Multispecific molecules targeting il-11 and tslp

Abstract

A multispecific molecule is provided, comprising a first binding domain capable of targeting 1L-11 and a second binding domain capable of targeting TSLP, the multispecific molecule being capable of treating diseases, and the use of the multispecific molecule as a drug is also provided.

Description

Multispecific molecules targeting IL-11 and TSLP Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a multispecific molecule targeting IL-11 and TSLP and its use. Background Art

[0002] Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine. Mature hTSLP consists of 131 amino acid residues with a typical four-stranded α-helical bundle structure. It is primarily produced in epithelial cells, smooth muscle cells, keratinocytes, stromal cells, fibroblasts, mast cells (MC), monocytes / macrophages, granulocytes, and dendritic cells (DC). As an important allergic factor secreted by epithelial cells, TSLP induces Th2 responses through interactions with locally infiltrating DCs and T cells. TSLP can also inhibit the Th1 pathway, shifting the inflammatory response from Th1 to Th2, playing a crucial role in coordinating and balancing the inflammatory response.

[0003] The TSLP receptor complex is a heterodimer composed of the TSLP receptor (TSLPR) and the IL-7 receptor α (IL-7Ra), both of which are highly expressed on myeloid dendritic cells. TSLP binds to TSLPR on the cell membrane and then to IL-7Rα, forming a stable TSLP-TSLPR-IL7Ra receptor complex. The intracellular domain of the TSLPR receptor in this complex recruits and activates JAK2, which, in conjunction with JAK1 recruited by IL7Rα, activates downstream signaling molecules. After TSLP binds to surface receptors on myeloid dendritic cells, dendritic cells secrete IL-8 and eotaxin-2 to recruit neutrophils and eosinophils, and TARC and MDC to recruit Th2 cells. Furthermore, TSLP-activated DCs induce CD4+ T cells to differentiate into Th2 cells. Th2 cells are capable of producing IL4, IL-5, IL-13, and TNF. These cytokines promote the production of IgE, eosinophils, and mucus, initiating allergic reactions and triggering diseases such as asthma and atopic dermatitis.

[0004] Interleukin-11 (IL-11), discovered in 1990, is a member of the IL-6 family. It consists of 178 amino acids and has a molecular weight of approximately 19 kDa. IL-11 is expressed by a variety of cell types, including leukocytes, fibroblasts, and epithelial cells; its expression is regulated by numerous cytokines, including TGF-β, IL-1β, IL-22, IL-17F, IFN-γ, TNF-α, and COX-2. IL-11 activates downstream signaling pathways by binding to the α subunit of the IL-11 receptor (IL-11Rα), which in turn forms a 2:2:2 complex (IL-11 / IL-11Rα / GP130) with the glycoprotein β receptor subunit (GP130).

[0005] The most clearly studied physiological function of IL-11 is its promotion of megakaryocyte expansion, which in turn promotes platelet production. In addition to hematopoiesis, another major physiological function of IL-11 is osteogenesis; osteoblasts constitutively express IL-11 and IL-11Rα. Recent studies have demonstrated that it is an important downstream regulator of TGF-β. In polarized cells, IL-11 secretion leads to cellular dysfunction and can trigger apoptosis, while also blocking regeneration. In stromal cells, IL-11 triggers extracellular matrix production, invasion, and migration of myofibroblasts. IL-11-activated fibroblasts and myofibroblasts secrete cytokines and chemokines and are strongly proinflammatory. Numerous basic studies have demonstrated that inhibition of IL-11 can protect normal tissues, combat fibrosis, and reduce matrix-driven inflammation.

[0006] TSLP and IL-11 share a common mechanism in their effects on inflammation and fibrosis. Studies have found that TSLP can promote fibrosis during the tissue repair process. If the fibrosis stage continues to progress unchecked, the result of fibrotic disease will lead to extensive tissue remodeling and the formation of permanent scar tissue.

[0007] While the development of drugs targeting IL-11 and TSLP is of great significance, it also faces numerous challenges, such as preclinical evaluation models, low expression levels, poor stability, complex processes, and wide variability in quality control. Therefore, there is an urgent need to develop multispecific molecules with excellent multispecificity, good efficacy, and ease of preparation to better treat related diseases. Summary of the Invention

[0008] The present application provides a multispecific molecule comprising a first binding domain capable of targeting IL-11 protein and a second binding domain capable of targeting TSLP protein, wherein the multispecific molecule has one or more of the following properties: (1) the ability to bind to 1×10- 9M or less KD value for binding to TSLP protein, wherein the KD value is determined by surface plasmon resonance; (2) capable of binding to TSLP protein at a KD value of 1×10 -9 The present invention relates to a multispecific molecule that binds to IL-11 protein with a KD value of M or less, wherein the KD value is determined by surface plasmon resonance; (3) can block the binding of TSLP to the receptor TSLPR / IL7Ra complex; (4) can inhibit the production of TARC (thymus and activation-regulated chemokine); (5) can inhibit cell fibrosis; and (6) blocks the TH2 pro-inflammatory effects induced by TSLP and IL11. The multispecific molecules provided herein have better therapeutic activity, stability or immunogenicity than the IL-11 and / or TSLP antibodies available in the prior art.

[0009] In one aspect, the present application provides a multispecific molecule comprising a first binding domain that targets IL-11 and a second binding domain that targets TSLP.

[0010] In certain embodiments, the first binding domain capable of targeting IL-11 comprises an antibody or an antigen-binding fragment thereof.

[0011] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and dAb fragment.

[0012] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, a fully human antibody, a heavy chain antibody, and / or a nanobody.

[0013] In certain embodiments, the first binding domain capable of targeting IL-11 comprises HCDR3, HCDR2 and HCDR1 of the heavy chain variable region VH; wherein the amino acid sequence of the HCDR3 is shown in SEQ ID NO:7, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO:5.

[0014] In certain embodiments, the first binding domain capable of targeting IL-11 comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:4.

[0015] In certain embodiments, the first binding domain capable of targeting IL-11 is a VHH.

[0016] In certain embodiments, the VHH comprises the amino acid sequence shown in SEQ ID NO:4.

[0017] In certain embodiments, the first binding domain capable of targeting IL-11 comprises the amino acid sequence shown in SEQ ID NO:4.

[0018] In certain embodiments, the first binding domain capable of targeting IL-11 comprises an antibody heavy chain variable region VH and / or an antibody light chain variable region VL.

[0019] In certain embodiments, the first binding domain capable of targeting IL-11 may be a sequence known in the prior art.

[0020] In certain embodiments, the first binding domain capable of targeting IL-11 may be the sequence described in CN113056481A.

[0021] In certain embodiments, the first binding domain capable of targeting IL-11 comprises HCDR3, HCDR2 and HCDR1 of the heavy chain variable region VH; wherein the HCDR1 can be selected from the sequences shown in SEQ ID NO: 64, 65, 95, 104, 110, 116, the HCDR2 can be selected from the sequences shown in SEQ ID NO: 66, 72, 73, 74, 96, 105, 111, 117, and the HCDR3 can be selected from the sequences shown in SEQ ID NO: 67, 68, 97, 106, 112, 118.

[0022] In certain embodiments, the first binding domain capable of targeting IL-11 comprises LCDR3, LCDR2 and LCDR1 of the light chain variable region VL; wherein the LCDR1 can be selected from the sequences shown in SEQ ID NO: 69, 70, 71, 98, 101, 107, 113, 119, the LCDR2 can be selected from the sequences shown in SEQ ID NO: 72, 73, 99, 102, 108, 114, 120, and the LCDR3 can be selected from the sequences shown in SEQ ID NO: 74-94, 100, 103, 109, 115, 121.

[0023] In certain embodiments, the first binding domain capable of targeting IL-11 comprises an antibody heavy chain variable region VH, and the VH may comprise an amino acid sequence as shown in SEQ ID NOs: 122-134.

[0024] In certain embodiments, the first binding domain capable of targeting IL-11 comprises an antibody light chain variable region VL, and the VL may comprise an amino acid sequence as shown in SEQ ID NOs: 135-177.

[0025] In some embodiments, the first binding domain capable of targeting IL-11 comprises an antibody heavy chain constant region.

[0026] In some embodiments, the first binding domain capable of targeting IL-11 comprises an antibody light chain constant region.

[0027] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody or an antigen-binding fragment thereof.

[0028] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and dAb.

[0029] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, a fully human antibody, a nanobody and / or a heavy chain antibody.

[0030] In certain embodiments, the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 19, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 20, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 21.

[0031] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, wherein the amino acid sequence of VH is shown in SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:53.

[0032] In certain embodiments, the second binding domain capable of targeting TSLP comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL; wherein the amino acid sequence of the LCDR1 is shown in SEQ ID NO:22, the amino acid sequence of the LCDR2 is shown in SEQ ID NO:23 (GAR), and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:24.

[0033] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody light chain variable region VL, wherein the amino acid sequence of VL is shown in SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:57.

[0034] In certain embodiments, the second binding domain capable of targeting TSLP comprises an amino acid sequence selected from any one of the following groups of VH and VL:

[0035] 1) VH: SEQ ID NO: 53, VL: SEQ ID NO: 57;

[0036] 2) VH: SEQ ID NO: 49, VL: SEQ ID NO: 54;

[0037] 3) VH: SEQ ID NO: 49, VL: SEQ ID NO: 55;

[0038] 4) VH: SEQ ID NO: 49, VL: SEQ ID NO: 56;

[0039] 5) VH: SEQ ID NO: 50, VL: SEQ ID NO: 54;

[0040] 6) VH: SEQ ID NO: 50, VL: SEQ ID NO: 55;

[0041] 7) VH: SEQ ID NO: 50, VL: SEQ ID NO: 56;

[0042] 8) VH: SEQ ID NO: 51, VL: SEQ ID NO: 54;

[0043] 9) VH: SEQ ID NO: 51, VL: SEQ ID NO: 55;

[0044] 10) VH: SEQ ID NO: 51, VL: SEQ ID NO: 56;

[0045] 11) VH: SEQ ID NO: 52, VL: SEQ ID NO: 54;

[0046] 12) VH: SEQ ID NO: 52, VL: SEQ ID NO: 55; and

[0047] 13) VH: SEQ ID NO: 52, VL: SEQ ID NO: 56.

[0048] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain constant region.

[0049] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

[0050] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody light chain constant region.

[0051] In certain embodiments, the antibody light chain constant region is derived from a human Ig kappa constant region.

[0052] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain comprising the amino acid sequences shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.

[0053] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody light chain comprising the amino acid sequences shown in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

[0054] In certain embodiments, the second binding domain capable of targeting TSLP may be a sequence known in the prior art.

[0055] In certain embodiments, the second binding domain capable of targeting TSLP can be Tezepelumab.

[0056] In certain embodiments, the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 178, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 179, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 180.

[0057] In certain embodiments, the second binding domain capable of targeting TSLP comprises LCDR3, LCDR2 and LCDR1 of the light chain variable region VL; wherein the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 181, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 182, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 183.

[0058] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 184.

[0059] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:185.

[0060] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain constant region.

[0061] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody light chain constant region.

[0062] In certain embodiments, the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 186, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 187, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 188.

[0063] In certain embodiments, the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 189, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 190, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 191.

[0064] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:192.

[0065] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:193.

[0066] In certain embodiments, the second binding domain capable of targeting TSLP is directly or indirectly linked to the first binding domain targeting IL-11.

[0067] In certain embodiments, the C-terminus of the first binding domain capable of targeting IL-11 is linked to the N-terminus of the second binding domain targeting TSLP.

[0068] In certain embodiments, the C-terminus of the first binding domain capable of targeting IL-11 is linked to the N-terminus of the heavy chain variable region VH of the second binding domain targeting TSLP.

[0069] In certain embodiments, the N-terminus of the first binding domain capable of targeting IL-11 is linked to the C-terminus of the second binding domain targeting TSLP.

[0070] In certain embodiments, the N-terminus of the first binding domain capable of targeting IL-11 is linked to the C-terminus of the heavy chain constant region of the second binding domain targeting TSLP.

[0071] In certain embodiments, the first binding domain capable of targeting IL-11 and the second binding domain capable of targeting TSLP are connected via a linker.

[0072] In certain embodiments, the linker comprises a peptide linker.

[0073] In certain embodiments, the linker comprises an amino acid sequence of (GGGGS)n, wherein n is any positive integer between 0 and 10.

[0074] In certain embodiments, the linker comprises the amino acid sequence shown as GGGGSGGGGS (SEQ ID NO: 3).

[0075] In certain embodiments, the multispecific molecule comprises two first binding domains capable of targeting IL-11.

[0076] In certain embodiments, the two first binding domains capable of targeting IL-11 are directly or indirectly linked to the N-termini of the two heavy chains of the second binding domain targeting TSLP.

[0077] In certain embodiments, the two first binding domains capable of targeting IL-11 are directly or indirectly linked to the C-termini of the two heavy chains of the second binding domain targeting TSLP.

[0078] In certain embodiments, the multispecific molecule comprises a first polypeptide chain and a second polypeptide chain.

[0079] In certain embodiments, the first polypeptide chain comprises a heavy chain comprising the first binding domain capable of targeting IL-11 and the second binding domain capable of targeting TSLP.

[0080] In certain embodiments, the N-termini of the heavy chains of the first polypeptide chain comprising the first binding domain capable of targeting IL-11 and the second binding domain capable of targeting TSLP are directly or indirectly linked.

[0081] In certain embodiments, the C-termini of the heavy chains of the first polypeptide chain comprising the first binding domain capable of targeting IL-11 and the second binding domain capable of targeting TSLP are directly or indirectly linked.

[0082] In certain embodiments, the first polypeptide chain comprising the first binding domain capable of targeting IL-11 and the heavy chain comprising the second binding domain capable of targeting TSLP are connected by a linker.

[0083] In certain embodiments, the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63.

[0084] In certain embodiments, the second polypeptide chain comprises the light chain of the second binding domain.

[0085] In certain embodiments, the second polypeptide chain comprises the amino acid sequences shown in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

[0086] In certain embodiments, the multispecific molecule comprises two of the first polypeptide chains and two of the second polypeptide chains.

[0087] In certain embodiments, the first polypeptide chain and the second polypeptide chain are linked by a disulfide bond.

[0088] On the other hand, the present application provides a pharmaceutical combination comprising a first antibody that targets IL-11 and a second antibody that targets TSLP.

[0089] In certain embodiments, the first antibody and the second antibody are present in the form of a mixture.

[0090] In certain embodiments, the first antibody and the second antibody each exist independently.

[0091] In certain embodiments, the first antibody and the second antibody may be combined in a certain ratio.

[0092] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, a fully human antibody, a nanobody, and / or a heavy chain antibody.

[0093] In certain embodiments, the first antibody comprises HCDR3, HCDR2 and HCDR1 of the heavy chain variable region VH; wherein the amino acid sequence of the HCDR3 is shown in SEQ ID NO:7, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:6, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO:5.

[0094] In certain embodiments, the first antibody comprises an antibody heavy chain variable region VH, and the amino acid sequence of VH is shown in SEQ ID NO:4.

[0095] In certain embodiments, the first antibody comprises an antibody heavy chain constant region that is derived from an IgG heavy chain constant region.

[0096] In certain embodiments, the first antibody is a nanobody comprising the amino acid sequence shown in SEQ ID NO:4.

[0097] In certain embodiments, the first antibody may be an antibody disclosed in patent WO2023 / 143556A1.

[0098] In certain embodiments, the first antibody capable of targeting IL-11 may be a sequence known in the prior art.

[0099] In certain embodiments, the first antibody capable of targeting IL-11 may be the sequence described in CN113056481A.

[0100] In certain embodiments, the first antibody capable of targeting IL-11 comprises HCDR3, HCDR2 and HCDR1 of the heavy chain variable region VH; wherein the HCDR1 can be selected from the sequences shown in SEQ ID NO: 64, 65, 95, 104, 110, 116, the HCDR2 can be selected from the sequences shown in SEQ ID NO: 66, 72, 73, 74, 96, 105, 111, 117, and the HCDR3 can be selected from the sequences shown in SEQ ID NO: 67, 68, 97, 106, 112, 118.

[0101] In certain embodiments, the first antibody capable of targeting IL-11 comprises LCDR3, LCDR2 and LCDR1 of the light chain variable region VL; wherein the LCDR1 can be selected from the sequences shown in SEQ ID NO: 69, 70, 71, 98, 101, 107, 113, 119, the LCDR2 can be selected from the sequences shown in SEQ ID NO: 72, 73, 99, 102, 108, 114, 120, and the LCDR3 can be selected from the sequences shown in SEQ ID NO: 74-94, 100, 103, 109, 115, 121.

[0102] In certain embodiments, the first antibody capable of targeting IL-11 comprises an antibody heavy chain variable region VH, and the VH may comprise an amino acid sequence as shown in SEQ ID NOs: 122-134.

[0103] In certain embodiments, the first antibody capable of targeting IL-11 comprises an antibody light chain variable region VL, and the VL may comprise an amino acid sequence as shown in SEQ ID NOs: 135-177.

[0104] In some embodiments, the first antibody capable of targeting IL-11 comprises an antibody heavy chain constant region.

[0105] In some embodiments, the first antibody capable of targeting IL-11 comprises an antibody light chain constant region.

[0106] In certain embodiments, the second antibody comprises HCDR3, HCDR2 and HCDR1 of the heavy chain variable region VH; wherein the amino acid sequence of the HCDR3 is shown in SEQ ID NO:21, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:20, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO:19.

[0107] In certain embodiments, the second antibody comprises an antibody heavy chain variable region VH, wherein the amino acid sequence of VH is selected from SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

[0108] In certain embodiments, the second antibody comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL; wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:22, the amino acid sequence of LCDR2 is shown in SEQ ID NO:23 (GAR), and the amino acid sequence of LCDR3 is shown in SEQ ID NO:24.

[0109] In certain embodiments, the second antibody comprises an antibody light chain variable region VL, wherein the amino acid sequence of VL is selected from SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57.

[0110] In certain embodiments, the second antibody comprises an amino acid sequence selected from any one of the following groups of VH and VL:

[0111] 1) VH: SEQ ID NO: 53, VL: SEQ ID NO: 57;

[0112] 2) VH: SEQ ID NO: 49, VL: SEQ ID NO: 54;

[0113] 3) VH: SEQ ID NO: 49, VL: SEQ ID NO: 55;

[0114] 4) VH: SEQ ID NO: 49, VL: SEQ ID NO: 56;

[0115] 5) VH: SEQ ID NO: 50, VL: SEQ ID NO: 54;

[0116] 6) VH: SEQ ID NO: 50, VL: SEQ ID NO: 55;

[0117] 7) VH: SEQ ID NO: 50, VL: SEQ ID NO: 56;

[0118] 8) VH: SEQ ID NO: 51, VL: SEQ ID NO: 54;

[0119] 9) VH: SEQ ID NO: 51, VL: SEQ ID NO: 55;

[0120] 10) VH: SEQ ID NO: 51, VL: SEQ ID NO: 56;

[0121] 11) VH: SEQ ID NO: 52, VL: SEQ ID NO: 54;

[0122] 12) VH: SEQ ID NO: 52, VL: SEQ ID NO: 55; and

[0123] 13) VH: SEQ ID NO: 52, VL: SEQ ID NO: 56.

[0124] In certain embodiments, the second antibody may be an antibody known in the art.

[0125] In certain embodiments, the second antibody is Tezepelumab.

[0126] In certain embodiments, the second antibody capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 178, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 179, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 180.

[0127] In certain embodiments, the second antibody capable of targeting TSLP comprises LCDR3, LCDR2 and LCDR1 of the light chain variable region VL; wherein the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 181, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 182, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 183.

[0128] In certain embodiments, the second antibody capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 184.

[0129] In certain embodiments, the second antibody capable of targeting TSLP comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:185.

[0130] In certain embodiments, the second antibody capable of targeting TSLP comprises an antibody heavy chain constant region.

[0131] In certain embodiments, the second antibody capable of targeting TSLP comprises an antibody light chain constant region.

[0132] In certain embodiments, the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 186, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 187, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 188.

[0133] In certain embodiments, the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 189, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 190, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 191.

[0134] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:192.

[0135] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:193.

[0136] In another aspect, the present application also provides one or more isolated nucleic acid molecules encoding the multispecific molecule.

[0137] On the other hand, the present application also provides a vector comprising the nucleic acid molecule.

[0138] On the other hand, the present application also provides a cell comprising the nucleic acid molecule or the vector.

[0139] On the other hand, the present application also provides a method for preparing the multispecific molecule, which comprises culturing the cell under conditions that allow the multispecific molecule to be expressed.

[0140] On the other hand, the present application also provides a pharmaceutical composition comprising the multispecific molecule, the drug combination, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable adjuvant.

[0141] On the other hand, the present application also provides the use of the multispecific molecule, the drug combination, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a drug for preventing, alleviating and / or treating a disease or condition.

[0142] In certain embodiments, the disease and / or disorder comprises an IL-11-associated disease.

[0143] In certain embodiments, the disease and / or condition comprises a TSLP-associated disease.

[0144] In certain embodiments, the disease and / or condition comprises a fibrotic disease, an inflammatory disease, or a tumor.

[0145] On the other hand, the present application also provides a method for blocking the binding of TSLP to the receptor TSLPR / IL7Ra complex, which comprises administering the multispecific molecule and / or the drug combination.

[0146] On the other hand, the present application also provides a method for inhibiting the production of TARC (thymus and activation-regulated chemokine), which comprises administering the multispecific antibody and / or the drug combination.

[0147] On the other hand, the present application also provides the use of the multispecific molecule, the drug combination, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a drug for preventing, alleviating and / or treating a disease or condition.

[0148] In certain embodiments, the disease and / or disorder comprises an IL-11-associated disease.

[0149] In certain embodiments, the disease and / or condition comprises a TSLP-associated disease.

[0150] In certain embodiments, the disease and / or condition comprises a fibrotic disease, an inflammatory disease, or a tumor.

[0151] In another aspect, the present application provides a method for preventing, alleviating and / or treating a disease or condition, comprising administering the multispecific molecule, drug combination, nucleic acid molecule, vector, cell and / or pharmaceutical composition described herein to a patient in need thereof.

[0152] In certain embodiments, in the methods described above, the disease and / or condition comprises an IL-11-related disease.

[0153] In certain embodiments, in the methods described herein, the disease and / or condition comprises a TSLP-related disease.

[0154] In certain embodiments, in the methods described above, the disease and / or condition comprises a fibrotic disease, an inflammatory disease, or a tumor.

[0155] On the other hand, the present invention provides the multispecific molecule, the drug combination, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition for preventing, alleviating and / or treating a disease or condition.

[0156] In certain embodiments, the disease and / or disorder comprises an IL-11-associated disease.

[0157] In certain embodiments, the disease and / or condition comprises a TSLP-associated disease.

[0158] In certain embodiments, the disease and / or condition comprises a fibrotic disease, an inflammatory disease, or a tumor.

[0159] On the other hand, the multispecific molecule, the drug combination, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition provided in the present application have better effects than IL-11 and / or TSLP antibodies.

[0160] In certain embodiments, the IL-11 antibody may have a sequence as described in CN113056481A.

[0161] In certain embodiments, the IL-11 antibody comprises HCDR3, HCDR2 and HCDR1 of the heavy chain variable region VH; wherein the HCDR1 can be selected from the sequences shown in SEQ ID NOs: 64, 65, 95, 104, 110, and 116, the HCDR2 can be selected from the sequences shown in SEQ ID NOs: 66, 72, 73, 74, 96, 105, 111, and 117, and the HCDR3 can be selected from the sequences shown in SEQ ID NOs: 67, 68, 97, 106, 112, and 118.

[0162] In certain embodiments, the IL-11 antibody comprises LCDR3, LCDR2 and LCDR1 of the light chain variable region VL; wherein the LCDR1 can be selected from the sequences shown in SEQ ID NOs: 69, 70, 71, 98, 101, 107, 113, and 119, the LCDR2 can be selected from the sequences shown in SEQ ID NOs: 72, 73, 99, 102, 108, 114, and 120, and the LCDR3 can be selected from the sequences shown in SEQ ID NOs: 74-94, 100, 103, 109, 115, and 121.

[0163] In certain embodiments, the IL-11 antibody comprises an antibody heavy chain variable region VH, and the VH may comprise an amino acid sequence as shown in SEQ ID NOs: 122-134.

[0164] In certain embodiments, the IL-11 antibody comprises an antibody light chain variable region VL, and the VL may comprise an amino acid sequence as shown in SEQ ID NOs: 135-177.

[0165] In certain embodiments, the TSLP antibody may be Tezepelumab.

[0166] In certain embodiments, the TSLP antibody comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 178, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 179, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 180.

[0167] In certain embodiments, the TSLP antibody comprises LCDR3, LCDR2 and LCDR1 of the light chain variable region VL; wherein the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 181, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 182, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 183.

[0168] In certain embodiments, the TSLP antibody comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:184.

[0169] In certain embodiments, the TSLP antibody comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:185.

[0170] In certain embodiments, the TSLP antibody comprises an antibody heavy chain constant region.

[0171] In certain embodiments, the TSLP antibody comprises an antibody light chain constant region.

[0172] In certain embodiments, the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 186, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 187, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 188.

[0173] In certain embodiments, the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 189, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 190, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 191.

[0174] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:192.

[0175] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:193.

[0176] In certain embodiments, the effect may be a better affinity for IL-11 and / or TSLP.

[0177] In certain embodiments, the effect may be an inhibitory effect on IL-11 and / or TSLP.

[0178] In certain embodiments, the effect may be a better therapeutic effect on diseases associated with IL-11 and / or TSLP.

[0179] In certain embodiments, the effect may be an anti-fibrotic effect.

[0180] In certain embodiments, the effect may be an effect of inhibiting the production of TARC (thymus and activation-regulated chemokine).

[0181] In certain embodiments, the effect may be an anti-inflammatory effect.

[0182] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0183] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0184] FIG1A shows the structures of antibodies I700019, I700020, and I700021 described in this application.

[0185] FIG1B shows the structures of antibodies I700022, I700023, and I700024 described in this application.

[0186] FIG2A shows the reducing and non-reducing SDS-PAGE images of the TSLP / IL-11 bispecific antibodies I700019, I700020, and I700021 described in the present application.

[0187] FIG2B shows the reducing and non-reducing SDS-PAGE images of the TSLP / IL-11 bispecific antibodies I700022, I700023, and I700024 described in the present application.

[0188] FIG3 shows the flow cytometry binding results of the TSLP antibody described in this application and BaF3-huTSLP-3G11 cells.

[0189] FIG4 shows the results of the TSLP antibody described in the present application blocking the binding of TSLP to the receptor TSLPR / IL7Ra complex.

[0190] FIG5 shows the results of the TSLP antibody described in the present application blocking the H_TSLP reporter cell line cell activation caused by TSLP.

[0191] FIG6 shows that the IL-11 antibody described in the present application blocks IL-11-mediated activation of hIL11Effector Reporter Cells.

[0192] FIG7 shows the result of the human TSLP antibody described in the present application blocking the production of TARC by PBMC stimulated by TSLP.

[0193] FIG8 shows the detection results of the inhibitory effect of TSLP / IL-11 bispecific antibody on cell fibrosis activity.

[0194] FIG9 shows the detection results of TSLP / IL-11 bispecific antibody blocking TSLP and IL-11-induced TH2 pro-inflammatory effect. DETAILED DESCRIPTION

[0195] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0196] Definition of terms

[0197] In this application, the term "multispecific molecule" generally refers to an antibody having a variable region that recognizes more than one epitope on one or more antigens. Multispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains (such as monoclonal antibodies, chimeric antibodies, humanized antibodies and fully human antibodies), antibody fragments (such as Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and dAb), diabodies, bispecific diabodies and triabodies, and antibody fragments that have been covalently or non-covalently linked. In certain embodiments, a multispecific antibody can be a "bispecific antibody" that recognizes two different epitopes on the same or different antigens.

[0198] In this application, the term "IL-11" generally refers to a stromal cell-derived cytokine that belongs to the IL-6 superfamily. The term "IL-11" encompasses "full-length," unprocessed IL-11 as well as any form of IL-11 produced by cell processing. In this application, the term "IL-11" includes mutants, fragments, variants, isoforms, and homologs thereof.

[0199] As used herein, the term "TSLP," also known as "thymic stromal lymphopoietin," is an interleukin-7 (IL-7)-like cytokine. Mature hTSLP consists of 131 amino acid residues and exhibits a typical four-stranded α-helical bundle structure. The term "TSLP" encompasses "full-length," unprocessed TSLP, as well as any form of TSLP produced by cellular processing. As used herein, the term "TSLP" encompasses full-length, wild-type TSLP, as well as its mutants, fragments, variants, isoforms, and homologs.

[0200] In this application, the term "drug combination" generally refers to a combination comprising at least two active ingredients / therapeutic agents. In some embodiments, each active ingredient / therapeutic agent can be prepared as an independent formulation (solid, liquid, gel, etc.), in some embodiments, each active ingredient / therapeutic agent can be present in different containers, and can also be formulated into a desired formulation simultaneously or separately with a suitable carrier when needed; in some embodiments, each active ingredient / therapeutic agent can be from different sources; in some embodiments, each active ingredient / therapeutic agent can be present in the form of a mixture.

[0201] In this application, the term "isolated" generally refers to a substance obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been altered, or that the substance has been separated from its natural environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal and has not been separated is considered isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0202] In the present application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained by artificial means from a natural state. The "isolated antigen-binding protein" may comprise a portion that binds to an antigen and, optionally, a framework or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding portion to the antigen. The antigen-binding protein may comprise, for example, a protein framework region (FR) derived from an antibody or an alternative protein framework region or an artificial framework region having a transplanted CDR or CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions comprising, for example, mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein and fully synthetic framework regions comprising, for example, biocompatible polymers. Examples of antigen binding proteins include, but are not limited to, human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, dAb, IgD antibody; IgE antibody; IgM antibody; IgG1 antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody and fragments thereof.

[0203] In this application, the term "CDR", also known as "complementarity determining region", generally refers to a region in an antibody variable domain whose sequence is highly variable and / or forms a structurally defined loop. Typically, an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). In certain embodiments, naturally occurring camel antibodies consisting only of heavy chains can function normally and stably in the absence of light chains. Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat / Chothia, etc. These coding systems are known in the art. For example, the CDRs of the multispecific molecule can be divided according to the IMGT numbering system. For example, the CDRs of the multispecific molecule can be divided according to the Kabat numbering system. For example, the CDRs of the multispecific molecule can be divided according to the Chothia numbering system. For example, in the multispecific molecule, the CDRs of the first binding domain / antibody capable of targeting IL-11 can be divided according to the Kabat numbering system. For example, in the multispecific molecule, the CDRs of the second binding domain / antibody capable of targeting TSLP can be divided according to the IMGT numbering system.

[0204] In this application, the term "FR" generally refers to the more highly conserved portion of an antibody variable domain, which is referred to as a framework region. Typically, the variable domains of natural heavy and light chains each comprise four FR regions, i.e., four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).

[0205] In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy chain and / or light chain. The variable domains of the heavy and light chains may be referred to as "V H ” and “V L ” (or “VH” and “VL”, respectively). These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0206] In this application, the term "VHH" generally refers to an antibody comprising the variable antigen-binding domain of a heavy chain antibody. VHHs may also be referred to as nanobodies (Nb), heavy chain antibodies, and / or single domain antibodies. For example, the VHH may bind to IL-11. For example, the VHH may be specific for IL-11.

[0207] Throughout this application, the terms "polypeptide molecule," "polypeptide," and "peptide" are used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two covalently linked moieties. Each moiety can be a polypeptide with a distinct property. This property can be a biological property, such as in vitro or in vivo activity. It can also be a simple chemical or physical property, such as binding to a target molecule or catalysis of a reaction. The two moieties can be directly linked by a single peptide bond or through a peptide linker.

[0208] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length in isolated form, either deoxyribonucleotides or ribonucleotides, or analogs thereof, isolated from their natural environment or artificially synthesized.

[0209] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can be used to transform, transduce, or transfect a host cell, allowing the genetic material elements it carries to be expressed in the host cell. For example, vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Types of animal viruses used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. Vectors may also include components that assist in their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0210] In this application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been a recipient of a subject's plasmid or vector, including nucleic acid molecules of the present invention or vectors of the present invention. Cells may include the offspring of a single cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original mother cell (in the form of total DNA complement or in the genome). Cells may include cells transfected in vitro with the vectors described herein. Cells may be bacterial cells (e.g., Escherichia coli), yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells. In certain embodiments, cells are mammalian cells. In certain embodiments, mammalian cells are HEK293 cells.

[0211] In this application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or condition. The pharmaceutical composition may comprise the isolated antigen-binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and / or the cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also comprise a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes, but is not limited to, liquid, frozen and lyophilized compositions.

[0212] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, such as sodium; and / or nonionic surfactants.

[0213] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0214] In the present application, the proteins, polypeptides and / or amino acid sequences involved should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the proteins or polypeptides.

[0215] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to the CD73 protein). For example, the functional variant may comprise a protein or polypeptide having an amino acid change by at least 1, for example, 1-30, 1-20 or 1-10, for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0216] In the present application, the homolog can be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to a CD73 protein).

[0217] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology percentage ratio " in the following manner: two sequences to be compared are compared in a comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, the number of matching positions is divided by the total number of positions (that is, window size) in the comparison window, and the result is multiplied by 100, to produce the sequence homology percentage ratio.Comparison carried out in order to determine the sequence homology percentage ratio can be realized by various ways known in the art.

[0218] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0219] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0220] Detailed Description of the Invention

[0221] IL-11 Antibody / Isolated IL-11 Antigen Binding Protein

[0222] In one aspect, the present application provides IL-11 antibodies / isolated IL-11 antigen binding proteins.

[0223] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise at least one CDR in the antibody heavy chain variable region VH, the amino acid sequence of which is shown in SEQ ID NO: 4.

[0224] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise HCDR3, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 7.

[0225] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise HCDR2, and the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 6.

[0226] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise HCDR1, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO:5.

[0227] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise HCDR3, HCDR2 and HCDR1, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 7, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 5.

[0228] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise at least one CDR in the VH described herein. The CDR may be obtained by dividing according to any division method.

[0229] For example, the CDRs of the IL-11 antibody / isolated IL-11 antigen binding protein can be divided according to Kabat.

[0230] In this application, the antibody framework region FR refers to the portion of the antibody variable region that is present between the more divergent (i.e., hypervariable) CDRs. Such framework regions are typically referred to as frameworks 1 to 4 (FR1, FR2, FR3, and FR4) and provide a framework for presenting six CDRs (three from the heavy chain and three from the light chain) in three-dimensional space to form an antigen-binding surface.

[0231] In the present application, the IL-11 antibody / isolated IL-11 antigen binding protein may comprise H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the amino acid sequence of the H-FR1 is as shown in SEQ ID NO: 8.

[0232] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the amino acid sequence of the H-FR2 is shown in SEQ ID NO:9.

[0233] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the amino acid sequence of the H-FR3 is shown in SEQ ID NO:10.

[0234] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the amino acid sequence of the H-FR4 is shown in SEQ ID NO:11.

[0235] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise a variable region VH, and the amino acid sequence of the VH is shown in SEQ ID NO:4.

[0236] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.

[0237] For example, the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0238] For example, the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a nanobody and / or a heavy chain antibody.

[0239] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise VHH, and the amino acid sequence of VHH is shown in SEQ ID NO: 4.

[0240] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the isolated antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region. In certain embodiments, the isolated antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region and subjected to amino acid mutations.

[0241] In the present application, the IL-11 antibody / isolated IL-11 antigen binding protein may be a sequence known in the prior art.

[0242] In the present application, the IL-11 antibody / isolated IL-11 antigen binding protein may be the sequence described in CN113056481A.

[0243] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein may comprise HCDR3, HCDR2 and HCDR1 of the heavy chain variable region VH; wherein the HCDR1 may be selected from the sequences shown in SEQ ID NOs: 64, 65, 95, 104, 110, and 116, the HCDR2 may be selected from the sequences shown in SEQ ID NOs: 66, 72, 73, 74, 96, 105, 111, and 117, and the HCDR3 may be selected from the sequences shown in SEQ ID NOs: 67, 68, 97, 106, 112, and 118.

[0244] In the present application, the IL-11 antibody / isolated IL-11 antigen binding protein may comprise LCDR3, LCDR2 and LCDR1 of the light chain variable region VL; wherein, the LCDR1 may be selected from the sequences shown in SEQ ID NOs: 69, 70, 71, 98, 101, 107, 113, 119, the LCDR2 may be selected from the sequences shown in SEQ ID NOs: 72, 73, 99, 102, 108, 114, 120, and the LCDR3 may be selected from the sequences shown in SEQ ID NOs: 74-94, 100, 103, 109, 115, 121.

[0245] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein comprises an antibody heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NOs: 122-134.

[0246] In the present application, the IL-11 antibody / isolated IL-11 antigen-binding protein comprises an antibody light chain variable region VL, and the VL may comprise an amino acid sequence as shown in SEQ ID NOs: 135-177.

[0247] In the present application, the IL-11 antibody / isolated IL-11 antigen binding protein comprises an antibody heavy chain constant region.

[0248] In the present application, the IL-11 antibody / isolated IL-11 antigen binding protein comprises an antibody light chain constant region.

[0249] In addition, it should be noted that the isolated antigen-binding proteins described herein may include heavy chain and / or light chain sequences that have one or more conservative sequence modifications thereto. The so-called "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are the replacement of amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described herein can be replaced with other amino acid residues from the same side chain group. Those skilled in the art will appreciate that some conservative sequence modifications will not eliminate antigen binding.

[0250] TSLP Antibody / Isolated TSLP Antigen Binding Protein

[0251] In another aspect, the present application provides TSLP antibodies / isolated TSLP antigen binding proteins.

[0252] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein comprises HCDR3, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 21.

[0253] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may further comprise HCDR2, the amino acid sequence of which is shown in SEQ ID NO: 20.

[0254] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may further comprise HCDR1, the amino acid sequence of which is shown in SEQ ID NO:19.

[0255] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise HCDR3, HCDR2, and HCDR1. For example, the amino acid sequence of HCDR3 of the isolated antigen-binding protein is shown in SEQ ID NO: 21, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 20, and the amino acid sequence of HCDR1 is shown in SEQ ID NO: 19.

[0256] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise LCDR3, the amino acid sequence of which is shown in SEQ ID NO: 24.

[0257] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may further comprise LCDR2, the amino acid sequence of which is shown in SEQ ID NO: 23 (GAR).

[0258] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may further comprise LCDR1, the amino acid sequence of which is shown in SEQ ID NO: 22.

[0259] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise LCDR3, LCDR2, and LCDR1. For example, the amino acid sequence of LCDR3 of the isolated antigen-binding protein is shown in SEQ ID NO: 24, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 23 (GAR), and the amino acid sequence of LCDR1 is shown in SEQ ID NO: 22.

[0260] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1. For example, the amino acid sequence of HCDR3 of the isolated antigen-binding protein described herein is shown in SEQ ID NO:21, the amino acid sequence of HCDR2 is shown in SEQ ID NO:20, the amino acid sequence of HCDR1 is shown in SEQ ID NO:19, the amino acid sequence of LCDR3 is shown in SEQ ID NO:24, the amino acid sequence of LCDR2 is shown in SEQ ID NO:23 (GAR), and the amino acid sequence of LCDR1 is shown in SEQ ID NO:22.

[0261] In the present application, the TSLP antibody / isolated TSLP antigen binding protein may comprise H-FR1, the C-terminus of the H-FR1 may be directly or indirectly connected to the N-terminus of the HCDR1, and the amino acid sequence of the H-FR1 may be as shown in SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.

[0262] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise H-FR2, which may be located between the HCDR1 and the HCDR2, and the amino acid sequence of the H-FR2 may be as shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31.

[0263] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise H-FR3, the H-FR3 may be located between the HCDR2 and the HCDR3, and the amino acid sequence of the H-FR3 may be as shown in SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36.

[0264] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise H-FR4, the N-terminus of the H-FR4 may be connected to the C-terminus of the HCDR3, and the amino acid sequence of the H-FR4 may be as shown in SEQ ID NO:37 and SEQ ID NO:38.

[0265] In the present application, the TSLP antibody / isolated TSLP antigen binding protein may comprise L-FR1, the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1, and the amino acid sequence of the L-FR1 may be as shown in SEQ ID NO:39 and SEQ ID NO:40.

[0266] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise L-FR2, which may be located between the LCDR1 and the LCDR2. The amino acid sequence of the L-FR2 may be as shown in SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43.

[0267] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise L-FR3, which may be located between the LCDR2 and the LCDR3, and the amino acid sequence of the L-FR3 may be as shown in SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47.

[0268] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise L-FR4, the N-terminus of the L-FR4 may be connected to the C-terminus of the LCDR3, and the amino acid sequence of the L-FR4 may be as shown in SEQ ID NO:48.

[0269] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise a VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

[0270] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise a VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:57.

[0271] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise the VH and VL.

[0272] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:53, and the VL may comprise the amino acid sequence shown in SEQ ID NO:57.

[0273] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:49, and the VL may comprise the amino acid sequence shown in SEQ ID NO:54.

[0274] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:49, and the VL may comprise the amino acid sequence shown in SEQ ID NO:55.

[0275] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:49, and the VL may comprise the amino acid sequence shown in SEQ ID NO:56.

[0276] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:50, and the VL may comprise the amino acid sequence shown in SEQ ID NO:54.

[0277] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:50, and the VL may comprise the amino acid sequence shown in SEQ ID NO:55.

[0278] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:50, and the VL may comprise the amino acid sequence shown in SEQ ID NO:56.

[0279] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:51, and the VL may comprise the amino acid sequence shown in SEQ ID NO:54.

[0280] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:51, and the VL may comprise the amino acid sequence shown in SEQ ID NO:55.

[0281] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:51, and the VL may comprise the amino acid sequence shown in SEQ ID NO:56.

[0282] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:52, and the VL may comprise the amino acid sequence shown in SEQ ID NO:54.

[0283] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:52, and the VL may comprise the amino acid sequence shown in SEQ ID NO:55.

[0284] For example, the VH may comprise the amino acid sequence shown in SEQ ID NO:52, and the VL may comprise the amino acid sequence shown in SEQ ID NO:56.

[0285] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise at least one CDR of the VH described herein. In the present application, the isolated antigen-binding protein may comprise at least one CDR of the VL described herein. The CDRs may be obtained by dividing according to any division method.

[0286] For example, the CDRs of the TSLP antibody / isolated TSLP antigen binding protein can be divided by IMGT.

[0287] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3 in the VH described herein, and the VH may comprise an amino acid sequence selected from SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

[0288] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise LCDR1, LCDR2 and LCDR3 in the VL described herein, and the VL may comprise an amino acid sequence selected from SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57.

[0289] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may include an antibody heavy chain constant region. The antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region.

[0290] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may include an antibody light chain constant region. The antibody light chain constant region may be derived from a human Igκ constant region.

[0291] In certain embodiments, the antigen-binding fragment may include a Fab, a Fab', an Fv fragment, a F(ab')2, a F(ab)2, a scFv, a di-scFv and / or a dAb.

[0292] In certain embodiments, the antibody may include a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a nanobody and / or a heavy chain antibody.

[0293] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may be an antibody known in the prior art.

[0294] In the present application, the TSLP antibody / isolated TSLP antigen binding protein is Tezepelumab.

[0295] In the present application, the TSLP antibody / isolated TSLP antigen binding protein may comprise HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 178, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 179, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 180.

[0296] In the present application, the TSLP antibody / isolated TSLP antigen binding protein may comprise LCDR3, LCDR2 and LCDR1 of the light chain variable region VL; wherein the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 181, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 182, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 183.

[0297] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:184.

[0298] In the present application, the TSLP antibody / isolated TSLP antigen-binding protein may comprise an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:185.

[0299] In the present application, the TSLP antibody / isolated TSLP antigen binding protein may comprise an antibody heavy chain constant region.

[0300] In the present application, the TSLP antibody / isolated TSLP antigen binding protein may comprise an antibody light chain constant region.

[0301] In certain embodiments, the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 186, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 187, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 188.

[0302] In certain embodiments, the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; wherein the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 189, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 190, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 191.

[0303] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:192.

[0304] In certain embodiments, the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:193.

[0305] Multispecific molecules

[0306] In another aspect, the present application provides a multispecific molecule comprising a first binding domain capable of binding to IL-11. In the present application, the first binding domain of the multispecific antibody may comprise the IL-11 antibody / isolated IL-11 antigen binding protein described above.

[0307] In the present application, the multispecific antibody may further include a second targeting moiety, which may include a second binding domain capable of binding to TSLP. In the present application, the second binding domain of the multispecific antibody may include the TSLP antibody / isolated TSLP antigen-binding protein described above.

[0308] In the present application, the first binding domain capable of binding to IL-11 and the second binding domain capable of binding to TSLP comprise Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv, and / or dAb. For another example, the first binding domain capable of binding to IL-11 and the second binding domain capable of binding to TSLP are selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a single-chain antibody, a nanobody, and / or a heavy-chain antibody.

[0309] In the present application, the multispecific molecule may be a bispecific antibody. A bispecific antibody (bsAb) may be a non-natural antibody that can simultaneously target two different antigens or proteins, block one or more signaling pathways, and stimulate a specific immune response. Its specificity and bifunctionality play an increasingly important role in tumor immunotherapy, and it has become a research hotspot in the field of antibody engineering for treating tumors in the world today. Studies have shown that bispecific antibodies mainly mediate the killing of tumors by immune cells in tumor immunotherapy; they combine dual targets, block signaling pathways, and exert unique or overlapping functions, which can effectively prevent drug resistance; they have strong specificity, targeting, and reduced off-target toxicity; they effectively reduce treatment costs and other advantages. Therefore, the use of bispecific antibody drugs can reduce the chance of tumor cell escape, eliminate tumor cells, and improve efficacy. Bispecific antibodies can be prepared through dual hybridoma cells, chemical coupling, recombinant genes, etc. Among them, recombinant gene technology has strong flexibility in terms of binding sites and production. The structures of bispecific antibodies mainly include bispecific antibodies with Fc fragments (IgG-like bispecific antibodies, which have Fc-mediated effector functions) and bispecific antibodies without Fc fragments (non-IgG-like bispecific antibodies, which exert their effects through antigen binding and have advantages such as small molecular weight and low immunogenicity).

[0310] In the present application, in the multispecific molecule, the first binding domain capable of targeting IL-11 and the second binding domain targeting TSLP can be directly or indirectly connected. For example, the C-terminus of the first binding domain capable of targeting IL-11 is connected to the N-terminus of the second binding domain targeting TSLP. For example, the C-terminus of the first binding domain capable of targeting IL-11 is connected to the N-terminus of the heavy chain variable region VH of the second binding domain targeting TSLP. For example, the N-terminus of the first binding domain capable of targeting IL-11 is connected to the C-terminus of the second binding domain targeting TSLP. For example, the N-terminus of the first binding domain capable of targeting IL-11 is connected to the C-terminus of the heavy chain constant region of the second binding domain targeting TSLP. For example, the first binding domain capable of targeting IL-11 is connected to the second binding domain targeting TSLP via a linker. Wherein, the linker may comprise a peptide linker. For example, the linker may comprise an amino acid sequence of (GGGGS)n, wherein n is any positive integer between 0 and 10. For example, the linker comprises the amino acid sequence shown as GGGGSGGGGS (SEQ ID NO: 3).

[0311] In the present application, the multispecific molecule may comprise two first binding domains capable of targeting IL-11, wherein the two first binding domains capable of targeting IL-11 are directly or indirectly linked to the N-termini of the two heavy chains of the second binding domain targeting TSLP. For example, the two first binding domains capable of targeting IL-11 are directly or indirectly linked to the C-termini of the two heavy chains of the second binding domain targeting TSLP. For example, the two first binding domains capable of targeting IL-11 are directly or indirectly linked to the N-termini of the two heavy chains of the second binding domain targeting TSLP.

[0312] In the present application, the multispecific molecule may comprise a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain may comprise a heavy chain comprising the first binding domain capable of targeting IL-11 and the second binding domain capable of targeting TSLP, and the second polypeptide chain may comprise a light chain comprising the second binding domain. In the present application, the multispecific molecule may comprise two first polypeptide chains and two second polypeptide chains. For example, the N-terminus of the heavy chain comprising the first binding domain capable of targeting IL-11 and the second binding domain capable of targeting TSLP of the first polypeptide chain is directly or indirectly connected. For example, the C-terminus of the heavy chain comprising the first binding domain capable of targeting IL-11 and the second binding domain capable of targeting TSLP of the first polypeptide chain is directly or indirectly connected. For example, the heavy chain comprising the first binding domain capable of targeting IL-11 and the second binding domain capable of targeting TSLP of the first polypeptide chain is connected by a linker. For example, the first polypeptide chain may comprise an amino acid sequence selected from SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63, or an IL-11 antibody / antigen-binding fragment known in the art. For example, the second polypeptide chain may comprise an amino acid sequence as set forth in SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, or a TSLP antibody / antigen-binding fragment known in the art.

[0313] In the present application, the structure of the multispecific molecule can be VHH-IgG, that is, the N-terminus or C-terminus of both heavy chains of an IgG antibody targeting the TSLP antigen are connected to the VHH fragment of another nanobody targeting the IL-11 antigen. In the present application, the structure of the multispecific molecule can be a configuration in which the VHH is connected to the N-terminus of the IgG antibody, or a configuration in which the VHH is connected to the C-terminus of the IgG antibody heavy chain. For example, the structure of the multispecific molecule can be as shown in Figures 1A and 1B. For example, the structure of the multispecific molecule can be as shown in antibodies I700019, I700020, I700021, I700022, I700023, and I700024.

[0314] Drug combinations

[0315] In another aspect, the present application provides a pharmaceutical combination comprising a first antibody that targets IL-11 and a second antibody that targets TSLP.

[0316] In the present application, the first antibody capable of targeting IL-11 may include the IL-11 antibody / isolated IL-11 antigen-binding protein described above, or may be one or more of the currently known IL-11 antibodies, such as those described in WO2023 / 143556A1 or CN113056481A.

[0317] In the present application, the second antibody capable of targeting TSLP may include the TSLP antibody / isolated TSLP antigen-binding protein described above, or may be one or more currently known TSLP antibodies, such as Tezepelumab or the antibody disclosed in patent CN102782149B.

[0318] In the present application, the pharmaceutical combination may refer to a product produced by the mixing or combination of more than one active ingredient, and includes fixed and non-fixed combinations of active ingredients. A fixed combination may refer to an active ingredient (e.g., the IL-11 antibody, TSLP antibody, and multispecific molecule described herein) and one or more combination partners being administered to a patient simultaneously in the form of a single entity or dosage. A non-fixed combination may refer to an active ingredient and one or more combination partners being administered to a patient simultaneously, jointly, or sequentially (without a specific time limit) as separate entities, wherein such administration provides therapeutically effective levels of the two compounds in the patient's body. The latter also applies to cocktail therapies, such as the administration of three or more active ingredients.

[0319] Isolated nucleic acid molecule(s), vector(s) and cells

[0320] In another aspect, the present application also provides one or more isolated nucleic acid molecules. The one or more nucleic acid molecules can encode the multispecific molecules described in the present application. For example, each of the one or more nucleic acid molecules can encode the entire multispecific molecule, or a portion thereof (e.g., a first binding domain targeting IL-11, a second binding domain targeting TSLP, an IL-11 antibody, a TSLP antibody, HCDR1-3, LCDR1-3, VL, VH, one or more of a light chain or a heavy chain).

[0321] The nucleic acid molecules described herein can be isolated. For example, they can be produced or synthesized by the following methods: (i) in vitro amplification, such as by polymerase chain reaction (PCR) amplification, (ii) by cloning and recombination, (iii) purification, such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesis, such as by chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0322] In the present application, nucleic acids encoding the multispecific antibodies can be prepared by various methods known in the art, including but not limited to, restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. For specific operations, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.

[0323] In another aspect, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. Each vector may contain one or more of the nucleic acid molecules described herein. Furthermore, the vectors may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Furthermore, the vectors may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequences are adjustable elements. The specific structure of the expression control sequences may vary depending on the function of the species or cell type, but typically include 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as a TATA box, a capping sequence, a CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence functionally linked to a nucleic acid for transcriptional control. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. In the present application, suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6RNA promoter, CMV promoter and artificial hybrid promoters thereof (such as CMV), wherein a certain portion of the promoter may be fused to a certain portion of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene promoters, which may or may not include other introns. One or more nucleic acid molecules described herein may be operably connected to the expression control element. The vector may include, for example, a plasmid, a cosmid, a virus, a phage or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

[0324] In another aspect, the application provides cells, which may include one or more nucleic acid molecules and / or one or more vectors described herein. In certain embodiments, each or each cell may include one or more nucleic acid molecules or vectors described herein. In certain embodiments, each or each cell may include multiple (e.g., 2 or more) or multiple (e.g., 2 or more) nucleic acid molecules or vectors described herein. For example, the vectors described herein may be introduced into the cells, such as eukaryotic cells, such as cells from plants, fungi or yeast cells, etc. The vectors described herein may be introduced into the cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0325] Preparation method

[0326] In another aspect, the present application provides a method for producing the multispecific molecule. The method may include culturing the cells described herein under conditions that allow for expression of the multispecific molecule. For example, the method may be performed using an appropriate culture medium, at an appropriate temperature and for an appropriate time, as is known to those skilled in the art.

[0327] Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. In the present application, the antibody is an IgG antibody, and the IgG1 subtype is used. Similarly, any class of light chain can be used in the compounds and methods herein. Specifically, kappa, lambda chains, or variants thereof can be used in the compounds and methods herein.

[0328] The sequences of the DNA molecules of the multispecific molecules or fragments thereof of the present invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form a single-chain antibody.

[0329] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated cells by conventional methods.

[0330] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then connecting them, very long fragments of sequence can be obtained. The nucleic acid molecule can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0331] The present application also relates to vectors comprising the above-mentioned appropriate nucleic acid molecules and appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable protein expression. The host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. For example, animal cells can include (but are not limited to): CHO-S, CHO-K1, and HEK-293 cells.

[0332] The steps of transforming cells with recombinant DNA described herein can be performed using techniques well known in the art. The resulting transformants can be cultured using conventional methods, and the transformants express the polypeptides encoded by the nucleic acid molecules of the present application. Depending on the cells used, the cells are cultured in conventional culture medium under appropriate conditions. Typically, the transformed cells are cultured under conditions suitable for expression of the multispecific molecules of the present application. The multispecific antibodies of the present application can then be purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are conventional separation and purification methods well known to those skilled in the art.

[0333] The resulting monoclonal antibodies or multispecific molecules can be identified using conventional means. For example, the binding specificity of the monoclonal antibodies or multispecific antibodies can be determined using immunoprecipitation or in vitro binding assays such as flow cytometry (FACS), radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

[0334] Pharmaceutical composition

[0335] In another aspect, the present application also provides a composition. In some cases, the composition can be a pharmaceutical composition comprising the multispecific antibody of the present application and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium. The formulated pharmaceutical composition can be administered via conventional routes.

[0336] The pharmaceutical compositions described herein can be used to directly bind to IL-11 or TSLP protein molecules and thus can be used to prevent and treat diseases associated with IL-11 or TSLP. The pharmaceutical compositions described herein can contain a safe and effective amount of the antigen-binding protein described herein and a pharmaceutically acceptable adjuvant (which may include a carrier or excipient). The pharmaceutical formulation should be compatible with the route of administration.

[0337] The multispecific antibodies or pharmaceutical compositions described herein can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the etiology of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to medical practitioners.

[0338] Uses and methods

[0339] On the other hand, the present application also provides a method for blocking the binding of TSLP to the receptor TSLPR / IL7Ra complex, which comprises administering the multispecific molecule and / or the drug combination.

[0340] On the other hand, the present application also provides a method for inhibiting the production of TARC (thymus and activation-regulated chemokine), which comprises administering the multispecific antibody and / or the drug combination.

[0341] In another aspect, the present application provides uses of the multispecific molecules, pharmaceutical compositions, nucleic acid molecules, vectors, cells, and / or pharmaceutical compositions in the preparation of medicaments. The medicaments are used to prevent, alleviate, and / or treat diseases or conditions, such as IL-11-related diseases, such as TSLP-related diseases. In some cases, the diseases or conditions may be fibrotic diseases, inflammatory diseases, or tumors.

[0342] In another aspect, the present application provides a method for preventing, alleviating, and / or treating a disease or condition, comprising administering to a patient in need thereof the multispecific molecule, the drug combination, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition described herein. In some cases, the disease or condition may be an IL-11-related disease, a TSLP-related disease, a fibrotic disease, an inflammatory disease, or a tumor.

[0343] In another aspect, the present application provides multispecific molecules, drug combinations, nucleic acid molecules, vectors, cells, and / or pharmaceutical compositions for preventing, alleviating, and / or treating a disease or condition. In some cases, the disease or condition may be an IL-11-related disease, a TSLP-related disease, a fibrotic disease, an inflammatory disease, or a tumor.

[0344] Without intending to be bound by any theory, the following examples are merely intended to illustrate the multispecific molecules, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0345] Example

[0346] Example 1 Sequence Design of the Heavy and Light Chains of the TSLP / IL-11 Bispecific Antibody

[0347] The TSLP / IL-11 bispecific antibody in this application, in which the TSLP-targeting monoclonal antibody is derived from hybridoma technology, is obtained by immunizing mice with human TSLP protein, selecting the best mice to obtain spleen cells and fusing them with SP2 / 0 myeloma cells. After monoclonal screening and functional activity and affinity testing, candidate molecules are obtained and then humanized. In addition, the VHH nano single-chain antibody sequence targeting IL-11 is derived from alpacas immunized with human IL-11 protein and obtained through a phage display technology platform.

[0348] The structure of the TSLP / IL-11 bispecific antibody in this application belongs to VHH-IgG, that is, the N-terminus or C-terminus of the two heavy chains of an IgG antibody targeting the TSLP antigen are connected to the VHH fragment of another nanobody targeting the IL-11 antigen.

[0349] Among them, antibodies I700019, I700020, and I700021 are in a configuration in which VHH is linked to the N-terminus of an IgG antibody (as shown in FIG1A ). This type of bispecific antibody structure is abbreviated as IL11-VHH-Linker-TSLP-IgG. Antibodies I700022, I700023, and I700024 are in a configuration in which VHH is linked to the C-terminus of the IgG heavy chain (as shown in FIG1B ). This type of bispecific antibody structure is abbreviated as TSLP-IgG-Linker-IL-11-VHH.

[0350] The full-length heavy chain sequence of I700019, SEQ ID NO: 58:

[0351] The full-length heavy chain sequence of I700020, SEQ ID NO: 59:

[0352] The full-length heavy chain sequence of I700021, SEQ ID NO: 60:

[0353] Note: The underlined and bold parts are CDR regions

[0354] The full-length heavy chain sequence of I700022, SEQ ID NO: 61:

[0355] The full-length heavy chain sequence of I700023, SEQ ID NO: 62:

[0356] The full-length heavy chain sequence of I700024, SEQ ID NO: 63:

[0357] The full-length sequence of the light chain of I700019, I700021, I700022, and I700024, SEQ ID NO: 18:

[0358] I700020, full-length light chain sequence of I700023, SEQ ID NO: 17: Note: The underlined and bold parts are the CDR regions, as follows:

[0359] Example 2 Expression and purification of TSLP / IL-11 bispecific antibody

[0360] The expression vector obtained in Example 1 was amplified in E. coli, and sufficient plasmid was prepared using an endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., #DP117) for transient transfection to express the bispecific antibody. The host cells used for expression were CHO-S cells (Thermo Fisher Scientific, #R80007). The two heavy chain vectors and the light chain vector were mixed with polyetherimide (PEI, Polysciences, #24765-1) to form liposome complexes. The complexes were then transfected into CHO-S cells and cultured in an incubator for 5-7 days. The cell culture supernatant was collected by centrifugation and purified using a Protein A affinity chromatography column to obtain the bispecific antibody. Protein aggregation was further assessed by size exclusion chromatography.

[0361] The expression, purification, and testing of the TSLP / IL-11 bispecific antibodies are shown in Figures 2A and 2B and Table 1. The results demonstrate that the SEC purity of all six TSLP / IL-11 bispecific antibodies exceeded 98%. Reducing and non-reducing SDS-PAGE images revealed no degradation of either the heavy or light chains of the six TSLP / IL-11 bispecific antibodies, demonstrating excellent purity and stability.

[0362] Table 1. SEC purity data of TSLP / IL-11 bispecific antibodies

[0363] Example 3 Detection of biological activity of humanized anti-human TSLP antibodies

[0364] 3.1 Detection of the binding activity of anti-human TSLP antibodies to TSLP-positive cells by flow cytometry

[0365] BaF3-huTSLP-3G11 cells were collected and resuspended in 1% BSA / PBS, and the cells were diluted to 1×10 6 cells / mL, 20 μL was added to each well of a 96-well U-shaped plate (Greiner, 780201). Anti-TSLP antibody was diluted to 60 μg / mL with 1% BSA / PBS, and then diluted 3-fold to 10 concentrations, for a total of 11 concentration gradients.

[0366] Anti-TSLP antibodies of varying dilutions were added to a 96-well U-shaped plate at 20 μL per well and incubated at room temperature for 30 minutes. At the end of the incubation period, 100 μL of 1% BSA / PBS was added to the 96-well plate, followed by centrifugation at 300 g (Thermo Fisher Scientific, Sorvall ST16 / 16R) for 3 minutes to collect the cells. The supernatant was removed and the wash step repeated twice. Next, 20 μL of a 1:200 dilution of R-Phycoerythrin AffiniPure Goat Anti-Human IgG, Fcγ Fragment antibody was added to the 96-well plate and incubated at room temperature for 15 minutes. At the end of the incubation period, 100 μL of 1% BSA / PBS was added to the 96-well plate, followed by centrifugation at 300 g for 3 minutes to collect the cells. The supernatant was removed and this step repeated three times. 1% BSA / PBS was added to a 96-well U-shaped plate to resuspend the cells, with 100 μL per well, and the fluorescence signal value was read using a flow cytometer.

[0367] The experimental results are shown in FIG3 . The anti-TSLP antibody I700021 disclosed herein substantially maintains the binding activity of the parent antibody 900792.

[0368] 3.2 Detection of the blocking activity of anti-human TSLP antibodies on TSLP-TSLPR / IL7Ra interaction

[0369] Biotin-TSLP (Acro, TSP-H82EB-200) was diluted to 1.6 μg / mL with 1% BSA / PBS. Anti-TSLP antibody was diluted to 120 μg / mL with 1% BSA / PBS and then diluted 2-fold to 10 concentrations, for a total of 11 concentration gradients. Biotin-TSLP was mixed with anti-TSLP antibody at different concentrations at a ratio of 1:1 and 20 μL was added to each well of a 96-well U-shaped plate (Greiner, 780201). Baf3-huTSLP / IL7Ra-3H9-2 cells were collected, resuspended in 1% BSA / PBS, and diluted to 1×10 6cells / mL, 20 μL was added to each well of a 96-well U-shaped plate and incubated at room temperature for 30 minutes. At the end of incubation, 100 μL of 1% BSA / PBS was added to each well of the 96-well U-shaped plate, and the cells were centrifuged at 300 g for 3 minutes to collect the cells. The supernatant was removed and the above washing step was repeated once. Then, 20 μL of a 1:200 diluted SA-APC antibody (BD, 554067) was added to each well of the 96-well U-shaped plate, mixed thoroughly, and incubated at room temperature for 30 minutes. At the end of incubation, 100 μL of 1% BSA / PBS was added to each well of the 96-well U-shaped plate, and the cells were centrifuged at 300 g for 3 minutes to collect the cells. The supernatant was removed and this step was repeated once. The cells were resuspended in 100 μL of 1% BSA / PBS to each well of the 96-well U-shaped plate and the fluorescence signal was read using a flow cytometer.

[0370] The experimental results are shown in FIG4 , showing that the anti-TSLP antibody I700021 disclosed herein substantially maintains a blocking activity similar to that of the parent antibody 900792.

[0371] 3.3 Anti-human TSLP antibody blocks TSLP-induced H_TSLP reporter cell line activation

[0372] H_TSLP reporter cell line (GM-C15572 from Jiman Biotechnology) was collected and resuspended in RPMI 1640 medium (Gibco Cat#A1049101) containing 10% FBS (Gibco, 10099141C). The cells were diluted to 3×10 6 cells / mL, and 33 μL was added to each well of a 96-well white plate (Corning, 3599). The anti-TSLP antibody was diluted to 100 μg / mL with 10% FBS RPMI 1640 medium, and then diluted 10-fold to 8 concentrations, for a total of 9 concentration gradients. Antibodies with different concentration gradients were added to the 96-well white plate at 33 μL per well and incubated at room temperature for 1 hour. Human TSLP (R127A, R130A) protein (Acro, Cat#TSP-H52Ha) was diluted to 4 ng / mL with 10% FBS RPMI 1640 medium, and 33 μL was added to each well of the 96-well white plate. The 96-well plate was placed in a 37°C, 5% CO2 incubator and cultured for 23 hours. After the incubation, the 96-well white plate was removed and equilibrated at room temperature for 15 minutes. One-Glo reagent (Promega, E6120) was added to each well at 100 μL, and the plate was analyzed using a microplate reader (MD i3x) for full-wavelength fluorescence detection.

[0373] The experimental results are shown in Figure 5. The anti-human TSLP antibodies can inhibit the binding of Human TSLP (R127A, R130A) Protein to huTSLPR on the H_TSLP Reporter Cell Line reporter gene cell line, and the biological activity of the anti-human TSLP antibodies in inhibiting the binding of TSLP to TSLPR is comparable to that of the parental TSLP antibodies.

[0374] 3.4 Anti-IL-11 Antibody Blocks IL-11-Mediated Activation of hIL11 Effector Reporter Cells

[0375] The hIL11Effector Reporter Cell (Nanjing Kebai Biotechnology, CBP74114) reporter cell line is a luciferase reporter cell line constructed based on the JAK1-STAT3 intracellular signaling pathway. IL-11 binds to the IL-11R receptor and recruits gp130 to form a ternary complex, activating JAK. This phosphorylates STAT3, which then translocates to the nucleus and activates luciferase expression. Luminescence produced by luciferase-catalyzed luciferin substrates represents the activation of the signaling pathway and can therefore be used to evaluate the in vitro efficacy of IL-11-related drugs.

[0376] Collect hIL11Effector Reporter Cell cells, resuspend them in DMEM medium (Gibco, 11965092) containing 10% FBS (Gibco, 10099141C), dilute the cells to 1.2×106 cells / mL, add 50 μL per well to a 96-well white plate (Corning, 3599), and place the 96-well white plate in a 37°C, 5% CO2 incubator for overnight culture. Dilute the anti-IL-11 antibody to 120 μg / mL with DMEM medium containing 10% FBS, and then dilute it 3-fold to 10 concentrations, for a total of 11 concentration gradients. Add 25 μL of antibodies of different concentration gradients to each well of a 96-well white plate. Dilute Human IL-11 (novoprotein, C006) to 1 ng / mL with DMEM0 medium containing 10% FBS and add 25 μL per well to the above 96-well white plate. The 96-well plate was placed in a 37°C, 5% CO2 incubator for 6 hours. After incubation, the 96-well plate was removed and allowed to equilibrate at room temperature for 15 minutes. 100 μL of Bright-Glo reagent (Promega, E2620) was added to each well and the plate was analyzed using a microplate reader (MD i3x) for full-wavelength fluorescence detection.

[0377] The experimental results are shown in Figure 6. The IC50 of 700077 (anti-IL-11 monoclonal antibody) in blocking IL-11-induced hIL11Effector Reporter Cell activation is 10.41 μg / mL. Correspondingly, the IC50 of I700021 in blocking reporter gene cell line activation is 3.457 μg / mL. Therefore, the anti-IL-11 and TSLP bispecific antibody I700021 disclosed herein basically maintains a blocking activity similar to that of the parent IL-11 monoclonal antibody 700077.

[0378] 3.5 Anti-human TSLP antibody blocks TSLP-stimulated PBMC production of TARC

[0379] TSLP can stimulate dendritic cells or monocytes to produce the chemokine TARC (thymus and activation-regulated chemokine), thereby recruiting type 2 helper T cells and causing a type 2 inflammatory response. TARC is not produced in the resting state. In an in vitro culture system, dendritic cells and monocytes in PBMCs are stimulated with TSLP, and the amount of TARC produced is measured. Adding anti-TSLP antibodies to neutralize the added TSLP blocks TARC production, and the effect is dose-dependent with a gradient of anti-TSLP antibody concentrations, thus evaluating the neutralizing activity of the antibody.

[0380] Experimental method: Anti-TSLP antibody was diluted to 2 μg / mL, then diluted 2-fold to a total of 7 concentrations, and 50 μL / well was added to a 96-well plate for a final concentration of 0.5 μg / mL. TSLP was diluted to 10 ng / mL and added to the above wells at 50 μL / well. Mix and incubate for 30 minutes, for a final concentration of 2.5 ng / mL. Cells were revived, centrifuged at 500 g for 5 minutes, and the supernatant was removed. The cells were resuspended in complete medium and counted, and the density was adjusted to 2x10 6 cells / mL, 100 μL / well was added to the cell plate. The incubated antigen-antibody mixture was added to the cells at 100 μL / well. The cells were cultured at 37°C in a 5% CO2 incubator for 48 hours, and the supernatant was collected by centrifugation. TARC content in the cell culture supernatant was determined by ELISA.

[0381] The experimental results are shown in Figure 7 , showing that I700021 can effectively inhibit the level of CCL17 produced by PBMCs stimulated by TSLP, and the IC50 of the effect is comparable to that of the parental TSLP monoclonal antibody.

[0382] 3.6 I700021 Antibody Affinity Testing

[0383] 3.6.1 Affinity Detection of Antibody I700021 for Human, Monkey, Rat, and Mouse TSLP

[0384] Test materials and instruments:

[0385] Experimental method (SPR):

[0386] The sample chamber and flow cell temperature of the Biacore 8k were set to 25°C, and the data collection frequency was 10 Hz. Anti-His antibodies were amino-coupled and immobilized on a Series S Sensor Chip CM5 using the Amine Coupling Kit to produce the Anti-His Series CM5 chip. Human, monkey, rat, and mouse TSLP were captured using the Anti-His Series CM5 chip as ligands. Antibody samples were diluted with HBS-EP+, pH 7.4 buffer to a gradient concentration of 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.3125 nM, and 0.78 nM as analytes, and HBS-EP+, pH 7.4 buffer was used as the zero concentration for background subtraction. A multi-cycle kinetics method was used with an analyte flow rate of 30 μL / min, an association time of 120 s, a dissociation time of 600 s, and a regeneration time of 60 s with 10 mM glycine (pH 1.5) at 50 μL / min. Data were analyzed in 1:1 binding mode and Fit Local Kinetics mode.

[0387] Table 2. Affinity test results of I700021 antibody and human, monkey, rat and mouse TSLP Note: NB refers to No Binding

[0388] The affinity constants (KD(M)) of the humanized anti-TSLP / IL-11 bispecific monoclonal antibody I700021 of the present invention for human TSLP were 10 -11 It has a strong affinity for human antigens and an affinity for monkey TSLP of 10 -09 Furthermore, it did not bind to rat and mouse TSLP.

[0389] 3.6.2 Affinity Detection of I700021 Antibody to Human and Mouse IL-11

[0390] Test materials and instruments:

[0391] Experimental method (SPR):

[0392] The sample chamber and flow cell temperature of the Biacore T200 were set to 25°C, and the data collection frequency was 10 Hz. Antibody samples were captured to 300 RU on a Series S Protein A chip as ligands. Human and mouse IL-11 antigens were diluted with HBS-EP+, pH 7.4 buffer to a series of gradient concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.56 nM, 0.78 nM, and 0.39 nM as analytes, and HBS-EP+, pH 7.4 buffer was used as the zero concentration for background subtraction. A multi-cycle kinetic method was used with an analyte flow rate of 30 μL / min, an association time of 120 s, a dissociation time of 600 s, and a regeneration of 10 mM glycine (pH 1.5) at 50 μL / min for 60 s. Data were analyzed in 1:1 binding mode and Fit Local Kinetics mode.

[0393] Table 3. Affinity test results of I700021 antibody and human and mouse IL-11 Note: NB refers to No Binding

[0394] The affinity constants (KD(M)) of the humanized anti-TSLP / IL-11 bispecific monoclonal antibody I700021 of the present invention for human and mouse IL-11 were 10 -09 It has a strong affinity for human and mouse antigens.

[0395] 3.6.3 Affinity Detection of I700021 Antibody and Rat IL-11

[0396] Test materials and instruments:

[0397] Experimental method (BLI):

[0398] Kinetic analysis was performed using an SA sensor loaded with 5 μg / mL biotinylated sample as the ligand and a gradient-diluted Rat IL-11 solution as the analyzer. Kinetic constants were analyzed using a 1:1 binding model using a global fit and a temperature of 30°C, a shaking rate of 1000 rpm, an association of 360 s and a dissociation of 360 s.

[0399] Table 4. Affinity test results of I700021 antibody and rat IL-11 Note: NB refers to No Binding

[0400] The affinity constants (KD(M)) for various species showed that the affinity of the humanized anti-TSLP / IL-11 bispecific monoclonal antibody I700021 of the present invention for rat IL-11 was 10 -10 Order of magnitude, with extremely strong affinity for rat antigens.

[0401] 3.6.4 Affinity Detection of I700021 Antibody and Monkey IL-11

[0402] Test materials and instruments:

[0403] Experimental method (SPR):

[0404] The sample chamber and flow cell temperature of the Biacore 8k were set at 25°C, and the data collection frequency was 10 Hz. Cynomolgus IL-11 was immobilized by amino coupling on a Series S Sensor Chip CM5 using the Amine Coupling Kit. Monkey IL-11 was diluted in HBS-EP+, pH 7.4 buffer to a gradient concentration of 20 nM, 10 nM, 5 nM, 2.5 nM, and 1.25 nM as the analyte, with HBS-EP+, pH 7.4 buffer used as the zero concentration for background subtraction. A single-cycle kinetic method was used with an analyte flow rate of 30 μL / min, an association time of 120 s, a dissociation time of 600 s, and a regeneration time of 10 mM glycine (pH 1.5) at 50 μL / min for 60 s. Data were analyzed in 1:1 binding mode and Fit Local Kinetics mode.

[0405] Table 5. Affinity test results of I700021 antibody and monkey IL-11 Note: NB refers to No Binding

[0406] The affinity constant (KD(M)) results showed that the affinity of the humanized anti-TSLP / IL-11 bispecific monoclonal antibody I700021 of the present invention to monkey IL-11 was 10 -09 Order of magnitude, with extremely strong affinity for monkey antigens.

[0407] 3.7 Detection of the inhibitory effect of I700021 antibody on the fibrotic activity of MRC-5 cells

[0408] Transforming growth factor-β (TGF-β) can induce phenotypic changes in normal fibroblasts, leading to differentiation and production of extracellular matrix and other substances, thereby promoting further tissue fibrosis. Interleukin-11 (IL-11) has also been widely reported to be produced downstream of the TGF-β pathway and plays a key role in promoting fibrosis, generating inflammation and implicated in various allergic diseases, chronic inflammatory conditions, autoimmune diseases, and cancer. Following TGF-β induction, MRC-5 (human embryonic lung fibroblast) cells differentiate into myofibroblasts and upregulate the expression of fibrosis-related genes such as Fibronectin (FN) and α-SMA (encoded by the gene ACTA2). To investigate the potential efficacy of the I700021 antibody in inhibiting fibrosis-related diseases, we used MRC-5 cells as a vector and tested its ability to block the expression of the extracellular matrix component FN and the myofibroblast marker gene ACTA2 after TGF-β induction.

[0409] Experimental method: MRC-5 cells in the logarithmic growth phase in the culture flask were resuspended and 5×10 5 The number of cells was cultured in a six-well plate for 8 hours. After the cells adhered, the culture medium (without FBS) was replaced and starvation treatment was performed for 16 hours. The relevant reagents were prepared with complete culture medium containing FBS, with a final concentration of TGF-β of 10 ng / mL and a final concentration of antibody of 100 nM. The blank group (Ctrl group) was treated with complete culture medium, and each test condition was repeated three times. The original starvation culture medium was discarded from the starved cells, and the above-prepared solution was added to the cells. After incubation in an incubator for 24 hours, the cells were collected into RNase-free EP tubes and centrifuged to obtain a cell pellet. After total RNA was extracted according to the instructions of the RNeasy Micro kit, the extracted RNA was purified using a gDNA eraser. Reverse transcription was performed according to the instructions of the PrimeScriptTM RT reagent Kit to obtain cDNA. cDNA was amplified by PCR using Fast Start Universal SYBR Green Master (Rox) as a fluorescent marker, human GAPDH as a housekeeping gene, and primers for FN and ACTA2. Gene expression was expressed as fold change and calculated using the 2^-ΔΔCt formula, where each value was normalized to the mean of the control group.

[0410] The expression levels of fibrosis-related genes FN and ACTA2 in MRC-5 cells were used as evaluation criteria to detect the inhibitory abilities of antibody I700021, parental Anti-TSLP monoclonal antibody, and Anti-IL-11 monomer on TGF-β-induced fibrosis.

[0411] The experimental results, as shown in Figure 8, show that TGF-β induction significantly increased the expression levels of both genes, while the three tested antibodies significantly inhibited the expression of both genes. Furthermore, the bispecific antibody I700021 significantly inhibited the gene expression levels of ACTA2 and Fibronectin more strongly than the two parental monoclonal antibodies.

[0412] 3.8 Detection of the I700021 Antibody Blocking TSLP- and IL-11-Induced TH2 Proinflammatory Effect

[0413] This study aimed to evaluate the biological activity of the I700021 antibody on TSLP- and IL-11-induced Th2 cytokine release in a PBMC system. The study found that IL-11 promotes IL-33 production by PBMCs, while combined stimulation with TSLP and IL-33 more effectively induces PBMCs to produce Th2 cytokines, including IL-5 and IL-13. The I700021 antibody inhibits Th2 cytokine production by blocking the interaction of IL-11 and TSLP with PBMCs. The ability of the antibody to block TSLP- and IL-11-induced Th2 cytokine release was then evaluated.

[0414] Experimental method: PBMC cells were adjusted to a cell suspension density of 2×10 6 cells / mL, and plated into 96-well U-bottom plates at 50 μL / well, i.e., 1×10 cells / mL per well. 5 Cells. Human TSLP stock solution was diluted to a 40 ng / mL working solution in assay medium and added to the sample wells of the 96-well plate at the end of the incubation at a rate of 50 μL / well (final concentration 10 ng / mL). Human IL-11 stock solution was diluted to a 400 ng / mL working solution in assay medium and added to the sample wells of the 96-well plate at the end of the incubation at a rate of 50 μL / well (final concentration 100 ng / mL). The test antibody was diluted to 600 nM in assay medium (final concentration 150 nM). The 96-well U-bottom plate was then incubated in a 37°C, 5% CO2 incubator for 3 days. Anti-CD3 antibody stock solution was diluted to a 4 μg / mL working solution in assay medium and added to the washed cells. The cells were then incubated in a 37°C, 5% CO2 incubator for another day. 25 μL of the supernatant was collected for cytokine analysis. TH2 cytokines were detected using flow cytometry using the LEGENDplex kit. GraphPad Prism 8 software was used to plot the working concentration of the samples versus the converted TH2 cytokine concentration.

[0415] The experimental results, as shown in Figure 9, show that combined stimulation with TSLP and IL-11 can induce PBMCs to produce large amounts of TH2 cytokines (including IL-5, IL-13, and IL-4). Furthermore, compared to the two parental monoclonal antibodies, the bispecific antibody can produce even more TH2 cytokines (including IL-5, IL-13, and IL-4), demonstrating superior inhibitory efficacy.

Claims

1. A multispecific molecule comprising a first binding domain capable of targeting IL-11 and a second binding domain capable of targeting TSLP, said multispecific molecule having one or more of the following properties: (1) capable of binding to the TSLP protein with a KD value of 1×10 -9 M or lower, wherein the KD value is determined by surface plasmon resonance; (2) capable of binding to IL-11 protein with a KD value of 1×10 -9 M or lower, wherein the KD value is determined by surface plasmon resonance method; (3) capable of blocking the binding of TSLP to the receptor TSLPR / IL7Ra complex; and (4) capable of inhibiting the production of TARC (thymus and activation regulated chemokine).

2. The multispecific molecule according to claim 1, wherein the first binding domain capable of targeting IL-11 comprises an antibody or an antigen-binding fragment thereof.

3. The multispecific molecule according to any one of claims 1-2, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, VHH, and dAb fragment.

4. The multispecific molecule according to any one of claims 1-2, wherein the antibody is selected from the group consisting of: monoclonal antibody, single-chain antibody, chimeric antibody, multispecific antibody, humanized antibody, fully human antibody, nanobody, and / or heavy-chain antibody.

5. The multispecific molecule according to any one of claims 1-4, wherein the first binding domain capable of targeting IL-11 comprises HCDR3, HCDR2, and HCDR1 of the heavy chain variable region VH; wherein, The amino acid sequence of the HCDR3 is as shown in SEQ ID NO:7, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:6, and the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:

5.

6. The multispecific molecule according to any one of claims 1-5, wherein the first binding domain capable of targeting IL-11 comprises the heavy-chain variable region VH of an antibody, and the VH comprises the amino acid sequence shown in SEQ ID NO:

4.

7. The multispecific molecule according to any one of claims 1-6, wherein the first binding domain capable of targeting IL-11 is a VHH.

8. The multispecific molecule according to claim 7, the VHH comprises the amino acid sequence shown in SEQ ID NO:

4.

9. The multispecific molecule according to any one of claims 1-8, wherein the first binding domain capable of targeting IL-11 comprises the amino acid sequence shown in SEQ ID NO:

4.

10. The multispecific molecule according to any one of claims 1-4, the first binding domain capable of targeting IL-11 is selected from the sequences known in the prior art.

11. The multispecific molecule according to any one of claims 1-4, 10, the first binding domain capable of targeting IL-11 comprises the heavy-chain variable region VH and / or the light-chain variable region VL of an antibody.

12. The multispecific molecule according to any one of claims 1-4, 10-11, wherein the first binding domain capable of targeting IL-11 comprises HCDR3, HCDR2 and HCDR1 of the heavy chain variable region VH; wherein, The HCDR1 may be selected from the sequences as shown in SEQ ID NO:64, 65, 95, 104, 110, 116, the HCDR2 may be selected from the sequences as shown in SEQ ID NO:66, 72, 73, 74, 96, 105, 111, 117, and the HCDR3 may be selected from the sequences as shown in SEQ ID NO:67, 68, 97, 106, 112, 118.

13. The multispecific molecule according to any one of claims 1-4, 10-12, wherein the first binding domain capable of targeting IL-11 comprises LCDR3, LCDR2, and LCDR1 of the variable light chain region VL; wherein, The LCDR1 can be selected from the sequences shown in SEQ ID NO: 69, 70, 71, 98, 101, 107, 113, 119; the LCDR2 can be selected from the sequences shown in SEQ ID NO: 72, 73, 99, 102, 108, 114, 120; and the LCDR3 can be selected from the sequences shown in SEQ ID NO: 74 - 94, 100, 103, 109, 115, 121.

14. The multispecific molecule according to any one of claims 1 - 4, 10 - 13, wherein the first binding domain capable of targeting IL-11 comprises the variable region of the antibody heavy chain VH, and the VH may comprise the amino acid sequences shown in SEQ ID NO: 122 - 134.

15. The multispecific molecule according to any one of claims 1 - 4, 10 - 14, wherein the first binding domain capable of targeting IL-11 comprises the variable region of the antibody light chain VL, and the VL may comprise the amino acid sequences shown in SEQ ID NO: 135 - 177.

16. The multispecific molecule according to any one of claims 1 - 4, 10 - 15, wherein the first binding domain capable of targeting IL-11 comprises the constant region of the antibody heavy chain.

17. The multispecific molecule according to any one of claims 1 - 4, 10 - 16, wherein the first binding domain capable of targeting IL-11 comprises the constant region of the antibody light chain.

18. The multispecific molecule according to any one of claims 1 - 17, wherein the second binding domain capable of targeting TSLP comprises an antibody or an antigen-binding fragment thereof.

19. The multispecific molecule according to any one of claims 2 - 18, wherein the antigen-binding fragment is selected from the group consisting of: Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, VHH and dAb.

20. The multispecific molecule according to any one of claims 2 - 19, wherein the antibody is selected from the group consisting of: monoclonal antibody, single-chain antibody, chimeric antibody, multispecific antibody, humanized antibody and fully human antibody.

21. The multispecific molecule according to any one of claims 1-20, wherein the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH; wherein, The amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 19, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 20, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

21.

22. The multispecific molecule according to any one of claims 1 - 21, wherein the second binding domain capable of targeting TSLP comprises the variable region of the antibody heavy chain VH, and the amino acid sequence of the VH is as shown in SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52 or SEQ ID NO:

53.

23. The multispecific molecule according to any one of claims 1-22, wherein the second binding domain capable of targeting TSLP comprises LCDR1, LCDR2, and LCDR3 of the variable light chain region VL; wherein, The amino acid sequence of the LCDR1 is as shown in SEQ ID NO:22, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:23(GAR), and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:

24.

24. The multispecific molecule according to any one of claims 1-23, wherein the second binding domain capable of targeting TSLP comprises the variable light chain region VL of an antibody, and the amino acid sequence of the VL is as shown in SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56 or SEQ ID NO:

57.

25. The multispecific molecule according to any one of claims 1-24, wherein the second binding domain capable of targeting TSLP comprises the amino acid sequences of any one group of VH and VL selected from the following: 1) VH: SEQ ID NO:53, VL: SEQ ID NO:57; 2) VH: SEQ ID NO:49, VL: SEQ ID NO:54; 3) VH: SEQ ID NO:49, VL: SEQ ID NO:55; 4) VH: SEQ ID NO:49, VL: SEQ ID NO:56; 5) VH: SEQ ID NO:50, VL: SEQ ID NO:54; 6) VH: SEQ ID NO:50, VL: SEQ ID NO:55; 7) VH: SEQ ID NO:50, VL: SEQ ID NO:56; 8) VH: SEQ ID NO:51, VL: SEQ ID NO:54; 9) VH: SEQ ID NO:51, VL: SEQ ID NO:55; 10) VH: SEQ ID NO:51, VL: SEQ ID NO:56; 11) VH: SEQ ID NO:52, VL: SEQ ID NO:54; 12) VH: SEQ ID NO:52, VL: SEQ ID NO:55; and 13) VH: SEQ ID NO:52, VL: SEQ ID NO:

56.

26. The multispecific molecule according to any one of claims 1-25, wherein the second binding domain capable of targeting TSLP comprises the constant heavy chain region of an antibody.

27. The multispecific molecule according to claim 26, wherein the constant heavy chain region of the antibody is derived from the constant heavy chain region of human IgG.

28. The multispecific molecule according to any one of claims 26-27, wherein the constant heavy chain region of the antibody is derived from the constant heavy chain region of human IgG1.

29. The multispecific molecule according to any one of claims 1-28, wherein the second binding domain capable of targeting TSLP comprises the constant light chain region of an antibody.

30. The multispecific molecule according to claim 29, wherein the constant light chain region of the antibody is derived from the constant kappa chain region of human Ig.

31. The multispecific molecule according to any one of claims 1 - 30, wherein the second binding domain capable of targeting TSLP comprises an antibody heavy chain, and the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:

15.

32. The multispecific molecule according to any one of claims 1 - 31, wherein the second binding domain capable of targeting TSLP comprises an antibody light chain, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:

18.

33. The multispecific molecule according to any one of claims 1 - 32, wherein the second binding domain capable of targeting TSLP can be a sequence known in the prior art.

34. The multispecific molecule according to any one of claims 1 - 33, wherein the second binding domain capable of targeting TSLP can be Tezepelumab.

35. The multispecific molecule according to any one of claims 1-33, wherein the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH; wherein, The amino acid sequence of the HCDR1 is as shown in SEQ ID NO:178, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:179, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

180.

36. The multispecific molecule according to any one of claims 1-33, 35, wherein the second binding domain capable of targeting TSLP comprises LCDR3, LCDR2 and LCDR1 of the variable light chain region VL; wherein, The amino acid sequence of the LCDR1 is as shown in SEQ ID NO:181, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:182, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:

183.

37. The multispecific molecule according to any one of claims 1 - 33, 35 - 36, wherein the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:

184.

38. The multispecific molecule according to any one of claims 1 - 33, 35 - 37, wherein the second binding domain capable of targeting TSLP comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:

185.

39. The multispecific molecule according to any one of claims 1 - 33, 35 - 38, wherein the second binding domain capable of targeting TSLP comprises an antibody heavy chain constant region.

40. The multispecific molecule according to any one of claims 1 - 33, 35 - 39, wherein the second binding domain capable of targeting TSLP comprises an antibody light chain constant region.

41. The multispecific molecule according to any one of claims 1-33, 35-40, wherein the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region; wherein, The amino acid sequence of the HCDR1 is as shown in SEQ ID NO:186, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:187, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

188.

42. The multispecific molecule according to any one of claims 1-33, 35-41, wherein the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region; wherein, The amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 189, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 190, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

191.

43. The multispecific molecule according to any one of claims 1-33, 35-42, wherein the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:

192.

44. The multispecific molecule according to any one of claims 1-33, 35-43, wherein the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:

193.

45. The multispecific molecule according to any one of claims 1-44, wherein the first binding domain capable of targeting IL-11 and the second binding domain targeting TSLP are directly or indirectly linked.

46. The multispecific molecule according to any one of claims 1-45, wherein the C-terminus of the first binding domain capable of targeting IL-11 is linked to the N-terminus of the second binding domain targeting TSLP.

47. The multispecific molecule according to claim 46, wherein the C-terminus of the first binding domain capable of targeting IL-11 is linked to the N-terminus of the heavy chain variable region VH of the second binding domain targeting TSLP.

48. The multispecific molecule according to any one of claims 1-47, wherein the N-terminus of the first binding domain capable of targeting IL-11 is linked to the C-terminus of the second binding domain targeting TSLP.

49. The multispecific molecule according to claim 48, wherein the N-terminus of the first binding domain capable of targeting IL-11 is linked to the C-terminus of the heavy chain constant region of the second binding domain targeting TSLP.

50. The multispecific molecule according to any one of claims 1-49, wherein the first binding domain capable of targeting IL-11 and the second binding domain targeting TSLP are linked by a linker.

51. The multispecific molecule according to claim 50, wherein the linker comprises a peptide linker.

52. The multispecific molecule according to any one of claims 50-51, wherein the linker comprises the amino acid sequence of (GGGGS)n, where n is any positive integer from 0 to 10.

53. The multispecific molecule according to any one of claims 50-52, wherein the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 3).

54. The multispecific molecule according to any one of claims 50-53, wherein the multispecific molecule comprises two first binding domains capable of targeting IL-11.

55. The multispecific molecule according to claim 54, wherein the two first binding domains capable of targeting IL-11 are directly or indirectly linked to the N-termini of the two heavy chains of the second binding domain targeting TSLP respectively.

56. The multispecific molecule according to claim 55, wherein the two first binding domains capable of targeting IL-11 are directly or indirectly connected to the C-terminus of the two heavy chains of the second binding domain capable of targeting TSLP, respectively.

57. The multispecific molecule according to any one of claims 1-56, which comprises a first polypeptide chain and a second polypeptide chain.

58. The multispecific molecule according to claim 57, wherein the first polypeptide chain comprises the first binding domain capable of targeting IL-11 and the heavy chain of the second binding domain capable of targeting TSLP.

59. The multispecific molecule according to any one of claims 58-59, wherein in the first polypeptide chain, the N-terminus of the first binding domain capable of targeting IL-11 and the heavy chain of the second binding domain capable of targeting TSLP are directly or indirectly connected.

60. The multispecific molecule according to any one of claims 58-60, wherein in the first polypeptide chain, the C-terminus of the first binding domain capable of targeting IL-11 and the heavy chain of the second binding domain capable of targeting TSLP are directly or indirectly connected.

61. The multispecific molecule according to any one of claims 57-60, wherein the first polypeptide chain comprises the first binding domain capable of targeting IL-11 and the heavy chain of the second binding domain capable of targeting TSLP are connected by a linker.

62. The multispecific molecule according to any one of claims 57-61, wherein the first polypeptide chain comprises an amino acid sequence selected from any one of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:

63.

63. The multispecific molecule according to any one of claims 57-62, wherein the second polypeptide chain comprises the light chain of the second binding domain.

64. The multispecific molecule according to any one of claims 57-63, wherein the light chain of the second polypeptide chain comprises an amino acid sequence selected from any one of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:

18.

65. The multispecific molecule according to any one of claims 57-64, which comprises two of the first polypeptide chains and two of the second polypeptide chains.

66. The multispecific molecule according to any one of claims 57-65, wherein the first polypeptide chain and the second polypeptide chain are connected by a disulfide bond.

67. A pharmaceutical combination, which comprises a first antibody capable of targeting IL-11 and a second antibody capable of targeting TSLP.

68. The pharmaceutical combination according to claim 67, wherein the first antibody and the second antibody are present in a mixture.

69. The pharmaceutical combination according to claim 67 or 68, wherein the first antibody and the second antibody exist independently of each other.

70. The pharmaceutical combination according to any one of claims 67 - 69, wherein the antibody is selected from the group consisting of: monoclonal antibody, single-chain antibody, chimeric antibody, multispecific antibody, humanized antibody, fully human antibody, nanobody and / or heavy-chain antibody.

71. The pharmaceutical combination according to any one of claims 67 - 70, wherein the first antibody comprises HCDR3, HCDR2, and HCDR1 of the heavy chain variable region VH; wherein, The amino acid sequence of the HCDR3 is as shown in SEQ ID NO:7, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:6, and the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:

5.

72. The pharmaceutical combination according to any one of claims 67 - 71, wherein the first antibody comprises an antibody heavy-chain variable region VH, and the amino acid sequence of the VH is as shown in SEQ ID NO:

4.

73. The pharmaceutical combination according to any one of claims 67 - 72, wherein the heavy-chain variable region is VHH.

74. The pharmaceutical combination according to any one of claims 67 - 73, wherein the first antibody targeting IL-11 comprises an antibody heavy-chain constant region, and the antibody heavy-chain constant region is derived from an IgG heavy-chain constant region.

75. The pharmaceutical combination according to any one of claims 67 - 74, wherein the first antibody targeting IL-11 is a nanobody, and it comprises the amino acid sequence shown in SEQ ID NO:

4.

76. The pharmaceutical combination according to any one of claims 67 - 70, wherein the first antibody targeting IL-11 can be an antibody known in the prior art.

77. The pharmaceutical combination according to any one of claims 67 - 70, 76, wherein the first antibody targeting IL-11 comprises an antibody heavy-chain variable region VH and / or an antibody light-chain variable region VL.

78. The multispecific molecule according to any one of claims 67-70, 76-77, wherein the first antibody targeting IL-11 comprises the HCDR3, HCDR2, and HCDR1 of the heavy chain variable region VH; wherein, The HCDR1 can be selected from the sequences shown in SEQ ID NO:64, 65, 95, 104, 110, 116, the HCDR2 can be selected from the sequences shown in SEQ ID NO:66, 72, 73, 74, 96, 105, 111, 117, and the HCDR3 can be selected from the sequences shown in SEQ ID NO:67, 68, 97, 106, 112, 118.

79. The multispecific molecule according to any one of claims 67-70, 76-78, wherein the first antibody targeting IL-11 comprises LCDR3, LCDR2, and LCDR1 of the light chain variable region VL; wherein, The LCDR1 can be selected from the sequences shown in SEQ ID NO:69, 70, 71, 98, 101, 107, 113, 119, the LCDR2 can be selected from the sequences shown in SEQ ID NO:72, 73, 99, 102, 108, 114, 120, and the LCDR3 can be selected from the sequences shown in SEQ ID NO:74 - 94, 100, 103, 109, 115, 121.

80. The multispecific molecule according to any one of claims 67 - 70, 76 - 79, wherein the first antibody targeting IL-11 comprises an antibody heavy-chain variable region VH, and the VH can comprise the amino acid sequence shown in SEQ ID NO:122 - 134.

81. The multispecific molecule according to any one of claims 67-70, 76-80, wherein the first antibody targeting IL-11 comprises an antibody light chain variable region VL, and the VL may comprise an amino acid sequence as shown in SEQ ID NOs: 135-177.

82. The multispecific molecule according to any one of claims 67-70, 76-81, wherein the first antibody capable of targeting IL-11 comprises an antibody heavy chain constant region.

83. The multispecific molecule according to any one of claims 67-70, 76-82, wherein the first antibody capable of targeting IL-11 comprises an antibody light chain constant region.

84. The pharmaceutical combination according to any one of claims 67-83, wherein the second antibody comprises HCDR3, HCDR2 and HCDR1 of the heavy chain variable region VH; wherein, The amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 21, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 20, and the amino acid sequence of the HCDR1 is as shown in SEQ ID NO:

19.

85. The pharmaceutical combination according to any one of claims 67-84, wherein the second antibody comprises an antibody heavy chain variable region VH, and the amino acid sequence of the VH is optionally selected from SEQ ID NOs: 49, 50, 51, 52, and 53.

86. The pharmaceutical combination according to any one of claims 67 - 85, wherein the second antibody comprises LCDR1, LCDR2 and LCDR3 of the variable light chain region VL; wherein, The amino acid sequence of the LCDR1 is as shown in SEQ ID NO: 22, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO: 23(GAR), and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:

24.

87. The pharmaceutical combination according to any one of claims 67-86, wherein the second antibody comprises an antibody light chain variable region VL, and the amino acid sequence of the VL is optionally selected from SEQ ID NOs: 54, 55, 56, and 57.

88. The pharmaceutical combination according to any one of claims 67-87, wherein the second antibody comprises an amino acid sequence of any one group of VH and VL selected from the following: 1) VH: SEQ ID NO: 53, VL: SEQ ID NO: 57; 2) VH: SEQ ID NO: 49, VL: SEQ ID NO: 54; 3) VH: SEQ ID NO: 49, VL: SEQ ID NO: 55; 4) VH: SEQ ID NO: 49, VL: SEQ ID NO: 56; 5) VH: SEQ ID NO: 50, VL: SEQ ID NO: 54; 6) VH: SEQ ID NO: 50, VL: SEQ ID NO: 55; 7) VH: SEQ ID NO: 50, VL: SEQ ID NO: 56; 8) VH: SEQ ID NO: 51, VL: SEQ ID NO: 54; 9) VH: SEQ ID NO: 51, VL: SEQ ID NO: 55; 10) VH: SEQ ID NO:51, VL: SEQ ID NO:56; 11) VH: SEQ ID NO:52, VL: SEQ ID NO:54; 12) VH: SEQ ID NO:52, VL: SEQ ID NO:55; and 13) VH: SEQ ID NO:52, VL: SEQ ID NO:

56.

89. The pharmaceutical combination according to claims 67 - 83, wherein the second antibody can be an antibody known in the prior art.

90. The pharmaceutical combination according to claims 67 - 83, 89, wherein the second antibody is Tezepelumab.

91. The pharmaceutical combination according to any one of claims 67-83, 89, wherein the second antibody capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH; wherein, The amino acid sequence of the HCDR1 is as shown in SEQ ID NO:178, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:179, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

180.

92. The pharmaceutical combination according to any one of claims 67-83, 89, 91, wherein the second antibody capable of targeting TSLP comprises LCDR3, LCDR2 and LCDR1 of the variable light chain region VL; wherein, The amino acid sequence of the LCDR1 is as shown in SEQ ID NO:181, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO:182, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO:

183.

93. The pharmaceutical combination according to any one of claims 67 - 83, 89, 91 - 92, wherein the second antibody capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:

184.

94. The pharmaceutical combination according to any one of claims 67 - 83, 89, 91 - 93, wherein the second antibody capable of targeting TSLP comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:

185.

95. The pharmaceutical combination according to any one of claims 67 - 83, 89, 91 - 94, wherein the second antibody capable of targeting TSLP comprises an antibody heavy chain constant region.

96. The pharmaceutical combination according to any one of claims 67 - 83, 89, 91 - 95, wherein the second antibody capable of targeting TSLP comprises an antibody light chain constant region.

97. The pharmaceutical combination according to any one of claims 67-83, 89, wherein the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; wherein, The amino acid sequence of the HCDR1 is as shown in SEQ ID NO:186, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:187, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

188.

98. The pharmaceutical combination according to any one of claims 67-83, 89, 97, wherein the second binding domain capable of targeting TSLP comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region; wherein, The amino acid sequence of the HCDR1 is as shown in SEQ ID NO:189, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO:190, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO:

191.

99. The pharmaceutical combination according to any one of claims 67 - 83, 89, 97 - 98, the second binding domain capable of targeting TSLP comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:

192.

100. The pharmaceutical combination according to any one of claims 67 - 83, 89, 99 - 100, wherein the second binding domain capable of targeting TSLP comprises the variable region VH of an antibody heavy chain, and the VH comprises the amino acid sequence shown in SEQ ID NO:

193.

101. An isolated nucleic acid molecule or molecules encoding a multispecific molecule according to any one of claims 1 - 66.

102. A vector comprising the nucleic acid molecule according to claim 101.

103. A cell comprising the nucleic acid molecule according to claim 101 or the vector according to claim 102.

104. A method for preparing a multispecific molecule according to any one of claims 1 - 66, the method comprising culturing the cell according to claim 103 under conditions such that the multispecific molecule according to any one of claims 1 - 66 is expressed.

105. A pharmaceutical composition comprising a multispecific molecule according to any one of claims 1 - 66, a pharmaceutical combination according to any one of claims 67 - 100, the nucleic acid molecule according to claim 101, the vector according to claim 102, and / or the cell according to claim 103, and optionally a pharmaceutically acceptable adjuvant.

106. A method for blocking the binding of TSLP to the receptor TSLPR / IL7Ra complex, the method comprising administering a multispecific molecule according to any one of claims 1 - 66 and / or a pharmaceutical combination according to any one of claims 67 - 100.

107. A method for inhibiting the production of TARC (thymus and activation-regulated chemokine), the method comprising administering a multispecific antibody according to any one of claims 1 - 66 and / or a pharmaceutical combination according to any one of claims 67 - 100.

108. Use of a multispecific molecule according to any one of claims 1 - 66, a pharmaceutical combination according to any one of claims 67 - 100, the nucleic acid molecule according to claim 101, the vector according to claim 102, the cell according to claim 103, and / or the pharmaceutical composition according to claim 105 in the manufacture of a medicament for preventing, alleviating, and / or treating a disease or disorder.

109. The use according to claim 108, wherein the disease and / or disorder comprises an IL-11 related disease.

110. The use according to any one of claims 108 - 109, wherein the disease and / or disorder comprises a TSLP related disease.

111. The use according to any one of claims 108 - 110, wherein the disease and / or disorder comprises a fibrotic disease, an inflammatory disease, or a tumor.

112. A method for preventing, alleviating, and / or treating a disease or disorder, which comprises administering to a patient in need thereof a multispecific molecule according to any one of claims 1 - 66, a pharmaceutical combination according to any one of claims 67 - 100, the nucleic acid molecule according to claim 101, the vector according to claim 102, the cell according to claim 103, and / or the pharmaceutical composition according to claim 105.

113. The method according to claim 112, wherein the disease and / or disorder comprises an IL-11 related disease.

114. The method according to any one of claims 112-113, wherein the disease and / or disorder comprises a TSLP related disease.

115. The method according to any one of claims 112-114, wherein the disease and / or disorder comprises a fibrotic disease, an inflammatory disease or a tumor.

116. The multispecific molecule according to any one of claims 1-66, the pharmaceutical combination according to any one of claims 67-100, the nucleic acid molecule according to claim 101, the vector according to claim 102, the cell according to claim 103 and / or the pharmaceutical composition according to claim 105, for preventing, alleviating and / or treating a disease or disorder.

117. The multispecific molecule, pharmaceutical combination, nucleic acid molecule, vector, cell and / or pharmaceutical composition according to claim 116, wherein the disease and / or disorder comprises an IL-11 related disease.

118. The multispecific molecule, pharmaceutical combination, nucleic acid molecule, vector, cell and / or pharmaceutical composition according to any one of claims 116 or 117, wherein the disease and / or disorder comprises a TSLP related disease.

119. The multispecific molecule, pharmaceutical combination, nucleic acid molecule, vector, cell and / or pharmaceutical composition according to any one of claims 116-118, wherein the disease and / or disorder comprises a fibrotic disease, an inflammatory disease or a tumor.