Dimeric immunoadhesins, pharmaceutical compositions and uses

Abstract

This invention relates to the field of biomedical engineering technology, and discloses compositions and methods related to soluble dimer immunoadhesins. The dimer immunoadhesins comprise dimerized first and second polypeptide chains. The first polypeptide chain has a general structural formula of Z1-Z2, and the second polypeptide chain has a general structural formula of Y1-Y2. Z1 is (i) an extracellular domain of a first cell surface receptor or a functional variant or fragment thereof, or (ii) a first cytokine or a functional variant or fragment thereof; Z2 is a dimerized domain of an immunoglobulin constant region or a functional variant or fragment thereof. Y1 is an extracellular domain of a second cell surface receptor or a functional variant or fragment thereof, or (ii) a second cytokine or a functional variant or fragment thereof. Y2 is a dimerized domain of an immunoglobulin constant region or a functional variant or fragment thereof. The dimer protein can be used for the treatment and prevention of infertility-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, specifically to a dimer immunoadhesive, a pharmaceutical composition thereof as an active component, and its pharmaceutical uses, particularly for the treatment of infertility and related diseases. Background Technology

[0002] T cells have many important membrane molecules on their surface, which play a vital role in T cell activation, proliferation, differentiation, and effector function. Based on their functions, they can be mainly divided into the following categories: (1) TCR-CD3 complex, which enables T cells to recognize antigen peptide-MHC molecule complexes on antigen-presenting cells and transmit activation signals into the cell; (2) CD4 and CD8 molecules, which respectively assist the TCR of CD4+ and CD8+ T cells in recognizing antigens and participating in the transmission of T cell activation signals; (3) Co-stimulatory molecules, such as CD28, CTLA-4, ICOS, and PD-1, which transmit second signals for T cells; (4) Other surface molecules, mainly including cytokine receptors related to T cell activation, proliferation, and differentiation, as well as adhesion molecules that facilitate cell-cell interactions.

[0003] TIGIT protein (UniProtKB ID: Q495A1) is a newly discovered costimulatory molecule with immunosuppressive effects. In 2005, Abbas et al. (Abbas AR, Baldwin D, Ma Y, et al..Genes&Immunity,2005,6(4):319-331.) sequenced activated human T cells in order to find new immune costimulatory or inhibitory molecules and further studied some protein molecules with immunoregulatory-like domains. As a result, they discovered a new molecule expressed on T cells and NK cells. This molecule has an immunoglobulin-like domain, a transmembrane region and an immunoreceptor protein tyrosine inhibitory motif (ITIM), and was therefore named TIGIT (T cell immunoglobulin and ITIM domain) (Xin Y, Harden K, Gonzalez LC, et al..Nature Immunology,2009,10(1):48-57.). Shortly thereafter, other laboratories also discovered the molecule using different methods, naming it WUCAM (Boles KS, Vermi W, Facchetti F, et al., 2010, 39(3): 695-703.), Vstm3 (Levin SD, Taft DW, Brandt CS, et al. Vstm3 is a member of the CD28 family and an important modulator of T-cell function. [J]. European Journal of Immunology, 2011, 41(4): 902-915.) or Vsig9 (Stanietsky N, Mandelboim O. Paired NK cell receptors controlling NK cytotoxicity [J]. Febs Letters, 2010, 584(24): 4895-4900.).

[0004] In existing technologies, the soluble fragment of TIGIT protein has been shown in basic research to have a certain cellular-level inhibitory effect on antigen presentation by antigen-presenting cells such as dendritic cells (Xin Y, Harden K, Gonzalez LC, et al. Nature Immunology, 2009, 10(1):48-57.), and can be used to treat autoimmune diseases such as lupus nephritis (Liu S, Sun L, Wang C, et al. Clinical immunology. 2019; 203:72-80.).

[0005] However, many cell surface receptors are known to exist in soluble forms similar to TIGIT proteins. These soluble receptors correspond to the ligand-binding domains of their cell surface counterparts. For example, naturally occurring soluble cytokine receptors inhibit cytokine responses and act as transport proteins. Furthermore, it has been found that dimerizing soluble receptor peptides using fusion proteins can enhance the binding properties of these soluble receptors, making them therapeutically useful antagonists of their corresponding ligands. A representative example of such dimerized fusions is immunoadhesins. (See, for example, Sledziewski et al., U.S. Patents 5,155,027 and 5,567,584; Jacobs et al., U.S. Patent 5,605,690; Wallner et al., U.S. Patent 5,914,111; and Ashkenazi and Chamow, Curr. Opin. Immunol. 9:195-200, 1997).

[0006] However, in recurrent miscarriage, dysregulation of maternal-fetal immune factors is also a key link in the pathological progression of the disease (Trowsdale J, Betz AG. Nat Immunol. 2006; 7:241-6.), but due to the unique nature of intrauterine maternal-fetal immunity, the therapeutic value of these immune adhesins remains unclear. The invention described in this article clarifies the application value of this type of drug. Summary of the Invention

[0007] The purpose of this invention is, based on the above research background, to investigate whether soluble dimer immunoadhesins can be used to treat recurrent miscarriage and threatened miscarriage mediated by maternal-fetal immune disorders, and to describe the specific structure, preparation method and uses of dimer immunoadhesins, that is, to provide dimer immunoadhesins, their preparation method and uses.

[0008] In a first aspect, the present invention provides a soluble dimerized immunoadhesive. The dimerized immunoadhesive comprises a dimerized first and second polypeptide chain, the first polypeptide chain having a general structural formula of Z1-Z2, and the second polypeptide chain having a general structural formula of Y1-Y2. Z1 is (i) an extracellular domain of a first cell surface receptor or a functional variant or fragment thereof, or (ii) a first cytokine or a functional variant or fragment thereof; Z2 is a dimerized domain or a functional variant or fragment thereof. Y1 is (i) an extracellular domain of a second cell surface receptor or a functional variant or fragment thereof, or (ii) a second cytokine or a functional variant or fragment thereof; Y2 is a dimerized domain or a functional variant or fragment thereof.

[0009] In certain embodiments of the aforementioned polypeptide chain or dimer immunoadhesins (where Z1 is the extracellular domain of a first cell surface receptor or a functional variant or fragment thereof, and / or Y1 is the extracellular domain of a second cell surface receptor or a functional variant or fragment thereof), the first cell surface receptor and / or the second cell surface receptor are each selected from: 4-1BB; ACTH receptor; activin receptor; BLTR (leukotriene B4 receptor); BMP receptor; C3a receptor; C5a receptor; CCR1; CCR2; CCR3; CCR4; CCR5; CCR6; CCR7; CCR8; CCR9; CD19; CD22; CD27; CD28; CD30; CD40; CD7 0; CD80; CD86; CD96; CD200R; CTLA-4; CD226; CD274; CD273; CD275; CD276; CD278; CD279; VSTM3 (TIGIT, B7R1); CD112; CD155; B7H6; NKp30; ICAM; VLA-4; VCAM; CT-1 receptor; CX3CR1; CXCR1; CXCR2; CXCR3; CXCR4; CXCR5; D6; DARC; DcR3; DR4; DR5; DcR1; DcR2; ECRF3; Fas; fMLP receptor; G-CSF receptor; GIT receptor; GM-CSF receptor; growth Hormone receptors; HVEM; BTLA; Interferon-α receptor; Interferon-β receptor; Interferon-γ receptor; IL-1 receptor type I; IL-1 receptor type II; IL-10 receptor; IL-11 receptor; IL-12 receptor; IL-13 receptor; IL-15 receptor; IL-16 receptor (CD4); IL-17 receptor A; IL-17 receptor B; IL-17 receptor C; IL-17 receptor D; IL-17 receptor E; IL-18 receptor; IL-2 receptor; IL-3 receptor; IL-4 receptor; IL-5 receptor; IL-6 receptor; IL-7 receptor; IL-9 receptor; IL-20 receptor A; IL-20 receptor B; IL-21 receptor; IL-22 receptor A IL-22 receptor B; IL-28 receptor A; IL-27 receptor A; IL-31 receptor A; BCMA; TACI; BAFF receptor; immunomodulatory brain signaling protein receptor CD72; Kaposi's sarcoma-associated herpesvirus GPCR; lipoxygenin A4 receptor; lymphotoxin β receptor; lysophospholipid growth factor receptor; neurokinin 1; μ, δ, and κ opioid receptors for endorphins; oncokinin M receptor; osteopontin receptor; osteoprotegerin; Ox40; OX40L; PACAP and VIP receptors; PAF receptor; poxvirus; IFNα / β receptor homologues; poxvirus IFNγ receptor homologues; poxvirus IL-1β receptor homologues; poxvirus membrane-bound G protein-coupled receptor homologues;Chemokine-binding proteins secreted by poxviruses; poxvirus TNF receptor homologues; prolactin receptor; RANK; RON receptor; SCF receptor; somatostatin receptor; T1 / ST2; TGF-β receptor; TNF receptors (e.g., p60 and p80); TNFRSF19; TPO receptor; US28; XCR1; erythropoietin receptor; growth hormone receptor; leukemia inhibitory factor receptor; and C-kit receptor.

[0010] In cases where Z1 and Y1 are both extracellular domains of cell surface receptors or their functional variants or fragments, the first and second can be the same as or different from the cell surface receptor.

[0011] In other embodiments, where Z1 is a first cytokine or a functional variant or fragment thereof and / or Y1 is a second cytokine or a functional variant or fragment thereof, the first cytokine and / or the second cytokine are each selected from: α-MSH; 9E3 / cCAF; ACTH; activin; AK155; angiogenesis inhibitor; Apo2L / TRAIL; APRIL; BAFF (BLys); BLR1 ligand / BCA-1 / BLC / CXCL13; BMP family; BRAK; calcitonin gene-associated peptide (CGRP); CC chemokine of molluscum contagiosum virus; CCL27; CCL28; CD100 / Sema4D; CD27 ligand CD30 ligand; CD40 ligand; CKβ8-1 / MPIF-1 / CCL23; CLF / CLC; CSF-1; CT-1; CTAP-III, βTG and NAP-2 / / CXCL7; CXCL16; defensins; ELC / MIP-3β / Exodus-3 / CCL19; ENA-78 / CXCL5; endorphins; endostatin; eosinophil chemokine 2 / MPIF-2 / CCL24; eosinophil chemokine / CCL11; erythropoietin; Exodus-1 / LARC / MIP-3α(SCYA20); Fas ligand; Flt-3 ligand; fMLP; Frac talkine / CX3CL1; G-CSF; GCP-2 / CXCL6; GM-CSF; growth hormone; HCC-1 / CCL14; HCC-4 / CCL16; high-velocity swimming family box 1 (HMGB1); human cathelicidin antimicrobial peptide LL-37; I-309 / CCL1; IFNα, IFNβ and IFNω ligands; IFNγ; IL-1α; IL-1β; IL-10; IL-11; IL-12; IL-13; IL-15; IL-16; IL-17A; IL-17B; IL-17C; IL-17D; IL-17E; IL-17F; IL-18; IL-1Ra IL-2; IL-27; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8 / CXCL8; IL-9; IP-10 / CXCL10; IL-19; IL-20; IL-21; IL-22; IL-23; IL-24; IL-26; IL-31; Keratinocyte growth factor; KSHV-associated IL-6 ligand; Leptin; Leukocyte inducer 1 / HCC-2 / MIP-1δ / CCL15; Leukotriene B4; LIGHT; Lipoxygen; Lymphocyte chemokine / XCL1; Lymphotoxin α and β; Lysophospholipid growth factor; Macrophage-derived chemokine; Macrophage stimulating protein (MSP);MCP-1 / CCL2, MCP-2 / CCL8, MCP-3 / CCL7, MCP-4 / CCL13, and MCP-5 / CCL12; methoxyestradiol; MGSA / GRO / CXCL1, CXCL2, and CXCL3; MIF; MIG / CXCL9; MIP-1α / CCL3 and MIP-1β / CCL4; MIP-1γ / MRP-2 / CCF18 / CCL9 / 10; MuC10 / CCL6; oncokinetic protein M; osteopontin; parapoxetine (sheep infectious stomatitis virus) IL-10 homologue; PARC / DC-CCK1 / AMAC-1 / CCL18; PDGF-A; PDGF-B; PDGF-C; PDGF-D; platelet activating factor; platelet factor 4 / CXCL4; related to epidermal growth factor. Poxvirus growth factors; complement regulatory proteins secreted by poxviruses; vascular endothelial growth factor (VEGF) homologues of sheep infectious stomatitis virus; prolactin; RANK ligands; RANTES / CCL5; S100A12; SDF-1 / CXCL12; SERP-1; secretory poxvirus serine protease inhibitor; SLC(6Ckine) / Exodus-2 / TCA-4 / CCL21; somatostatin; stem cell factor; substance P; TARC / CCL17; TCA3 / mouse CCL1; TECK / CCL25; TGFβ; thrombopoietin; TNFα; TSG-6; TWEAK; vaccinia virus brain signaling proteins; vCXC-1 and vCXC-2; VEGF; VIP and PACAP; and IL-10 variants of the virus.

[0012] When Z1 and Y1 are both cytokines or their functional variants or fragments, the first and second cytokines can be the same or different.

[0013] The particularly suitable dimerizing domains used in the aforementioned dimer immunoadhesins include the immunoglobulin heavy chain constant region. For example, in specific variations, the dimerizing domains Z2 and Y2 are Fc fragments of IgG, such as human immunoglobulin γ1 Fc fragments. When Z1 differs from Y1, the dimerizing domains Z2 and Y2 can be engineered to increase specific heterodimerization, such as Knob-in-hole, ART-Ig with altered charge polarity, BiMab, and other bispecific antibody constant region construction methods (review article Brinkmann U, Kontermann R E.mAbs, 2017, 9(2):182-212.).

[0014] In some embodiments of the dimer immunoadhesins described above, the dimerizing domains Z2 and Y2 include peptide linkers consisting of 15-32 amino acid residues, of which 1-8 (e.g., 2) are cysteine ​​residues. In specific variations, Z2 and Y2 include immunoglobulin hinge regions or variants thereof. For example, in one embodiment, Z2 and YY include immunoglobulin hinge variants (e.g., human immunoglobulin γ1 hinge variants) in which the cysteine ​​residue corresponding to Fc fragment 220 is replaced by a serine residue. Particularly suitable peptide linkers used according to the dimerizing domains Z2 and Y2 described above include peptide linkers that contain a plurality of glycine residues and optionally at least one serine residue.

[0015] In some embodiments of the present invention, the dimerizing domains Z2 and Y2 may be active variants of the human immunoglobulin Fc fragment, such as the Fc domains of IgG2, IgG3, or IgG4. In some embodiments, Fc mutants may be further employed to reduce the biological activities of immunoglobulins such as ADCC and complement binding, such as the LALA-PG mutant, L235E, E318A, K320A, and K322A mutants.

[0016] In a preferred embodiment of the invention, each of Z1 and Y1 is an extracellular domain of TIGIT (VSTM3, B7R1) or a functional variant or fragment thereof. For example, in specific variations of soluble dimeric immunoadhesins having the above-described general formulas Z1-Z2 and Y1-Y2, the amino acid sequences of Z1 and Y1 have at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% identity with the amino acid sequence of the human TIGIT protein shown in SEQ ID NO.1.

[0017] In a specific preferred embodiment, in a particular variation of the dimer immunoadhesin comprising formulas Z1-Z2 and Y1-Y2 (where each of Z1 and Z2 is an extracellular domain of TIGIT or a functional variant or fragment thereof), the dimer immunoadhesin comprises an amino acid sequence selected from the following: the human TIGIT immunoadhesin amino acid sequence shown in SEQ ID NO:2.

[0018] In a specific preferred embodiment, in a particular variation of the dimer immunoadhesin comprising formulas Z1-Z2 and Y1-Y2 (where each of Z1 and Z2 is an extracellular domain of TIGIT or a functional variant or fragment thereof), the dimer immunoadhesin comprises an amino acid sequence selected from the following: the amino acid sequence of the human TIGIT immunoadhesin LALA-PG mutant shown in SEQ ID NO:3.

[0019] In a preferred embodiment of the invention, Z1 is the extracellular domain of TIGIT or a functional variant or fragment thereof. Y1 is the extracellular domain of CTLA4 or a functional variant or fragment thereof. For example, in specific variations of soluble dimer immunoadhesins having the above-described general formulas Z1-Z2 and Y1-Y2, the amino acid sequence of Z1 has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% identity with the amino acid sequence of the ABATACEPTN terminal domain shown in SEQ ID NO.4. The amino acid sequence of Y1 has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% identity with the amino acid sequence of the ABATACEPTN terminal domain shown in SEQ ID NO.4.

[0020] In a specific preferred embodiment, in a particular variation of the soluble dimeric immunoadhesin comprising formulas Z1-Z2 and Y1-Y2 (Z1 is the extracellular domain of TIGIT or a functional variant or fragment thereof; Y1 is the extracellular domain of CTLA4 or a functional variant or fragment thereof), the two polypeptide chains of the soluble dimeric immunoadhesin comprise an amino acid sequence selected from the following: (a) the Z1-Z2 polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:5, and (b) the Y1-Y2 polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:6.

[0021] In a preferred embodiment of the invention, Z1 is the extracellular domain of TIGIT or a functional variant or fragment thereof. Y1 is the cytokine IL-10 or a functional variant or fragment thereof. For example, in specific variations of soluble dimeric immunoadhesins having the above-described general formulas Z1-Z2 and Y1-Y2, the amino acid sequence of Z1 has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% identity with the amino acid sequence shown in SEQ ID NO. 7. The amino acid sequence of Y1 has at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% identity with the amino acid sequence shown in SEQ ID NO. 7.

[0022] In a specific preferred embodiment, in a particular variation of the soluble dimeric immunoadhesin comprising formulas Z1-Z2 and Y1-Y2 (Z1 is the extracellular domain of TIGIT or a functional variant or fragment thereof; Y1 is the cytokine IL-10 or a functional variant or fragment thereof), the two polypeptide chains of the soluble dimeric immunoadhesin comprise an amino acid sequence selected from the following: (a) Z1 is the amino acid sequence shown in SEQ ID NO:5, and (b) Y1 is the mutant amino acid sequence shown in SEQ ID NO:8.

[0023] In addition, the present invention provides a polynucleotide encoding the aforementioned dimer immunoadhesin. In one related aspect, the present invention provides a vector comprising such a polynucleotide. For example, in some embodiments, the present invention provides an expression vector comprising the following operatively linked elements: a transcription promoter; a DNA region encoding the aforementioned dimer immunoadhesin; and a transcription terminator.

[0024] In other related aspects, the present invention provides cultured cells comprising such a vector, and methods for producing the polypeptides or dimers disclosed above. For example, in some embodiments, the cultured cells according to the invention comprise an expression vector comprising elements operatively linked as follows: a transcription promoter; a DNA segment encoding the aforementioned dimeric immunoadhesin; and a transcription terminator; and wherein the cells express the dimeric immunoadhesin encoded by the DNA segment. In certain variations of the method for preparing dimeric immunoadhesins, the method comprises: (i) culturing cells comprising the expression vector disclosed above, wherein the cells express the dimeric immunoadhesin encoded by the DNA segment and produce the encoded dimeric immunoadhesin; and (ii) recovering the soluble dimeric immunoadhesin. Similarly, in certain variations of the method for preparing dimers, the method comprises: (i) culturing cells comprising the expression vector disclosed above, wherein the cells express the dimeric immunoadhesin encoded by the DNA segment and produce the encoded dimeric immunoadhesin as a dimer; and (ii) recovering the dimer.

[0025] A second aspect of the invention provides a pharmaceutical composition comprising the aforementioned soluble dimer immunoadhesin and at least one pharmaceutically acceptable carrier. These formulations, thereby ensuring more stable therapeutic efficacy, maintain the conformational integrity of the amino acid core sequence of the TIGIT immunoadhesin disclosed herein, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation).

[0026] Under normal circumstances, liquid formulations can be stored stably at 2℃-8℃ for at least one year, while lyophilized formulations remain stable at 30℃ for at least six months. The formulations can be commonly used in the pharmaceutical industry, such as suspensions, injections, or lyophilized formulations, with injections or lyophilized formulations being preferred.

[0027] For the aqueous or lyophilized formulations of the dimer immunoadhesive disclosed in this invention, pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonic adjusters, and buffers. Surfactants include nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleic, or octadecyl sarcosine; Pluronics; MONAQUAT™, etc., and their addition should minimize the particulate tendency of the bifunctional, bispecific antibody protein. Solution stabilizers can be sugars, including reducing and non-reducing sugars; amino acids, including monosodium glutamate or histidine; alcohols, including one or a combination of triols, higher sugar alcohols, propylene glycol, and polyethylene glycol, and the amount of solution stabilizer added should be such that the final formulation remains stable within a time considered stable by those skilled in the art. Isotonic adjusters can be one of sodium chloride or mannitol. Buffers can be one of TRIS, histidine buffer, or phosphate buffer.

[0028] A third aspect of the invention provides the use of the aforementioned dimer immunoadhesin, specifically its use in the preparation of medicaments for the treatment and prevention of infertility-related diseases, wherein the medicament uses the soluble immunoadhesin protein disclosed above as an active component. The method of administration includes administering an effective amount of the soluble immunoadhesin protein to a subject (human or animal) with infertility-related diseases, or prophylactically administering an effective amount of the soluble immunoadhesin protein to a healthy subject at risk of infertility.

[0029] In some preferred embodiments of the present invention, infertility-related diseases suitable for use with the soluble immunoadhesins disclosed herein include maternal-fetal immune tolerance disorders and gynecological reproductive inflammation-related diseases. The former includes recurrent spontaneous abortion, threatened abortion, or failure of assisted reproductive technology treatment; the latter includes pelvic inflammatory disease, decreased endometrial receptivity, endometritis, endometrial polyps, intrauterine adhesions, decreased endometrial glands, endometrial fibrosis, amenorrhea, abnormal uterine bleeding, adenomyosis and endometriosis, reproductive system infections, and uterine fibroids.

[0030] Through a classic maternal-fetal immune tolerance disorder abortion model validation experiment, dimeric immunoadhesin can significantly reduce the abortion rate; through an endometrial injury model validation experiment, dimeric immunoadhesin treatment can effectively alleviate endometrial damage caused by suction curettage, and can also effectively alleviate the formation of fibrotic tissue in the endometrium and subendometrium, and improve uterine receptivity.

[0031] The beneficial protections and effects of this invention are as follows:

[0032] The dimer immunoadhesin, pharmaceutical composition, and uses provided by this invention are simple to construct and express. Experiments have demonstrated that they have good therapeutic effects on infertility diseases such as maternal-fetal immune tolerance disorders and gynecological reproductive inflammation. By using them alone or in combination with other drugs for related diseases, they can effectively treat related diseases caused by maternal-fetal immune dysregulation and have broad clinical application prospects. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the structure of TIGIT immunoadhesive.

[0034] Figure 2 The effects of various soluble dimeric immunoadhesins on the secretion of IL-10 and TNFα by decidual dendritic cells;

[0035] Figure 3 To investigate the therapeutic effects of various soluble dimeric immunoadhesins in an immune-mediated spontaneous abortion mouse model;

[0036] Figure 4 The effect of soluble dimer immunoadhesin on the expression of T helper cells in mouse para-aortic lymph nodes;

[0037] Figure 5 The results show the effects of soluble dimeric immunoadhesins on LIF and OSM, endometrial receptivity markers, in mice after conception.

[0038] Figure 6 The results show the effect of soluble dimer immunoadhesins on the degree of endometrial fibrosis in mice. Detailed Implementation

[0039] The following examples and experimental cases further illustrate the present invention and should not be construed as limiting the invention. The examples do not include detailed descriptions of conventional methods, such as those used for constructing vectors and plasmids, inserting genes encoding proteins into such vectors and plasmids, or introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press.

[0040] Example 1. Construction and expression of soluble dimer immunoadhesins

[0041] like Figure 1As shown, soluble dimer immunoadhesin is a dimer carrying antibody IgG Fc. The construction and expression methods of the dimer immunoadhesin itself are routine experimental techniques in the field, and are briefly described below:

[0042] (1) The whole-gene synthesis of soluble dimeric immunoadhesins TIGIT-Fc-wt (containing two polypeptide chains, the amino acid and nucleotide sequences of each polypeptide chain are shown in SEQ ID NO:2 and SEQ ID NO:9); TIGIT-Fc-LALA-PG (containing two polypeptide chains, the amino acid and nucleotide sequences of each polypeptide chain are shown in SEQ ID NO:3 and SEQ ID NO:10); TIGIT / CTLA4-Fc (containing two polypeptide chains, the amino acid and nucleotide sequences of the first polypeptide chain are shown in SEQ ID NO:5 and SEQ ID NO:11, and the amino acid and nucleotide sequences of the second polypeptide chain are shown in SEQ ID NO:6 and SEQ ID NO:12); TIGIT / IL10-Fc (containing two polypeptide chains, the amino acid and nucleotide sequences of the first polypeptide chain are shown in SEQ ID NO:5 and SEQ ID NO:11, and the amino acid and nucleotide sequences of the second polypeptide chain are shown in SEQ ID NO:8 and SEQ ID NO:13).

[0043] (2) Expression and purification of fusion protein

[0044] Soluble dimeric immunoadhesins were expressed according to the methods described in the literature (Finck B K. Science, 265.; Mihara M et al. Journal of Clinical Investigation. 2000; 106: 91-101; Yu X, et al. Nature Immunology. 2009; 10: 48-57. Liu S, et al. Clin Immunol. 2019 Jun; 203: 72-80.).

[0045] Example 2. Biacore Analysis

[0046] The affinity of each immunoadhesin was detected using a Biacore T100 (GE Healthcare) according to the method described in the literature (Bruhns P. et al. Blood, 2009, 113(16): 3716-3725.). The specific affinity values ​​are shown in Table 1.

[0047] Table 1. Biacore analysis results (unit: nM)

[0048]

[0049] Example 3. Effects of Dimer Immunoadhesin on Decidual Immune Cells

[0050] Dendritic cells (DCs) (CD1c positive) were isolated from decidual tissue from individuals who had terminated pregnancy for non-medical reasons. The isolation and screening methods were the same as those in the literature (Guo PF, et al. Blood, 2010, 116(12):2061-2069.). The DCs were divided into a negative control group (control IgG, 10 μg / mL), a dimeric immunoadhesin treatment group (dimeric immunoadhesin, 10 μg / mL), and an LPS treatment group (100 ng / mL). After 48 hours, the levels of interleukin-10 (IL-10) and tumor necrosis factor-α (TNFα) were detected using the same methods as in the literature (Guo PF, et al. Blood, 2010, 116(12):2061-2069.).

[0051] Test results as follows Figure 2 As shown, the dimer immunoadhesin can significantly increase IL-10 secretion levels without increasing TNFα levels, confirming that the dimer immunoadhesin can exert immune tolerance through DCs.

[0052] Example 4. Therapeutic effect of dimeric immunoadhesin on spontaneous abortion model

[0053] A stress-induced abortion model was established using female CBA / J mice and male DBA / 2J mice. This abortion model is a classic research model of maternal-fetal immune tolerance disorder. The establishment method, experimental method and observation time points are the same as those in the literature (Blois SM, et al.. Nature Medicine, 2007, 13(12):1450-1457.).

[0054] Immediately after confirming vaginal pregnancy, mice were divided into a negative control group, a stress group, and a dimeric immunoadhesin treatment group. The negative control group and the stress group were treated with control IgG. The experimental methods were referenced in (Blois SM, et al. Nature Medicine, 2007, 13(12): 1450-1457.), and embryonic development was monitored. All drugs were administered at a concentration of 20 μg per mouse via intraperitoneal injection daily.

[0055] Experimental results are as follows Figure 3 The results showed that the miscarriage rate in each treatment group was significantly lower than that in the stress-induced miscarriage group, indicating that the use of dimeric immunoadhesive has a good therapeutic effect.

[0056] Example 5. Effects of Dimer Immunoadhesin on T Helper Cells

[0057] Para-aortic lymph nodes were isolated from mice in the control group, stress group, and TIGIT-Fc-LALA-PG dimer immunoadhesin treatment group, and the level of Foxp3-positive T helper lymphocytes was detected. The isolation and detection methods were the same as those in the literature (Kim BJ, et al. Proceedings of the National Academy of Sciences, 2015, 112(5):1559-1564). The results are as follows: Figure 4 As shown, the results indicate that administration of TIGIT-Fc-LALA-PG dimer immunoadhesin can effectively increase the level of Foxp3-positive T helper lymphocytes.

[0058] Example 6. Effect of Dimer Immunoadhesin on Endometrial Receptivity in Mice After Endometrial Injury

[0059] An endometrial injury model was established in ICR mice using negative pressure suction curettage. Eight-week-old mice were divided into a suction curettage group, suction curettage + dimer immunoadhesive treatment groups, and a blank control group, with ten mice in each group. The model establishment methods for the suction curettage group and the suction curettage + dimer immunoadhesive treatment group were the same as those in the literature (Wang Yanpeng, et al. Journal of Zhejiang University: Medical Edition, 2017(46):191.).

[0060] After establishing the model, each treatment group began receiving medication, with all drugs administered at a concentration of 20 μg per mouse via intraperitoneal injection daily. The suction curettage group received a control antibody. Medication was stopped for one week after two weeks, and the estrus period was determined based on vaginal smears. Males and females were caged at a 1:1 ratio that evening, and vaginal plugs were examined at 7:00 AM the following morning; the presence of plugs indicated 0.5 days of gestation. Endometrial receptivity was assessed in each group using the same method as in the literature, employing ELISA to detect LIF (leukemia inhibitory factor) and OSM (tumor suppressor) levels in tissues. The endometrial receptivity window in mice was approximately 4 days after conception. The expression of endometrial receptivity markers LIF and OSM in mice after conception showed the following results: Figure 5 As shown, the results indicate that dimeric immunoadhesin therapy can effectively alleviate endometrial damage caused by suction curettage.

[0061] Example 7. Effect of Dimer Immunoadhesin on Intrauterine Adhesions in Mice

[0062] Eight-week-old ICR mice were randomly divided into three groups: an intrauterine adhesion group, an intrauterine adhesion + dimer immunoadhesive treatment group, and a blank control group. Ten mice were in each group. The intrauterine adhesion group and the intrauterine adhesion + dimer immunoadhesive treatment group underwent intrauterine adhesion modeling. The modeling method was as follows: The mice were fasted for 12 hours the night before surgery, but allowed free water. After anesthesia and routine disinfection of the lower abdomen, a midline incision was made to expose the Y-shaped uterus. Using a 1mL syringe, a needle was inserted into the uterine cavity at the pelvic division point, and 50 μL of 25% phenol gel was slowly injected towards both ovaries.

[0063] After model establishment, the abdomen was closed in layers, and the surgical area was disinfected. Following model establishment, the control group was injected with saline, while the drug-treated groups began drug administration. The intraperitoneal adhesion group received control antibody treatment. All drugs were administered at a concentration of 20 μg per mouse intraperitoneally daily for 18 consecutive days. Mice were then sacrificed to assess the degree of uterine fibrosis. Figure 6 The results showed that dimeric immunoadhesin therapy could effectively alleviate the formation of fibrotic tissue in the endometrium and subendometrium.

[0064] In summary, the study demonstrated good therapeutic effects on maternal-fetal immune tolerance disorders and uterine receptivity-related diseases in a spontaneous abortion mouse model, which is beneficial for subsequent clinical trials.

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

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[0074]

[0075] sequence list <110> Fengchao Pharmaceutical Technology (Shanghai) Co., Ltd. <120> Dimeric immunoadhesins, pharmaceutical compositions and uses <130> Claims, Description <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 120 <212> PRT <213> Artificial sequence <400> 1 Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys 1 5 10 15 Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30 Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45 Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60 Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn 65 70 75 80 Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95 Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110 His Gly Ala Arg Phe Gln Ile Pro 115 120 <210> 2 <211> 352 <212> PRT <213> Artificial sequence <400> 2 Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys 1 5 10 15 Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30 Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45 Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60 Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn 65 70 75 80 Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95 Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110 His Gly Ala Arg Phe Gln Ile Pro Glu Pro Lys Ser Cys Asp Lys Thr 115 120 125 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 130 135 140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 165 170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 260 265 270 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 3 <211> 352 <212> PRT <213> Artificial sequence <400> 3 Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys 1 5 10 15 Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30 Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45 Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60 Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn 65 70 75 80 Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95 Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110 His Gly Ala Arg Phe Gln Ile Pro Glu Pro Lys Ser Cys Asp Lys Thr 115 120 125 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 130 135 140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 165 170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 260 265 270 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 4 <211> 125 <212> PRT <213> Artificial sequence <400> 4 Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile 1 5 10 15 Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val 20 25 30 Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys 35 40 45 Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser 50 55 60 Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln 65 70 75 80 Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu 85 90 95 Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile 100 105 110 Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln 115 120 125 <210> 5 <211> 352 <212> PRT <213> Artificial sequence <400> 5 Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys 1 5 10 15 Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30 Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45 Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60 Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn 65 70 75 80 Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95 Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110 His Gly Ala Arg Phe Gln Ile Pro Glu Pro Lys Ser Cys Asp Lys Thr 115 120 125 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 130 135 140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 165 170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu 245 250 255 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 260 265 270 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 6 <211> 357 <212> PRT <213> Artificial sequence <400> 6 Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile<​​Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val 20 25 30 Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys 35 40 45 Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser 50 55 60 Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln 65 70 75 80 Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu 85 90 95 Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile 100 105 110 Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys 115 120 125 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 130 135 140 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 145 150 155 160 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 165 170 175 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 180 185 190 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 195 200 205 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 210 215 220 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 225 230 235 240 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 245 250 255 Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln 260 265 270 Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 275 280 285 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 290 295 300 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 305 310 315 320 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 325 330 335 缬氨酸与谷氨酸、丙氨酸、亮氨酸、组氨酸、天冬酰胺、组氨酸、酪氨酸、苏氨酸、谷氨酰胺、赖氨酸、丝氨酸、亮氨酸、丝氨酸 340 345 350 亮氨酸、丝氨酸、脯氨酸、甘氨酸、赖氨酸 355 <210> 7 <211> 160 <212> PRT <213> Artificial sequence <400> 7 丝氨酸、脯氨酸、甘氨酸、谷氨酰胺、甘氨酸、苏氨酸、谷氨酰胺、丝氨酸、谷氨酸、天冬酰胺、丝氨酸、半胱氨酸、苏氨酸、组氨酸、苯丙氨酸、脯氨酸 1 5 10 15 甘氨酸、天冬酰胺、亮氨酸、脯氨酸、天冬酰胺、甲硫氨酸、亮氨酸、精氨酸、天冬氨酸、亮氨酸、精氨酸、天冬氨酸、丙氨酸、苯丙氨酸、丝氨酸、精氨酸 20 25 30 缬氨酸、赖氨酸、苏氨酸、苯丙氨酸、苯丙氨酸、谷氨酰胺、甲硫氨酸、赖氨酸、天冬氨酸,谷氨酰胺、亮氨酸、天冬氨酸、天冬酰胺、亮氨酸、亮氨酸、亮氨酸 35 40 45 赖氨酸、谷氨酸、丝氨酸、亮氨酸、亮氨酸、谷氨酸、天冬氨酸、苯丙氨酸、赖氨酸、甘氨酸、酪氨酸、亮氨酸、甘氨酸、半胱氨酸、谷氨酰胺、丙氨酸 50 55 60 亮氨酸、丝氨酸、谷氨酸、甲硫氨酸、异亮氨酸、谷氨酰胺、苯丙氨酸、酪氨酸、亮氨酸、谷氨酸、谷氨酸、缬氨酸、甲硫氨酸、脯氨酸、谷氨酰胺、丙氨酸 65 70 75 80 谷氨酸、天冬酰胺、谷氨酰胺天冬氨酸、脯氨酸、天冬氨酸、异亮氨酸、赖氨酸、丙氨酸、组氨酸、缬氨酸、天冬酰胺、丝氨酸、亮氨酸、甘氨酸、谷氨酸 85 90 95 天冬酰胺、亮氨酸、赖氨酸,苏氨酸、亮氨酸、精氨酸、亮氨酸、精氨酸、亮氨酸、精氨酸、精氨酸、半胱氨酸、组氨酸、精氨酸、苯丙氨酸、亮氨酸 100 105 110 Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe 115 120 125 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp 130 135 140 Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 145 150 155 160 <210> 8 <211> 392 <212> PRT <213> Artificial sequence <400> 8 Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro 1 5 10 15 Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30 Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45 Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala i 50 55 60 Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala 65 70 75 80 Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95 Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105 110 Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe 115 120 125 Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp 130 135 140 Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn 145 150 155 160 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 165 170 175 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 180 185 190 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 195 200 205 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 210 215 220 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 225 230 235 240 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 245 250 255 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 260 265 270 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 275 280 285 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr 290 295 300 Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser 305 310 315 320 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 325 330 335 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 340 345 350 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 355 360 365 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 370 375 380 Ser Leu Ser Leu Ser Pro Gly Lys 385 390 <210> 9 <211> 1056 <212> DNA <21 <400> 9 atgatgaccg gcaccatcga gaccaccggc aacatcagcg ccgagaaggg cggcagcatc 60 atcctgcagt gccacctgag cagcaccacc gcccaggtga cccaggtgaa ctgggagcag 120 caggaccagc tgctggccat ctgcaacgcc gacctgggct ggcacatcag ccccagcttc 180 aaggacagag tggcccccgg ccccggcctg ggcctgaccc tgcagagcct gaccgtgaac 240 gacaccggcg agtacttctg catctaccac acctaccccg acggcaccta caccggcaga 300 atcttcctgg aggtgctgga gagcagcgtg gccgagcacg gcgccagatt ccagatcccc 360 gagcccaaga gctgcgacaa gacccacacc tgccccccct gccccgcccc cgagctgctg 420 ggcggcccca gcgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcaga 480 acccccgagg tgacctgcgt ggtggtggac gtgagccacg aggaccccga ggtgaagttc 540 aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcccag agaggagcag 600 tacaacagca cctacagagt ggtgagcgtg ctgaccgtgc tgcaccagga ctggctgaac 660 ggcaaggagt acaagtgcaa ggtgagcaac aaggccctgc ccgcccccat cgagaagacc 720 atcagcaagg ccaagggcca gcccagagag ccccaggtgt acaccctgcc ccccagcaga 780 gacgagctga ccaagaacca ggtgagcctg acctgcctgg tgaagggctt ctaccccagc 840 gacatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 900 cccgtgctgg acagcgacgg cagcttcttc ctgtacagca agctgaccgt ggacaagagc 960 agatggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 1020 tacacccaga agagcctgag cctgagcccc ggcaag 1056 <210> 10 <211> 1056 <212> DNA <213> Artificial sequence <400> 10 atgatgaccg gcaccatcga gaccaccggc aacatcagcg ccgagaaggg cggcagcatc 60 atcctgcagt gccacctgag cagcaccacc gcccaggtga cccaggtgaa ctgggagcag 120 caggaccagc tgctggccat ctgcaacgcc gacctgggct ggcacatcag ccccagcttc 180 aaggacagag tggcccccgg ccccggcctg ggcctgaccc tgcagagcct gaccgtgaac 240<00005gacaccggcg agtacttctg catctaccac acctaccccg acggcaccta caccggcaga 300 atcttcctgg aggtgctgga gagcagcgtg gccgagcacg gcgccagatt ccagatcccc 360 gagcccaaga gctgcgacaa gacccacacc tgccccccct gccccgcccc cgaggccgcc 420 ggcggcccca gcgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcaga 480 acccccgagg tgacctgcgt ggtggtggac gtgagccacg aggaccccga ggtgaagttc 540 aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcccag agaggagcag 600 tacaacagca cctacagagt ggtgagcgtg ctgaccgtgc tgcaccagga ctggctgaac 660 ggcaaggagt acaagtgcaa ggtgagcaac aaggccctgg gcgcccccat cgagaagacc 720 atcagcaagg ccaagggcca gcccagagag ccccaggtgt acaccctgcc ccccagcaga 780 gacgagctga ccaagaacca ggtgagcctg acctgcctgg tgaagggctt ctaccccagc 840 gacatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 900 cccgtgctgg acagcgacgg cagcttcttc ctgtacagca agctgaccgt ggacaagagc 960 agatggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 1020 tacacccaga agagcctgag cctgagcccc ggcaag 1056 <210> 11 <211> 1056 <212> DNA <213> Artificial sequence <400> 11 atgatgaccg gcaccatcga gaccaccggc aacatcagcg ccgagaaggg cggcagcatc 60 atcctgcagt gccacctgag cagcaccacc gcccaggtga cccaggtgaa ctgggagcag 120 caggaccagc tgctggccat ctgcaacgcc gacctgggct ggcacatcag ccccagcttc 180 aaggacagag tggcccccgg ccccggcctg ggcctgaccc tgcagagcct gaccgtgaac 240 gacaccggcg agtacttctg catctaccac acctaccccg acggcaccta caccggcaga 300 atcttcctgg aggtgctgga gagcagcgtg gccgagcacg gcgccagatt ccagatcccc 360 gagcccaaga gctgcgacaa gacccacacc tgccccccct gccccgcccc cgagctgctg 420 ggcggcccca gcgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcaga 4~80 acccccgagg tgacctgcgt ggtggtggac gtgagccacg aggaccccga ggtgaagttc 540 aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcccag agaggagcag 600 tacaacagca cctacagagt ggtgagcgtg ctgaccgtgc tgcaccagga ctggctgaac 660 ggcaaggagt acaagtgcaa ggtgagcaac aaggccctgc ccgcccccat cgagaagacc 720 atcagcaagg ccaagggcca gcccagagag ccccaggtgt gcaccctgcc ccccagcaga 780 gacgagctga ccaagaacca ggtgagcctg agctgcgccg tgaagggctt ctaccccagc 840 gacatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 900 cccgtgctgg acagcgacgg cagcttcttc ctggtgagca agctgaccgt ggacaagagc 960 agatggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 1020 tacacccaga agagcctgag cctgagcccc ggcaag 1056 [[ID=**19**]]<210> 12 [[ID=**21**]]<211> 1071 [[ID=**23**]]<212> DNA [[ID=**25**]]<213> Artificial sequence [[ID=**27**]]<400> 12 [[ID=**29**]]atgcacgtgg cccagcccgc cgtggtgctg gccagcagca gaggcatcgc cagcttcgtg 60 tgcgagtacg ccagccccgg caaggccacc gaggtgagag tgaccgtgct gagacaggcc 120 gacagccagg tgaccgaggt gtgcgccgcc acctacatga tgggcaacga gctgaccttc 180 ctggacgaca gcatctgcac cggcaccagc agcggcaacc aggtgaacct gaccatccag 240 ggcctgagag ccatggacac cggcctgtac atctgcaagg tggagctgat gtaccccccc 300 ccctactacc tgggcatcgg caacggcacc cagatctacg tgatcgaccc cgagccctgc 360 cccgacagcg accaggagcc caagagctgc gacaagaccc acacctgccc cccctgcccc 420 gcccccgagc tgctgggcgg ccccagcgtg ttcctgttcc cccccaagcc caaggacacc 480 ctgatgatca gcagaacccc cgaggtgacc tgcgtggtgg tggacgtgag ccacgaggac 540 cccgaggtga agttcaactg gtacgtggac ggcgtggagg tgcacaacgc caagaccaag 600 cccagagagg agcagacaa cagcacctac agagtggtga gcgtgctgac cgtgctgcac 660 caggactggc tgaacggcaa ggagtacaag tgcaaggtga gcaacaaggc cctgcccgcc 720 cccatcgaga agaccatcag caaggccaag ggccagccca gagagcccca ggtgtacacc 780 ctgcccccct gcagagacga gctgaccaag aaccaggtga gcctgtggtg cctggtgaag 840 ggcttctacc ccagcgacat cgccgtggag tgggagagca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggacagc gacggcagct tcttcctgta cagcaagctg 960 accgtggaca agagcagatg gcagcagggc aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc ctgagcctga gccccggca g <210> 13 <211> 1176 <212> DNA <213> Artificial sequence (Artificial sequence) <400> 13 agccccggcc agggcaccca gagcgagaac acttccccgg caacctgccc aacatgctga gagacctgag agacgccttc agcagagtga agaccttctt ccagatgag gaccagctgg acaacctgct gctgaaggag agcctgctgg aggacttcaa gggctacctg ggctgccagg ccctgagcga gatgatccag ttctacctgg aggaggtgat gccccaggcc 240 gagaaccagg accccgacat caaggcccac gtgaacagcc tgggcgagaa cctgaagacc ctgagactga gactgagaag atgccacaga ttcctgccct gcgagaacaa gagcaaggcc 360 gtggagcagg tgaagaacgc cttcaacaag ctgcaggaga agggcatcta caaggccatg 420 agcgagttcg acatcttcat caactacatc gaggcctaca tgaccatgaa gatcagaaac 480 gagcccaaga gctgcgacaa gacccacacc tgccccccct gccccgcccc cgagctgctg 540 ggcggcccca gcgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcaga 600 acccccgagg tgacctgcgt ggtggtggac gtgagccacg aggaccccga ggtgaagttc 660 aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcccag agaggagcag 720 tacaacagca cctacagagt ggtgagcgtg ctgaccgtgc tgcaccagga ctggctgaac 780 ggcaaggagt acaagtgcaa ggtgagcaac aaggccctgc ccgcccccat cgagaagacc 840 atcagcaagg ccaagggcca gcccagagag ccccaggtgt acaccctgcc cccctgcaga 900 gacgagctga ccaagaacca ggtgagcctg tggtgcctgg tgaagggctt ctaccccagc 960 gacatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 1020 cccgtgctgg acagcgacgg cagcttcttc ctgtacagca agctgaccgt ggacaagagc 1080 agatggcagc agggcaacgt gttcagctgc agcgtgatgc acgaggccct gcacaaccac 1140 tacacccaga agagcctgag cctgagcccc ggcaag 1176

Claims

1. The use of soluble dimeric immunoadhesin in the preparation of drugs for the treatment and prevention of recurrent spontaneous abortion and gynecological reproductive inflammatory diseases, characterized in that, The recurrent spontaneous abortion refers to recurrent spontaneous abortion due to maternal-fetal immune tolerance disorder; The gynecological reproductive inflammation-related diseases mentioned are endometrial damage or endometrial fibrosis. The soluble dimer immunoadhesive comprises a dimerized first polypeptide chain and a second polypeptide chain. The soluble dimer immunoadhesin is selected from any of the following: (1) The amino acid sequence of each polypeptide chain is as shown in SEQ ID NO:2; (2) The amino acid sequence of each polypeptide chain is shown in SEQ ID NO:3; (3) The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO:5, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO:

5. As shown in SEQ ID NO:6; (4) The amino acid sequence of the first polypeptide chain is shown in SEQ ID NO:5, and the amino acid sequence of the second polypeptide chain is shown in SEQ ID NO:

5. As shown in SEQ ID NO:8.

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