WO2024027599A1 - 预测直肠癌对an0025联合放疗/放化疗(rt/crt)治疗敏感性的生物标志物 - Google Patents

预测直肠癌对an0025联合放疗/放化疗(rt/crt)治疗敏感性的生物标志物 Download PDF

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WO2024027599A1
WO2024027599A1 PCT/CN2023/109888 CN2023109888W WO2024027599A1 WO 2024027599 A1 WO2024027599 A1 WO 2024027599A1 CN 2023109888 W CN2023109888 W CN 2023109888W WO 2024027599 A1 WO2024027599 A1 WO 2024027599A1
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patient
radiotherapy
chemoradiotherapy
crt
rectal cancer
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PCT/CN2023/109888
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English (en)
French (fr)
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梁洪铭
卢煦阳
劳特米尔克内森
爱金逊罗伯特
汤凯扬
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杭州阿诺生物医药科技有限公司
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Priority to CN202380010282.0A priority Critical patent/CN117242348A/zh
Publication of WO2024027599A1 publication Critical patent/WO2024027599A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to the biological field and involves a series of biomarkers that can be used to predict the sensitivity of rectal cancer patients to AN0025 combined radiotherapy/chemoradiotherapy (RT/CRT) treatment.
  • RT/CRT radiotherapy/chemoradiotherapy
  • Rectal cancer is one of the common malignant tumors in middle-aged and elderly people, and it most commonly occurs over the age of 50. With the changes in people's eating habits and structure and the aging of the population, its incidence rate is on the rise.
  • the main treatment methods for rectal cancer also include targeted therapy, gene therapy, traditional Chinese medicine treatment, immunotherapy, etc. At present, surgery is still the first choice treatment and the only means to cure rectal cancer.
  • surgery is still the first choice treatment and the only means to cure rectal cancer.
  • some patients cannot undergo surgical treatment, and some patients cannot undergo repeated surgical treatment.
  • radiotherapy or chemoradiotherapy is the main treatment method, with the purpose of improving the patient's survival rate and quality of life.
  • AN0025 is a highly selective and highly active antagonist of prostaglandin E2 (PGE2) receptor 4 (EP4) developed by Arnold Pharmaceuticals. It can inhibit the signaling pathway mediated by PGE2-EP4.
  • Myeloid cells are the most important immune cells infiltrating tumors, mainly including tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC). TAM and MDSC are differentiated from immature monocytes.
  • EP4 is one of the four PGE2 receptors. It is expressed on myeloid cells and plays a key role in promoting the differentiation of monocytes into TAMs and MDSCs in the tumor microenvironment.
  • the signaling pathway composed of prostaglandin E2 (PGE2) and its subtype 4 receptor (EP4) can not only promote the occurrence of cancer, but also form a cancer microenvironment that promotes cancer development and suppresses immunity.
  • cAMP produced by the activation of the PGE2/EP4 signaling pathway in T cells can activate the cAMP-PKA signaling pathway, and this process can effectively inhibit the function of T cells.
  • the PGE2/EP4 signaling pathway can also negatively regulate the maturation of DCs and induce the formation of a variety of immunosuppressive cells including M2 macrophages and myeloid suppressor cells (MDSC) ( S et al. Front Immunol. 2019;10:475).
  • MDSC myeloid suppressor cells
  • the compound (AN0025) with the structure of formula (I) and its pharmaceutically acceptable salts is a highly selective small molecule inhibitor targeting E-type prostaglandin receptor 4.
  • AN0025 can effectively reduce immunosuppressive cells in the tumor microenvironment by inhibiting the PGE2/EP4 signaling pathway.
  • AN0025 has been used in multiple clinical strategies.
  • FIH first in human
  • AN0025 has demonstrated certain single-agent efficacy
  • AN0025 has shown great efficacy in combination with radiotherapy and chemotherapy. Strong clinical efficacy (Wyrwicz et.al, Poster#540, ESMO2019).
  • the present invention provides a series of biomarker applications, aiming to solve the problem of predicting the sensitivity of rectal cancer patients to AN0025 combined radiotherapy/chemoradiotherapy (RT/CRT), so as to screen out more suitable treatment options. group of patients with rectal cancer to further improve the targeting of therapy.
  • RT/CRT radiotherapy/chemoradiotherapy
  • the invention provides a method of detecting IL-1 alpha, IL-4, IL-18, IL-27a, EGF, HGF, VEGF-D, SCF, IFN-gamma, MCP-1 in blood samples of patients with rectal cancer.
  • the invention provides IL-1 alpha, IL-4, IL-18, IL-27a, EGF, HGF, VEGF-D, SCF, IFN-gamma, MCP-1 (CCL2 ) or CCL11
  • (Eotaxin) as a biomarker in the preparation of products for predicting the patient's sensitivity to AN0025 combined radiotherapy/chemoradiotherapy (RT/CRT).
  • the above application provided by the present invention includes the following steps:
  • Step 1 Centrifuge the peripheral blood samples to be tested from patients with rectal cancer to collect plasma
  • Step 2 Detect IL-1 alpha, IL-4, IL-18, IL-27a, EGF, HGF, VEGF-D, SCF, IFN-gamma, MCP-1 (CCL2) or CCL11 (Eotaxin) in the plasma concentration;
  • Step 3 If the plasma detection concentration is higher than the reference value, the rectal cancer patient is considered to be sensitive to AN0025 combined radiotherapy/chemoradiotherapy (RT/CRT) treatment; otherwise, the patient is insensitive.
  • RT/CRT radiotherapy/chemoradiotherapy
  • the present invention provides the use of CD8, CD3&CD8, CD3&CD8&PD-1 in tumor tissue samples of rectal cancer patients as biomarkers for predicting the sensitivity of the patient to AN0025 combined radiotherapy/chemoradiotherapy (RT/CRT) treatment.
  • RT/CRT radiotherapy/chemoradiotherapy
  • the present invention provides CD8, CD3&CD8, CD3&CD8&PD-1 in tumor tissue samples of rectal cancer patients as biomarkers for preparing the patient's sensitivity to AN0025 combined radiotherapy/chemoradiotherapy (RT/CRT). application in the product.
  • RT/CRT radiotherapy/chemoradiotherapy
  • the above-mentioned application provided by the present invention includes the following steps:
  • Step 1 Process the tumor tissue samples from rectal cancer patients according to the ELISE method
  • Step 2 Detect the expression levels of CD3, CD8, and PD-1 in the tissue sample
  • Step 3 If the detected expression levels of CD8, CD3&CD8, CD3&CD8&PD-1 are higher than the reference value, the rectal cancer patient is considered to be sensitive to AN0025 combined with radiotherapy/chemoradiotherapy (RT/CRT); otherwise, the patient is insensitive.
  • RT/CRT radiotherapy/chemoradiotherapy
  • the present invention provides a method for predicting the sensitivity of colorectal patients to AN0025 combined with radiotherapy/chemoradiotherapy (RT/CRT), including selecting the above-mentioned blood biomarkers IL-1 alpha, IL-4 of the patient , one or more of IL-18, IL-27a, EGF, HGF, VEGF-D, SCF, IFN-gamma, MCP-1 (CCL2) or CCL11 (Eotaxin), and/or such as the patient's tumor tissue One or more of the biomarkers CD8, CD3&CD8, CD3&CD8&PD-1.
  • RT/CRT radiotherapy/chemoradiotherapy
  • the present invention also provides a method for treating patients with rectal cancer, comprising the following steps:
  • Step 1 According to the method of claim 7, predict the sensitivity of colorectal patients to AN0025 combined with radiotherapy/chemoradiotherapy (RT/CRT);
  • Step 2 If the rectal cancer patient is predicted to be treated with AN0025 combined with radiotherapy/chemoradiotherapy (RT/CRT) If the sensitivity is high, a therapeutically effective dose of AN0025 combined with radiotherapy/chemoradiotherapy (RT/CRT) will be administered to the patient with rectal cancer.
  • RT/CRT radiotherapy/chemoradiotherapy
  • the present invention provides a method of treating a rectal cancer patient in need thereof, comprising: administering to the rectal cancer patient a therapeutically effective amount of AN0025 combined with radiotherapy/chemoradiotherapy (RT/CRT), wherein the The patient was predicted to be sensitive to AN0025 combined with radiotherapy/chemoradiotherapy (RT/CRT) by the aforementioned method.
  • RT/CRT radiotherapy/chemoradiotherapy
  • the present invention provides a method of treating a rectal cancer patient in need thereof, comprising: administering a therapeutically effective amount of AN0025 combined with radiotherapy/chemoradiotherapy (RT/CRT) to the rectal cancer patient, wherein, Detection concentrations of IL-1 alpha, IL-4, IL-18, IL-27a, EGF, HGF, VEGF-D, SCF, IFN-gamma, MCP-1 (CCL2) or CCL11 (Eotaxin) in the patient's blood samples is higher than its reference value, and/or the expression of CD8, CD3&CD8, CD3&CD8&PD-1 in the patient's tumor tissue sample is higher than its reference value.
  • RT/CRT radiotherapy/chemoradiotherapy
  • the "reference value” refers to the specific value with which the detection value is compared to determine whether the patient is responsive or sensitive to AN0025 combined with radiotherapy/chemoradiotherapy (RT/CRT) treatment, which can be obtained, for example, through clinical statistical analysis.
  • RT/CRT radiotherapy/chemoradiotherapy
  • the present invention also provides a kit for predicting the sensitivity of rectal cancer patients to AN0025 combined with radiotherapy/chemoradiotherapy (RT/CRT), which kit includes the patient's blood biomarker as described above. one or more, and/or one or more biomarkers of the patient's tumor tissue as described above.
  • R/CRT radiotherapy/chemoradiotherapy
  • Figure 1 shows the content of each biomarker in the blood samples of patients with complete pathological response (pCR)/complete clinical response (cCR) and no complete response (non-CR), with the Y-axis unit pg/mL.
  • Figure 2 shows the proportion of positive cells for each biomarker in total cells in tumor tissue samples from patients with complete pathological response (pCR)/complete clinical response (cCR) and no complete response (non-CR).
  • the Y-axis represents the percentage. .
  • biomarker is equivalent to a biomarker or a molecular marker, which is any gene or protein whose expression level in a tissue or cell is altered compared to the expression level of a normal or healthy cell or tissue, ( used alone or in combination with other qualitative terms such as breast cancer marker, breast cancer-specific marker, control marker, exogenous marker, endogenous marker) refers to something that is measurable, calculable, or otherwise obtainable, and is associated with any Parameters related to molecules or combinations of molecules that can be used as indicators of biological and/or chemical states.
  • marker refers to a parameter associated with one or more biological molecules (i.e., “biomarker”) such as natural or synthetically produced nucleic acids (i.e., individual genes, as well as coding and non-coding DNA and RNA) and proteins (e.g., peptides, polypeptides).
  • biomarker in the present invention also includes reference to a single parameter that can be calculated or otherwise obtained by considering expression data from two or more different markers.
  • IL-1 alpha/IL-4/IL-18/IL-27a/EGF/HGF/VEGF-D/SCF/IFN-gamma/MCP-1(CCL2)/CCL11(Eotaxin) of the present invention is a blood sample biomarker substances, including IL-1 alpha/IL-4/IL-18/IL-27a/EGF/HGF/VEGF-D/SCF/IFN-gamma/MCP-1(CCL2)/CCL11( Eotaxin) protein sequence, also encompasses the amino acid sequence of its variants and/or homologs, and proteins with at least 80%, at least 85%, at least 90% or at least 95% homology to fragments of that sequence, provided that the variant Body proteins (including isotypes), homologous proteins and/or fragments are affected by one or more IL-1 alpha/IL-4/IL-18/IL-27a/EGF/HGF/VEGF-D/SCF/IFN -gamma/MCP-1(CCL2)/CCL
  • CD3/CD8/PD-1 of the present invention is a tissue sample biomarker, covering, for example, the CD3/CD8/PD-1 protein sequence included in databases such as Uniprot, and also covering the amino acid sequences of its variants and/or homologues, and proteins with fragments of that sequence having at least 80%, at least 85%, at least 90% or at least 95% homology, provided that variant proteins (including isotypes), homologous proteins and/or fragments are affected by one or Recognized by multiple CD3/CD8/PD-1 specific antibodies.
  • an amino acid or nucleic acid sequence has a certain degree of identity with an amino acid or nucleic acid sequence as described herein
  • the skilled person can use means and methods well known in the art, such as alignment, either manually or by using Computer programs known in the art or described herein.
  • the term "identical” or “percent identity” in the context of two or more amino acid or nucleic acid sequences means as measured using sequence comparison algorithms known in the art or by manual alignment and visual inspection.
  • two or more sequences or subsequences that are identical, or a specified percentage of amino acid residues or nucleotides are identical are considered to have, for example, 60% when compared and aligned over a comparison window or over a specified region for maximum correspondence to 95% or more Sequences with large sequence identity are essentially identical.
  • sequences with large sequence identity are essentially identical.
  • sequences with large sequence identity are essentially identical.
  • sequences with large sequence identity are essentially identical.
  • the described identity exists over a region of at least about 15 to 25 amino acids or nucleotides in length, more preferably about 50 to 100 amino acids or nucleotides in length.
  • Rectal cancer in the present invention refers to malignant tumors of the digestive tract occurring in the colon or rectum. Rectal cancer occurs when the cells lining the colon behave abnormally or grow into polyps. If polyps are not treated, they can turn into cancer. It includes but is not limited to various pathological types such as adenocarcinoma and squamous cell carcinoma.
  • Radiotherapy refers to the medical use of ionizing radiation, especially for the treatment of cancer.
  • the medical use of ionizing radiation in cancer treatment results in the reduction and/or killing of cancer cells in a subject.
  • Radiation therapy can be administered by any means known to those skilled in the art. Examples of radiation utilized in radiotherapy include, but are not limited to, photon radiation, ionizing radiation, or charged particle radiation, such as X-rays or protons. Examples of radiation therapy include, but are not limited to: external beam radiation therapy or teletherapy; brachytherapy or sealed beam source therapy; and whole body radioactive isotope therapy or unsealed source radiation therapy.
  • “Chemotherapy” in the present invention also known as “chemotherapy” refers to the use of chemotherapeutic agents to treat cancer subjects, wherein the chemotherapeutic agents include those commonly used for cancer patients so far, such as taxanes, paclitaxel, Docetaxel, cabazitaxel, gemcitabine, carboplatin, cisplatin, oxaliplatin, fluorouracil, capecitabine, or tegafur, or any functional analog thereof.
  • the chemotherapeutic agent of the invention is selected from capecitabine or leucovorin/5-fluoropyrimidine/oxaliplatin (mFOLFOX-6).
  • Radiotherapy in the present invention refers to administering radiotherapy and chemotherapy to treat cancer patients at the same time or at different time points.
  • Treatment refers to reducing, inhibiting, and/or reversing the progression of a disease (eg, tumor/cancer) in a subject in need thereof.
  • treatment includes any indication of successful treatment or improvement of a disease, including any objective or subjective parameter, such as alleviation; alleviation; reduction of symptoms or making the injury, pathology or disorder more tolerable to the subject; delaying or slowing the rate of progression, etc. Measurement of treatment or improvement may be based, for example, on the results of physical, pathological, and/or diagnostic examinations known in the art.
  • Treatment may also refer to reducing the onset or risk of a disease, or reducing the recurrence of the disease (eg, prolonging the time to recurrence) compared to what would occur if the measure was not taken. In the medical field, this treatment is also called “prophylaxis.”
  • subject refers to a mammalian subject, and in particular a human subject, including male or female subjects, and includes newborn, infant, toddler, adolescent, adult or geriatric subjects, and further includes various human races and ethnicities, e.g. Caucasian, African and Asian races.
  • salts refers to relatively non-toxic inorganic or organic acid salts of the compounds of formula I of the present invention. This These salts may be prepared in situ during the final isolation and purification of the compound, or by reacting the free form of the purified compound with a suitable organic or inorganic acid, respectively, and isolating the salt so formed.
  • Representative acid salts include, but are not limited to, acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate/carbonate, bisulfate/sulfate , borate, dextrorotary camphorsulfonate, citrate, cyclosulfonate, ethanedisulfonate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconic acid Salt, glucuronate, hexafluorophosphate, hypobenrate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactic acid Salt, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthylate, 2-naphthalenesulfonate, nicotinate, nitrate, orotic acid Salt, oxalate
  • pharmaceutically acceptable and “pharmaceutically acceptable” are used interchangeably and refer to those generally accepted by those skilled in the pharmaceutical art.
  • pharmaceutically acceptable salts pharmaceutically acceptable carriers, etc.
  • an “effective amount” or “therapeutically effective amount” refers to an amount effective in treating a disease as documented through clinical testing and evaluation, patient observation, and the like. “Effective amount” may further mean an amount that causes a detectable change in biological or chemical activity. Detectable changes can be detected and/or further quantified by one skilled in the art familiar with the relevant mechanisms or methods. Additionally, an “effective amount” may mean an amount that maintains a desired physiological state (i.e., reduces or prevents significant decline and/or promotes improvement of a condition). "Effective amount” may further refer to a therapeutically effective amount.
  • WO2012039972 specifically describes the compound of formula I of the present invention.
  • the compound or its pharmaceutically acceptable salt and its preparation process are disclosed in the Examples of WO2012/039972, which is incorporated herein by reference in its entirety.
  • Negative controls should be species- and isotype-matched nonspecific immunoglobulins diluted in PBS-T.
  • the washing step is the same as step 4 of antigen coating.
  • the washing step is the same as step 4 of antigen coating.
  • biomarkers were detected, including: BDNF, EGF, CCL11 (Eotaxin), FGF-2, GM-CSF, GRO alpha (CXCL1), HGF, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8(CXCL8), IL -9, IL-10, IL-12p70, IL-13, IL-15, IL-17A (CTLA-8), IL-18, IL-21, IL-22, IL-23, IL-27a, IL- 31, IP-10 (CXCL10), LIF, MCP-1 (CCL2), MIP-1 alpha (CCL3), MIP-1 beta (CCL4), NGF beta, PDGF-
  • Tissue samples were obtained from a multi-center clinical trial conducted in the United States, with the trial registration number ClinicalTrials.gov ID: NCT03152370.
  • Pretreatment Screening or baseline. Available archived tumor material may be submitted as a pretreatment biopsy, provided that local pathology review as defined in the laboratory manual meets minimum requirements. If archived tumor material is unavailable or does not meet minimum requirements, a new tumor biopsy must be performed according to local institutional practice; 2) before radiotherapy (week 2 [days 10-14]); 3) at the end of treatment (optional day 10-14) 10 weeks [+/-2]); 4) During surgery (weeks 14-16).
  • Negative controls should be species- and isotype-matched nonspecific immunoglobulins diluted in PBS-T.
  • the washing step is the same as step 4 of antigen coating.
  • the washing step is the same as step 4 of antigen coating.
  • Statistical analysis is used to compare whether there are differences in the expression of specific biomarkers between the patient group that achieves the best response and the patient group that has no response. A total of 6 different combinations of different types of biomarkers were tested, including: CD163, CD3, CD4, CD8, PD-1, and PD-L1.
  • test results are shown in Figure 2: The combination of these three biomarkers in the tumor tissues of patients with complete pathological response (pCR) or complete clinical response (cCR) was statistically significantly higher than that of patients with no complete response (non-CR). patient group. Among them, patients who test positive for CD8, patients who test positive for a combination of CD3 and CD8, and patients who test positive for CD3, CD8, and PD-1 can achieve better results during treatment. This also suggests that the three combinations of CD8, CD3&CD8, and CD3&CD8&PD-1 can be used to predict the efficacy of patient populations.
  • one or more of the blood biomarkers as shown in Figure 1 and one or more of the tumor tissue biomarkers as shown in Figure 2 of patients with rectal cancer can be selected in combination to further improve The accuracy of predicting the sensitivity of this rectal cancer patient to AN0025 combined with radiotherapy/chemoradiotherapy (RT/CRT).
  • RT/CRT radiotherapy/chemoradiotherapy

Abstract

本发明提供了直肠癌患者血液样本中IL-1 alpha、IL-4、IL-18、IL-27a、EGF、HGF、VEGF-D、SCF、IFN-gamma、MCP-1(CCL2)或CCL11(Eotaxin)以及肿瘤组织样本中CD8，CD3&CD8，CD3&CD8&PD-1作为预测该患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的生物标志物的应用及其步骤，以及使用该应用预测直肠患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的方法和治疗直肠癌患者的方法。

Description

预测直肠癌对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的生物标志物 技术领域
本发明涉及生物领域，涉及一系列生物标志物，可以用于预测直肠癌患者对AN0025联合放疗/放化疗(RT/CRT)治疗的敏感性。
背景技术
直肠癌是中老年人常见的恶性肿瘤之一，好发于50岁以上。随着人们饮食习惯和结构的改变及人口的老龄化，其发病率呈上升趋势。直肠癌的主要治疗方式除了手术、放化疗之外，还有靶向治疗、基因治疗、中医治疗、免疫治疗等。目前外科手术仍是首选治疗，也是根治直肠癌的唯一手段。但有些患者不能够采用手术治疗，也有些患者无法进行反复的手术治疗，对于这一类的患者，放疗或放化疗是主要的治疗方法，其目的是提高患者生存率和生活质量。
AN0025是阿诺医药开发的一款***素E2(PGE2)受体4(EP4)的高选择性、高活性拮抗剂，可抑制PGE2-EP4介导的信号通路。髓细胞是肿瘤浸润的最主要的免疫细胞，主要包括肿瘤相关巨噬细胞(TAM)和髓源性抑制细胞(MDSC)，TAM和MDSC从未成熟单核细胞分化而来。EP4为4种PGE2受体中的1种，在髓细胞上表达，且在促进肿瘤微环境中单核细胞分化为TAM和MDSC的过程中起关键作用。
在癌症中，***素E2(PGE2)和其亚型4受体(EP4)组成的信号通路，不仅能够促进癌症的发生，而且能够形成促进癌症发展，免疫抑制的癌症微环境。T细胞中PGE2/EP4信号通路激活产生的cAMP能够激活cAMP-PKA信号通路，这个过程能够有效地抑制T细胞的功能。PGE2/EP4信号通路也能够负调控DC的成熟，诱导包括M2巨噬细胞以及髓系抑制细胞(MDSC)在内的多种免疫抑制细胞的形成(S et al.Front Immunol.2019；10:475)。
式(I)结构的化合物(AN0025)及其药学上可接受的盐是一种具有高度选择性的，针对E型***素受体4的小分子抑制剂。在临床前研究中，AN0025通过抑制PGE2/EP4信号通路，能够在肿瘤微环境中有效的减少免疫抑制细胞。AN0025已经运用到多个临床策略中。在针对多种晚期实体瘤FIH(first in human)I期临床实验中，AN0025已经展现出了一定的单药药效；并且在直肠癌的新辅助治疗中，AN0025与放化疗联用表现出很强的临床药效(Wyrwicz et.al,Poster#540,ESMO2019)。
临床上直肠癌患者对于AN0025联合放疗/放化疗(RT/CRT)治疗敏感性差异较大。因此需要在血浆及组织标本中寻找能够预测患者治疗敏感性的一种或几种生物标志物。
发明内容
基于上述内容，本发明提供了一系列的生物标志物应用，旨在解决预测直肠癌患者对于AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的问题，以筛选出更适合使用该治疗方案的直肠癌患者群组，进一步提高疗法的针对性。
为实现上述发明目的，本发明采用的技术方案如下。本发明所有的数据和样本均来自一项在美国进行的试验登记号为ClinicalTrials.gov ID：NCT03152370的临床试验，并且关于该临床试验相关的全部已***息通过引用的形式并入到本发明中。
在一个方面，本发明提供了一种直肠癌患者血液样本中IL-1 alpha、IL-4、IL-18、IL-27a、EGF、HGF、VEGF-D、SCF、IFN-gamma、MCP-1(CCL2)或CCL11(Eotaxin)作为预测该患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的生物标志物的应用。
在一个方面，本发明提供了直肠癌患者血液样本中IL-1 alpha、IL-4、IL-18、IL-27a、EGF、HGF、VEGF-D、SCF、IFN-gamma、MCP-1(CCL2)或CCL11 (Eotaxin)作为生物标志物在制备用于预测该患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的产品中的应用。
在一个方面，本发明提供的上述应用包括如下步骤：
步骤1：将直肠癌患者的外周血待测样本，进行离心处理收集血浆；
步骤2：检测所述血浆中IL-1 alpha、IL-4、IL-18、IL-27a、EGF、HGF、VEGF-D、SCF、IFN-gamma、MCP-1(CCL2)或CCL11(Eotaxin)的浓度；
步骤3：如果血浆检测浓度高于参考值，则认为该直肠癌患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感；反之，则不敏感。
在另一个方面，本发明提供了直肠癌患者肿瘤组织样本中CD8，CD3&CD8，CD3&CD8&PD-1作为预测该患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的生物标志物的应用。
在另一个方面，本发明提供了直肠癌患者肿瘤组织样本中CD8，CD3&CD8，CD3&CD8&PD-1作为生物标志物在制备用于预测该患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的产品中的应用。
在另一个方面，本发明提供的上述应用包括如下步骤：
步骤1：将直肠癌患者的肿瘤组织样本按照ELISE法进行处理；
步骤2:检测所述组织样本中CD3，CD8，PD-1的表达量；
步骤3：如果检测CD8，CD3&CD8，CD3&CD8&PD-1表达量高于参考值，则认为该直肠癌患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感；反之，则不敏感。
在又一个方面，本发明提供了一种预测结直肠患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的方法，包括选择上述该患者血液生物标志物IL-1 alpha、IL-4、IL-18、IL-27a、EGF、HGF、VEGF-D、SCF、IFN-gamma、MCP-1(CCL2)或CCL11(Eotaxin)中的一种或者多种，和/或如该患者肿瘤组织生物标志物CD8，CD3&CD8，CD3&CD8&PD-1中的一种或者多种。
在又一个方面，本发明还提供了一种治疗直肠癌患者的方法，包括如下步骤：
步骤1：根据权利要求7所述的方法，预测结直肠患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性；
步骤2：如果预测该直肠癌患者对AN0025联合放疗/放化疗(RT/CRT)治疗 敏感性，则对该直肠癌患者施用治疗有效量的AN0025联合放疗/放化疗(RT/CRT)。
在另一个方面，本发明提供一种治疗对其有需要的直肠癌患者的方法，包括：对所述直肠癌患者施用治疗有效量的AN0025联合放疗/放化疗(RT/CRT)，其中，所述患者通过前述方法被预测对AN0025联合放疗/放化疗(RT/CRT)治疗敏感。
在又一个方面，本发明提供一种治疗对其有需要的直肠癌患者的方法，包括：对所述直肠癌患者施用治疗有效量的AN0025联合放疗/放化疗(RT/CRT)，其中，所述患者血液样本中IL-1 alpha、IL-4、IL-18、IL-27a、EGF、HGF、VEGF-D、SCF、IFN-gamma、MCP-1(CCL2)或CCL11(Eotaxin)的检测浓度高于其参考值，和/或所述患者肿瘤组织样本中CD8，CD3&CD8，CD3&CD8&PD-1的表达量高于其参考值。其中，“参考值”是指将检测值与其对比以判断患者是否对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性有响应或者敏感的特定数值，可以例如通过临床统计分析获得。
在又另一个方面，本发明还提供一种预测直肠癌患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性试剂盒，所述试剂盒包括如上所述的该患者血液生物标志物一种或者多种，和/或如上所述的该患者肿瘤组织生物标志物一种或者多种。
附图说明
图1示出了完全病理缓解(pCR)/完全临床缓解(cCR)以及未见完全缓解(non-CR)患者血液样本中各生物标志物的含量，Y轴单位pg/mL。
图2示出了完全病理缓解(pCR)/完全临床缓解(cCR)以及未见完全缓解(non-CR)患者肿瘤组织样本中各生物标志物的阳性细胞占全部细胞的比例，Y轴表示百分比。
具体实施方式
术语定义：
除非本文中另外指出，本文所用术语具有在其所属领域的常规含义。
在本发明中，“生物标志物”等同于生物标志物或分子标志物，是在组织或细胞中的表达水平与正常或健康细胞或组织的表达水平相比发生改变的任何基因或蛋白，(单独使用或与其他定性术语组合例如乳腺癌标志物、乳腺癌特异性标志物、对照标志物、外源标志物、内源标志物)指可测量、可计算或可以其他方式获得的，与任何分子或分子组合相关，可用作生物和/或化学状态的指示物的参数。在本发明中，“标志物”指与一个或多个生物分子相关的参数(即“生物标志物”)例如天然或人工合成产生的核酸(即个体基因，以及编码和非编码DNA和RNA)和蛋白(例如肽、多肽)。本发明中的“标志物”还包括指可通过考虑来自两个或多个不同标志物的表达数据计算或以其他方式获得的单个参数。
本领域技术人员将认识到，本发明的实用性并不局限于对本发明的标志物基因的任何特定变体的基因表达进行定量。
本发明的IL-1 alpha/IL-4/IL-18/IL-27a/EGF/HGF/VEGF-D/SCF/IFN-gamma/MCP-1(CCL2)/CCL11(Eotaxin)为血液样本生物标志物，涵盖例如uniprot等数据库中收录的IL-1 alpha/IL-4/IL-18/IL-27a/EGF/HGF/VEGF-D/SCF/IFN-gamma/MCP-1(CCL2)/CCL11(Eotaxin)蛋白序列，还涵盖与其变体和/或同源物的氨基酸序列，以及该序列的片段具有至少80％、至少85％、至少90％或至少95％同源性的蛋白质，前提是变体蛋白质(包括同等型)、同源物蛋白质和/或片段受到一种或多种IL-1 alpha/IL-4/IL-18/IL-27a/EGF/HGF/VEGF-D/SCF/IFN-gamma/MCP-1(CCL2)/CCL11(Eotaxin)特异性抗体识别。
本发明的CD3/CD8/PD-1为组织样本生物标志物，涵盖例如uniprot等数据库中收录的CD3/CD8/PD-1蛋白序列，还涵盖与其变体和/或同源物的氨基酸序列，以及该序列的片段具有至少80％、至少85％、至少90％或至少95％同源性的蛋白质，前提是变体蛋白质(包括同等型)、同源物蛋白质和/或片段受到一种或多种CD3/CD8/PD-1特异性抗体识别。
为了测定一种氨基酸或核酸序列与如本文中描述的氨基酸或核酸序列是否具有某种程度的同一性，技术人员可使用本领域公知的手段和方法，例如比对，或是手工或是通过使用本领域已知的或本文中描述的计算机程序。依照本发明，术语“相同”或“百分比同一性”在两种或更多种氨基酸或核酸序列的语境中指如使用本领域已知的序列比较算法或通过手工比对和目视检查测量的，在比较窗口上或在指定区域上为最大对应进行比较和比对时，两种或更多种序列或亚序列相同，或规定百分比的氨基酸残基或核苷酸相同，认为具有例如60％至95％或更 大序列同一性的序列是基本相同的。此类定义也适用于测试序列的互补物。优选地，所描述的同一性在长度为至少约15至25个氨基酸或核苷酸的区域上、更优选在长度为约50至100个氨基酸或核苷酸的区域上存在。
本发明的“直肠癌”，指发生于结肠或直肠部位的消化道恶性肿瘤。当结肠内壁细胞异常表现或生长为息肉时，直肠癌就会发生。如果息肉未得到治疗，它们可转变成癌。其包含但不限于腺癌、鳞癌等各类病理类型。
本发明的“放疗”，又称“放射疗法”，是指电离辐射的医学使用，尤其用于治疗癌症。优选地，在癌症治疗中电离辐射的医学使用导致受试对象体内癌细胞的减少和/或杀死。放射疗法可以通过所属领域技术人员了解的任何方式施用。放射疗法中所利用的放射的实例包括(但不限于)光子辐射、电离辐射或带电粒子辐射，例如X光或质子。放射疗法的实例包括(但不限于)：体外放射疗法或远距离放射疗法；短距离放射疗法或密封射束源疗法；和全身放射性同位素疗法或非密封源放射疗法。
本发明的“化疗”又称“化学疗法”，是指使用化学治疗剂治疗癌症对象，其中化学治疗剂包括目前为止常见的用于癌症患者的化学治疗剂，例如可以是紫杉烷、紫杉醇、多西他赛、卡巴他赛、吉西他滨、卡铂、顺铂、奥沙利铂、氟尿嘧啶、卡培他滨、或替加氟(tegafor)、或其任何功能类似物。优选地，本发明的化学治疗剂选自卡培他滨或者亚叶酸/5-氟嘧啶/奥沙利铂(mFOLFOX-6)。
本发明的“放化疗”是指为患者同时或者在不同时间点施用放射疗法和化学疗法治疗癌症患者。
“治疗”、“处理”和“医治”是指在有需要的对象中减轻、抑制和/或逆转疾病(如肿瘤/癌症)的发展。术语“治疗”包括疾病的成功治疗或改善的任何迹象，包括任何客观或主观参数，例如减轻；缓和；症状减少或使受试对象更容易容忍损伤、病理或病症；延迟或减缓发展速率等。治疗或改善的测量可基于例如所属领域已知的身体检查、病理学检查和/或诊断检查的结果。治疗也可指与在不采取该措施的情况下将会发生的相比，减少疾病的发病或发作风险，或减少疾病复发(例如延长复发时间)。在医学领域，这种治疗也被称为“预防”。
术语“对象”是指哺乳动物对象，并且尤其是人类对象，包括雄性或雌性对象，并且包括新生儿、婴儿、幼儿、青少年、成人或老年人对象，并且进一步包括各种人种和种族，例如高加索人种、非洲人种和亚洲人种。
术语“可药用盐”是指本发明式I化合物的相对无毒的无机或有机酸盐。这 些盐可以在化合物的最终分离和纯化期间原位制备，或者通过将游离形态的经纯化化合物分别与合适的有机酸或无机酸反应并分离如此形成的盐而制备。代表性的酸盐包括(但不限于)乙酸盐、己二酸盐、天冬氨酸盐、苯甲酸盐、苯磺酸盐、碳酸氢盐/碳酸盐、硫酸氢盐/硫酸盐、硼酸盐、右旋樟脑磺酸盐、柠檬酸盐、环磺酸盐、乙二磺酸盐、乙磺酸盐、甲酸盐、富马酸盐、葡庚糖酸盐、葡糖酸盐、葡糖醛酸盐、六氟磷酸盐、海苯酸盐、盐酸盐/氯化物、氢溴酸盐/溴化物、氢碘酸盐/碘化物、羟乙基磺酸盐、乳酸盐、苹果酸盐、马来酸盐、丙二酸盐、甲磺酸盐、甲基硫酸盐、萘酸盐(naphthylate)、2-萘磺酸盐、烟酸盐、硝酸盐、乳清酸盐、草酸盐、棕榈酸盐、双羟萘酸盐、磷酸盐/磷酸氢盐/磷酸二氢盐、焦谷氨酸盐、糖酸盐、硬脂酸盐、琥珀酸盐、丹宁酸盐、酒石酸盐、甲苯磺酸盐、三氟乙酸盐和昔萘酸盐。在一个实施方案中，药学上可接受的盐是盐酸盐/氯化物盐。
本文中，术语“可药用”和“药学上可接受”可互换使用，是指制药领域技术人员一般接受的类型。例如可药用盐、可药用载体等。
术语“有效量”或“治疗有效量”是指如通过临床测试和评估、患者观察等所记录的，可有效治疗疾病的量。“有效量”可进一步表示引起生物或化学活性的可检测到的变化的量。可检测到的变化可以由熟悉相关机制或方法的所属领域技术人员检测和/或进一步定量。此外，“有效量”可表示维持期望生理状态(即减少或预防显著的衰退和/或促进病症的改善)的量。“有效量”可进一步指治疗有效量。
WO2012039972具体阐述了本发明的式I化合物，该化合物或其药学上可接受的盐和其制备工艺公开于WO2012/039972的实施例中，其以全文引用方式并入本文中。
为了可以更充分地理解本文中描述的发明，列出以下实施例。应理解这些实施例仅用于说明性目的，而不应解释为以任何方式限制本发明。
具体实施例
实施例1：血液肿瘤标志物
材料和方法
材料：血液样本来自于一项在美国进行的多中心临床试验，试验登记号为ClinicalTrials.gov ID：NCT03152370。
样本采集：
全血样本、用于mRNA分析的血液和PD血浆样本收集如下：基线期，第3周(第15天，放疗开始前)，第3周(第19天，放疗结束)，第5周(第29天，化疗开始/AN0025治疗中期)，第7周(第43天化疗中期)，第10周(第70天，AN0025治疗结束)和第14-16周(手术前)。
实验方法：
采用ELISA法：
首先抗原包被
1.采用碳酸盐-碳酸氢盐缓冲液或PBS中制备适当浓度的抗原溶液。
2.微量滴定板的每个孔中加入0.2ml上述溶液。
3. 37℃孵育30分钟，或4℃孵育过夜(需完全覆盖)。
4.去除包被液，用PBS-T洗涤三次。
其次一抗反应
1.用PBS-T稀释单克隆一抗。最佳浓度应使用滴定法来确定。
2.每孔加入0.2ml稀释的单克隆抗体。阴性对照应该是在PBS-T中稀释的物种和同种型匹配的非特异性免疫球蛋白。
3.室温孵育2小时。
4.洗涤步骤同抗原包被的步骤4。
最后二抗结合
1.用PBS-T稀释酶结合的二抗。每孔加入0.2ml该溶液。最佳浓度应使用滴定法来确定。
2.室温孵育2小时。
3.洗涤步骤同抗原包被的步骤4。
计算/评估方法：
采用统计分析对比达到最佳疗效的患者群组和疗效欠佳患者群组，在特定生物标志物的表达上是否有差异性。总共检测了45项不同种类的生物标志物，包括：BDNF，EGF，CCL11(Eotaxin)，FGF-2，GM-CSF，GRO alpha(CXCL1)， HGF，IFN alpha，IFN gamma，IL-1 alpha，IL-1 beta，IL-1RA，IL-2，IL-4，IL-5，IL-6，IL-7，IL-8(CXCL8)，IL-9，IL-10，IL-12p70，IL-13，IL-15，IL-17A(CTLA-8)，IL-18，IL-21，IL-22，IL-23，IL-27a，IL-31，IP-10(CXCL10)，LIF，MCP-1(CCL2)，MIP-1 alpha(CCL3)，MIP-1 beta(CCL4)，NGF beta，PDGF-BB，PlGF-1，RANTES(CCL5)，SCF，SDF-1 alpha，TNF alpha，TNF beta，VEGF-A，VEGF-D。
实验结果
最终在血液样本中，我们发现如下11种生物标志物在最佳疗效患者群组(完全病理缓解或完全临床缓解)与疗效欠佳患者群组(未见完全缓解)之间有差异(达到统计学显著性水平0.10)，可以预示患者对疗法的敏感性：IL-1alpha，IL-4，IL-18，IL-27a，EGF，HGF，VEGF-D，SCF，IFN-gama，CCL2，CCL-11。
试验结果如图1所示，完全病理缓解(pCR)或完全临床缓解(cCR)患者血清中的这11种生物标志物含量统计学上显著低于未见完全缓解(non-CR)患者组。其中，在pCR及cCR患者中，这11种生物标志物检测值均显著低于未达到完全缓解(non-CR)的患者。因此提示这11种生物标志物可以用来预测患者是否可以获得pCR/cCR。
实施例2：组织肿瘤标志物
材料和方法
材料：组织样本来自于一项在美国进行的多中心临床试验，试验登记号为ClinicalTrials.gov ID：NCT03152370。
样本采集：
受试者将进行如下肿瘤活检采集样本：1)预处理(筛选或基线)。可用存档的肿瘤材料可以作为预处理活检提交，前提是实验室手册中定义的当地病理学审查满足最低要求。如果存档的肿瘤材料不可用或不符合最低要求，则必须根据当地机构惯例进行新的肿瘤活检；2)放疗前(第2周[第10-14天])；3)治疗结束(可选第10周[+/-2])；4)在手术中(第14-16周)。
实验方法：
采用ELISA法：
首先抗原包被
1.采用碳酸盐-碳酸氢盐缓冲液或PBS中制备适当浓度的抗原溶液。
2.微量滴定板的每个孔中加入0.2ml上述溶液。
3. 37℃孵育30分钟，或4℃孵育过夜(需完全覆盖)。
4.去除包被液，用PBS-T洗涤三次。
其次一抗反应
1.用PBS-T稀释单克隆一抗。最佳浓度应使用滴定法来确定。
2.每孔加入0.2ml稀释的单克隆抗体。阴性对照应该是在PBS-T中稀释的物种和同种型匹配的非特异性免疫球蛋白。
3.室温孵育2小时。
4.洗涤步骤同抗原包被的步骤4。
最后二抗结合
1.用PBS-T稀释酶结合的二抗。每孔加入0.2ml该溶液。最佳浓度应使用滴定法来确定。
2.室温孵育2小时。
3.洗涤步骤同抗原包被的步骤4。
计算/评估方法：
采用统计分析对比达到最佳疗效的患者群组和无疗效患者群体，在特定生物标志物的表达上是否有差异性。总共检测了6项不同种类的生物标志物的不同组合，包括：CD163，CD3，CD4，CD8，PD-1，PD-L1。
实验结果
最终在组织样本中，我们发现如下3种生物标志物组合在最佳疗效患者群组(完全病理缓解或完全临床缓解)与疗效欠佳患者群组(未见完全缓解)之间有差异(达到统计学显著性水平0.10)，可以预示患者对疗法的敏感性：CD8，CD3&CD8，CD3&CD8&PD-1。其中“CD8”表示CD8检测阳性，“CD3&CD8”表示CD3及CD8检测均为阳性，“CD3&CD8&PD-1”表示CD3、CD8及PD-1检测均为阳性。
试验结果如图2所示：完全病理缓解(pCR)或完全临床缓解(cCR)患者肿瘤组织中的这3类生物标志物组合在统计学上显著高于未见完全缓解(non-CR) 患者组。其中，CD8检测阳性的患者，CD3及CD8组合检测阳性的患者，以及CD3、CD8、PD-1检测均阳性的患者，能在治疗中获得更好的疗效。这也提示CD8，CD3&CD8，CD3&CD8&PD-1这3种组合可以用来预测患者人群的疗效。
在实际应用中，可以组合选用直肠癌患者的如图1所述的血液生物标志物的一种或多种以及如图2所述的肿瘤组织生物标志物的一种或多种，以进一步提高预测该直肠癌患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的准确度。

Claims (13)

  1. 直肠癌患者血液样本中IL-1 alpha、IL-4、IL-18、IL-27a、EGF、HGF、VEGF-D、SCF、IFN-gamma、MCP-1(CCL2)或CCL11(Eotaxin)作为预测该患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的生物标志物的应用。
  2. 直肠癌患者血液样本中IL-1 alpha、IL-4、IL-18、IL-27a、EGF、HGF、VEGF-D、SCF、IFN-gamma、MCP-1(CCL2)或CCL11(Eotaxin)作为生物标志物在制备用于预测该患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的产品中的应用。
  3. 如权利要求1或者2所述的应用，包括如下步骤：
    步骤1：将直肠癌患者的外周血待测样本，进行离心处理收集血浆；
    步骤2：检测所述血浆中IL-1 alpha、IL-4、IL-18、IL-27a、EGF、HGF、VEGF-D、SCF、IFN-gamma、MCP-1(CCL2)或CCL11(Eotaxin)的浓度；
    步骤3：如果血浆检测浓度高于参考值，则认为该直肠癌患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感；反之，则不敏感。
  4. 直肠癌患者肿瘤组织样本中CD8，CD3&CD8，CD3&CD8&PD-1作为预测该患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的生物标志物的应用。
  5. 直肠癌患者肿瘤组织样本中CD8，CD3&CD8，CD3&CD8&PD-1作为生物标志物在制备用于预测该患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的产品中的应用。
  6. 如权利要求4或者5所述的应用，包括如下步骤：
    步骤1：将直肠癌患者的肿瘤组织样本按照ELISE法进行处理；
    步骤2:检测所述组织样本中CD3，CD8，PD-1的表达量；
    步骤3：如果检测CD8，CD3&CD8，CD3&CD8&PD-1表达量高于参考值，则认为该直肠癌患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感；反之，则 不敏感。
  7. 一种预测结直肠患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性的方法，包括选择如权利要求1-3任一所述的该患者血液生物标志物一种或者多种，和/或如权利要求4-6任一所述的该患者肿瘤组织生物标志物一种或者多种。
  8. 一种治疗直肠癌患者的方法，包括如下步骤：
    步骤1：根据权利要求7所述的方法，预测结直肠患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性；
    步骤2：如果预测该直肠癌患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性，则对该直肠癌患者施用治疗有效量的AN0025联合放疗/放化疗(RT/CRT)。
  9. 一种治疗对其有需要的直肠癌患者的方法，包括：对所述直肠癌患者施用治疗有效量的AN0025联合放疗/放化疗(RT/CRT)，其中，所述患者通过权利要求7所述的方法被预测对AN0025联合放疗/放化疗(RT/CRT)治疗敏感。
  10. 一种治疗对其有需要的直肠癌患者的方法，包括：对所述直肠癌患者施用治疗有效量的AN0025联合放疗/放化疗(RT/CRT)，其中，所述患者血液样本中IL-1 alpha、IL-4、IL-18、IL-27a、EGF、HGF、VEGF-D、SCF、IFN-gamma、MCP-1(CCL2)或CCL11(Eotaxin)的检测浓度高于其参考值，和/或所述患者肿瘤组织样本中CD8，CD3&CD8，CD3&CD8&PD-1的表达量高于其参考值。
  11. 一种预测直肠癌患者对AN0025联合放疗/放化疗(RT/CRT)治疗敏感性试剂盒，所述试剂盒包括如权利要求1-3任一所述的该患者血液生物标志物一种或者多种，和/或如权利要求4-6任一所述的该患者肿瘤组织生物标志物一种或者多种。
  12. 如前述任一权利要求所述的方法或者应用或者试剂盒，其中AN0025联合放疗/放化疗(RT/CRT)中的AN0025为式(I)结构的化合物或其药学上可接受的盐、同位素异构体、立体异构体。
  13. 如前述任一权利要求所述的方法或者应用或者试剂盒，其中AN0025联合放疗/放化疗(RT/CRT)中的放化疗包括放疗和化疗，其中化疗选自卡培他滨或者亚叶酸/5-氟嘧啶/奥沙利铂中的一个或多个。
PCT/CN2023/109888 2022-08-04 2023-07-28 预测直肠癌对an0025联合放疗/放化疗(rt/crt)治疗敏感性的生物标志物 WO2024027599A1 (zh)

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