TGF [beta] 1 binding molecules, GARP-TGF [beta] 1 binding molecules and medical uses thereof

Abstract

Relates to TGF [beta] 1 binding molecules, GARP-TGF [beta] 1 binding molecules and medical uses thereof. In particular to a TGF [beta] 1 binding molecule, a GARP-TGF [beta] 1 binding molecule, a pharmaceutical composition containing the same, and a preparation method and medical application of the GARP-TGF [beta] 1 binding molecule. The invention also relates to a method for treating and / or preventing a disease (such as a tumor or cancer) by using the TGF [beta] 1 binding molecule, the GARP-TGF [beta] 1 binding molecule and the pharmaceutical composition containing the TGF [beta] 1 binding molecule and the GARP-TGF [beta] 1 binding molecule.

Description

TGFβ1 binding molecules, GARP-TGFβ1 binding molecules and medical uses thereof

[0001] The present 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, and the entire contents of the foregoing patent applications are incorporated into the present disclosure by reference. Technical Field

[0002] The present disclosure relates to the field of biomedicine, and specifically to TGFβ1 binding molecules, pharmaceutical compositions comprising TGFβ1 binding molecules, as well as medical uses, preparation methods, and methods for preventing or treating diseases related to the TGFβ1 signaling pathway. Background Art

[0003] TGFβ (Transforming growth factor β) is a class of multi-effect growth factors, including TGFβ1, TGFβ2 and TGFβ3. They regulate many important physiological processes, such as cell growth, differentiation, proliferation, apoptosis and matrix production. Studies have found that TGFβ can inhibit tumor growth in the early stages of tumor development, but promote tumor growth in the late stages of tumor progression (Liu S, Ren J, Ten Dijke P. Signal Transduct Target Ther. 2021 Jan 8; 6(1): 8.). An important mechanism by which TGFβ promotes the growth of late-stage tumors is to inhibit immune cells in the tumor microenvironment. Among them, TGFβ1, rather than TGFβ2 and TGFβ3, is a key factor in promoting the growth of late-stage tumors (Constance J Martin, et al. Sci Transl Med. 2020 Mar 25; 12(536): eaay8456.). TGFβ1 produced by tumor cells, regulatory T cells (Treg) and suppressive phagocytes can directly promote the activity of Treg cells and inhibit effector T cells and antigen-presenting cells, thereby achieving the effect of suppressing immunity.

[0004] Currently, anti-PD-1 (Programmed Cell Death-1) antibodies, tumor immune checkpoint inhibitors, have achieved great success in tumor immunotherapy, but the overall response rate of patients is only about 20%. The combination of anti-TGFβ1 antibodies and anti-PD-1 antibodies can significantly overcome tumor immune evasion and enhance the efficacy of anti-PD-1 antibodies in tumor models such as EMT-6, MBT-2, Cloudman S91, CT26, and MC38. However, pan-TGFβ antibodies targeting mature TGFβ1 have shown dose-dependent in vivo toxicity in experiments. Targeting the pro / latent TGFβ1 complex inhibits the production of TGFβ1 at the source, which has better efficacy in animal models than targeting mature TGFβ1 and has significantly reduced toxicity.

[0005] The production of TGFβ1 is tightly regulated by a multi-step process. In the TGFβ1 precursor protein, the mature TGF-β1 domain at its C-terminus is covalently or non-covalently bound to a domain at its N-terminus called the latency associated peptide (LAP). Because LAP prevents mature TGF-β1 from binding to its receptor, it is usually inactive. Such a TGFβ1 precursor protein can further form a large latent complex with GARP, LRRC33, LTBP1, LTBP3, etc. In the tumor microenvironment, GARP-TGFβ1 is mainly produced by Treg and tumor cells, LRRC33-TGFβ1 is mainly produced by immunosuppressive phagocytes, and LTBP1-TGFβ1 and LTBP3-TGFβ1 are mainly present in the tumor stroma. At present, there are no antibodies targeting the TGFβ1 precursor protein or the TGFβ1 complex on the market worldwide. There is still a need in the art for a compound that can bind to TGFβ1 precursor protein or TGFβ1 complex with high affinity and block its activation, so as to be used for the treatment of TGFβ-related diseases such as tumors and fibrosis.

[0006] Summary of the Invention

[0007] The present disclosure provides TGFβ1 binding molecules, GARP-TGFβ1 binding molecules, pharmaceutical compositions comprising the aforementioned binding molecules, as well as medical uses, preparation methods, and methods for preventing or treating diseases related to the TGFβ signaling pathway, particularly methods for preventing or treating cancer or tumors.

[0008] TGFβ1 binding molecules

[0009] 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).

[0010] In some embodiments, the VH comprises HCDR1, HCDR2 and HCDR3 of the amino acid sequence shown in SEQ ID NO: 11, and / or the VL comprises LCDR1, LCDR2 and LCDR3 of the amino acid sequence shown in any one of SEQ ID NOs: 12, 21-35.

[0011] The above-mentioned 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 scheme.

[0012] In some embodiments, the CDRs may also be defined by other numbering systems, for example, according to the IMGT, Chothia, AbM, or Contact numbering systems. Exemplary examples include HCDR1, HCDR2, and HCDR3 in the VH of SEQ ID NO: 11, and LCDR1, LCDR2, and LCDR3 in the VL of SEQ ID NO: 35, as defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems.

[0013] Table 1. HCDR1, HCDR2, and HCDR3 in the VH of SEQ ID NO: 11

[0014] Table 2. LCDR1, LCDR2, LCDR3 in VL of SEQ ID NO: 35

[0015] In some embodiments, the TGFβ1 binding molecule comprises VH and VL as shown below:

[0016] The VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and / or the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 38, 64, 18.

[0017] Wherein, the amino acid sequence shown in SEQ ID NO: 38 is RASQX1ISX2YLN, 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;

[0018] The amino acid sequence shown in 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.

[0019] In some embodiments, the amino acid sequence shown in SEQ ID NO:38 is RASQX1ISX2YLN, X1 is selected from F, Y or A, and X2 is selected from D or P; the amino acid sequence shown in SEQ ID NO:64 is X3ASX4LX5S, 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.

[0020] In some embodiments, the TGFβ1 binding molecule, wherein:

[0021] The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 13,

[0022] The amino acid sequence of the HCDR2 is shown in SEQ ID NO: 14,

[0023] The amino acid sequence of the HCDR3 is shown in SEQ ID NO: 15,

[0024] The amino acid sequence of the LCDR1 is shown in any one of SEQ ID NOs: 16, 36, 39, 41-42, 44, 46, 49, 52, 54, 56-59,

[0025] The amino acid sequence of the LCDR2 is shown in any one of SEQ ID NOs: 17, 37, 40, 43, 45, 47-48, 50-51, 53, and 55, and / or

[0026] The amino acid sequence of the LCDR3 is shown in SEQ ID NO: 18.

[0027] In some embodiments, the TGFβ1 binding molecule, wherein

[0028] a) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 59, 40, and 18;

[0029] b) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 36-37 and 18;

[0030] c) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 39-40 and 18;

[0031] d) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 41, 40, and 18;

[0032] e) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 42-43 and 18;

[0033] f) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 44-45 and 18;

[0034] g) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 46-47 and 18;

[0035] h) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 46, 48, and 18;

[0036] j) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 49-50 and 18;

[0037] k) the VH comprises HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 46, 51, and 18;

[0038] 1) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 52-53 and 18;

[0039] m) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 54-55 and 18;

[0040] n) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 56, 53, and 18;

[0041] o) the VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 57, 45, and 18;

[0042] p) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 58, 50, 18; or,

[0043] q) the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 13-15, and the VL comprises LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 16-18.

[0044] In some embodiments, the TGFβ1 binding molecule comprises a VH and a VL, wherein:

[0045] Any HCDR contained in the VH has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any of the aforementioned HCDRs; and / or, any LCDR contained in the VL has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any of the aforementioned HCDRs.

[0046] In some specific embodiments, the amino acid mutations in the HCDR or LCDR are conservative substitutions.

[0047] In some embodiments, the present disclosure provides a TGFβ1 binding molecule comprising any one or a combination of any several (e.g., 2, 3, 4, 5, or 6) of the aforementioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0048] In some embodiments, the TGFβ1 binding molecule is an anti-TGFβ1 antibody or an antigen-binding fragment thereof.

[0049] 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 of the foregoing.

[0050] 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. In some specific embodiments, the anti-TGFβ1 antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.

[0051] In some specific embodiments, the anti-TGFβ1 antibody or antigen-binding fragment thereof is affinity matured. The affinity matured anti-TGFβ1 antibody or antigen-binding fragment thereof comprises one or more mutations in VH and / or VL. Exemplarily, the mutations in VH comprise mutations at any one or any combination of the following positions: 99, 100, 53, 56, 58, 31, 33, 95, 100. Exemplarily, the mutations in VL comprise mutations at any one or any combination of the following positions: 24, 28, 31, 32, 50, 53, 55, 92, 93, 94. The positions of the above mutations are numbered according to the Kabat numbering convention.

[0052] In some embodiments, the TGFβ1 binding molecule comprises VH and VL, wherein,

[0053] The VH comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 80% sequence identity thereto; and / or,

[0054] The VL comprises an amino acid sequence as shown in any one of SEQ ID NOs: 12, 21-35, or an amino acid sequence having at least 80% sequence identity thereto.

[0055] In some embodiments, the TGFβ1 binding molecule comprises a VH and a VL, wherein: the VH has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to any of the aforementioned VHs; and / or, the VL has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to any of the aforementioned VLs.

[0056] In some embodiments, the present disclosure provides a TGFβ1-binding molecule comprising any one or a combination of any two of the aforementioned VH and VL.

[0057] In some embodiments, the TGFβ1 binding molecules provided herein 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 of the foregoing. 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 sites of the above mutations are numbered according to EU numbering conventions.

[0058] In some embodiments, the TGFβ1 binding molecule further comprises a light chain constant region and / or a heavy chain constant region.

[0059] In some embodiments, the light chain constant region is derived from a kappa light chain, a lambda light chain, or a variant of any of the foregoing. In some embodiments, the light chain constant region is derived from a human kappa light chain, a human lambda light chain, or a variant of any of the foregoing.

[0060] In some specific embodiments, the light chain constant region is derived from a human kappa light chain or a variant thereof.

[0061] In some specific embodiments, the light chain constant region is derived from a murine kappa light chain or a variant thereof.

[0062] In some specific embodiments, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity thereto.

[0063] In some embodiments, the heavy chain constant region is derived from IgG1, IgG2, IgG3, IgG4, or a variant of any of the foregoing. In some embodiments, the heavy chain constant region is derived from human IgG1, human IgG2, human IgG3, human IgG4, or a variant of any of the foregoing.

[0064] In some specific embodiments, the heavy chain constant region is derived from human IgG4 or a variant thereof.

[0065] In some specific embodiments, the human IgG4 variant comprises a mutation that reduces or eliminates Fc effector function. Exemplarily, the human IgG4 variant comprises the mutation S228P. The sites of the above mutations are numbered according to EU numbering conventions.

[0066] In some specific embodiments, the heavy chain constant region is derived from murine IgG2 or a variant thereof.

[0067] In some specific embodiments, the murine IgG2 variant comprises a mutation that reduces or eliminates ADCC efficacy. Exemplarily, the murine IgG2 variant comprises at least one of the following mutations: L234A / L235E / G237A / D327Q / A330S / P331S. The sites of the aforementioned mutations are numbered according to EU numbering conventions.

[0068] In some embodiments, the immunoglobulin Fc region is an Fc region with reduced or eliminated effector function, for example, a mutation that reduces or eliminates ADCC effect. Exemplarily, the mutation that reduces or eliminates ADCC effect comprises at least one of the following:

[0069] 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 above mutations are numbered according to EU numbering conventions.

[0070] In some specific embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 20, or an amino acid sequence having at least 80% sequence identity thereto.

[0071] In some embodiments, the TGFβ1 binding molecule comprises a heavy chain and a light chain, wherein:

[0072] The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 66, or an amino acid sequence having at least 80% sequence identity thereto; and / or,

[0073] The light chain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 65, 67-81, or an amino acid sequence having at least 80% sequence identity thereto.

[0074] In some embodiments, the TGFβ1 binding molecule comprises a heavy chain and a light chain, wherein:

[0075] The heavy chain comprises the amino acid sequence shown in SEQ ID NO: 82, or an amino acid sequence having at least 80% sequence identity thereto; and / or,

[0076] The light chain comprises the amino acid sequence shown in SEQ ID NO: 83, or an amino acid sequence having at least 80% sequence identity thereto.

[0077] In some embodiments, the TGFβ1 binding molecule comprises a heavy chain and a light chain, wherein: the heavy chain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to any of the aforementioned heavy chains; and / or the light chain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid mutations compared to any of the aforementioned light chains.

[0078] In some specific embodiments, the amino acid mutations in the heavy chain and / or light chain are not conservative substitutions.

[0079] In some embodiments, the TGFβ1 binding molecules of the present disclosure comprise any one or a combination of any two of the aforementioned heavy chains and light chains.

[0080] In the context of the present disclosure, "at least 80%" encompasses 80% and above, for example 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.

[0081] In some embodiments, the aforementioned TGFβ1 binding molecules specifically bind to TGFβ1 precursor protein or TGFβ1 complex.

[0082] In some embodiments, the TGFβ1 in the TGFβ1 complex is present as a TGFβ1 precursor protein.

[0083] In some embodiments, the TGFβ1 complex comprises any of the following:

[0084] (2) LTBP1-TGFβ1 complex,

[0085] (3) LTBP3-TGFβ1 complex,

[0086] (4) LRRC33-TGFβ1 complex,

[0087] (5)GARP-TGFβ1 complex.

[0088] As used in this disclosure, "TGFβ1" or "TGFβ1 protein" is understood in a broad sense, including the proprotein form (also known as pro-TGFβ1) or latent form (latent TGFβ1) of transforming growth factor-β1 (TGFβ1), or mature TGFβ1 after release.

[0089] In some embodiments, "TGFβ1" or "TGFβ1 protein" as used in the present disclosure refers to "TGFβ1 precursor protein."

[0090] In some embodiments, the TGFβ1 precursor protein comprises: (i) a mature TGFβ1 domain, (ii) a potency-associated peptide (LAP).

[0091] Exemplarily, the TGFβ1 precursor protein is proTGFβ1 or latent TGFβ1, wherein the mature TGFβ1 domain in proTGFβ1 is covalently bound to latency-associated peptides (LAPs), and the mature TGFβ1 domain in latent TGFβ1 is non-covalently bound to latency-associated peptides (LAPs).

[0092] In the present disclosure, "pro-TGFβ1" or "latent TGFβ1" may be used interchangeably.

[0093] During translation, latent TGFβ1 (also known as the small latent complex (SLC)) becomes linked to a "presenting molecule" via a disulfide bridge to form a large latent complex (LLC), including, for example, GARP to form a GARP-TGFβ1 complex. TGFβ1 present in the GARP-TGFβ1 complex can be in a latent form (latent TGFβ1) or a precursor form (proTGFβ1).

[0094] In some embodiments, the TGFβ1 precursor protein or TGFβ1 complex further comprises a protein fragment, a functional variant, etc. of any of the aforementioned proteins or protein complexes.

[0095] Exemplarily, "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, alpha 1 helical regions, alpha 2 helical regions, RGD sequence regions, trigger loop regions, extracellular domains, transmembrane domains, intracellular domains, and the like.

[0096] Exemplarily, "variant" or "functional variant" refers to a protein or protein complex comprising one or more amino acid substitutions, deletions and / or added amino acids and having comparable biological activity.

[0097] Exemplarily, the variant of the TGFβ1 precursor protein comprises a mutation in amino acid position 4 according to natural numbering relative to the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the variant of the TGFβ1 precursor protein comprises a mutation C4S, wherein the mutation site is a site according to natural numbering relative to the amino acid sequence shown in SEQ ID NO: 6.

[0098] In some embodiments, the TGFβ1 complex is a complex formed by TGFβ1 precursor protein and other types of proteins or their protein fragments and functional variants, for example, a complex formed with LTBP1S, LTBP4, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4, etc.

[0099] In some embodiments, the TGFβ1 precursor protein comprises an amino acid sequence as 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.

[0100] Exemplarily, the TGFβ1 complex is a GARP-TGFβ1 complex comprising GARP and a TGFβ1 precursor protein; wherein:

[0101] GARP comprises the 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;

[0102] And / or, the TGFβ1 protein comprises the 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.

[0103] In some embodiments, the TGFβ1 binding molecule is an anti-TGFβ1 antibody or an antigen-binding fragment thereof that specifically binds to an antigenic epitope of a TGFβ1 precursor protein or a TGFβ1 complex. In some embodiments, the anti-TGFβ1 antibody or antigen-binding fragment thereof does not prevent TGFβ1 from binding to an integrin. For example, in some embodiments, the anti-TGFβ1 antibody or antigen-binding fragment thereof does not mask the integrin binding site of TGFβ1.

[0104] In some embodiments, the TGFβ1 binding molecule is present at 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M or lower K D Binding to a TGFβ1 precursor protein or a TGFβ1 complex (eg, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, LRRC33-TGFβ1 complex, GARP-TGFβ1 complex, etc.) In some embodiments, the TGFβ1 precursor protein is included in the TGFβ1 complex.

[0105] In some embodiments, the TGFβ1 binding molecule does not bind or binds very weakly to the TGFβ2 protein or TGFβ2 complex. For example, the anti-TGFβ1 antibody or antigen-binding fragment thereof does not specifically bind or binds very weakly to at least one of the following: mature TGFβ2, TGFβ2 precursor protein, GARP-TGFβ2 complex, LTBP-TGFβ2 complex, etc.

[0106] In some embodiments, the TGFβ1 binding molecule does not bind or binds very weakly to the TGFβ3 protein or TGFβ3 complex. For example, the anti-TGFβ1 antibody or antigen-binding fragment thereof does not specifically bind or binds very weakly to at least one of the following: mature TGFβ3, TGFβ3 precursor protein, GARP-TGFβ3 complex, LTBP-TGFβ3 complex, etc.

[0107] In the present disclosure, "TGFβ2 protein" should be understood broadly, covering mature TGFβ2 as well as inactive precursor protein forms such as pro-TGFβ2 and latent TGFβ2. "Pro-TGFβ2" and "latent TGFβ2" can be used interchangeably.

[0108] In the present disclosure, "TGFβ3 protein" should be understood broadly, covering mature TGFβ3 as well as inactive precursor protein forms such as pro-TGFβ3 and latent TGFβ3. "Pro-TGFβ3" and "latent TGFβ3" can be used interchangeably.

[0109] In some embodiments, the TGFβ1 binding molecules of the present disclosure inhibit TGFβ1 activity. For example, the TGFβ1 binding molecules bind to TGFβ1 precursor protein (original / latent TGFβ1) or TGFβ1 complex (e.g., GARP-TGFβ1 complex, LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, LRRC33-TGFβ1 complex), thereby selectively inhibiting the activity of TGFβ1 (e.g., inhibiting the activation of TGFβ1, and / or inhibiting the release of mature TGFβ1; and / or, inhibiting TGFβ1 signal transduction). Furthermore, the activity of TGFβ2 and / or TGFβ3 is not selectively inhibited, for example, the activation of TGFβ1 is selectively inhibited, but the activation of TGFβ2 and / or TGFβ3 is not inhibited. Wherein, the TGFβ1 in the TGFβ1 complex exists in the form of TGFβ1 precursor protein.

[0110] In some embodiments, the TGFβ1 binding molecules of the present disclosure have improved safety (eg, reduced in vivo toxicity and / or adverse effects).

[0111] In some embodiments, the TGFβ1 binding molecules of the present disclosure further have at least one of the following properties: inhibition of regulatory T (T reg ) cells’ immunosuppressive activity, inhibiting tumor growth and fibrosis.

[0112] In some embodiments, the TGFβ1 binding molecule is an anti-TGFβ1 antibody or an antigen-binding fragment thereof, further Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody and multispecific antibody (bispecific antibody, double-chain antibody (diabody), three-chain antibody (triabody) and four-chain antibody (tetrabody), tandem two-scFv, tandem three-scFv), for example, specifically scFv, Fv, Fab or Fab' fragment.

[0113] In some embodiments, anti-TGFβ1 antibodies or antigen-binding fragments thereof are provided that bind to or compete for binding to the same epitope of a TGFβ1 precursor protein or TGFβ1 complex (e.g., a human or mouse GARP-TGFβ1 complex, a LTBP-TGFβ1 complex, or a LRRC33-TGFβ1 complex) as the aforementioned TGFβ1-binding molecules. In some embodiments, the TGFβ1 in the TGFβ1 complex exists in the form of a TGFβ1 precursor protein.

[0114] In some embodiments, anti-TGFβ1 antibodies or antigen-binding fragments thereof are provided that block the binding of the aforementioned TGFβ1-binding molecules to a TGFβ1 precursor protein or a TGFβ1 complex (e.g., a human or mouse GARP-TGFβ1 complex, a LTBP-TGFβ1 complex, or a LRRC33-TGFβ1 complex). In some embodiments, the TGFβ1 in the TGFβ1 complex exists in the form of a TGFβ1 precursor protein.

[0115] In some embodiments, an anti-TGFβ1 antibody or antigen-binding fragment thereof is provided, wherein the binding of the antibody to a TGFβ1 protein or a TGFβ1 complex (e.g., a human or mouse GARP-TGFβ1 complex, a LTBP-TGFβ1 complex, or a LRRC33-TGFβ1 complex) is blocked by the aforementioned TGFβ1 binding molecules. In some embodiments, the TGFβ1 in the TGFβ1 complex exists in the form of a TGFβ1 precursor protein.

[0116] GARP-TGFβ1 binding molecule

[0117] In a second aspect, the present disclosure provides a GARP-TGFβ1 binding molecule comprising at least one immunoglobulin single variable domain that binds to a TGFβ1 complex.

[0118] 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. Exemplarily, the TGFβ1 precursor protein is selected from pro-TGFβ1 or latent TGFβ1. In this disclosure, "pro-TGFβ1" and "latent TGFβ1" can be used interchangeably.

[0119] In some embodiments, the present disclosure provides GARP-TGFβ1 binding molecules comprising at least one immunoglobulin single variable domain that binds to a GARP-TGFβ1 complex.

[0120] In some embodiments, the immunoglobulin single variable domain comprises three complementarity determining regions CDR1, CDR2, and CDR3, wherein:

[0121] a) the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence shown in SEQ ID NO: 84;

[0122] b) the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the amino acid sequence shown in SEQ ID NO: 90;

[0123] c) the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence shown in SEQ ID NO: 91; or

[0124] d) The immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in SEQ ID NO: 92.

[0125] The above-mentioned 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 numbering system. Exemplarily, CDR1, CDR2 and CDR3 defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems are provided below.

[0126] Table 3. CDR1, CDR2 and CDR3 in the sequence shown in SEQ ID NO: 84

[0127] In some embodiments, the immunoglobulin single variable domain comprises three complementarity determining regions CDR1, CDR2, and CDR3, wherein:

[0128] CDR1 comprises the amino acid sequence shown in SEQ ID NO:85;

[0129] CDR2 comprises the amino acid sequence shown in SEQ ID NO: 86; and / or,

[0130] CDR2 comprises the amino acid sequence shown in SEQ ID NO:87.

[0131] In some embodiments, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 shown in SEQ ID NOs: 85-87.

[0132] In some embodiments, the CDR1 comprised by the 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: 85; and / or,

[0133] The CDR2 comprised by the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared with the amino acid sequence shown in SEQ ID NO: 86; and / or,

[0134] The CDR3 comprised in the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared with the amino acid sequence shown in SEQ ID NO: 87.

[0135] In some specific embodiments, the amino acid mutations in CDR1, CDR2, or CDR3 are conservative substitutions.

[0136] 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.) of the aforementioned immunoglobulin single variable domains. In some embodiments, the GARP-TGFβ1 binding molecule comprises two or more immunoglobulin single variable domains, wherein any two immunoglobulin single variable domains may be the same or different.

[0137] In some embodiments, the GARP-TGFβ1 binding molecule may comprise any of the aforementioned complete immunoglobulin single variable domains; it may also comprise a functional portion of any of the aforementioned 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-FR4.

[0138] In some embodiments, the variant of the functional portion 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% or at least 90% sequence homology therewith, for example, it may be a polypeptide that retains the TGFβ1 complex binding function of CDR3 and has a certain sequence homology therewith, for example, a polypeptide that has at least 80% or at least 90% sequence homology with any of the above-mentioned CDR3s.

[0139] In some embodiments, the immunoglobulin single variable domain is a VHH. Exemplarily, the immunoglobulin single variable domain is a camelid VHH, a fully human VHH, or a humanized VHH.

[0140] In some specific embodiments, the immunoglobulin single variable domain is a humanized VHH.

[0141] In some embodiments, the human germline template of the humanized VHH is selected from at least one of 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.

[0142] In some embodiments, the humanized VHH comprises at least one back mutation as shown below: 23T, 29Y, 30C, 37Y, 44E, 45R, 47F, 71Q, 74A, 75R, 78G, 81E, 93K and 94T. In some specific embodiments, the framework region of the humanized VHH comprises at least one back mutation as described above.

[0143] The above-mentioned back mutation sites are numbered according to the Kabat numbering rules.

[0144] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 84, 90-92, or an amino acid sequence having at least 80% sequence identity thereto.

[0145] In some embodiments, the amino acid sequence comprised by the immunoglobulin single variable domain has one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) amino acid mutations compared to the sequence shown in any one of SEQ ID NOs: 84, 90-92.

[0146] In some specific embodiments, the amino acid mutations in the immunoglobulin single variable domain are conservative substitutions.

[0147] In some embodiments, the GARP-TGFβ1 binding molecule further comprises an immunoglobulin Fc region. Exemplarily, 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.

[0148] In some embodiments, the immunoglobulin Fc region comprises a mutation that reduces or eliminates Fc effector function. Exemplarily, the immunoglobulin Fc region comprises the mutation: S228P. The positions of the above mutations are numbered according to EU numbering conventions.

[0149] In some embodiments, the immunoglobulin Fc region comprises the amino acid sequence shown in SEQ ID NO: 88, or an amino acid sequence having at least 80% sequence identity thereto.

[0150] In some embodiments, the immunoglobulin Fc region is derived from mouse IgG2.

[0151] In some embodiments, the immunoglobulin Fc region is an Fc region with reduced or eliminated effector function, for example, a mutation that reduces or eliminates the ADCC effect. Exemplarily, the mutation that reduces or eliminates the ADCC effect comprises at least one of the following: L234A / L235E / G237A / D327Q / A330S / P331S (IgG2a); N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG2a); gG4); 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 above mutations are numbered according to EU numbering convention.

[0152] In some embodiments, the GARP-TGFβ1 binding molecule is an antibody or antigen-binding fragment thereof that binds to the GARP-TGFβ1 complex.

[0153] In some embodiments, the GARP-TGFβ1 binding molecule comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 89, 93-95, 106, or an amino acid sequence having at least 80% or at least 90% sequence identity thereto.

[0154] In some embodiments, the GARP-TGFβ1 binding molecule comprises one or more (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) amino acid mutations compared to the amino acid sequence shown in any one of SEQ ID NOs: 89, 93-95, 106, and has the functional activity of specifically binding to the GARP-TGFβ1 complex.

[0155] In some specific embodiments, the amino acid mutations in the GARP-TGFβ1 binding molecule are conservative substitutions.

[0156] In the context of the present disclosure, "at least 80%" encompasses 80% and above, for example 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.

[0157] In some embodiments, the aforementioned 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 signal transduction.

[0158] In some embodiments, the GARP-TGFβ1 binding molecule does not bind to free mature TGFβ1.

[0159] In some embodiments, the GARP-TGFβ1 binding molecule does not bind or very weakly binds to TGFβ2 protein or TGFβ2 complex. For example, the TGFβ1 binding molecule does not bind or very weakly binds to mature TGFβ2, TGFβ2 precursor protein, GARP-TGFβ2 complex, LTBP-TGFβ2 complex, etc.

[0160] In some embodiments, the GARP-TGFβ1 binding molecule does not bind or very weakly binds to TGFβ3 protein or TGFβ3 complex. For example, the GARP-TGFβ1 binding molecule does not bind or very weakly binds to mature TGFβ3, TGFβ3 precursor protein, GARP-TGFβ3 complex, LTBP-TGFβ3 complex, etc.

[0161] In the present disclosure, the term "TGFβ2 protein" should be broadly interpreted, encompassing mature TGFβ2, as well as inactive precursor forms such as pro-TGFβ2 and latent TGFβ2. The term "TGFβ3 protein" should be broadly interpreted, encompassing mature TGFβ3, as well as inactive precursor forms such as pro-TGFβ3 and latent TGFβ3.

[0162] In some embodiments, the aforementioned GARP-TGFβ1 binding molecules are capable of selectively inhibiting TGFβ1 activity, but not inhibiting the activity of TGFβ2 and / or TGFβ3. For example, the GARP-TGFβ1 binding molecules selectively inhibit the activation of TGFβ1, but not the activation of 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).

[0163] In some embodiments of the disclosure, mature TGFβ1, mature TGFβ2, and / or mature TGFβ3 are in a free state in the cell.

[0164] In some embodiments, the GARP-TGFβ1 binding molecule specifically binds to a GARP-TGFβ1 complex, wherein the GARP-TGFβ1 complex comprises:

[0165] a) Glycoprotein A repeat dominant sequence (GARP);

[0166] b) the mature TGFβ1 domain; and,

[0167] c) Latency-associated peptide (LAP).

[0168] In some embodiments, the GARP comprises the amino acid sequence as shown 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 as shown 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 as shown in SEQ ID NO: 102 or 105, or an amino acid sequence having at least 90% sequence identity thereto.

[0169] In some embodiments, the GARP-TGFβ1 binding molecule is present at 10 -7 M, 10 -8 M, 10 -9 M or lower K D Binds to the GARP-TGFβ1 complex or its fragments.

[0170] In some embodiments, the GARP-TGFβ1 binding molecule has the following properties: inhibits TGFβ1 activity, inhibits the immunosuppressive activity of regulatory T cells, and / or inhibits tumor growth.

[0171] In some embodiments, the GARP-TGFβ1 binding molecule is an anti-GARP-TGFβ1 antibody or an antigen-binding fragment thereof; in some specific embodiments, it is a chimeric antibody, a camelid antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof.

[0172] In some embodiments, the GARP-TGFβ1 binding molecule is an anti-GARP-TGFβ1 antibody or an antigen-binding fragment thereof; in some specific embodiments, the anti-GARP-TGFβ1 antibody or an 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 peptibodies, domain antibodies and multispecific antibodies (bispecific antibodies, diabodies, triabodies and tetrabodies, tandem two-scFv, tandem three-scFv), for example, scFv, Fv, Fab or Fab' fragments.

[0173] In some embodiments, anti-GARP-TGFβ1 antibodies or antigen-binding fragments thereof are provided that bind to or compete for binding to the same epitope of a GARP-TGFβ1 complex (eg, a human or mouse GARP-TGFβ1 complex) as the aforementioned GARP-TGFβ1 binding molecules.

[0174] In some embodiments, anti-GARP-TGFβ1 antibodies or antigen-binding fragments thereof are provided that block the binding of the aforementioned GARP-TGFβ1 binding molecules to GARP-TGFβ1 (eg, human or mouse GARP-TGFβ1 complex).

[0175] In some embodiments, an anti-GARP-TGFβ1 antibody or antigen-binding fragment thereof is provided, whose binding to a GARP-TGFβ1 complex (eg, a human or murine GARP-TGFβ1 complex) is blocked by a GARP-TGFβ1 binding molecule as described above.

[0176] In some embodiments, the GARP-TGFβ1 complex comprises (i) GARP, and (ii) TGFβ1 precursor protein. Exemplarily, in the GARP-TGFβ1 complex:

[0177] GARP comprises the 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;

[0178] And / or, the TGFβ1 protein comprises the 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.

[0179] Protein binding molecules

[0180] In some embodiments, the present disclosure provides TGFβ1 binding molecules.

[0181] 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.

[0182] As used herein, "TGFβ1" or "TGFβ1 protein" is understood in a broad sense to include the proprotein form (also known as proTGFβ1) 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 TGFβ1 precursor protein.

[0183] In some embodiments, the TGFβ1 precursor protein comprises: (i) a mature TGFβ1 domain, (ii) a potency-associated peptide (LAP).

[0184] Exemplarily, the TGFβ1 precursor protein is proTGFβ1 or latent TGFβ1, wherein the mature TGFβ1 domain in proTGFβ1 is covalently bound to latency-associated peptides (LAPs), and the mature TGFβ1 domain in latent TGFβ1 is non-covalently bound to latency-associated peptides (LAPs).

[0185] In the present disclosure, "pro-TGFβ1" or "latent TGFβ1" may be used interchangeably.

[0186] Illustratively, the mature TGFβ1 domain comprises the amino acid sequence shown in SEQ ID NO: 101 or 104, or an amino acid sequence having at least 80% sequence identity thereto.

[0187] Illustratively, the LAP comprises the amino acid sequence shown in SEQ ID NO: 102 or 105, or an amino acid sequence having at least 80% sequence identity thereto.

[0188] Exemplarily, the TGFβ1 precursor protein comprises an amino acid sequence as 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.

[0189] In some embodiments, the TGFβ1 precursor protein or TGFβ1 complex further comprises a protein fragment, a functional variant, etc. of any of the aforementioned proteins or protein complexes.

[0190] In some embodiments, the TGFβ1 precursor protein or TGFβ1 complex is derived from any vertebrate, including mammals such as primates (eg, humans) and other species (eg, mice, rats, guinea pigs, rabbits, dogs, pigs, sheep, etc.).

[0191] Exemplarily, "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, alpha 1 helical regions, alpha 2 helical regions, RGD sequence regions, trigger loop regions, extracellular domains, transmembrane domains, intracellular domains, and the like.

[0192] Exemplarily, "variant" or "functional variant" refers to a protein or protein complex comprising one or more amino acid substitutions, deletions and / or added amino acids and having comparable biological activity.

[0193] Exemplarily, the variant of the TGFβ1 precursor protein comprises a mutation in amino acid position 4 according to natural numbering relative to the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the variant of the TGFβ1 precursor protein comprises a mutation C4S, wherein the mutation site is a site according to natural numbering relative to the amino acid sequence shown in SEQ ID NO: 6.

[0194] In some embodiments, the TGFβ1 precursor protein or TGFβ1 complex may or may not contain a leader sequence. Exemplarily, the leader sequence is a signal peptide sequence. In some embodiments, the TGFβ1 precursor protein or TGFβ1 complex may or may not contain a tag sequence. Exemplarily, the tag sequence is a His-tag, AVI-tag, myc-tag, fluorescent tag, etc.

[0195] In some embodiments, the TGFβ1 complex comprises a TGFβ1 protein and a second protein selected from any of the following: glycoprotein A repeat dominant sequence (GARP), latent TGF-β-binding proteins (LTBP; for example, LTBP1, LTBP1S, LTBP2, LTBP3, LTBP4), fibrillin (for example, fibrillin-1, fibrillin-2, fibrillin-3, fibrillin-4), LRRC33 (Leucine-Rich Repeat-Containing Protein 33), or a variant or protein fragment of any of the foregoing.

[0196] Exemplarily, "protein fragment" or "fragment" includes but is not limited to an extracellular domain, a transmembrane domain or an intracellular domain, etc.

[0197] 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, TGFβ1 in the TGFβ1 complex exists in the form of a TGFβ1 precursor protein.

[0198] Exemplarily, the GARP-TGFβ1 complex comprises:

[0199] a) Glycoprotein A repeat dominant sequence (GARP);

[0200] b) the mature TGFβ1 domain; and,

[0201] c) Latency-associated peptide (LAP).

[0202] In some embodiments, the GARP comprises the amino acid sequence as shown 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 as shown 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 as shown in SEQ ID NO: 102 or 105, or an amino acid sequence having at least 80% sequence identity thereto.

[0203] In some embodiments, the GARP-TGFβ1 complex is formed by GARP and TGFβ1 precursor protein.

[0204] In some specific embodiments, the GARP-TGFβ1 complex is formed by the extracellular region of GARP and the TGFβ1 precursor protein.

[0205] In the present disclosure, "GARP" can be a wild-type GARP of natural origin, or a functional variant of GARP (e.g., comprising one or more modifications, truncations, and / or mutations compared to wild-type GARP). In some embodiments, GARP can be full-length or a partial domain thereof (e.g., an extracellular domain, a transmembrane domain, or an intracellular domain). For example, natural origin refers to any vertebrate source, including mammals such as primates (e.g., humans) and other species (e.g., mice, rats, guinea pigs, rabbits, dogs, pigs, sheep, etc.).

[0206] In some embodiments, GARP may or may not include a leader sequence. Exemplarily, the leader sequence is a signal peptide sequence. In some embodiments, GARP may include one or more tag sequences, such as a His-tag, AVI-tag, myc-tag, fluorescent tag, etc. In some embodiments, GARP may not include a tag sequence.

[0207] Illustratively, the extracellular region of GARP comprises the amino acid sequence shown in SEQ ID NO: 100 or 103, or an amino acid sequence having at least 80% sequence identity thereto.

[0208] Exemplarily, GARP comprises an amino acid sequence as shown 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.

[0209] In some embodiments, the protein binding molecule that binds to the TGFβ1 precursor protein or the TGFβ1 complex is selected from an antibody or an antigen-binding fragment thereof. Exemplarily, the antibody or antigen-binding fragment thereof includes, but is not limited to, any of the following: a linear antibody, a single-chain antibody (scFv), a single-domain antibody (sdAb), a nanobody, a peptibody, a domain antibody, and a multispecific antibody (bispecific antibody, diabody, triabody, and tetrabody, tandem two-scFv, tandem three-scFv), Fab, Fv, sFv, Fab', and F(ab')2.

[0210] In some embodiments, antibodies or antigen-binding fragments thereof are provided that specifically bind to an epitope of the TGFβ1 precursor protein or TGFβ1 complex. In some embodiments, the epitope is available for binding by the antibody or antigen-binding fragment thereof when TGFβ1 is in the form of a precursor protein or in a complex with GARP, LTBP1, LTBP3, and / or LRRC33. In some embodiments, the epitope is available due to a conformational change in TGFβ1 when complexed with GARP, LTBP, and / or LRRC33. In some embodiments, the epitope bound by the antibody or antigen-binding fragment thereof in TGFβ1 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 prevent TGFβ1 from binding to integrins. For example, in some embodiments, the antibody or antigen-binding fragment thereof does not mask the integrin binding site of TGFβ1.

[0211] In some embodiments, the protein-binding molecule does not bind or very weakly binds to the TGFβ2 protein or TGFβ2 complex. Exemplarily, the protein-binding molecule does not specifically bind to at least one of the following: mature TGFβ2, TGFβ2 precursor protein, GARP-TGFβ2 complex, LTBP-TGFβ2 complex, etc.

[0212] In some embodiments, the protein-binding molecule does not bind or very weakly binds to the TGFβ3 protein or TGFβ3 complex. Exemplarily, the protein-binding molecule does not specifically bind to at least one of the following: mature TGFβ3, TGFβ3 precursor protein, GARP-TGFβ3 complex, LTBP-TGFβ3 complex, etc.

[0213] In the present disclosure, "TGFβ2 protein" should be understood broadly, covering mature TGFβ2 as well as inactive precursor protein forms such as pro-TGFβ2 and latent TGFβ2. "Pro-TGFβ2" and "latent TGFβ2" can be used interchangeably.

[0214] In the present disclosure, "TGFβ3 protein" should be understood broadly, covering mature TGFβ3 as well as inactive precursor protein forms such as pro-TGFβ3 and latent TGFβ3. "Pro-TGFβ3" and "latent TGFβ3" can be used interchangeably.

[0215] In some embodiments, mature TGFβ1, mature TGFβ2, and / or mature TGFβ3 are present in a free state in the cell.

[0216] In some embodiments, the protein binding molecule specifically binds to a TGFβ1 precursor protein or a TGFβ1 complex (e.g., a GARP-TGFβ1 complex, a LTBP1-TGFβ1 complex, a LTBP3-TGFβ1 complex, a LRRC33-TGFβ1 complex, etc.), and can selectively inhibit the activity of TGFβ1, for example, inhibiting the activation of TGFβ1, and / or inhibiting the release of mature TGFβ1, and / or inhibiting TGFβ1 signal transduction.

[0217] polynucleotides

[0218] The present disclosure provides polynucleotides encoding any of the aforementioned protein binding molecules. In some embodiments, the present disclosure provides polynucleotides encoding any of the aforementioned TGFβ1 binding molecules and GARP-TGFβ1 binding molecules.

[0219] In some embodiments, the present disclosure provides a polynucleotide encoding the TGFβ1 binding molecule of the first aspect of the present disclosure. In some embodiments, the present disclosure provides a polynucleotide encoding the GARP-TGFβ1 binding molecule of the second aspect of the present disclosure.

[0220] In some embodiments, the polynucleotides of the present disclosure may be RNA, DNA, or cDNA.

[0221] In some embodiments, the polynucleotides of the present disclosure are isolated polynucleotides.

[0222] In some embodiments, the polynucleotides of the present disclosure may also be in the form of a vector, may be present in a vector and / or may be part of a vector, which may be a eukaryotic vector, a prokaryotic vector, a viral vector, such as a plasmid, a cosmid, a YAC or a viral vector, etc. The vector may be, in particular, an expression vector, i.e., a vector that provides for 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). The expression vector typically comprises at least one polynucleotide of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., a promoter, an enhancer, a terminator, etc.). It is common knowledge for those skilled in the art to select the elements and their sequences for expression in a specific host. Regulatory elements and other elements that are useful or necessary for the expression of the protein binding molecules, TGFβ1 binding molecules, and GARP-TGFβ1 binding molecules of the present disclosure are, for example, promoters, enhancers, terminators, integrons, selection markers, leader sequences, and reporter genes.

[0223] The polynucleotides of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0224] In some embodiments, the polynucleotides and vectors disclosed herein can be used to prepare TGFβ1 binding molecules. In some embodiments, the polynucleotides and vectors disclosed herein are used to express TGFβ1 binding molecules in vitro or in vivo, and the TGFβ1 binding molecules bind to TGFβ1 precursor protein or TGFβ1 complex for various purposes such as detection, diagnosis, treatment, and regulation.

[0225] In some embodiments, the polynucleotides and vectors disclosed herein can be used to prepare GARP-TGFβ1 binding molecules. In some embodiments, the polynucleotides and vectors disclosed herein are used to express GARP-TGFβ1 binding molecules in vitro or in vivo, and the GARP-TGFβ1 binding molecules bind to the GARP-TGFβ1 complex for various purposes such as detection, diagnosis, treatment, and regulation.

[0226] host cells

[0227] The present disclosure provides host cells that express one or more protein binding molecules of the present disclosure. In some embodiments, the present disclosure provides host cells that comprise any of the aforementioned polynucleotides or vectors; or, alternatively, the host cells express any of the aforementioned TGFβ1 binding molecules or GARP-TGFβ1 binding molecules.

[0228] In some embodiments, the host cell expresses the TGFβ1 binding molecule of the first aspect of the present disclosure. In some embodiments, the host cell expresses the GARP-TGFβ1 binding molecule of the second aspect of the present disclosure.

[0229] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0230] Exemplary bacterial cells include, for example, cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0231] Exemplary fungal cells include cells of species of the genera Trichoderma, Neurospora, and Aspergillus; or cells of species of the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0232] Illustratively, mammalian cells such as monkey kidney CV1 line (COS-7), human embryonic kidney line (293 or 293T cells), baby hamster 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 liver cells (BRL3A), human lung cells (W138), human liver cells (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 (as described, for example, in Mather et al., Annals of Medicine, 1994). 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.

[0233] The present disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0234] The cells of the present disclosure are incapable of developing into complete plant or animal individuals.

[0235] Production or preparation method

[0236] The present disclosure provides methods of making the protein binding molecules of the present disclosure.

[0237] In some embodiments, the present disclosure provides a method for preparing the TGFβ1 binding molecule of the first aspect, comprising the following steps:

[0238] a. forming a TGFβ1 precursor protein or a TGFβ1 complex comprising the TGFβ1 precursor protein as an antigenic protein;

[0239] b. Construction of human antibody phage library;

[0240] c. Using the phage library to screen TGFβ1 binding molecules that specifically bind to the antigen protein.

[0241] In some embodiments, the method for preparing a TGFβ1 binding molecule further comprises the following steps:

[0242] d. Perform affinity maturation on the TGFβ1 binding molecule.

[0243] The above preparation method is also a method for screening TGFβ1 binding molecules that specifically bind to TGFβ1 precursor protein or a TGFβ1 complex containing the TGFβ1 precursor protein.

[0244] In some embodiments, the present disclosure provides a method for preparing the GARP-TGFβ1 binding molecule of the second aspect, comprising the following steps:

[0245] a. forming a complex comprising human GARP and human TGFβ1 precursor protein as an antigen protein;

[0246] b. Immunizing animals with the antigen protein to construct a phage library;

[0247] c. Using the phage library to screen for GARP-TGFβ1 binding molecules, wherein the GARP-TGFβ1 binding molecules comprise immunoglobulin single variable domains that bind to the GARP-TGFβ1 complex.

[0248] In some embodiments, the method for preparing a GARP-TGFβ1 binding molecule further comprises the following steps:

[0249] d. Humanize the GARP-TGFβ1 binding molecule.

[0250] In some specific embodiments, step b is further:

[0251] Camels are immunized with the antigen protein, peripheral blood of the immunized camels is collected, nucleic acid is extracted, and a phage library is constructed using the nucleic acid.

[0252] The above preparation method is also a method for screening GARP-TGFβ1 binding molecules that specifically bind to the GARP-TGFβ1 complex.

[0253] In other embodiments, the present disclosure provides a method for preparing any of the aforementioned TGFβ1 binding molecules or GARP-TGFβ1 binding molecules, comprising:

[0254] - culturing the host cell of the present disclosure under conditions that allow expression of the TGFβ1 binding molecule or GARP-TGFβ1 binding molecule of the present disclosure; and

[0255] - recovering the target protein expressed by the host cell from the culture; and

[0256] - Optionally, further purification and / or modification of the protein of interest of the present disclosure is included.

[0257] The TGFβ1-binding molecules or GARP-TGFβ1-binding molecules of the present disclosure can be produced intracellularly in the cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

[0258] Methods and reagents for recombinantly producing proteins or polypeptides, such as specifically suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, and the like, are known in the art. Similarly, isolation and purification techniques suitable for producing target proteins, such as binding molecules or antibodies, of the present disclosure are well known to those skilled in the art. Methods for producing and purifying antibodies are well known in the art and can be found in the Cold Spring Harbor Laboratory Manual (Chapters 5-8 and 15). The engineered antibodies of the present disclosure can also be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can be stably transfected into 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 fluid secreting the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0259] However, the TGFβ1 binding molecules or GARP-TGFβ1 binding molecules of the present 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.

[0260] Pharmaceutical composition

[0261] The present disclosure provides pharmaceutical compositions comprising any one or a combination thereof selected from the following: the protein binding molecules or polynucleotides encoding the same as described above. In some embodiments, the pharmaceutical compositions comprise a prophylactically or therapeutically effective amount of a TGFβ1 binding molecule or GARP-TGFβ1 binding molecule as described above, or a polynucleotide or vector encoding the same.

[0262] In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, diluents, buffers, or vehicles.

[0263] In some embodiments, the pharmaceutical composition can be formulated into any dosage form known in the medical art, with the dosage form selected depending on the intended mode of administration and therapeutic use.

[0264] In some embodiments, the pharmaceutical composition further comprises an immune checkpoint inhibitor. For example, immune checkpoint inhibitors include, but are not limited to, one or more inhibitors of PD-1, PD-L1, and PD-L2. In some embodiments, the pharmaceutical composition further comprises an anti-PD-1 antibody or an antigen-binding fragment thereof.

[0265] The TGFβ1 binding molecules or GARP-TGFβ1 binding molecules disclosed herein can inhibit TGFβ1 activity and the immunosuppressive activity of regulatory T cells. The combination of TGFβ1 binding molecules or GARP-TGFβ1 binding molecules with anti-PD-1 antibodies can relieve the tumor immunosuppressive microenvironment. The disclosed embodiments found that the combination of TGFβ1 binding molecules or GARP-TGFβ1 binding molecules with anti-PD-1 antibodies significantly improved the therapeutic effect in tumor treatment.

[0266] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the TGFβ1 binding molecule per unit dose. In other specific embodiments, the pharmaceutical composition may contain 0.1-2000 mg of the TGFβ1 binding molecule per unit dose; in some specific embodiments, 1-1000 mg.

[0267] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the GARP-TGFβ1 binding molecule per unit dose. In other specific embodiments, the pharmaceutical composition may contain 0.1 to 2000 mg of the GARP-TGFβ1 binding molecule per unit dose; in some specific embodiments, 1 to 1000 mg.

[0268] Combination therapy

[0269] The present disclosure provides the use of any protein-binding molecule or its encoding polynucleotide in combination with an immune checkpoint inhibitor for treating diseases associated with the TGFβ signaling pathway. Exemplarily, the immune checkpoint inhibitor includes but is not limited to one or more inhibitors of PD-1, PD-L1, and PD-L2.

[0270] In some embodiments, provided is a use of a TGFβ1 binding molecule in combination with an immune checkpoint inhibitor for treating a disease associated with the TGFβ signaling pathway.

[0271] In some embodiments, provided is a use of a TGFβ1 binding molecule in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof for treating a disease associated with the TGFβ signaling pathway.

[0272] In some embodiments, provided is a use of a GARP-TGFβ1 binding molecule in combination with an immune checkpoint inhibitor for treating diseases associated with the TGFβ signaling pathway.

[0273] In some embodiments, provided is a use of a GARP-TGFβ1 binding molecule in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof for treating a disease associated with the TGFβ signaling pathway.

[0274] The term "TGFβ signaling pathway-associated disease" 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 level of one or more TGFβ family proteins. Diseases associated with the TGFβ signaling pathway may include, but are not limited to, tumors or cancer.

[0275] In some embodiments, the disease associated with the TGFβ signaling pathway is cancer. The present disclosure provides use of a TGFβ1 binding molecule or a GARP-TGFβ1 binding molecule in combination with an anti-PD-1 antibody or an antigen-binding fragment thereof for treating cancer.

[0276] TGFβ1 binding molecules or GARP-TGFβ1 binding molecules significantly improve the immunosuppressive microenvironment of tumors, and when used in combination with anti-PD-1 antibodies, they exert significantly improved therapeutic effects in tumor treatment.

[0277] 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, head and neck cancer.

[0278] Reagent kit (or medicine box)

[0279] The present disclosure provides a kit or a medicine box comprising one or more containers, each of which independently contains any one or a combination thereof selected from the following: a protein binding molecule of the present disclosure (e.g., the TGFβ1 binding molecule or GARP-TGFβ1 binding molecule), or a polynucleotide or vector encoding the same.

[0280] Methods for preventing and treating diseases and pharmaceutical uses

[0281] The present disclosure provides protein-binding molecules, or their encoding polynucleotides, vectors, pharmaceutical compositions, and methods for preventing, treating, and alleviating diseases or symptoms.

[0282] In some embodiments, the present disclosure provides for use of a TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition in at least one of the following:

[0283] (1) for inhibiting TGFβ1 activity; for example, inhibiting TGFβ1 activation, inhibiting the release of mature TGFβ1 from the TGFβ1 complex, and / or inhibiting TGFβ1 signal transduction;

[0284] (2) preparing a drug for inhibiting TGFβ1 activity;

[0285] (3) used to inhibit the immunosuppressive activity of regulatory T cells;

[0286] (4) preparing a drug for inhibiting the immunosuppressive activity of regulatory T cells;

[0287] (5) Used for preventing or treating diseases or conditions related to the TGFβ signaling pathway.

[0288] In some embodiments, the present disclosure provides methods for preventing or treating diseases or disorders associated with the TGFβ signaling pathway, comprising administering to a subject a preventively or therapeutically effective amount of a TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition.

[0289] In some embodiments, the present disclosure provides a method for inhibiting TGFβ1 activity in vitro, 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.

[0290] In some embodiments, the present disclosure provides a method for inhibiting TGFβ1 activity in vivo, comprising administering to a subject a TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition. In some specific embodiments, an inhibitory effective amount of a TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition is administered to the subject.

[0291] In some specific embodiments, the subject suffers from a disease or disorder associated with the TGFβ signaling pathway.

[0292] The term "TGFβ signaling pathway-associated disease" 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 level of one or more TGFβ family proteins. Diseases associated with the TGFβ signaling pathway may include, but are not limited to, fibrosis-related diseases or conditions, tumors, or cancer.

[0293] In some embodiments, the disease associated with the TGFβ signaling pathway is cancer.

[0294] In some embodiments, the disease or disorder associated with the TGFβ signaling pathway is fibrosis.

[0295] In some embodiments, a method for preventing or treating cancer is provided, comprising administering a preventively or therapeutically effective amount of the components shown in 1) and 2) below:

[0296] 1) anti-TGFβ1 antibody or antigen-binding fragment thereof, or encoding polynucleotide, vector, or pharmaceutical composition;

[0297] 2) Immune checkpoint inhibitors.

[0298] Illustratively, the PD-1 signaling pathway inhibitor includes, but is not limited to, one or more inhibitors 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.

[0299] 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, head and neck cancer.

[0300] In some embodiments, the TGFβ1 binding molecules or pharmaceutical compositions of the present disclosure may be administered by any suitable method known in the art, and administration may be systemic or local.

[0301] In some embodiments, the dosage regimen can be adjusted to obtain the optimal intended response (e.g., therapeutic or preventive response). For example, the dosage can be a single dose, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the therapeutic situation.

[0302] In some embodiments, the present disclosure provides for use of a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition in at least one of the following:

[0303] (1) for inhibiting TGFβ1 activity; for example, inhibiting TGFβ1 activation, inhibiting the release of mature TGFβ1 from the TGFβ1 complex, and / or inhibiting TGFβ1 signal transduction;

[0304] (2) preparing a drug for inhibiting TGFβ1 activity;

[0305] (3) used to inhibit the immunosuppressive activity of regulatory T cells;

[0306] (4) preparing a drug for inhibiting the immunosuppressive activity of regulatory T cells;

[0307] (5) Used for preventing or treating diseases or conditions related to the TGFβ signaling pathway.

[0308] In some embodiments, the present disclosure provides methods for preventing or treating diseases or disorders associated with the TGFβ signaling pathway, comprising administering to a subject a preventively or therapeutically effective amount of a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition.

[0309] In some embodiments, the present disclosure provides a method for preventing or treating a disease associated with the TGFβ signaling pathway, comprising administering to a subject a preventively or therapeutically effective amount of a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector or pharmaceutical composition; and a preventively or therapeutically effective amount of an immune checkpoint inhibitor.

[0310] Illustratively, immune checkpoint inhibitors include, but are not limited to, one or more inhibitors 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.

[0311] In some embodiments, the present disclosure provides a method for inhibiting TGFβ1 activity in vitro, comprising administering a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition in vitro. In some specific embodiments, an inhibitory effective amount of the GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition is administered.

[0312] In some embodiments, the present disclosure provides a method for inhibiting TGFβ1 activation or inhibiting the release of mature TGFβ1 from the GARP-TGFβ1 complex in vitro, comprising: administering a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition in vitro. In some specific embodiments, an inhibitory effective amount of the GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition is administered.

[0313] In some embodiments, the present disclosure provides a method for inhibiting TGFβ1 activity in vivo, comprising administering to a subject a GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition. 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.

[0314] In some embodiments, the present disclosure provides a method for inhibiting TGFβ1 activation or inhibiting the release of mature TGFβ1 from the GARP-TGFβ1 complex in vivo, 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 the GARP-TGFβ1 binding molecule, encoding polynucleotide, vector, or pharmaceutical composition is administered to the subject.

[0315] In some specific embodiments, the subject suffers from a disease or disorder associated with the TGFβ signaling pathway.

[0316] In some embodiments, the disease associated with the TGFβ signaling pathway is cancer.

[0317] 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, head and neck cancer.

[0318] In some embodiments, the GARP-TGFβ1 binding molecules or pharmaceutical compositions of the present disclosure can be administered by any suitable method known in the art, and administration can be systemic or local.

[0319] In some embodiments, the dosage regimen can be adjusted to obtain the optimal intended response (e.g., therapeutic or preventive response). For example, the dosage can be a single dose, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the therapeutic situation.

[0320] definition

[0321] In order to make the present disclosure more easily understood, certain technologies and sciences are specifically defined below. Unless otherwise explicitly defined in the present disclosure, all other technologies and sciences used in the present disclosure have the meanings commonly understood by those skilled in the art in the art to which the present disclosure belongs.

[0322] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be construed to have an inclusive sense rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to."

[0323] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0324] "About" and "approximately" refer to values ​​within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, between 0.5% and 1%, and in this disclosure, each instance of a number or numerical range preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0325] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0326] The term "transforming growth factor-β" refers to a family of structurally and functionally related polypeptide growth factors involved in numerous biological pathways. In addition to TGF-β, this family also includes activins, inhibins, growth and differentiation factors (GDFs), and bone morphogenetic proteins (BMPs). TGFβ has three isoforms: TGFβ1, TGFβ2, and TGFβ3, which are widely expressed in virtually all cell types in mammals.

[0327] Unlike other cytokines, members of the TGFβ superfamily are not secreted as active growth factors, but rather as dimeric precursor proteins consisting of an N-terminal prodomain and a C-terminal growth factor domain. ProTGFβ1 is separated from its homodimeric prodomain (also known as latency-associated peptide (LAP)) by cleavage with furin. However, the growth factor and LAP remain non-covalently bound, forming a latent complex that is unable to bind to its receptor and induce signal transduction. During translation, latent TGFβ1 (also known as the small latent complex (SLC)) becomes linked to a "presenting molecule" via a disulfide bridge, forming a large latent complex (LLC). These molecules allow proTGFβ1 to exist in specific cell or tissue contexts. Two cysteines near the N-terminus of latent TGFβ1 are linked to appropriately positioned cysteines on the presenting molecule. The identity of the presenting molecule depends on the environment and the cell type producing the latent TGFβ1. For example, fibroblasts secrete latent TGFβ1 that is tethered to TGFβ-binding proteins (LTBPs), which then bind to proteins in the extracellular matrix (ECM) (i.e., fibronectin, fibrillin-1) to link 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 linked to the transmembrane protein GARP, and a protein closely related to GARP, 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): p. 1129-39 and TA Springer, Int. BMP Conference 2016).

[0328] Within the scope of the present disclosure, TGFβ1 protein should be understood in the broadest sense. The term covers natural forms of TGFβ1 in nature, naturally occurring variants, as well as artificially expressed forms, functional variants, and the like. When the context does not specifically state otherwise, TGFβ1 covers the scope of mature TGFβ1, TGFβ1 precursor protein, and protein epitopes thereof. TGFβ1 precursor protein covers pro-TGFβ1, latent TGFβ1, and fragments thereof. TGFβ1 sequences can be obtained from GenBank, UniProt, and the like. For example, human pro-TGFβ1 (Uniprot: P01137), mouse pro-TGFβ1 (Uniprot: P04202).

[0329] In the context of the present disclosure, "pro-TGFβ1" and "latent TGFβ1" are used interchangeably.

[0330] As used herein, the term "TGFβ1 complex" refers to a complex formed by TGFβ1 and a protein molecule including but not limited to GARP, LRRC33, LTBP3 or LTBP1 through a disulfide bridge, 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).

[0331] The term "functional variant" includes but is not limited to homologs, fragments, truncations, mutants, modifications, etc. of the wild-type protein. The functional variant of the protein has improved, reduced or maintained protein activity compared to the wild-type protein.

[0332] The term "binding molecule" encompasses any molecule that can specifically bind to an antigen or antigenic epitope, such as an antibody, an antigen-binding fragment thereof, or a conjugate or fusion protein thereof as defined in the present disclosure.

[0333] "Antibody" is used in the broadest sense to encompass various 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), as long as they exhibit the desired antigen-binding activity. An antibody may refer to an immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Immunoglobulins differ in their antigenicity due to the amino acid composition and arrangement order of their heavy chain constant regions. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, corresponding to μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, different subclasses can be formed based on 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 classified as either κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either kappa or lambda chains. The approximately 110 amino acids near the N-terminus of both the heavy and light chains of antibodies vary greatly in sequence and constitute the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region consists of three hypervariable regions (CDRs) and four framework regions (FRs) whose sequences are relatively conserved. These three hypervariable regions determine the specificity of the antibody and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0334] For the determination or definition of CDRs, the definitive delineation of CDRs and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of 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.

[0335] 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 positions 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 produced by the Oxford Molecular Group for modeling antibody structure (see, e.g., Martin et al., 1989, Proc Natl 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 uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from the primary sequence (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," 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). Other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used herein, CDR may refer to a CDR defined by any method known in the art (including a combination of methods). The correspondence between various numbering systems is well known to those skilled in the art, and is exemplified by the following Table 4.

[0336] Table 4. Relationships between CDR numbering systems

[0337] A "domain" of a polypeptide or protein refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein. In general, a domain is responsible for a single functional property of a protein and in many cases can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0338] An "immunoglobulin variable domain" is an immunoglobulin domain that essentially consists of four "framework regions," referred to in the art 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 separated by three "complementarity determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity for an antigen by having an antigen-binding site.

[0339] "Antibody framework (FR)" refers to the portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain.

[0340] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the VH / VL interactions required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, e.g., camelized human VH), IgNAR, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs), and antibodies that are VH domains or derived from VH domains. TM ) and (single domain) antibodies (such as dAbs) as the VL domain or derived from the VL domain TM ). Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0341] "VHH domain", also known as heavy chain single domain antibody, VHH, V HH domain, VHH antibody fragment, VHH antibody, nanobody, is a variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., an "antibody lacking a light chain") (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 as "VH domain" in this disclosure) and the light chain variable domain (referred to as "VL domain" in this disclosure) present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to an epitope without the need for other antigen-binding domains (this is in contrast to the VH or VL domain in conventional tetrapeptide chain structure antibodies, in which case the epitope is recognized by both the VL domain and the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. H H domain", "VHH antibody fragment", "VHH antibody", "Nanobody" and "Nanobody domain" are used interchangeably. "VHH domain" includes but is not limited to natural antibodies produced by camelids, antibodies produced by camelids that are then humanized, or fully human antibodies obtained by phage display technology. The total number of amino acid residues in a VHH domain will generally be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: 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, 17 June, 2009 and WO94 / 04678.

[0342] As is well 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 numbered according to Kabat may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions include Chothia, IMGT, and AbM.

[0343] "Humanized antibodies," also known as CDR-grafted antibodies, are antibodies produced by transplanting non-human CDR sequences into the variable region framework of a human antibody. This can overcome the strong immune response induced by chimeric antibodies due to the large amount of non-human protein components they carry. To avoid a decrease in activity while also reducing immunogenicity, minimal reverse mutations can be performed on the fully human antibody variable region to maintain activity. Examples of "humanization" include "humanizing" a VHH domain derived from Camelidae by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at corresponding positions in a VH domain of a conventional human tetrapeptide chain antibody (also referred to as "sequence optimization" in this disclosure; in addition to humanization, "sequence optimization" may also encompass other modifications to the sequence by one or more mutations that provide 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. In addition, in order to avoid a decrease in activity caused by a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0344] "Fully human antibodies" or "fully humanized antibodies" include antibodies having variable and constant regions of human germline immunoglobulin sequences. The fully human antibodies of 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). "Fully human antibodies" do not include "humanized antibodies."

[0345] When "competition" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it means competition between antigen binding proteins, which is determined by the following assay: the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen. Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli 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 and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1992).

[0014] The present invention relates to a method for the determination of competitive inhibition by direct solid-phase labeling of antigen-binding proteins (RIA) using an I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, the assay involves the use of purified antigen that binds to an unlabeled test antigen-binding protein and a labeled reference antigen-binding protein (the antigen being on a solid surface or on the surface of a cell). Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is 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 a reference antigen binding protein; and antigen binding proteins that bind to an epitope that is sufficiently proximal to the epitope bound by the reference antigen binding protein that the two epitopes sterically interfere with each other's binding. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of the reference antigen binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0346] 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 compete with each other or cross-compete for antigen binding. High-throughput methods for obtaining antibodies that bind to the same epitope based on their cross-competition are described in International Patent Publication WO03 / 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 the antigen protein.

[0347] The term "Glycoprotein-A Repetitions Predominant (GARP)" is a protein with a single transmembrane structure. "GARP" is also known as Leucine-rich Repeat 32 (LeucinRichRepeatContaining32, LRRC32) and belongs to the Leucine-rich repeat family. GARP can be expressed on the cell surface of activated Tregs and form a complex with TGF-β precursors (eg, latent TGF-β). GARP sequences can be obtained from GenBank, UniProt, etc. For example, human GARP (LRRC32, Uniprot: Q14392), mouse GARP (LRRC32, Uniprot: G3XA59).

[0348] In the context of this disclosure, GARP should be understood in the broadest sense. The term encompasses GARP in its native form and naturally occurring variants, as well as artificially expressed forms and functional variants. Unless otherwise specified in the context, GARP encompasses the entire protein, the extracellular domain, and its epitopes in the context of antigen-antibody interactions.

[0349] The terms "GARP-TGFβ1 complex", "GARP / TGFβ1 protein" and "GARP / TGFβ1" refer to a protein complex comprising a precursor protein of the transforming growth factor-β1 (TGFβ1) protein and a glycoprotein-A repeat-based protein (GARP). In some embodiments, the proprotein form or latent form of the TGFβ1 protein may be referred to as a "pro / latent TGFβ1 protein". In some embodiments, the GARP-TGFβ1 complex comprises a GARP covalently linked to a TGFβ1 precursor protein (pro / latent TGFβ1 protein) through one or more disulfide bonds. In other embodiments, the GARP-TGFβ1 complex comprises a GARP non-covalently linked to a TGFβ1 precursor protein (pro / latent TGFβ1 protein). In some embodiments, the GARP-TGFβ1 complex is a naturally occurring complex, such as a GARP-TGFβ1 complex in a cell.

[0350] The term "LTBP" refers to latent transforming growth factor β binding proteins. LTBP is an important component of the extracellular matrix (ECM), and its primary function is related to regulating fibrillin and transforming growth factor β (TGF-β). In mammals, there are four known LTBPs, LTBP1-4, each with multiple splice variants (Robertson, IB et al., Matrix Biol, 2015. 47: p. 44-53).

[0351] The terms "GARP-TGFβ1 complex", "GARP / TGFβ1 protein", and "GARP / TGFβ1" refer to a protein complex comprising a precursor protein form or a latent form of the transforming growth factor-β1 (TGFβ1) protein and a glycoprotein-A repeat-based protein (GARP). In some embodiments, the proprotein form or the latent form of the TGFβ1 protein may be referred to as a "pro / latent TGFβ1 protein". In some embodiments, the GARP-TGFβ1 complex comprises a GARP covalently linked to a TGFβ1 precursor protein (pro / latent TGFβ1 protein) through one or more disulfide bonds. In other embodiments, the GARP-TGFβ1 complex comprises a GARP non-covalently linked to a TGFβ1 precursor protein (pro / latent TGFβ1 protein). In some embodiments, the GARP-TGFβ1 complex is a naturally occurring complex, such as a GARP-TGFβ1 complex in a cell.

[0352] The term "LTBP1-TGFβ1 complex" refers to a protein complex comprising a precursor protein of the transforming growth factor β1 (TGFβ1) protein and a latent TGFβ-binding protein (e.g., LTBP1, LTBP3). In some embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 covalently linked to the TGFβ1 precursor protein via one or more disulfide bonds. In other embodiments, the LTBP1-TGFβ1 complex comprises LTBP1 non-covalently linked to the TGFβ1 precursor protein. In some embodiments, the LTBP1-TGFβ1 complex is a naturally occurring complex, such as an LTBP1-TGFβ1 complex in a cell.

[0353] The term "antigen" refers to a molecule used to immunize an immunocompetent vertebrate to produce antibodies that recognize the antigen, or to screen an expression library (e.g., a phage, yeast, or ribosome display library). In the present disclosure, antigens are defined more broadly to include target molecules specifically recognized by antibodies, as well as a portion or mimetic of a molecule used in an immunization process for producing antibodies or in a library screening for selecting antibodies. For example, antibodies that bind to the human GARP-TGFβ1 complex of the present disclosure, as well as truncated variants and other variants of the human GARP-TGFβ1 complex are referred to as antigens.

[0354] The term "epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope generally comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitope is bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0355] "Specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. For example, when a human GARP-TGFβ1 complex or its epitope is used as an analyte and an antibody is used as a ligand, the antibody binds to the epitope at a specific binding rate of less than 10 -7 M or even smaller equilibrium dissociation constant (K D) binds to a predetermined antigen or an epitope thereof, and its affinity for binding to the predetermined antigen or an epitope thereof is at least twice that of its affinity for binding to a nonspecific antigen (such as BSA, etc.) other than the predetermined antigen (or its epitope) or a closely related antigen. "Antibody that recognizes an antigen" may be used interchangeably with "specifically binding antibody" in this disclosure.

[0356] "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 portion thereof and an antigen). The binding affinity between two molecules can be quantified by determining the dissociation constant (KD). KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, a surface plasmon resonance (SPR) method (Biacore). The rate constants corresponding to the association and dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. K D Through equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362). For example, K can be determined using a double filtration nitrocellulose filter binding assay such as that 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 exemplified elsewhere in this disclosure. The binding kinetics and binding affinity of an antibody can also be determined by standard assays known in the art, such as surface plasmon resonance (SPR), for example, by using Biacore TM The K of each antibody / antigen complex can be compared by comparing the K D The K values ​​can be used to compare the binding affinities associated with different molecular interactions, for example, the binding affinities of different antibodies for a given antigen. Similarly, the specificity of an interaction can be determined and compared by determining and comparing the K values ​​of the interactions of interest (e.g., the specific interaction between an antibody and an antigen). D The K value is compared with the K value of non-target interactions (such as control antibodies known not to bind antigen). D The value is evaluated.

[0357] The terms "conservative substitution" and "conservative replacement" refer to substitution 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. In addition, 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 non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit a specific change in properties.

[0358] The terms "homology," "identity," or "sequence identity" refer to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent 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 x 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0359] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably to refer to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0360] The term "host cell" includes individual cells or cell cultures that may be or have been recipients of vectors for incorporating polynucleotide inserts. Host cells include progeny of a single host cell, and due to natural, accidental or intentional mutations, the progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present invention. "Cell," "cell line," and "cell culture" are used interchangeably, and all such names include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included.

[0361] The terms "inhibit" or "block" are 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 an epitope may be located on the prodomain of a TGFβ protein (e.g., TGFβ1), a growth factor, or other epitopes that result in reduced growth factor activity when bound by an antibody. Inhibitory antibodies of the present invention include, but are not limited to, TGFβ1-inhibitory antibodies.

[0362] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0363] The term "pharmaceutical composition" refers to a mixture containing one or more active ingredients described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredients and thereby exerting their biological activity.

[0364] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable vehicle" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react 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 / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th edition Mack Publishing, 2000).

[0365] "Cancer," "cancerous," "proliferative disorder," and "tumor" as referred to in this disclosure are not mutually exclusive.

[0366] The terms "administer," "apply," and "treat" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, for example, for therapeutic, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "apply," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, they refer to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0367] The term "treatment" means administering a therapeutic agent, such as a fusion protein or insulin analog comprising any of the present disclosure, to a subject who has, is suspected of having, or is predisposed to having one or more diabetes or hyperglycemia-related diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, a therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in a treated subject or population, by preventing or delaying the onset of symptoms or complications, alleviating symptoms or complications, or eliminating the disease, condition, or disorder to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although the embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating the symptoms of the target disease in a subject, they should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test. The patient to be treated is a mammal, and preferably a human.

[0368] The terms "prevent," "prevent," and "prevent" refer to reducing the risk or incidence, or eliminating or slowing the progression of one or more conditions, symptoms, complications, or disorders.

[0369] The terms "subject" and "patient" refer to mammals, particularly primates, and especially humans. BRIEF DESCRIPTION OF THE DRAWINGS

[0370] Figure 1 shows the results of detecting the inhibitory effects of anti-TGFβ1 antibodies (H27, SL2-2, SL2-9, SL2-12, SL2-19, and SL2-22) on the activity of human or mouse TGFβ1 complexes (human proTGFβ1, GARP-TGFβ1 complex, LRRC33-TGFβ1 complex, and mouse GARP-TGFβ1 complex). In Figure 1 , A shows the inhibitory effects of the antibodies on human proTGFβ1 activation, B shows the inhibitory effects of the antibodies on human GARP-TGFβ1 complex activation, C shows the inhibitory effects of the antibodies on human LRRC33-TGFβ1 complex activation, and D shows the inhibitory effects of the antibodies on mouse GARP-TGFβ1 complex activation.

[0371] Figure 2 shows the results of an experiment detecting the binding of anti-TGFβ1 antibodies (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).

[0372] Figures 3A-3C show the results of the combination of anti-TGFβ1 antibody and anti-PD-1 antibody (RMP1-14-mIgG2a-FcS + SL2-22-mIgG2a-FcS) in inhibiting tumor growth in the mouse EMT-6 model. Figure 3A is a graph of mouse tumor volume, Figure 3B is a graph of mouse survival rate, and Figure 3C is a graph of mouse body weight.

[0373] Figures 4A-4C show the results of anti-TGFβ1 antibody and anti-PD-1 antibody combined with RMP1-14-mIgG2a-FcS + SL2-22-mIgG2a-FcS in inhibiting tumor growth in the mouse CT26 model. Figure 4A is a graph of mouse tumor volume, Figure 4B is a graph of mouse survival rate, and Figure 4C is a graph of mouse body weight.

[0374] Figures 5A-5E show the results of the anti-TGFβ1 antibody (SL2-22-mIgG2a-FcS) inhibiting fibrosis in a mouse idiopathic fibrosis model. Figure 5A shows the weight change curve of mice in the pulmonary fibrosis model, Figure 5B shows the survival rate curve of mice in the pulmonary fibrosis model, Figure 5C shows the lung weight and hydroxyproline content of mice in the pulmonary fibrosis model at the end of the experiment, Figure 5D shows the lung sections of mice at the end of the experiment, and Figure 5E shows the collagen area ratio by Masson staining.

[0375] Figure 6 shows the inhibition of GARP-TGFβ1 complex activation by anti-GARP-TGFβ1 single domain antibodies detected by LN229 assay. LN229 cells were transfected with human or mouse pro-TGFβ1 and human or mouse GARP expression plasmids. HepG2CAGA12-luc luciferase reporter cells were used to detect the inhibition of human or mouse GARP-TGFβ1 complex activation by Abbv-151, C19, C19-3, C19-7, and C19-8. Figure 6A shows the inhibitory effect of the antibodies on human GARP-TGFβ1 complex activation, and Figure 6B shows the inhibitory effect of the antibodies on mouse GARP-TGFβ1 complex activation.

[0376] Figure 7 shows the results of a binding experiment between C19-8 and HEK293E cells expressing human or mouse GARP-TGFβ1 complex. HEK293E cells were transfected with the plasmids listed in Table 3, and C19-8 and Abbv-151 were added, and the cells were stained and detected to obtain the results.

[0377] Figures 8A-8C show the results of C19-8 combined with anti-PD-1 antibodies (RMP1-14-mIgG2a-FcS + C19-8-mIgG2a-FcS) inhibiting tumor growth in the mouse EMT-6 model. Figure 8A is a graph of mouse tumor volume, Figure 8B is a graph of mouse survival rate, and Figure 8C is a graph of mouse weight.

[0378] Figures 9A-9C show the results of C19-8 combined with anti-PD-1 antibodies (RMP1-14-mIgG2a-FcS + C19-8-mIgG2a-FcS) inhibiting tumor growth in the mouse CT26 model. Figure 9A is a graph of mouse tumor volume, Figure 9B is a graph of mouse survival rate, and Figure 9C is a graph of mouse body weight. DETAILED DESCRIPTION

[0379] Example

[0380] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of the present disclosure.

[0381] Experimental methods in the disclosed embodiments or test examples, where specific conditions are not specified, generally followed conventional conditions or those recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0382] Example 1. Preparation of antigens and detection proteins

[0383] Using human pro-TGFβ1 (Uniprot: P01137), human GARP (LRRC32, Uniprot: Q14392), mouse pro-TGFβ1 (Uniprot: P04202) and mouse GARP (LRRC32, Uniprot: G3XA59) as templates, the amino acid sequences of related proteins for screening and detection were designed.

[0384] >Full-length amino acid sequence of human original TGFβ1:

[0385] (Note: The italicized part is the signal peptide; the underlined part is the latency-associated peptide (LAP); the italicized bold part is the TGFβ1 domain

[0386] >Mature human TGFβ1 (mature transforming growth factor beta-1):

[0387] > Human LAP:

[0388] >Human GARP full-length amino acid sequence:

[0389] (Note: The italicized part is the signal peptide; the underlined part is the extracellular domain; the italicized bold part is the transmembrane domain; the italicized underlined part is the intracellular domain)

[0390] >Human GARP extracellular domain:

[0391] >Full-length amino acid sequence of mouse pro-TGFβ1:

[0392] (Note: The italicized part is the signal peptide; the underlined part is the potential-associated peptide (LAP); the italicized bold part is the TGFβ1 domain)

[0393] >Mature mouse TGFβ1:

[0394] Mouse LAP:

[0395] >Full-length amino acid sequence of mouse GARP:

[0396] (Note: The italicized part is the signal peptide; the underlined part is the extracellular region; the italicized bold part is the transmembrane region; the italicized underlined part is the intracellular region)

[0397] > Mouse GARP extracellular domain:

[0398] 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 co-expressed in ExpiCHO cells (ThermoFisher, A29127) and purified to obtain a protein complex; the human TGFβ1 C4S protein 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 co-expressed in ExpiCHO cells (ThermoFisher, A29127) and purified to obtain a protein complex; the mouse TGFβ1 C4S protein is shown in SEQ ID NO: 10:

[0399] >Human GARP-avi-his amino acid sequence:

[0400] (Note: The underlined part is the extracellular domain of human GARP; the italic part is the (G4S)2 linker; For Avi tag; His tag)

[0401] >Human TGFβ1-Native amino acid sequence:

[0402] >Human TGFβ1 C4S amino acid sequence:

[0403] (Note: is a His tag; the italic part is a (G4S)2 linker; Avi tag; the underlined portion is the full-length human TGFβ1 protein, in which the fourth amino acid C is mutated to S)

[0404] Mouse GARP-avi-his amino acid sequence:

[0405] (Note: The underlined part is the mouse GARP extracellular domain; the italicized part is the (G4S)2 linker; The lower curve is the His tag)

[0406] > Mouse TGFβ1 - natural amino acid sequence:

[0407] Mouse TGFβ1 C4S amino acid sequence:

[0408] (Note: is a His tag; the italic part is a (G4S)2 linker; The underlined portion is the full-length mouse TGFβ1 protein, in which the fourth amino acid C is mutated to S)

[0409] The above sequences can be expressed, purified and isolated by conventional methods in the art.

[0410] Example 2. Screening of anti-TGFβ1 monoclonal antibodies that specifically bind to human TGFβ1 complex

[0411] Antibodies with high affinity to human TGFβ1 complex were obtained by screening human antibody phage libraries. There are 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 Fab library; 4: fully human scFV library), both were donated by Shanghai Hengrui Medicine Co., Ltd. 10 μg 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 reacted at room temperature for half an hour. The beads were washed three times with 1× PBS and then blocked with 2% skim milk for 1 hour at room temperature. The human antibody phage display library was simultaneously blocked and depleted with 2% skim milk and 1 mg of Dynabeads M-280 streptavidin. The treated phage library was then added to the antigen-bound beads and incubated at room temperature for 1 hour. The cells were washed 10 times with 1×PBST (containing 0.05% Tween-20), pH 7.4 solution to remove unbound phages. After washing twice more with 1×PBS, the phages specifically bound to human GARP / TGFβ1 were eluted with 0.5 mL of trypsin (1 mg / mL) and infected with Escherichia coli TG1 in the logarithmic growth phase. 2YT (containing 2% glucose) resistant plates were grown overnight. Phages were scraped from the plates to produce and purify phages for the next round of screening.

[0412] The same screening process was repeated two rounds. To identify antibodies that cross-bind to the mouse TGFβ1 complex, biotinylated mouse TGFβ1_C4S (SEQ ID NO: 10) was used as the screening antigen in the second round. In the third round, biotinylated human GARP-TGFβ1 complex (comprising SEQ ID NOs: 5 and 6) was used as the screening antigen. Positive clones were enriched after three rounds of screening.

[0413] 6 × 92 clones were selected from the enriched clones and packaged into monoclonal phage for phage ELISA assay. 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), respectively, and incubated overnight at 4°C. The plates were washed three times with 1× PBST and blocked with 2% BSA at 37°C for 1 hour. After three washes with PBST, phage supernatant diluted in blocking buffer was added and incubated at room temperature for 1 hour. The plates were washed six times with PBST, and anti-M13 HRP (Cat No. 11973-MM05T-H, Sino Biological) was added and incubated at room temperature for 1 hour. The plates were washed three times with PBST, and 100 μL of TMB chromogenic substrate was added. The reaction was terminated with 100 μL of 1 M sulfuric acid, and the absorbance was read at 450 nm using a SpectraMax M5 microplate reader. Clones with OD450 readings greater than three times that of the negative control in the ELISA binding test were sequenced and analyzed, yielding 43 specific antibody sequences. SPR experiments showed that antibodies H5, H14, H16, H17, H19, H20, H21, H23, H24, H27, H28, H30, H32, H34, and H39 were able to bind to the TGFβ1 complex. However, cell function experiments revealed that only H27 had a strong inhibitory function, binding well to both the human and mouse GARP-TGFβ1 complex and human and mouse TGFβ1_C4S (i.e., the TGFβ1 precursor protein).

[0414] The sequence of H27 is shown below.

[0415] >H27 VH

[0416] >H27 VL

[0417] In the above sequences SEQ ID NO: 11 and 12, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italicized sequences are FR sequences, and the underlined sequences are CDR1, CDR2, and CDR3 sequences. The numbering convention of the human antibodies provided in the present disclosure is Kabat, and Table 5 shows the CDR sequences.

[0418] Table 5. CDR sequences of H27 antibody

[0419] Example 3. Construction, expression and purification of complete monoclonal antibodies

[0420] 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 the 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 the pTT5 expression vector containing the human IgG4-S228P (CH1-CH2-CH3) heavy chain constant region (SEQ ID NO: 20).

[0421] >Human kappa light chain constant region:

[0422] >Human IgG4-S228P (CH1-CH2-CH3) heavy chain constant region:

[0423] The complete H27 antibody sequence is as follows:

[0424] >H27 light chain:

[0425] >H27 heavy chain:

[0426] The cloned light chain and heavy chain plasmids were paired and co-transfected into HEK293E cells (donated by Shanghai Hengrui Medicine Co., Ltd.) or ExpiCHO (Cat No. A29127, ThermoFisher) cells. The cell culture supernatant was harvested 5 days after transfection (37 degrees, HEK29E3 cells) or 10-12 days (32 degrees, expiCHO cells), centrifuged at 4000 rpm for 20 minutes, and the supernatant was filtered using a 0.45 μM filter. The first step of affinity purification was then performed using a MabSelectSure LX column (GE Healthcare). The culture supernatant was passed through a MabSelectSure LX column equilibrated with PBS, washed with PBS, and the target protein was eluted with 0.1 M Glycine acidic eluent at pH 3.0, neutralized with 1 M MES (pH 6.0), and finally finely purified using a HiTrap SP HP ion column. After detection, the target antibody was obtained.

[0427] Example 4. Affinity determination of TGFβ1 monoclonal antibody

[0428] The affinity of the antibodies to human / mouse GARP / TGFβ1 and human / mouse TGFβ1_C4S was determined using Biacore T200 (GE Healthcare).

[0429] A certain amount of the test antibody was affinity-captured using a Protein A biosensor chip (Cat No.#29127556, GE). A series of gradient dilutions of the human or mouse GARP-TGFβ1 complex, TGFβ1_C4S antigen, was then passed over the chip surface at a flow rate of 50 μL / min and a dissociation time of 5 minutes. After each cycle, the chip was regenerated using glycine-HCl (Cat.#BR-1003-54, GE) at pH 1.5. The reaction buffer was HBS-EP+ buffer solution (Cat No.#BR-1006-69, GE) diluted to 1x with distilled water (pH 7.4).

[0430] Biacore T200 was used to detect the reaction signal in real time to obtain the binding and dissociation curves. The obtained data were fitted with the Langmuir 1:1 binding model using BIAevaluation version 4.1, GE software to obtain the affinity values. The results are shown in Table 6.

[0431] Table 6. Affinity data of H27

[0432] Example 5. Affinity maturation of TGFβ1 monoclonal antibody H27

[0433] The H27 antibody molecule underwent three-dimensional structural simulation. Based on human germline mutation hotspots and three-dimensional structural simulation results, key amino acid residues in the framework and CDR regions were selected and four random mutation phage libraries were constructed (Table 7). Phage library display technology was used to screen for functional antibodies with improved affinity. New amino acid residues obtained from different libraries were combined and validated to obtain functional antibodies with improved affinity and function.

[0434] Table 7. H27 affinity maturation library design

[0435] Note: “ / ” indicates residues without mutation

[0436] Four random mutation phage libraries were screened using biotinylated human GARP-TGFβ1 (comprising SEQ ID NOs: 5 and 6) as the screening antigen. After 3-4 rounds of screening, the obtained clones were sequenced and analyzed. Combined with the phage ELISA results, 62 antibody sequences were selected for cloning and construction. The antibody light chain variable region VL was cloned into the 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 the pTT5 expression vector containing the human hIgG4-S228P (CH1-CH2-CH3) heavy chain constant region (SEQ ID NO: 20).

[0437] 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 the cells. After purification using MabSelect Sure, affinity determination was performed according to the method of Example 4.

[0438] Antibodies with affinity greater than 5-fold higher than H27, as determined by SPR protein interaction, were all derived from the second random mutagenesis library, whose heavy chains were the H27 heavy chains. Their light chain variable region sequences are shown in Table 8 (the underlined regions are CDR regions, using Kabat numbering). Additionally, their full-length light chain sequences and CDR combinations are shown in Tables 9 and 10, respectively.

[0439] Table 8. Light chain variable region sequences after affinity maturation of TGFβ1 monoclonal antibody H27

[0440] Table 9. Light chain sequences of TGFβ1 monoclonal antibody H27 after affinity maturation

[0441] Table 10. Light chain CDR sequences of TGFβ1 monoclonal antibody H27 after affinity maturation

[0442] 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:

[0443] LCDR1 is RASQX1ISX2YLN (SEQ ID NO: 38), 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;

[0444] LCDR2 is X3ASX4LX5S (SEQ ID NO: 64), 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.

[0445] SL2-2, SL2-9, SL2-12, SL2-19, and SL2-22 have the following general structures:

[0446] LCDR1 is RASQX1ISX2YLN (SEQ ID NO: 38), wherein X1 is selected from F, Y or A, and X2 is selected from D or P;

[0447] LCDR2 is X3ASX4LX5S (SEQ ID NO: 64), wherein 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.

[0448] Example 6. Affinity determination of TGFβ1 affinity-matured monoclonal antibodies

[0449] The negative control for this example is HBS-EP, and the positive controls are Ab6 (Scholar rock, WO2020014460A1) and Abbv-151 (Abbvie, US10793627B2). Ab6 binds to the GARP-TGFβ1 complex, the LRRC33-TGFβ1 complex, the LTBP3-TGFβ1 complex, and the LTBP1-TGFβ1 complex, with the binding epitope being entirely on TGFβ1 (Martin et al., 2020); whereas the binding epitope of Abbv-151 includes both GARP and TGFβ1 in the GARP-TGFβ1 complex and does not bind to either GARP or TGFβ1 alone (Streel et al., 2020). The sequences of Ab6 and Abbv-151 are as follows:

[0450] >Ab6 antibody heavy chain sequence:

[0451] >Ab6 antibody light chain sequence:

[0452] >Abbv-151 antibody heavy chain sequence:

[0453] >Abbv-151 antibody light chain:

[0454] The affinity of several affinity matured monoclonal antibodies to human and mouse GARP-TGFβ1 complexes was determined using Biacore T200 (GE Healthcare) according to the method of Example 4. The affinity determination results are shown in Tables 11 and 12.

[0455] Table 11. Affinity data of H27 affinity matured antibodies

[0456] Table 12. Affinity data of SL2-22

[0457] Example 7. TGFβ1 monoclonal antibody inhibits the function of GARP-TGFβ1 complex in vitro

[0458] 2×10 6LN229 cells (Procell, CL-0578) were transferred to T75 culture flasks (Nunc) and 24 hours later, 293fectin was used to TM Human or mouse pro-TGFβ1 was transfected alone, or human or mouse pro-TGFβ1 and GARP or LRRC33 plasmids were co-transfected using Invitrogen transfection reagent. After 24 h, cells were plated at 1×10 4 The cells were transferred to a white opaque 96-well cell culture plate (PerkinElmer). After 24 hours, the culture medium was aspirated and HepG2CAGA12-luc luciferase reporter cells resuspended in DMEM + 0.5% BSA were added. H27, H27 affinity matured antibody and control antibody Ab6 were also added in serial dilutions. After 20 hours of mixed culture, ONE-Glo TM After a 5-minute incubation with luciferase reagent, chemiluminescence was read on a SpectraMax M5 multi-functional microplate reader. Luciferase activity in the vehicle-treated group was normalized to 1. Relative activity = luciferase activity in the antibody-treated group / vehicle control group. Graphs were constructed using Prism 9 using a nonlinear fit and a three-parameter logarithmic inhibitor-response model.

[0459] The results, as shown in Figures 1A and 1B, show that affinity-matured SL2-2, SL2-9, SL2-12, SL2-19, and SL2-22 exhibited higher inhibitory activity against pro-TGFβ1 and each TGFβ1 complex than the parent H27. SL2-2, SL2-9, SL2-12, SL2-19, and SL2-22 exhibited comparable inhibitory activity against human pro-TGFβ1, the GARP-TGFβ1 complex, and the mouse GARP-TGFβ1 complex as the positive control Ab6, and were superior to Ab6 in inhibiting the human LRRC33-TGFβ1 complex.

[0460] Example 8. Binding experiment of TGFβ1 monoclonal antibody SL2-22 to cells expressing different TGFβ complexes

[0461] HEK293E cells were prepared into 1×10 6 / mL, take 5mL and put it into a 50mL culture tube, and use 293fectin TM Reagents were used to transfect the plasmids listed in Table 13. After 48 h, the cells were washed twice with 1 x PBS and 1 x 10 5Place cells in a 96-well round-bottom cell culture plate (Nunc). Add 100 μL of 10 nM antibody or control antibody in 1 x PBS and stain at room temperature for 1 hour. After washing three times with 1 x PBS, add 100 μL of FITC-labeled anti-human Fc antibody and stain at room temperature for 30 minutes. After staining, wash three times with 1 x PBS. Add 7-AAD and incubate for 5 minutes. After washing three times with 1 x PBS, analyze by flow cytometry. Analyze the number of FITC-positive cells in live cells using FlowJo, calculate the percentage of FITC-positive cells to the total number of live cells, and plot the graph.

[0462] Table 13. Plasmid structures

[0463] The results are shown in Figure 2. Both SL2-22 and Ab6 bind 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), and do not bind to TGFβ2 or the GARP-TGFβ2 complex (corresponding to TGFβ2+GARP in Figure 2). The difference is that Ab6 also binds to human TGFβ3 and the GARP-TGFβ3 complex (corresponding to TGFβ3+GARP in Figure 2), while SL2-22 only specifically binds to the TGFβ1 complex.

[0464] Example 9. Epitope competition study of TGFβ1 antibodies

[0465] The negative control of this example was HBS-EP, and the positive controls were Ab6 and Abbv-151.

[0466] The epitope competition between the anti-human TGFβ1 antibody SL2-22 and a positive control antibody and the human GARP-TGFβ1 complex was studied using Biacore T200 (GE Healthcare).

[0467] Anti-His tag antibodies were coupled to the CM5 sensor chip (Cat. 29-1049-88, GE) using an amino coupling kit (Cat No. BR-1000-50, GE) and a His capture kit (Cat No. 28-9950-56, GE) according to the kit instructions, so that the surface response value reached approximately 10,000 RU, and then blocked with ethanolamine for use.

[0468] The steps for epitope competition studies (using dual-mode samples) are as follows (antibodies are paired in pairs):

[0469] 1) Antigen capture: human GARP-TGFβ1 complex

[0470] 2) First antibody: concentration 100 μg / ml, flow rate 50 μL / min, 60 seconds, HBS-EP buffer as control

[0471] 3) Second antibody: concentration 100 μg / ml, flow rate 50 μL / min, 60 seconds, HBS-EP buffer as control

[0472] 4) Analysis of the sensorgrams showed that SL2-22 partially competed with Ab6 for the binding epitope of the human GARP-TGFβ1 complex, while SL2-22 and Abbv151 did not compete at all for the binding epitope of the human GARP-TGFβ1 complex (Table 14).

[0473] Table 14. Determination of epitope competition between SL2-22 and positive antibodies Ab6 and Abbv151

[0474] (Note: “×” indicates no competition at all)

[0475] Example 10. TGFβ1 monoclonal antibody SL2-22 inhibits tumor growth in the mouse EMT-6 model

[0476] To detect the tumor suppressor activity of SL2-22 in vivo, BALB / c mice (female, 6 weeks old, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were adaptively housed for 1 week and numbered and weighed on the day of the experiment. Mouse breast cancer EMT-6 cells (Procell, CL-0573) in the logarithmic growth phase were collected, resuspended in PBS, and diluted to 5×10 6 The concentration of 0.1 mL / mouse was inoculated subcutaneously on the right flank of BALB / c mice. On the 3rd day after inoculation, when the average tumor volume reached about 60 mm 3When the tumor volume was moderate, mice with moderate individual tumor volume were selected and randomly divided into groups as shown in Table 15. mIgG2a-FcS is mIgG2a Fc with L234A / L235E / G237A / D327Q / A330S / P331S with ADCC effect removed, which serves as an isotype control; RMP1-14-mIgG2a-FcS is the variable region of the anti-PD1 antibody RMP1-14 (sequences 285 (heavy chain variable region) and 286 (light chain variable region) without the signal peptide in WO2018223182A1; WO2018223182A1 is incorporated herein by reference in its entirety) and mIgG2a Fc with L234A / L235E / G237A / D327Q / A330S / P331S with ADCC effect removed. SL2-22-mIgG2a-FcS is an antibody fused with the variable region of SL2-22 and the mIgG2a heavy chain constant region with L234A / L235E / G237A / D327Q / A330S / P331S and the mouse kappa light chain constant region, with the following sequences:

[0477] >SL2-22-mIgG2a-FcS antibody heavy chain:

[0478] >SL2-22-mIgG2a-FcS antibody light chain:

[0479] The drug was administered on the day of grouping, at the same molar dose, twice a week for a total of 7 times, by intraperitoneal injection. The body weight and tumor volume of the mice were measured twice a week, and the tumor volume was calculated as TV = L 长 ×L 短 2 The tumor volume of each group was expressed as mean ± standard deviation, and statistical analysis was performed using two-way ANOVA. The tumor inhibition rate (%TGI) was calculated using the formula: %TGI = [1-(T-T0) / (C-C0)] × 100%.

[0480] The results are shown in Figures 3A, 3B, and Table 15. The combination of SL2-22-mIgG2a-FcS and the anti-PD-1 antibody RMP1-14-mIgG2a-FcS significantly inhibited EMT-6 tumor growth in mice compared to RMP1-14-mIgG2a-FcS alone (p = 0.0127, Two-way ANOVA) and prolonged animal survival (p = 0.0323, Log-rank (Mantel-Cox) test). During treatment, the mice tolerated the treatment well, with no significant weight loss (Figure 3C).

[0481] Table 15. Dosage regimen, tumor inhibition effect and median survival of SL2-22 in the EMT-6 model

[0482] Note: The molar concentration of mIgG2a-FcS, RMP1-14-mIgG2a-FcS, and SL2-22-mIgG2a-FcS is the same, approximately 68 μmol / kg.

[0483] Example 11. TGFβ1 Antibody SL2-22 Inhibits Tumor Growth in Mouse CT26 Model

[0484] To test the tumor suppressor activity of SL2-22 in vivo, BALB / c mice (female, 6 weeks old, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were adaptively housed for 1 week and numbered and weighed on the day of the experiment. Mouse colon cancer cells CT26 (National Biomedical Laboratory Cell Resource Bank, 1101MOU-PUMC000275) in the logarithmic growth phase were collected and resuspended in PBS at 3×10 6 The concentration of 0.1 mL / mouse was inoculated subcutaneously on the right flank of BALB / c mice. On the 8th to 9th day after inoculation, when the average tumor volume reached about 60 mm 3 At the same time, mice with moderate tumor volumes were selected and randomly divided into groups as shown in Table 16. Dosing began on the day of grouping, with the same molar dose, twice a week for a total of 6 doses, and the administration route was intraperitoneal injection. The body weight and tumor volume of mice were measured twice a week. The tumor volume was calculated as TV = L 长 ×L 短 2 The tumor volume of each group was expressed as mean ± standard deviation, and the tumor inhibition rate (%TGI) was calculated using the formula: %TGI = [1-(T-T0) / (C-C0)] × 100%.

[0485] The results, as shown in Figures 4A and 4B and Table 16, show 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. During treatment, the mice tolerated the treatment well, with no significant weight loss (Figure 4C).

[0486] Table 16. Dosage regimen, tumor inhibition effect and median survival of SL2-22 in the CT26 model

[0487] Note: The molar concentration of mIgG2a-FcS, RMP1-14-mIgG2a-FcS and SL2-22-mIgG2a-FcS is the same, approximately 68 μmol / kg

[0488] Example 12. TGFβ1 monoclonal antibody SL2-22 inhibits fibrosis progression in a mouse idiopathic fibrosis model

[0489] To test the in vivo inhibitory effect of the TGFβ1 antibody SL2-22 on fibrosis, 8-week-old male C57BL / 6J mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and acclimated for 1 week. The mice were numbered and weighed on the day of the experiment. On the day of modeling, the mice were numbered and weighed and randomly divided into groups of 15 mice each according to body weight. The specific groupings 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 intratracheally using a blunt-end needle at a dose of 2 U / kg, with approximately 50 μL per animal. The drug was then administered twice weekly for a total of 3 weeks, according to the aforementioned groupings. On day 22 of the experiment, bronchoalveolar lavage fluid and lung tissue were collected for analysis of immune cell infiltration, lung tissue hydroxyproline levels, and Masson staining.

[0490] Table 17. Experimental groups and drug administration

[0491] The SL2-22-mIgG2a-Fc administration group significantly improved the overall body weight and survival rate of the animals (Figures 5A and 5B), and reduced the hydroxyproline level in the lung tissue by 40.44% compared with the modeling group (Figure 5C); at the same time, it significantly reduced the collagen deposition and fibrosis levels in the lungs after bleomycin modeling, by 46.51% compared with the modeling group (Figures 5D and 5E).

[0492] Example 13. Screening and preparation of single-domain antibodies (VHH) against human and mouse GARP-TGFβ1

[0493] 1. Camel Immunization and Library Construction

[0494] Human GARP / TGFβ1 protein (a protein complex obtained by co-expressing and purifying the proteins represented by SEQ ID NOs: 5 and 6) was used as an antigen to immunize a Bactrian camel. Freund's complete adjuvant was mixed with the antigen at a 1:1 volume ratio, and the camel was immunized subcutaneously at multiple sites every two weeks. The first immunization dose was 200 μg of protein, followed by four subsequent immunizations at a dose of 100 μg protein each, for a total of five immunizations. The titer of the human GARP / TGFβ1 protein was then measured. When the titer met the criteria, peripheral blood was collected from the camel, lymphocytes were isolated, and the cells were lysed with Trizol. RNA was extracted and reverse transcribed to obtain cDNA for the construction of a phage library.

[0495] 2. Screening of single domain antibodies (VHH)

[0496] 20 μg of biotinylated human GARP / TGFβ1 protein was bound to 100 μL of Dynabeads. TM After incubation at 37°C for 1 hour with M-280 streptavidin, block with 2% skim milk at room temperature for 1 hour, add the aforementioned phage library, and allow to react at room temperature for 1 hour. Wash 9 times with PBST (PBS containing 0.05% Tween-20) to remove unbound phage. Phages that specifically bind to human GARP / TGFβ1 protein were eluted with 1 mg / mL trypsin and infected with Escherichia coli TG1 in logarithmic phase growth to produce and purify the first round of positive phages. Based on the positive phages screened in the first round, single domain antibodies (VHHs) with high affinity for mouse GARP-TGFbβ1 complex protein were obtained in the second round of screening.

[0497] From the positive clones screened and enriched, 96 monoclonal colonies were selected and packaged into phage-encapsulated single-chain antibody (scFv) for phage ELISA testing. ELISA plates were coated with 2 μg / mL of human GARP-TGFβ1 complex protein, and phage supernatant diluted in blocking buffer was added. Anti-M13 HRP-labeled antibody was used for detection. Clones with an OD450 / background value >5 in the ELISA binding assay were sequenced. The C19-VHH sequence is shown below.

[0498] >C19-VHH

[0499] In SEQ ID NO: 84, the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italics in the sequence are FR sequences, and the underlined sequences are CDR1, CDR2, and CDR3 sequences. The numbering convention for the anti-GARP-TGFβ1 single domain antibodies provided herein is Kabat.

[0500] Table 18. CDR sequences of C19 VHH

[0501] 3. Preparation of Complete Antibodies

[0502] The C19-VHH sequence was fused to the following human IgG4-Fc (CH2-CH3, containing S228P) fragment to obtain C19.

[0503] >hIgG4-Fc(S228P)

[0504] >C19

[0505] The C19 sequence was cloned into the mammalian expression vector pTT5 and transfected into HEK293E or ExpiCHO (ThermoFisher, A29127) cells. The cell culture supernatant was harvested by centrifugation at 4000 rpm for 20 min 5 days after transfection (HEK293E cells, 37°C) or 10-12 days after transfection (ExpiCHO cells, 32°C) and filtered with a 0.45 μM filter. The first step of affinity purification was then performed using a MabSelectSure LX column (GE Healthcare). The culture supernatant was passed through a MabSelectSure LX column equilibrated with PBS, washed with PBS, and the target protein was eluted with 0.1 M Glycine acidic eluent at pH 3.0, neutralized with 1 M Tris-HCl (pH 8.0), and finally finely purified using a HiTrap Q HP ion column. After detection, the target antibody was obtained.

[0506] SPR and cell function experiments showed that C19 has good inhibitory function and binds well to both human and mouse GARP-TGFβ1 complexes.

[0507] Example 14. Humanized transformation of anti-GARP-TGFβ1 single domain antibody

[0508] By performing three-dimensional homology modeling on the selected TGFβ1-specific single-domain antibody C19 and comparing the results with the V-base human germline sequence database and the IMGT human antibody heavy chain variable region germline gene database, the heavy chain variable region germline gene IGHV3-23, which shows high sequence homology to C19, was selected as the template for FR1, FR2, and FR3. IGJH4 was used as the template for FR4. The CDRs of the camelid single-domain antibody were transplanted into the corresponding human template, resulting in a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. To preserve the original activity of the humanized single-domain antibody, a series of backmutations were performed in the framework regions. The humanized VHH framework regions contain at least one amino acid mutation selected from the following: 23T, 29Y, 30C, 37Y, 44E, 45R, 47F, 71Q, 74A, 75R, 78G, 81E, 93K, and 94T. The above amino acid positions are according to the Kabat numbering.

[0509] The obtained humanized sequence is as follows, the CDR region is underlined, and the coding rule is Kabat coding:

[0510] >C19-hu3

[0511] >C19-hu7

[0512] >C19-hu8

[0513] The method in Example 2 was used to construct a full antibody sequence fused with the humanized single-domain antibody VHH and the Fc region of hIgG4. The obtained humanized complete antibody sequence is as follows:

[0514] >C19-3

[0515] >C19-7

[0516] >C19-8

[0517] Example 15. Affinity determination of anti-GARP-TGFβ1 single domain antibody and GARP-TGFβ1 complex

[0518] The negative control of this example is HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4). The positive control is Abbv-151 (Abbvie, US10793627B2). Its sequence is as follows:

[0519] >Abbv-151 antibody heavy chain:

[0520] >Abbv-151 antibody light chain:

[0521] The affinity of C19 and its humanized antibody, positive control antibody Abbv-151 to human GARP-TGFβ1 complex (formed by the protein complex of SEQ ID NO: 5 and 6) or mouse GARP-TGFβ1 complex (formed by the protein complex of SEQ ID NO: 8 and 9) was measured by Biacore T200 (GE Healthcare). The antibody to be tested was captured on the chip surface using a Series S sensor chip Protein A (GE Healthcare, 29127556). Then, different concentrations of human or mouse GARP-TGFβ1 complex were passed over the chip surface. The reaction signal was detected in real time to obtain the binding and dissociation curves, and the binding constant was obtained by fitting. The solution used in the experiment was HBS-EP solution. At the end of each experimental cycle, the chip was regenerated with pH 1.5 Glycine (GE Healthcare, BR-1003-54) solution. The affinity results of the antibodies are shown in Table 19. The results showed that the affinity of the antibodies C19, C19-3, C19-7 and C19-8 obtained by the present disclosure screening to the human GARP-TGFβ1 complex was comparable to that of the positive control Abbv-151, except that they also had a certain binding to the mouse GARP-TGFβ1 complex.

[0522] Table 19. Affinity determination of C19 and C19 humanized antibody to human or mouse GARP / TGFβ1

[0523] (Note: “ / ” means no combination)

[0524] Example 16. Anti-GARP / TGFβ1 single domain antibody inhibits GARP / TGFβ1 function in vitro

[0525] 2 x 10 6 LN229 cells (Procell, CL-0578) were transferred to T75 culture flasks (Nunc) and 24 hours later, 293fectin was used to TM (Invitrogen) transfection reagent was used to transfect human or mouse pro-TGFβ1 alone, or co-transfect human and mouse pro-TGFβ1 and GARP plasmids. After 24 h, cells were plated at 1×10 4The cells were transferred to a white opaque 96-well cell culture plate (PerkinElmer). After 24 hours, the culture medium was aspirated and HepG2CAGA12-luc luciferase reporter cells resuspended in DMEM + 0.5% BSA were added. At the same time, serially diluted C19, C19 humanized antibody, and control antibody Abbv-151 were added. After 20 hours of mixed culture, ONE-Glo TM After a 5-minute incubation with luciferase reagent, chemiluminescence was read on a SpectraMax M5 multi-functional microplate reader. Luciferase activity in the vehicle-treated group was normalized to 1. Relative activity = luciferase activity in the antibody-treated group / vehicle control group. Graphs were constructed using Prism 9 using a nonlinear fit and a three-parameter logarithmic inhibitor-response model.

[0526] The results are shown in Figures 6A and 6B . C19, C19-3, C19-7, and C19-8 were comparable to the positive control Abbv-151 in inhibiting the activity of the human GARP-TGFβ1 complex, except that they also inhibited the mouse GARP-TGFβ1 complex.

[0527] Example 17. Binding experiment of anti-GARP-TGFβ1 single domain antibody C19-8 to cells expressing GARP-TGFβ1

[0528] HEK293E cells were prepared into 1×10 6 / mL, take 5mL and put it into a 50mL culture tube, and use 293fectin TM Reagents were used to transfect the plasmids listed in Table 20. After 48 h, the cells were washed twice with 1× PBS and 1×10 5 Place cells in a 96-well round-bottom cell culture plate (Nunc). Add 100 μL of 10 nM antibody or control antibody prepared in 1× PBS and stain at room temperature for 1 hour. After washing three times with 1× PBS, add 100 μL of FITC-labeled anti-human Fc antibody and stain at room temperature for 30 minutes. After staining, wash three times with 1× PBS. Add 7-AAD and incubate for 5 minutes. After washing three times with 1× PBS, analyze by flow cytometry. Analyze the number of FITC-positive cells in living cells using FlowJo, calculate the percentage of FITC-positive cells to the total number of living cells, and plot the graph.

[0529] Table 20. Plasmid structures

[0530] As shown in Figure 7, both C19-8 and Abbv-151 bound to the human GARP-TGFβ1 complex (corresponding to TGFβ1 + GARP in Figure 7). However, C19-8 also bound 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) or the TGFβ3 complex (TGFβ3 + GARP).

[0531] Example 18. Epitope competition study between anti-GARP / TGFβ1 single domain antibody C19-8 and Ab6 and Abbv-151

[0532] The negative control for this example was HBS-EP, and the positive controls were Ab6 (Scholar rock, WO2020014460A1, incorporated herein by reference in its entirety) 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 being entirely on TGFβ1 (Martin et al., 2020); whereas the binding epitope of Abbv-151 includes both GARP and TGFβ1 in the GARP-TGFβ1 complex and does not bind to either GARP or TGFβ1 alone (Streel et al., 2020). The sequence of Ab6 is as follows:

[0533] >Ab6 antibody heavy chain:

[0534] >Ab6 antibody light chain:

[0535] Epitope competition between C19-8 and the positive antibodies Abbv-151 and Ab6 and human GARP / TGFβ1 was determined using a Biacore T200 (GE Healthcare) instrument. A Series S sensor chip CM5 (GE Healthcare, Br100530) was coupled to an anti-histidine tag antibody using a His Capture Kit (GE Healthcare, 28-9950-56). The antigen protein human GARP-TGFβ1 complex was captured on the chip surface. Antibodies of the same concentration (500 nM) were then sequentially passed over the chip surface, using the combinations shown in Table 21. st The antibody is the first antibody to flow through, 2 nd The antibody is the second antibody to flow through.

[0536] Table 21. Antibody combinations

[0537] Epitope competition curves were obtained by real-time detection of reaction signals using a Biacore instrument. The experimental solution used was HBS-EP solution. At the end of each experimental cycle, the chip was regenerated with pH 1.5 Glycine (GE Healthcare, BR-1003-54) solution. The results of the antibody epitope competition assay are shown in Table 22.

[0538] The results showed that C19-8 completely competed with Ab6 and did not compete at all with Abbv-151. The aforementioned in vitro inhibition and cell binding experiments indicate that C19-8, like Abbv-151, inhibits the GARP-TGFβ1 complex, but binds to a different epitope.

[0539] Table 22. Determination of epitope competition between C19-8 and positive antibodies Abbv-151 and Ab6

[0540] (Note: “√” indicates perfect competition; “×” indicates no competition)

[0541] Example 19. Anti-GARP-TGFβ1 single domain antibody C19-8 inhibits tumor growth in the mouse EMT-6 model

[0542] To detect the tumor suppressive activity of C19-8 in vivo, BALB / c mice (female, 6 weeks old, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were adaptively housed for 1 week and numbered and weighed on the day of the experiment. Mouse breast cancer EMT-6 cells (Procell, CL-0573) in the logarithmic growth phase were collected, resuspended in PBS, and diluted to 5×10 6 The concentration of 0.1 mL / mouse was inoculated subcutaneously on the right flank of BALB / c mice. On the 3rd day after inoculation, when the average tumor volume reached about 60 mm 3 At the same time, mice with moderate tumor volumes were selected and randomly divided into groups as shown in Table 23.

[0543] Among them, the positive drug M7824 is PD-L1 / TGFβ-trap (Merck KGaA, sequence 3 (heavy chain) and sequence 1 (light chain) in WO2018029367A1 (incorporated into this disclosure by reference in its entirety)); mIgG2a-FcS is an mIgG2a with L234A / L235E / G237A / D327Q / A330S / P331S that removes the ADCC effect Fc, here as an isotype control; RMP1-14-mIgG2a-FcS is the variable region of the anti-PD1 antibody RMP1-14 (sequences 285 (heavy chain variable region) and 286 (light chain variable region) of WO2018223182A1 (incorporated herein by reference in their entirety) without the signal peptide) and the mIgG2a heavy chain constant region with L234A / L235E / G237A / D327Q / A330S / P331S and mouse kappa light chain constant region with ADCC removed. The antibody is fused together; C19-8-mIgG2a-FcS is the variable region of C19-8 and the mIgG2a heavy chain Fc with L234A / L235E / G237A / D327Q / A330S / P331S and ADCC removed. The sequence is as follows:

[0544] >C19-8-mIgG2a-FcS:

[0545] The drug was administered on the day of grouping, at the same molar dose, twice a week for a total of 7 times, by intraperitoneal injection. The body weight and tumor volume of the mice were measured twice a week, and the tumor volume was calculated as TV = L 长 ×L 短 2 The tumor volume of each group was expressed as mean ± standard deviation, and statistical analysis was performed using two-way ANOVA. The tumor inhibition rate (%TGI) was calculated using the formula: %TGI = [1-(T-T0) / (C-C0)] × 100%.

[0546] The results are 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 EMT-6 tumor growth in mice compared to RMP1-14-mIgG2a-FcS alone (p = 0.0017, Two-way ANOVA) and prolonged animal survival (p = 0.0157, Log-rank (Mantel-Cox) test). During the treatment, the mice tolerated the treatment well and did not experience significant weight loss (Figure 8C).

[0547] Table 23. Dosage regimen, tumor inhibition effect and median survival of C19-8 in the EMT-6 model

[0548] Note: The molar concentration of mIgG2a-FcS, RMP1-14-mIgG2a-FcS, C19-8-mIgG2a-FcS and M7824 is the same, approximately 68 μmol / kg.

[0549] Example 9. Anti-GARP-TGFβ1 single domain antibody C19-8 inhibits tumor growth in the mouse CT26 model

[0550] To test the tumor suppressor activity of C19-8 in vivo, BALB / c mice (female, 6 weeks old, Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were adaptively housed for 1 week and numbered and weighed on the day of the experiment. Mouse colon cancer cells CT26 (National Biomedical Laboratory Cell Resource Bank, 1101MOU-PUMC000275) in the logarithmic growth phase were collected and resuspended in PBS at 3×10 6 The concentration of 0.1 mL / mouse was inoculated subcutaneously on the right flank of BALB / c mice. On the 8th to 9th day after inoculation, when the average tumor volume reached about 60 mm 3 At the same time, mice with moderate tumor volumes were selected and randomly divided into groups as shown in Table 24. Dosing began on the day of grouping, with the same molar dose, twice a week for a total of 6 doses, and the administration route was intraperitoneal injection. The body weight and tumor volume of mice were measured twice a week. The tumor volume was calculated as TV = L 长 ×L 短 2 The tumor volume of each group was expressed as mean ± standard deviation, and the tumor inhibition rate (%TGI) was calculated using the formula: %TGI = [1-(T-T0) / (C-C0)] × 100%.

[0551] 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 mice tolerated the treatment well, with no significant weight loss (Figure 9C).

[0552] Table 24. Dosage regimen, tumor inhibition effect and median survival of C19-8 in the CT26 model

[0553] Note: The molar concentration of mIgG2a-FcS, RMP1-14-mIgG2a-FcS and C19-8-mIgG2a-FcS is the same, approximately 68 μmol / kg

[0554] Although specific embodiments of the present disclosure have been described above, those skilled in the art will appreciate that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.

Claims

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 of SEQ ID NO: 11, and the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence of any one of SEQ ID NO: 12, 21-35; the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the VH comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 13-15, respectively, and the VL comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 38, 64 and 18, respectively; Preferably, the TGFβ1 binding molecule comprises: HCDR1 as shown in SEQ ID NO:13, HCDR2 as shown in SEQ ID NO: 14, HCDR3 as shown in SEQ ID NO: 15, LCDR1 as shown in any one of SEQ ID NOs: 16, 36, 39, 41-42, 44, 46, 49, 52, 54 and 56-59, LCDR2 as shown in any one of SEQ ID NOs: 17, 37, 40, 43, 45, 47-48, 50-51, 53 and 55, and LCDR3 as shown in SEQ ID NO:

18.

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

3. The TGFβ1 binding molecule according to any one of claims 1 to 2, wherein: The VH comprises the amino acid sequence shown 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 as shown in any one of SEQ ID NOs: 12 and 21-35, or an amino acid sequence having at least 90% sequence identity thereto.

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

5. The TGFβ1 binding molecule according to any one of claims 1 to 4, comprising a heavy chain and a light chain, wherein: The heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 66, or having at least 90% sequence identity thereto; and the light chain comprises an amino acid sequence as shown 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 as shown in SEQ ID NO: 82, or a sequence having at least 90% sequence identity thereto. and the light chain comprises an amino acid sequence as shown in SEQ ID NO: 83, or an amino acid sequence having at least 90% sequence identity thereto.

6. The TGFβ1 binding molecule according to any one of claims 1 to 5, which is 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 selected from scFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab' or F(ab')2.

7. The TGFβ1 binding molecule according to any one of claims 1 to 6, which has at least one of the following properties: (1) binds to TGFβ1 precursor protein and / or binds to TGFβ1 complex, (2) does not bind to TGFβ2 or TGFβ2 complex, (3) does not bind to TGFβ3 or TGFβ3 complex, (4) Inhibit TGFβ1 activity, (5) Inhibition of regulatory T (T reg ) cells’ immunosuppressive activity, (6) Inhibit tumor growth, (7) Inhibit fibrosis; Preferably, the inhibiting TGFβ1 activity comprises: inhibiting TGFβ1 activation, inhibiting the release of mature TGFβ1 from the TGFβ1 complex, and / or inhibiting TGFβ1 signaling; Preferably, the TGFβ1 in the TGFβ1 complex exists as a TGFβ1 precursor protein, and the TGFβ1 precursor protein preferably comprises a mature TGFβ1 domain and a potency-associated peptide (LAP); Preferably, the TGFβ1 complex is selected from the group consisting of LTBP1-TGFβ1 complex, LTBP3-TGFβ1 complex, LRRC33-TGFβ1 complex and / or GARP-TGFβ1 complex.

8. A GARP-TGFβ1 binding molecule comprising at least one immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 of the amino acid sequence shown 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 systems; Preferably, the CDR1, CDR2, and CDR3 comprise the amino acid sequences shown in SEQ ID NOs: 85, 86, and 87, respectively.

9. The GARP-TGFβ1 binding molecule according to claim 8, wherein The immunoglobulin single variable domain is humanized, affinity matured, T cell epitope removed, antibody deamidation reduced and / or antibody isomerization reduced; Preferably, the human germline template used in the humanization process is selected from IGHV3-23 and / or IGJH4.

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

11. The GARP-TGFβ1 binding molecule according to any one of claims 8 to 10, further comprising an immunoglobulin Fc region; preferably, the Fc region is derived from IgG1, IgG2, IgG3, IgG4 or a variant of any of the foregoing; more preferably, it is derived from human IgG4 or a variant thereof, wherein the variant comprises the mutation 228P. 12 . The GARP-TGFβ1 binding molecule according to claim 11 , comprising an amino acid sequence as shown in any one of SEQ ID NOs: 89, 93-95 and 106, or an amino acid sequence having at least 90% sequence identity thereto.

13. The GARP-TGFβ1 binding molecule according to any one of claims 8 to 12, which is an antibody or an 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 camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof.

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

15. The GARP-TGFβ1 binding molecule according to any one of claims 8 to 14, which has at least one of the following properties: (1) Binding to the GARP-TGFβ1 complex; (2) does not bind to TGFβ2 protein or TGFβ2 complex; (3) does not bind to TGFβ3 protein or TGFβ3 complex; (4) does not bind to free mature TGFβ1; (5) Inhibit TGFβ1 activity; (6) Inhibition of regulatory T (T reg ) The immunosuppressive activity of cells; (7) Inhibit tumor growth; Preferably, the inhibiting TGFβ1 activity comprises: Inhibit TGFβ1 activation, inhibit the release of mature TGFβ1 from the GARP-TGFβ1 complex, and / or inhibit TGFβ1 signaling; Preferably, the GARP-TGFβ1 complex comprises (i) GARP, and (ii) TGFβ1 precursor protein, wherein the TGFβ1 precursor protein preferably comprises a mature TGFβ1 domain and a potency-associated peptide (LAP). 16 . A polynucleotide encoding the TGFβ1 binding molecule according to claim 1 , or the GARP-TGFβ1 binding molecule according to claim 8 .

17. A vector comprising the polynucleotide according to claim 16.

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 pastoris, Chinese hamster ovary cell or human embryonic kidney 293 cell.

19. A pharmaceutical composition comprising the TGFβ1 binding molecule according to any one of claims 1 to 7, the GARP-TGFβ1 binding molecule according to any one of claims 8 to 15, the polynucleotide according to claim 16, or the vector according to claim 17; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients, diluents or adjuvants; 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.

20. A method for preparing the TGFβ1 binding molecule according to any one of claims 1 to 7, wherein: The method comprises: expressing the 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 the step of purifying the TGFβ1 binding molecule.

21. A method for preparing the GARP-TGFβ1 binding molecule according to any one of claims 8 to 15, wherein: The method comprises: expressing the GARP-TGFβ1 binding molecule according to 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 the step of purifying the GARP-TGFβ1 binding molecule.

22. Use 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 in the preparation of a drug for a disease or symptom associated with the TGFβ signaling pathway; 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.

23. A method for preventing or treating a disease or condition associated with the TGFβ signaling pathway, comprising administering to a subject a preventively 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 the subject a preventive or therapeutically effective amount of 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; and a preventive 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; 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.

24. A method for inhibiting TGFβ1 activity in vitro or in a subject, comprising: 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 is administered in vitro or in vivo.

25. Use of a TGFβ1 binding molecule or a GARP-TGFβ1 binding molecule in combination with an immune checkpoint inhibitor in the preparation of a drug for treating cancer; 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.

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