0127, Two-way ANOVA) and prolonged animal survival (p=0.0323, Log-rank (Mantel-Cox) test) compared with RMP1-14-mIgG2a-FcS alone. The mice tolerated the treatment well and showed no significant weight loss during the course of treatment (Figure 3C).

[0318] [Table 15]

[0319] Example 11. Inhibition of tumor growth in the murine CT26 model by TGFβ1 antibody SL2-22 To detect the tumor-inhibitory activity of SL2-22 in vivo, BALB / c mice (female, 6 weeks old, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were adapted for breeding for 1 week and then numbered and weighed on the day of the experiment. Logarithmically growing mouse colon cancer cells CT26 (National Biomedical Experimental Cell Resource Library, 1101MOU-PUMC000275) were harvested and resuspended in PBS at 3 × 10 6 The virus was inoculated subcutaneously into the right rib of BALB / c mice at a concentration of 0.1 mL / mouse at a concentration of 100 cells / mL. The average tumor volume was approximately 60 mm at 8-9 days after inoculation. 3When the tumor volume reached 100 mg / kg, mice with an appropriate individual tumor volume were selected and randomly divided into groups as shown in Table 16. Administration began on the day of grouping, with the same molar dose administered twice a week for a total of six doses, administered intraperitoneally. The body weight and tumor volume of the mice were measured twice a week, and the tumor volume was calculated using the formula TV = L 長 ×L 短 2 The tumor volume of each group was expressed as the mean ± standard deviation, and the tumor inhibition rate (%TGI) was calculated using the formula: %TGI = [1-(T-T0) / (C-C0)] × 100%.

[0320] The results, as shown in Figures 4A and 4B and Table 16, demonstrate that the combination of SL2-22-mIgG2a-FcS and the anti-PD-1 antibody RMP1-14-mIgG2a-FcS inhibited CT26 tumor growth in mice and prolonged animal survival. The mice tolerated the treatment well and showed no significant weight loss during the course of treatment (Figure 4C).

[0321] [Table 16]

[0322] Example 12. TGFβ1 monoclonal antibody SL2-22 inhibits fibrosis progression in a mouse idiopathic fibrosis model To test for the in vivo inhibition of fibrosis progression by the TGFβ1 antibody SL2-22, male, 8-week-old C57BL / 6J mice purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. were used. After one week of adaptation, mice were numbered and weighed on the day of the experiment. On the day of model construction, mice were numbered and weighed and randomly assigned to groups of 15 mice per group according to their weight distribution. The specific group assignments are shown in Table 17 below. On day 1 of the experiment, the antibody drug or vehicle control was administered intraperitoneally according to body weight. Two hours later, bleomycin was instilled into the trachea using a flat-head needle at a dose of 2 U / kg (approximately 50 μL per animal). The mice were then administered the drug twice weekly for a total of three weeks according to the group assignment. On day 22 of the experiment, bronchoalveolar lavage fluid and lung tissue samples were collected for analysis of immune cell infiltration, lung tissue hydroxyproline levels, and Masson staining analysis.

[0323] [Table 17] The SL2-22-mIgG2a-Fc administration group significantly improved the overall body weight and survival rate of the animals (Figures 5A and 5B), reduced lung tissue hydroxyproline levels by 40.44% compared to the model construction group (Figure 5C), and significantly reduced intrapulmonary collagen deposition and fibrosis levels after bleomycin model construction by 46.51% compared to the model construction group (Figures 5D and 5E).

[0324] Example 13. Screening and preparation of anti-human and mouse GARP-TGFβ1 single domain antibodies (VHH) 1. Camel Immunization and Library Construction Bactrian camels were immunized with human GARP / TGFβ1 protein (a protein complex obtained by co-expressing and purifying the proteins represented by SEQ ID NOs: 5 and 6) as an antigen. Complete Freund's adjuvant and the antigen were mixed at a 1:1 ratio, and the camels were immunized subcutaneously at multiple sites every two weeks. The initial immunization dose was 200 μg of protein, followed by four immunization doses of 100 μg protein each, for a total of five immunizations. The titer was then measured with human GARP / TGFβ1 protein. If the titer was acceptable, camel peripheral blood was collected, lymphocytes were isolated, Trizol cells were lysed, RNA was extracted, and cDNA was reverse transcribed to construct a phage library.

[0325] 2. Screening of single domain antibodies (VHH) 100 μL Dynabeads TM 20 μg of biotinylated human GARP / TGFβ1 protein bound to M-280 streptavidin was incubated at 37°C for 1 hour, followed by blocking with 2% nonfat milk at room temperature for 1 hour. The phage library was then added and incubated at room temperature for 1 hour. To remove unbound phages, the plates were washed nine times with PBST (PBS containing 0.05% Tween-20). Phages specifically binding to human GARP / TGFβ1 protein were eluted with 1 mg / mL trypsin and infected log-phase-grown E. coli TG1. Positive phages from the first round were then purified. Based on the positive phages from the first round, a second round of screening yielded single-domain antibodies (VHHs) with high affinity for the mouse GARP-TGFβ1 complex protein.

[0326] Ninety-six monoclonal colonies were selected from the enriched positive clones and packaged into phage single-chain antibodies for phage ELISA testing. ELISA plates were coated with 2 μg / mL of human GARP-TGFβ1 complex protein, and phage supernatant diluted with blocking solution was added and detected with anti-M13 HRP-labeled antibody. Clones with an OD450 / background value >5 in the ELISA binding test were sequenced to obtain their sequences. The C19-VHH sequences are shown below.

[0327] >C19-VHH [ka] In SEQ ID NO: 84, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italics in the sequence indicate FR sequences, and the underlines indicate CDR1, CDR2, and CDR3 sequences, respectively. The numbering convention for the anti-GARP-TGFβ1 single domain antibodies provided by the present disclosure is all Kabat.

[0328] [Table 18]

[0329] 3. Complete Antibody Preparation The C19-VHH sequence was fused to the following human IgG4-Fc (CH2-CH3 containing S228P) fragment to obtain C19.

[0330] >hIgG4-Fc(S228P) [ka] >C19 [ka] The C19 sequence was cloned into the mammalian expression vector pTT5 and transfected into HEK293E or ExpiCHO (ThermoFisher, A29127) cells. On day 5 (HEK293E cells, 37°C) or day 10–12 (ExpiCHO cells, 32°C) after transfection, the cells were centrifuged at 4000 rpm for 20 min, and the cell culture supernatant was collected and filtered through a 0.45 μM filter. The first affinity purification step was then performed using a MabSelectSure LX column (GE Healthcare). The culture supernatant was loaded onto a PBS-equilibrated MabSelectSure LX column and washed with PBS. The target protein was eluted with an acidic elution solution containing 0.1 M glycine at pH 3.0, neutralized with 1 M Tris-HCl (pH 8.0), and finally purified using a HiTrap Q HP ion column. Target antibodies were obtained by detection.

[0331] SPR and cellular function experiments showed that C19 had relatively good inhibitory function and bound well to both human and mouse GARP-TGFβ1 complexes.

[0332] Example 14. Humanized modification of anti-GARP-TGFβ1 single domain antibody Three-dimensional structural homology modeling was performed on the selected TGFβ1-specific single-domain antibody C19. Alignment with the V-base human germline sequence database and the IMGT human antibody heavy chain variable region germline gene database was then performed. Based on the results, the heavy chain variable region germline gene IGHV3-23, which has high homology to the C19 sequence, was selected as the template for FR1, FR2, and FR3, and IGJH4 was selected as the template for FR4. The CDRs of the camel-derived single-domain antibody were then grafted onto the corresponding human template to form the variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. To maintain the original activity of the single-domain antibody after humanization, a series of back mutations were performed in the framework regions. The humanized VHH framework regions comprise at least one amino acid mutation selected from 23T, 29Y, 30C, 37Y, 44E, 45R, 47F, 71Q, 74A, 75R, 78G, 81E, 93K and 94T, where the amino acid positions are numbered according to Kabat.

[0333] The resulting humanized sequences are as follows, with the CDR regions underlined and the numbering convention according to Kabat:

[0334] >C19-hu3 [ka] >C19-hu7 [ka] >C19-hu8 [ka] A complete antibody sequence was constructed in which a humanized single-domain antibody VHH was fused with the Fc region of hIgG4 using the method described in Example 2. The resulting humanized complete antibody sequence is as follows: >C19-3 [ka] >C19-7 [ka] >C19-8 [ka]

[0335] Example 15. Measurement of the affinity of anti-GARP-TGFβ1 single domain antibodies for the GARP-TGFβ1 complex The negative control in this example was HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4). The positive control was Abbv-151 (Abbvie, US10793627B2). Its sequence is as follows:

[0336] >Abbv-151 antibody heavy chain: [ka] >Abbv-151 antibody light chain: [ka] The affinity of C19, its humanized antibody, and the positive control antibody Abbv-151 for the human GARP-TGFβ1 complex (formed by complexing with the proteins shown in SEQ ID NOS: 5 and 6) or the mouse GARP-TGFβ1 complex (formed by complexing with the proteins shown in SEQ ID NOS: 8 and 9) was measured using a Biacore T200 (GE Healthcare) instrument. The antibody to be detected was captured on the chip surface using a Series S sensor chip Protein A (GE Healthcare, 29127556). Different concentrations of human or mouse GARP-TGFβ1 complex were then flowed over the chip surface, and the response signal was detected in real time. The binding / dissociation curve was then obtained, and the binding force constant was calculated by fitting. The solution used in the experiment was HBS-EP solution. After each experimental cycle, the chip was regenerated with a pH 1.5 Glycine (GE Healthcare, BR-1003-54) solution. The antibody affinity results are shown in Table 19. The results showed that antibodies C19, C19-3, C19-7, and C19-8 obtained by the screening of the present disclosure have affinity for the human GARP-TGFβ1 complex comparable to that of the positive control Abbv-151, but differ in that they also bind to the mouse GARP-TGFβ1 complex to some extent.

[0337] [Table 19]

[0338] Example 16. Inhibition of GARP / TGFβ1 function in vitro by anti-GARP / TGFβ1 single domain antibodies 2×10 6 LN229 cells (Procell, CL-0578) were transferred to a T75 culture flask (Nunc) and transfected with 293- TM Human or mouse pro-TGFβ1 was transfected singly using the (Invitrogen) transfection reagent, or human and mouse pro-TGFβ1 and GARP plasmid were co-transfected. After 24 h, cells were cultured at 1 × 10 4The cells were transferred to a white opaque 96-well cell culture plate (PerkinElmer) at 1 / well. After 24 h, the medium was aspirated, and HepG2 CAGA12-luc luciferase reporter cells resuspended in DMEM + 0.5% BSA were added. Five-fold diluted C19, C19 humanized antibody, and control antibody Abbv-151 were simultaneously added. After 20 h of co-culture, ONE-Glo TM Luciferase reagent was added, and after 5 minutes of incubation, chemiluminescence values ​​were read using a multifunction plate reader (SpectraMax M5). Luciferase activity in the solvent-treated group was normalized to 1, and relative activity was calculated as luciferase activity in the antibody-treated group divided by the solvent control group. Reaction models were plotted using Prism9 with nonlinear fitting and a three-parameter logarithmic inhibitor model.

[0339] The results, as shown in Figures 6A and 6B, showed that C19, C19-3, C19-7, and C19-8 were comparable to the positive control Abbv-151 in inhibiting human GARP-TGFβ1 complex activity, except that they also inhibited the mouse GARP-TGFβ1 complex.

[0340] Example 17. Binding experiment of anti-GARP-TGFβ1 single domain antibody C19-8 to cells expressing GARP-TGFβ1 1 × 10 HEK293E cells 6 5 mL of the solution was placed in a 50 mL culture tube and incubated with 293fectin TM The cells were transfected with the plasmids listed in Table 20 using the reagents. After 48 h, the cells were washed twice with 1x PBS and transfected with 1x10 5Cells were placed in a 96-well round-bottom cell culture plate (Nunc) at 100 μL per well. 10 nM antibody or control antibody (100 μL, prepared in 1x PBS) was added and stained at room temperature for 1 hour. After washing three times with 1x PBS, 100 μL of FITC-labeled anti-human Fc antibody was added and stained at room temperature for 30 minutes. After staining, the cells were washed three times with 1x PBS. 7-AAD was added and incubated for 5 minutes. After washing three times with 1x PBS, cells were detected by flow cytometry. The number of FITC-positive cells among live cells was analyzed using Flowjo, and the percentage of FITC-positive cells out of the total number of live cells was calculated and plotted.

[0341] [Table 20-1] [Table 20-2]

[0342] The results, as shown in Figure 7, showed that both C19-8 and Abbv-151 bound to the human GARP-TGFβ1 complex (corresponding to TGFβ1 + GARP in Figure 7), with the difference that C19-8 also bound to some extent to the mouse GARP-TGFβ1 complex (corresponding to mTGFβ1 + mGARP in Figure 7). Furthermore, neither C19-8 nor Abbv-151 bound to the TGFβ2 complex (TGFβ2 + GARP) nor the TGFβ3 complex (TGFβ3 + GARP).

[0343] Example 18. Epitope competition study of anti-GARP / TGFβ1 single domain antibody C19-8 with Ab6 and Abbv-151 The negative control in this example was HBS-EP, and the positive controls were Ab6 (Scholar rock, WO2020014460A1, the entire text of which is incorporated herein by reference) and Abbv-151. Ab6 binds to the GARP-TGFβ1 complex, the LRRC33-TGFβ1 complex, the LTBP1-TGFβ1 complex, and the LTBP3-TGFβ1 complex, with the binding epitope located entirely on TGFβ1 (Martin et al., 2020). Meanwhile, the binding epitope of Abbv-151 encompasses GARP and TGFβ1 in the GARP-TGFβ1 complex, and does not bind to GARP or TGFβ1 alone (Street et al., 2020). The sequence of Ab6 is as follows: >Ab6 antibody heavy chain: [ka] >Ab6 antibody light chain: [ka] The competition between C19-8 and the positive antibodies Abbv-151 and Ab6 for the human GARP / TGFβ1 epitope was measured using a Biacore T200 (GE Healthcare) instrument. The chip was coupled to an anti-histidine tag antibody using a Series S sensor chip CM5 (GE Healthcare, Br100530), and the reagent kit used was a His capture reagent kit (GE Healthcare, 28-9950-56). The antigen protein human GARP-TGFβ1 complex was captured on the chip surface, and then antibodies at the same concentration (500 nM) were passed over the chip surface in sequence according to the combinations in Table 21. st The antibody is the first flow antibody, nd The antibody was a second stream antibody.

[0344] [Table 21]

[0345] The reaction signal was detected in real time using a Biacore device, thereby obtaining an epitope competition curve. The solution used in the experiment was HBS-EP solution. At the end of each experimental cycle, the chip was regenerated with a pH 1.5 Glycine (GE Healthcare, BR-1003-54) solution. The measurement results of antibody epitope competition are shown in Table 22.

[0346] The results showed that C19-8 completely competed with Ab6 but not with Abbv-151. Taken together, the above in vitro inhibition and cell binding experiments demonstrated that C19-8, like Abbv-151, inhibits only the GARP-TGFβ1 complex, but is an antibody with a different binding epitope.

[0347] [Table 22]

[0348] Example 19. Inhibition of tumor growth by anti-GARP-TGFβ1 single domain antibody C19-8 in the murine EMT-6 model To detect the tumor-inhibitory activity of C19-8 in vivo, BALB / c mice (female, 6 weeks old, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were adapted for one week and then numbered and weighed on the day of the experiment. Logarithmically growing mouse breast cancer cells EMT-6 (Procell, CL-0573) were harvested, resuspended in PBS, and cultured at 5 × 10 6 The virus was inoculated subcutaneously into the right rib of BALB / c mice at a concentration of 0.1 mL / mouse at a concentration of 0.1 mL / mouse. Three days after inoculation, the average tumor volume was approximately 60 mm. 3 When the tumor volume reached 100 mg / kg, mice with an appropriate individual tumor volume were selected and randomly assigned to groups as shown in Table 23.

[0349] Here, the positive drug M7824 is a PD-L1 / TGFβ-trap (Merck KGaA, Sequence 3 (heavy chain) and Sequence 1 (light chain) in WO2018029367A1 (the entire text of which is incorporated herein by reference)), and mIgG2a-FcS is an mIgG2a with L234A / L235E / G237A / D327Q / A330S / P331S residues, from which the ADCC effector has been removed, as an isotype control. RMP1-14-mIgG2a-FcS contains the variable region of the anti-PD1 antibody RMP1-14 (sequences 285 (heavy chain variable region) and 286 (light chain variable region) from which the signal peptide has been removed, as described in WO2018223182A1 (the entire text of which is incorporated herein by reference)) and the ADCC effector-removed sequence L234A / L235E / G237A / D327Q / A3 C19-8-mIgG2a-FcS is an antibody in which the variable region of C19-8 is fused with an mIgG2a heavy chain constant region having L234A / L235E / G237A / D327Q / A330S / P331S and a mouse kappa light chain constant region, and the ADCC effector has been removed and the sequences are as follows: >C19-8-mIgG2a-FcS: [ka] EVQLLESGGGLVQPGGSLRLSCTASGFTYCEYDMSWYRQAPGKEREFVSRIASDGRTSYVDSVKGRFTISQDNSKNTGYLEMNSLRAEDTAVYYCKTEAVKYSGNWCVAAPGFAYWGQGTLVTVSS PTIKPCPPCKCPAPNAEGAPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKQLPSSI ERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 106) The administration started on the day of grouping, and the same molar dose was administered twice a week for a total of seven times. The administration method was intraperitoneal injection. The body weight and tumor volume of the mice were measured twice a week, and the tumor volume was calculated using the formula TV = L 長 ×L 短 2The tumor volume of each group was expressed as the mean ± standard deviation, and statistical analysis was performed using two-way ANOVA to calculate the tumor inhibition rate (%TGI), which was calculated using the formula: %TGI = [1 - (T - T0) / (C - C0)] × 100%.

[0350] As shown in Figures 8A and 8B, the combination of C19-8-mIgG2a-FcS and the anti-PD-1 antibody RMP1-14-mIgG2a-FcS significantly inhibited mouse EMT-6 tumor growth (p=0.0017, Two-way ANOVA) and prolonged animal survival (p=0.0157, Log-rank (Mantel-Cox) test) compared with RMP1-14-mIgG2a-FcS alone. The mice tolerated the treatment well and showed no significant weight loss during the course of treatment (Figure 8C).

[0351] [Table 23]

[0352] Example 9. Inhibition of tumor growth by anti-GARP-TGFβ1 single domain antibody C19-8 in the murine CT26 model To detect the tumor-inhibitory activity of C19-8 in vivo, BALB / c mice (female, 6 weeks old, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were adapted for breeding for 1 week and then numbered and weighed on the day of the experiment. Logarithmically growing mouse colon cancer cells CT26 (National Biomedical Experimental Cell Resource Library, 1101MOU-PUMC000275) were harvested and resuspended in PBS at 3 × 10 6 The virus was inoculated subcutaneously into the right rib of BALB / c mice at a concentration of 0.1 mL / mouse at a concentration of 100 cells / mL. The average tumor volume was approximately 60 mm at 8-9 days after inoculation. 3 When the tumor volume reached 100 mg / kg, mice with an appropriate individual tumor volume were selected and randomly divided into groups as shown in Table 24. Administration began on the day of grouping, with the same molar dose administered twice a week for a total of six doses, administered intraperitoneally. The body weight and tumor volume of the mice were measured twice a week, and the tumor volume was calculated using the formula TV = L 長 ×L 短 2The tumor volume of each group was expressed as the mean ± standard deviation, and the tumor inhibition rate (%TGI) was calculated using the formula: %TGI = [1-(T-T0) / (C-C0)] × 100%.

[0353] As shown in Figures 9A and 9B, the combination of C19-8-mIgG2a-FcS and the anti-PD-1 antibody RMP1-14-mIgG2a-FcS inhibited CT26 tumor growth in mice and prolonged animal survival. The treatment was well tolerated by the mice, with no significant weight loss (Figure 9C).

[0354] [Table 24]

[0355] Although specific embodiments of the present disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples and that various changes and modifications can be made to these embodiments without departing from the principles and spirit of the present disclosure. Accordingly, the scope of the present disclosure is limited by the appended claims.

Claims

1. 1. A TGFβ1 binding molecule comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence shown in SEQ ID NO: 11, and the VL comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 12, 21 to 35, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; Preferably, the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 38, 64 and 18, respectively; Preferably, the TGFβ1 binding molecule is HCDR1 represented by SEQ ID NO: 13, HCDR2 represented by SEQ ID NO: 14, HCDR3 represented by SEQ ID NO: 15, LCDR1 represented by any one of SEQ ID NOs: 16, 36, 39, 41 to 42, 44, 46, 49, 52, 54, and 56 to 59; LCDR2 represented by any one of SEQ ID NOs: 17, 37, 40, 43, 45, 47 to 48, 50 to 51, 53 and 55; and comprising an LCDR3 represented by SEQ ID NO: 18; TGFβ1 binding molecule.

2. a) the VH comprises HCDR1, HCDR2 and HCDR3 represented by SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 represented by SEQ ID NOs: 59, 40 and 18, respectively; b) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 36 to 37 and 18, respectively; c) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 39 to 40 and 18, respectively; d) the VH comprises HCDR1, HCDR2 and HCDR3 represented by SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 represented by SEQ ID NOs: 41, 40 and 18, respectively; e) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 42 to 43 and 18, respectively; f) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 44 to 45 and 18, respectively; g) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 46 to 47 and 18, respectively; h) the VH comprises HCDR1, HCDR2 and HCDR3 represented by SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 represented by SEQ ID NOs: 46, 48 and 18, respectively; j) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 49 to 50 and 18, respectively; k) the VH comprises HCDR1, HCDR2 and HCDR3 represented by SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 represented by SEQ ID NOs: 46, 51 and 18, respectively; l) the VH comprises HCDR1, HCDR2 and HCDR3 represented by SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 represented by SEQ ID NOs: 52 to 53 and 18, respectively; m) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 54 to 55 and 18, respectively; n) the VH comprises HCDR1, HCDR2 and HCDR3 represented by SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 represented by SEQ ID NOs: 56, 53 and 18, respectively; o) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 57, 45 and 18, respectively; p) the VH comprises HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NOs: 58, 50 and 18, respectively; or q) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13 to 15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 16 to 18, respectively; The TGFβ1 binding molecule of claim 1.

3. the VH comprises the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 90% sequence identity thereto; and, The VL comprises an amino acid sequence represented by any one of SEQ ID NOs: 12 and 21 to 35, or an amino acid sequence having at least 90% sequence identity thereto; A TGFβ1-binding molecule according to any one of claims 1 to 2.

4. The antibody further comprises an immunoglobulin Fc region, preferably the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, IgG4 or a variant of any one of them, more preferably the immunoglobulin Fc region is derived from human IgG4 or a variant thereof, and the variant comprises the mutation 228P. A TGFβ1-binding molecule according to any one of claims 1 to 3.

5. a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 66, or having at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 65, 67-81, or having at least 90% sequence identity thereto; or the heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 82, or having at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence set forth in SEQ ID NO: 83, or having at least 90% sequence identity thereto; A TGFβ1-binding molecule according to any one of claims 1 to 4.

6. an anti-TGFβ1 antibody or an antigen-binding fragment thereof; Preferably, the anti-TGFβ1 antibody is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody, preferably a humanized antibody, more preferably a humanized antibody modified by affinity maturation; Preferably, the antigen-binding fragment is an scFv, a dsFv, or a (dsFv) ２ , dsFv-dsFv', Fv fragment, Fab, Fab' or F(ab') ２ Selected from A TGFβ1-binding molecule according to any one of claims 1 to 5.

7. At least one of the following characteristics: (1) binding to the TGFβ1 precursor protein and / or binding to the TGFβ1 complex; (2) non-binding to TGFβ2 or TGFβ2 complexes; (3) non-binding to TGFβ3 or the TGFβ3 complex; (4) inhibition of TGFβ1 activity; and (5) Controllability T (T ｒｅｇ ) inhibiting the immunosuppressive activity of cells; (6) inhibition of tumor growth; and (7) Inhibition of fibrosis, Preferably, said inhibition of TGFβ1 activity comprises inhibition of TGFβ1 activation, inhibition of release of mature TGFβ1 from TGFβ1 complexes, and / or inhibition of TGFβ1 signaling; Preferably, the TGFβ1 in said TGFβ1 complex is present as a TGFβ1 precursor protein, said TGFβ1 precursor protein preferably comprising a mature TGFβ1 domain and a latency-associated peptide (LAP); Preferably, the TGFβ1 complex is selected from an LTBP1-TGFβ1 complex, an LTBP3-TGFβ1 complex, an LRRC33-TGFβ1 complex, and / or a GARP-TGFβ1 complex. A TGFβ1-binding molecule according to any one of claims 1 to 6.

8. A GARP-TGFβ1 binding molecule comprising at least one immunoglobulin single variable domain, said immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of the amino acid sequence set forth in any one of SEQ ID NOs: 84 and 90-92, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; Preferably, the CDR1, CDR2, and CDR3 comprise the amino acid sequences set forth in SEQ ID NOs: 85, 86, and 87, respectively. GARP-TGFβ1 binding molecule.

9. the immunoglobulin single variable domain has been modified by humanization, affinity maturation, removal of T-cell epitopes, reduction of antibody deamidation and / or reduction of antibody isomerization; Preferably, the human germline template used in the humanization process is selected from IGHV3-23 and / or IGJH4; The GARP-TGFβ1 binding molecule of claim 8.

10. the immunoglobulin single variable domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 84 and 90-92 or having at least 90% sequence identity thereto, and preferably the immunoglobulin single variable domain is a VHH; The GARP-TGFβ1 binding molecule of any one of claims 8 to 9.

11. further comprising an immunoglobulin Fc region, preferably said Fc region being derived from IgG1, IgG2, IgG3, IgG4 or a variant of any one of said, more preferably from human IgG4 or a variant thereof, said variant comprising the mutation 228P; The GARP-TGFβ1 binding molecule of any one of claims 8 to 10.

12. An amino acid sequence having at least 90% sequence identity thereto, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 89, 93 to 95, and 106; The GARP-TGFβ1 binding molecule of claim 11.

13. an antibody or antigen-binding fragment thereof that binds to the GARP-TGFβ1 complex; Preferably, the antibody or antigen-binding fragment thereof is selected from a linear antibody, a single chain antibody, a nanobody, a peptide antibody, a domain antibody, a multispecific antibody or an antigen-binding fragment thereof; Preferably, the antibody or antigen-binding fragment thereof is a camelid antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof. The GARP-TGFβ1 binding molecule of any one of claims 8 to 12.

14. 1. A GARP-TGFβ1 binding molecule comprising an immunoglobulin single variable domain that binds to a GARP-TGFβ1 complex, said GARP-TGFβ1 complex comprising: a TGFβ1 precursor protein and a glycoprotein A repeat dominant sequence (GARP), wherein the TGFβ1 precursor protein comprises a mature TGFβ1 domain and a latency-associated peptide (LAP); Preferably, the GARP comprises the amino acid sequence set forth in SEQ ID NO: 100 or 103, or an amino acid sequence having at least 90% sequence identity thereto, the mature TGFβ1 domain comprises the amino acid sequence set forth in SEQ ID NO: 101 or 104, or an amino acid sequence having at least 90% sequence identity thereto, and / or the LAP comprises the amino acid sequence set forth in SEQ ID NO: 102 or 105, or an amino acid sequence having at least 90% sequence identity thereto. GARP-TGFβ1 binding molecule.

15. At least one of the following characteristics: (1) binding to the GARP-TGFβ1 complex; (2) non-binding to TGFβ2 protein or TGFβ2 complex; (3) non-binding to TGFβ3 protein or TGFβ3 complex; (4) non-binding to free mature TGFβ1; (5) inhibition of TGFβ1 activity; (6) Controllability T (T ｒｅｇ ) inhibiting the immunosuppressive activity of cells; (7) inhibiting tumor growth; Preferably, said inhibition of TGFβ1 activity comprises inhibition of TGFβ1 activation, inhibition of release of mature TGFβ1 from the GARP-TGFβ1 complex, and / or inhibition of TGFβ1 signaling; Preferably, the GARP-TGFβ1 complex comprises (i) GARP, and (ii) a TGFβ1 precursor protein, the TGFβ1 precursor protein preferably comprising a mature TGFβ1 domain and a latency-associated peptide (LAP); The GARP-TGFβ1 binding molecule of any one of claims 8 to 14.

16. A polynucleotide encoding a TGFβ1 binding molecule according to any one of claims 1 to 7 or a GARP-TGFβ1 binding molecule according to any one of claims 8 to 15. Polynucleotide.

17. 17. The polynucleotide of claim 16, vector.

18. 18. A host cell comprising the polynucleotide of claim 16 or the vector of claim 17, Preferably, the host cell is a bacterial, yeast or mammalian cell, more preferably, the host cell is Escherichia coli, Pichia yeast, Chinese hamster ovary cell or human embryonic kidney 293 cell. host cell.

19. A pharmaceutical composition comprising a TGFβ1 binding molecule according to any one of claims 1 to 7, a GARP-TGFβ1 binding molecule according to any one of claims 8 to 15, a polynucleotide according to claim 16, or a vector according to claim 17; Preferably, the pharmaceutical composition further comprises one or more medicament excipients, diluents or additives, Preferably, the pharmaceutical composition further comprises an immune checkpoint inhibitor, Preferably, the pharmaceutical composition further comprises an anti-PD-1 antibody or an antigen-binding fragment thereof. Pharmaceutical compositions.

20. 8. A method for preparing a TGFβ1 binding molecule according to any one of claims 1 to 7, said method comprising: expressing a TGFβ1-binding molecule according to any one of claims 1 to 7 in a host cell; and isolating the TGFβ1 binding molecule from the host cell; Optionally, the method further comprises purifying the TGFβ1 binding molecule. method.

21. 16. A method for preparing a GARP-TGFβ1 binding molecule according to any one of claims 8 to 15, said method comprising: expressing the GARP-TGFβ1 binding molecule of any one of claims 8 to 15 in a host cell; and isolating the GARP-TGFβ1 binding molecule from the host cell; Optionally, the method further comprises purifying the GARP-TGFβ1 binding molecule. method.

22. 20. Use of a TGFβ1 binding molecule according to any one of claims 1 to 7, a GARP-TGFβ1 binding molecule according to any one of claims 8 to 15, a polynucleotide according to claim 16, a vector according to claim 17, or a pharmaceutical composition according to claim 19 in the preparation of a medicament for a disease or condition associated with the TGFβ signaling pathway, comprising: Preferably, the disease associated with the TGFβ signaling pathway is selected from cancer or fibrosis; Preferably, the cancer is selected from lung cancer, intestinal cancer, kidney cancer, bladder cancer, liver cancer, stomach cancer, breast cancer, colon cancer, cervical cancer, prostate cancer and head and neck cancer; use.

23. A method for preventing or treating a disease or condition associated with the TGFβ signaling pathway, comprising administering to a subject a prophylactically or therapeutically effective amount of the TGFβ1-binding molecule of any one of claims 1 to 7, the GARP-TGFβ1-binding molecule of any one of claims 8 to 15, the polynucleotide of claim 16, the vector of claim 17, or the pharmaceutical composition of claim 19; Or, The method comprises administering to a subject a prophylactically or therapeutically effective amount of a TGFβ1-binding molecule of any one of claims 1 to 7 or a GARP-TGFβ1-binding molecule of any one of claims 8 to 15, and a prophylactically or therapeutically effective amount of an immune checkpoint inhibitor; Preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof; Preferably, the disease or condition associated with the TGFβ signaling pathway is selected from cancer or fibrosis, and preferably, the cancer is selected from lung cancer, intestinal cancer, kidney cancer, bladder cancer, liver cancer, gastric cancer, breast cancer, colon cancer, cervical cancer, prostate cancer, and head and neck cancer. method.

24. 1. A method of inhibiting TGFβ1 activity in vitro or in vivo in a subject, comprising:

20. A method for treating a cancer, comprising administering in vitro or in vivo a TGFβ1 binding molecule according to any one of claims 1 to 7, a GARP-TGFβ1 binding molecule according to any one of claims 8 to 15, a polynucleotide according to claim 16, a vector according to claim 17, or a pharmaceutical composition according to claim 19. method.

25. 1. Use of a TGFβ1 binding molecule or a GARP-TGFβ1 binding molecule in combination with an immune checkpoint inhibitor in the preparation of a medicament for treating cancer, comprising: wherein the TGFβ1 binding molecule is as defined in any one of claims 1 to 7 and the GARP-TGFβ1 binding molecule is as defined in any one of claims 8 to 15; Preferably, the immune checkpoint inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof; Preferably, the cancer is selected from lung cancer, intestinal cancer, kidney cancer, bladder cancer, liver cancer, stomach cancer, breast cancer, colon cancer, cervical cancer, prostate cancer and head and neck cancer; use.

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