CN117159714A - Application of VCAM1-CD49d signal pathway inhibitor in preparation of medicines for treating tumors - Google Patents
Application of VCAM1-CD49d signal pathway inhibitor in preparation of medicines for treating tumors Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, in particular to application of a VCAM1-CD49d signal pathway inhibitor in preparation of a medicine for treating tumors. According to the research of the invention, the knockdown of the tumor Vcam1 gene can promote the infiltration and immune response capacity of inKT cells and enhance the anti-tumor effect mediated by the inKT cells; the use of VCAM1 antibody blocking therapy or CD49d antibody blocking therapy in combination with iNKT cell therapy synergistically inhibits tumor growth progression. The results prove that the VCAM1-CD49d signal channel provides important basis for regulating and controlling new targets of inKT cell infiltration and anti-tumor functions and a new scheme for improving relevant treatment effects of inKT cells, and has important clinical application prospect and value.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of a VCAM1-CD49d signal pathway inhibitor in preparation of a medicine for treating tumors.
Background
Cancer is one of the diseases with highest global mortality. Current approaches for cancer treatment mainly include chemotherapy and radiotherapy, but chemotherapy and radiotherapy have limited effectiveness and serious adverse effects. Thus, new methods for treating cancer that are more effective and safer are needed. The immunotherapy can prevent tumor growth and metastasis by twisting tumor microenvironment immunosuppression state to make tumor immune system exert anti-tumor effect. Therefore, immunotherapy has become the most promising tumor treatment strategy. Currently, immunotherapy involves mainly inhibition of immune checkpoints, adoptive transfer of immune cells, and treatment with Chimeric Antigen Receptor (CAR) cells in three directions (Nelson, lukacs et al 2021).
iNKT cells are a class of innate T lymphocytes. iNKT cells interact with a variety of immune cells, regulating homeostasis and balance in the body (Brennan, brigl et al 2013). Along with the deep research, iNKT cells are found to be involved in the regulation of various diseases such as viral infection, inflammation, tumor and the like. Based on iNKT cell immune monitoring effect and direct or indirect anti-tumor function, iNKT cell immunotherapy has a good application prospect (Fujii and Shimizu 2019). However, studies have demonstrated that accumulation of lactic acid, impaired lipid synthesis, and abnormal glycolysis in the tumor microenvironment lead to abnormal function of iNKT cells within the tumor, thereby preventing iNKT cells from exerting antitumor function (Xie, zhu et al 2016, fu, zhu et al 2019, fu, he et al 2020). Furthermore, the number of iNKT cells in tumor tissue is positively correlated with prognosis of tumor patients, suggesting that iNKT cell infiltration is one of the factors affecting the therapeutic effect of iNKT cells (Tang, liu et al 2021). Therefore, the research of dynamic regulation and control mechanisms affecting the infiltration and activation of iNKT cells and the discovery of key molecules for regulation and control will help to provide a new therapeutic scheme for improving the immune therapeutic effect of iNKT cells.
Disclosure of Invention
In view of this, the present invention provides the use of inhibitors of the VCAM1-CD49d signaling pathway in the manufacture of a medicament for the treatment of tumors.
In order to achieve the above object, the present invention provides the following technical solutions:
the VCAM1-CD49d signal path is used as a target for treating tumor or the VCAM1-CD49d signal path and lymphocyte immunotherapy are combined to be used as the target for treating tumor to prepare medicaments;
the lymphocyte includes at least one of iNKT cell, CD8T cell, NK cell, and MAIT cell.
Use of an inhibitor targeting the VCAM1-CD49d signaling pathway alone or in combination with a lymphocyte immunizing agent in the manufacture of a medicament for the prevention and/or treatment of a tumor;
the lymphocyte immunizing agent comprises a lymphocyte and/or a lymphocyte agonist;
the lymphocyte comprises at least one of iNKT cell, CD8T cell, NK cell, and MAIT cell;
the lymphocyte agonists comprise alpha GalCer, PBS57, alpha GC acC8, alpha GC acC20:2 and OCH.
In the invention, the inhibitor is at least one of 1) to 3):
1) Substances that inhibit the protein activity of the VCAM1-CD49d signaling pathway-associated protein;
2) Substances that degrade VCAM1-CD49d signaling pathway-associated proteins;
3) Substances that inhibit the synthesis of proteins associated with the VCAM1-CD49d signaling pathway;
4) Agents that knock-out or knock-down genes associated with the VCAM1-CD49d signaling pathway.
Any form of inhibition, silencing, knocking out and expression reduction modes or substances of the VCAM1-CD49d signal path related proteins or related genes (such as VCAM1 and CD49 d) can enhance the effect of the inKT cells on inhibiting tumor growth, and the combination of the novel anti-tumor agent and the inKT cell treatment can synergistically inhibit the growth process of tumors, so that the anti-tumor effect is better and more remarkable.
In some embodiments, the VCAM1-CD49d signal pathway-associated protein is VCAM1 and/or CD49d;
the VCAM1-CD49d signal path related genes are VCAM1 and/or ITGA4.
In some embodiments, the agent of 1) to 3) is an antibody or small molecule drug that targets the VCAM1-CD49d signaling pathway. In some embodiments, the inhibitor that targets the VCAM1-CD49d signaling pathway is an anti-VCAM 1 antibody and/or an anti-CD 49d antibody; the small molecule drug is Firategrast, ATL, TCS2314, surfactin C1 or CAM741. Wherein Firategrast, ATL and TCS2314 are targeted CD49d antagonist drugs; surfacin C1 and CAM741 are targeted VCAM1 antagonist drugs.
In some embodiments, in the formulation of 4) the knockout or knockdown of the VCAM1-CD49d signaling pathway related gene, the knockout or knockdown of the gene is accomplished using gene editing techniques common in the art, including but not limited to lentiviral transfection systems, CRISPR-mediated gene editing systems. In the specific embodiment of the invention, a lentivirus transfection system is used for knocking out genes.
According to the research, the invention discovers that knocking down the tumor Vcam1 obviously enhances the infiltration and anti-tumor functions of inKT cells, CD8T cells and NK cells in tumor tissues; knocking down tumor Vcam1 further enhances iNKT cell therapeutic effects; in MC38 and B16F10 tumor models, CD49d antibody blocking therapy or VCAM1 antibody blocking therapy is used for enhancing inKT treatment effect and synergistically inhibiting the growth process of tumors; the VCAM1 can be used as a new target for treating tumors and inKT cell immunotherapy tumors. In addition, the combination of CD49d antibody blocking therapy and inKT cell therapy synergistically inhibit the growth process of tumors, which indicates that CD49d can also be used as a novel target for treating tumors and inKT cell immunotherapy tumors.
In the present invention, the treatment of tumors comprises at least one of the following:
enhancing infiltration of at least one of iNKT cells, CD8T cells, NK cells in the tumor tissue;
reducing the volume and weight of the tumor and inhibiting the growth process of the tumor;
enhancing the immune response capacity of iNKT cells, and promoting the iNKT cells to produce IFN- γ;
improving the indirect activation ability of iNKT cells to CD8T cells and NK cells, enhancing the immune response of CD8T cells and NK cells, and promoting IFN- γ production.
In the invention, the tumor comprises at least one of colon cancer, melanoma, low-grade glioma and liver cancer.
In the present invention, the lymphocytes include at least one of iNKT cells, CD8T cells, NK cells, and MAIT cells.
The invention also provides a composition comprising lymphocytes and an antibody that targets the VCAM1-CD49d signaling pathway; or an antibody comprising lymphocytes, lymphocyte agonists, and targeting VCAM1-CD49d signaling pathway;
the antibodies of the VCAM1-CD49d signal pathway are anti-VCAM 1 antibodies and/or anti-CD 49d antibodies; the lymphocytes include iNKT cells, CD8T cells, NK cells, and MAIT cells.
The invention also provides an anti-tumor drug, which comprises the composition and a pharmaceutically acceptable carrier.
The invention also provides a method of treating a tumor comprising: administering an inhibitor that targets the VCAM1-CD49d signaling pathway;
or comprises: lymphocytes, lymphocyte agonists, and inhibitors targeting the VCAM1-CD49d signaling pathway are administered.
Wherein the administration of an inhibitor of a VCAM1-CD49d signaling pathway comprises an antibody or small molecule drug targeting the VCAM1-CD49d signaling pathway; the antibody may in particular be an anti-VCAM 1 antibody and/or an anti-CD 49d antibody. Further, the lymphocyte comprises at least one of iNKT cell, CD8T cell, NK cell, and MAIT cell. The lymphocyte agonists comprise alpha GalCer, PBS57, alpha GCacC8, alpha GC acC20:2 and OCH.
According to the research of the invention, the knockdown of the tumor Vcam1 gene can promote the infiltration and immune response capacity of inKT cells and enhance the anti-tumor effect mediated by the inKT cells; the use of VCAM1 antibody blocking therapy or CD49d antibody blocking therapy in combination with iNKT cell therapy synergistically inhibits tumor growth progression. The results prove that the VCAM1-CD49d signal channel provides important basis for regulating and controlling new targets of inKT cell infiltration and anti-tumor functions and a new scheme for improving relevant treatment effects of inKT cells, and has important clinical application prospect and value.
Drawings
FIG. 1 shows that knockdown of tumor Vcam1 enhances immune cell infiltration and responsiveness; (A) Knocking down the tumor tissue and transferring iNKT cells, endogenous iNKT cells, CD8T cells and NK cell proportion statistics map of the control tumor tissue; (B) Knocking down tumor tissues and transferring iNKT cells, endogenous iNKT cells, CD8T cells, NK cells IFN-positive proportion statistics of control tumor tissues;
FIG. 2 shows that knockdown of tumor Vcam1 synergistically enhances inKT cell therapy; (A) MC38 solid tumor model with knocked-down Vcam1 gene is combined with inKT cell transfusion and alpha GC treatment flow chart; (B) tumor solid plots of mice from different treatment groups on day 32 tumor-bearing; (C) Tumor weight statistics of mice in different treatment groups on day 32 with tumors; (D) a statistical plot of tumor growth in mice from different treatment groups;
FIG. 3 shows that CD49d or VCAM1 antibody blocking treatment enhances MC38 tumor inKT treatment effect; (A) CD49d antibody blocking combined iNKT cell therapy MC38 tumor model flow chart; (B) tumor solid plots of mice from different treatment groups on day 30 tumor-bearing; (C) Tumor weight statistics of mice in different treatment groups on day 30 with tumor; (D) a statistical plot of tumor growth in mice from different treatment groups; (E) Statistics of the positive proportion of iNKT cells, CD8T cells, NK cells IFN- γ in tumors of mice of different treatment groups on day 30 of tumor-bearing; (F) VCAM1 antibody blocking combined iNKT cell therapy MC38 tumor model flow diagram; (G) tumor growth statistics of mice from different treatment groups; (H) tumor weight statistics of mice from different treatment groups on day 29;
FIG. 4 shows that CD49d or VCAM1 antibody blocking treatment enhances the therapeutic effect of B16F10 tumor inKT; (A) CD49d or VCAM1 antibody blocking combined inKT cell therapy model flow chart for B16F10 tumor; (B) tumor solid plots of mice from different treatment groups on day 20 tumor-bearing; (C) Tumor weight statistics of mice in different treatment groups on day 20 with tumors; (D) a statistical plot of tumor growth in mice from different treatment groups; (E) Statistics of positive proportion of iNKT cells, CD8T cells, NK cells IFN- γ in tumor-bearing day 20 mice of different treatment groups;
FIG. 5 shows blocking VCAM1 signaling to promote activation of human iNKT cells; (A) an in vitro three-system experimental flow diagram of the humanized cell; (B) Statistics of IFN-gamma positive proportion of human inKT cells in different treatment groups; (C) The IFN-. Gamma.production by the different treatment groups of human iNKT cells is schematically shown.
Detailed Description
The invention provides application of VCAM1-CD49d signal pathway inhibitor in preparing a medicament for treating tumors. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Wild Type (WT) mice: purchased from Jiangsu Jizhikang biotechnology Co., ltd;
Vα14 Tg cxcr6 Gfp mice: the university of chicago, U.S. a. Bendelac professor problem group gifts;
MC38: wang Yocai, university of science and technology, teaches that the subject group gives away;
B16F10: wang Yocai, university of science and technology, teaches that the subject group gives away;
hela hcd1d: the university of chicago, U.S. a. Bendelac professor problem group gifts;
MDA-MB-231.HVCAM1: the inventor constructs;
NTC MC38: the inventor constructs;
shVcam1 MC38: the inventor constructs;
αGC：Avanti Polar Lipid，86700；
zombie reactive dye: biolegend,423106;
anti-murine CD49d antibody: bioxcell, BE0071;
anti-murine VCAM1 antibody: bioxcell, BE0027;
rat IgG isotype control antibody: bioxcell, BE0090;
PerCP/Cy5.5 anti-murine CD45.2 antibody: biolegend,109828;
APC/Cy7 anti-murine NK1.1 antibody: biolegend,108724;
pacific Blue anti-murine TCR beta antibody: biolegend,108724;
BV510 anti-murine B220 antibody: biolegend,103248;
BV605 anti-murine CD8 a antibody: biolegend,100743;
PE/Cy7 anti-murine IFN-gamma antibody: biolegend,505826;
anti-human VCAM1 antibody: biolegend,305802;
mouse IgG isotype control antibody: biolegend,400101;
FITC anti-human CD3 antibody: biolegend,317306;
PerCP/Cy5.5 anti-human IFN-gamma antibody: biolegend,502526;
APC murine CD1d-PBS57 tetramer and APC human CD1d-PBS57 tetramer: are all provided by the university of emery tetramer research and development center in the national institutes of health.
The hela.hcd1d cell line is an artificial mimetic antigen presenting cell, a cell line obtained by over-expressing human CD1d on HeLa cells, wherein CD1d can capture activator aGC and thereby activate NKT.
In the present invention, MDA-MB-231.HVCAM1 is a cell line for over-expressing human VCAM1 gene (full length gene) on human MDA-MB-231 cells, wherein the vector used for over-expression is (pcDNA3.1). shVcam1 MC38 refers to a cell line obtained by knocking down the Vcam1 gene on a mouse colon cancer cell line MC38 by using shRNA technology. NTC MC38 is a knockdown control group: a cell line transfected with shRNA without target gene (none target control cells transfected with scramble shRNA). All three cell lines described above can be constructed and obtained according to methods conventional in the art.
The invention is further illustrated by the following examples:
example 1 tumor VCAM1 inhibits infiltration of effector cells and the ability to respond to immunity
1. Amplification of mouse iNKT-GFP cells
(1)Vα14 Tgcxcr6 Gfp Mouse spleen tissue was ground to a single cell suspension via a 200 mesh copper mesh. 1500r/min, and centrifuging for 5min. The pellet was lysed by adding erythrocyte lysate for 2min and the lysis was stopped by adding 10% fetal bovine serum-1640 medium.
(2) 1500r/min, and centrifuging for 5min. The sediment is added into 10% fetal bovine serum-1640 culture medium and is evenly mixed. Cell density was adjusted to 2X 10 6 Per ml, and 100IU/ml IL-2, 100ng/ml alpha GC was added to the cell suspension.
(3) Placed at 37 ℃ and 5% CO 2 The cell culture was incubated in an incubator for 4 days.
(4) All cell suspensions were collected, 1500r/min and centrifuged for 5min. The pellet was resuspended in 10% fetal bovine serum-1640 medium containing 100IU/ml IL-2 and the cell density was adjusted to 1X 10 6 /ml。
(5) When the cell density exceeds 2X 10 6 At/ml, the cells were plated in a half-liquid-change mode.
(6) The dead cells were removed by Ficoll solution on days 10-14 of culture. The cell suspension and Ficoll liquid are added according to the volume of 1:1 at the upper and lower parts, the centrifugal rising rate is 1, the dropping rate is 0, 500g and the centrifugal is carried out for 15min at room temperature. The intermediate buffy coat after centrifugation expands iNKT-GFP cells.
2. shRNA lentivirus transfected knockdown MC38 cell Vcam1 gene
(1) HEK-293T cells cultured in a 10cm dish and with a cell density of 80% -90% were selected.
(2) 500. Mu.l of Opti-MEM, 12. Mu.g of VCAM1 shRNA plasmid or the Scramble shRNA control plasmid, 3.5. Mu.g of pMD2G and psPAX2. Uniformly mixing to prepare a reaction liquid 1. Preheating for 5min at 37 ℃; mu.l of Opti-MEM and 66. Mu.g of PEI were mixed uniformly to prepare a reaction solution 2. (VCAM 1 shRNA sequence: CCAGATCCTTAATACTGTTTA)
(3) Slowly adding the reaction solution 1 into the reaction solution 2, uniformly mixing, and standing at room temperature for 30min. The mixture was slowly and evenly added dropwise to a 10cm dish.
(4) After 6-8 hours, the medium was changed, and 10ml of 5% FBS-DMEM medium was added. After culturing for 72 hours, collecting cell supernatant, and filtering the supernatant by a 0.22 mu m filter, wherein the obtained supernatant is virus stock solution.
(5) MC38 cells which are cultured in a 10cm dish and have a cell density of 80% -90% are selected, the cell supernatant is removed, 4ml of virus stock solution is added, and Polybrene with a final concentration of 8 mug/ml is added. After 6-8 h of infection, 8ml of 10% FBS-DMEM complete medium was added uniformly.
(6) 48h after infection, cells were passaged and knockdown efficiency was detected. Puromycin was added at a final concentration of 1. Mu.g/ml at cell passage to maintain the trait. The knockdown Vcam1 group is shVcam1 MC38 cells, and the negative control group is NTC MC38 cells.
3. Knocking down tumor Vcam1 gene enhances infiltration and immune response capacity of intratumoral iNKT cells, CD8T cells and NK cells
(1) Knocking down the Vcam1 gene increases tumor cell apoptosis. Therefore, the experiment is carried out to plant different numbers of tumor cells when carrying tumor, so as to ensure that the sizes of tumor tissues are consistent when the experiment is carried out. WT mouse lotus 1X 10 6 NTC MC38 cells and 1.5X10 6 shVcam1 MC38 cells.
(2) Tumor-bearing day 8, intravenous infusion 2X 10 7 And (3) amplifying the iNKT-GFP cells.
(3) On day 10 of tumor bearing, αgc (2 μg/dose) was injected intraperitoneally.
(4) After 4h of intraperitoneal injection, mice were sacrificed and spleen tissues and tumor tissues of the mice were removed.
(5) Spleen tissue was ground to a single cell suspension via a 200 mesh copper mesh. 1500r/min, and centrifuging for 5min. The pellet was lysed by adding erythrocyte lysate for 2min and 1% bovine serum albumin-PBS solution was added to terminate the split red. Centrifuging for 5min, and precipitating to obtain spleen lymphocyte.
(6) Tumor tissue was minced and digested for 45min with 0.1% collagenase IV-PBS solution in a shaker at 37℃at 200 r/min. Digestion was stopped by adding 1% bovine serum albumin-PBS solution. Vortex shaking to mix tissue suspension, passing through 200 mesh copper mesh, collecting cell suspension, centrifuging at 2000r/min for 10min. An appropriate amount of 40% percoll solution was added according to the amount of precipitation and mixed well. Half the volume of the 40% percoll solution of 70% percoll solution was slowly added to the lower layer of 40% percoll solution along the tube wall. At room temperature, the centrifugal rising rate is 6, the falling rate is 2, 2000r/min and the centrifugal force is 20min. The intermediate tunica albuginea layer is tumor lymphocytes.
(7) Collecting the lymphocytes extracted from (5) and (6), carrying out surface staining to mark iNKT cells, CD8T cells and NK cells, and carrying out intracellular staining to detect the expression of IFN-gamma of various cells.
Based on the indirect activation of CD8T cells and NK cells by iNKT cells, activated intratumoral iNKT cells promote activation of CD8T cells and NK cells following in vivo injection of mouse lipid antigen αgc. Knocking down tumor Vcam1 resulted in increased infiltration of intratumoral transfusion iNKT cells (GFP positive), endogenous iNKT cells (GFP negative), CD8T cells and NK cells, and an increase in cell proportion (fig. 1A). Meanwhile, knocking down tumor Vcam1 promotes iNKT cell activation and cytokine IFN- γ production, improves indirect activation ability of iNKT cells to CD8T cells and NK cells, increases CD8T cell and NK cell immune response and IFN- γ production (fig. 1B).
Example 2 knockdown of tumor Vcam1 in combination with inKT cell therapy to enhance the anti-tumor effect of inKT cells and inhibit the growth process of tumors
(1) WT mouse lotus 1X 10 6 NTC MC38 cells and 1.5X10 6 shVcam1 MC38 cells.
(2) Tumor-bearing mice were randomized on day 8:
a first group: tumor-bearing NTC MC38 cells, given PBS solution control treatment;
second group: tumor-bearing NTC MC38 cells, iNKT cell transfusion and αgc treatment;
third group: tumor-bearing shVcam1 MC38 cells are subjected to PBS solution control treatment;
fourth group: tumor-bearing shVcam1 MC38 cells, iNKT cell transfusion and αgc treatment were given.
(3) Tumor-bearing day 8, iNKT cell-treated tail mice were infused intravenously 2×10 7 The iNKT-GFP cells were individually expanded and the mice of the control group were infused with equal volumes of PBS solution at their tail veins.
(4) On day 10 of tumor bearing, iNKT cell treated mice were intraperitoneally injected with αgc (2 μg/mouse) and control mice were intraperitoneally injected with the same volume of PBS solution.
(5) Tumor length, width, volume = length x width x height (1/2 width) were measured every other day starting on day 8 of tumor loading.
(6) Tumor-bearing day 32, when tumor volume of mice appeared to exceed 2000mm 3 Is terminated and the tumor weights of mice in the different treatment groups are counted.
Treatment and observation of tumor growth in mice by group (FIG. 2A), inKT cell treatment was effective to inhibit the progress of tumor growth, and decrease the weight of tumor (FIGS. 2B-D). Knocking down tumor Vcam1 synergistically enhanced iNKT cell therapy, further inhibiting tumor growth (fig. 2B-D).
Example 3 in MC38 tumor model, CD49d antibody blocking therapy or VCAM1 antibody blocking therapy was effective in enhancing inKT cell therapy effect, synergistically inhibiting the growth process of tumor
1. CD49d antibody blocking treatment improves inKT cell treatment effect and cooperatively inhibits growth process of MC38 solid tumor
(1) WT mouse lotus 1X 10 6 And MC38 cells.
(2) Tumor-bearing mice were randomized on day 7:
a first group: iNKT cell transfusion and αgc treatment, treatment with rat IgG isotype control antibody;
second group: iNKT cell transfusion and αgc treatment, anti-murine CD49d antibody treatment was administered.
(3) On day 8 of tumor bearing, mice in each group were infused 2X 10 by tail vein infusion 7 And (3) amplifying the iNKT-GFP cells.
(4) Starting from day 9 of tumor-bearing, mice were intraperitoneally injected with anti-mouse CD49d antibody (10 mg/kg) or rat IgG isotype control antibody (10 mg/kg) on every two days in groups, and were subjected to drug treatment on days 9, 12, 15, 18, 21, 24, and 27, respectively.
(5) On day 10 of tumor bearing, mice in each group were intraperitoneally injected with αgc (2 μg/mouse).
(6) Tumor length, width, volume = length x width x height (1/2 width) were measured every other day starting on day 7 of tumor loading.
(7) Tumor-bearing day 30, when tumor volume of mice appeared to exceed 2000mm 3 The treatment was terminated, the tumor weights of the mice of the different treatment groups were counted and tumor tissue lymphocytes were extracted.
(8) Collecting the extracted tumor tissue lymphocytes, carrying out surface staining for marking iNKT cells, CD8T cells and NK cells, and carrying out intracellular staining for detecting the expression condition of IFN-gamma of each cell.
Tumor growth was observed in groups of mice treated in groups (FIG. 3A), blocking CD49D signaling effectively enhanced the treatment effect of iNKT cells, reduced tumor weight, and further inhibited tumor growth progression (FIGS. 3B-D). Through flow cytometry detection, it was found that the CD49d antibody blocking treatment significantly improved the anti-tumor effect of iNKT cells, enhanced the indirect activation ability of iNKT cells to CD8T cells and NK cells, and further improved the anti-tumor ability of CD8T cells and NK cells (fig. 3E).
2. VCAM1 antibody blocking treatment improves inKT cell treatment effect, and synergistically inhibits growth progress of MC38 solid tumor
(1) WT mouse lotus 1X 10 6 And MC38 cells.
(2) Tumor-bearing mice were randomized on day 7:
a first group: iNKT cell transfusion and αgc treatment, treatment with rat IgG isotype control antibody;
second group: iNKT cell transfusion and αgc treatment, anti-murine VCAM1 antibody treatment was administered.
(3) On day 8 of tumor bearing, mice in each group were infused 2X 10 by tail vein infusion 7 And (3) amplifying the iNKT-GFP cells.
(4) Starting from day 9 of tumor-bearing, mice were intraperitoneally injected with anti-mouse VCAM1 antibody (10 mg/kg) or rat IgG isotype control antibody (10 mg/kg) on every two days in groups, and were subjected to drug treatment on days 9, 12, 15, 18, 21, 24, and 27, respectively.
(5) On day 10 of tumor bearing, mice in each group were intraperitoneally injected with αgc (2 μg/mouse).
(6) Tumor length, width, volume = length x width x height (1/2 width) were measured every other day starting on day 8 of tumor loading.
(7) Tumor-bearing day 29 when tumor volume in mice appeared to exceed 2000mm 3 The treatment was terminated and the tumor weights of mice from different treatment groups were counted.
The tumor growth of mice in different treatment groups was treated and observed in groups (fig. 3F), blocking VCAM1 signaling effectively enhanced iNKT cell therapy, reduced tumor weight, and further inhibited tumor growth progression (fig. 3G-H).
Example 4 blocking of VCAM1-CD49d signaling in B16F10 tumor model effectively enhances inKT cell therapy and synergistically inhibits tumor growth progression
(1) WT mouse lotus 2X 10 5 B16F10 cells.
(2) Tumor-bearing mice were randomized on day 8:
a first group: iNKT cell transfusion and αgc treatment, treatment with rat IgG isotype control antibody;
second group: iNKT cell transfusion and αgc treatment, anti-murine CD49d antibody treatment;
third group: iNKT cell transfusion and αgc treatment, anti-murine VCAM1 antibody treatment was administered.
(3) On day 8 of tumor bearing, mice in each group were infused 2X 10 by tail vein infusion 7 And (3) amplifying the iNKT-GFP cells.
(4) Starting from day 9 of tumor-bearing, mice were intraperitoneally injected with anti-mouse CD49d antibody (10 mg/kg), anti-mouse VCAM1 antibody (10 mg/kg) or rat IgG isotype control antibody (10 mg/kg) on every two days in groups, and were subjected to drug treatment on days 9, 12, 15, and 18, respectively.
(5) On day 10 of tumor bearing, mice in each group were intraperitoneally injected with αgc (2 μg/mouse).
(6) Tumor length, width, volume = length x width x height (1/2 width) were measured every other day starting on day 7 of tumor loading.
(7) Tumor-bearing day 20 when tumor volume of mice appeared to exceed 2000mm 3 The treatment was terminated, the tumor weights of the mice of the different treatment groups were counted and tumor tissue lymphocytes were extracted.
(8) Collecting the extracted tumor tissue lymphocytes, carrying out surface staining for marking iNKT cells, CD8T cells and NK cells, and carrying out intracellular staining for detecting the expression condition of IFN-gamma of each cell.
Tumor growth was observed in groups of mice treated with either CD49D antibody or VCAM1 antibody blocking the VCAM1-CD49D signal, which enhanced the treatment effect of the iNKT cells, decreased tumor weight, and further inhibited tumor growth (FIGS. 4B-D). Through flow cytometry detection, the VCAM1 antibody blocking treatment or the CD49d antibody blocking treatment obviously improves the anti-tumor effect of the inKT cells, enhances the indirect activation capability of the inKT cells on the CD8T cells and the NK cells, and further improves the anti-tumor capability of the CD8T cells and the NK cells (figure 4E).
Example 5 inhibition of activation of human iNKT cells by human tumor VCAM1
1. Expansion of human iNKT cells
(1) The proportion of iNKT cells in human tissue and blood is low. Therefore, in this experiment, a large number of human iNKT cells were obtained by in vitro amplification of human peripheral blood PBMCs. Ficoll liquid separates human peripheral blood PBMC, and centrifugation at room temperature at 1 rise rate and 0, 500g drop rate for 15min. The intermediate buffy coat after centrifugation was PBMC.
(2) PBMC were washed twice with 10% fetal bovine serum-1640 human lymphocyte complete medium, 1500r/min and centrifuged for 5min.
(3) The sediment is added with 10 percent of fetal bovine serum-1640 complete culture medium of human lymphocytes and is evenly mixed. Cell density was adjusted to 4X 10 6 Per ml, and 200IU/ml IL-2, 1. Mu.g/ml alpha GC was added to the cell suspension.
(4) Placed at 37 ℃ and 5% CO 2 The cell culture was incubated in an incubator for 4 days.
(5) All cell suspensions were collected, 1500r/min and centrifuged for 5min. 10% fetal bovine serum-1640 human lymphocyte complete medium containing 200IU/ml IL-2 was resuspended and pelleted and the cell density was adjusted to 1X 10 6 /ml。
(6) When the cell density exceeds 2X 10 6 At/ml, the cells were plated in a half-liquid-change mode.
(7) The dead cells were removed by Ficoll solution on days 10-14 of culture.
2. Blocking human tumor VCAM1 signaling effectively promotes activation of human inKT cells and production of cytokine IFN-gamma
(1) The log phase MDA-MB-231.HVCAM1 cells and HeLa. HCD1d cells were digested.
(2) Adding 1X 10 per packet 5 MDA-MB-231.HVCAM1 cells and 1X 10 5 Individual hela.hcd1d cells were plated evenly and added in groups either αgc or equal volumes of PBS solution at a final concentration of 1 μg/ml.
A first group: PBS, amplified human iNKT cells, heLa. HCD1d cells, PBS negative control
Second group: αgc, expanded human iNKT cells, hela.hcd1d cells, PBS negative control
Third group: αgc, expanded human iNKT cells, hela. Hcd1d cells, MDA-MB-231.Hvcam1 cells, mouse IgG isotype control antibodies
Fourth group: αgc, expanded human iNKT cells, hela.hcd1d cells, MDA-MB-231.hvcam1 cells, anti-human VCAM1 antibodies
(3) Culturing for 10-12 hr, removing supernatant, adding 2×10 5 Human iNKT cells were individually expanded and anti-human VCAM1 antibody, mouse IgG isotype control antibody or PBS solution of equal volume was added in groups to a final concentration of 5 μg/ml.
(4) After 12h of co-culture system treatment, cells were collected and examined for IFN-. Gamma.production by human iNKT cells.
The co-culture system was added with different types of cells and treatment drugs in order and groups (fig. 5A). The production of the human iNKT cytokine IFN- γ was examined by flow cytometry in different treatment groups. It was found statistically that co-culturing a tumor cell line overexpressing human VCAM1 with human iNKT cells effectively inhibited activation of human iNKT cells (fig. 5B-C), blocking VCAM1-CD49d signaling with VCAM1 antibodies significantly restored the immune response of human iNKT cells, promoting production of cytokine IFN- γ by human iNKT cells (fig. 5B-C).
Reference to the literature
1.Brennan,P.J.,M.Brigl and M.B.Brenner(2013)."Invariant natural killer T cells:an innate activation scheme linked to diverse effector functions."Nat Rev Immunol 13(2):101-117.
2.Fu,S.,K.He,C.Tian,H.Sun,C.Zhu,S.Bai,J.Liu,Q.Wu,D.Xie,T.Yue,Z.Shen,Q.Dai,X.Yu,S.Zhu,G..Liu,R.Zhou,S.Duan,Z.Tian,T.Xu,H.Wang and L.Bai(2020)."Impaired lipid biosynthesis hinders anti-tumor efficacy of intratumoral iNKT cells."Nat Commun 11(1):438.
3.Fu,S.,S.Zhu,C.Tian,S.Bai,J.Zhang,C.Zhan,D.Xie,L.Wang,Z.Li,J.Li,H.Zhang,R.Zhou,Z.Tian,T.Xu and L.Bai(2019)."Immunometabolism regulates TCR recycling and iNKT cell functions."Sci Signal 12(570).
4.Fujii,S.I.and K.Shimizu(2019)."Immune Networks and Therapeutic Targeting of iNKT Cells in Cancer."Trends Immunol 40(11):984-997.
5.Nelson,A.,J.D.Lukacs and B.Johnston(2021)."The Current Landscape of NKT Cell Immunotherapy and the Hills Ahead."Cancers(Basel)13(20).
6.Tang,R.,X.Liu,C.Liang,J.Hua,J.Xu,W.Wang,Q.Meng,J.Liu,B.Zhang,X.Yu and S.Shi(2021)."Deciphering the Prognostic Implications of the Components and Signatures in the Immune Microenvironment of Pancreatic Ductal Adenocarcinoma."Front Immunol 12:648917.
7.Xie,D.,S.Zhu and L.Bai(2016)."Lactic acid in tumor microenvironments causes dysfunction of NKT cells by interfering with mTOR signaling."Sci China Life Sci 59(12):1290-1296.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (12)
- Application of VCAM1-CD49d signal path as target for treating tumor or application of VCAM1-CD49d signal path combined lymphocyte immunotherapy as target for treating tumor in preparation of medicines ；The lymphocyte includes at least one of iNKT cell, CD8T cell, NK cell, and MAIT cell.
- 2. Use of an inhibitor targeting the VCAM1-CD49d signaling pathway alone or in combination with a lymphocyte immunizing agent in the manufacture of a medicament for the prevention and/or treatment of a tumor;the lymphocyte immunizing agent comprises a lymphocyte and/or a lymphocyte agonist;the lymphocyte comprises at least one of iNKT cell, CD8T cell, NK cell, and MAIT cell;the lymphocyte agonists comprise alpha GalCer, PBS57, alpha GC acC8, alpha GC acC20:2 and OCH.
- 3. The use according to claim 2, wherein the inhibitor is at least one of 1) to 4):1) Substances that inhibit the protein activity of the VCAM1-CD49d signaling pathway-associated protein;2) Substances that degrade VCAM1-CD49d signaling pathway-associated proteins;3) Substances that inhibit the synthesis of proteins associated with the VCAM1-CD49d signaling pathway;4) Agents that knock-out or knock-down genes associated with the VCAM1-CD49d signaling pathway.
- 4. The use according to claim 3, wherein the VCAM1-CD49d signal pathway-related protein is VCAM1 and/or CD49d;the VCAM1-CD49d signal path related genes are VCAM1 and/or ITGA4.
- 5. The use according to claim 3, wherein the substance of 1) to 3) is an antibody or a small molecule drug targeting the VCAM1-CD49d signaling pathway.
- 6. The use according to claim 5, wherein the antibody is an anti-VCAM 1 antibody and/or an anti-CD 49d antibody; the small molecule drug is Firategrast, ATL, TCS2314, surfactin C1 or CAM741.
- 7. The use according to claim 3, wherein the formulation in 4) comprises a lentiviral transfection system, a CRISPR mediated gene editing system.
- 8. The use according to any one of claims 1 to 7, wherein the treatment of a tumour comprises at least one of:enhancing infiltration of at least one of iNKT cells, CD8T cells, NK cells in the tumor tissue;reducing the volume and weight of the tumor and inhibiting the growth process of the tumor;enhancing the immune response capacity of iNKT cells, and promoting the iNKT cells to produce IFN- γ;improving the indirect activation ability of iNKT cells to CD8T cells and NK cells, enhancing the immune response of CD8T cells and NK cells, and promoting IFN- γ production.
- 9. The use according to any one of claims 1 to 8, wherein the tumour comprises at least one of colon cancer, melanoma, low grade glioma, liver cancer.
- 10. The use according to any one of claims 1 to 9, wherein said lymphocytes comprise at least one of iNKT cells, CD8T cells, NK cells, MAIT cells.
- 11. A composition comprising lymphocytes and an antibody that targets the VCAM1-CD49d signaling pathway; or an antibody comprising lymphocytes, lymphocyte agonists, and targeting VCAM1-CD49d signaling pathway;the antibodies of the VCAM1-CD49d signal pathway are anti-VCAM 1 antibodies and/or anti-CD 49d antibodies;the lymphocyte comprises at least one of iNKT cell, CD8T cell and NK cell.
- 12. An antitumor agent comprising the composition of claim 11 and a pharmaceutically acceptable carrier.
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