Method for Treating Cancer Using Immune Checkpoint Inhibitor

Abstract

The present invention relates to a method for treating cancer using an immune checkpoint inhibitor, more specifically, a method for determining the expression of an immune checkpoint protein and a lymphatic or myeloid cell-specific protein in circulating tumor cells isolated from blood, selecting a patient who is determined to have positive expression of an immune checkpoint protein and negative expression of a lymphatic or myeloid cell-specific protein as a patient to whom an immune anticancer drug can be applied, and administering an immune checkpoint inhibitor to the patient to treat cancer. The method for treating cancer using circulating tumor cells according to the present invention is highly accurate and commercially useful because it determines the expression of an immune checkpoint protein and a lymphatic or myeloid cell-specific protein simultaneously by liquid biopsy and then removes false-positive circulating tumor cells to select patients.

Description

[Technical Field]

[0001] The present invention relates to a method for treating cancer using an immune checkpoint inhibitor, and more specifically, to a method for treating cancer by confirming the expression of immune checkpoint proteins and lymphoid or myeloid cell-specific proteins in circulating tumor cells isolated from blood, selecting patients who are confirmed to have positive expression of immune checkpoint proteins and negative expression of lymphoid or myeloid cell-specific proteins as patients to whom an immune anticancer drug can be applied, and administering the immune checkpoint inhibitor to said patients. [Background technology]

[0002] Cancer is one of the most deadly threats to human health. In the United States, cancer affects approximately 1.3 million new cases each year and is the second leading cause of death after heart disease, accounting for one in four deaths. Cancer is also expected to surpass cardiovascular disease as the leading cause of death within five years. Solid tumors account for the majority of these deaths.

[0003] Although various methods for treating cancer have been tried, the overall 5-year survival rate for all cancers has only improved by about 10% over the past 20 years, as the clinical outcomes and prognoses of individual patients are not always consistent with the reported data. Therefore, by predicting the response to therapeutic drugs and prognosis of cancer patients in anti-cancer treatment, we can provide the most appropriate treatment for cancer patients with heterogeneous characteristics, avoid unnecessary treatment-related toxicity, and ultimately improve treatment efficacy.

[0004] In response to this need, active research has recently been conducted on companion diagnostics, which are approved diagnostics that can select appropriate targeted anticancer drugs and treatment methods based on the results of a systematic analysis of a patient's personal factors. Companion diagnostics can provide clear clinical evidence for a doctor's prescription and recommend appropriate treatment options to patients, thereby not only improving the efficiency of cancer treatment but also reducing the misuse of targeted anticancer drugs and contributing to the soundness of national health insurance finances. Currently, the companion diagnostics market is growing in the treatment fields of breast cancer, lung cancer, colorectal cancer, gastric cancer, and melanoma, with breast cancer and lung cancer in particular expected to drive market growth. As pharmaceutical companies seek to reduce new drug development costs and demand for targeted therapies increases, the global companion diagnostics market is growing at a rapid rate every year.

[0005] Immunotherapy, on the other hand, harnesses a patient's own immune system to attack cancer regardless of its origin. The immune system is regulated by a network of checks and balances that evolved to attack foreign invaders such as bacteria and viruses. However, cancers can evade the immune system by expressing proteins, such as PD-L1 and PD-L2, that inhibit the immune system from attacking cancer cells.

[0006] In particular, the interaction between tumor cells and T cells involves contact between the major histocompatibility complex (MHC) on the tumor cell and the T cell receptor (TCR) on the T cell. Upon contact between the MHC and the T cell receptor, the T cell is activated and the tumor cell is destroyed.

[0007] Tumor cells can evade T cell immune surveillance when they express the immune checkpoint protein PD-L1 on their surface. When present, PD-L1 binds to PD-1 expressed by T cells and blocks T cell activation, thereby inhibiting T cell immune surveillance.

[0008] Immune checkpoint inhibitors have been developed that can block the PD-L1 / PD-1 interaction. These drugs allow the T cell immune surveillance mechanism to function normally again, allowing tumor cells to be destroyed by the subject's normal immune response. Blocking CTLA-4 on T cells can also have a similar effect (Hodi et al., N. Engl. J. Med. Vol. 363, pp. 711-23, 2010).

[0009] The key to effective use of immune checkpoint inhibitors is determining whether a specific cancer patient will respond to the drug. If an antibody that binds to PD-L1 or PD-1 and acts as an immune checkpoint inhibitor is administered to a patient whose tumor cells do not express PD-L1, the treatment will be ineffective. To date, the response rate to such immune checkpoint inhibitors has been limited to 20-30% (Herbst RS, et al. The New England Journal of Medicine. Vol. 383(14), pp. 1328-39, 2020).

[0010] The currently recommended method for measuring PD-L1 expression is to calculate a PD-L1 tumor proportion score (TPS) from patient tissue cells. For example, for pembrolizumab (Keytruda) and nivolumab (Opdivo), which are PD-1-specific antibodies, to be effective, PD-L1 expression must be detected at a certain percentage or higher in patient tissue tests. To be eligible for health insurance coverage, Opdivo requires PD-L1 expression of 10% or higher, Keytruda requires 50% or higher, and atezolizumab (Tecentriq) requires PD-L1 expression of 5% or higher on both tumor cells and tumor-infiltrating immune cells.

[0011] However, obtaining cancer cells through tumor biopsy to investigate PD-L1 expression has serious drawbacks, including pain and discomfort for the patient, the inability to investigate abnormalities in isolated tumor tissue, and the possibility that protein expression profiles may change over time in the tumor microenvironment. Furthermore, re-biopsy also poses invasiveness and other complex issues. Furthermore, the patient identification criteria for each immune checkpoint inhibitor vary, making it difficult to select the most effective treatment for each patient.

[0012] To overcome these drawbacks, liquid biopsies containing circulating tumor cells (CTCs), circulating cell-free DNA (cfDNA), and exosomes have recently emerged as a novel solution (Rijavec E, et al., Cancers. Vol. 12(1):17, 2020). Blood-based biopsies offer an improvement over tissue biopsies in that they allow for real-time sequential tracking of cells shed by tumors or otherwise separated.

[0013] Blood samples are more easily obtained and can be obtained more frequently from patients. Furthermore, changes in protein expression profiles can be monitored over time. Circulating tumor cells (CTCs) are one cancer-associated cell type that can be easily isolated from peripheral blood and used as an alternative to tumor cells obtained from tissue biopsies. CTCs are tumor cells that are isolated from solid tumors into the bloodstream. CTCs can be found in the blood of patients with carcinoma, sarcoma, neuroblastoma, and melanoma. Identifying additional cell types that can be obtained from blood-based biopsies is important for further developing the application of this technology to identify cancer patients who would benefit from treatment with immune checkpoint inhibitors.

[0014] Against this technical background, the present inventors have made extensive efforts to develop a CTC-based cancer treatment method. As a result, they have confirmed that by simultaneously confirming the expression of immune checkpoint proteins and lymphatic or immune cell-specific proteins, and administering an immune checkpoint inhibitor to patients whose expression of immune checkpoint proteins is positive and whose expression of lymphatic or immune cell-specific proteins is negative, false-positive patients can be excluded and only patients who respond to the therapeutic agent can be administered, thereby improving the efficacy of cancer treatment, and have completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0015] An object of the present invention is to provide a method for selecting patients for the application of immune checkpoint inhibitors. Another object of the present invention is to provide a method for treating cancer. Another object of the present invention is to provide a method for determining sensitivity to immune checkpoint inhibitors. Another object of the present invention is to provide a method for selecting an immune anti-cancer therapy for cancer patients. [Means for solving the problem]

[0016] To achieve the above object, the present invention provides a method for selecting patients for the administration of immune anti-cancer drugs, comprising the steps of: (a) confirming the expression of immune checkpoint proteins and lymphoid- or myeloid-specific proteins in isolated circulating cancer cells; and (b) classifying patients confirmed to be positive for the expression of immune checkpoint proteins and negative for the expression of lymphoid- or myeloid-specific proteins as patients to whom the immune anti-cancer drugs can be administered. The present invention also provides The present invention provides a method for treating cancer, comprising: (a) confirming the expression of an immune checkpoint protein and a lymphoid- or myeloid-specific protein in isolated circulating cancer cells; and (b) administering an immune checkpoint inhibitor to a patient confirmed to be positive for the expression of an immune checkpoint protein and negative for the expression of a lymphoid- or myeloid-specific protein.

[0017] The present invention also provides The present invention provides a method for determining susceptibility to immune checkpoint inhibitors, comprising: (a) confirming the expression of an immune checkpoint protein and a lymphoid- or myeloid-specific protein in isolated circulating cancer cells; and (b) determining that the cancer is susceptible to the immune checkpoint inhibitor if the expression of the immune checkpoint protein is confirmed to be positive and the expression of the lymphoid- or myeloid-specific protein is confirmed to be negative.

[0018] The present invention also provides The present invention provides a method for selecting an immune anti-cancer therapy for a cancer patient, comprising: (a) confirming the expression of an immune checkpoint protein and a lymphoid or myeloid lineage-specific protein in isolated circulating cancer cells; and (b) selecting an immune checkpoint inhibitor as a therapeutic therapy when the expression of the immune checkpoint protein is confirmed to be positive and the expression of the lymphoid or myeloid lineage-specific protein is confirmed to be negative.

[0019] The present invention also provides Provided is a pharmaceutical composition for treating cancer, comprising an immune checkpoint inhibitor, for treating an individual whose expression is positive for an immune checkpoint protein and whose expression is negative for a lymphoid- or myeloid-specific protein.

[0020] The present invention also provides the use of an immune checkpoint inhibitor for the manufacture of a therapeutic medicament for treating an individual who is positive for expression of an immune checkpoint protein and negative for expression of a lymphoid or myeloid-specific protein. [Brief explanation of the drawings]

[0021] [Figure 1] 1 shows fluorescent images of circulating tumor cells and immune cells identified according to an embodiment of the present invention. [Figure 2] Fluorescence images of circulating tumor cells (A) expressing circulating tumor cell markers and PD-L1, as confirmed by one embodiment of the present invention, and false positive cells (myeloid cells, (B)) expressing both the markers and CD18. [Figure 3] 1 shows a fluorescent image (A) confirming the expression of CD16 and CD18 from a myeloid cell line according to one embodiment of the present invention, and a graph (B) showing the quantification thereof. [Figure 4] 1 shows a fluorescent image (A) confirming the expression of CD11b and CD18 from a myeloid cell line according to one embodiment of the present invention, and a graph (B) showing the quantification thereof. [Figure 5] 1A shows a fluorescent image confirming the expression of CD16 and CD18 from a patient's blood sample according to an embodiment of the present invention, and FIG. 1B shows a graph quantifying the same. [Figure 6] PD-L1 expression ratio scores were compared between tissue samples and CD18- / CD45- CTCs according to one embodiment of the present invention. (A) is a Bland-Altman plot, and (B) is a scatter plot. [Figure 7] 1 shows the results of an analysis of the correlation between the number of CTCs and the size of cancer tissue according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures described below are those well known and commonly employed in the art.

[0023] Terms such as "first," "second," "A," and "B" may be used to describe various components, but the components are not limited by these terms and are used solely to distinguish one component from another. For example, a first component may be designated a "second component," and similarly, a second component may be designated a "first component" without departing from the scope of the technology described below. The term "and / or" includes a combination of multiple related and described items or any of multiple related and described items.

[0024] In the terms used in this specification, singular expressions should be understood to include plural expressions unless the context clearly indicates otherwise, and terms such as "comprise" should be understood to mean the presence of a stated feature, number, step, operation, component, part, or combination thereof, and not to exclude the possible presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0025] Furthermore, in performing a method or method of operation, the steps constituting the method may be performed out of the order specified, unless the context clearly dictates a particular order. That is, the steps may be performed in the same order as specified, substantially simultaneously, or in the opposite order.

[0026] In the present invention, the term "antibody" is used in the broadest sense and includes various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0027] As used herein, the term "biomarker" refers to an indicator that can be detected in a sample and that can act as a predictive, diagnostic, and / or prognostic indicator of a disease or disorder (e.g., cancer) characterized by specific molecular, pathological, histological, and / or clinical features.

[0028] As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung; bladder cancer (e.g., urothelial bladder cancer (UBC), muscle-invasive bladder cancer (MIBC), and BCG-refractory non-muscle-invasive bladder cancer (NMIBC)); kidney or renal cancer (e.g., renal cell carcinoma (RCC)); urinary tract cancer; breast cancer (e.g., estrogen receptor (ER-), progesterone receptor (PR-), and HER2 (H)-dependent cancers). HER2+ breast cancer and triple-negative breast cancer (TNBC) that are ER2-negative; prostate cancer such as castration-resistant prostate cancer (CRPC); peritoneal cancer; hepatocellular carcinoma; gastric or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; liver tumors; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine carcinoma; salivary gland carcinoma; prostate cancer; vulvar cancer; thyroid cancer; liver carcinoma; anal carcinoma; penile carcinoma melanoma; melanoma, including superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-resected cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloid leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as phagocytosis, edema (e.g., associated with brain tumors), Meigs syndrome, abnormal vascular proliferation associated with brain tumors, head and neck cancer, and related metastases.

[0029] In the present invention, the terms "treat" and "treatment" have their common and ordinary meanings, including one or more of completely or partially removing a tumor or cancer from a subject, reducing the size of a tumor in a subject, killing tumor or cancer cells in a subject, and improving symptoms of cancer or a tumor in a subject. Treating refers to removing, reducing, killing, or improving by about 1% to about 100% compared to a subject not administered an immune checkpoint inhibitor. Desirably, removal, reduction, killing, or improvement is about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, or about 1%. Treatment results can be permanent or can continue to progress over a period of days (e.g., 1, 2, 3, 4, 5, 6, or 7 days), weeks (e.g., 1, 2, 3, or 4 weeks), months (e.g., 1, 2, 3, 4, 5, 6, or more months), or years (e.g., 1, 2, 3, 4, 5, 6, or more years).

[0030] As used herein, the terms "inhibit," "inhibiting," and "inhibition" have their common and ordinary meanings, including one or more of preventing, delaying, blocking, arresting, or arresting the establishment of cancer or tumor, the development of cancer or tumor, or the growth and metastasis of cancer or tumor. Inhibition refers to about 1% to about 100% inhibition relative to a subject not administered an immune checkpoint inhibitor. Desirably, inhibition is about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, or about 1%. The inhibition method can be performed on a subject before, simultaneously with, or after the onset of clinical symptoms of cancer or tumor. Thus, the subject may be a subject suffering from cancer or tumor, or may simply be a subject predisposed to developing cancer or tumor. The results of the inhibition can be permanent or can continue and progress over a period of days (e.g., 1, 2, 3, 4, 5, 6, or 7 days), weeks (e.g., 1, 2, 3, or 4 weeks), months (e.g., 1, 2, 3, 4, 5, 6, or more months), or years (e.g., 1, 2, 3, 4, 5, 6, or more years).

[0031] In the present invention, the term "administration" refers to a method of providing a dosage of a compound or composition. The compounds and / or compositions used in the methods described herein can be administered, for example, intravenously (e.g., by intravenous infusion), subcutaneously, intramuscularly, intradermally, transdermally, intraarterially, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subconjunctivally, intravesically, mucosally, intrapericardially, intraumbilically, intraocularly, orally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion to directly bathe target cells, by catheter, by lavage, in a cream, or in a lipid composition. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).

[0032] Immune checkpoint inhibitors and pharmaceutical formulations containing immune checkpoint inhibitors can be administered to a subject on different schedules, depending on the specific purpose or goal of the method, the age and size of the subject, and the general health of the subject, to name a few factors to consider. Generally, immune checkpoint inhibitors and pharmaceutical formulations can be administered once, twice, three times, four times, five times, six times, or more times over the course of treatment or inhibition. The timing between each dose in the dosing schedule can range from days, weeks, months, or years, including once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more weeks. The same amount of immune checkpoint inhibitor can be administered at each dose in the dosing schedule, or the amount at each dose can vary. The identity of the immune checkpoint inhibitor at each dose in a dosing schedule may also vary or may remain the same.

[0033] In each method of the present invention, the immune checkpoint inhibitor or a pharmaceutical formulation containing the immune checkpoint inhibitor is administered to a subject in a "therapeutically effective amount." The therapeutically effective amount will vary from subject to subject. However, a therapeutically effective amount is an amount sufficient to achieve the goal or objective of the method, whether that goal or objective is to inhibit or treat. By way of example, a therapeutically effective amount of an immune checkpoint inhibitor used in the methods of the present invention is typically about 0.1 μg to about 10,000 μg of immune checkpoint inhibitor per kg of body weight of the subject receiving the peptide. Therapeutically effective amounts also include about 0.5 μg to about 5,000 μg, about 1 μg to about 500 μg, about 10 μg to about 200 μg, about 1 μg to about 800 μg, about 10 μg to about 1,000 μg, about 50 μg to about 5,000 μg, about 50 μg to about 500 μg, about 100 μg to about 1,000 μg, about 250 μg to about 2,500 μg, about 500 μg to about 2,000 μg, about 10 μg to about 800 μg, about 1 μg to about 300 μg, and about 10 μg to about 300 μg of immune checkpoint inhibitor per kg of subject body weight.

[0034] In the case of Keytruda, a treatment used in conventional immune-mediated anti-cancer therapy, the Phase 2 / 3 clinical trial "KEYNOTE-010" was conducted on previously treated patients with PD-L1 expression of 1% or higher. The results showed that the higher the PD-L1 expression rate, the better the therapeutic effect of Keytruda, and that the treatment response was relatively clearly determined by PD-L1 expression rate, providing a good example of how PD-L1 can function as a marker for immune-mediated anti-cancer drugs. On the other hand, a retrospective analysis of data from the Phase 3 clinical trial "Checkmate-057" in previously treated advanced non-squamous non-small cell lung cancer showed similar objective response rates (ORR) of 31%, 36%, and 37% in the Opdivo group with PD-L1 expression of 1%, 5%, and 10% or higher.

[0035] In addition, in the OAK clinical trial of patients with locally advanced or metastatic non-small cell lung cancer who had failed conventional platinum-based chemotherapy, Tecentriq was reported to extend overall survival (OS) by approximately four months compared to the conventional chemotherapy drug docetaxel, regardless of PD-L1 expression, indicating that Tecentriq is effective in both PD-L1-positive and -negative patients. In the PACIFIC phase 3 clinical trial of patients with stage 3 non-small cell lung cancer who had not progressed after simultaneous platinum-based chemotherapy and radiation therapy, Imfinzi was reported to approximately double progression-free survival (PFS) compared to placebo, and subgroup analysis showed consistent efficacy regardless of PD-L1 expression.

[0036] Ultimately, under current circumstances, even if PD-1 / PD-L1 immune checkpoint inhibitors are administered, dramatic therapeutic effects are not seen in all cancer patients, and there are questions about the clinical usefulness of immunohistochemistry (IHC) diagnostic equipment for measuring the PD-L1 expression rate of these therapeutic agents.

[0037] For example, approximately 28% of patients who respond to treatment will be diagnosed with a false negative PD-L1 expression rate, resulting in missed opportunities for treatment, and approximately 42% of patients who do not respond to treatment will be mistakenly diagnosed with a false positive, potentially compromising the appropriateness and economic viability of drug administration.

[0038] In the present invention, we aimed to develop a biomarker that can efficiently remove cells that show false positives and a method that can effectively select patients to whom immune anti-cancer therapy can be applied, and confirmed that immune anti-cancer therapy can be selected with high accuracy when the expression of immune checkpoint proteins and lymphoid or myeloid lineage-specific proteins is confirmed simultaneously.

[0039] In one example of the present invention, it was confirmed that when CTCs isolated from isolated blood were stained with PD-L1 and CD18 antibodies to confirm their expression levels, false-positive cells could be effectively removed compared to a method that only confirmed PD-L1 expression (Figures 1 and 2). Therefore, in one aspect, the present invention comprises: Regarding the method for selecting patients for the application of immunotherapy, the method includes the following steps: (a) confirming the expression of immune checkpoint proteins and lymphoid or myeloid lineage-specific proteins from isolated circulating cancer cells; and (b) Classifying patients who are confirmed to be positive for immune checkpoint protein expression and negative for lymphoid or myeloid lineage-specific protein expression as patients to whom an immune anticancer drug can be administered.

[0040] In the present invention, the expression of the protein being "positive" means that the presence of the protein in circulating cancer cells in the blood is confirmed by various methods known to those skilled in the art, preferably, but not limited to, by fluorescence intensity.

[0041] In the present invention, the expression of the proteins can be determined to be positive by taking into consideration the number of cells expressing each or all of the proteins, the expression intensity (fluorescence intensity) of each protein, and determining based on a specific cut-off value, or by determining whether the expression level is high or low compared to a reference population, but is not limited thereto.

[0042] In the present invention, the expression of the protein being "negative" means that the presence of the protein in circulating cancer cells in the blood cannot be confirmed by various methods known to those skilled in the art, and preferably, but not limited to, can be confirmed by fluorescence intensity.

[0043] In the present invention, the expression of the proteins can be determined to be negative by taking into consideration the number of cells that do not express each or all of the proteins, the expression intensity (fluorescence intensity) of each protein, and determining based on a specific cut-off value, or by determining whether the expression level is high or low compared to a reference population, but is not limited thereto.

[0044] In the present invention, the immune checkpoint protein may be any protein involved in an immune checkpoint-associated signaling pathway, and may preferably be selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, BY-H4, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, PD-L1, TIM-3, VISTA, and SIGLEC7, more preferably from the group consisting of PD-1, PD-L1, and CTLA-4, and most preferably, PD-L1, but is not limited to these.

[0045] In the present invention, the lymphoid or myeloid specific protein may be any protein expressed in lymphoid or myeloid cells without limitation, and preferably includes CD18, CD29, CD61, CD104, ITGB5, ITGB6, ITGB7, ITGB8, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD11D, It may be selected from the group consisting of CD103, CD11a, CD11b, CD51, CD41, CD11c, CD64, CD32, CD16a, CD16b, CD23, CD89, FcRn, FcεRI and Fcα / μR, more preferably selected from the group consisting of CD18, CD16, CD29, CD61, CD104, CD32, CD11b and CD64, most preferably, CD18, but is not limited to these. In the present invention, the isolated circulating cancer cells may be isolated by the following steps: (i) obtaining blood from a cancer patient; and (ii) isolating cancer cells from the blood using a biochip.

[0046] In the present invention, circulating tumor cells (CTCs) refer to tumor cells found in the peripheral blood of patients with malignant tumors. CTCs are extremely rare, and the amount of available specimens is severely limited. Techniques for detecting and characterizing CTCs include, but are not limited to, multiplex reverse transcription quantitative polymerase chain reaction (RT-PCR), imaging-based approaches, microfiltration, and microchip devices. CTCs can act as tumor biological markers, which can facilitate personalized treatment and post-treatment follow-up in liquid biopsy samples. CTCs can also be used as targets for understanding tumor biology and tumor cell dissemination, but are not limited to these.

[0047] The biochip is a hybrid device fabricated in the form of a conventional semiconductor chip by integrating and combining biologically derived substances such as DNA, proteins, enzymes, antibodies, microorganisms, animal and plant cells and organs, and nerve cells at high density on a solid substrate made of an inorganic material such as a semiconductor. It is a tool or device that uses the inherent functions of biomolecules to obtain biological information such as gene expression patterns, gene binding, and protein distribution, or to increase the speed of biochemical processes, reactions, or information processing. The high-density microchip refers to a biochip that can separate substances of a specific size based on the working principle of the biochip. The high-density microchip can capture circulating cancer cells in the blood with a recovery rate of approximately 90% within 10 minutes based on the size difference of blood cells.

[0048] In one embodiment of the present invention, the pore size of the high-density microchip may be preferably 5.5 to 8.5 μm, more preferably 6.5 to 7.5 μm. If the pore size is smaller than 5.5 μm, red blood cells and white blood cells cannot pass through the chip and are trapped on the chip, preventing their removal. If the pore size is larger than 8.5 μm, the pore size is larger than the size of circulating cancer cells, preventing them from passing through the chip and being selectively collected. In one embodiment of the present invention, the pore shape of the high-density microchip may be circular, rectangular, or elliptical, preferably rectangular.

[0049] In one embodiment of the present invention, the pores of the high-density microchips may be arranged in a regular pattern. In another embodiment of the present invention, the high-density microchips may be made of stainless steel, nickel, aluminum, or copper. The pores may be formed by etching using MEMS (Micro-electro Mechanical Systems) technology.

[0050] The distance between two adjacent pores is narrower than the diameter of a circulating cancer cell. According to one embodiment of the present invention, the distance between two pores may be 45 to 65% of the diameter of a circulating cancer cell. The high-density microchip is not deformed by the pressure of blood or a solution flowing through the passage. After passing through the pores, blood is discharged to the outside, and the circulating cancer cells in the blood cannot pass through the pores and remain on the surface. Non-target cells, i.e., red blood cells, which have a higher deformability than circulating cancer cells, easily pass through the pores.

[0051] After filtering the circulating cancer cells, the circulating cancer cells can be discharged by supplying a solution in the reverse or forward direction. According to one embodiment of the present invention, the solution can be supplied in the reverse direction to minimize damage to the circulating cancer cells. The solution can be supplied using a syringe, syringe pump, plunger pump, etc. According to one embodiment of the present invention, the solution can be composed of a diluent for diluting blood, water, and a diluting acid. The circulating cancer cells discharged by supplying the solution can be easily collected by collecting them in a container, such as a test tube or a culture dish.

[0052] On the other hand, circulating cancer cells with diameters of 7.5 to 15 μm pass through an 8 μm diameter tube when a pressure of approximately 100 mmHg is applied. Circulating cancer cells with diameters of 15 μm passing through an 8 μm diameter tube exhibit a deformation rate of approximately 53%. When backwashing, i.e., flowing a solution in the opposite direction of the blood flow to expel circulating cancer cells with diameters of 7.5 μm, the distance between two pores is preferably 4 μm or less, considering the deformation rate of circulating cancer cells. For example, non-small cell lung cancer and breast cancer cells are known to reach diameters of approximately 40 μm. Considering the deformation rate of circulating cancer cells with diameters of 40 μm during backwashing, the distance between two pores is preferably 21 μm or less. When circulating cancer cells with diameters of 7.5 μm are present between two pores spaced more than 4 μm apart, the circulating cancer cells may be deformed by the flow of the solution and may not detach from the surface. Furthermore, cancer cells with a diameter of 40 μm may not fall off from the surface between two pores if the distance between them exceeds 21 μm. In the high-density microchip according to the present invention, the distance between two pores is 45 to 65% of the diameter of the circulating cancer cells, taking into account the pressure of the solution. Circulating cancer cells with a diameter of 7.5 μm may not fall off from the surface between two pores by backwashing if the distance exceeds 65%, i.e., approximately 4.9 μm. Circulating cancer cells with a diameter of 40 μm may not fall off from the surface between two pores by backwashing if the distance exceeds 65%, i.e., more than 26 μm. Increasing the pressure of the solution to forcibly remove circulating cancer cells adhering to the surface between two pores may damage the circulating cancer cells, resulting in a reduced collection rate of viable circulating cancer cells. If the spacing for circulating cancer cells with a diameter of 7.5 μm is less than 45%, i.e., less than about 3.375 μm, there is a high risk that the high-density microchip will be damaged by the flow of blood and solution.

[0053] In one embodiment of the present invention, the sample can be repeatedly passed through the high-density microchip. Specifically, after circulating cancer cells are separated once by the high-density microchip, the separated circulating cancer cells can be loaded onto the high-density microchip again and separated, and the separation process can be repeated.

[0054] In one embodiment of the present invention, the separation of circulating cancer cells using the high-density microchip can be performed by loading a solution containing circulating cancer cells into the high-density microchip and then separating the circulating cancer cells using gravity rather than by applying a specific artificial pressure. The separation of circulating cancer cells using the high-density microchip according to the present invention can maintain the state of the circulating cancer cells in the same state as they were in the patient's body by minimizing damage to the circulating cancer cells caused by artificial pressure.

[0055] In one embodiment of the present invention, the high-density microchip may be coated with a specific substance to minimize damage to circulating cancer cells caused by the high-density microchip during separation, to facilitate repeated use of the high-density microchip, or to improve the recovery rate of circulating cancer cells. Specifically, the specific substance may be an antibody capable of specifically binding to circulating cancer cells, or any biomaterial that does not physically or chemically damage the cells. According to one embodiment of the present invention, the specific substance may be bovine serum albumin (BSA) or an antibody. The antibody may be, for example, an anti-epithelial cell adhesion molecule antibody (anti-EpCAM antibody), an anti-cytokeratin antibody (anti-CK antibody), or the like. According to a preferred embodiment of the present invention, the specific substance may be bovine serum albumin (BSA).

[0056] The BSA (Bovine Serum Albumin) solution refers to bovine serum albumin. It is a protein with a molecular weight of approximately 66.4 kDa and is abundant in most animals. In biochemistry and biology, BSA can be added as a nutrient for cells during cell culture. It is also widely used as a standard for obtaining calibration curves in protein quantification. It can also be added to supplement the protein concentration in a solution when using restriction enzymes, which require small amounts of enzyme (protein). It can also be used in various biochemical experiments (Western blot, immunocytochemistry, ELISA, etc.) to prevent nonspecific binding, i.e., the attachment of antibodies to undesired proteins or undesired locations, before attaching specific antibodies to proteins to be detected.

[0057] According to one embodiment of the present invention, peripheral blood can be reacted with a high-density microchip coated with the BSA solution using centrifugation to remove other biopolymers except for circulating cancer cells. According to one embodiment of the present invention, separation using the high-density microchip can be performed by gravity, specifically at atmospheric pressures of 1000 to 1020 hPa, preferably at atmospheric pressures of 1000 to 1015 hPa, and more preferably at atmospheric pressures of 1000 to 1013 hPa.

[0058] According to one embodiment of the present invention, the BSA solution may be coated on the upper surface, the lower surface, or the inner surface of the pores of the high-density microchip, preferably on all of the upper surface, the lower surface, and the inner surface of the pores of the high-density microchip.

[0059] According to one embodiment of the present invention, the BSA solution coating may be performed at a concentration of 0.05 to 0.15%. According to a preferred embodiment of the present invention, the BSA solution coating may be performed at a concentration of 0.08 to 0.012%.

[0060] According to one embodiment of the present invention, the BSA solution coating can be applied for 5 to 15 minutes. According to a preferred embodiment of the present invention, the BSA solution coating can be applied for 8 to 12 minutes.

[0061] In the present invention, the expression of the immune checkpoint protein and lymphoid or myeloid lineage-specific protein may be confirmed by performing the following steps:

[0062] (1) reacting a fluorescent label that specifically binds to circulating cancer cells in the blood with a fluorescent label that specifically binds to an immune checkpoint protein; and a fluorescent label that specifically binds to a lymphoid or myeloid lineage-specific protein; (2) receiving optical images for each of a plurality of wavelength ranges, targeting circulating cancer cells reacted with the fluorescent label; immune checkpoint proteins; and lymphoid- or myeloid-specific proteins; (3) measuring the fluorescence intensities of the circulating cancer cells in the blood, immune checkpoint proteins, and lymphoid or myeloid lineage-specific proteins from the optical images of all or part of the multiple wavelength ranges, and performing primary filtering; (4) measuring the morphology of the circulating cancer cells in the blood from the optical images of all or part of the wavelength ranges, and performing secondary filtering; and (5) A step of measuring the morphology of the circulating cancer cells in the blood from an integrated image obtained by merging all or part of the optical images for each of the multiple wavelength ranges, and performing third-order filtering.

[0063] In the present invention, the fluorescent label that specifically binds to circulating cancer cells in the blood refers to a fluorescent substance that can specifically bind to circulating cancer cells themselves or to substances present inside or outside the cells. According to one embodiment of the present invention, the fluorescent label that can identify circulating cancer cells in the blood can specifically bind to cell nuclei or proteins, DNA, RNA, etc. present inside or outside the cells. Specifically, DAPI can be used as a fluorescent label for cell nuclei, fluorescent labels that specifically bind to the intermediate filament protein vimentin can be used, fluorescent labels that specifically bind to epithelial cell adhesion molecule (EpCAM) and cytokeratin (CK) can be used, and fluorescent labels that specifically bind to CD45, which is used as a means for removing non-target cells, can be used. The fluorescent label that specifically binds to circulating cancer cells in the blood can be composed of nucleotides, oligonucleotides, peptides, polypeptides, nucleic acids, or proteins, and can also be a protein-based antibody. The fluorescent label that specifically binds to circulating cancer cells in the blood can be any substance that specifically binds to circulating cancer cells in the blood and enables them to be identified.

[0064] The optical image may be generated by an imaging system. According to one embodiment of the present invention, the imaging system may be a cell imaging system, which places stained cells on a platform such as a glass slide and observes and photographs the cells at various wavelengths. The cell imaging system may include an automatic cell counting module, a fluorescence intensity analysis module, and a cytology-based cell classification / recognition module. Furthermore, as user convenience functions, a measurement function, an automatic report generation function, and a database management function may be included as a user interface. Such a cell imaging system may include a digital image analysis device for distinguishing between cells, culture medium, debris, etc., and identifying and counting the cells desired by the user. The cell imaging system according to one embodiment of the present invention can accurately identify and count fluorescently labeled or marker-bound target cells from an optical image.

[0065] The optical image may be an optical image captured by light reflected from an object. A cell optical image may be output by a cell imaging device and may be an image of cells and debris against a background. The optical image may be provided as a single image file constructed by stitching multiple divided images for each specific wavelength range. Here, the optical images for multiple wavelength ranges may include a blue wavelength range image, a green wavelength range image, and a red wavelength range image. The blue wavelength range optical image is particularly useful for identifying the nuclei of circulating cancer cells in the blood, and the green and red wavelength range optical images are particularly useful for identifying the cell membranes of circulating cancer cells in the blood. In addition, specific fluorescent labels or markers can be identified using various color wavelength ranges.

[0066] If the primary or secondary filtering does not achieve the desired level of discrimination between circulating cancer cells, immune checkpoint proteins, and lymphoid or myeloid cell-specific proteins, data filtering of the optical image data can be performed again, followed by primary or secondary filtering, or multiple data filtering steps. After primary or secondary filtering, the image data can be saved and output.

[0067] In addition, a primary filtering step and a secondary filtering step may be performed on an optical image for a first wavelength range, and then one or more of the primary filtering step and the secondary filtering step may be further performed on an optical image for a second wavelength range different from the first wavelength range.

[0068] For example, the first wavelength range may be a blue wavelength range, and the second wavelength range may be a green or red wavelength range. In this case, the nuclei of cancer cells circulating in the blood may be identified by performing a first filtering process and a second filtering process on the blue wavelength range image. Furthermore, the cell membranes of cancer cells circulating in the blood may be identified by performing the first filtering process on one or more of the green wavelength range image and the red wavelength range image. Through such primary and secondary filtering, cells can be accurately identified from the optical image. For example, the green and red wavelength range images enhance the discrimination of target cells, such as white blood cells and cancer cells.

[0069] Meanwhile, in the secondary filtering process, the morphology of circulating cancer cells in the blood is measured, where the morphology of circulating cancer cells in the blood can include one or more of cell area, cell diameter, and circularity.

[0070] In one embodiment of the present invention, the fluorescence intensity measurement for immune checkpoint proteins and lymphoid or myeloid cell-specific proteins is performed by measuring the fluorescence intensity from an optical image of all or part of multiple wavelength ranges emitted from fluorescent labels that specifically bind to the immune checkpoint proteins and lymphoid or myeloid cell-specific proteins, and primary filtering can be performed on the measured fluorescence intensity.

[0071] Looking more specifically at the process of measuring the fluorescence intensity of circulating cancer cells in the blood, in addition to immune checkpoint proteins and lymphoid or myeloid cell-specific proteins, and performing primary filtering, the size of the cells is measured in an optical image of all or part of multiple wavelength ranges, and then a polygonal or circular area that is larger than the measured cell size by a predetermined percentage or amount is set, and the fluorescence intensity of the cells within this area is measured to perform primary filtering.

[0072] In the tertiary filtering step, the morphology of circulating cancer cells in the blood is measured from an integrated image obtained by merging all or part of the optical images for each of the plurality of wavelength ranges, and then tertiary filtering is performed. Here, the morphology of circulating cancer cells in the blood may include one or more of cell area, cell size, and circularity.

[0073] The tertiary filtering can be performed on all circulating cancer cells in the blood, or can be performed as a supplement to identify circulating cancer cells that are difficult to identify in the primary and secondary filtering processes. If additional identification is required, separate data filtering or multiple tertiary filtering processes can be performed.

[0074] The image analysis may be performed by a computer program, which may be implemented using one or more general-purpose or special-purpose computers, such as a processor, controller, arithmetic logic unit (ALU), digital signal processor, microcomputer, field programmable gate array (FPGA), programmable logic unit (PLU), microprocessor, or any other device capable of executing and responding to instructions.

[0075] In the present invention, the fluorescent label that specifically binds to the immune checkpoint protein may be selected from the group consisting of, but is not limited to, an antibody specific to PD-1, an antibody specific to PD-L1, and an antibody specific to CTLA-4.

[0076] In the present invention, the fluorescent label that specifically binds to the lymphoid or myeloid protein may be selected from the group consisting of, but is not limited to, an antibody specific to CD18, an antibody specific to CD16, an antibody specific to CD29, an antibody specific to CD61, an antibody specific to CD104, an antibody specific to CD32, an antibody specific to CD11b, and an antibody specific to CD64.

[0077] In the present invention, the immune checkpoint inhibitor can be any substance that can inhibit the function of an immune checkpoint protein, and may be a protein, a compound, a natural product, DNA, RNA, a peptide, etc., preferably an antibody, more preferably a monoclonal antibody, and even more preferably a human antibody, a humanized antibody, or a chimeric antibody.

[0078] In the present invention, the immune checkpoint inhibitor may be characterized as being any one or more selected from the group consisting of a PD-L1 antagonist, a PD-1 antagonist, and a CTLA-4 antagonist, but is not limited thereto.

[0079] As used herein, the term "PD-1 antagonist" refers to a molecule that reduces, blocks, inhibits, abolishes, or prevents signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, e.g., PD-L1 and / or PD-L2. In some embodiments, a PD-1 antagonist is a molecule that inhibits the binding of PD-1 to its binding partner. In a specific embodiment, a PD-1 antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 antagonists include anti-PD-1 antibodies and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, small molecule antagonists, polynucleotide antagonists, and other molecules that reduce, block, inhibit, abolish, or prevent signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, the PD-1 antagonist reduces negative signals mediated by or through cell surface proteins expressed on T lymphocytes and other cells that mediate signaling through PD-1 or PD-L1, such that dysfunctional T-cells become non-dysfunctional. In some embodiments, the PD-1 antagonist is an anti-PD-1 antibody.

[0080] As used herein, the term "PD-L1 antagonist" refers to a molecule that reduces, blocks, inhibits, abolishes, or prevents signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, e.g., PD-1 or B7-1. In some embodiments, a PD-L1 antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific embodiment, a PD-L1 antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abolish, or prevent signal transduction resulting from the interaction of PD-L1 with its binding partners, e.g., PD-1 or B7-1. In one embodiment, the PD-L1 antagonist reduces negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that mediate signaling through PD-L1, such that dysfunctional T-cells become less dysfunctional (e.g., enhance effector responses to antigen recognition). In some embodiments, the PD-L1 antagonist is an anti-PD-L1 antibody.

[0081] As used herein, the term "CTLA4 antagonist" refers to a molecule that reduces, blocks, inhibits, abolishes, or prevents signal transduction via the interaction of CTLA4 with one or more of its binding partners, e.g., B7-1. In some embodiments, a CTLA4 antagonist is a molecule that inhibits the binding of CTLA4 to its binding partner. In a specific embodiment, a CTLA4 antagonist inhibits the binding of CTLA4 to B7-1. In some embodiments, CTLA4 antagonists include anti-CTLA4 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abolish, or prevent signal transduction via the interaction of CTLA4 with its binding partner, e.g., B7-1. In one embodiment, the CTLA4 antagonist reduces negative co-stimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that mediate signaling through CTLA4, such that dysfunctional T cells become non-dysfunctional (e.g., enhance effector responses to antigen recognition). In some embodiments, the CTLA4 antagonist is an anti-CTLA4 antibody.

[0082] In the present invention, the immune checkpoint inhibitor may be any one or more selected from the group consisting of pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Lybtayo), atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), ipilimumab (Yervoy), and tremelimumab, but is not limited to these.

[0083] In the present invention, the cancer may be selected from the group consisting of lung cancer, kidney cancer, bladder cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, gastric carcinoma, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis sarcoma, Merkel cell carcinoma, or hematological malignancies, preferably lung cancer, most preferably non-small cell lung cancer, but is not limited to these.

[0084] From another viewpoint, the present invention provides: Regarding cancer treatment methods, which include the following steps: (a) confirming the expression of immune checkpoint proteins and lymphoid or myeloid lineage-specific proteins from isolated circulating cancer cells; and (b) administering an immune checkpoint inhibitor when expression of the immune checkpoint protein is positive and expression of the lymphoid or myeloid-specific protein is negative. From another viewpoint, the present invention provides: A method for determining susceptibility to an immune checkpoint inhibitor, comprising the steps of: (a) confirming the expression of immune checkpoint proteins and lymphoid or myeloid lineage-specific proteins from isolated circulating cancer cells; and (b) determining that the cell is sensitive to an immune checkpoint inhibitor when the expression of the immune checkpoint protein is positive and the expression of the lymphoid or myeloid-specific protein is negative.

[0085] From another viewpoint, the present invention provides: Regarding the method for selecting an immune anti-cancer therapy for cancer patients, the method includes the following steps: (a) confirming the expression of immune checkpoint proteins and lymphoid or myeloid lineage-specific proteins from isolated circulating cancer cells; and (b) selecting an immune checkpoint inhibitor as a therapeutic therapy when the expression of the immune checkpoint protein is positive and the expression of the lymphoid or myeloid-specific protein is negative.

[0086] From another viewpoint, the present invention provides: The present invention relates to a pharmaceutical composition for treating cancer, comprising an immune checkpoint inhibitor, for treating an individual whose expression is positive for an immune checkpoint protein and whose expression is negative for a lymphoid or myeloid lineage-specific protein.

[0087] From another viewpoint, the present invention provides: The present invention relates to the use of an immune checkpoint inhibitor for the manufacture of a therapeutic agent for treating an individual who is positive for the expression of an immune checkpoint protein and negative for the expression of a lymphoid or myeloid lineage-specific protein. [Example]

[0088] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0089] statistical analysis Patient-derived data were used to compare baseline and demographic characteristics. All values ​​were expressed as mean ± standard deviation or percentage. Continuous variables were compared using a T-test, and categorical variables were compared using a Chi-square test. Accuracy and weighted kappa were used to compare PD-L1 positivity between tissue and blood samples. Bland-Altman plots were used to analyze the concordance of PD-L1 expression as a continuous variable between CTCs and tissue samples. Pearson correlation coefficients were used to analyze the relationship between tumor burden and CTC count. Baseline clinical tumor burden was measured using the sum of the longest diameters of target lesions (maximum of 2 per organ and total of 5) according to the Response Evaluation Criteria in Solid Tumors in CT version 1.1. A p-value of less than 0.05 was considered statistically significant. Statistical analyses were performed using IBM SPSS version 25.0 (IBM Corp., Armonk, NY, USA).

[0090] Example 1. Blood CTC isolation This study was approved by the Institutional Research Board (IRB), and tissue and blood samples from 29 lung cancer patients (Table 1) and 30 inflammation patients (Table 2) were used with prior consent.

[0091] [Table 1]

[0092] [Table 2] The basic information of the patients is summarized in Table 3.

[0093] [Table 3] In addition, the molecular diagnostic information related to PD-L1 expression obtained during recruitment of the lung cancer patient group is summarized in Table 4.

[0094] [Table 4]

[0095] SmartBiopsy from the patient's blood TM Cell Isolation kit (Cytogen) and SmartBiopsy TM CTCs were isolated using a cell isolator (Cytogen) according to the manufacturer's instructions. TM The high-density microchip used in the cell isolator uses a chip with a pore size of 5 μm, allowing target cells larger than 5 μm to be collected on the chip. The pore size of the microchip can be adjusted, making it possible to selectively collect cells of a specific size (US 10,502,668, KR10-1254675).

[0096] Smart Biopsy TM Cell Isolator (CIS030) - Smart Biopsy TM The Cell Isolator is a device that recognizes the height of the target object (peripheral blood mononuclear cell, PBMC) from the solution and automatically mixes, transports, and filters it using an ADP (Air Displacement Pipette). The system of this device consists of an XY Main Axis, ADP, Chip Handler, and Consumable Table. Basically, Smart Biopsy TM The Cell Isolator is driven using software according to manual procedures.

[0097] Example 2. Marker selection for false-positive filtering of isolated CTCs 2-1. CTC staining method Smart Biopsy TMIF Stainer (CST030) - This automated immunostaining equipment can stain a total of 12 slides simultaneously. The system consists of an XY main axis module, a 1ml pipette (ADP) module, a covering module, a consumable parts module, a vision parts module, and a consumable table. Smart Biopsy TM The Cell Isolator uses in-house developed software and is operated according to the manual. Staining method for circulating tumor cells

[0098] [Smart Biopsy TM IF Stainer method] 1. Smart Biopsy TM Place the slide with fixed cells and the necessary antibodies (e.g., EpCAM, Vimentin, PD-L1, CD18, CD45 antibodies) into the buffer tube in the IF Stainer equipment, and operate according to the equipment manual. 2. The processes of washing, blocking, primary antibody / secondary antibody staining, and mounting are performed automatically, and take a total of 6 hours (based on 12 slides). 3. Smart Biopsy TM All staining processes using the IF Stainer are performed according to the following [Manual Method].

[0099] [Manual method] 1. Treat the sample (slide) with 50 μl of 0.2% Triton X-100 for 10 minutes, then wash with PBS three times for 5 minutes. 2. To reduce non-specific binding, proceed with blocking using 1% BSA for 1 hour. 3. Dilute the necessary antibodies (e.g., anti-CD45 for negative selection of immune cells, anti-CD18 for negative selection of lymphoid cells, and PD-L1 antibody for positive selection) in 1% BSA and treat for 1 hour (primary antibody treatment).

[0100] 4. Dilute two secondary antibodies with different wavelengths ('goat anti-mouse Alexa Fluor 647', 'goat anti-rabbit Alexa Fluor 546') in 1% BSA and store at room temperature for 1 hour. (Secondary antibody treatment) 5. To inhibit cross-reaction with lung cancer circulating tumor cell-specific antibodies (PD-L1), a blocking process is performed for 1 hour. 6. Store the circulating tumor cell marker "Anti-EpCAM Alexa Fluor 488 conjugated" antibody at room temperature for 1 hour. 7. Wash with PBS three times for 5 minutes. 8. After DAPI staining, proceed with the mounting process using a cover glass.

[0101] 2-2. Marker selection for CTC filtering To more clearly identify CTCs in the blood, lymphoid lineage cells were removed using CD45, a known marker for lymphoid lineage cells. In addition, to remove the remaining false-positive cells (myeloid lineage) after excluding CD45-positive cells, the expression levels of CD18 and CD16, known as specific markers, and CD18 and CD11b were confirmed using immunofluorescence staining (IF) in inflammatory cell lines.

[0102] The cell lines used were Jurkat (KCLB 40152) human T lymphocyte cells, THP-1 (KCLB 40202) human monocytic cells, and KG-1 (KCLB 10246) human myeloblast cells. Antibody information for each antigen is shown in Table 5 below. Samples with the CD18 / CD16 and CD18 / CD11b combinations were stained and confirmed on 10 slides each.

[0103] [Table 5]

[0104] The secondary antibodies used were Thermo Fisher's anti-rabbit IgG, Alexa Fluor 546 (A-11010 / 2 mg / mL) and anti-mouse IgG, Alexa Fluor 546 (A-11003 / 2 mg / mL).

[0105] As a result, as shown in Figures 3 and 4, it was confirmed that CD18 is not only more highly expressed than CD16 and CD11b, but also important as a specific marker. Furthermore, the same experiment was repeated using PBMCs from the patient's blood, and as a result, it was confirmed that the expression level of CD18 was higher, as shown in Figure 5. Therefore, CD18 was selected as a marker for CTC filtering together with CD45 and used to remove false positive cells.

[0106] Example 3. Selected marker-based CTC staining The cells (approximately 500 to 10,000 or more) isolated by the isolator were subjected to a cytospin process, which is a cell centrifugation method, and fixed on a slide for staining.

[0107] The isolated CTCs were characterized using CD45 and CD18 markers for negative selection of immune cells and EpCAM / Vimentin and PD-L1 markers for positive selection of CTCs.

[0108] The antibodies used were EpCAM / Vimentin, PD-L1, CD18, and CD45. The antibody information is shown in Table 6 below. Finally, DAPI (4',6-diamidino-2-phenylindole), which can stain cell nuclei, was added and analyzed using SmartBiopsy. TM Cell staining proceeded using IF Stainer (CST030, Cytogen) according to the manufacturer's instructions.

[0109] [Table 6]

[0110] Example 4. CTC fluorescence image analysis and confirmation of CTC isolation accuracy SmartBiopsy TM Images of slides loaded with fluorescently labeled CTCs were taken using a Cell Image Analyzer (Cytogen), and the number and fluorescence intensity of EpCAM / Vimentin, PD-L1, and CD18-expressing cells were measured (Figures 1 and 2).

[0111] We examined the expression of CTC markers and PD-L1 using blood samples from patients with inflammation and lung cancer, and found that there were false positive cells expressing CTC markers and PD-L1 (Figures 1 and 2).

[0112] Such false-positive cells are thought to be myeloid cells expressing CD16 / 18. Therefore, in the present invention, false-positive cells were eliminated by using the CD16 / 18 marker as a negative marker or a leukocyte-specific selective marker. Therefore, CD18(-) and PD-L1(+) cells were defined as pure PD-L1 expressing cells with false positives removed.

[0113] Furthermore, CD18 / CD45-negative CTCs were isolated from 21 of 29 lung cancer patients and 4 of 30 infected patients. As shown in Table 7, the sensitivity of CTC isolation for lung cancer diagnosis was confirmed to be 72.4% and the specificity was 86.7%.

[0114] [Table 7]

[0115] Example 5. Correlation analysis of PD-L1 expression levels between CTCs and tissue samples The results of PD-L1 expression analysis in tissue samples are shown in Table 4. Patients with PD-L1-positive CTCs selected from PBMCs of 29 lung cancer patients according to the criteria in Example 4 were identified.

[0116] As a result, as shown in Table 8, it was confirmed that 14 out of 29 patients showed positive results in all tissue samples and blood samples, and 7 patients showed negative results in all samples.

[0117] [Table 8]

[0118] Therefore, it was confirmed that the expression of PD-L1 in tumor tissue can be predicted based on the number of PD-L1-positive CTCs in the blood, with an accuracy of 72.4% and a sensitivity of 82.4%.

[0119] In addition, for 21 patients with CTCs in their blood, the circulating tumor proportion score (CTPS) of PD-L1 was calculated using the following formula 1, and the tumor proportion score (TPS) was calculated using the following formula 2 and compared.

[0120]

number

[0121]

number

[0122] As a result, as shown in Figure 6, it was confirmed that the higher the TPS value, the higher the CTPS value, and when the TPS was negative, the CTPS value was relatively low. Example 6. Tumor burden prediction using CTPS

[0123] When tumor size and CTPS values ​​were compared in tissue samples from lung cancer patients, a Pearson correlation coefficient of 0.445 was obtained, as shown in Figure 7, confirming a positive correlation between the two comparison groups.

[0124] Although certain aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined by the appended claims and their equivalents. [Industrial Applicability]

[0125] The method for treating cancer using circulating tumor cells according to the present invention is highly accurate and commercially viable, as it simultaneously confirms the expression of immune checkpoint proteins and lymphoid or myeloid cell-specific proteins in a liquid biopsy, and then selects patients by removing false-positive circulating tumor cells. Therefore, the method of the present invention is useful for the diagnosis and treatment of cancer.

Claims

1. A method for selecting patients for the application of immunotherapy to anticancer drugs, comprising the following steps: a) detecting the expression of PD-L1 protein and CD18 protein in circulating cancer cells; and b) Classifying patients who are confirmed to have positive PD-L1 protein expression and negative CD18 protein expression as patients eligible for immunotherapy.

2. The method of claim 1, wherein the isolated circulating cancer cells are isolated by performing the following steps: (i) Isolating circulating cancer cells from a blood sample from a cancer patient using a biochip.

3. The method of claim 1, wherein the expression of PD-L1 protein and CD18 protein is confirmed by performing the following steps: (1) reacting circulating cancer cells with a fluorescent label that specifically binds to circulating cancer cells; a fluorescent label that specifically binds to PD-L1 protein; and a fluorescent label that specifically binds to CD18 protein; (2) receiving optical images for each of a plurality of wavelength ranges of the circulating cancer cells reacted with the fluorescent label; the PD-L1 protein; and the CD18 protein; (3) measuring the fluorescence intensities of the cancer cells, PD-L1 protein, and CD18 protein from the optical images of all or part of the multiple wavelength ranges, and performing primary filtering; (4) measuring the morphology of the cancer cells from the optical images of all or part of the multiple wavelength ranges and performing secondary filtering; and (5) A step of measuring the morphology of the circulating cancer cells in the blood from an integrated image obtained by merging all or part of the optical images for each of the multiple wavelength ranges, and performing third-order filtering.

4. The method of claim 3, wherein the fluorescent label that specifically binds to circulating cancer cells in the blood is selected from the group consisting of an antibody specific to vimentin, an antibody specific to EpCAM, and an antibody specific to CK (cytokeratin).

5. 4. The method of claim 3, wherein the fluorescent label that specifically binds to PD-L1 protein is an antibody specific to PD-L1.

6. The method according to claim 3, wherein the fluorescent label that specifically binds to the CD18 protein is an antibody specific to CD18.

7. The method according to claim 1, wherein the immunological anticancer agent is an antibody.

8. The method according to claim 1, wherein the immunological anticancer agent is a monoclonal antibody.

9. The method of claim 1, wherein the immunoanticancer agent is a human antibody, a humanized antibody, or a chimeric antibody.

10. The method according to claim 1, wherein the immunological anticancer agent is at least one selected from the group consisting of a PD-L1 antagonist, a PD-1 antagonist, and a CTLA-4 antagonist.

11. 2. The method of claim 1, wherein the anticancer immune agent is any one or more selected from the group consisting of pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Lybtayo), atezolizumab (Tecentriq), avelumab (Bavencio), durvalumab (Imfinzi), ipilimumab (Yervoy), and tremelimumab.

12. 2. The method of claim 1, wherein the cancer is selected from the group consisting of lung cancer, kidney cancer, bladder cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, gastric carcinoma, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycosis sarcoma, Merkel cell carcinoma, or hematological malignancies.

13. 13. The method of claim 12, wherein the cancer is lung cancer.

14. 13. The method of claim 12, wherein the cancer is non-small cell lung cancer.

15. A pharmaceutical composition for treating cancer, comprising an immune checkpoint inhibitor, for treating an individual whose expression of PD-L1 protein is positive and whose expression of CD18 protein is negative.

16. An in vitro method for determining susceptibility to an immune checkpoint inhibitor, comprising the steps of: (a) confirming the expression of PD-L1 protein and CD18 protein in isolated circulating cancer cells; and (b) determining that the isolated circulating cancer cells are sensitive to an immune checkpoint inhibitor if PD-L1 protein expression is positive and CD18 protein expression is negative.

Images