CN116926193B - Tumor immunotherapy prognosis evaluation preparation and application of ANO1 targeting agent in preparation of tumor prognosis improving medicine - Google Patents
Tumor immunotherapy prognosis evaluation preparation and application of ANO1 targeting agent in preparation of tumor prognosis improving medicine Download PDFInfo
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Abstract
The invention discloses a tumor immunotherapy prognosis evaluation preparation and application of an ANO1 targeting reagent in preparation of a medicine for improving tumor prognosis. The invention reveals a brand new value of ANO1 as a target point and a new function of the existing targeted ANO1 inhibitor, and finally proves great potential of ANO1 as a tumor immunotherapy curative effect marker and a molecular target point in digestive tract tumor.
Description
Technical Field
The invention relates to the technical field of tumor markers and biological medicines, in particular to a tumor immunotherapy prognosis evaluation preparation and application of a reagent targeting ANO1 in preparation of medicines for improving tumor prognosis.
Background
The lack of tumor therapeutic targets is a long-felt problem for the development of anti-tumor drugs. In recent years, targeted therapies typified by HER2 targeting and immunotherapy typified by anti-PD1 (anti-PD-1) have made great progress in various tumors typified by digestive system tumors, but lack of effective therapeutic markers, difficulty in distinguishing benefited populations, and patients often develop primary and secondary drug resistance. Thus, there is a need to find targets with pharmaceutical value and develop concomitant diagnostic means and reverse drug resistance strategies for immunotherapy.
Disclosure of Invention
The invention aims to provide a tumor immunotherapy prognosis evaluation preparation and application of an ANO1 targeting agent in preparation of a medicine for improving tumor prognosis.
ANO1 (DOG 1/TMEM 16A), also known as transmembrane protein 16A, is a structural protein of calcium-activated chloride channel, and the coding Gene (Gene ID:55107 in NCBI) is located in the CCNDA-EMS1 locus of human chromosome 11q 13. ANO1 is highly expressed in a variety of digestive tract tumors including esophageal cancer, gastric cancer, colon cancer, rectal cancer, and gastrointestinal stromal tumors, and is significantly more expressed in tumor tissue than in paracancerous tissue. A few researches on ANO1 report on the promotion effect of the ANO1 on tumor proliferation and the promotion effect of the ANO1 on tumor metastasis, but the reports on the in-vitro level of a cell line are mainly used, the reports on a small number of immunodeficiency animals are assisted, and the research report on the ANO1 serving as a treatment target point is not yet seen. The invention firstly proves the value of ANO1 as an immune therapy accompanying diagnostic marker by carrying out identification on a patient sample and systematic research on a cell in vitro level, an immune deficiency animal level/an immune sound animal level/a PDX model level, and reveals the potential of ANO1 as a drug therapy target for single application or for reversing the drug resistance of targeted HER2 therapy and immune therapy, wherein the depth and the breadth of the research are far beyond those of the prior research on ANO 1.
To achieve the object of the present invention, in a first aspect, the present invention provides an application of an ANO1 gene in preparing a prognostic evaluation preparation or a drug resistance evaluation preparation for tumor immunotherapy, designing a detection product capable of detecting the expression of ANO1 and its product at the gene level or the protein level according to the protein encoded by the ANO1 gene or the ANO1 gene, and using the detection product as the prognostic evaluation preparation or the drug resistance evaluation preparation for tumor immunotherapy.
In a second aspect, the present invention provides a therapeutic and prognostic agent for tumour immunotherapy, said agent being capable of detecting ANO1 expression and products thereof at the gene level or protein level.
Further, the reagents include, but are not limited to, primers, probes, gene chips, or antibodies to ANO 1.
In a third aspect, the present invention provides the use of ANO1 as a tumor immunotherapy efficacy and prognosis biomarker in the construction of a tumor prognosis evaluation model or a tumor resistance evaluation model by detecting the gene/protein expression level of ANO1 in a patient as a tumor immunotherapy efficacy and prognosis evaluation biomarker, or as a tumor resistance marker, and using it in the construction of a tumor immunotherapy efficacy and prognosis evaluation model, or in the construction of a tumor resistance evaluation model.
Such tumors include, but are not limited to, esophageal cancer, gastric cancer, colon cancer, rectal cancer, gastrointestinal stromal tumor, melanoma, breast cancer.
In a fourth aspect, the present invention provides the use of an agent targeting ANO1, said agent targeting ANO1 comprising a functional expression inhibitor of ANO1, said functional expression inhibitor being a substance capable of inhibiting the expression or activity of the ANO1 gene or protein at the transcriptional or translational level, said inhibitor being selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low-molecular compounds, peptides, antibodies, etc., for the manufacture of a medicament for improving tumor prognosis or reversing tumor resistance.
The tumor drug resistance comprises an anti-tumor drug, a tumor immunotherapy drug and the like which take HER2 as a therapeutic target point.
Preferably, the anti-tumor drug with HER2 as a therapeutic target includes, but is not limited to trastuzumab.
Preferably, the tumor immunotherapeutic agent comprises an anti-PD-1 immunotherapeutic agent; such anti-PD-1 immunotherapeutic agents include, but are not limited to Pembrolizumab, nivolumab, sintilimab, tislelizumab, camrelizumab, atezolizumab, envolimab.
In a fifth aspect, the invention provides an anti-neoplastic agent, said agent comprising an agent that targets ANO1, or said agent comprising an agent that targets ANO1 and a FAP inhibitor;
Wherein the agent that targets ANO1 comprises a nucleic acid interfering sequence that targets ANO1 and/or an ANO1 inhibitor.
Preferably, the nucleic acid interfering sequence targeted to inhibit human ANO1 is as shown in SEQ ID NO:1-2 or SEQ ID NO:3-4 (human ANO1 interfering sequence sh-1-ANO1 or sh-2-ANO 1).
Preferably, the nucleic acid interfering sequence targeted to inhibit mouse ANO1 is shown as SEQ ID NO 5-6 or SEQ ID NO 7-8 (mouse ANO1 interfering sequence sh-1-ANO1 or sh-2-ANO 1).
Preferably, ANO1 inhibitors include CaCCinh-A01 (CAI) and Benzbromarone (BBR).
Preferably, the FAP inhibitor comprises Tablabostat mesylate.
Wherein, the FAP Gene has Gene ID 2191 in NCBI. Tumor types for which the above antitumor drugs are suitable include, but are not limited to, esophageal cancer, gastric cancer, colon cancer, rectal cancer, gastrointestinal stromal tumor and the underlying sub-divided individual tissues, pathologies, gene molecule subtypes.
In a sixth aspect, the invention provides an anti-tumor pharmaceutical composition for reversing trastuzumab secondary resistance, comprising the above-described nucleic acid interfering sequence targeted to inhibit ANO1 and the above-described ANO1 inhibitor.
Tumor types for which the above antitumor pharmaceutical composition is suitable include, but are not limited to, gastric cancer, breast cancer and the underlying sub-divided individual tissues, pathologies, gene molecule subtypes.
In a seventh aspect, the present invention provides an anti-tumor pharmaceutical composition for enhancing the therapeutic effect of anti-PD-1 immunotherapy or reversing anti-PD-1 resistance therapy, the pharmaceutical composition comprising the above-described ANO1 targeting agent, or the pharmaceutical composition comprising the above-described ANO1 targeting agent and the above-described FAP inhibitor;
wherein the agent that targets ANO1 comprises a nucleic acid interfering sequence that targets ANO1 and/or an ANO1 inhibitor as described above.
Tumor types for which the above antitumor pharmaceutical composition is suitable include, but are not limited to, esophageal cancer, gastric cancer, colon cancer, rectal cancer, gastrointestinal stromal tumor and the underlying sub-divided individual tissues, pathologies, gene molecule subtypes.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the invention provides application of ANO1 as a tumor immunotherapy curative effect and prognosis biomarker, and further provides clinical application of targeted ANO1 in tumor therapy and tumor prognosis improving medicines, which comprises the following specific steps:
Traditional markers for immunotherapy include MSI/MMR status, EBV status and CPS score related to PD-L1, but in digestive tract tumor, the above index covers less population, and has poor effect on indication of therapeutic effect of immunotherapy. The invention systematically defines the value of ANO1 as a biomarker for evaluating the curative effect and prognosis of various tumor immunotherapy and a drug-resistant marker for the first time, overcomes the defect of the related index of prognosis of the existing tumor therapy, and has good clinical value.
The tumor targets of the digestive tract are rare, and more targets for medicine use need to be explored. Some previous studies suggest that ANO1 knockdown can inhibit proliferation of cell lines at levels in vitro, but lack evidence of levels in animal models in vivo, and do not systematically reveal the potential of ANO1 as a target. The invention systematically researches the inhibition effect of inhibiting the growth and metastasis of the ANO1 in various tumor cells, CDX models, PDX models and various derivative drug resistant models, and reveals the brand new value of the ANO1 as an independent drug administration target and the potential of independent application of the existing targeted ANO1 inhibitor. In addition, the invention proves that the ANO1 serving as a target point can reverse drug resistance in an immune treatment primary and secondary drug resistance model and further enhance the curative effect in an immune treatment sensitive model for the first time, and reveals the brand new value of the ANO1 serving as an immune treatment combined drug target point and the potential of the application of the existing targeted ANO1 inhibitor combined immune treatment drug.
Drawings
FIG. 1 is an identification of ANO1 amplification or expression positives of the present invention as immunotherapeutic markers. Wherein a is north edema queue 1: ANO1 amplification predicts poor PFS/OS for immunotherapy; b is MSK queue: ANO1 amplification predicts poor PFS/OS for immunotherapy; c is north edema queue 2: ANO1 protein expresses positive predictive immunotherapy poor PFS/OS; d is north edema queue 2: ANO1 protein expresses positive predictive immunity treatment secondary drug resistance.
FIG. 2 is a chemical formula of 2 targeted inhibitors of ANO1 of the present invention, left CaCCinh-A01, right Benzbromarone.
FIG. 3 is an identification of the in vitro proliferation of a knockdown ANO 1-inhibited tumor cell line and in vivo proliferation of a CDX model of the present invention.
FIG. 4 is an identification of the ANO1 inhibitors of the present invention inhibiting proliferation of tumor cell lines in vitro and growth of PDX models.
FIG. 5 is an identification of secondary resistance to trastuzumab in the ANO1 inhibitor reversal gastric cancer PDX model of the present invention.
FIGS. 6-8 are illustrations of the identification of the targeting ANO1 to enhance the therapeutic efficacy of anti-PD1 immunotherapy and to reverse the resistance of anti-PD1 immunotherapy according to the invention. Wherein, figure 6 is shRNA knockdown of ANO1 to enhance anti-PD1 (AP) efficacy, reversing primary resistance; FIG. 7 shows that ANO1 inhibitors CaCCinh-A01 (CAI) and Benzbromarone (BBR) enhance anti-PD1 (AP) efficacy, reversing primary resistance; FIG. 8 is a graph showing that ANO1 inhibitor CaCCinh-A01 (CAI) reverses anti-PD1 (AP) secondary resistance.
FIG. 9 is a chemical formula of FAP inhibitor Tablabostat mesylate of the present invention.
Fig. 10 is an identification of the shRNA knockdown of ANO1 in combination with FAP inhibitors of the invention to significantly enhance the efficacy of anti-PD 1.
Detailed Description
The invention relates to application of ANO1 serving as a biomarker and a target point in medicines for treating tumor diseases. The method specifically relates to the following steps:
(1) The use of amplification or high expression of ANO1 as a poor prognosis for tumor immunotherapy and an immunotherapeutic drug resistance marker; (2) Use of a nucleic acid interfering sequence and an inhibitor targeted to inhibit ANO1 alone for the treatment of a tumor; (3) Use of a combination of an ANO 1-targeting nucleic acid interfering sequence and an inhibitor for reversing tumor-targeted HER2 therapeutic resistance; (4) The application of the ANO1 targeting nucleic acid interference sequence and the inhibitor in combination therapy to enhance the curative effect of anti-PD1 immunotherapy and reverse the drug resistance of the anti-PD1 immunotherapy; (5) Use of an ANO 1-targeting nucleic acid interfering sequence and an inhibitor in combination with a FAP inhibitor for reversing anti-PD1 immunotherapeutic resistance.
To clarify the potential of ANO1 as a marker and target, the invention explores in patient cohorts and uses in vivo models of cells, CDX, PDX, immunodeficient mice and immunorobust mice to develop preclinical transformation studies. The inventor designs a targeted interference sequence aiming at ANO1, and discloses a brand new value of taking ANO1 as a target point and a new function of the existing targeted ANO1 inhibitor aiming at ANO1 inhibitors (CaCCinh-A01 and Benzbromarone), targeted HER2 drugs, immunotherapeutic drugs (anti-PD 1) and FAP inhibitors (Tablabostat mesylate) existing on the market, and finally proves great potential of taking ANO1 as a curative effect marker and a molecular target point in digestive tract tumors. The method comprises the following steps:
1. Analysis of multiple cohorts of data from the Beijing university tumor Hospital and the Stoneketjen center confirmed that ANO1 amplification or protein expression positivity corresponds poorly to immunotherapy irPFS/irOS, and that the immunotherapy was secondary to drug resistance, as shown in FIG. 1.
2. In cell lines, CDX and PDX models, the interference sequence of ANO1 is knocked down or ANO1 inhibitors CaCCinh-A01 and Benzbromarone are used for inhibiting the proliferation of tumors in vivo and in vitro, and good tumor inhibition effects are shown as shown in figures 3-5.
FIG. 3 shows that shRNA knockdown ANO1 inhibited proliferation of tumor cell lines in vitro and CDX model in vivo.
FIG. 4 shows that ANO1 inhibitors CaCCinh-A01 (CAI) and Benzbromarone (BBR) inhibit proliferation of tumor cell lines in vitro, exhibiting dose dependence; meanwhile, the growth of a gastric cancer and gastrointestinal stromal tumor PDX model can be inhibited.
FIG. 5 shows that ANO1 inhibitor CaCCinh-A01 (CAI) reverses Trastuzumab (Trastuzumab) secondary resistance to gastric cancer PDX model.
3. Trastuzumab resistance was reversed using the ANO1 inhibitor CaCCinh-a01 in a HER 2-targeted mouse CDX model of trastuzumab resistance, as shown in fig. 5.
4. The interference sequence knockdown of ANO1 in an immune-sound mouse CDX model is proved, or ANO1 inhibitors CaCCinh-A01 and Benzbromarone are used, so that the sensitivity of anti-PD1 immunotherapy can be enhanced, and the anti-PD1 immunotherapy resistance can be reversed, as shown in figures 6-8.
5. The interference sequence knockdown of ANO 1in combination with FAP inhibitor Tablabostat mesylate (labeled i) was demonstrated in an immunorobust mouse CDX model to further reverse anti-PD1 immunotherapeutic resistance, as shown in FIG. 10.
The invention adopts the following technical scheme:
First, the present invention provides the use of ANO1 amplification or high expression as a concomitant diagnostic index for guiding clinical decisions, judging poor prognosis of tumor immunotherapy, including progression free survival PFS (irPFS)/total survival OS (irOS), and immunotherapy secondary resistance (acquired resistance), as shown in FIG. 1.
According to the use, the definition of ANO1 amplification includes the amplification of ANO1 gene detected by DNA detection means such as PCR, next Generation Sequencing (NGS) and FISH. The definition of ANO1 high expression includes the high level of ANO1 protein or transcriptome expression detected by protein detection means such as Western Blot and RNA detection means such as qPCR and RNA-sequencing. ANO1 positive is defined as positive expression of ANO1 in tissues detected by protein detection means such as IHC.
According to the use, the tumor types for which ANO1 amplification or high expression as a concomitant diagnostic index is useful for guiding clinical decisions include esophageal cancer, gastric cancer, colon cancer, rectal cancer, gastrointestinal stromal tumor, melanoma and individual tissues, pathologies, gene molecule subtypes subdivided thereunder.
Depending on the use, immunotherapeutic drugs for which ANO1 amplification or high expression is a concomitant diagnostic index for guiding clinical decisions include Pembrolizumab, nivolumab, sintilimab, tislelizumab, camrelizumab, atezolizumab, envolimab, ipilimumab and various single and combination regimens related thereto.
Second, the present invention provides the use of a nucleic acid interfering sequence and an inhibitor targeted to inhibit ANO1 as a medicament, alone for the prevention and treatment of tumors (FIGS. 3-5).
According to the use, the interfering sequences of ANO1 include specific interfering sequences of human and murine origin.
According to the use, inhibitors of ANO1 include CaCCinh-A01 (CAI) and Benzbromarone (BBR), as shown in FIG. 2.
According to the use, tumor types for which targeted inhibition of ANO1 is suitable for the treatment of tumors include esophageal cancer, gastric cancer, colon cancer, rectal cancer, gastrointestinal stromal tumor and individual tissues, pathologies, gene molecule subtypes subdivided thereunder.
Third, the present invention provides the use of a nucleic acid interfering sequence and an inhibitor targeted to inhibit ANO1 as a medicament for reversing trastuzumab secondary resistance (fig. 5).
According to the use, the interfering sequences of ANO1 include specific interfering sequences of human and murine origin. Inhibitors of ANO1 include CaCCinh-A01 (CAI) and Benzbromarone (BBR), as shown in FIG. 2.
According to the use, tumor types for which targeted inhibition of ANO1 is useful for reversing trastuzumab secondary resistance include gastric cancer, breast cancer and the underlying subdivision of individual tissue, pathology, gene molecule subtypes.
Fourth, the present invention provides the use of ANO 1-targeting nucleic acid interfering sequences and inhibitors for combination therapy, enhancing the efficacy of anti-PD1 immunotherapy, and reversing the resistance of anti-PD1 immunotherapy (FIGS. 6-8).
According to the use, the interfering sequences of ANO1 include specific interfering sequences of human and murine origin. Inhibitors of ANO1 include CaCCinh-A01 and Benzbromarone, as shown in FIG. 2. Drugs involved in Anti-PD1 immunotherapy include Pembrolizumab, nivolumab, sintilimab, tislelizumab, camrelizumab, atezolizumab, envolimab, ipilimumab, and the like.
According to the application, tumor types applicable to targeted inhibition of ANO1 for enhancing the curative effect of anti-PD1 immunotherapy and reversing the resistance of anti-PD1 immunotherapy include esophageal cancer, gastric cancer, colon cancer, rectal cancer, gastrointestinal stromal tumor and tissues, pathology and gene molecule subtypes subdivided below.
Fifth, the present invention provides the use of ANO 1-targeting nucleic acid interfering sequences and inhibitors in combination with FAP inhibitors for reversing anti-PD1 immunotherapeutic resistance (FIG. 10).
According to the use, the interfering sequences of ANO1 include specific interfering sequences of human and murine origin. Inhibitors of ANO1 include CaCCinh-A01 and Benzbromarone, as shown in FIG. 2. The FAP inhibitor was Tablabostat mesylate as shown in figure 9. Drugs involved in Anti-PD1 immunotherapy include Pembrolizumab, nivolumab, sintilimab, tislelizumab, camrelizumab, atezolizumab, envolimab, ipilimumab, and the like.
According to the use, tumor types for which the targeted inhibition of ANO1 combined with FAP inhibitors is suitable for reversing anti-PD1 immunotherapeutic resistance include esophageal cancer, gastric cancer, colon cancer, rectal cancer, gastrointestinal stromal tumor and individual tissues, pathology and molecular subtypes subdivided thereunder.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
The experimental methods involved in the following examples are as follows:
1. Identification of the markers: the DNA/RNA/protein condition is identified by means of PCR, next-generation sequencing, qPCR, RNA-sequencing, western Blot, IHC and the like, and then the ANO1 is analyzed by means of Kaplan-Meier, COX multifactor analysis, T test and the like to predict the curative effect of immunotherapy and the value of the marker as an accompanying diagnosis in combination with the treatment information of patients.
2. Cell model dosing: IC 50 values were calculated by setting a gradient of 0 μM,1 μM,10 μM,25 μM,50 μM,100 μM, and administering AGS/MGC803/HGC27/LOVO/KYSE-180/KYSE-510/MC38 cells while measuring the effect of the drug using CCK8 method.
3. Animal model nodulation:
1) Model of human CDX-transplanted tumor: after knocking down ANO1, 5×10 6 HGC27/MGC803 cells were suspended in a 100 μL system, inoculated subcutaneously into 5-week-old female BALB-C-nude mice, and tumor volumes (formula V=L×W 2 ×0.5, where V, volume; L, length; W, tumor width) and changes in mouse body weight were measured every 3 days to observe the effect of ANO1 knockdown on mouse engrafting tumor growth. Mice were sacrificed when tumor volume exceeded 2000mm 3;
2) Murine CDX engraftment tumor model: after knocking down ANO1, 5×10 6 CT26/MC38 cells were suspended in a 100. Mu.L system, and inoculated subcutaneously into 5-week-old female BALB-C/C57BL-6J mice, respectively, and tumor volumes (formula V=L×W 2 ×0.5, where V, volume, L, length, W, tumor width) and weight changes of the mice were measured every 3 days to observe the effect of ANO1 knocking down on the growth of the transplanted tumors of the mice. Mice were sacrificed when tumor volume exceeded 2000mm 3;
3) PDX model: patient-derived tissues such as gastric cancer and gastrointestinal stromal tumor were inoculated subcutaneously into 5-week-old NOD/SCID mice to establish primary PDX. After 5 passages, the PDX model is available;
4) And (3) establishing a drug resistance model: long-term low-dose trastuzumab induction was performed on a PDX model established for a patient source sensitive to trastuzumab, and individual passages were selected in which tumor inhibition was poor. Obtaining a PDX model of trastuzumab secondary drug resistance after about half a year; long-term low-dose anti-PD1 induction is carried out aiming at an MC38-CDX model sensitive to anti-PD1 immunotherapy, and individual passages with poor tumor inhibition effect are selected. And obtaining a CDX model of anti-PD1 immunotherapy secondary drug resistance after about half a year.
4. Animal model dosing:
anti-mouse-PD-1,3mg/kg, 2 times per week for 3 weeks, intraperitoneal injection;
CAI,50mg/kg, administered 3 weeks apart, intraperitoneally;
BBR,50mg/kg, is administered every other day for 3 weeks, and is orally administered;
Talabostat mesylate, 10 μg per mouse, 2 times per day for 3 weeks, orally;
The CAI, BBR, talabostat mesylate used in the examples below were all purchased from MCE. The goods number is as follows: CAI (HY-100611), BBR (HY-B1135), talabostat mesylate (HY-13233A).
The antibodies used for protein level detection were ANO1 Rabbit p A10498, abclonal.
EXAMPLE 1 use of amplified or high expression of ANO1 as a marker for poor prognosis of tumor immunotherapy and resistance to immunotherapy
Detecting ANO1 conditions in tumor tissues of a patient receiving immunotherapy according to a second generation sequencing (NGS) technology, and judging ANO1 amplification; ANO1 in tumor tissue of a patient receiving immunotherapy is stained using an immunohistochemical staining method, ANO1 expression was judged to be positive (where-/+ was defined as negative, definition of +/+++. Positive). For ANO1 amplified/unamplified, and ANO1 low/high expressing populations, the prognosis differences (PFS/OS) were analyzed by Kaplan-Meier method, and drug resistance (primary and secondary) was analyzed by t-test and chi-square test.
EXAMPLE 2 use of nucleic acid interfering sequences and inhibitors targeted to inhibit ANO1 alone for the treatment of tumors
The inventor establishes a large-scale mouse subcutaneous transplantation tumor (PDX) model based on patient tumor tissues by using real-time endoscope biopsy trace advanced gastric cancer tissues in the early stage, establishes a PDX tissue library, confirms the high consistency of the PDX tissues and the patient tumor characteristics, and the PDX model becomes an optimal model for preclinical research and also provides an optimal platform for constructing a drug-resistant PDX model. The PDX tissue library consists of PDX tissues derived from tumor tissues of a plurality of patients, and each PDX tissue is prepared by the following method: patient tumor tissue was inoculated subcutaneously into mice (5 week old female NOD/SCID mice weighing about 15-16g, purchased from Fukang Biotechnology Co., ltd.) until tumor volume reached a size of about 150-200mm 3, and the tumor tissue was taken and frozen.
Performing immunohistochemical staining from the established PDX model library, and selecting PDX models positive and negative in ANO1 expression for performing pharmacodynamic experiment verification; the same administration time standard was followed unless the volume exceeded 2000mm 3;
First group (ctrl control group): corn oil was administered once daily by gavage (oral administration) in a single volume of 100 μl for 3 weeks;
Second group (CAI inhibitor group): CAI is given by intraperitoneal injection once daily, wherein the single volume is 100 μl, and the single dose is "CAI 50mg/kg" for 3 weeks;
Third group (BBR inhibitor group): the BBR is administrated by gastric lavage (oral administration) once every two days, wherein the single volume is 100 μl, and the single dose is 'BBR 50 mg/kg', for 3 weeks;
Tumor volumes were measured every 2 days, and the results are shown in fig. 4 (E, F, G), and the ANO1 inhibitor single drug had a certain antitumor effect on the ANO 1-positive PDX model.
In addition, a cell line in which ANO1 is stably knocked down is established based on an interference sequence of ANO1 (mouse ANO1 interference sequence sh-1-ANO1 or sh-2-ANO1, the sequences of which are shown as SEQ ID NO:5-6 or SEQ ID NO: 7-8), and subcutaneous oncolysis is carried out. Tumor volumes were measured every 2 days and the results are shown in fig. 3, which demonstrate that interfering with the expression of cell ANO1 can inhibit tumor cell proliferation.
Example 3 use of ANO 1-targeting inhibitors in combination for reversing trastuzumab resistance to tumor-targeted HER2
In the early stage, a trastuzumab-sensitive PDX model (TS) is established for a patient with HER2 targeting trastuzumab sensitivity, a trastuzumab drug-resistant PDX model (TR) is established through long-term low-dose drug induction, PDX tissues of the drug-resistant model are revived and passaged, and then the tissues are trimmed into tissue blocks with the same size, subcutaneous tumor formation is carried out, and grouping administration is carried out until the tumor volume is as long as about 100-150mm 3. The same administration time standard was followed unless the volume exceeded 2000mm 3;
first group (ctrl control group): corn oil is administered by intraperitoneal injection once daily, wherein the single volume is 100 μl for 3 weeks;
Second group (CAI inhibitor group): CAI is given by intraperitoneal injection once daily, wherein the single volume is 100 μl, and the single dose is "CAI 50mg/kg" for 3 weeks;
tumor volumes were measured every 2 days, and as a result, as shown in fig. 5, the ano1 inhibitor had a certain anti-tumor effect on HER 2-resistant PDX model.
EXAMPLE 4 use of ANO 1-targeting nucleic acid interfering sequences and inhibitors for combination therapy, enhancing anti-PD1 immunotherapeutic efficacy, and reversing anti-PD1 immunotherapeutic resistance
Establishing a mouse colon cancer cell line with stable knockout of ANO1 based on an interference sequence of ANO1 (mouse ANO1 interference sequence sh-1-ANO1 or sh-2-ANO1, and the sequences are shown as SEQ ID NO:5-6 or SEQ ID NO: 7-8), performing subcutaneous tumor formation, performing grouping administration until the tumor volume is up to about 100-150mm 3, and conforming to the same administration time standard unless the tumor volume exceeds 2000mm 3;
First group (ctrl control group): physiological saline is administered by intraperitoneal injection once daily, and the single volume is 100 μl for 3 weeks;
Second group (ANO 1 knockdown control group): physiological saline is administered by intraperitoneal injection once daily, and the single volume is 100 μl for 3 weeks;
Third group (anti-PD-1 immune control group): intraperitoneal injection is carried out once every three days, the single volume is 100 μl, and the single dose is 'anti-PD-3 mg/kg', and the continuous period is 3 weeks;
Fourth group (anti-PD-1 immunoknockdown group): intraperitoneal injection is carried out once every three days, the single volume is 100 μl, and the single dose is 'anti-PD-3 mg/kg', and the continuous period is 3 weeks;
tumor volumes were measured every 2 days, and the results are shown in fig. 6-8, which demonstrate that interfering with the expression of the mouse colon cancer cell ANO1 can enhance the sensitivity of immunotherapy.
The inventors based on wild-type mouse colon cancer cell lines and performed subcutaneous oncology, and group administration was performed until the tumor volume was as large as about 100-150mm 3.
First group (ctrl control group): the corn oil is orally taken once a day, the single volume is 100 μl, and the period is 3 weeks;
Second group (CAI inhibitor group): CAI is given by intraperitoneal injection once daily, wherein the single volume is 100 μl, and the single dose is "CAI 50mg/kg" for 3 weeks;
Third group (BBR inhibitor group): the BBR is administrated by gastric lavage (oral administration) once every two days, wherein the single volume is 100 μl, and the single dose is 'BBR 50 mg/kg', for 3 weeks;
Fourth group (anti-PD-1 immunotherapeutic group): intraperitoneal injection is carried out once every three days, the single volume is 100 μl, the single dose is 'anti-PD-3 mg/kg', and the continuous period is 3 weeks
Fifth group (CAI inhibitor combined anti-PD-1 treatment group): CAI is given by intraperitoneal injection once daily, wherein the single volume is 100 μl, and the single dose is "CAI 50mg/kg"; the anti-PD-1 is injected into the abdominal cavity once every three days, the single volume is 100 mu l, the single dose is 'anti-PD-3 mg/kg', and the continuous period is 3 weeks;
Sixth group (BBR inhibitor in combination with anti-PD-1 treatment group): the BBR is administrated by gastric lavage (oral administration) once every two days, wherein the single volume is 100 μl, and the single dose is 'BBR 50 mg/kg'; the anti-PD-1 is injected into the abdominal cavity once every three days, the single volume is 100 mu l, the single dose is 'anti-PD-3 mg/kg', and the continuous period is 3 weeks;
tumor volumes were measured every 2 days and the results are shown in fig. 6-8, demonstrating that the ANO1 inhibitors could be used in synergy with immunotherapy.
EXAMPLE 5 use of ANO 1-targeting nucleic acid interfering sequences and inhibitors in combination with FAP inhibitors for reversing anti-PD1 immunotherapeutic resistance
The inventor of the invention adopts a long-term intermittent induction method to simulate the administration of patients for about 12 months, constructs an MC-38/C57 subcutaneous tumor model of anti-PD-1 drug resistance, and complies with the same administration time standard unless the volume exceeds 2000mm 3;
First group (ctrl control group): physiological saline is administered by intraperitoneal injection once daily, and the single volume is 100 μl for 3 weeks;
Second group (CAI inhibitor group): CAI is given by intraperitoneal injection once daily, wherein the single volume is 100 μl, and the single dose is "CAI 50mg/kg" for 3 weeks;
Third group (anti-PD-1 immunotherapeutic group): intraperitoneal injection is carried out once every three days, the single volume is 100 μl, the single dose is 'anti-PD-3 mg/kg', and the continuous period is 3 weeks
Fourth group (CAI inhibitor combined anti-PD-1 treatment group): CAI is given by intraperitoneal injection once daily, wherein the single volume is 100 μl, and the single dose is "CAI 50mg/kg"; the anti-PD-1 is injected into the abdominal cavity once every three days, the single volume is 100 mu l, the single dose is 'anti-PD-3 mg/kg', and the continuous period is 3 weeks;
tumor volumes were measured every 2 days and the results are shown in fig. 10, demonstrating that the ANO1 inhibitor can be used as a follow-up treatment option for anti-PD-1 resistance.
The mouse colon cancer cell line CT26 is an MSS type cell line and is insensitive to the primary of anti-PD-1, and based on the MSS type cell line and the MSS type cell line, the inventor utilizes ANO1 to interfere with the combined administration of the cell line and the anti-PD-1 and the tumor fibroblast inhibitor, and discovers the treatment mode of the primary drug resistance of the immunotherapy.
First group (ctrl control group): physiological saline is administered by intraperitoneal injection once daily, and the single volume is 100 μl for 3 weeks;
Second group (ANO 1 knockdown control group): physiological saline is administered by intraperitoneal injection once daily, and the single volume is 100 μl for 3 weeks;
third group (anti-PD-1 immunotherapy control): intraperitoneal injection is carried out once every three days, the single volume is 100 μl, the single dose is 'anti-PD-3 mg/kg', and the continuous period is 3 weeks
Fourth group (anti-PD-1 immunotherapeutic knockdown group): intraperitoneal injection is carried out once every three days, the single volume is 100 μl, the single dose is 'anti-PD-3 mg/kg', and the continuous period is 3 weeks
Fifth group (FAP inhibitor control group): mice were perfused Talabostat mesylate (orally) once every two days with a single volume of 100 μl and a single dose of "10 μg" for 3 consecutive weeks;
sixth group (FAP inhibitor knockdown group): mice were perfused Talabostat mesylate (orally) once every two days with a single volume of 100 μl and a single dose of "10 μg" for 3 consecutive weeks;
seventh group (FAP inhibitor combined anti-PD-1 control group): the mice were perfused Talabostat mesylate (orally) once every two days, with a single volume of 100 μl, a single dose of "10 μg", and were intraperitoneally injected once every three days, with a single volume of 100 μl, a single dose of "anti-PD-3mg/kg", for 3 consecutive weeks;
Eighth group (FAP inhibitor combined anti-PD-1 knockdown group): the mice were perfused Talabostat mesylate (orally) once every two days, with a single volume of 100 μl, a single dose of "10 μg", and were intraperitoneally injected once every three days, with a single volume of 100 μl, a single dose of "anti-PD-3mg/kg", for 3 consecutive weeks;
The experimental results are shown in fig. 10, which indicate that the targeted inhibition of ANO1 combined with FAP inhibitor can further reverse the drug resistance of tumors to anti-PD-1. Fig. 10 shows that shRNA knockdown of ANO1 in combination with FAP inhibitor Talabostat mesylate significantly enhanced anti-PD1 (AP) efficacy.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (3)
1. Application of an ANO1 targeting agent and a nucleic acid interference sequence in preparing an anti-tumor drug for reversing anti-PD-1 drug resistance treatment;
The ANO1 targeting agents include CaCCinh-A01 and Benzbromarone;
The nucleic acid interference sequence is a nucleic acid interference sequence for targeted inhibition of ANO 1; wherein, the nucleic acid interference sequence of the targeted inhibition human ANO1 is shown as SEQ ID NO 1-2 or SEQ ID NO 3-4; the nucleic acid interference sequence of the targeted inhibition mouse ANO1 is shown as SEQ ID NO 5-6 or SEQ ID NO 7-8;
anti-PD-1 treatments include Pembrolizumab, nivolumab, sintilimab, tislelizumab, camrelizumab, atezolizumab, envolimab;
the tumor is colon cancer.
2. Application of an ANO1 targeting agent and a nucleic acid interference sequence in preparing an anti-tumor medicament for enhancing the curative effect of an FAP inhibitor;
The ANO1 targeting agents include CaCCinh-A01 and Benzbromarone;
The nucleic acid interference sequence is a nucleic acid interference sequence for targeted inhibition of ANO 1; wherein, the nucleic acid interference sequence of the targeted inhibition human ANO1 is shown as SEQ ID NO 1-2 or SEQ ID NO 3-4; the nucleic acid interference sequence of the targeted inhibition mouse ANO1 is shown as SEQ ID NO 5-6 or SEQ ID NO 7-8;
the FAP inhibitor is Tablabostat mesylate;
the tumor is colon cancer.
3. Application of targeted ANO1 inhibition nucleic acid interference sequence, anti-PD1 preparation and FAP inhibitor in preparing antitumor drugs;
Wherein, the nucleic acid interference sequence of the targeted inhibition human ANO1 is shown as SEQ ID NO 1-2 or SEQ ID NO 3-4; the nucleic acid interference sequence of the targeted inhibition mouse ANO1 is shown as SEQ ID NO 5-6 or SEQ ID NO 7-8;
the FAP inhibitor is Tablabostat mesylate;
the tumor is colon cancer.
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| CN113117088A (en) * | 2021-02-25 | 2021-07-16 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Use of calcium-activated chloride channel inhibitors in tumor immunotherapy |
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2023
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| CN102154482A (en) * | 2011-02-16 | 2011-08-17 | 北京大学 | Application of calcium activated chloride channels ANO1/TMEM16A in diagnosis and treatment of prostate cancer |
| WO2013052480A1 (en) * | 2011-10-03 | 2013-04-11 | The Board Of Regents Of The University Of Texas System | Marker-based prognostic risk score in colon cancer |
| WO2021202755A2 (en) * | 2020-03-31 | 2021-10-07 | Cedars-Sinai Medical Center | Biomarker panels for stratification of response to immune checkpoint blockade in cancer |
| CN113117088A (en) * | 2021-02-25 | 2021-07-16 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Use of calcium-activated chloride channel inhibitors in tumor immunotherapy |
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| ANO1: More Than Just Calcium-Activated Chloride Channel in Cancer;Guo et al.;《Frontiers in oncology》;20220606;第12卷;文章号922838第1-13页 * |
| Inhibition of calcium-activated cholride channel ANO1 suppresses proliferation and induces apoptosis of epithelium originated cancer cells;Guan et al.;《Oncotarget》;20161008;第7卷(第48期);第78619-78630页 * |
| TMEM16A overexpression contributes to tumor invasion and poor prognosis of human gastric cancer through TGF-beta signaling.;Liu et al.;《Oncotarget》;20150510;第6卷(第13期);第11585–11599页 * |
| TMEM16A/ANO1 suppression improves response to antibody-mediated targeted therapy of EGFR and HER2/ERBB2;Kulkarni et al.;《Genes, chromosomes & cancer》;20170630;第56卷(第6期);第460-471页 * |
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| Tumor copy-number alterations predict response to immune-checkpoint-blockade in gastrointestinal cancer;Lu et al.;《Journal for immunotherapy of cancer》;20200831;第8卷(第2期);文章号e000374第1-11页 * |
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