DIMER IMMUNOADHESIN, PHARMACEUTICAL COMPOSTION AND USE THEREOF

Abstract

A soluble dimeric immunoadhesin includes a dimerized first polypeptide chain and a dimerized second polypeptide chain. The first polypeptide chain has a general formula of Z1-Z2, and the second polypeptide chain has a general 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 dimerization 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 dimerization domain of an immunoglobulin constant region or a functional variant or fragment thereof. A dimeric protein can be used for the treatment and prevention of infertility-related diseases.

Claims

1. A soluble dimeric immunoadhesin, comprising a dimerized first polypeptide chain and a dimerized second polypeptide chain, wherein the first polypeptide chain has a general formula of Z1-Z2, and the second polypeptide chain has a general formula of Y1-Y2, wherein 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 dimerization 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; and Y2 is a dimerization domain or a functional variant or fragment thereof.

2. The soluble dimeric immunoadhesin according to claim 1, wherein, the Z1 and the Y1 are the same or different extracellular domains or functional variants or fragments thereof, and each being any one selected from the group consisting of: 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; CD70; 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 receptor; 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 semaphoring receptor CD72; Kaposi's sarcoma-associated herpesvirus GPCR; lipoxin A4 receptor; lymphotoxin β receptor; lysophospholipid growth factor receptor; neurokinin 1; μ-, δ-, and κ-opioid receptors of endorphins; oncostatin M receptor; osteopontin receptor; osteoprotegerin; Ox40; OX40L; PACAP and VIP receptors; PAF receptor; poxvirus; IFNα/β receptor homologs; poxvirus IFNγ receptor homologs; poxvirus IL-10 receptor homologs; poxvirus membrane-bound G protein-coupled receptor homologs; poxvirus-secreted chemokine binding protein; poxvirus TNF receptor homologs; prolactin receptor; RANK; RON receptor; SCF receptor; somatostatin receptor; T1/ST2; TGF-β receptor; TNF receptor; TNFRSF19; TPO receptor; US28; XCR1; erythropoietin receptor; growth hormone receptor; leukemia inhibitory factor receptor; and C-kit receptor.

3. The soluble dimeric immunoadhesin according to claim 1, wherein. the Z1 and the Y1 are the same or different cytokines or functional variants or fragments thereof, and each being any one selected from the group consisting of: α-MSH; 9E3/cCAF; ACTH; activin; AK155; angiogenesis inhibitor; Apo2L/TRAIL; APRIL; BAFF; BLR1 ligand/BCA-1/BLC/CXCL13; BMP family; BRAK; calcitonin gene-related peptide; molluscum contagiosum virus CC chemokine; 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 chemotactic factor 2/MPIF-2/CCL24; eosinophil chemotactic factor/CCL11; erythropoietin; Exodus-1/LARC/MIP-3α; Fas ligand; Flt-3 ligand; fMLP; Fractalkine/CX3CL1; G-CSF; GCP-2/CXCL6; GM-CSF; growth hormone; HCC-1/CCL14; HCC-4/CCL16; high-mobility group box 1; 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; leukotaxin 1/HCC-2/MIP-1δ/CCL15; leukotriene B4; LIGHT; lipoxin; chemotactic factor for lymphocyte (ChFL)/XCL1; lymphotoxins a and (3; lysophospholipid growth factor; macrophage-derived chemokine; macrophase-stimulating protein; 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-1a/CCL3 and MIP-1β/CCL4; MIP-1γ/MRP-2/CCF18/CCL9/10; MuC10/CCL6; oncostatin M; osteopontin; parapoxvirus IL-10 homologs; PARC/DC-CCK1/AMAC-1/CCL18; PDGF-A; PDGF-B; PDGF-C; PDGF-D; platelet activating factor; platelet factor 4/CXCL4; poxvirus growth factor related to epidermal growth factor; poxvirus-secreted complement regulatory protein; poxvirus vascular endothelial growth factor homologs of orf virus; prolactin; RANK ligand; RANTES/CCL5; S100A12; SDF-1/CXCL12; SERP-1, secreted poxvirus serine protease inhibitor; SLC/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 semaphorin; vCXC-1 and vCXC-2; VEGF; VIP and PACAP; and viral IL-10 variants.

4. The soluble dimeric immunoadhesin according to claim 1, wherein, the Z2 and the Y2 are Fc fragments of IgG or Fc mutants that change biological activity thereof, or heterodimeric IgG-Fc fragments constructed using Knob-in-holes technology, ART-Ig technology that changes charge polarity, or BiMab technology, and flexible linker can be added if necessary.

5. The soluble dimeric immunoadhesin according to claim 1, wherein, when each of the Z1 and the Y1 is an extracellular domain of TIGIT or a functional variant or fragment thereof, amino acid sequences of the Z1 and the Y1 are at least 90% identical to an amino acid sequence shown in SEQ ID NO: 1; and the soluble dimeric immunoadhesin has an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.

6. The soluble dimeric immunoadhesin according to claim 1, wherein, the Z1 is an extracellular domain of TIGIT or a functional variant or fragment thereof, the Y1 is an extracellular domain of CTLA4 or a functional variant or fragment thereof, an amino acid sequence of the Z1 is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1, and an amino acid sequence of the Y1 is at least 90% identical to an amino acid sequence shown in SEQ ID NO: 4; and the Z1-Z2 polypeptide chain comprises an amino acid sequence shown in SEQ ID NO: 5, and the Y1-Y2 polypeptide chain comprises an amino acid sequence shown in SEQ ID NO: 6.

7. The soluble dimeric immunoadhesin according to claim 1, wherein, the Z1 is an extracellular domain of TIGIT or a functional variant or fragment thereof, the Y1 is cytokine IL-10 or a functional variant or fragment thereof, the amino acid sequence of the Z1 is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence of the Y1 is at least 90% identical to an amino acid sequence shown in SEQ ID NO: 7; and the Z1-Z2 polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 5, and the Y1-Y2 polypeptide chain comprises an amino acid sequence shown in SEQ ID NO: 8.

8. A pharmaceutical composition comprising the soluble dimeric immunoadhesin according to claim 1, further comprising a medically acceptable pharmaceutical carrier.

9. Use of the soluble dimeric immunoadhesin according to claim 1 in the preparation of a medicine for the treatment and prevention of infertility-related diseases.

10. The use of the soluble dimeric immunoadhesin in the preparation of a medicine for the treatment and prevention of infertility-related diseases according to claim 9, wherein, the infertility-related diseases comprise diseases related to maternal-fetal immune tolerance disorder or gynecological reproductive inflammation.

11. The use of the soluble dimeric immunoadhesin in the preparation of a medicine for the treatment and prevention of infertility-related diseases according to claim 10, wherein, the diseases related to maternal-fetal immune tolerance disorder comprise recurrent spontaneous abortion, threatened abortion, or treatment failure of assisted reproductive technology; and the diseases related to gynecological reproductive inflammation comprise pelvic inflammatory disease, decreased endometrial receptivity, endometritis, endometrial polyps, intrauterine adhesions, reduction of endometrial glands, endometrial fibrosis, amenorrhea, abnormal uterine bleeding, adenomyosis and endometriosis, reproductive system infection or hysteromyoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 is a schematic diagram of the structure of TIGIT immunoadhesin;

[0035] FIG. 2 illustrates an effect of a plurality of soluble dimeric immunoadhesins on the secretion of IL-10 and TNFα in decidual dendritic cells;

[0036] FIG. 3 illustrates a therapeutic effect of administration of a plurality of soluble dimeric immunoadhesins in a mouse model of immune spontaneous abortion;

[0037] FIG. 4 illustrates an effect of soluble dimeric immunoadhesin on the expression of T helper cells in mouse para-aortic lymph nodes;

[0038] FIG. 5 illustrates an effect of soluble dimeric immunoadhesin on pregnant mouse endometrial receptivity markers LIF and OSM;

[0039] FIG. 6 illustrates an effect of soluble dimeric immunoadhesin on the degree of endometrial fibrosis in mice.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] The following examples and experimental examples further illustrate the present disclosure, and should not be construed as limiting the present disclosure. The examples do not include detailed descriptions of conventional methods, such as those used for constructing vectors and plasmids, those for inserting protein-encoding genes into such vectors and plasmids, or those for introducing plasmids into host cells. Such methods are well known to those of ordinary skills in the art, and have been described in a plurality of publications, including Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2.sup.nd edition, Cold spring Harbor Laboratory Press.

Example 1. Construction and Expression of Soluble Dimeric Immunoadhesins

[0041] As shown in FIG. 1, soluble dimeric immunoadhesin is a dimer with antibody IgG Fc. The method for constructing and expressing dimeric immunoadhesin itself is a conventional experimental technique in the field. A briefly description is as follows:

[0042] (1) Full gene synthesis was used to synthesize soluble dimeric immunoadhesins: TIGIT-Fc-wt (containing two polypeptide chains; the amino acid sequence and nucleotide sequence 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 sequence and nucleotide sequence 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 sequence and nucleotide sequence of the first polypeptide chain are shown in SEQ ID NO: 5 and SEQ ID NO: 11, and those 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 sequence and nucleotide sequence of the first polypeptide chain are shown in SEQ ID NO: 5 and SEQ ID NO: 11, and those of the second polypeptide chain are shown in SEQ ID NO: 8 and SEQ ID NO: 13).

[0043] (2) Expression and Purification of Fusion Proteins

[0044] The soluble dimeric immunoadhesins were expressed according to the method as 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 June; 203:72-80).

Example 2. Biacore Analysis

[0045] Biacore T100 (GE Healthcare) was used to detect the affinity of each immunoadhesin according to the method in the literature (Bruhns P. et al. Blood, 2009, 113(16): 3716-3725). The specific values of the detected affinity are shown in Table 1.

TABLE-US-00001 TABLE 1 Biacore analysis results (in nM) TIGIT- TIGIT-Fc- TIGIT/ TIGIT/ Parameter Fc-wt LALA-PG CTLA4-Fc IL10-Fc Affinity/kinetics 1.55 1.84 2.39 2.41 of human CD155 Affinity/kinetics 2.07 2.15 3.55 3.21 of mouse CD155 Affinity/kinetics — — 2.51 — of B7 Affinity/kinetics — — — 0.91 of anti-IL 10

Example 3. The Effect of Dimeric Immunoadhesin on Decidual Immune Cells

[0046] Dendritic cells (DCs) (CD1c positive) were isolated from human decidual tissue that terminated pregnancy for non-medical reasons. Isolation and screening methods were the same as those in the literature (Guo P F, et al. Blood, 2010, 116(12): 2061-2069). The DC cells were divided into a negative control group (control IgG, 10 μg/mL), a dimeric immunoadhesin treatment group (dimeric immunoadhesin, 10 μg/mL), and LPS treatment group (100 ng/mL). The levels of interleukin 10 (IL-10) and tumor necrosis factor α (TNFα) were detected after 48 h. The detection method was the same as that in the literature (Guo P F, et al. Blood, 2010, 116(12): 2061-2069).

[0047] The detection results are shown in FIG. 2, showing that the dimeric immunoadhesin can significantly increase the secretion level of IL-10 without increasing the level of TNFα, and demonstrating that the dimeric immunoadhesin can exert immune tolerance through DCs.

Example 4. Therapeutic Effect of Dimeric Immunoadhesin on Spontaneous Abortion Model

[0048] Female CBA/J mice and male DBA/2J mice were used to establish a stress abortion model. This abortion model was a classic research model of maternal-fetal immune tolerance disorder. Its establishment method, experimental method and observation time points were the same as those in the literature (Blois S M, et al. Nature Medicine, 2007, 13(12): 1450-1457).

[0049] The mice were divided into a negative control group, a stress group, and a dimeric immunoadhesin treatment group immediately after confirming that vaginal plugs were pregnant. The negative control group and the stress group were treated with control IgG. The experimental method referred to the literature (Blois S M, et al. Nature Medicine, 2007, 13(12):1450-1457), and embryonic development was detected. All drugs were intraperitoneally administered at a concentration of 20 μs per mouse per day.

[0050] The experimental results are shown in FIG. 3. The abortion rate of each treatment group is significantly lower than that of the stress abortion group, indicating that the use of dimeric immunoadhesin has a good therapeutic effect.

Example 5. The Effect of Dimeric Immunoadhesin on T Helper Cells

[0051] The para-aortic lymph nodes were separated from the mice in the control group, the stress group and the TIGIT-Fc-LALA-PG dimeric immunoadhesin treatment group, and the levels of Foxp3-positive T helper lymphocytes therein were detected. The separation and detection methods were the same as those in the literature (Kim B J, et al. Proceedings of the National Academy of Sciences, 2015, 112(5): 1559-1564). The results are shown in FIG. 4, and the results show the administration of TIGIT-Fc-LALA-PG dimeric immunoadhesin can effectively increase the level of Foxp3-positive T helper lymphocytes.

Example 6. The Effect of Dimeric Immunoadhesin on Endometrial Receptivity after Endometrial Injury in Mice

[0052] An endometrial injury model was established in ICR mice by negative pressure uterine aspiration. The 8-week-old mice were divided into a uterine aspiration group, uterine aspiration+dimeric immunoadhesin treatment groups, and a blank control group. Each group contained ten mice. The modeling methods in the uterine aspiration group and the uterine aspiration+dimeric immunoadhesin treatment groups were the same as those in the literature (Wang Y P, et al. Journal of Zhejiang University: Medicine Edition, 2017(46): 191).

[0053] After the model was established, each administration group started to administer, and all drugs were intraperitoneally administered at a concentration of 20 μg per mouse per day. The uterine aspiration group was given a control antibody. Two weeks later, the drug was withdrawn for one week, and then the estrus was determined according to the vaginal smear. The male and female mice were caged at 1:1 that night, and the vaginal plug was checked at 7:00 a.m. the next morning. Those with vaginal plug were recorded as pregnant for 0.5 days. Each group was tested for endometrial receptivity. The test method was the same as that in the literature. The levels of leukemia inhibitory factor (LIF) and oncostatin (OSM) in the tissues were tested by enzyme-linked immunosorbent assay (ELISA). The window period of the endometrial receptivity in mice was about 4 days after conception. The expression of LIF and OSM, which are endometrial receptivity markers of the pregnant mice, is shown in FIG. 5. The results show that dimeric immunoadhesin therapy can effectively alleviate the endometrial injury caused by uterine aspiration.

Example 7. The Effect of Dimeric Immunoadhesin on Intrauterine Adhesions in Mice

[0054] The 8-week-old ICR mice were divided into an intrauterine adhesions group, an intrauterine adhesion+dimeric immunoadhesin treatment group, and a blank control group. Each group contained 10 mice. The intrauterine adhesions group and the intrauterine adhesions+dimeric immunoadhesin treatment group were subjected to intrauterine adhesion modelling. The modeling method was as follows: the night before the operation, the mice were deprived of food but not water for 12 h; after anesthesia, the lower abdomen was routinely sterilized, and a midline incision was made to expose the Y-shaped uterus; using a 1 mL syringe, a needle was inserted into the uterine cavity at the uterine pelvis, and facing both sides 50 μL of 25% phenol mucilage was slowly injected in the direction of each ovary.

[0055] After the modeling was completed, the abdomen was closed in layers and the field of operation was disinfected. After the model was established, the control group was injected with normal saline, each administration group started to administer, and the intrauterine adhesions group was given control antibody. All drugs were intraperitoneally administered at a concentration of 20 μg per mouse per day. The mice were sacrificed to evaluate the degree of uterine fibrosis in the mice 18 days after continuous administration. According to the results in FIG. 6, the dimeric immunoadhesin therapy can effectively relieve the formation of endometrial and subendometrial fibrotic tissue.

[0056] In summary, in the mouse model of spontaneous abortion, dimeric immunoadhesin has excellent therapeutic effects on maternal-fetal immune tolerance disorders and diseases related to decreased endometrial receptivity, which is conducive to the conduct of subsequent clinical trials.