Antibody fusion proteins targeting FAP and TGF [beta] and uses thereof

Abstract

The invention provides an antibody fusion protein. The antibody fusion protein can be specifically combined with FAP protein and block a TGF beta signal channel. The invention further provides a nucleic acid molecule for coding the antibody fusion protein, an expression vector for expressing the antibody fusion protein, a host cell and a preparation method thereof. The invention also provides application of the antibody fusion protein.

Description

Antibody fusion protein targeting FAP and TGFβ and its use Technical Field

[0001] The present invention provides a bifunctional antibody fusion protein targeting FAP and TGFβ. This antibody fusion protein can specifically bind to the FAP protein and block the TGFβ signaling pathway. The present invention further provides a nucleic acid molecule encoding the antibody fusion protein, an expression vector for expressing the antibody fusion protein, a host cell, and methods for preparing the same. The present invention also provides uses of the antibody fusion protein. Background Art

[0002] The pathological hallmark of fibrotic diseases is the accumulation of excessive fibrous connective tissue, such as collagen, in organs. Its etiology is generally caused by factors such as chemical damage, persistent infection, metabolism, and autoimmune reactions. It is an inevitable accompaniment of chronic inflammation and a key pathological feature that influences the pathological process. It also represents the progression of the disease to a more serious stage, greatly affecting the function of vital organs such as the heart, lungs, liver, and kidneys. Clinically, there are many diseases with fibrosis as a major pathological feature and process. Among the most common are HBV-induced cirrhosis, diabetes-induced renal fibrosis, and the unexplained idiopathic pulmonary fibrosis (IPF) and skin diseases. Fibrosis is a repair response to chronic damage and persistent inflammation. It may be a reversible process in its early stages, but as the disease progresses, it leads to structural damage to tissues and organ dysfunction. The fibrotic response is associated with changes in fibroblasts driven by TGFβ, including: activation of resting fibroblasts and excessive growth; phenotypic transformation to myofibroblasts; increased production of matrix proteins, triggering extracellular deposition of collagen; inhibition of MMP synthesis, induction of expression of the protease inhibitor TIMP-1, inhibition of matrix degradation by enzymes, upregulation of connective tissue growth factor (CTGF), and further promotion of fibroblast proliferation (ANNA BIERNACKA, et al. (2011). TGF-b signaling in fibrosis).

[0003] Fibroblast activation is a primary driver of fibrosis. Proteins involved in fibroblast activation include fibroblast-activation protein (FAP), alpha-smooth muscle actin (α-SMA), and vimentin. Because other molecules are also expressed in quiescent fibroblasts, pericytes, and vascular smooth muscle cells, FAP is highly specifically expressed only in activated fibroblasts. Therefore, FAP is a well-established molecular marker for fibrosis. FAP is also a key molecule that potentially drives the fibrotic process. FAP is a cell surface glycoprotein expressed on activated fibroblasts. While it is virtually undetectable in healthy adults, it is expressed in areas of active tissue repair, such as wound healing and fibrotic diseases (Pinak S. et al. (2006). "Fibroblast activation protein: a serine protease expressed at the remodeling interface in idiopathic pulmonary fibrosis," Human Pathology (2006) 37, 352–360).

[0004] TGF-β regulates numerous biological processes and is implicated in numerous diseases, including cardiovascular, fibrosis, reproductive, and cancer. The TGF-β signaling pathway is primarily mediated through the phosphorylation of Smad2 / 3. TGF-β produced by cells is inactive, and its activation requires a complex process: initially, TGF-β covalently binds to LTBP (Latent Transforming Growth Factor Beta Binding Protein) as a homodimer. TGF-β activation requires proteolysis by matrix proteases, followed by integrin-mediated conformational changes, ultimately releasing active TGF-β. TGF-β then binds to TGFβ-RII, which further recruits TGFβ-RI. This triad binding activates the catalytic activity of TGFβ-RI, which in turn triggers phosphorylation of serine residues on Smad2 and Smad3. Phosphorylated Smad2 and Smad3 form a trimeric complex with Smad4, which then enters the cell nucleus and regulates the transcription of target genes. The regulated target genes include collagens, fibronectin, tissue inhibitors of metalloproteinase (TIMP), α-SMA, etc. (Xiao-ming Meng, et al. (2016). TGF-β: the master regulator of fibrosis).

[0005] The pathological characteristics, mechanisms, and impacts on organ function of fibrosis are similar, including the cells involved in the pathological process, such as the activation of fibroblasts and the key role played by the TGF-β signaling pathway. Currently, there are no studies globally on bifunctional antibody fusion proteins targeting FAP and TGFβ.

[0006] This invention is the first to research and discover a bifunctional antibody fusion protein targeting FAP and TGFβ. This protein utilizes the targeting effect of FAP to deliver the TGFβR2 fusion protein to fibrotic tissue, achieving beneficial effects by antagonizing the TGFβ signaling pathway. Furthermore, the invention can also utilize the Fc effect of anti-FAP antibodies to kill and eliminate activated fibroblasts that produce fibrotic components of the extracellular matrix. This, in conjunction with TGF-β antagonism, can ameliorate tissue cell damage through multiple mechanisms, reducing and inhibiting the production of fibrotic components such as collagen.

[0007] Summary of the Invention

[0008] The present invention relates to a bifunctional antibody fusion protein targeting FAP and TGFβ.

[0009] In some aspects, the present invention relates to the following embodiments:

[0010] 1. A fusion protein molecule that specifically binds to human FAP and human TGFβ, such as human TGFβ1, TGFβ2 and / or TGFβ3.

[0011] 2. The fusion protein molecule of embodiment 1, comprising a first target binding region and a second target binding region, wherein the first target binding region specifically binds to FAP, and the second target binding region specifically binds to TGFβ.

[0012] 3. The fusion protein molecule of embodiment 2, wherein the first target binding region and the second target binding region are connected via a linker, or are directly connected without a linker, for example, the linker is (G4S) n , wherein n=1, 2, 3, 4, 5 or 6, for example, n=4.

[0013] 4. The fusion protein molecule of embodiment 2 or 3, wherein the first target binding region is derived from an anti-FAP antibody, such as F19 antibody or a humanized antibody thereof or 28H1 antibody or a humanized antibody thereof.

[0014] 5. The fusion protein molecule of any one of embodiments 2-4, wherein the first target binding region comprises three CDRs of the heavy chain variable region VH, HCDR1, HCDR2 and HCDR3, and three CDRs of the light chain variable region VL, LCDR1, LCDR2 and LCDR3, wherein

[0015] (i) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 7; and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 1;

[0016] (ii) the HCDR1, HCDR2, and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 13; and the LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 15;

[0017] (iii) the HCDR1, HCDR2, and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 21; and the LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 15;

[0018] (iv) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 30; and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 27; or

[0019] (v) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions or the three LCDR regions relative to the sequence of any one of (i) to (iv), for example, the amino acid change is a mutation that enhances antibody stability, such as a mutation to a deamidation site, such as present in HCDR2, which comprises a G56R substitution;

[0020] Preferably, the CDRs are identified by the Kabat numbering scheme.

[0021] 6. The fusion protein molecule of any one of embodiments 2-4, wherein the first target binding region comprises

[0022] (i) HCDR1 as set forth in SEQ ID NO: 10, HCDR2 as set forth in SEQ ID NO: 11, HCDR3 as set forth in SEQ ID NO: 12; LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 6; or

[0023] (ii) HCDR1 as set forth in SEQ ID NO: 10, HCDR2 as set forth in SEQ ID NO: 22, HCDR3 as set forth in SEQ ID NO: 12; LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 6;

[0024] (iii) HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 of the 28H1 antibody disclosed in WO2012020006; or

[0025] (iv) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions or the three LCDR regions relative to the sequence of any one of (i) or (ii) or (iii), for example, the amino acid change is a mutation that enhances antibody stability, such as a mutation to a deamidation site, such as present in HCDR2, which comprises a G56R substitution.

[0026] 7. The fusion protein molecule according to any one of embodiments 2-6, wherein the first target binding region comprises a heavy chain variable region VH, wherein the heavy chain variable region

[0027] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7, 13, 21 or 30; or

[0028] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 7, 13, 21 or 30.

[0029] 8. The fusion protein molecule according to any one of embodiments 2-7, wherein the first target binding region comprises a light chain variable region VL, wherein the light chain variable region

[0030] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1 or 15 or 27; or

[0031] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 1, 15 or 27.

[0032] 9. The fusion protein molecule according to any one of embodiments 2-8, comprising a heavy chain variable region VH and a light chain variable region VL, wherein

[0033] (i) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:1, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0034] (ii) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0035] (iii) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0036] (iv) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:30, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:27, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0037] 10. The fusion protein molecule according to any one of embodiments 2-9, wherein the first target binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL respectively comprise or consist of the amino acid sequences shown below:

[0038] (i).SEQ ID NO:7 and SEQ ID NO:1;

[0039] (ii).SEQ ID NO:13 and SEQ ID NO:15;

[0040] (iii). SEQ ID NO: 21 and SEQ ID NO: 15; or

[0041] (iv). SEQ ID NO:30 and SEQ ID NO:27.

[0042] 11. The fusion protein molecule of any one of embodiments 2-10, wherein the first target binding region further comprises an Fc or heavy chain constant region CH, for example,

[0043] The Fc region is an Fc region of IgG1, IgG2, IgG3 or IgG4, preferably an Fc region of IgG1, and optionally, the last amino acid K of the Fc region is deleted or mutated to A; or

[0044] The antibody heavy chain constant region CH is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1. Optionally, the last amino acid K of the heavy chain constant region is deleted or mutated to A.

[0045] 12. The fusion protein molecule of embodiment 11, wherein the heavy chain constant region CH

[0046] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8; or

[0047] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 8; or

[0048] The Fc region

[0049] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 40; or

[0050] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 40.

[0051] 13. The fusion protein molecule of any one of embodiments 2-12, wherein the first target binding region further comprises a light chain constant region, for example, the light chain constant region is a lambda or kappa light chain constant region.

[0052] 14. The fusion protein molecule of embodiment 13, wherein the light chain constant region

[0053] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2; or

[0054] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 2.

[0055] 15. The fusion protein molecule of any one of embodiments 2-14, wherein the first target binding region comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 9, 14, 23, 35, 41 or 42, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence.

[0056] 16. The fusion protein molecule of any one of embodiments 2-15, wherein the first target binding region comprises a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 3, 16 or 28, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence.

[0057] 17. The fusion protein molecule of embodiment 15 or 16, wherein the first target binding region comprises a heavy chain and a light chain, wherein

[0058] (i) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0059] (ii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0060] (iii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0061] (iv) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0062] (v) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 41 or 42, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0063] 18. The fusion protein molecule of embodiment 17, wherein the first target binding region comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise or consist of the amino acid sequence shown in the following SEQ ID NO:

[0064] (i).SEQ ID NO:9 and SEQ ID NO:3;

[0065] (ii).SEQ ID NO:14 and SEQ ID NO:16;

[0066] (iii). SEQ ID NO: 23 and SEQ ID NO: 16;

[0067] (iv). SEQ ID NO: 35 and SEQ ID NO: 16;

[0068] (v). SEQ ID NO: 41 and SEQ ID NO: 28; or

[0069] (vi). SEQ ID NO:42 and SEQ ID NO:28.

[0070] 19. The fusion protein molecule of any one of embodiments 2-18, wherein the first target binding region is a humanized antibody or a chimeric antibody.

[0071] 20. The fusion protein molecule of any one of embodiments 2-19, wherein the first target binding region is a monoclonal antibody.

[0072] 21. The fusion protein molecule of any one of embodiments 2-20, wherein the first target binding region is an antigen binding fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody) or linear antibody.

[0073] 22. The fusion protein molecule of any one of embodiments 2-21, wherein the second target binding region comprises a TGFβ receptor or a functional fragment thereof that specifically binds to TGFβ.

[0074] 23. The fusion protein molecule of embodiment 22, wherein the TGFβ receptor is a TGFβRII molecule, such as the TGFβRII molecule

[0075] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 34;

[0076] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 34; or

[0077] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 34.

[0078] 24. The fusion protein molecule of embodiment 22 or 23, wherein the functional fragment comprises ECD or is ECD.

[0079] 25. The fusion protein molecule of embodiment 24, wherein the ECD is the ECD of a wild-type TGFβ receptor, such as the ECD of a wild-type TGFβRII molecule, or is a variant ECD comprising a modification relative to the wild-type ECD, for example, the modification comprises a truncation or substitution of the N-terminus or C-terminus.

[0080] 26. The fusion protein molecule of embodiment 25, wherein

[0081] The substitution is selected from the group consisting of substituting G at position 20 of ECD with T, i.e., G20T; or substituting N at position 19 of ECD with E; or a combination of the two; or

[0082] The truncation comprises truncation of the N-terminus, for example, deletion of any several or all of the amino acids at positions 1-19 at the N-terminus, for example, deletion of amino acids 1-19 of ECD, or deletion of amino acids 4-19 of ECD, or deletion of amino acids 18-19 of ECD.

[0083] 27. The fusion protein molecule of any one of embodiments 24-26, wherein the ECD is the ECD of a TGFβRII molecule, such as the ECD of the TGFβRII molecule

[0084] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24, 37 or 39;

[0085] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 24, 37 or 39;

[0086] (iii) comprising or consisting of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 24, 37, or 39; preferably, the amino acid change is selected from the group consisting of T at position 20 (i.e., G20T), and / or E at position 19 (i.e., N19E);

[0087] (iv) comprising an amino acid sequence having a truncated N-terminus and / or C-terminus compared to any one of (i) to (iii), wherein preferably, the truncation refers to deletion of any several or all of the amino acids at positions 1 to 19 at the N-terminus, for example, deletion of amino acids 1 to 19 of the ECD, or deletion of amino acids 4 to 19 of the ECD, or deletion of amino acids 18 to 19 of the ECD.

[0088] 28. The fusion protein molecule according to any one of embodiments 1-27, comprising

[0089] (i) Chain A: anti-FAP antibody light chain;

[0090] (ii) Chain B: an anti-FAP antibody heavy chain linked to a TGFβ receptor or a functional fragment thereof at the N-terminus or C-terminus without a linker.

[0091] 29. The fusion protein molecule according to embodiment 28, wherein the fusion protein molecule consists of 2 chains A and 2 chains B.

[0092] 30. The fusion protein molecule of embodiment 28 or 29, wherein chain A comprises or consists of the amino acid sequence shown in SEQ ID NO:16 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0093] 31. The fusion protein molecule of any one of embodiments 28-30, wherein chain B comprises or consists of the amino acid sequence shown in SEQ ID NO: 25, 36 or 38, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0094] 32. The fusion protein molecule of embodiment 28 or 29, wherein

[0095] Chain A comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0096] Chain B comprises or consists of the amino acid sequence of SEQ ID NO: 25, 36 or 38, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0097] Chain A comprises, or consists of, the amino acid sequence of SEQ ID NO:28, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0098] Chain B comprises or consists of the amino acid sequence set forth in SEQ ID NO:29, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0099] 33. The fusion protein molecule of embodiment 32, wherein

[0100] Chain A comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, and chain B comprises or consists of the amino acid sequence shown in SEQ ID NO: 25, 36 or 38; or

[0101] Chain A comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and chain B comprises or consists of the amino acid sequence shown in SEQ ID NO: 29.

[0102] 34. An anti-FAP antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises

[0103] HCDR1 as shown in SEQ ID NO: 10, HCDR2 as shown in SEQ ID NO: 22, HCDR3 as shown in SEQ ID NO: 12; LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5 and LCDR3 as shown in SEQ ID NO: 6.

[0104] 35. An anti-FAP antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein

[0105] (i) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0106] (iii) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0107] 36. The anti-FAP antibody or antigen-binding fragment thereof of embodiment 34 or 35, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL respectively comprise or consist of the amino acid sequence shown below:

[0108] (i) SEQ ID NO: 13 and SEQ ID NO: 15; or

[0109] (ii). SEQ ID NO:21 and SEQ ID NO:15.

[0110] 37. The anti-FAP antibody or antigen-binding fragment thereof of any one of embodiments 34-36, wherein the anti-FAP antibody or antigen-binding fragment thereof further comprises an Fc or heavy chain constant region CH, for example,

[0111] The Fc region is an Fc region of IgG1, IgG2, IgG3 or IgG4, preferably an Fc region of IgG1, and optionally, the last amino acid K of the Fc region is deleted or mutated to A; or

[0112] The antibody heavy chain constant region CH is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1. Optionally, the last amino acid K of the heavy chain constant region is deleted or mutated to A.

[0113] 38. The anti-FAP antibody or antigen-binding fragment thereof according to embodiment 37, wherein the heavy chain constant region CH

[0114] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8; or

[0115] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 8; or

[0116] The Fc region

[0117] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 40; or

[0118] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 40.

[0119] 39. The anti-FAP antibody or antigen-binding fragment thereof of any one of embodiments 34-38, wherein the anti-FAP antibody or antigen-binding fragment thereof further comprises a light chain constant region, for example, the light chain constant region is a lambda or kappa light chain constant region.

[0120] 40. The anti-FAP antibody or antigen-binding fragment thereof according to embodiment 39, wherein the light chain constant region

[0121] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2; or

[0122] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 2.

[0123] 41. The anti-FAP antibody or antigen-binding fragment thereof of any one of embodiments 34-40, wherein the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 14, 23 or 35, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence.

[0124] 42. The anti-FAP antibody or antigen-binding fragment thereof of any one of embodiments 34-41, wherein the anti-FAP antibody or antigen-binding fragment thereof comprises a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 16, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence.

[0125] 43. The anti-FAP antibody or antigen-binding fragment thereof of embodiment 41 or 42, wherein the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein

[0126] (i) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0127] (ii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0128] (iii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0129] 44. The anti-FAP antibody or antigen-binding fragment thereof of embodiment 43, wherein the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise or consist of the amino acid sequence of the following SEQ ID NO:

[0130] (i).SEQ ID NO:14 and SEQ ID NO:16;

[0131] (ii). SEQ ID NO: 23 and SEQ ID NO: 16; or

[0132] (iii). SEQ ID NO: 35 and SEQ ID NO: 16;

[0133] 45. The fusion protein molecule of any one of embodiments 34-44, wherein the anti-FAP antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody.

[0134] 46. ​​The fusion protein molecule of any one of embodiments 34-45, wherein the anti-FAP antibody or its antigen-binding fragment is a monoclonal antibody.

[0135] 47. The fusion protein molecule of any one of embodiments 34-46, wherein the anti-FAP antibody or its antigen-binding fragment is an antigen-binding fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody) or linear antibody.

[0136] 48. A nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding the fusion protein molecule of any one of embodiments 1-33 or any one chain of the antibody of any one of embodiments 34-47.

[0137] 49. An expression vector comprising the nucleic acid molecule of embodiment 48, preferably, the expression vector is pCDNA, such as pCDNA3.1.

[0138] 50. A host cell comprising the nucleic acid molecule of embodiment 48 or the expression vector of embodiment 49, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293F cells or 293T cells or CHO-S cells.

[0139] 51. A method for preparing the fusion protein molecule of any one of embodiments 1-33 or the antibody of any one of embodiments 34-47, the method comprising culturing a host cell comprising the nucleic acid molecule of embodiment 48 or the expression vector of embodiment 49 under conditions suitable for expression of the chain of the fusion protein, and optionally recovering the fusion protein molecule or antibody from the host cell (or host cell culture medium).

[0140] 52. A pharmaceutical composition, drug or formulation comprising the fusion protein molecule of any one of embodiments 1-33 or the antibody of any one of embodiments 34-47 and optionally a pharmaceutical excipient.

[0141] 53. A pharmaceutical combination product comprising the fusion protein molecule of any one of embodiments 1-33 or the antibody of any one of embodiments 34-47, and one or more other therapeutic agents (e.g., anti-fibrotic agents, other antibodies, or small molecule drugs).

[0142] 54. A method for preventing or treating fibrosis (e.g., fibrosis of the lungs, kidneys, liver, or skin) in a subject, comprising administering to the subject an effective amount of the fusion protein molecule of any one of embodiments 1-33 or the antibody of any one of embodiments 34-47, or the pharmaceutical composition or formulation of embodiment 52; or the pharmaceutical combination product of embodiment 53.

[0143] 55. A method for reducing fibrotic lesions (e.g., fibrotic lesions in the lungs, kidneys, or liver, such as idiopathic pulmonary fibrosis, cirrhosis, scleroderma, or diabetic nephropathy) in a subject, comprising administering to the subject an effective amount of the fusion protein molecule of any one of embodiments 1-33 or the antibody of any one of embodiments 34-47, or the pharmaceutical composition or formulation of embodiment 52; or the pharmaceutical combination product of embodiment 53.

[0144] 56. A method for inhibiting fibroblast proliferation, such as activated fibroblasts, such as (activated) fibroblasts that specifically express FAP, in a subject, comprising administering to the subject an effective amount of the fusion protein molecule of any one of embodiments 1-33 or the antibody of any one of embodiments 34-47, or the pharmaceutical composition or formulation of embodiment 52; or the pharmaceutical combination product of embodiment 53.

[0145] 57. The method of any one of embodiments 54-56, wherein FAP expression is present or elevated in the subject's tissue or organ with fibrosis (e.g., compared to tissue / organ of a healthy subject, or compared to healthy tissue / organ of the same patient).

[0146] 58. The method of embodiment 57, wherein FAP expression is present or increased in fibroblasts in the subject's tissue or organ with fibrosis (e.g., compared to fibroblasts in tissue / organ of a healthy subject, or compared to fibroblasts in healthy tissue / organ of the same patient).

[0147] 59. The method of any one of embodiments 54-56, wherein the subject has FAP nucleic acid present in a tissue or organ with fibrosis or has elevated FAP nucleic acid (e.g., compared to a tissue / organ of a healthy subject, or compared to a healthy tissue / organ of the same patient).

[0148] 60. The method of embodiment 59, wherein FAP nucleic acid is present or has elevated FAP nucleic acid in fibroblasts of the subject's tissue or organ having fibrosis (e.g., compared to tissue / organ of a healthy subject, or compared to fibroblasts of healthy tissue / organ of the same patient). BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1: Relative binding activity test results of FAP antibody and human FAP recombinant protein (ELISA);

[0150] Figure 2: FACS analysis of the binding of FAP antibodies to 293T-cynomolgus monkey FAP cells (2A) and 293T-human FAP cells (2B);

[0151] Figure 3: FAP antibody and DPP4 antibody binding to DPP4 cells;

[0152] Figure 4: Detection results of optimized FAP antibody binding to 293T-cynomolgus monkey FAP cells (A) and 293T-human FAP cells (B) (FACS);

[0153] Figure 5: Detection results of huF19-LG-NGR-Trap binding to 293T-human FAP cells (FACS);

[0154] Figure 6: Reporter gene activity against FAP-Trap;

[0155] Figure 7: Pathological section staining of IPF model;

[0156] Figure 8: Fibrosis staining score (A) and fibrosis grading score (B) of IPF model;

[0157] Figure 9: Pathological section staining of UUO model;

[0158] Figure 10: UUO model fibrosis score and percentage;

[0159] Figure 11: Schematic diagram of the structure of the fusion protein targeting FAP and TGFβ.

[0160] Detailed description of the invention:

[0161] It should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0162] I. Definition

[0163] For the purpose of interpreting this specification, the following definitions will be used, and whenever appropriate, terms used in the singular may also include the plural, and vice versa. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0164] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0165] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0166] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0167] When "first" and "second" are mentioned herein, it is only to distinguish the two domains or two chains, but does not indicate the positions of the two domains in any way.

[0168] As used herein, the term "fibroblast activation protein (FAP)" refers to any native FAP from any vertebrate source, including mammals such as primates (e.g., humans, see NM_004460.2, or cynomolgus monkeys, see XM_005573320.2) and rodents (e.g., mice), unless otherwise indicated. The term encompasses "full-length," unprocessed FAP as well as any form of FAP resulting from processing in the cell. The amino acid sequence of an exemplary full-length human FAP protein can be found under accession number NM_004460.2, while an exemplary full-length cynomolgus monkey FAP protein sequence can be found under accession number XM_005573320.2. The term also encompasses naturally occurring variants of FAP, such as splice variants or allelic variants. Exemplary human and cynomolgus monkey amino acid sequences are shown in SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

[0169] As used herein, the terms "anti-FAP antibody," "anti-FAP," "FAP antibody," or "antibody that binds to FAP" refer to an antibody that is capable of binding to a FAP protein or a fragment thereof with sufficient affinity. In one embodiment, the extent of binding of an anti-FAP antibody to a non-FAP protein is less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or more of the binding of the antibody to FAP, as measured by radioimmunoassay (RIA) or biointerferometry or MSD assay. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), thin-layer interferometry (BLI), biointerferometry, MSD assay, surface plasmon resonance (SPR), or flow cytometry.

[0170] "TGFβR" or "TGFβ receptor" refers to a polypeptide having the amino acid sequence of wild-type human TGFβ receptor, or a polypeptide having a sequence substantially identical to the wild-type amino acid sequence.

[0171] "TGFβRII" or "TGFβ receptor II" refers to a polypeptide having the sequence of wild-type human TGFβ receptor type 2 isoform A (e.g., the amino acid sequence of NCBI Reference Sequence (RefSeq) Accession No. NP_001020018 or the amino acid sequence of Uniprot ID: P37173), or a polypeptide having the sequence of wild-type human TGFβ receptor type 2 isoform B (e.g., the amino acid sequence of NCBI RefSeq Accession No. NP_003233), or a polypeptide having a sequence substantially identical to such a wild-type amino acid sequence. TGFβRII can retain at least 75%, 80%, 85%, 90%, 95%, or 99% of the TGFβ-binding activity of the wild-type sequence, or can retain binding activity that is equal to or greater than that of the wild-type sequence. In one embodiment, the TGFβRII of the invention comprises the amino acid sequence of SEQ ID NO: 34, or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 34.

[0172] "Extracellular domain" or "ECD" refers to the extracellular domain of TGFβR, which can bind to TGF-β and block its activity. In some embodiments, the ECD is determined based on the Topological domain of Uniprot. In some embodiments, the ECD described herein also encompasses variant ECDs that retain the function of the original extracellular domain but undergo certain modifications. In some embodiments, the modification is a substitution or truncation. In some embodiments, the substitution is, for example, a substitution of G at position 20 with T, i.e., G20T; or, for example, a substitution of N at position 19 with E. In some embodiments, the truncation is an N-terminal truncation, or a C-terminal truncation, thereby obtaining a truncated N-terminus or a truncated C-terminus. In some embodiments, the truncation is an N-terminal truncation, for example, deletion of any or all of amino acids at positions 1-19 at the N-terminus, for example, deletion of amino acids 1-19 of the ECD, or deletion of amino acids 4-19 of the ECD, or deletion of amino acids 18-19 of the ECD. In some embodiments, the ECD of TGFβRII generally comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 24, 37, or 39, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence set forth in SEQ ID NO: 24, 37, or 39.

[0173] The term "functional fragment" of a protein includes a portion of a protein that is a portion or segment of an intact or complete protein having fewer amino acid residues than the intact or complete protein, but still retains the desired protein function.

[0174] The terms "whole antibody," "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to a naturally occurring glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDRs) interposed with relatively conserved regions (framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions. In some embodiments, the antibody heavy chain constant region HC of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1. The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen binding fragment.

[0175] The term "antigen-binding fragment" is a portion or segment of an intact or complete antibody that has fewer amino acid residues than the intact or complete antibody and that is capable of binding to an antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, and single-domain antibodies (sdAbs). The Fab fragment is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. For example, Fab fragments can be obtained by digesting an intact antibody with papain. In addition, digesting an intact antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab', a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antibody fragments, see: Fundamental Immunology, edited by W.E. Paul, Raven Press, NY (1993)). The Fv fragment consists of the VL and VH domains of a single antibody arm. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by independent genes, recombinant methods can be used to connect them via a synthetic linker peptide that enables the two domains to be produced as a single protein chain, in which the VL and VH regions are paired to form a single-chain Fv (scFv). The antibody fragment can be obtained by chemical methods, recombinant DNA methods, or protease digestion.

[0176] "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and a constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen binding site. Herein, the Fab chain comprising the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; accordingly, the Fab chain comprising the light chain constant region CL is also referred to as the "Fab light chain."

[0177] The term "target" refers to the object to which a binding molecule (such as a fusion protein) is bound. The target can be an antigen, a ligand, or a receptor.

[0178] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. Additionally, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with an antibody molecule.

[0179] As used herein, the term "target binding region" refers to the portion of a multispecific binding molecule, such as a fusion protein, that binds a specific target or antigen. The target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the remainder of the bispecific antibody molecule and may or may not bind to its target as a separate entity. The target binding region can also be a receptor or a ligand, or a domain of a receptor that is capable of binding a ligand. When the target binding region is an antibody, the "target binding region" is also referred to as an "antigen binding region."

[0180] When referring to "the target binding region is derived from an antibody", it means that the binding domain constituting the target binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen, for example, the heavy chain variable region and / or light chain variable region of the target binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the target binding region are CDRs of the antibody.

[0181] The term "derived from" means that the fragment in the target binding region is substantially identical to the fragment of the antibody from which it is derived, but has a mutation, such as a substitution, deletion, or addition, at one or more sites. In a specific embodiment, the mutation is not in the CDR of the antibody.

[0182] The term "immunoglobulin" refers to a protein with the structure of a naturally occurring antibody. For example, the IgG class immunoglobulin is a heterotetrameric glycoprotein of approximately 150,000 daltons consisting of two light chains and two heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL). The heavy chains of immunoglobulins can be assigned to one of five classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be divided into one of two types, called κ and λ, based on the amino acid sequence of their constant domains. IgG immunoglobulins essentially consist of two Fab molecules connected by an immunoglobulin hinge region and two dimerized Fc regions (Fc dimers).

[0183] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions.

[0184] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0185] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0186] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). Unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position of the Kabat numbering system according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0187] In one embodiment, the HCDRs and LCDRs in the antibodies of the invention are defined according to the Kabat scheme.

[0188] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment schemes may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment schemes may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment scheme rules or combinations).

[0189] The term "Fc domain" or "Fc region" or "Fc fragment" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD classes of antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE classes of antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also known as the EU index) as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) (https: / / pubmed.ncbi.nlm.nih.gov / 5257969 / ), see also http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. As used herein, the term "Fc domain" or "Fc region" or "Fc fragment" does not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, as well as the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region, such as EPKSS or EPKSC. In some embodiments, the heavy chain constant region Fc suitable for the present invention is derived from an antibody heavy chain constant region, such as the constant region of human IgG1, IgG2, IgG3, or IgG4, preferably the constant region of IgG1.

[0190] Examples of "effector functions" of immunoglobulins include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B-cell receptor), and B-cell activation.

[0191] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and / or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.

[0192] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a mouse monoclonal antibody) while being less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their human counterparts (i.e., replacing the constant region and portions of the variable region not involved in binding with the corresponding portions of a human antibody).

[0193] As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0194] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) indicates that the dissociation constant is the dissociation rate constant and the association rate constant (K dis and K on ). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0195] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative changes or be conservatively modified variants.

[0196] For polypeptide sequences, "conservative changes" include replacements, deletions or additions to the polypeptide sequence, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing mutually conservative replacements: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative sequence change" is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the antigen of interest relative to the parent antibody or binding protein.

[0197] "Single-chain variable fragment" or "scFv" is used herein to refer to a single-chain antibody fragment comprising a heavy chain variable domain VH and a light chain variable domain VL connected by a linker, wherein VH and VL pair to form an antigen binding site.

[0198] In this article, antibody constant region or antibody constant domain, including CH1, CL and Fc domains and CH2, CH3 and optional CH4 domains constituting the Fc domain, can be selected according to the expected function of the antibody molecule. For example, the constant region can be an IgA, IgD, IgE, IgG or IgM region, in particular an immunoglobulin constant domain of human IgG, for example, a constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably a constant domain of human IgG1. As an example, the Fab fragment of an antibody can comprise the CH and CL constant regions from IgG1. As another example, the Fc region of an antibody can comprise the CH2 and CH3 domains from IgG1. The immunoglobulin constant region can have a native sequence or a variant sequence.

[0199] As used herein, the term "joint" refers to any molecule that enables the direct connection of the different parts of a bispecific binding molecule. The example of a covalently linked joint between different molecular parts includes a peptide joint and a non-protein polymer, including but not limited to a copolymer of polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or polyethylene glycol, polypropylene glycol. In some embodiments, the joint is a peptide joint (also referred to as a "connecting peptide"), which refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or a hinge region from an immunoglobulin, for connecting the amino acid sequence of the first part of the binding molecule to the second part of the binding molecule. For example, a peptide joint can connect the first target binding region of the binding molecule to the second target binding region. For example, a peptide joint can also connect a part of an antibody to another part of the antibody, such as connecting a light chain variable region to a heavy chain variable region. Preferably, the peptide joint has a length that is sufficient to connect two entities in a manner such that they maintain their conformations relative to each other so as not to hinder the desired activity. Useful linkers also include glycine-alanine polymers, glycine-serine polymers, alanine-serine polymers and other flexible linkers. In one embodiment, the linker is, for example, a glycine-serine polymer, such as (G4S) n (SEQ ID NO:43), wherein n=an integer from 1 to 10, such as 1, 2, 3, 4, 5 or 6.

[0200] In yet another embodiment, the connecting peptide is a hinge region or a portion of a hinge region from an immunoglobulin, including a native hinge region or a portion thereof, or a mutated hinge region or a portion thereof. In one embodiment, the connecting peptide is, for example, a hinge region or a portion thereof (e.g., EPKSC (SEQ ID NO: 44)) of an immunoglobulin (e.g., IgG, e.g., IgG1, IgG2, IgG3, or IgG4) or a mutated hinge region or a portion thereof, e.g., EPKSS (SEQ ID NO: 45). Alternatively, a computer program can be used to simulate the three-dimensional structure of proteins and peptides, or by phage display methods, to rationally design suitable flexible connecting peptides.

[0201] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0202] The term "vector" when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences that are operatively linked to the nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which recombinant polynucleotides are incorporated.

[0203] The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0204] The term "treat," ...

[0205] The term "preventing" includes the inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition.

[0206] As used herein, the term "therapeutic agent" encompasses any substance effective in preventing or treating fibrosis, eg, a fibrotic lesion, including but not limited to anti-fibrotic agents, other antibodies, or small molecule drugs.

[0207] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

[0208] The term "effective amount" refers to that amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0209] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the fusion protein, composition, or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject.

[0210] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0211] The term "fibrosis", also known as fibrotic scar formation, is a process in which connective tissue replaces normal parenchymal tissue to an uncontrolled degree, with excessive and disordered deposition of extracellular matrix. Fibrosis is generally caused by factors such as chemical damage, persistent infection, and autoimmune response. Fibrosis is initially a reversible process in which extracellular matrix components (such as collagen produced by fibroblasts) accumulate excessively and disorderly. As the degree continues to deepen, it will seriously affect the normal physiological functions of various tissues or organs. This type of fibrosis is also called "fibrotic lesions" or "fibrotic diseases" of organs or tissues. Fibrosis can arise from various injuries or diseases, can occur as a complication of various diseases, and is a pathological feature of many diseases.

[0212] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0213] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0214] The term "drug combination or drug combination product or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) a fusion protein of the present invention, and (ii) other therapeutic agents) are administered to a patient as separate entities simultaneously, without specific time restrictions, or at the same or different time intervals, sequentially, wherein such administration provides prophylactically or therapeutically effective levels of two or more active agents in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0215] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0216] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. In some embodiments, the tissue sample is fibrotic tissue. The tissue sample may contain compounds that are not naturally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0217] II. Fusion Protein

[0218] In some embodiments, the fusion protein molecules of the present invention or fragments thereof bind to FAP (eg, human FAP) with high affinity. In some embodiments, the fusion protein molecules of the present invention or fragments thereof do not bind to or bind to DPP4, eg, human DPP4, with low affinity.

[0219] In some embodiments, the fusion protein molecules of the present invention or fragments thereof bind to TGFβ (eg, human TGFβ, eg, human TGFβ1, TGFβ2, and / or TGFβ3) with higher affinity.

[0220] In some embodiments, the fusion protein molecule of the present invention or a fragment thereof binds to FAP (e.g., human FAP) with high affinity and binds to TGFβ (e.g., human TGFβ, e.g., human TGFβ1, TGFβ2 and / or TGFβ3) with higher affinity. Optionally, the fusion protein of the present invention also binds to TGFβ (e.g., human TGFβ, e.g., human TGFβ1, TGFβ2 and / or TGFβ3) with lower affinity than it binds to FAP.

[0221] In some embodiments, the binding affinity of the fusion protein molecules or fragments thereof of the present invention to human FAP is comparable to or greater than that of known anti-FAP antibodies. In some embodiments, the binding affinity of the fusion protein molecules or fragments thereof of the present invention to human TGFβ is greater than that of known fusion protein molecules. In some embodiments, the affinity of the fusion protein molecules or fragments thereof is determined by biointerferometry.

[0222] In some embodiments, the fusion protein molecule of the present invention or its fragment has an equilibrium dissociation constant (K D ) binds to human TGFβ1, the K D Less than or equal to about 100 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM.

[0223] In some embodiments, the fusion protein molecule of the present invention or its fragment has an equilibrium dissociation constant (K D ) binds to human FAP, the K D Less than or equal to about 1 nM, 0.5 nM, 0.1 nM, 50 pM, 10 pM, 5 pM, 2 pM, or 1 pM.

[0224] In some embodiments, the molecules of the invention or fragments thereof are capable of blocking TGFβ (eg, TGFβ1, 2, or 3) signaling.

[0225] In some embodiments, the molecules of the invention or fragments thereof are capable of targeting FAP or targeting fibroblasts, such as activated fibroblasts.

[0226] In some embodiments, the molecules of the invention or fragments thereof are more stable.

[0227] In some embodiments, the molecules of the invention or fragments thereof are capable of treating fibrosis, eg, inhibiting fibrotic lesions, eg, activated fibroblasts expressing FAP.

[0228] In some embodiments, the molecules of the present invention or fragments thereof can be used to treat fibrosis. In some embodiments, the molecules of the present invention or fragments thereof can effectively inhibit fibrotic lesions.

[0229] In some embodiments, a fusion protein of the invention comprises a first binding specificity for FAP and a second binding specificity for TGFβ.

[0230] Therefore, one aspect of the present invention relates to a fusion protein comprising a first target binding region and a second target binding region, wherein the first target binding region specifically binds FAP and the second target binding region specifically binds TGFβ.

[0231] In some embodiments, the first target binding region and the second target binding region are connected via a linker, or are directly connected without a linker.

[0232] In some embodiments, the first target binding region is derived from an anti-FAP antibody, such as the F19 antibody or a humanized antibody thereof (such as the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, such as the 28H1 antibody, or an antigen-binding fragment thereof.

[0233] The first target binding region of the fusion protein suitable for the present invention can comprise an anti-FAP antibody or an antigen-binding fragment thereof of the present invention, or consist of the same, as long as it can specifically bind to FAP, including but not limited to, for example, full-length antibodies, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH or heavy chain antibodies that specifically bind to FAP.

[0234] In some embodiments, the antigen binding region is derived from an anti-FAP antibody, such as the F19 antibody or a humanized antibody thereof (such as the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, such as the 28H1 antibody.

[0235] In some embodiments, the antigen binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of an anti-FAP antibody, such as the F19 antibody, or a humanized antibody thereof (such as the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, such as the 28H1 antibody.

[0236] In some embodiments, the antigen binding region comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of an anti-FAP antibody, e.g., the F19 antibody, or a humanized antibody thereof (e.g., the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, e.g., the 28H1 antibody.

[0237] In some embodiments, the antigen binding region comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of an anti-FAP antibody, e.g., the F19 antibody, or a humanized antibody thereof (e.g., the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, e.g., the 28H1 antibody.

[0238] In some embodiments, the antigen binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of an anti-FAP antibody, such as the F19 antibody, or a humanized antibody thereof (such as the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, such as the 28H1 antibody.

[0239] In some embodiments, the antigen binding region comprises the heavy chain variable region of an anti-FAP antibody, such as the F19 antibody, or a humanized antibody thereof (such as the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, such as the 28H1 antibody.

[0240] In some embodiments, the antigen binding region comprises a light chain variable region of an anti-FAP antibody, such as the F19 antibody, or a humanized antibody thereof (such as the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, such as the 28H1 antibody.

[0241] In some embodiments, the antigen binding region comprises the heavy chain variable region and the light chain variable region of an anti-FAP antibody, such as the F19 antibody, or a humanized antibody thereof (such as the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, such as the 28H1 antibody.

[0242] In some embodiments, the antigen binding region comprises the heavy chain of an anti-FAP antibody, such as the F19 antibody, or a humanized antibody thereof (such as the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, such as the 28H1 antibody.

[0243] In some embodiments, the antigen binding region comprises a light chain of an anti-FAP antibody, such as the F19 antibody, or a humanized antibody thereof (such as the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, such as the 28H1 antibody.

[0244] In some embodiments, the antigen binding region comprises the heavy and light chains of an anti-FAP antibody, such as the F19 antibody, or a humanized antibody thereof (such as the anti-FAP humanized antibody described herein), or an anti-FAP antibody disclosed in WO2012020006, such as the 28H1 antibody.

[0245] The second target binding region of the fusion protein suitable for the present invention may comprise any protein that specifically binds to TGFβ or a functional fragment thereof, such as a TGFβ receptor, such as TGFβRII, or a functional domain thereof, such as the extracellular region of TGFβ.

[0246] III. Anti-FAP Antibodies

[0247] In one aspect, the present invention provides a FAP antibody having a binding affinity to FAP, such as a higher binding affinity than known FAP antibodies.

[0248] In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention bind to FAP (e.g., human FAP or monkey FAP, such as cynomolgus monkey FAP) with higher affinity. In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention are capable of binding to both human FAP and monkey FAP, such as cynomolgus monkey FAP.

[0249] In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention bind to FAP expressed by cells. In some embodiments, the affinity of the anti-FAP antibodies to FAP expressed by cells is determined by flow cytometry.

[0250] In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention have better stability, for example, better stability than known FAP antibodies.

[0251] In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.

[0252] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0253] In some embodiments, an anti-FAP antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0254] In some aspects, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH). In some aspects, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention comprise a light chain variable region (VH). In some aspects, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0255] In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region, CH. In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region, CL. In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region, CH, and a light chain constant region, CL.

[0256] In some embodiments, the heavy chain variable region of an anti-FAP antibody of the invention is

[0257] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7, 13 or 21, 30; or

[0258] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 7, 13, 21, or 30; or

[0259] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 7, 13 or 21, 30, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0260] In some embodiments, the light chain variable region of an anti-FAP antibody of the invention is

[0261] (i) comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1, 15, 27; or

[0262] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 1, 15, or 27; or

[0263] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 1, 15, 27, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0264] In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region of the anti-FAP antibodies of the present invention, HCDR1, HCDR2, and HCDR3 are selected from

[0265] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 13 or 21, or

[0266] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions relative to the sequence of any one of (i),

[0267] For example, the CDRs are determined by the Kabat protocol.

[0268] In some embodiments, the anti-FAP antibodies of the present invention have three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3 selected from

[0269] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 15, or

[0270] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions relative to the sequence of any one of (i),

[0271] For example, the CDRs are determined by the Kabat protocol.

[0272] In some embodiments, the anti-FAP antibody of the present invention comprises three complementarity determining regions (HCDRs) contained in a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7, 13, 21 or 30 and three complementarity determining regions (LCDRs) contained in a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, 15 or 27.

[0273] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 10, or HCDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 10.

[0274] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 11 or 22, or HCDR2 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 11 or 22.

[0275] In some embodiments, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 12, or HCDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 12.

[0276] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 4.

[0277] In some embodiments, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, or consists of the amino acid sequence, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 5.

[0278] In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 6.

[0279] In some embodiments of the present invention, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 as described above.

[0280] In some embodiments of the present invention, the anti-FAP antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.

[0281] In some embodiments of the present invention, the anti-FAP antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein

[0282] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 10,

[0283] HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 11 or 22,

[0284] HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 12,

[0285] LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, or consists of the amino acid sequence,

[0286] LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5, and

[0287] LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0288] In some specific embodiments of the present invention, the anti-FAP antibody or antigen-binding fragment thereof of the present invention comprises HCDR1 as shown in SEQ ID NO:10, HCDR2 as shown in SEQ ID NO:11 or 22, HCDR3 as shown in SEQ ID NO:12; LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5 and LCDR3 as shown in SEQ ID NO:6.

[0289] In some embodiments of the present invention, the anti-FAP antibody or antigen-binding fragment thereof comprises VH and VL, wherein

[0290] The VH comprises the amino acid sequence shown in SEQ ID NO: 7, 13, 21 or 30, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of the amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO: 1 or 15 or 27, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of the amino acid sequence.

[0291] In some specific embodiments of the present invention, the anti-FAP antibody or antigen-binding fragment thereof of the present invention comprises VH and VL, wherein VH and VL respectively comprise or consist of the amino acid sequences shown below:

[0292] SEQ ID NO: 13 and SEQ ID NO: 15;

[0293] SEQ ID NO: 22 and SEQ ID NO: 15;

[0294] SEQ ID NO: 7 and SEQ ID NO: 1;

[0295] SEQ ID NO:30 and SEQ ID NO:27.

[0296] In some embodiments of the present invention, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain. In some embodiments of the present invention, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention comprise a light chain. In some embodiments of the present invention, the anti-FAP antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain.

[0297] In some specific embodiments, the heavy chain of the anti-FAP antibody comprises the amino acid sequence of SEQ ID NO: 9, 14, 23, 35, 41 or 42, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0298] In some specific embodiments, the light chain of the anti-FAP antibody comprises the amino acid sequence of SEQ ID NO: 3, 16 or 28, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0299] In some specific embodiments, the anti-FAP antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 9, 14, 23, 35, 41 or 42, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain comprises, or consists of, an amino acid sequence as set forth in SEQ ID NO: 3, 16 or 28, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0300] In some specific embodiments, the anti-FAP antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, 14, 23, 35, 41 or 42, and wherein the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, 16 or 28.

[0301] In some specific embodiments, the anti-FAP antibody comprises a heavy chain and a light chain, wherein

[0302] (i) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0303] (ii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0304] (iii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0305] (iv) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0306] (v) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 41 or 42, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0307] In some specific embodiments, the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise or consist of the amino acid sequence shown in the following SEQ ID NO:

[0308] (i).SEQ ID NO:9 and SEQ ID NO:3;

[0309] (ii).SEQ ID NO:14 and SEQ ID NO:16;

[0310] (iii). SEQ ID NO: 23 and SEQ ID NO: 16;

[0311] (iv). SEQ ID NO: 35 and SEQ ID NO: 16;

[0312] (v). SEQ ID NO: 41 and SEQ ID NO: 28; or

[0313] (vi). SEQ ID NO:42 and SEQ ID NO:28.

[0314] In one embodiment of the present invention, the amino acid changes of the anti-FAP antibodies described herein include amino acid substitutions, insertions, or deletions. In preferred embodiments, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0315] In one embodiment of the present invention, the amino acid changes of the anti-FAP antibodies of the present invention are mutations that enhance antibody stability, such as mutations to deamidation sites, such as those present in the CDRs of the anti-FAP antibodies, such as HCDR2 of the anti-FAP antibodies, which comprises a G56R substitution.

[0316] In some embodiments, the anti-FAP antibodies or antigen-binding fragments thereof of the invention have one or more of the following properties:

[0317] (i) exhibiting the same or similar binding affinity and / or specificity for FAP as the antibodies of the present invention;

[0318] (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the invention to FAP;

[0319] (iii) binds to the same or overlapping epitope as an antibody of the invention;

[0320] (iv) competing with the antibodies of the present invention for binding to FAP;

[0321] (v) possess one or more biological properties of an antibody of the invention.

[0322] In some embodiments, the anti-FAP antibody of the present invention is an antibody in the form of IgG1, an antibody in the form of IgG2, an antibody in the form of IgG3, or an antibody in the form of IgG4, for example, an antibody in the form of IgG1. In some embodiments, the light chain constant region of the anti-FAP antibody of the present invention is a lambda or kappa light chain constant region, for example, a kappa light chain constant region.

[0323] In some embodiments, the anti-FAP antibody is a monoclonal antibody.

[0324] In some embodiments, the anti-FAP antibody is humanized.

[0325] In some embodiments, the anti-FAP antibody is a chimeric antibody.

[0326] In one embodiment, the anti-FAP antibodies of the present invention also encompass antibody fragments thereof (e.g., antigen-binding fragments), preferably antibody fragments selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), or linear antibodies.

[0327] IV. TGFβ receptor or functional fragment thereof

[0328] Any TGFβ receptor or functional fragment thereof known in the art is suitable for the fusion protein molecule of the present invention, as long as it can specifically bind to TGFβ. In some embodiments, the TGFβ receptor is a TGFβRII molecule.

[0329] In some embodiments, the second target binding region of the fusion protein molecule of the present invention comprises a TGFβ receptor, or is a TGFβ receptor. In some embodiments, the second target binding region of the fusion protein molecule of the present invention comprises a TGFβRII molecule, or is a TGFβRII molecule.

[0330] In some embodiments, the TGFβRII molecules of the invention are

[0331] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 34;

[0332] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 34; or

[0333] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 34.

[0334] In some embodiments, the second target binding region of the fusion protein molecule of the present invention comprises a functional fragment of a TGFβ receptor, or is a functional fragment of a TGFβ receptor. In some embodiments, the second target binding region of the fusion protein molecule of the present invention comprises a functional fragment of a TGFβRII molecule, or is a functional fragment of a TGFβRII molecule. In some embodiments, the functional fragment of a TGFβ receptor comprises or is the extracellular functional domain (extracellular region, ECD) of a TGFβ receptor.

[0335] In some embodiments, the ECD is the ECD of a wild-type TGFβ receptor, such as the ECD of a wild-type TGFβRII molecule. In some embodiments, the ECD is a variant ECD that comprises modifications, such as truncations or substitutions, relative to the wild-type ECD. In some embodiments, the substitution is, for example, a substitution of G at position 20 of the ECD with a T, i.e., G20T; or, for example, a substitution of N at position 19 of the ECD with an E, i.e., N19E, or a combination of the two. In some embodiments, the truncation is an N-terminal truncation, or a C-terminal truncation, thereby obtaining a truncated N-terminus or a truncated C-terminus. In some embodiments, the truncation is an N-terminal truncation, such as a deletion of any or all of amino acids at positions 1-19 at the N-terminus, such as a deletion of amino acids 1-19 of the ECD, or a deletion of amino acids 4-19 of the ECD, or a deletion of amino acids 18-19 of the ECD.

[0336] In some embodiments, the ECD of the TGFβRII molecule

[0337] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 24;

[0338] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 24;

[0339] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 24; or

[0340] (iv) comprising an amino acid sequence having a truncated N-terminus and / or C-terminus compared to any one of (i) to (iii), wherein preferably, the truncation refers to deletion of any several or all of the amino acids at positions 1 to 19 at the N-terminus, for example, deletion of amino acids 1 to 19 of the ECD, or deletion of amino acids 4 to 19 of the ECD, or deletion of amino acids 18 to 19 of the ECD.

[0341] In some embodiments, the ECD of a TGFβRII molecule of the invention

[0342] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 37, preferably the amino acid sequence comprises a T at position 20 (i.e., comprises G20T);

[0343] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 37;

[0344] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 37, preferably the amino acid sequence comprises a T at position 20 (i.e., comprises G20T); or

[0345] (iv) comprising an amino acid sequence having a truncated N-terminus and / or C-terminus compared to any one of (i) to (iii), wherein preferably, the truncation refers to deletion of any several or all of the amino acids at positions 1 to 19 at the N-terminus, for example, deletion of amino acids 1 to 19 of the ECD, or deletion of amino acids 4 to 19 of the ECD, or deletion of amino acids 18 to 19 of the ECD.

[0346] In some embodiments, the ECD of a TGFβRII molecule of the invention

[0347] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 39, preferably the amino acid sequence comprises E at position 19 (i.e., comprises N19E);

[0348] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 39;

[0349] (iii) an amino acid sequence comprising or consisting of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 39, preferably the amino acid sequence comprises E at position 19 (i.e., comprises N19E); or

[0350] (iv) comprising an amino acid sequence having a truncated N-terminus and / or C-terminus compared to any one of (i) to (iii), wherein preferably, the truncation refers to deletion of any several or all of the amino acids at positions 1 to 19 at the N-terminus, for example, deletion of amino acids 1 to 19 of the ECD, or deletion of amino acids 4 to 19 of the ECD, or deletion of amino acids 18 to 19 of the ECD.

[0351] V. Exemplary Fusion Proteins

[0352] In some embodiments, the fusion protein molecule of the present invention comprises

[0353] (i) a first target binding region that specifically binds to FAP, comprising an anti-FAP antibody or an antigen-binding fragment thereof;

[0354] (ii) a second target binding region that specifically binds to TGFβ, which comprises a TGFβ receptor or a functional fragment thereof, for example, the TGFβ receptor is a TGFβRII molecule, for example, the functional fragment is an ECD.

[0355] In some embodiments, (i) and (ii) are connected via a linker. In some embodiments, (i) and (ii) are directly connected without a linker.

[0356] In some embodiments, the first target binding region comprises an Fc region. The Fc region fragment suitable for use in the fusion protein of the present invention can be any antibody Fc region. The Fc region can include a native sequence Fc region and a variant Fc region. The native sequence Fc domain encompasses various naturally occurring immunoglobulin Fc sequences, such as various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). For example, the Fc region of an antibody of the present invention can include two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. In some embodiments, the antibody Fc region can also have an IgG hinge region or a portion of an IgG hinge region at the N-terminus, such as an IgG1 hinge region or a portion of an IgG1 hinge region. The hinge region can contain mutations. In some embodiments, the hinge region can be EPKSS or EPKSC. Preferably, the Fc region of an antibody of the present invention comprises, from N-terminus to C-terminus, CH2-CH3, or, from N-terminus to C-terminus, hinge region-CH2-CH3. In some embodiments, the Fc region suitable for use in the fusion protein molecule of the present invention is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3, or human IgG4 Fc. In one embodiment, the Fc region comprises or consists of the amino acid sequence of SEQ ID NO:40, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or greater identity thereto.

[0357] The Fc region in the antibody or fusion protein of the present invention can be mutated to obtain desired properties. In some embodiments, the lysine at the C-terminus of the Fc region is replaced, for example, with alanine to prevent the fusion protein from breaking.

[0358] In one embodiment, the first target binding region is a full-length antibody and the one or more second target binding regions are linked to the N-terminus and / or C-terminus of the heavy and / or light chains, respectively, of the full-length antibody (via or without a linker).

[0359] In one embodiment, the first target binding region is a full-length antibody and the one or more second target binding regions are linked to the N-terminus and / or C-terminus of the heavy chain of the full-length antibody (via or without a linker), respectively.

[0360] In one embodiment, the first target binding region is a full-length antibody and the one or more second target binding regions are each linked to the C-terminus of the heavy chain of the full-length antibody (via or without a linker).

[0361] In one embodiment, the second target binding region comprises or is a functional fragment of a TGFβ receptor (eg, a TGFβRII molecule), such as an ECD.

[0362] In a specific embodiment, the fusion protein molecule of the present invention comprises

[0363] (i) Chain A: anti-FAP antibody light chain;

[0364] (ii) Chain B: an anti-FAP antibody heavy chain linked at the N-terminus or C-terminus (via or without a linker) to the ECD of a TGFβ receptor (e.g., a TGFβRII molecule).

[0365] In one embodiment, the fusion protein molecule of the present invention consists of two A chains and two B chains.

[0366] In embodiments of the invention, the anti-FAP antibody light chain in chain A is as defined above. In some embodiments of the invention, the anti-FAP antibody heavy chain in chain B is as defined above. In some embodiments of the invention, the ECD of the TGFβRII receptor (e.g., TGFβRII molecule) is as defined above.

[0367] In some specific embodiments of the present invention, chain A of the fusion protein molecule of the present invention:

[0368] A method of producing a polynucleotide comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of the amino acid sequence.

[0369] In some specific embodiments of the present invention, chain B of the fusion protein molecule of the present invention:

[0370] A method of producing a polynucleotide comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 25, 36 or 38, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0371] In some specific embodiments of the present invention, chain A of the fusion protein molecule of the present invention:

[0372] A method of producing a polynucleotide comprising the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of the amino acid sequence.

[0373] In some specific embodiments of the present invention, chain B of the fusion protein molecule of the present invention:

[0374] A method of producing a polynucleotide comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of the amino acid sequence.

[0375] V. Nucleic acids of the present invention and host cells containing the same

[0376] In one aspect, the present invention provides nucleic acids encoding any of the above antibodies or fusion protein molecules. For example, the nucleic acids of the present invention comprise nucleic acids encoding the amino acid sequences shown in SEQ ID NO: 13, 14, 15, 16, 21, 23, 25, 29, 35, 36 or 38, or nucleic acids encoding amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequences shown in SEQ ID NO: 13, 14, 15, 16, 21, 23, 25, 29, 35, 36 or 38.

[0377] As will be appreciated by those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences. Nucleic acid sequences encoding molecules of the present invention can be produced by methods well known in the art, for example, by de novo solid phase DNA synthesis, or by PCR amplification.

[0378] In one aspect, the present invention provides nucleic acids encoding any of the above antibodies or any antibody chains. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human FAP antigen (and / or monkey (eg, cynomolgus monkey)) binding ability.

[0379] In another aspect, the present invention provides nucleic acids encoding any of the above fusion proteins. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of exhibiting human or monkey (e.g., cynomolgus monkey) FAP antigen binding ability. In one embodiment, the nucleic acids encoding the various chains of the fusion protein may be in the same vector or in different vectors. In yet another embodiment, the nucleic acids encoding the various chains of the fusion protein may be introduced into the same or different host cells for expression. Therefore, in some embodiments, the method for producing the fusion protein of the present invention comprises the steps of culturing a host cell containing the nucleic acids encoding the various chains under conditions suitable for expressing the various chains of the molecule to produce the fusion protein of the present invention.

[0380] In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is, for example, a pcDNA vector, such as pcDNA3.1.

[0381] In one embodiment, a host cell comprising the nucleic acid or the vector is provided, for example, a vector for cloning or expressing an anti-FAP antibody or fusion protein. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells such as CHO cells (e.g., CHO-S, such as ExpiCHO-S) or 293 cells (e.g., 293F or HEK293 cells) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, for example, a bacterium, such as Escherichia coli.

[0382] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NS0, and Sp2 / 0. Suitable mammalian host cell lines for producing antibodies are known in the art.

[0383] VI. Production and Purification of Antibodies or Fusion Proteins of the Invention

[0384] In one embodiment, the present invention provides a method for preparing an antibody or fusion protein molecule of the present invention or a fragment thereof (preferably an antigen-binding fragment), wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding the antibody or fusion protein molecule of the present invention or a fragment thereof, and optionally isolating the antibody or fusion protein or a fragment thereof. In a certain embodiment, the method further comprises recovering the antibody or fusion protein molecule of the present invention or a fragment thereof from the host cell.

[0385] In one embodiment, a method for preparing an antibody or fusion protein molecule of the invention is provided, wherein the method comprises culturing a host cell comprising nucleic acids encoding each chain of the antibody or fusion protein, e.g., chain A and chain B (optionally in one vector or in two vectors), or an expression vector comprising the nucleic acid, under conditions suitable for expression of the antibody or fusion protein, as provided above, and optionally recovering the antibody or fusion protein from the host cell (or host cell culture medium).

[0386] In order to recombinantly produce the antibody or fusion protein molecule of the present invention, the nucleic acid encoding the chain of the antibody or fusion protein is separated and inserted into one or more vectors for further cloning and / or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0387] The antibody or fusion protein molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and such factors, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0388] VII. Assay

[0389] The antibodies or fusion proteins provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art. In one aspect, the antibodies or fusion proteins of the invention are tested for their FAP and / or TGFβ binding activity, for example, by known methods such as biointerference.

[0390] The present invention also provides assays for identifying antibodies or fusion proteins with biological activity. Biological activity can include, for example, binding to FAP or TGFβ (e.g., binding to human FAP or TGFβ), blocking TGFβ (e.g., TGFβ1, 2, or 3) signaling, reversing TGFβ (e.g., TGFβ1, 2, or 3) inhibition of T cells, and / or activating T cells, inhibiting fibroblasts or inhibiting fibrotic lesions, etc.

[0391] In certain embodiments, the antibodies or fusion proteins of the invention are tested for such biological activities.

[0392] Cells for use in any of the above in vitro assays include cell lines that naturally express FAP or TGFβ or are engineered to express or overexpress FAP or TGFβ. Such cells also include cell lines that express FAP or TGFβ and are transfected with DNA encoding FAP or TGFβ that does not normally express FAP or TGFβ.

[0393] VIII. Pharmaceutical Compositions and Pharmaceutical Formulations

[0394] In some embodiments, the present invention provides a composition comprising any of the antibodies or fusion protein molecules described herein or fragments thereof, preferably a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an antibody or fusion protein molecule of the present invention or fragment thereof, and a combination of one or more other therapeutic agents.

[0395] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising the antibodies or fusion protein molecules or fragments thereof described herein, or compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising polynucleotides encoding the fusion antibodies or fusion protein molecules or fragments thereof described herein. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0396] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0397] For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0398] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0399] Pharmaceutical formulations comprising the antibodies described herein can be prepared by mixing the antibodies or fusion protein molecules of the present invention having the desired degree of purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0400] The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient, the active ingredients being required for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, such as anti-fibrotic agents, chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, or small molecule drugs, etc. The active ingredients are suitably combined in an amount effective for the intended use.

[0401] IX. Drug Combinations and Kits

[0402] In some embodiments, the present invention also provides a drug combination or a drug combination product comprising an antibody or fusion protein molecule of the present invention or a fragment thereof, and one or more other therapeutic agents (e.g., anti-fibrotic agents, chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, or small molecule drugs, etc.).

[0403] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0404] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0405] - a first container containing a pharmaceutical composition comprising an antibody or fusion protein molecule as described herein, or a fragment thereof;

[0406] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0407] X. Therapeutic Uses

[0408] In one aspect, the present invention provides a method for preventing or treating fibrosis (eg, fibrotic lesions) in a subject, comprising administering to the subject an effective amount of the antibody or fusion protein molecule, or fragment thereof, pharmaceutical composition, pharmaceutical combination, or kit of the present invention.

[0409] In some embodiments, the patient or patient tissue / organ has (e.g., elevated levels, such as nucleic acid or protein levels) a FAP (e.g., compared to a tissue / organ of a healthy subject, or compared to a healthy tissue / organ of the same patient).

[0410] In a specific embodiment, the antibody or fusion protein molecule of the present invention or fragment thereof is capable of inhibiting the proliferation of fibroblasts, such as activated fibroblasts, such as (activated) fibroblasts that specifically express FAP.

[0411] In some embodiments, the patient or fibroblasts of a tissue / organ of the patient have (e.g., elevated levels, such as nucleic acid or protein levels, of) FAP (e.g., compared to a tissue / organ of a healthy subject, or compared to fibroblasts of a healthy tissue / organ of the same patient).

[0412] In a specific embodiment, the antibody or fusion protein molecule of the present invention or its fragment can reduce fibrosis, such as fibrotic lesions. In some embodiments, the fibrosis is selected from fibrosis of the lungs, kidneys, liver, or skin. In some embodiments, the fibrotic lesion is selected from fibrotic lesions of the lungs, kidneys, or liver. In some embodiments, the fibrotic lesion is selected from idiopathic pulmonary fibrosis, cirrhosis, scleroderma, or diabetic nephropathy.

[0413] The subject can be a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., an individual suffering from a disease described herein or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein or at risk of suffering from a disease described herein. In certain embodiments, the subject receives or has received other treatments, e.g., anti-fibrotic treatment.

[0414] In other aspects, the present invention provides the use of an antibody or fusion protein molecule or fragment thereof or a pharmaceutical composition or pharmaceutical combination or a kit in the production or preparation of a medicament for the purposes described herein, such as for preventing or treating the relevant diseases or conditions mentioned herein.

[0415] In some embodiments, the antibody or fusion protein molecule or fragment thereof or pharmaceutical composition or pharmaceutical combination or kit of the invention delays the onset of a disorder and / or symptoms associated with the disorder.

[0416] In some embodiments, the antibodies or fusion protein molecules or fragments thereof or pharmaceutical compositions of the present invention can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the purposes described herein, such as for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0417] In some embodiments, the therapeutic agent is selected from an anti-fibrotic agent, other antibody, or a small molecule drug.

[0418] Such combination therapies encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations), and separate administration, in which case administration of the antibody or fusion protein molecule of the invention, or fragment thereof, can occur before, concurrently with, and / or after administration of the other therapeutic and / or agent.

[0419] The route of administration of the pharmaceutical composition is according to known methods, for example, orally, by intravenous injection, intraperitoneally, intracerebral (intraparenchymal), intracerebroventricular, intramuscularly, intraocularly, intraarterially, intraportally or intralesionally; by sustained release system or by implant device. In certain embodiments, the composition can be administered by bolus injection or by continuous infusion or by implant device.

[0420] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example

[0421] Example 1: FAP antibody humanization

[0422] 1.1 Preparation for FAP antibody humanization

[0423] The F19 antibody is a murine antibody that specifically binds to FAP and has cross-species activity with humans and monkeys (WO1999057151A2). In this example, the humanized antibody F19 was used as a candidate molecule.

[0424] Mouse anti-F19 was humanized using CDR grafting technology. Briefly, the F19VH (SEQ ID No: 7) and F19VL (SEQ ID No: 1) sequences were searched and aligned in the IMGT database to obtain the human germline gene sequences IGHV1-8*02 and IGKV1-16*01, which were highly homologous to the F19 heavy chain variable region and light chain variable region, respectively. These sequences were used as the framework for the humanized antibody variable regions. The CDRs of mouse anti-F19 were transplanted into the corresponding humanized antibody heavy and light chain variable region frameworks, respectively, to form the humanized anti-FAP antibody variable regions. To maintain the affinity of the mouse antibody for FAP, the obtained humanized antibody variable regions needed to be backmutated.

[0425] The humanized heavy chain variable region sequence (huF19-LG-VH) (SEQ ID No: 13) and light chain variable region sequence (huF19-LG-VL) (SEQ ID No: 15) of the FAP antibody were obtained, see Table 1, which also lists the back mutation sites of the humanized sequence.

[0426] Table 1: Heavy and light chain variable region sequences of the humanized anti-FAP antibody huF19-LG

[0427] The variable region sequence of the humanized control antibody (sibrotuzumab) is from patent WO9957151, wherein the heavy chain variable region sequence is the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region sequence is the amino acid sequence shown in SEQ ID NO: 19.

[0428] The variable region amino acid sequences of huF19-LG and the control antibody sibrotuzumab were submitted to GenScript Biotech for codon optimization and gene synthesis. The genes encoding the VH and VL regions of the antibodies were inserted into the expression vector pcDNA3.1(+), which contains the gene encoding the human IgG1 heavy chain constant region (SEQ ID No: 8) and the kappa light chain constant region (SEQ ID No: 2). This yielded plasmids expressing the anti-FAP antibody huF19-LG and the full-length heavy chain of sibrotuzumab, and plasmids expressing the anti-FAP antibody huF19-LG and the full-length light chain of sibrotuzumab, respectively. See the sequence listing for huF19-LG (heavy chain: SEQ ID NO: 14; light chain: SEQ ID NO: 16) and the full-length heavy and light chains of sibrotuzumab (heavy chain: SEQ ID NO: 18; light chain: SEQ ID NO: 20).

[0429] Use ExpiCHO according to the manufacturer's product manual. TM The plasmids containing the full-length heavy and light chain sequences obtained above were co-transfected into ExpiCHO-S cells using an expression system (ThermoFisher, Cat# A29133) to express the full-length anti-FAP humanized antibody huF19-LG and the control antibody sibrotuzumab. Cells were cultured for 10-12 days after transfection. When cell viability dropped to 60% to 70%, the supernatant was collected and the antibody expressed in the supernatant was purified using the MabSelect Sure Protein A affinity chromatography system (GE healthcare).

[0430] The purified antibody was concentrated, sterile filtered, and the purity of the antibody was confirmed by size exclusion chromatography (SEC). Specifically, 100mM sodium phosphate + 100mM Na2SO4 (pH 7.0) was used as the running buffer, and 20μg of sample was injected onto the TSK G3000SWXL column. Run for 30 minutes. The collected effluent was measured using an Agilent 1220 HPLC, and the data was analyzed using OpenLAB software. The humanized anti-FAP antibody huF19-LG and the control antibody sibrotuzumab have a purity of >90%, have high purity and integrity, and can be used for subsequent experiments.

[0431] 1.2 Humanized FAP antibody ELISA activity detection

[0432] An ELISA assay was used to determine the relative binding activity of the antibodies to recombinant human FAP.

[0433] Human FAP protein (Sino Biological, Cat. No. 10464-H07H) was immobilized on a 96-well plate by incubation overnight in PBS buffer at 4°C. The plate was then blocked by incubation at 37°C with PBS containing 1% BSA for 1 hour. After blocking, the plate was washed three times with PBST (PBS containing 0.05% Tween 20). Three-fold serial dilutions of anti-FAP antibody (huF19-LG, Sibrotuzumab) starting at 2 nM were prepared in binding buffer (PBS containing 0.05% Tween 20 and 0.5% BSA) and incubated with the immobilized protein at 37°C for 1 hour. After binding, the plates were washed three times with PBST, incubated for 1 hour at 37°C with peroxidase-labeled goat anti-human IgG, F(ab')2 fragment specific (Jackson Immuno Research, Cat. No. 109-035-097) diluted 1 / 15,000 in binding buffer, washed again, developed with TMB and stopped with 1M H2SO4. 50 (nM) and representative binding curves are shown in Figure 1 .

[0434] The results showed that the humanized antibody huF19-LG specifically bound to human FAP and displayed comparable binding ability to the control antibody sibrotuzumab.

[0435] 1.3 Flow cytometric binding assay of humanized FAP antibody

[0436] Cell binding experiments were performed to determine whether the humanized antibody of the present invention could bind to the human FAP protein stably expressed on the surface of 293T cells. TMThe sequences encoding human FAP protein (NM_004460.2, SEQ ID NO: 31) and cynomolgus macaque FAP protein (XM_005573320.2, SEQ ID NO: 32) were cloned into the PIRES-puro3 (Clontech, Catalog No.: 631619) expression vector using 2000 (Invitrogen, Catalog No.: 11668019) transfection reagent, and used to transfect 293T cells, respectively. 293T cells were then screened with 0.3 μg / mL puromycin (Gibco, Catalog No.: A1113802) to obtain 293T cells highly expressing human FAP (293T-human FAP) or 293T cells highly expressing cynomolgus macaque FAP (293T-cynomolgus macaque FAP).

[0437] A serial dilution of human FAP antibody (huF19-LG, Sibrotuzumab) (initial concentration: 200 nM, 4-fold serial dilutions, 8 total) was added to the cells and incubated at 4°C for 30 minutes. After washing the cells twice with PBS, the fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific (Jackson Immuno Research, Cat. No. 109-116-098) was added and incubated at 4°C for 30 minutes. After washing the cells twice with PBS and resuspending them, the fluorescence signal was detected using a BD Accuri C5 flow cytometer (BD Bioscience). The results are shown in Figure 2.

[0438] As shown in the results of Figure 2 , huF19-LG can bind to human FAP protein and cynomolgus monkey FAP protein expressed on engineered 293T cells, and its binding ability is comparable to that of the control antibody.

[0439] 1.4 Binding of FAP humanized antibodies to DPP4

[0440] Cell binding experiments were performed to determine whether the humanized antibody of the present invention could bind to the human DPP4 protein stably expressed on the surface of engineered 293T cells.

[0441] First, Lipofectamine TMThe human DPP4 protein sequence (NM_001935.3, SEQ ID NO: 33) was cloned into the expression vector PIRES-puro3 (Clontech, Catalog No.: 631619) using 2000 (Invitrogen, Cat#11668019) transfection reagent and transfected into 293T cells. 293T cells were then screened with 0.3 μg / mL puromycin (Gibco, Cat#A1113802) to obtain 293T cells that highly express human DPP4 (293T-DPP4 cells).

[0442] The anti-human FAP antibody to be tested (huF19-LG, Sibrotuzumab) was diluted to 10 μg / mL and 1 μg / mL in PBS, and then resuspended in 293T-DPP4 cells and incubated at 4°C for 30 minutes. After washing the cells twice with PBS, the fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson Immuno Research, Catalog No. 109-116-098) was added and incubated at 4°C for 30 minutes. After washing the cells twice with PBS and resuspending them, the fluorescence signal was detected using a BD Accuri C5 flow cytometer (BD Bioscience). FITC anti-human CD26 (DPP4) (BioLegend, Catalog No. 302704) was used as a positive control to detect DPP4 protein expression in 293T-DPP4 cells. The antibody was diluted 1:100 in PBS, and the cell pellet was resuspended and incubated at 4°C for 30 minutes. After washing the cells twice with PBS, they were resuspended in 100 μL of PBS and the fluorescence signal was detected using a BD Accuri C5 (BD Bioscience) flow cytometer. The results are shown in Figure 3.

[0443] From the results shown in FIG3 , it can be seen that huF19-LG cannot bind to the human DPP4 protein expressed on 293T cells.

[0444] 1.5 Physicochemical Analysis and Stability Testing of Humanized Anti-FAP Antibodies

[0445] The huF19-LG antibody was subjected to a 40°C stability test, with ELISA (see Example 1.2) and SEC (see Example 1.1) testing at 0 weeks (0W), 2 weeks (2W), and 4 weeks (4W). The results are as follows:

[0446] The above results indicate that purity decreased during the sample preparation process, with a corresponding slight decrease in activity. This is presumably due to molecular instability caused by deamidation of the asparagine residue in the CDR. This position will be modified in the future.

[0447] Example 2: Sequence modification of FAP antibodies

[0448] 2.1 Deamidation site mutation

[0449] The heavy chain CDR site was mutated G56R (Kabat), and the mutated full-length antibody sequence was eukaryotically expressed using the same method as in Example 1. The mutated antibody was named huF19-LG-NGR, and the variable region amino acid sequence was as follows:

[0450] 2.2 Flow cytometry detection of mutated antibody molecules

[0451] Cell binding assays were performed to determine whether the mutated antibodies of the present invention could bind to human or cynomolgus macaque FAP proteins stably expressed on the surface of 293T cells. 293T cells overexpressing human FAP or cynomolgus macaque FAP were prepared as described in Example 1.3. Serial dilutions of antibodies huF19-LG-NGR and huF19-LG (initial concentration: 200 nM, 4-fold serial dilutions, 8 total) were added to the cells and incubated at 4°C for 30 minutes. The cells were washed twice with PBS, followed by the addition of a fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat# 109-116-098), and incubated at 4°C for 30 minutes. The cells were washed twice with PBS, resuspended, and the fluorescence signal was detected using a BD Accuri C5 flow cytometer (BD Bioscience).

[0452] From the results shown in Figure 4 , it can be seen that huF19-LG-NGR can bind to human FAP protein and cynomolgus monkey FAP protein expressed on engineered 293T cells, and its binding ability is comparable to that of the antibody before mutation.

[0453] 2.3 Stability testing of mutated antibodies

[0454] The activity and purity of the molecule were confirmed by SEC during the stability assay. The specific method is described in Example 1.5. The results are shown in the table below.

[0455] The above results indicate that the purity of the mutated antibody huF19-LG-NGR did not change significantly, and the stability of the mutated antibody was enhanced. HuF19-LG-NGR was subsequently used to construct an antibody fusion protein.

[0456] Example 3: Antibody fusion protein

[0457] 3.1 Antibody fusion protein molecules based on huF19-LG-NGR

[0458] 3.1.1 Molecular structure optimization and construction

[0459] The present invention constructed a fusion protein, LG-NGR-Trap, as shown in Figure 11 . The FAP antibody portion is derived from huF19-LG-NGR, and the Trap portion is derived from the extracellular region of TGFβR2 (SEQ ID No: 24, SEQ ID No: 37 (G20T), and SEQ ID No: 39 (N19E)). The anti-FAP antibody portion targets cells expressing FAP, while the Trap portion blocks the binding of free TGFβ molecules to TGFβR2 on cells. Furthermore, the antibody can kill target cells through antibody-dependent cellular cytotoxicity (ADCC).

[0460] The present application used standard construction methods to obtain the antibody fusion proteins LG-NGR-Trap, LG-NGR-TrapE (N19E), and LG-NGR-TrapGT (G20T) as shown in Figure 11. Their amino acid sequences are listed in the sequence listing. This indicates that the sequence includes, from N-terminus to C-terminus, the sequence derived from the FAP humanized antibody huF19-LG-NGR and the extracellular region sequence of TGFβR2.

[0461] The antibody fusion protein molecule shown in Figure 11 was constructed as follows:

[0462] LG-NGR-Trap heavy chain: The nucleotide sequence encoding the variable region of the anti-FAP antibody heavy chain was amplified and cloned into the human IgG1 heavy chain constant region (SEQ ID NO: 8) pcDNA3.1(+) vector. The nucleotide sequence encoding the TGFβR2 extracellular region (SEQ ID NO: 24) was ligated to the C-terminus of the constant region, and the final lysine in the Fc region was replaced with an alanine to prevent cleavage. This yielded an expression vector for the LG-NGR-Trap heavy chain. The amino acid sequence of the LG-NGR-Trap heavy chain is shown in SEQ ID NO: 25, which includes, from N-terminus to C-terminus, the following sequence: LG-NGR-VH-heavy chain constant region (terminal K mutated to A)-TGFβ extracellular region.

[0463] Based on the LG-NGR-Trap heavy chain, the Trap portion was mutated to N19E or G20T, and the linker (G4S) 4 was introduced during amplification to obtain the LG-NGR-TrapE heavy chain (SEQ ID NO: 38 (N19E)) and the LG-NGR-TrapGT heavy chain (SEQ ID NO: 36 (G20T)).

[0464] LG-NGR-Trap light chain: The gene encoding the huF19-LG light chain variable region was inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human kappa light chain constant region (SEQ ID No: 2) to generate the LG-NGR-Trap light chain (i.e., the full-length huF19-LG light chain) plasmid. The amino acid sequence of the LG-NGR-Trap light chain is shown in SEQ ID NO: 16.

[0465] The above expression vectors (plasmids containing nucleic acids encoding the LG-NGR-Trap, LG-NGR-TrapE, and LG-NGR-TrapGT heavy chains and the huF19-LG light chain, respectively) were co-transfected into ExpiCHO-S cells and expressed under appropriate conditions to produce the antibody fusion protein shown in Figure 11. The expression, purification, and preliminary analysis procedures were the same as in Example 1.1.

[0466] 3.1.2 Physicochemical and stability testing of antibody fusion proteins

[0467] 3.1.2.1 The purity of the obtained antibody fusion protein LG-NGR-Trap was confirmed by size exclusion chromatography (see Example 1 for details). LG-NGR-Trap had a purity >95% and could be used for subsequent experiments.

[0468] 3.1.2.2 A stability test was performed on the antibody fusion protein LG-NGR-Trap at pH 4.5 for 1 day, and the purity was also confirmed by size exclusion chromatography.

[0469] The results in Table 7 show that under these conditions, the main peak of LG-NGR-Trap on SEC remained basically unchanged, indicating that the molecule is relatively stable.

[0470] Table 7 Antibody fusion protein stability conditions and purity

[0471] 3.1.3 Flow cytometric detection of antibody fusion proteins

[0472] A cell binding assay was performed to determine whether the antibody fusion protein of the present invention could bind to human FAP protein stably expressed on the surface of engineered 293T cells. 293T cells expressing human FAP were grown as described in Example 1.3. A serial dilution of LG-NGR-Trap (initial concentration 150 nM, 4-fold aliquots, 8 total) was added to the cells and incubated at 4°C for 30 minutes. The cells were washed twice with PBS, followed by the addition of the fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat# 109-116-098) and incubation at 4°C for 30 minutes. The cells were washed twice with PBS, resuspended, and the fluorescence signal was detected using a BD Accuri C5 flow cytometer (BD Bioscience).

[0473] From the results shown in FIG5 , it can be seen that LG-NGR-Trap can bind well to the human FAP protein expressed on the engineered 293T cells.

[0474] 3.1.4 Affinity Detection of Antibody Fusion Proteins with Human / Cynomolgus Monkey FAP and TGFβ

[0475] In this experiment, the binding affinity of the antibody fusion protein LG-NGR-Trap to human FAP protein (Sino biological, Catalog No.: 10464-H07H), human TGFβ1 protein (Sinobiological, Catalog No.: 10804-HNAC), human TGFβ2 protein (Novoprotein, Catalog No.: CJ79), and human TGFβ3 protein (Novoprotein, Catalog No.: CJ44) was detected using ForteBio Octet RED96 according to the manufacturer's instructions.

[0476] Briefly, an AHC sensor (ForteBio, Catalog No.: 18-5060) was placed in Running Buffer (1X PBS Hyclone, Catalog No.: SH30256.01, containing 0.02% Tween 20, pH 7.0) and pre-equilibrated at room temperature for 10 min. Kinetic assays were performed in a 96-well plate as follows: a) equilibrate the baseline with Running Buffer for 180 seconds; b) add the antibody fusion protein LG-NGR-Trap diluted in Running Buffer to a final concentration of 5 μg / mL and allow to solidify for 200 seconds; c) equilibrate the baseline with Running Buffer for 300 seconds; d) add the following concentrations of human FAP, TGFβ1, TGFβ2, and TGFβ3 diluted in Running Buffer to each well: 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM, and 0.39 nM; allow association for 200 seconds and dissociation for 600 seconds. Data were fitted and calculated using a 1:1 binding model using Fortebio Data Analysis software.

[0477] Table 10 summarizes the binding affinity of the antibody fusion protein LG-NGR-Trap to human FAP protein. Table 11 summarizes the binding affinity of the antibody fusion protein LG-NGR-Trap to human TGFβ1, TGFβ2 and TGFβ3 proteins.

[0478] Table 10 Binding affinity of LG-NGR-Trap to human FAP protein

[0479] Table 11 Binding affinity of LG-NGR-Trap to human TGFβ1, TGFβ2 and TGFβ3 proteins

[0480] Tables 10-11 show that the antibody fusion protein constructed in this application can specifically bind to human FAP protein and TGFβ1, TGFβ2, and TGFβ3 proteins. The KD value of the antibody fusion protein obtained in this application for TGFβ protein is two orders of magnitude lower than that for FAP protein. The high affinity of FAP can effectively reduce the side effects of targeting TGFβ.

[0481] 3.2 Alternative Molecules for Pharmacological Efficacy

[0482] 3.2.1 Construction of alternative molecules with drug efficacy

[0483] Because huF19-LG-NGR cannot bind to mouse proteins, a surrogate antibody was constructed during the efficacy evaluation phase to bind to mouse FAP. This surrogate antibody was derived from 28H1 in WO2012020006. For detailed construction methods, see Examples 1 and 3.1. 28H1 was substituted for huF19-LG-NGR, and the resulting molecule was named 28H1-Trap.

[0484] 3.2.2 Reporter gene assay

[0485] This example examined the activation of the 293T / SBE-luc2P reporter gene system by an anti-FAP-Trap antibody fusion protein. 293T cells were transfected with a plasmid containing the SBE-luc2P reporter gene element (plasmid: pGL4.48[luc2P / SBE / Hygro] Vector, Promega, Catalog No. E367A). Hygromycin (Gibco, Catalog No. 10687010) pressure selection and limiting dilution were used to isolate a monoclonal cell line stably expressing the SBE-luc2P reporter gene element. Exogenously added human TGFβ1 (Sino Biological, Catalog No. 10804-HNAC) binds to endogenously expressed TGFβR on the 293T cell surface, initiating a signaling cascade to form a SMAD complex. The SMAD complex then enters the cell nucleus, leading to activation of the SBE reporter gene element and generating a fluorescent signal. The anti-FAP-Trap antibody fusion protein can bind to human TGFβ1, thereby blocking the activation of the SBE reporter gene element. The specific steps are as follows:

[0486] 293T / SBE-luc2P cells were collected by digestion and centrifugation, and the supernatant was discarded. The cell pellet was resuspended in DMEM (Gibco, Catalog No.: 11995-065) complete medium containing 10% inactivated fetal bovine serum (Gibco, Catalog No.: 10099-141, inactivated in a 56°C water bath for 30 min). The cell suspension was counted using a Countstar cell counter, and the cell suspension was diluted to 8 × 10 5cells / mL. A TGFβ1 protein working solution was prepared at 2× the working concentration, for a final working concentration of 0.4 ng / mL, using the above culture medium. The fusion proteins to be tested (LG-NGR-Trap and 28H1-Trap, LG-NGR-TrapGT, and LG-NGR-TrapE) were prepared at 2× the final assay concentration using the TGFβ1 protein working solution. The final assay concentrations of the antibodies to be tested were 0.145 pM, 0.434 pM, 1.301 pM, 3.902 pM, 5.853 pM, 8.779 pM, 13.169 pM, 19.753 pM, and 29.630 pM. The 293T / SBE-luc2P cell suspension was plated onto a 384-well white assay plate (Corning, Cat. No. 3570) at 25 μL / well. Serial antibody dilutions were added at 25 μL / well. Place in a 37°C, 5% CO2, saturated humidity incubator and incubate overnight (18-20 hours). Add 25 μL One-Glo TM Luciferase Assay Reagent (Promega, Catalog No.: E6130) was mixed, and the luminescent signal was detected using the fluorescence detection module of a multifunctional microplate reader (Tecan, Model: F200PRO) at room temperature.

[0487] The results are shown in Figure 6 and the table below:

[0488] LG-NGR-Trap, LG-NGR-TrapGT, and LG-NGR-TrapE showed no significant difference in their inhibitory effects on the TGFβ activation pathway compared to the control 28H1-Trap. Furthermore, it was confirmed that 28H1-Trap binds to human FAP at the cellular level (same as in Example 3.1.3). Therefore, 28H1-Trap can mimic LG-NGR-Trap in in vivo efficacy experiments in mice.

[0489] Example 4: IPF Animal Efficacy Experiment

[0490] Animal Efficacy: The bilateral idiopathic pulmonary fibrosis model induced by intratracheal instillation of bleomycin in mice provided by Kase Bio was used to evaluate the efficacy of the alternative antibody fusion protein 28H1-Trap on pulmonary fibrosis.

[0491] 8-9 week old C57BL / 6 mice (Cavensberg (Suzhou) Co., Ltd.) were dosed starting on the day of modeling. Bleomycin was then instilled intratracheally to induce pulmonary fibrosis. For the next three weeks, intraperitoneal administration was performed twice weekly, and body weights were measured every two days. Specific drugs and dosages are shown in the table below:

[0492] Table 12

[0493] At the end of the experiment, mice were euthanized. The lungs of 15 surviving animals (6 in the normal group) were collected and fixed with formalin perfusion. Lung tissue pathology sections, Masson staining, and imaging (Figures 7A-H) were performed, and pulmonary fibrosis grading scores were performed. Pathological staining is shown in Figure 7, where black arrows indicate intact alveolar structures and dark interstitial areas indicate collagen accumulation. The results showed that the high-dose 28H1-Trap group (C&G) can reduce the degree of pulmonary fibrosis compared with the model group (B&F model group) (A&E: normal group, B&F: model group, C&G: 28H1-Trap, 20 mg / kg, D&H: 28H1-Trap, 3 mg / kg).

[0494] The results of pulmonary fibrosis staining are shown in Figure 8A, demonstrating that 28H1-Trap reduced pulmonary fibrosis at high doses compared to the model group. The results of pulmonary fibrosis grading (Ashcroft score) are shown in Figure 8B, demonstrating that 28H1-Trap reduced pulmonary fibrosis compared to the model group in a dose-dependent manner.

[0495] Example 5: UUO Animal Efficacy Experiment

[0496] Animal Efficacy: A left ureteral ligation mouse model of renal interstitial fibrosis was used to evaluate the efficacy of the surrogate antibody fusion protein 28H1-Trap on renal fibrosis.

[0497] C57BL / 6J mice aged 8-9 weeks (Shanghai Jihui Experimental Animal Breeding Co., Ltd.) will be randomly divided into groups according to the weight of the animals. The day of modeling is day 0. After the mice in groups 2-4 are anesthetized with isoflurane, the abdominal cavity is opened through the midline of the abdomen, the left ureter of the animal is ligated, the abdomen is closed, and the skin of the animal is sutured layer by layer. The drugs are given by intraperitoneal injection on the day of modeling. For animals in group 1, after anesthesia with isoflurane, the abdominal cavity is opened through the midline of the abdomen, the abdomen is immediately closed, and the skin of the animal is sutured layer by layer. For the next 10 days, the drug is administered intraperitoneally every other day, and the body weight is weighed every two days. The specific drugs and dosages are shown in the table below:

[0498] Table 13

[0499] The animals were intraperitoneally injected with the test substance on days 0, 2, 4, 6, and 8, and subcutaneously injected with meloxicam as an analgesic on days 1 to 3. Animal body weight was recorded daily, and fibrosis was scored in the UUO-side kidney using Masson staining.

[0500] On the 10th day after modeling, the animals were anesthetized with sodium pentobarbital and dissected. After confirming that the animals were fully anesthetized, the abdominal cavity was opened along the midline of the abdomen, and the inferior vena cava was used to collect endpoint blood. The chest cavity was opened along the thoracic diaphragm and sternal costal cartilage to expose the heart, the inferior vena cava was severed, and pre-cooled saline was perfused through the left ventricle for systemic perfusion. After the blood stain on the kidney faded, the UUO side kidney was taken, the renal membrane was carefully peeled off, and the kidney was divided into left and right halves along the renal hilum. The left half of the kidney was quickly frozen in liquid nitrogen and then transferred to -80°C for cryopreservation; the right half of the kidney was immersed in 200 mL of 10% formalin for more than 3 days and fixed for Masson staining (Figure 9) to evaluate the degree of renal fibrosis (see Figure 10).

[0501] The pathological results of Masson staining (Figure 9) and the renal fibrosis score (Figure 10) indicated that the test substance 28H1-Trap had a significant improvement effect on renal cortical fibrosis, and 28H1-Trap was dose-dependent.

Claims

1. A fusion protein molecule that specifically binds to human FAP and human TGFβ, such as human TGFβ1, TGFβ2 and / or TGFβ3.

2. The fusion protein molecule of claim 1, comprising a first target binding region and a second target binding region, wherein the first target binding region specifically binds to FAP and the second target binding region specifically binds to TGFβ.

3. The fusion protein molecule of claim 2, wherein the first target binding region and the second target binding region are connected via a linker, or are directly connected without a linker, for example, the linker is (G4S) n , wherein n=1, 2, 3, 4, 5 or 6, for example, n=4.

4. The fusion protein molecule of claim 2 or 3, wherein the first target binding region is derived from an anti-FAP antibody, such as F19 antibody or a humanized antibody thereof or 28H1 antibody or a humanized antibody thereof.

5. The fusion protein molecule of any one of claims 2 to 4, wherein the first target binding region comprises three CDRs of the heavy chain variable region VH, HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable region VL, LCDR1, LCDR2, and LCDR3, wherein (i) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 7; and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 1; (ii) the HCDR1, HCDR2, and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 13; and the LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 15; (iii) the HCDR1, HCDR2, and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 21; and the LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 15; (iv) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 30; and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 27; or (v) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions or the three LCDR regions relative to the sequence of any one of (i) to (iv), for example, the amino acid change is a mutation that enhances antibody stability, such as a mutation to a deamidation site, such as present in HCDR2, which comprises a G56R substitution; Preferably, the CDRs are identified by the Kabat numbering scheme.

6. The fusion protein molecule of any one of claims 2 to 4, wherein the first target binding region comprises (i) HCDR1 as set forth in SEQ ID NO: 10, HCDR2 as set forth in SEQ ID NO: 11, HCDR3 as set forth in SEQ ID NO: 12; LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 6; or (ii) HCDR1 as set forth in SEQ ID NO: 10, HCDR2 as set forth in SEQ ID NO: 22, HCDR3 as set forth in SEQ ID NO: 12; LCDR1 as set forth in SEQ ID NO: 4, LCDR2 as set forth in SEQ ID NO: 5, and LCDR3 as set forth in SEQ ID NO: 6; (iii) HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 of the 28H1 antibody disclosed in WO2012020006; or (iv) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions or the three LCDR regions relative to the sequence of any one of (i) or (ii) or (iii), for example, the amino acid change is a mutation that enhances antibody stability, such as a mutation to a deamidation site, such as present in HCDR2, which comprises a G56R substitution.

7. The fusion protein molecule of any one of claims 2 to 6, wherein the first target binding region comprises a heavy chain variable region VH, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7, 13, 21 or 30; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 7, 13, 21 or 30.

8. The fusion protein molecule of any one of claims 2 to 7, wherein the first target binding region comprises a light chain variable region VL, wherein the light chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1 or 15 or 27; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 1, 15 or 27.

9. The fusion protein molecule of any one of claims 2 to 8, comprising a heavy chain variable region VH and a light chain variable region VL, wherein (i) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:1, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (ii) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (iii) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (iv) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:30, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:27, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

10. The fusion protein molecule of any one of claims 2 to 9, wherein the first target binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL respectively comprise or consist of the amino acid sequence shown below: (i).SEQ ID NO:7 and SEQ ID NO:1; (ii).SEQ ID NO:13 and SEQ ID NO:15; (iii). SEQ ID NO: 21 and SEQ ID NO: 15; or (iv). SEQ ID NO:30 and SEQ ID NO:

27.

11. The fusion protein molecule of any one of claims 2 to 10, wherein the first target binding region further comprises an Fc or heavy chain constant region CH, for example, The Fc region is an Fc region of IgG1, IgG2, IgG3 or IgG4, preferably an Fc region of IgG1, and optionally, the last amino acid K of the Fc region is deleted or mutated to A; or The antibody heavy chain constant region CH is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1. Optionally, the last amino acid K of the heavy chain constant region is deleted or mutated to A.

12. The fusion protein molecule of claim 11, wherein the heavy chain constant region CH (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 8; or The Fc region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 40; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO:

40.

13. The fusion protein molecule of any one of claims 2 to 12, wherein the first target binding region further comprises a light chain constant region, for example, the light chain constant region is a lambda or kappa light chain constant region.

14. The fusion protein molecule of claim 13, wherein the light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO:

2.

15. The fusion protein molecule of any one of claims 2-14, wherein the first target binding region comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 9, 14, 23, 35, 41 or 42, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence.

16. The fusion protein molecule of any one of claims 2-15, wherein the first target binding region comprises a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 3, 16 or 28, or comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence.

17. The fusion protein molecule of claim 15 or 16, wherein the first target binding region comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (ii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (iii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (iv) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (v) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 41 or 42, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

18. The fusion protein molecule of claim 17, wherein the first target binding region comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise or consist of the amino acid sequence shown in the following SEQ ID NO: (i).SEQ ID NO:9 and SEQ ID NO:3; (ii).SEQ ID NO:14 and SEQ ID NO:16; (iii). SEQ ID NO: 23 and SEQ ID NO: 16; (iv). SEQ ID NO: 35 and SEQ ID NO: 16; (v). SEQ ID NO: 41 and SEQ ID NO: 28; or (vi). SEQ ID NO:42 and SEQ ID NO:

28.

19. The fusion protein molecule of any one of claims 2 to 18, wherein the first target binding region is a humanized antibody or a chimeric antibody.

20. The fusion protein molecule of any one of claims 2-19, wherein the first target binding region is a monoclonal antibody.

21. The fusion protein molecule of any one of claims 2-20, wherein the first target binding region is an antigen binding fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody) or linear antibody.

22. The fusion protein molecule of any one of claims 2 to 21, wherein the second target binding region comprises a TGFβ receptor or a functional fragment thereof that specifically binds to TGFβ.

23. The fusion protein molecule of claim 22, wherein the TGFβ receptor is a TGFβRII molecule, such as the TGFβRII molecule (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 34; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 34; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:

34.

24. The fusion protein molecule of claim 22 or 23, wherein the functional fragment comprises or is an ECD.

25. The fusion protein molecule of claim 24, wherein the ECD is the ECD of a wild-type TGFβ receptor, e.g., the ECD of a wild-type TGFβRII molecule, or is a variant ECD comprising a modification relative to the wild-type ECD, e.g., the modification comprises a truncation or substitution of the N-terminus or C-terminus.

26. The fusion protein molecule of claim 25, wherein The substitution is selected from the group consisting of substituting G at position 20 of ECD with T, i.e., G20T; or substituting N at position 19 of ECD with E; or a combination of the two; or The truncation comprises truncation of the N-terminus, for example, deletion of any several or all of the amino acids at positions 1-19 at the N-terminus, for example, deletion of amino acids 1-19 of ECD, or deletion of amino acids 4-19 of ECD, or deletion of amino acids 18-19 of ECD.

27. The fusion protein molecule of any one of claims 24-26, wherein the ECD is the ECD of a TGFβRII molecule, such as the ECD of the TGFβRII molecule (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 24, 37 or 39; (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 24, 37 or 39; (iii) comprising or consisting of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 24, 37, or 39; preferably, the amino acid change is selected from the group consisting of T at position 20 (i.e., G20T), and / or E at position 19 (i.e., N19E); (iv) comprising an amino acid sequence having a truncated N-terminus and / or C-terminus compared to any one of (i) to (iii), wherein preferably, the truncation refers to deletion of any or all of amino acids 1 to 19 at the N-terminus, For example, amino acids 1-19 of the ECD are deleted, or amino acids 4-19 of the ECD are deleted, or amino acids 18-19 of the ECD are deleted.

28. The fusion protein molecule of any one of claims 1 to 27, comprising (i) Chain A: anti-FAP antibody light chain; (ii) Chain B: an anti-FAP antibody heavy chain linked to a TGFβ receptor or a functional fragment thereof at the N-terminus or C-terminus without a linker.

29. The fusion protein molecule of claim 28, wherein the fusion protein molecule consists of two chains A and two chains B.

30. The fusion protein molecule of claim 28 or 29, wherein chain A comprises or consists of the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

31. The fusion protein molecule of any one of claims 28-30, wherein chain B comprises or consists of the amino acid sequence shown in SEQ ID NO: 25, 36 or 38, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

32. The fusion protein molecule of claim 28 or 29, wherein Chain A comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and Chain B comprises or consists of the amino acid sequence of SEQ ID NO: 25, 36 or 38, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or Chain A comprises, or consists of, the amino acid sequence of SEQ ID NO:28, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and Chain B comprises or consists of the amino acid sequence set forth in SEQ ID NO:29, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

33. The fusion protein molecule of claim 32, wherein Chain A comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, and chain B comprises or consists of the amino acid sequence shown in SEQ ID NO: 25, 36 or 38; or Chain A comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and chain B comprises or consists of the amino acid sequence shown in SEQ ID NO:

29.

34. An anti-FAP antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises HCDR1 as shown in SEQ ID NO: 10, HCDR2 as shown in SEQ ID NO: 22, HCDR3 as shown in SEQ ID NO: 12; LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5 and LCDR3 as shown in SEQ ID NO:

6.

35. An anti-FAP antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or a sequence consisting of the amino acid sequence and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (iii) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO:15, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

36. The anti-FAP antibody or antigen-binding fragment thereof of claim 34 or 35, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL respectively comprise or consist of the amino acid sequence shown below: (i) SEQ ID NO: 13 and SEQ ID NO: 15; or (ii). SEQ ID NO:21 and SEQ ID NO:

15.

37. The anti-FAP antibody or antigen-binding fragment thereof of any one of claims 34-36, wherein the anti-FAP antibody or antigen-binding fragment thereof further comprises an Fc or heavy chain constant region CH, e.g., The Fc region is an Fc region of IgG1, IgG2, IgG3 or IgG4, preferably an Fc region of IgG1, and optionally, the last amino acid K of the Fc region is deleted or mutated to A; or The antibody heavy chain constant region CH is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG1. Optionally, the last amino acid K of the heavy chain constant region is deleted or mutated to A.

38. The anti-FAP antibody or antigen-binding fragment thereof of claim 37, wherein the heavy chain constant region CH (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 8; or The Fc region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 40; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO:

40.

39. The anti-FAP antibody or antigen-binding fragment thereof of any one of claims 34-38, wherein the anti-FAP antibody or antigen-binding fragment thereof further comprises a light chain constant region, eg, the light chain constant region is a lambda or kappa light chain constant region.

40. The anti-FAP antibody or antigen-binding fragment thereof of claim 39, wherein the light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO:

2.

41. The anti-FAP antibody or antigen-binding fragment thereof of any one of claims 34-40, wherein the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 14, 23, or 35, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said amino acid sequence.

42. The anti-FAP antibody or antigen-binding fragment thereof of any one of claims 34-41, wherein the anti-FAP antibody or antigen-binding fragment thereof comprises a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 16, or comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

43. The anti-FAP antibody or antigen-binding fragment thereof of claim 41 or 42, wherein the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (ii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (iii) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

44. The anti-FAP antibody or antigen-binding fragment thereof of claim 43, wherein the anti-FAP antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise or consist of the amino acid sequence shown in the following SEQ ID NO: (i).SEQ ID NO:14 and SEQ ID NO:16; (ii). SEQ ID NO: 23 and SEQ ID NO: 16; or (iii). SEQ ID NO: 35 and SEQ ID NO: 16; 45. The fusion protein molecule of any one of claims 34-44, wherein the anti-FAP antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody.

46. ​​The fusion protein molecule of any one of claims 34-45, wherein the anti-FAP antibody or antigen-binding fragment thereof is a monoclonal antibody.

47. The fusion protein molecule of any one of claims 34-46, wherein the anti-FAP antibody or antigen-binding fragment thereof is an antigen-binding fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody) or linear antibody.

48. A nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding the fusion protein molecule of any one of claims 1-33 or any one chain of the antibody of any one of claims 34-47.

49. An expression vector comprising the nucleic acid molecule according to claim 48, preferably, the expression vector is pCDNA, such as pCDNA3.

1.

50. A host cell comprising the nucleic acid molecule of claim 48 or the expression vector of claim 49, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293F cells or 293T cells or CHO-S cells.

51. prepare the method for the described fusion protein molecule of any one among claim 1-33 or the antibody described in any one among claim 34-47, described method comprises, under the condition of the chain expression of being fit to described fusion protein, cultivate the host cell that comprises the described nucleic acid molecule of claim 48 or the described expression vector of claim 49, and optionally reclaim described fusion protein molecule or antibody from described host cell (or host cell culture medium).

52. A pharmaceutical composition, a drug or a preparation comprising the fusion protein molecule according to any one of claims 1 to 33 or the antibody according to any one of claims 34 to 47 and optionally a pharmaceutically acceptable excipient.

53. A pharmaceutical combination product comprising the fusion protein molecule of any one of claims 1-33 or the antibody of any one of claims 34-47, and one or more other therapeutic agents (eg, anti-fibrotic agents, other antibodies, or small molecule drugs).

54. A method for preventing or treating fibrosis (e.g., fibrosis of the lungs, kidneys, liver, or skin) in a subject, comprising administering to the subject an effective amount of the fusion protein molecule of any one of claims 1-33 or the antibody of any one of claims 34-47, or the pharmaceutical composition or formulation of claim 52; or the pharmaceutical combination product of claim 53.

55. A method for reducing fibrotic lesions (e.g., fibrotic lesions in the lungs, kidneys, or liver, such as idiopathic pulmonary fibrosis, cirrhosis, scleroderma, or diabetic nephropathy) in a subject, comprising administering to the subject an effective amount of the fusion protein molecule of any one of claims 1-33 or the antibody of any one of claims 34-47, or the pharmaceutical composition or formulation of claim 52; or the pharmaceutical combination product of claim 53.

56. A method for inhibiting fibroblast proliferation, such as activated fibroblasts, such as (activated) fibroblasts that specifically express FAP, in a subject, comprising administering to the subject an effective amount of the fusion protein molecule of any one of claims 1-33 or the antibody of any one of claims 34-47, or the pharmaceutical composition or formulation of claim 52; or the pharmaceutical combination product of claim 53.

57. The method of any one of claims 54-56, wherein FAP expression is present or increased in the subject's tissue or organ having fibrosis (e.g., compared to a tissue / organ of a healthy subject, or compared to a healthy tissue / organ of the same patient).

58. The method of claim 57, wherein FAP expression is present or increased in fibroblasts in a tissue or organ of the subject having fibrosis (e.g., compared to fibroblasts in a tissue / organ of a healthy subject, or compared to fibroblasts in a healthy tissue / organ of the same patient).

59. The method of any one of claims 54-56, wherein the subject has FAP nucleic acid present or elevated in a tissue or organ with fibrosis (e.g., compared to a tissue / organ of a healthy subject, or compared to a healthy tissue / organ of the same patient).

60. The method of claim 59, wherein FAP nucleic acid is present or elevated in fibroblasts of the subject's tissue or organ having fibrosis (e.g., compared to tissue / organ of a healthy subject, or compared to fibroblasts of healthy tissue / organ of the same patient).

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