CN115708865A - Application of TIGIT immunoadhesin in preparation of angiogenesis regulating product - Google Patents
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Abstract
The invention relates to the technical field of biomedical engineering, and discloses a new application of TIGIT immunoadhesin in preparation of a product for regulating angiogenesis. On the other hand, the TIGIT immunoadhesin can stimulate angiogenesis and enhance the healing of neogenetic tissues, provides a certain direction for treating vascular injury diseases, and expands the clinical application prospect and range of the TIGIT immunoadhesin.
Description
The application belongs to divisional application of 202011000490.7, about 22.09.2020 and entitled TIGIT immunoadhesin for regulating tumor immunity and regulating angiogenesis products. The priority number of the parent case is 202010862713.4, and the priority date is 2020.08.25.
Technical Field
The invention relates to the technical field of biomedical engineering, in particular to application of TIGIT immunoadhesin in regulating tumor immunity and regulating angiogenesis products.
Background
The TIGIT protein (UniProtKB No.: Q495 A1) is a member of the poliovirus receptor (PVR)/Nectin family, which consists of an extracellular immunoglobulin variable region (IgV) domain, a type 1 transmembrane domain, and an intracellular domain with a classical Immunoreceptor Tyrosine Inhibition Motif (ITIM) and an Immunoglobulin Tyrosine Tail (ITT) motif, and can be constructed as a TIGIT immunoadhesin by immunoglobulin Fc fusion techniques familiar to those skilled in the art.
TIGIT proteins are widely used, and the applicant has filed two patent applications according to research results: the invention patent CN109206523A discloses the preparation of TIGIT immunoadhesin and its use in autoimmune diseases; the invention patent CN110669139A discloses the use of TIGIT immunoadhesin in the regulation of maternal and fetal immune tolerance.
The tumor immune cycle refers to the process of developing an immune response by an appropriate means to kill cancer cells. Briefly, dendritic cells acquire antigen from dead cancer cells and then enter lymph nodes to activate T cells, which leave lymph nodes and enter the circulatory system, infiltrate through the vessel wall into the tumor microenvironment, recognize and kill tumor cells through specific receptors, and then continue to circulate. In the research of tumor immunity, TIGIT protein itself is mainly researched more, and TIGIT immunoadhesin formed by further performing genetic engineering on the TIGIT protein is researched less.
Disclosure of Invention
In the process of research, the applicant unexpectedly finds that the TIGIT immunoadhesin has an unexpected technical effect in the aspects of enhancing the application of immune cells to kill tumor cells and promoting and regulating angiogenesis.
The TIGIT immunoadhesin of the invention comprises a TIGIT natural protein domain with the ability of binding a natural ligand thereof or an outer protective domain of the TIGIT protein or a functional variant or a fragment thereof, and the TIGIT adhesin is detailed in a background technical document, is a construction method well known by a person skilled in the art and is not described in detail herein.
In some embodiments of the invention, the TIGIT immunoadhesin Fc domain of the invention further comprises a LALA-PG mutant to remove potential immunoglobulin activity from its Fc domain.
In a first aspect of the invention, there is provided the use of TIGIT immunoadhesin in the manufacture of an agent for enhancing the immune system and immune cells in the clearance of tumour or modulating angiogenesis.
Specifically, the TIGIT immunoadhesin application comprises the following steps: (1) The reagent can enhance the killing of tumor cells by immune cells, enhance the infiltration quantity of the immune cells in tumor tissues, enhance the recognition of the tumor cells by the immune cells, reduce the activity of immunosuppressive signals in the tumor tissues, reduce the epithelial-mesenchymal transition of the tumor cells mediated by the pressure of the immune cells and enhance the tumor removal function mediated by an immune checkpoint inhibitor. (2) Promoting angiogenesis and normalization of blood vessels, including recruitment of blood vessel supporting cells, normalization of blood vessel network, improvement of tissue blood supply, reduction of tissue hypoxia and reduction of tissue injury, mainly embodied in promoting proliferation and migration of vascular endothelial cells and lumen formation ability.
Tumors, as referred to herein, include solid tumors such as adenocarcinomas, leukemias, lymphomas, melanomas, sarcomas, and sources of tumor tissue include, but are not limited to, adrenal glands, gall bladder, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, skin, salivary glands, spleen, testis, thymus, thyroid, and uterus. In addition to the above-mentioned solid tumors, the present invention can also be applied to tumors of the central nervous system such as glioblastoma multiforme, astrocytoma, etc.; in addition, the tumors of the eye comprise basal cell carcinoma, squamous cell carcinoma, melanoma and the like, and also comprise tumors of endocrine glands, tumors of neuroendocrine system, tumors of gastrointestinal pancreatic endocrine system, tumors of reproductive system, tumors of blood system, head and neck, and the like. This is not further illustrated.
The ability of TIGIT immunoadhesins to modulate angiogenesis such that uses of the TIGIT immunoadhesins disclosed herein include a variety of clinical diseases or conditions for which it is desirable to promote angiogenesis. Such conditions include promotion of angiogenesis of regenerative tissue, ischemic limb disease, endometrial microvascular formation, promotion of decidua tissue microangiogenesis, cerebral ischemia, stimulation of hair growth, erectile dysfunction, arteriosclerosis, vasculitic necrotic disease.
In some embodiments of the invention, the clinical condition for which promotion of angiogenesis is desired includes stroke, macular degeneration, macular edema, lymphedema, blood-retinal barrier disruption, blood-brain barrier disruption, bacterially-induced regeneration of vascular injury, particularly including lung injury, kidney fibrosis, stroke, vascular dementia, macular degeneration, and vascular injury due to microvascular disease during diabetic complications (e.g., in the kidney, eye, skin, and/or extremities).
Because the TIGIT immunoadhesin disclosed by the invention has the capacity of stimulating angiogenesis, the TIGIT immunoadhesin can be further used for preparing products for enhancing wound healing. Such as accelerated wound closure time compared to wound healing in the absence of the TIGIT immunoadhesin, increased granulation tissue at the wound site compared to untreated, and/or enhanced neovascularization of the wound compared to untreated.
In one embodiment, the method or use of stimulating wound healing is for treating a diabetic ulcer. In other embodiments, the methods or uses for stimulating wound healing may be used in a variety of clinical situations involving wounds, including but not limited to decubitus ulcers, pressure ulcers, surgical incisions, traumatic tissue injuries, burns, and tissue transplantation.
In one embodiment, the clinical condition for which promotion of angiogenesis is desired further comprises normalization of blood vessels against tumor tissue to improve the hypoxic condition of the tumor tissue, reducing the risk of metastasis.
Thus, in a second aspect of the invention, there is provided the use of an agent which enhances the immune system and immune cells to eliminate tumours or to modulate angiogenesis in the preparation of a medicament for the treatment of tumours.
In a third aspect of the present invention, there is provided an enhancer or angiogenesis regulator for promoting the immune system and immune cells to eliminate tumors, which comprises TIGIT immunoadhesin, a nucleotide encoding the same, or a recombinant expression vector as a sole active ingredient, and a pharmaceutically acceptable carrier.
The polynucleotide encoding TIGIT immunoadhesin described above may be in the form of RNA or DNA, which includes cDNA and synthetic DNA, and which may be double-stranded or single-stranded. The coding sequence encoding a protein of the invention may vary due to the redundancy or degeneracy of the genetic code.
Polynucleotides encoding the TIGIT immunoadhesins of the invention may comprise the following: coding sequence for protein only, coding sequence for protein and additional coding sequences (such as leader or secretory sequences or preprotein sequences): coding sequence for a protein and non-coding sequences (such as introns or non-coding sequences 5 'and/or 3' to the coding sequence for a protein). Thus, the term "polynucleotide encoding a protein" encompasses not only polynucleotides that may include the coding sequence of the protein, but also polynucleotides that include additional coding and/or non-coding sequences.
In a fourth aspect of the invention, an anti-tumor pharmaceutical composition is provided. The composition comprises the TIGIT immunoadhesin disclosed in the present invention and at least one immune checkpoint inhibitor, and further comprises a pharmaceutically acceptable pharmaceutical carrier. In certain specific embodiments of the invention, the immune checkpoint inhibitor comprises a PD1 inhibitor, a PD-L1 inhibitor, and/or a CTLA4 inhibitor.
The TIGIT immunoadhesin, other composition components and pharmaceutically acceptable auxiliary materials of the invention form a pharmaceutical preparation composition together so as to exert curative effects more stably, and the preparations can ensure the conformation integrity of the amino acid core sequence of the TIGIT immunoadhesin disclosed by the invention and simultaneously protect the multifunctional group of protein to prevent the degradation (including but not limited to aggregation, deamination or oxidation).
In general, liquid formulations are stable for at least one year at 2 ℃ to 8 ℃ and lyophilized formulations are stable for at least six months at 30 ℃. The preparation can be suspension, injection, or lyophilized preparation, preferably injection or lyophilized preparation.
For the water injection or freeze-dried preparation of the TIGIT immunoadhesin disclosed by the invention, pharmaceutically acceptable auxiliary materials comprise one or a combination of a surfactant, a solution stabilizer, an isotonic regulator and a buffer solution. Wherein the surfactant comprises nonionic surfactant such as polyoxyethylene sorbitol fatty acid ester (Tween-20 or Tween-80); poloxamer (such as poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; tetradecyl, oleyl, or octadecyl sarcosine; pluronics; monaquatm, etc., in an amount that minimizes the tendency of the bifunctional bispecific antibody protein to granulate; the solution stabilizer can be saccharides including reducing saccharides and non-reducing saccharides, amino acids including monosodium glutamate or histidine, alcohols including one of trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol or combinations thereof, and should be added in an amount such that the final formulation remains stable for a period of time deemed stable by one skilled in the art; the isotonic regulator can be one of sodium chloride and mannitol; the buffer may be one of TRIS, histidine buffer, and phosphate buffer.
The preparation is a composition containing TIGIT immunoadhesin, and has obvious antitumor effect or angiogenesis stimulating effect after being administrated to animals including human beings. In particular, the compound is effective in treating diseases related to tumor or vascular injury and can be used as a medicine for the related diseases.
When the TIGIT immunoadhesin and the composition thereof of the present invention are administered to animals including human, the administration dosage varies depending on the age and body weight of the patient, the nature and severity of the disease, and the administration route, and the total administration amount cannot exceed a certain range with reference to the results of animal experiments and various cases. In particular, the dosage of intravenous injection is 1-1800 mg/day.
The invention has the following beneficial guarantee and effects:
the invention discloses a new application of TIGIT immunoadhesin in products for regulating and controlling tumor immunity and angiogenesis, and experiments prove that the TIGIT immunoadhesin can effectively enhance immune cell killing and identify tumor cells; enhancing the growth inhibition of immune cells on tumor tissue; improving immunosuppressive signals in tumor tissues and breaking tumor immunosuppressive microenvironment; meanwhile, the hypoxia condition of tumor tissues can be improved, the blood flow supply is increased, the tumor growth environment is changed, and tumor cells are hammered from two aspects. On the other hand, the TIGIT immunoadhesin can stimulate angiogenesis and enhance the healing of neogenetic tissues, provides a certain direction for treating vascular injury diseases, and expands the clinical application prospect and range of the TIGIT immunoadhesin.
Detailed Description
The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plastrons, methods of inserting genes encoding proteins into such vectors and plastrons, or methods of introducing plasmids into host cells. A Laboratory Manual,2 nd edition，Cold spring Harbor Laboratory Press。
EXAMPLE 1 preparation of TIGIT immunoadhesin
Two immunoadhesins, TIGIT-Fc-wt and TIGIT-FC-LALA-PG, were prepared separately as described in patent document CN 110669139A.
Example 2TIGIT immunoadhesin-mediated killing of tumor cells by immune cells
The killing effect of peripheral mononuclear cells (PBMC) on breast cancer cell line SK-BR-3, lung cancer cell line HCC827, gastric cancer cell line N87, ovarian cancer cell line SK-OV-3 in the presence of TIGIT immunoadhesin was evaluated according to the method of the literature [ Fu W, et al. Nature communications,2019,10 (1): 1-12 ]. The ratio of effector cells to target cells was 10, the drug concentration was 1 μ g/ml, the control IgG served as a negative control, no drug was added as a blank, and the cell killing rate was the final cell signal/blank cell signal for each treatment group. The results are shown in tables 1 to 4.
TABLE 1 killing of SK-BR-3 by PBMC
| Group of | Cell kill rate (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 2 killing of HCC827 by PBMC
| Group of | Cell kill rate (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 3 killing of N87 by PBMC
| Group of | Cell kill rate (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 4 killing of N87 by PBMC
| Group of | Cell kill rate (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
The experiments show that the TIGIT immunoadhesin provided by the invention remarkably enhances the killing activity of immune cells on tumor cells.
Further, the tumor cell lines described above in immunodeficient mice were modeled according to the literature [ Fu W, et al. Nature communications,2019,10 (1): 1-12.] method, and the killing effect of PBMC on tumor cells in an in vivo model was evaluated. For calculating the Tumor Growth Inhibition rate (TGI), the conventional literature is referred to for immune cell injection method and Tumor volume calculation method of mouse model. The mice were dosed once a week for 3 weeks with 40mg/kg tail vein injection, and the control IgG was negative control given to the saline group as a blank control. The results are shown in tables 5 to 8.
TABLE 5 evaluation of animal models of tumor-bearing and immunocyte-acting SK-BR-3 mice
| Group of | TGI (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 6HCC827 mice tumor bearing and immune cell effect animal model evaluation
| Group of | TGI (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 7 evaluation of animal models of tumor-bearing and immunocyte-acting in N87 mice
| Group of | TGI (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 8 evaluation of animal models of tumor-bearing and immune cell effects in SK-OV-3 mice
| Group of | TGI (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
The experiments show that in an in vivo model, the TIGIT immunoadhesin disclosed by the invention effectively enhances the growth inhibition of immune cells on tumor tissues.
Furthermore, tumor tissues after the completion of the mouse model experiment were isolated, and PDL1 expression in each tumor tissue was detected. Isolation of tumor tissue, detection of gene expression in tissue reference [ Hu S, et al, science translational mechanism, 2017,9 (380); yen W C, et al, clinical cancer research,2015,21 (9): 2084-2095. The results are shown in tables 9 to 12.
TABLE 9 relative expression of PDL1 protein in tissue after the completion of SK-BR-3 mouse tumor-bearing model
| Group of | Relative expression (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 10 relative expression of PDL1 protein in tissues after termination of the HCC827 mouse tumor-bearing model
| Group of | Relative expression (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 11 relative expression of PDL1 protein in tissues after termination of N87 mouse tumor-bearing model
| Group of | Relative expression (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 12 relative expression of PDL1 protein in tissue after completion of the SK-OV-3 mouse tumor-bearing model
| Group of | Relative expression (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
The experiments show that the TIGIT immunoadhesin disclosed by the invention effectively improves immunosuppressive signals in tissues, breaks tumor immunosuppressive microenvironment and is beneficial to immune cells to play a role in tumor tissues.
Further, the detection detects the expression level of E-cadherin and Vimentin in the tissues, and the two markers are epithelial-mesenchymal transition markers of the tissues. The results are shown in tables 13 to 20.
TABLE 13 relative expression of tissue E-cadherin proteins after the completion of the SK-BR-3 mouse tumor-bearing model
| Group of | Relative expression (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 14 relative expression of Vimentin protein in tissue after completion of SK-BR-3 mouse tumor-bearing model
| Group of | Relative expression (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 15 relative expression of tissue E-cadherin protein following termination of HCC827 mouse tumor-bearing model
| Group of | Relative expression (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 16 relative expression of tissue Vimentin protein after completion of SK-BR-3 mouse tumor-bearing model
TABLE 17 relative expression of tissue E-cadherin protein following termination of the N87 mouse tumor-bearing model
| Group of | Sex-pair expression (% blank control) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 18 relative expression of tissue Vimentin protein after termination of the N87 mouse tumor-bearing model
| Group of | Sex-pair expression (% blank control) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 19 relative expression of tissue E-cadherin protein after completion of SK-OV-3 mouse tumor-bearing model
| Group of | Sex-pair expression (% blank control) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 20 relative expression of tissue Vimentin protein after completion of SK-OV-3 mouse tumor-bearing model
| Group of | Sex-pair expression (% blank control) | SD | P value vs control IgG |
| ControlIgG | 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 |
These experiments show that the TIGIT immunoadhesin provided by the invention can obviously enhance the expression of E-cadherin in tumor tissues and reduce the expression level of Vimentin protein, namely, the TIGIT immunoadhesin can obviously inhibit epithelial-mesenchymal transition of tissues mediated by immune cells and treatment and reduce the risk of tumor metastasis.
Example 3TIGIT immunoadhesin effects on vascular and microcirculation within tumor tissue
Each of the tumor tissues in the above examples was further subjected to histochemical analysis to detect perfusion of blood vessels in the tissue and to detect hypoxia levels in the tissue by immunohistochemical methods as described in Hu S, et al science metabolic medicine,2017,9 (380); yen W C, et al, clinical cancer research,2015,21 (9): 2084-2095 ]. The results are shown in tables 21 to 24:
TABLE 21 tissue perfusion of blood vessels after completion of the SK-BR-3 mouse tumor-bearing model
TABLE 22 hypoxia situation of SK-BR-3 mice after tumor-bearing model is finished
| Group of | Hypoxia signal (% blank control) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 23HCC827 mouse tumor-bearing model tissue perfusion vessel status after termination
TABLE 24 hypoxia status after termination of tumor-bearing model in HCC827 mice
| Group of | Hypoxia signal (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
According to the experimental surfaces, the TIGIT immunoadhesin provided by the invention can be used for remarkably enhancing the perfusion of blood vessels in tumor tissues, stimulating the angiogenesis in the tumor tissues and relieving the tissue hypoxia level.
Example 4 Effect of TIGIT immunoadhesin on mouse allograft model
A mouse model under a normal immune state was established using a mouse colon cancer cell line MC38, and references were made to the establishment of the model [ Juneja V R, et al. Journal of Experimental Medicine,2017,214 (4): 895-904 ]. Mice were grouped and dosed according to the method of example 2, and immunoadhesin treatment was performed. At this time, the condition of the immune cells does not need to be evaluated by inputting human immune cells. TGI was calculated and the results are shown in table 25:
TABLE 25MC38 mouse tumor-bearing animal model evaluation
| Group of | TGI (% blank) | SD | P value vs control IgG |
| ControlIgG | 3.94 | 5.91 | |
| TIGIT-Fc-wt | 100 | - | P＜0.01 |
| TIGIT-FC-LALA-PG | 100 | - | P＜0.01 |
The results showed that the TIGIT immunoadhesin treatment group completely eliminated the tumor.
To further assess the in-tissue condition, treatment time was reduced and mice were sacrificed 1 week after treatment when TGI was as in table 26.
TABLE 26MC38 mouse tumor-bearing animal model evaluation for shortening treatment time
| Group of | TGI (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
At this point, tumor tissues were isolated according to the method of the above example, and the level of CD8 lymphocyte infiltration in the tissues was examined by immunohistochemical methods, as described in Demotte N, et al cancer research,2010,70 (19): 7476-7488 ], with the results shown in Table 27.
TABLE 27 infiltration of lymphocytes in MC38 tissues
The experiment shows that TIGIT immunoadhesin can effectively increase the infiltration of lymphocytes in tumor tissues.
Further, vascular perfusion and hypoxia in the tumor tissue were evaluated according to the methods described in the above examples, and the results are shown in tables 28 to 29.
TABLE 28MC38 mice tissue perfusion vessel status after tumor-bearing model is finished
TABLE 29MC38 mice hypoxia status after tumor-bearing model completion
| Group of | Hypoxia signal (% blank control) | SD | P value vs control IgG |
| ControlIgG | 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 |
According to the experimental surfaces, the TIGIT immunoadhesin provided by the invention can be used for remarkably enhancing the perfusion of blood vessels in tumor tissues, stimulating the angiogenesis in the tumor tissues and relieving the tissue hypoxia level.
Example 5 effects of TIGIT immunoadhesin on vascular endothelial cells
The effect of TIGIT immunoadhesin on HUVEC vascular endothelial cells was evaluated according to the method of the patent literature (CN 102884073A). The amount of immunoadhesin is 1. Mu.g/ml. VEGF was used as a positive control. The results are shown in tables 30 to 32.
TABLE 30 effects of TIGIT immunoadhesins on vascular endothelial cell proliferation
| Group of | Cell proliferation (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 31 Effect of TIGIT immunoadhesins on vascular endothelial cell migration
| Group of | Cell proliferation (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 32 Effect of TIGIT immunoadhesins on vascular endothelial cell luminal formation
| Group of | Cell lumen (every lumen every view) | SD | P value vs control IgG |
| Blank control | 1.750 | 0.500 | |
| ControlIgG | 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 |
These experiments show that the TIGIT immunoadhesin disclosed by the invention has the functions of promoting the proliferation, migration and lumen formation of vascular endothelial cells.
The in vivo angiogenic ability was further evaluated by testing matrigel plugs according to the literature (CN 102884073A) using the C57 mouse in vivo model, with 1mg of each of the control drug and immunoadhesin, and a 600. Mu.l volume of the drug and matrigel mixture. The results are shown in Table 33:
TABLE 33 Effect of TIGIT immunoadhesin in vivo model angiogenesis
The experimental surface shows that the TIGIT immunoadhesin disclosed by the invention has the effect of promoting angiogenesis in vivo.
Example 6TIGIT immunoadhesin combination
In order to further clarify the immune regulation effect of TIGIT immunoadhesin, the anti-PD-L1 antibody, the anti-PD-1 antibody and the anti-CTLA 4 antibody are adopted to be combined and applied in an MC38 tumor model. All antibody drugs were administered at a dose of 10mg/kg in one administration, according to the method of reference example 2. The dose of the combination was 5mg/ml for each of the two drugs, with n =10. Experimental methods for control antibodies and tumor clearance can be found in the references [ 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.)
TABLE 34 tumor clearance in mice
| Group of | Tumor clearance (%) |
| ControlIgG | 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 |
The experimental surface shows that the TIGIT immunoadhesin disclosed by the invention can mediate the elimination of partial tumor tissues, and can obviously enhance the anti-tumor effect after being combined with the anti-PD-L1 antibody, the anti-PD-1 antibody and the anti-CTLA 4 antibody.
Further, tumor models were re-established and the experiments were repeated, and PD-L1 expression was examined in each treatment group before tumors had regressed, as described in Jiao S, xia W, yamaguchi H, et al, clinical Cancer Research,2017,23 (14): 3711-3720.)
TABLE 35 expression levels of PD-L1 in various groups of tissues
The experimental surface shows that the TIGIT immunoadhesin disclosed by the invention is applied to a PD-1/PD-L1 inhibitor in combination in a tumor model of an immune normal mouse to reduce the loss of antigens in tissues caused by the PD-1/PD-L1 inhibitor and can enhance the anti-tumor effect of the PD-L1/PD-1 signal pathway inhibitor.
Example 7 effects of TIGIT immunoadhesins on angiogenesis in pathological models
(1) Effects on promoting angiogenesis and improving ischemic limb recovery in diabetic limb-ischemic mice
The disease model was modeled according to the patent literature (CN 103260628A), and the drug was administered for 4 weeks before testing, at a dose of 40mg/kg per week by intravenous injection. Each group n =6. Blood flow was assessed according to literature methods. The results are shown in Table 34:
TABLE 36 Effect of in vivo model angiogenesis
Physiological examinations were further performed according to literature procedures, and the results are given in table 35:
TABLE 37 Effect of ischemic hind limb recovery
| Group of | Score of | SD | P value vs control IgG |
| ControlIgG | 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 |
Animals were sacrificed and leg skin capillary density was assessed according to the patent literature methods and the results are shown in table 36:
surface 38 tissue capillaries
These experiments show that the TIGIT immunoadhesin disclosed by the invention effectively enhances the local blood flow level of mice with diabetic limb ischemia and effectively reduces complications.
(2) Effect on post-endometrial injury blood Guan Xin generation and healing
Further, a mouse endometrial injury model was established according to the method of patent document CN 110721196A. After 3 weeks of treatment with the TIGIT immunoadhesin disclosed in the present invention after successful modeling, mouse tissues were isolated and intimal capillary density was assessed using an image analyzer (xu Qing, et al, first university of medicine (2): 104-107.) with the results shown in table 37:
surface 39 tissue capillaries
Further establishing a thick pregnancy model of mouse endometrial injury according to a method of patent document CN 110721196A, then performing mouse pregnancy tissue evaluation, wherein the capillary density of the tissue and the final pregnancy outcome are shown in tables 38-39:
TABLE 40 decidua spiral arterial blood vessel
| Group of | Blood vessel density (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
TABLE 41 pregnancy rates for each group
| Group of | Pregnancy rate | P value vs control IgG |
| Normal mice | 100％ | |
| Model set | 20％ | |
| ControlIgG | 10％ | |
| TIGIT-Fc-wt | 90％ | P＜0.01 |
| TIGIT-FC-LALA-PG | 80％ | P＜0.01 |
These results show that the TIGIT immunoadhesin disclosed by the invention is effective in reducing endometrial injury, enhancing endometrial microvascular generation, enhancing decidua spiral arterial density and improving pregnancy outcome.
(3) Effect on corneal Damage model
According to the patent literature method CN 106265682A, rat corneal alkali burn is established as an animal model, the administration mode is that 50mg/kg of immunoadhesin and a control drug are intravenously injected every day, and the corneal injury area detection and evaluation are carried out by the reference literature method after 7 days of operation. The results are shown in Table 40:
table 42 area of corneal injury
| Group of | Area of injury (mm) 2 ) | SD | P value vs control IgG |
| Model (model) | 1.26 | 0.36 | |
| ControlIgG | 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 |
The experiment shows that the TIGIT immunoadhesin disclosed by the invention effectively reduces the corneal injury area and effectively improves the tissue healing.
(4) Effect on cardiac injury models
The rat model of left anterior descending branch ligation to heart failure was established according to literature procedures [ Li T, et al, the FASEB Journal,2019,33 (6): 7467-7478 ], with immunoadhesin given 2 times a week, each 50mg/kg tail vein injection. Heart tissue was isolated after 4 weeks for heart pathology examination and Masson staining to assess infarct size. The results are shown in Table 41:
TABLE 43 infarct size
| Group of | Area of injury (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
Tissue hypoxia histochemistry measurements were performed simultaneously, with the results shown in table 42:
TABLE 44 myocardial tissue hypoxia
| Group of | Hypoxia signal (% blank) | SD | P value vs control IgG |
| ControlIgG | 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 |
The experiment shows that the TIGIT immunoadhesin disclosed by the invention effectively reduces tissue damage after myocardial infarction and relieves the hypoxia condition.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Claims (10)
- Use of tigit immunoadhesin in the manufacture of a medicament for modulating angiogenesis.
- 2. Use according to claim 1, characterized in that:wherein the agent for regulating angiogenesis is an agent for promoting angiogenesis and normalization of blood vessels.
- 3. Use according to claim 2, characterized in that:wherein the agent for promoting angiogenesis and blood vessel normalization is an agent for promoting proliferation, migration and lumen-forming ability of vascular endothelial cells.
- 4. Use according to claim 3, characterized in that:wherein the agent that promotes angiogenesis and normalization of blood vessels is an agent that recruits vascular supporting cells, promotes normalization of vascular networks, improves tissue blood supply, reduces hypoxia, or reduces tissue damage.
- 5. Use according to claim 1, characterized in that:wherein, the angiogenesis regulating agent takes TIGIT immunoadhesin, nucleotide or recombinant expression vector for coding the same as a unique active ingredient, and also comprises a pharmaceutically acceptable carrier.
- 6. Use of the agent for regulating angiogenesis according to claim 1 for the preparation of an antitumor drug.
- 7. An anti-tumor drug, characterized in that the active ingredients of the drug comprise TIGIT immunoadhesin and at least one immune checkpoint inhibitor.
- 8. The antitumor agent as claimed in claim 7, wherein:wherein the immune checkpoint inhibitor comprises an anti-PD-L1 antibody, an anti-PD-1 antibody, or an anti-CTLA 4 antibody.
- 9. Use of the agent for regulating angiogenesis according to claim 1 for the preparation of a medicament for treating vascular injury repair or improving tissue hypoxia.
- 10. Use according to claim 9, characterized in that:the medicine for treating vascular injury repair or improving tissue hypoxia is a medicine for improving diabetic limb ischemia, endometrial injury and corneal injury and reducing tissue hypoxia after myocardial infarction.
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