WO2022042329A1 - Tigit免疫粘附素在调控肿瘤免疫及调节血管生成产品中的应用 - Google Patents

Tigit免疫粘附素在调控肿瘤免疫及调节血管生成产品中的应用 Download PDF

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WO2022042329A1
WO2022042329A1 PCT/CN2021/112374 CN2021112374W WO2022042329A1 WO 2022042329 A1 WO2022042329 A1 WO 2022042329A1 CN 2021112374 W CN2021112374 W CN 2021112374W WO 2022042329 A1 WO2022042329 A1 WO 2022042329A1
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tigit
tumor
immunoadhesin
cells
angiogenesis
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French (fr)
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傅文燕
胡适
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沣潮医药科技（上海）有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of biomedical engineering, in particular to the application of TIGIT immunoadhesin in regulating tumor immunity and regulating angiogenesis products
  • TIGIT protein (UniProtKB code: Q495A1) is a member of the poliovirus receptor (PVR)/Nectin family, which consists of an extracellular immunoglobulin variable (IgV) domain, a type 1 transmembrane domain and a classical immune
  • ITIM receptor tyrosine inhibitory motif
  • ITT immunoglobulin tyrosine tail
  • TIGIT protein has a wide range of uses, and the applicant has applied for two patent applications based on the research results:
  • Invention patent CN109206523A discloses the preparation of TIGIT immunoadhesin and its use in autoimmune diseases;
  • Invention patent CN110669139A discloses TIGIT immunoadhesin Use in modulating maternal-fetal immune tolerance.
  • the tumor immune cycle refers to the process of developing an immune response in an appropriate way to kill cancer cells. Briefly, dendritic cells acquire antigens from dying cancer cells, then enter the lymph nodes to activate T cells, the activated T cells leave the lymph nodes and enter the circulatory system, infiltrate the tumor microenvironment through the blood vessel wall, and the T cells pass specific The receptor recognizes and kills tumor cells before continuing the cycle. In the study of tumor immunity, there are more studies on the TIGIT protein itself, and less on the TIGIT immunoadhesin formed by further genetic engineering of the TIGIT protein.
  • TIGIT immunoadhesin has unexpected technical effects in the application of enhancing immune cells to kill tumor cells and promoting the regulation of angiogenesis.
  • TIGIT immunoadhesin described in the present invention refers to the domain of TIGIT native protein that has the ability to bind its natural ligand or the unprotected domain of TIGIT protein or its functional variants or fragments. It has been described in detail in , which is a construction method well known to those skilled in the art, and will not be repeated here.
  • the TIGIT immunoadhesin Fc domain of the present invention further comprises a LALA-PG mutant to remove the potential immunoglobulin activity of its Fc domain.
  • the first aspect of the present invention provides the use of TIGIT immunoadhesin in the preparation of a reagent for enhancing the immune system and immune cells to clear tumors or a reagent for regulating angiogenesis.
  • the uses of the TIGIT immunoadhesin described in the present invention include: (1) enhancing the killing of tumor cells by immune cells, enhancing the number of immune cells infiltrated in tumor tissue, enhancing the recognition of tumor cells by immune cells, and reducing the amount of tumor cells in the tumor tissue.
  • Reagents for immunosuppressive signaling activity reducing immune cell stress-mediated epithelial-mesenchymal transition of tumor cells, and sensitizing immune checkpoint inhibitor-mediated tumor clearance.
  • the tumors referred to in the present invention include solid tumors, such as adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma, and the source of tumor tissue includes but is not limited to adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract , heart, kidneys, liver, lungs, muscles, ovaries, pancreas, parathyroid glands, penis, prostate, skin, salivary glands, spleen, testes, thymus, thyroid and uterus.
  • solid tumors such as adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma
  • the source of tumor tissue includes but is not limited to adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract , heart, kidneys, liver, lungs, muscles, ovaries, pancreas, parathyroid glands,
  • tumors of the central nervous system such as glioblastoma, astrocytoma, etc.
  • tumors of the eye include basal cell carcinoma, squamous cell carcinoma, melanoma, etc., as well as endocrine tumors Adenocarcinoma, neuroendocrine system tumor, gastrointestinal pancreatic endocrine system tumor, reproductive system tumor, including hematological system tumor and head and neck tumor. I won't list them all here.
  • TIGIT immunoadhesins to modulate angiogenesis allows the use of TIGIT immunoadhesins disclosed herein to include a variety of clinical diseases or conditions in which promotion of angiogenesis is desired. Such conditions include promotion of vascularization of regenerative tissue, ischemic limb disease, endometrial microangiogenesis, promotion of decidual tissue microangiogenesis, cerebral ischemia, stimulation of hair growth, erectile dysfunction, arteriosclerosis, vascular necrosis disease.
  • the clinical conditions for which promotion of angiogenesis is desired include stroke, macular degeneration, macular edema, lymphedema, disruption of the blood-retinal barrier, disruption of the blood-brain barrier, bacterial-induced regeneration of vascular lesions, especially Include vascular damage due to microvascular lesions during lung injury, kidney injury, renal fibrosis, stroke, vascular dementia, macular degeneration, and diabetic complications (eg, in the kidneys, eyes, skin, and/or extremities).
  • the TIGIT immunoadhesin disclosed in the present invention has the ability to stimulate angiogenesis, it can be further used in the preparation of products for enhancing wound healing. As evidenced, for example, by accelerated wound closure time compared to wound healing in the absence of the TIGIT immunoadhesin, increased granulation tissue at the wound site compared to no treatment, and/or enhanced neovascularization of the wound compared to no treatment .
  • the method or use of stimulating wound healing is for the treatment of diabetic ulcers.
  • the methods or uses for stimulating wound healing can be used in a variety of clinical situations involving wounds, including but not limited to decubitus ulcers, pressure ulcers, surgical incisions, traumatic tissue injury, burns, and tissue transplantation .
  • the clinical condition in which it is desired to promote angiogenesis also includes normalization of blood vessels in tumor tissue to improve hypoxic conditions of tumor tissue and reduce the risk of metastasis.
  • the second aspect of the present invention provides the use of an agent for enhancing immune system and immune cells to clear tumors or an agent for regulating angiogenesis in the preparation of anti-tumor drugs.
  • the third aspect of the present invention provides an enhancer or angiogenesis-regulating agent for promoting the immune system and immune cells to clear tumors, characterized in that TIGIT immunoadhesin, a nucleotide encoding it or a recombinant expression vector are used as The sole active ingredient also includes a pharmaceutically acceptable carrier.
  • TIGIT immunoadhesin-encoding polynucleotides may be in the form of RNA or DNA, including cDNA and synthetic DNA, and may be double-stranded or single-stranded. Coding sequences encoding proteins of the invention may vary due to redundancy or degeneracy of the genetic code.
  • Polynucleotides encoding TIGIT immunoadhesins of the invention may include the following: protein-only coding sequences, protein-coding sequences and additional coding sequences (such as leader or secretory sequences or pre-protein sequences): protein-coding sequences and non-coding sequences (Non-coding sequences such as introns or 5' and/or 3' ends of protein coding sequences).
  • protein-only coding sequences such as leader or secretory sequences or pre-protein sequences
  • protein-coding sequences and non-coding sequences Non-coding sequences such as introns or 5' and/or 3' ends of protein coding sequences.
  • polynucleotide encoding a protein encompasses not only polynucleotides that may include coding sequences for proteins, but also polynucleotides that include additional coding and/or non-coding sequences.
  • the fourth aspect of the present invention provides an antitumor pharmaceutical composition.
  • the composition includes the TIGIT immunoadhesin disclosed in the present invention and at least one immune checkpoint inhibitor, and also includes a medically acceptable pharmaceutical carrier.
  • the immune checkpoint inhibitor comprises a PD1 inhibitor, a PD-L1 inhibitor, and/or a CTLA4 inhibitor.
  • TIGIT immunoadhesin of the present invention other combination components and pharmaceutically acceptable excipients together form a pharmaceutical preparation composition, so as to exert the curative effect more stably, and these preparations can ensure the amino acid core sequence of the TIGIT immunoadhesin disclosed in the present invention. Conformational integrity, while also protecting the multifunctional groups of the protein from degradation (including but not limited to condensation, deamination, or oxidation).
  • liquid formulations are stable at 2°C to 8°C for at least one year, and lyophilized formulations are stable at 30°C for at least six months.
  • the preparation can be suspension, water injection, freeze-dried and other preparations commonly used in the pharmaceutical field, preferably water injection or freeze-dried preparation.
  • pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonicity regulators and buffers.
  • the surfactants include non-ionic surfactants such as polyoxyethylene sorbitan fatty acid ester (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); lauryl sulfate Sodium; tetradecyl, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc.
  • the addition amount should minimize the granulation tendency of the bifunctional bispecific antibody protein
  • the solution stabilizer can be saccharides, Including reducing sugars and non-reducing sugars, amino acids including monosodium glutamate or histidine, alcohols including one or a combination of trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol, solution stabilizers
  • the amount of addition should make the final formed preparation maintain a stable state within the time that those skilled in the art think that it reaches stability; the iso
  • the above-mentioned preparation is a composition containing TIGIT immunoadhesin, and after administration to animals including humans, the antitumor effect or the effect of stimulating angiogenesis is obvious. Specifically, it is effective for the treatment of diseases related to tumor or vascular injury, and can be used as a drug for related diseases.
  • the dosage varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. Reference can be made to animals. According to the results of the experiment and various circumstances, the total dosage cannot exceed a certain range. Specifically, the dose for intravenous injection is 1 to 1800 mg/day.
  • the invention discloses a new application of TIGIT immunoadhesin in regulating tumor immunity and regulating angiogenesis products. It is verified by experiments that TIGIT immunoadhesin can effectively enhance the killing of immune cells and identify tumor cells; Growth inhibition; improve the immunosuppressive signal in tumor tissue, break the tumor immunosuppressive microenvironment; at the same time, it can also improve the hypoxia of tumor tissue, increase blood supply, change the tumor growth environment, and hammer tumor cells from two aspects. On the other hand, TIGIT immunoadhesin can stimulate angiogenesis, enhance the healing of new tissue, provide a certain direction for the treatment of vascular injury diseases, and expand the clinical application prospect and scope of TIGIT immunoadhesin.
  • TIGIT immunoadhesin mediates the killing of tumor cells by immune cells
  • PBMC peripheral blood mononuclear cells
  • the ratio of effector cells to target cells was 10:1, the drug concentration was 1 ⁇ g/ml, the control IgG was used as a negative control, and no drug was added as a blank control, and the cell killing rate was the final cell signal/blank control cell signal in each treatment group.
  • the results are shown in Table 1-4.
  • TIGIT immunoadhesin of the present invention significantly enhances the killing activity of immune cells to tumor cells.
  • TGI Tumor Growth Inhibition
  • TGI (% blank) SD P value vs control IgG Control IgG 7.90 1.19 TIGIT-Fc-wt 78.97 23.44 P ⁇ 0.01 TIGIT-FC-LALA-PG 64.16 14.67 P ⁇ 0.01
  • TGI (% blank) SD P value vs control IgG Control IgG 4.71 2.29 TIGIT-Fc-wt 67.07 5.70 P ⁇ 0.01 TIGIT-FC-LALA-PG 49.85 10.35 P ⁇ 0.01
  • TGI (% blank) SD P value vs control IgG Control IgG 7.93 3.82 TIGIT-Fc-wt 61.79 12.55 P ⁇ 0.01 TIGIT-FC-LALA-PG 67.14 7.77 P ⁇ 0.01
  • TIGIT immunoadhesin of the present invention effectively improves the immunosuppressive signal in the tissue, breaks the tumor immunosuppressive microenvironment, and is beneficial for the immune cells to play a role in the tumor tissue.
  • E-cadherin and Vimentin were detected, and these two markers are markers of epithelial-mesenchymal transition of the tissue. The results are shown in Table 13-20.
  • TIGIT immunoadhesin of the present invention significantly enhances the expression of E-cadherin in tumor tissue, reduces the expression level of Vimentin protein, that is, significantly inhibits tissue due to immune cells and treatment-mediated epithelial-mesenchymal transition, Reduced risk of tumor metastasis.
  • Example 3 The effect of TIGIT immunoadhesin on blood vessels and microcirculation in tumor tissue
  • Each tumor tissue in the above-mentioned embodiment was further subjected to histochemical analysis to detect the perfusion of blood vessels in the tissue, and at the same time, the level of tissue hypoxia was detected by immunohistochemical method, and the detection method was the same as the literature [Hu S, et al. Science translational medicine, 2017 , 9(380); Yen WC, et al. Clinical cancer research, 2015, 21(9):2084-2095.]. The results are shown in Table 21-24:
  • TIGIT immunoadhesin of the present invention significantly enhanced the perfusion of blood vessels in the tumor tissue, stimulated angiogenesis in the tumor tissue, and reduced the level of tissue hypoxia.
  • mice colon cancer cell line MC38 was used to establish a mouse model in a normal immune state. Reference for the establishment of the model [Juneja VR, et al. Journal of Experimental Medicine, 2017, 214(4):895-904.]. Mice were grouped and administered according to the method of Example 2, and immunoadhesin treatment was performed. At this time, there is no need to infuse human immune cells to evaluate the immune cell status. Calculate TGI, the results are shown in Table 25:
  • TGI (% blank) SD P value vs control IgG Control IgG 3.94 5.91 TIGIT-Fc-wt 100 - P ⁇ 0.01 TIGIT-FC-LALA-PG 100 - P ⁇ 0.01
  • mice were sacrificed after 1 week of treatment, and the TGI at this time was shown in Table 26.
  • TGI (% blank) SD P value vs control IgG Control IgG 9.13 4.31 TIGIT-Fc-wt 76.55 8.75 P ⁇ 0.01 TIGIT-FC-LALA-PG 65.91 16.69 P ⁇ 0.01
  • the tumor tissue was separated according to the method of the above-mentioned embodiment, and the infiltration level of CD8 lymphocytes in the tissue was detected by immunohistochemical method. 7488.], the results are shown in Table 27.
  • TIGIT immunoadhesin can effectively increase the infiltration of lymphocytes in tumor tissue.
  • vascular perfusion and hypoxia in the tumor tissue were evaluated according to the methods in the above examples, and the results are shown in Tables 28-29.
  • TIGIT immunoadhesin of the present invention significantly enhanced the perfusion of blood vessels in the tumor tissue, stimulated angiogenesis in the tumor tissue, and reduced the level of tissue hypoxia.
  • Example 5 The effect of TIGIT immunoadhesin on vascular endothelial cells
  • TIGIT immunoadhesin The effect of TIGIT immunoadhesin on HUVEC vascular endothelial cells was evaluated according to the method of the patent document (CN 102884073 A). The dosage of immunoadhesin was 1 ⁇ g/ml. VEGF was used as a positive control. The results are shown in Tables 30-32.
  • TIGIT immunoadhesin disclosed in the present invention has the functions of promoting the proliferation, migration and lumen formation of vascular endothelial cells.
  • anti-PD-L1 antibody anti-PD-1 antibody
  • anti-CTLA4 antibody anti-CTLA4 antibody
  • the dosage of all antibody drugs is 10 mg/kg in one-time administration, and the method refers to Example 2.
  • Control antibodies and experimental methods for tumor clearance can be found in the literature [Woo S R, Turnis M E, Goldberg M V, et al.. Cancer research, 2012, 72(4): 917-927.; Dixon K O, Schorer M, Nevin J, et al.. The Journal of Immunology, 2018, 200(8):3000-3007.]
  • TIGIT-Fc-wt TIGIT-FC-LALA-PG 20 Anti-PD-1 20 Anti-PD-L1 20 Anti-CTLA4 30 TIGIT-Fc-wt+Anti-PD-1 90 TIGIT-Fc-wt+Anti-PD-L1 100 TIGIT-Fc-wt+Anti-CTLA4 90
  • TIGIT immunoadhesin disclosed in the present invention can mediate the removal of some tumor tissues, and can significantly enhance the anti-tumor effect when used in combination with anti-PD-L1 antibody, anti-PD-1 antibody, and anti-CTLA4 antibody. .
  • the tumor model was re-established and the test was repeated.
  • the PD-L1 expression was detected in the tumor tissue of each treatment group.
  • the detection method can be found in the literature [Jiao S, Xia W, Yamaguchi H, et al.Clinical Cancer Research, 2017, 23(14):3711-3720.]
  • This experiment shows that the TIGIT immunoadhesin disclosed in the present invention is combined with PD-1/PD-L1 inhibitor in immune normal mouse tumor model to reduce the effect of PD-1/PD-L1 inhibitor.
  • the loss of antigen in tissues can sensitize the antitumor effect of PD-L1/PD-1 signaling pathway inhibitors.
  • Example 7 The effect of TIGIT immunoadhesin on angiogenesis in pathological models
  • mice were treated with TIGIT immunoadhesin disclosed in the present invention for 3 weeks, and then the mouse tissues were separated, and the intimal capillary density was evaluated by an image analyzer (Xu Qing, et al. Journal of Capital Medical University (2): 104- 107.), the results are shown in Table 37:
  • TIGIT immunoadhesin disclosed in the present invention can effectively reduce endometrial damage, enhance endometrial microangiogenesis, enhance decidual spiral artery density, and improve pregnancy outcome.
  • the rat corneal alkali burn was established as an animal model, and the administration method was intravenous injection of 50 mg/kg of the immunoadhesin and the control drug every day, and the corneal injury was carried out 7 days after the operation by referring to the literature method. Area detection and evaluation. The results are shown in Table 40:

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Abstract

涉及生物医药工程技术领域，公开了TIGIT免疫粘附素在调控肿瘤免疫及调节血管生成产品中的新用途，通过实验验证，TIGIT免疫粘附素能有效增强免疫细胞杀伤、识别肿瘤细胞；增强免疫细胞对肿瘤组织的生长抑制；改善肿瘤组织内免疫抑制信号，打破肿瘤免疫抑制微环境；同时还能改善肿瘤组织缺氧状况，增加血流供应，改变肿瘤生长环境，从两方面锤击肿瘤细胞。另一方面，TIGIT免疫黏附素可以刺激血管新生，增强新生组织愈合，为血管损伤性疾病的治疗提供了一定方向，扩大了TIGIT免疫粘附素的临床应用前景和范围。

Description

TIGIT免疫粘附素在调控肿瘤免疫及调节血管生成产品中的应用 技术领域
本发明涉及生物医药工程技术领域，具体涉及TIGIT免疫粘附素在调控肿瘤免疫、调节血管生成产品中的应用
背景技术
TIGIT蛋白(UniProtKB编号：Q495A1)是脊髓灰质炎病毒受体(PVR)/Nectin家族的成员，它由细胞外免疫球蛋白可变区(IgV)结构域，1型跨膜结构域和具有经典免疫受体酪氨酸抑制基序(ITIM)和免疫球蛋白酪氨酸尾(ITT)基序的细胞内结构域组成，可以通过本领域技术人员熟悉的免疫球蛋白Fc融合技术构建成为TIGIT免疫粘附素。
TIGIT蛋白用途广泛，申请人根据研究成果已经申请了两项专利申请：发明专利CN109206523A公开了TIGIT免疫粘附素的制备和其在自身免疫病中的用途；发明专利CN110669139A公开了TIGIT免疫粘附素在调节母胎免疫耐受方面的用途。
肿瘤免疫循环指的是通过适当的方式发展免疫反应以杀死癌细胞的过程。简言之，树突状细胞从死亡的癌细胞中获取抗原，然后进入淋巴结激活T细胞，激活的T细胞离开淋巴结进入循环系统，穿过血管壁浸润到肿瘤微环境中，T细胞通过特异性受体识别并杀死肿瘤细胞，然后再继续循环。在肿瘤免疫的研究中，主要对TIGIT蛋白本身的研究较多，而对于TIGIT蛋白进一步进行基因工程化形成的TIGIT免疫粘附素研究较少。
发明内容
申请人在研究的过程中意外的发现，TIGIT免疫粘附素在增强免疫细胞杀伤肿瘤细胞、促进调节血管生成的应用方面具有意想不到的技术效果。
本发明所述的TIGIT免疫粘附素，指包括TIGIT天然蛋白的具有结合其天然配体能力的结构域或TIGIT蛋白的保外结构域或其功能变体或片段，TIGIT黏附素在背景技术文件中已经进行详述，是本领域技术人员熟知的构建方法，此处不再赘述。
在本发明的一些实施方案中，本发明所述的TIGIT免疫粘附素Fc结构域还包含LALA-PG突变体，用以去除其Fc结构域所潜在的免疫球蛋白活性。
本发明第一方面，提供了TIGIT免疫粘附素在制备增强免疫系统和免疫细胞清除肿瘤试剂或调节血管生成试剂中的用途。
具体而言，本发明所述的TIGIT免疫粘附素用途包括：(1)增强免疫细胞杀伤肿瘤细胞、增强肿瘤组织内免疫细胞的浸润数量、增强免疫细胞对肿瘤细胞的识别、降低肿瘤组织内免疫抑制信号活性、降低免疫细胞压力介导的肿瘤细胞的上皮-间质转化、增敏免疫检查点抑制剂介导的肿瘤清除作用的试剂。(2)促进血管新生和血管正常化，包括募集血管支持细胞、使血管网路正常化、改善组织血供、降低组织缺氧、减少组织损伤，主要体现在促进血管内皮细胞增殖、迁移以及管腔形成能力方面。
本发明所指的肿瘤，包括实体肿瘤，如腺癌、白血病、淋巴瘤、黑色素瘤、肉瘤，肿瘤组织的来源包括但不限于肾上腺、胆囊、骨、骨髓、脑、乳腺、胆管、胃肠道、心脏、肾脏、肝脏、肺、肌肉、卵巢、胰腺、甲状旁腺、阴茎、前列腺、皮肤、唾液腺、脾脏、睾丸、胸腺、甲状腺和子宫。除了上述的实体肿瘤外，还可用于中枢神经系统的肿瘤如胶质细胞多样性瘤、星细胞瘤等；此外眼部的肿瘤包括基底细胞癌、鳞状细胞癌、黑色素瘤等，还包括内分泌腺肿瘤、神经内分泌系统肿瘤、胃肠道胰腺内分泌系统肿瘤，生殖系统肿瘤，括血液系统肿瘤及头颈部肿瘤等。这里不再一一列举。
TIGIT免疫黏附素调节血管生成的能力，使得本文所公开的TIGIT免疫黏附素的用途包括期望促进血管生成的多种临床疾病或病症。此类病症包括促进再生组织的血管形成、缺血性肢体疾病、子宫内膜微血管生成、促进蜕膜组织微血管生成、脑缺血、毛发生长的刺激、勃起功能障碍、动脉硬化、血管炎性坏死疾病。
在本发明的一些实施方案中，所述期望促进血管生成的临床病症包括中风、黄斑变性、黄斑水肿、淋巴水肿、血-视网膜屏障破坏、血脑屏障破坏、细菌诱导的血管损伤再生，特别是包括肺损伤、肾损伤、肾纤维化、中风、血管性痴呆、黄斑变性和糖尿病并发症(例如，肾脏、眼睛、皮肤和/或四肢中)过程中由于微血管病变造成的血管损伤。
由于本发明所公开的TIGIT免疫粘附素具有刺激血管新生的能力，其可以进一步用于增强创伤愈合的产品制备。例如与不存在所述TIGIT免疫粘附素时的创伤愈合相比创伤闭合时间加速、与未治疗相比创伤部位处的肉芽组织增加和/或与未治疗相比创伤的新血管形成增强来证明。
在一个实施方案中，刺激创伤愈合的方法或用途用于治疗糖尿病性溃疡。在其他实施方案中，用于刺激创伤愈合的方法或用途可以用于涉及创伤的多种临床情况，包括但不限于褥疮性溃疡、压迫性溃疡、手术切口、创伤性组织损伤、烧伤和组织移植。
在一个实施方案中，所述期望促进血管生成的临床病症还包括针对肿瘤组织的血管正常化，以改善肿瘤组织的缺氧状况、降低转移风险。
因此，本发明的第二方面，提供了增强免疫系统和免疫细胞清除肿瘤试剂或调节血管生成试剂在制备抗肿瘤药物中的用途。
本发明的第三方面，提供了一种促进免疫系统和免疫细胞清除肿瘤的增强剂或血管生成调节试剂，其特征在于，以TIGIT免疫粘附素、编码其的核苷酸或重组表达载体为唯一活性成分，还包括药学上可接受的载体。
上述编码TIGIT免疫粘附素的多核昔酸可以是RNA或DNA的形式，其包括cDNA和合成DNA，并且其可以是双链或单链的。编码本发明的蛋白的编码序列可由于遗传密码的冗余或简并性而变化。
编码本发明TIGIT免疫粘附素的多核苷酸可包括以下：仅蛋白的编码序列，蛋白的编码序列和额外编码序列(诸如前导或分泌序列或前蛋白序列)：蛋白的编码序列和非编码序列(诸如内含子或蛋白的编码序列的5’和/或3’端的非编码序列)。因此，术语“编码蛋白的多核苷酸”不仅涵盖可包括蛋白的编码序列的多核苷酸，而且涵盖包括额外编码和/或非编码序列的多核苷酸。
本发明的第四方面，提供了一种抗肿瘤药物组合物。该组合物包括本发明所公开的TIGIT免疫粘附素和至少一种免疫检查点抑制剂，还包括医学上可接受的药物载体。在本发明的某些具体的实施方案中，所述免疫检查点抑制剂包括PD1抑制剂、PD-L1抑制剂、和/或CTLA4抑制剂。
本发明的TIGIT免疫粘附素、其他组合成分和药学上可以接受的辅料一起组成药物制剂组合物，从而更稳定地发挥疗效，这些制剂可以保证本发明公开的TIGIT免疫粘附素氨基酸核心序列的构像完整性，同时还要保护蛋白质的多官能团，防止其降解(包括但不限于凝聚、脱氨或氧化)。
通常情况下，液体制剂可以在2℃-8℃条件下保存至少稳定一年，冻干制剂在30℃至少六个月保持稳定。制剂可为制药领域常用的混悬、水针、冻干等制剂，优选水针或冻干制剂。
对于本发明公开的上述TIGIT免疫粘附素的水针或冻干制剂，药学上可以接受的辅料包括表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或其组合。其中，表面活性剂包括非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80)；poloxamer(如poloxamer 188)；Triton；十二烷基硫酸钠(SDS)；月桂硫酸钠；十四烷基、亚油基或十八烷基肌氨酸；Pluronics；MONAQUATTM等，其加入量应使双功能双特异性抗体蛋白的颗粒化趋势最小；溶液稳定剂可以为糖类，包括还原性糖和非还原性糖，氨基酸类包括 谷氨酸单钠或组氨酸，醇类包括三元醇、高级糖醇、丙二醇、聚乙二醇之一或其组合，溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态；等渗调节剂可以为氯化钠、甘露醇之一；缓冲液可以为TRIS、组氨酸缓冲液、磷酸盐缓冲液之一。
上述制剂为包含TIGIT免疫粘附素的组合物，在对包括人在内的动物给药后，抗肿瘤效果或刺激血管新生方面的效果明显。具体来讲，对肿瘤或血管损伤相关病症的治疗有效，可以作为相关病症药物使用。
本发明中TIGIT免疫粘附素及其组合物在对包括人在内的动物给药时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。具体讲静脉注射的剂量是1～1800mg/天。
本发明的有益保障及效果：
本发明公开了TIGIT免疫粘附素在调控肿瘤免疫及调节血管生成产品中的新用途，通过实验验证，TIGIT免疫粘附素能有效增强免疫细胞杀伤、识别肿瘤细胞；增强免疫细胞对肿瘤组织的生长抑制；改善肿瘤组织内免疫抑制信号，打破肿瘤免疫抑制微环境；同时还能改善肿瘤组织缺氧状况，增加血流供应，改变肿瘤生长环境，从两方面锤击肿瘤细胞。另一方面，TIGIT免疫黏附素可以刺激血管新生，增强新生组织愈合，为血管损伤性疾病的治疗提供了一定方向，扩大了TIGIT免疫粘附素的临床应用前景和范围。
具体实施方式
以下实施例、实验例对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质拉的方法，将编码蛋白的基因插入到这样的载体和质拉的方法或将质粒引入宿主细胞的方法.这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2 ndedition，Cold spring Harbor Laboratory Press。
实施例1 TIGIT免疫粘附素的制备
按照专利文献CN110669139A中的描述，分别制备TIGIT-Fc-wt和TIGIT-FC-LALA-PG两种免疫粘附素。
实施例2 TIGIT免疫粘附素介导免疫细胞杀伤肿瘤细胞
按照文献[Fu W,et al.Nature communications,2019,10(1):1-12.]的方法，评估外周单核细胞(peripheral blood mononuclear cells，PBMC)在TIGIT免疫粘附素存在的情况下对乳腺癌细胞系SK-BR-3、肺癌细胞系HCC827、胃癌细胞系N87、卵巢癌细胞系SK-OV-3的杀伤效果。效应细胞和靶细胞的比率为10:1，药物浓度为1μg/ml，对照IgG作为阴性对照，不添加任何药物作为空白对照，细胞杀伤率为各处理组最终细胞信号/空白对照细胞信号。结果如表1-4。
表1 PBMC对SK-BR-3的杀伤作用
组别 细胞杀伤率(％空白对照) SD P值vs对照IgG
Control IgG 10.25 2.45  
TIGIT-Fc-wt 72.91 4.37 P＜0.01
TIGIT-FC-LALA-PG 45.65 3.19 P＜0.01
表2 PBMC对HCC827的杀伤作用
组别 细胞杀伤率(％空白对照) SD P值vs对照IgG
Control IgG 10.30 3.21  
TIGIT-Fc-wt 66.88 14.44 P＜0.01
TIGIT-FC-LALA-PG 48.67 10.80 P＜0.01
表3 PBMC对N87的杀伤作用
组别 细胞杀伤率(％空白对照) SD P值vs对照IgG
Control IgG 12.10 2.98  
TIGIT-Fc-wt 79.40 11.96 P＜0.01
TIGIT-FC-LALA-PG 52.56 6.64 P＜0.01
表4 PBMC对N87的杀伤作用
组别 细胞杀伤率(％空白对照) SD P值vs对照IgG
Control IgG 11.99 2.36  
TIGIT-Fc-wt 75.09 17.50 P＜0.01
TIGIT-FC-LALA-PG 47.19 11.80 P＜0.01
这些实验显示，本发明所述的TIGIT免疫粘附素显著的增强免疫细胞对肿瘤细胞的杀伤活性。
进一步的，按照文献[Fu W,et al.Nature communications,2019,10(1):1-12.]方法，建立免疫缺陷小鼠中上述肿瘤细胞系的模型，同时评估在体内模型中PBMC对肿瘤细胞的杀伤作用。计算肿瘤生长抑制率(Tumor Growth Inhibition，TGI)，小鼠模型的免疫细胞注射方法、肿瘤体积计算方法等均参考现有文献。每组7只小鼠，小鼠每周给药一次连续治疗3周，尾静脉注射40mg/kg，对照IgG为阴性对照，给与生理盐水组作为空白对照。结果如表5-8。
表5 SK-BR-3小鼠荷瘤及免疫细胞作用动物模型评价
组别 TGI(％空白对照) SD P值vs对照IgG
Control IgG 7.90 1.19  
TIGIT-Fc-wt 78.97 23.44 P＜0.01
TIGIT-FC-LALA-PG 64.16 14.67 P＜0.01
表6 HCC827小鼠荷瘤及免疫细胞作用动物模型评价
组别 TGI(％空白对照) SD P值vs对照IgG
Control IgG 4.71 2.29  
TIGIT-Fc-wt 67.07 5.70 P＜0.01
TIGIT-FC-LALA-PG 49.85 10.35 P＜0.01
表7 N87小鼠荷瘤及免疫细胞作用动物模型评价
组别 TGI(％空白对照) SD P值vs对照IgG
Control IgG 7.93 3.82  
TIGIT-Fc-wt 61.79 12.55 P＜0.01
TIGIT-FC-LALA-PG 67.14 7.77 P＜0.01
表8 SK-OV-3小鼠荷瘤及免疫细胞作用动物模型评价
组别 TGI(％空白对照) SD P值vs对照IgG
Control IgG 6.09 3.20  
TIGIT-Fc-wt 89.64 16.33 P＜0.01
TIGIT-FC-LALA-PG 71.80 19.90 P＜0.01
上述实验表明，在体内模型中，本发明所述的TIGIT免疫粘附素有效增强免疫细胞对肿瘤组织的生长抑制。
进一步的，将上述小鼠模型实验结束后的肿瘤组织进行分离，检测各肿瘤组织内的PDL1的表达。肿瘤组织的分离、组织内基因的表达检测参考文献[Hu S,et al.Science translational medicine,2017,9(380)；Yen W C,et al.Clinical cancer research,2015,21(9):2084-2095.]。结果如表9-12。
表9 SK-BR-3小鼠荷瘤模型结束后组织PDL1蛋白的相对表达
组别 相对表达(％空白对照) SD P值vs对照IgG
Control IgG 110.47 21.23  
TIGIT-Fc-wt 57.27 18.20 P＜0.01
TIGIT-FC-LALA-PG 41.18 19.24 P＜0.01
表10 HCC827小鼠荷瘤模型结束后组织PDL1蛋白的相对表达
组别 相对表达(％空白对照) SD P值vs对照IgG
Control IgG 124.05 36.89  
TIGIT-Fc-wt 9.94 3.11 P＜0.01
TIGIT-FC-LALA-PG 13.28 1.77 P＜0.01
表11 N87小鼠荷瘤模型结束后组织PDL1蛋白的相对表达
组别 相对表达(％空白对照) SD P值vs对照IgG
Control IgG 119.23 13.60  
TIGIT-Fc-wt 47.78 10.36 P＜0.01
TIGIT-FC-LALA-PG 29.20 4.80 P＜0.01
表12 SK-OV-3小鼠荷瘤模型结束后组织PDL1蛋白的相对表达
组别 相对表达(％空白对照) SD P值vs对照IgG
Control IgG 116.27 18.15  
TIGIT-Fc-wt 38.24 11.59 P＜0.01
TIGIT-FC-LALA-PG 10.68 2.06 P＜0.01
这些实验表明，本发明所述的TIGIT免疫粘附素有效改善组织内免疫抑制信号，打破肿瘤免疫抑制微环境，有利于免疫细胞在肿瘤组织发挥作用。
进一步的，检测检测组织内的E-cadherin和Vimentin表达水平，这两个标记物是组织的上皮-间质转化标志物。结果如表13-20。
表13 SK-BR-3小鼠荷瘤模型结束后组织E-cadherin蛋白的相对表达
组别 相对表达(％空白对照) SD P值vs对照IgG
Control IgG 100.34 7.59  
TIGIT-Fc-wt 213.21 22.79 P＜0.01
TIGIT-FC-LALA-PG 195.88 7.91 P＜0.01
表14 SK-BR-3小鼠荷瘤模型结束后组织Vimentin蛋白的相对表达
组别 相对表达(％空白对照) SD P值vs对照IgG
Control IgG 105.71 13.13  
TIGIT-Fc-wt 65.89 14.62 P＜0.01
TIGIT-FC-LALA-PG 63.60 8.28 P＜0.01
表15 HCC827小鼠荷瘤模型结束后组织E-cadherin蛋白的相对表达
组别 相对表达(％空白对照) SD P值vs对照IgG
Control IgG 108.52 33.08  
TIGIT-Fc-wt 173.79 44.09 P＜0.01
TIGIT-FC-LALA-PG 199.39 17.32 P＜0.01
表16 SK-BR-3小鼠荷瘤模型结束后组织Vimentin蛋白的相对表达
组别 相对表达(％空白对照) SD P值vs对照IgG
Control IgG 107.62 25.26  
TIGIT-Fc-wt 60.20 25.52 P＜0.01
TIGIT-FC-LALA-PG 47.74 18.41 P＜0.01
表17 N87小鼠荷瘤模型结束后组织E-cadherin蛋白的相对表达
组别 性对表达(％空白对照) SD P值vs对照IgG
Control IgG 110.82 45.91  
TIGIT-Fc-wt 204.62 18.22 P＜0.01
TIGIT-FC-LALA-PG 248.22 20.95 P＜0.01
表18 N87小鼠荷瘤模型结束后组织Vimentin蛋白的相对表达
组别 性对表达(％空白对照) SD P值vs对照IgG
Control IgG 104.88 28.88  
TIGIT-Fc-wt 47.10 11.68 P＜0.01
TIGIT-FC-LALA-PG 66.65 10.29 P＜0.01
表19 SK-OV-3小鼠荷瘤模型结束后组织E-cadherin蛋白的相对表达
组别 性对表达(％空白对照) SD P值vs对照IgG
Control IgG 101.18 29.62  
TIGIT-Fc-wt 173.51 44.16 P＜0.01
TIGIT-FC-LALA-PG 190.65 76.82 P＜0.01
表20 SK-OV-3小鼠荷瘤模型结束后组织Vimentin蛋白的相对表达
组别 性对表达(％空白对照) SD P值vs对照IgG
Control IgG 106.71 33.64  
TIGIT-Fc-wt 44.29 4.15 P＜0.01
TIGIT-FC-LALA-PG 22.41 10.19 P＜0.01
这些实验表明，本发明所述的TIGIT免疫黏附素显著增强肿瘤组织内E-cadherin的表达，减少Vimentin蛋白的表达水平，即显著的抑制组织由于免疫细胞和治疗介导的上皮-间质转化、减少了肿瘤转移的风险。
实施例3 TIGIT免疫粘附素对肿瘤组织内部血管和微循环影响
将上述实施例中的各肿瘤组织进一步进行组织化学分析，检测组织内灌注血管情况，同时用免疫组织化学方法检测组织缺氧水平，检测方法同文献[Hu S,et al.Science translational medicine,2017,9(380)；Yen W C,et al.Clinical cancer research,2015,21(9):2084-2095.]。结果如表21-24：
表21 SK-BR-3小鼠荷瘤模型结束后组织灌注血管情况
Figure PCTCN2021112374-appb-000001
表22 SK-BR-3小鼠荷瘤模型结束后组织缺氧情况
组别 缺氧信号(％空白对照) SD P值vs对照IgG
Control IgG 105.31 27.35  
TIGIT-Fc-wt 10.60 3.20 P＜0.01
TIGIT-FC-LALA-PG 8.17 6.06 P＜0.01
表23 HCC827小鼠荷瘤模型结束后组织灌注血管情况
Figure PCTCN2021112374-appb-000002
表24 HCC827小鼠荷瘤模型结束后组织缺氧情况
组别 缺氧信号(％空白对照) SD P值vs对照IgG
Control IgG 112.61 35.26  
TIGIT-Fc-wt 7.90 2.52 P＜0.01
TIGIT-FC-LALA-PG 8.72 1.88 P＜0.01
这些实验表面，本发明所述的TIGIT免疫黏附素显著增强肿瘤组织内血管的灌注，刺激肿瘤组织内的血管新生、减轻组织缺氧水平。
实施例4 TIGIT免疫粘附素对小鼠同种异体移植模型的影响
利用小鼠结肠癌细胞系MC38建立正常免疫状态下的小鼠模型，模型的建立参考文献[Juneja V R,et al.Journal of Experimental Medicine,2017,214(4):895-904.]。按照实施例2的方法进行小鼠分组和给药，进行免疫粘附素的处理。此时无需输入人免疫细胞评估免疫细胞情况。计算TGI，结果如表25：
表25 MC38小鼠荷瘤动物模型评价
组别 TGI(％空白对照) SD P值vs对照IgG
Control IgG 3.94 5.91  
TIGIT-Fc-wt 100 - P＜0.01
TIGIT-FC-LALA-PG 100 - P＜0.01
结果显示，TIGIT免疫粘附素的处理组全部清除肿瘤。
为了进一步评估组织内的情况，减少处理时间，在治疗1周后即处死小鼠，此时TGI如表26。
表26 MC38小鼠荷瘤动物模型缩短治疗时间评价
组别 TGI(％空白对照) SD P值vs对照IgG
Control IgG 9.13 4.31  
TIGIT-Fc-wt 76.55 8.75 P＜0.01
TIGIT-FC-LALA-PG 65.91 16.69 P＜0.01
此时按照上述实施例的方法分离肿瘤组织，以免疫组织化学的方法检测组织内的CD8淋巴细胞浸入水平，方法参考文献[Demotte N,et al.Cancer research,2010,70(19):7476-7488.]，结果如表27。
表27 MC38组织内淋巴细胞浸润
Figure PCTCN2021112374-appb-000003
Figure PCTCN2021112374-appb-000004
该实验显示，TIGIT免疫粘附素可以有效增加肿瘤组织内淋巴细胞的浸润。
进一步的，按照上述实施例中的方法评估肿瘤组织内的血管灌注和缺氧情况，结果如表28-29。
表28 MC38小鼠荷瘤模型结束后组织灌注血管情况
Figure PCTCN2021112374-appb-000005
表29 MC38小鼠荷瘤模型结束后组织缺氧情况
组别 缺氧信号(％空白对照) SD P值vs对照IgG
Control IgG 105.84 23.45  
TIGIT-Fc-wt 7.84 3.71 P＜0.01
TIGIT-FC-LALA-PG 9.74 3.32 P＜0.01
这些实验表面，本发明所述的TIGIT免疫黏附素显著增强肿瘤组织内血管的灌注，刺激肿瘤组织内的血管新生、减轻组织缺氧水平。
实施例5 TIGIT免疫粘附素对血管内皮细胞的作用
按照专利文献(CN 102884073 A)的方法评估TIGIT免疫粘附素对HUVEC血管内皮细胞的作用。免疫粘附素的用量为1μg/ml。以VEGF为阳性对照。结果如表30-32。
表30 TIGIT免疫粘附素对血管内皮细胞增殖的影响
组别 细胞增殖(％空白对照) SD P值vs对照IgG
Control IgG 91.29 10.54  
VEGF 159.96 15.85 P＜0.01
TIGIT-Fc-wt 219.27 37.63 P＜0.01
TIGIT-FC-LALA-PG 169.15 31.89 P＜0.01
表31 TIGIT免疫粘附素对血管内皮细胞迁移的影响
组别 细胞增殖(％空白对照) SD P值vs对照IgG
Control IgG 100.76 22.84  
VEGF 195.45 12.07 P＜0.01
TIGIT-Fc-wt 173.91 38.20 P＜0.01
TIGIT-FC-LALA-PG 179.26 32.39 P＜0.01
表32 TIGIT免疫粘附素对血管内皮细胞管腔形成的影响
组别 细胞管腔(个管腔每视野) SD P值vs对照IgG
空白对照 1.750 0.500  
Control IgG 2.000 0.816  
VEGF 14.250 2.217 P＜0.01
TIGIT-Fc-wt 13.250 1.893 P＜0.01
TIGIT-FC-LALA-PG 15.750 2.986 P＜0.01
这些实验显示，本发明所公开的TIGIT免疫粘附素具有促进血管内皮细胞增殖、迁移、管腔形成的功能。
进一步按照文献(CN 102884073 A)利用C57小鼠体内模型检测基质胶塞来评估体内血管形成能力，对照药物和免疫粘附素的用药量均为1mg，药物和基质胶混合物体积为600μl。结果如表33：
表33 TIGIT免疫粘附素体内模型血管生成的影响
Figure PCTCN2021112374-appb-000006
该实验表面，本发明所公开的TIGIT免疫粘附素具有促进体内血管生成的作用。
实施例6 TIGIT免疫粘附素联合应用
为了进一步明确TIGIT免疫粘附素的免疫调控作用，采用抗PD-L1抗体、抗PD-1抗体、抗CTLA4抗体进行MC38肿瘤模型中联合应用。所有抗体药物的给药剂量为一次性给药10mg/kg，方法参考实施例2。联合给药的剂量为两种药物各5mg/ml，n＝10。对照抗体和肿瘤清除的实验方法可以参考文献[Woo S R,Turnis M E,Goldberg M V,et al..Cancer research,2012,72(4):917-927.；Dixon K O,Schorer M,Nevin J,et al..The Journal of Immunology,2018,200(8):3000-3007.]
表34 小鼠肿瘤清除率
组别 肿瘤清除率(％)
Control IgG 0
TIGIT-Fc-wt 30
TIGIT-FC-LALA-PG 20
Anti-PD-1 20
Anti-PD-L1 20
Anti-CTLA4 30
TIGIT-Fc-wt+Anti-PD-1 90
TIGIT-Fc-wt+Anti-PD-L1 100
TIGIT-Fc-wt+Anti-CTLA4 90
该实验表面，本发明所公开的TIGIT免疫粘附素可以介导部分肿瘤组织的清除，而和抗PD-L1抗体、抗PD-1抗体、抗CTLA4抗体联合使用后可以显著的增强抗肿瘤效果。
进一步的，重新建立肿瘤模型并重复试验，再肿瘤未消退前即对各处理组肿瘤组织检测其PD-L1表达，检测方法可参考文献[Jiao S,Xia W,Yamaguchi H,et al.Clinical Cancer Research,2017,23(14):3711-3720.]
表35 各组组织内PD-L1的表达水平
Figure PCTCN2021112374-appb-000007
Figure PCTCN2021112374-appb-000008
该实验表面，本发明所公开的TIGIT免疫粘附素在免疫正常小鼠肿瘤模型中给和PD-1/PD-L1抑制剂联合应用药处理减少了PD-1/PD-L1抑制剂造成的组织内抗原丢失，可以增敏PD-L1/PD-1信号通路抑制剂的抗肿瘤作用。
实施例7 TIGIT免疫粘附素对病理模型中血管生成的作用
(1)对于糖尿病肢体缺血的小鼠的促进血管生成及改善缺血肢体恢复的影响
按照专利文献(CN 103260628 A)进行疾病模型造模，药物给与4周处理后进行检测，给药剂量为每周40mg/kg静脉注射一次。每组n＝6。按照文献方法评估血流。结果如表34：
表36 体内模型血管生成的影响
Figure PCTCN2021112374-appb-000009
进一步按照文献方法进行生理检查，结果如表35：
表37 缺血后肢恢复的影响
组别 分数 SD P值vs对照IgG
Control IgG 2.833 0.408  
TIGIT-Fc-wt 13.167 0.408 P＜0.01
TIGIT-FC-LALA-PG 13.500 0.548 P＜0.01
处死动物，按照专利文献方法评估腿部皮肤毛细血管密度，结果如表36：
表38 组织毛细血管
Figure PCTCN2021112374-appb-000010
这些实验显示，本发明所公开的TIGIT免疫粘附素有效增强糖尿病肢体缺血的小鼠的局部血流水平，有效减少并发症。
(2)对子宫内膜损伤后血管新生和愈合的影响
进一步的，按照专利文献CN 110721196 A的方法建立小鼠子宫内膜损伤模型。造模成功后利用本发明所公布的TIGIT免疫粘附素治疗3周后，分离小鼠组织，利用图像分析仪评估内膜毛细血管密度(许晴,等.首都医科大学学报(2):104-107.)，结果如表37：
表39 组织毛细血管
Figure PCTCN2021112374-appb-000011
进一步建立按照专利文献CN 110721196 A的方法建立小鼠子宫内膜损伤厚妊娠模型，然后进行小鼠妊娠组织评估，组织毛细血管密度、最终妊娠结局如表38-39：
表40 蜕膜螺旋动脉血管
组别 血管密度(％空白对照) SD P值vs对照IgG
Control IgG 112.92 20.64  
TIGIT-Fc-wt 1236.48 136.27 P＜0.01
TIGIT-FC-LALA-PG 1143.29 123.77 P＜0.01
表41 各组妊娠率
组别 怀孕率 P值vs对照IgG
正常小鼠 100％  
模型组 20％  
Control IgG 10％  
TIGIT-Fc-wt 90％ P＜0.01
TIGIT-FC-LALA-PG 80％ P＜0.01
这些结果显示本发明公开的TIGIT免疫粘附素有效减少子宫内膜损伤、增强子宫内膜微血管生成、增强蜕膜螺旋动脉密度，提高妊娠结局。
(3)对角膜损伤模型的影响
按照专利文献方法CN 106265682 A，建立大鼠大鼠角膜碱烧伤为动物模型，给药方式为每天静脉注射50mg/kg所述免疫粘附素和对照药物，术后7天参考文献方法进行角膜损伤面积检测评估。结果如表40：
表42 角膜损伤面积
组别 损伤面积(mm 2) SD P值vs对照IgG
模型 1.26 0.36  
Control IgG 1.51 0.26  
TIGIT-Fc-wt 0.37 0.08 P＜0.01
TIGIT-FC-LALA-PG 0.29 0.12 P＜0.01
该实验表明，本发明公开的TIGIT免疫粘附素有效减少角膜损伤面积，有效提高组织愈合。
(4)对心脏损伤模型的影响
按照文献方法[Li T,et al..The FASEB Journal,2019,33(6):7467-7478.]建立大鼠左前降支结扎至心衰模型，同时给与免疫粘附素，剂量为每周2次，每次50mg/kg尾静脉注射。4周后分离心脏组织进行心脏病理检查进行Masson染色，评估梗死面积。结果如表41：
表43 梗死面积
组别 损伤面积(％空白对照) SD P值vs对照IgG
Control IgG 121.78 39.19  
TIGIT-Fc-wt 55.73 11.66 P＜0.01
TIGIT-FC-LALA-PG 60.39 16.50 P＜0.01
同时进行组织缺氧组织化学检测，结果如表42：
表44 心肌组织缺氧情况
组别 缺氧信号(％空白对照) SD P值vs对照IgG
Control IgG 118.83 26.31  
TIGIT-Fc-wt 24.68 10.79 P＜0.01
TIGIT-FC-LALA-PG 9.47 2.35 P＜0.01
该实验表明，本发明公开的TIGIT免疫粘附素有效减少心梗后组织损伤，减轻组织缺氧状况。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。

Claims (10)

  1. TIGIT免疫粘附素在制备增强免疫系统和免疫细胞清除肿瘤试剂或调节血管生成试剂中的用途。
  2. 根据权利要求1所述的用途，其特征在于：
    其中，所述增强免疫系统和免疫细胞清除肿瘤试剂为增强免疫细胞杀伤肿瘤细胞、增强肿瘤组织内免疫细胞的浸润数量、降低肿瘤组织内免疫抑制信号活性、降低免疫细胞压力介导的肿瘤细胞的上皮-间质转化、增敏免疫检查点抑制剂介导的肿瘤清除作用的试剂。
  3. 根据权利要求1所述的用途，其特征在于：
    其中，所述调节血管生成试剂为促进血管新生和血管正常化的试剂。
  4. 根据权利要求3所述的用途，其特征在于：
    其中，所述促进血管新生和血管正常化的试剂为促进血管内皮细胞增殖、迁移以及管腔形成能力的试剂。
  5. 根据权利要求3所述的用途，其特征在于：
    其中，所述促进血管新生和血管正常化的试剂为募集血管支持细胞、促进血管网路正常化、改善组织血供、降低组织缺氧或减少组织损伤的试剂。
  6. 根据权利要求1所述的用途，其特征在于：
    其中，所述增强免疫系统和免疫细胞清除肿瘤试剂和调节血管生成试剂均以TIGIT免疫粘附素、编码其的核苷酸或重组表达载体为唯一活性成分，还包括药学上可接受的载体。
  7. 权利要求1所述的增强免疫系统和免疫细胞清除肿瘤试剂或调节血管生成试剂在制备抗肿瘤药物中的用途。
  8. 一种抗肿瘤药物，其特征在于，该药物的活性成分包括TIGIT免疫粘附素和至少一种免疫检查点抑制剂。
  9. 根据权利要求7所述的抗肿瘤药物，其特征在于：
    其中，所述免疫检查点抑制剂包括抗PD-L1抗体、抗PD-1抗体或抗CTLA4抗体。
  10. 权利要求1所述的调节血管生成试剂在制备治疗血管损伤修复或改善组织缺氧药物中的用途。
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