Immunotherapy sensitivity test

Abstract

The present invention relates to the immunotherapy sensitivity test for the prediction of anti-PD-L1 treatment resistance in non-small cell lung cancer.

Description

[0001] IMMUNOTHERAPY SENSITIVITY TEST

[0002] Technical Field

[0003] The invention relates to the immunotherapy sensitivity test for the prediction of anti-PD-L1 treatment resistance in non-small cell lung cancer.

[0004] State of the Art

[0005] As well as many existing or ongoing studies on cancer treatment, there is currently a developing field of predicting treatment efficacy. Since each cancer has unique characteristics like every human being, pre-determination of individualized treatment profiling has positive effects on both economic and patient comfort by preventing unnecessary treatment. In current applications, tumor samples taken from cancer patients are grown under appropriate conditions in the laboratory. Chemotherapy drugs to be administered in the clinic are tested on these cells and drug effectiveness is evaluated. In this way, the results of the treatment the patient will receive are simulated in the laboratory environment (Kim vd., 2008; Lehnhardt vd., 2005; Zhang ve Li, 2015). These tests are basically based on the same principle but have commercial use according to different application methods. Chemotherapy resistance test "CTR-Test, The Chemotherapy- Resistance Test" performed on patient biopsy samples or "ZTX ONCOLEADS" performed in in- vivo models constitute commercially available examples.

[0006] Recently, the search for new therapeutic strategies or agents to improve the survival rate in cancer patients has reached a promising point where immunotherapy, i.e. immune checkpoint blockade therapy, has shown successful and exciting clinical benefits. For immunotherapy in patients, the immune checkpoint inhibitor is designed to target an inhibitory immune checkpoint molecule such as the PD-L1 receptor, PD-1 or cytotoxic T-lymphocyte-associated antigen. Two antibodies against PD-1 (nivolumab and pembrolizumab) and two antibodies against PD-L1 (atezolizumab and durvalumab) have been approved by the FDA and / or the European Medicines Agency (EMA) for the treatment of NSCLC. Although immune checkpoint inhibitors are highly effective compared to classical chemotherapy, the fact that they are a high-budget treatment option and that some patient groups show resistance to treatment constitute the biggest difficulties in their application. Since the action strategy of immunotherapy drugs is based on T-lymphocytes recognizing and killing tumor cells, classical chemotherapy resistance tests do not apply to immunotherapy drugs. At this point, complex tests based on joint analyses of T-cells and tumor cells are needed to predict the therapeutic efficacy of immune checkpoint inhibitors.

[0007] As a result, due to the abovementioned disadvantages and the insufficiency of the current solutions regarding the subject matter, a development is required to be made in the relevant technical field.

[0008] Brief Description of the Invention

[0009] The present invention relates to an immunotherapy sensitivity test which fulfills the abovementioned requirements, eliminates all disadvantages and brings some additional advantages.

[0010] The present invention aims to solve the abovementioned disadvantages by being inspired from the current conditions.

[0011] The main object of the invention is to develop a complex test for the preliminary determination of Atezolizumab treatment effectiveness, one of the immune checkpoint inhibitors. The invention subject to the application has the feature and capacity to fill the gap in the state of the art. NSCLC cancer tumor and blood samples are co-cultured under laboratory conditions. And responses to immune checkpoint inhibitors Atezolizumab will be evaluated, and clinical treatment will be guided.

[0012] Together with the invention, the following are possible:

[0013] • Preventing unnecessary treatment by predicting the clinical response of atezolizumab (anti-PD-L1) treatment,

[0014] • Reducing the economic pressure created by cancer treatment expenditures on the healthcare system by preventing the unnecessary use of high-budget drugs such as atezolizumab,

[0015] • Since the tumor structure of each cancer is unique, ensuring individual evaluation of immunotherapy effectiveness.

[0016] The test of the invention is a test for the preliminary determination of the activity of Atezolizumab (anti-PD-L1), an immunotherapy drug, in NSCLC tumors. For testing, cancer cells obtained from tumor samples are grown in 3D culture conditions in laboratory conditions. Additionally, PBMC is isolated from blood samples. T-lymphocytes and cancer cells are placed in the same environment and Atezolizumab drugs are added to the medium to create a common culture environment. Finally, it is tested whether Atezolizumab added to the co-culture medium inhibits PD-L1 receptors and enables T-cells in PBMC to recognize and kill cancer cells. In addition, the results obtained are evaluated with statistical approaches to determine the effectiveness of the analysis.

[0017] The structural and characteristic features of the present invention will be understood clearly by the following drawings and the detailed description made with reference to these drawings and therefore the evaluation shall be made by taking these figures and the detailed description into consideration.

[0018] Figures to Help Understanding the Invention

[0019] Figure 1 shows the changing cell viability after Atezolizumab application. * Indicates statistically significant differences in between-group comparisons:* (p < 0.05), ** (p< 0.01 ) and *** (p < 0.001 ). Data are given as mean ± SD (n=3).

[0020] Figure 2 shows the altered caspase-3 / 7 activation after atezolizumab administration. * Indicates statistically significant differences in the comparison between groups: * (p < 0.05), ** (p< 0.01) and *** (p < 0.001 ). Data are given as mean ± SD (n=3).

[0021] Figure 3 shows the changing IFN-5 levels after Atezolizumab administration. * Indicates statistically significant differences in the comparison between groups: * (p < 0.05), ** (p< 0.01) and *** (p < 0.001 ). Data are given as mean ± SD (n=3).

[0022] Figure 4 shows the changed T cell proliferations after Atezolizumab administration. * Indicates statistically significant differences in the comparison between groups: * (p < 0.05), ** (p< 0.01) and *** (p < 0.001 ). Data are given as mean ± SD (n=3).

[0023] Figure 5 shows the steps of statistical evaluation in outline.

[0024] Figure 6 shows the statistical evaluation 1.

[0025] Figure 7 shows the statistical evaluation 2.

[0026] Figure 8 shows the statistical evaluation 3. Figure 9 shows the statistical evaluation 4.

[0027] Figure 10 shows the statistical evaluation 5.

[0028] Figure 11 shows the statistical evaluation 6.

[0029] Detailed Description of the Invention

[0030] In this detailed description, the preferred embodiments of the present invention are described by means of examples only for clarifying the subject matter.

[0031] The present invention relates to the immunotherapy sensitivity test for the prediction of anti-PD- L1 treatment resistance in non-small cell lung cancer. In the invention, PBMC is first isolated from blood (peripheral blood). Secondly, as a result of enzymatic digestion of tumor samples, single cell suspensions are made ready for culture. It is grown on membrane matrix-covered surfaces in a 3D culture environment (ex-vivo organoid culture) to best characterize the tumor structure. Cancer cells grown in a 3D culture environment with PBMC cells are cultured simultaneously in the same environment (Co-Culture). Atezolizumab (anti-PD-L1) is added to the co-culture medium at different concentrations to evaluate the T cell-mediated response. After atezolizumab (anti-PD-L1 ) treatment, cellular analyzes are performed in 4 stages. Said analyzes are as follows; T-Cell-Mediated Cell Death Analysis, T-Cells-Mediated Apoptosis Analysis, Determination of IFN- 6 Levels and T-Cell Proliferation Levels. After four-stage analysis, data that can predict the clinical response after Atezolizumab (anti-PD-L1 ) treatment are obtained. In the final processing step, the discriminatory power of the results is increased by integrating the data obtained after cellular analysis with basic statistical approaches and algorithms.

[0032] Isolation of PBMC (Peripheral Mononuclear Cells) from Peripheral Blood

[0033] Mononuclear cell isolation is performed from peripheral blood using Ficoll (1.077 g / ml) from blood samples. The separated cells are used in the relevant co-culture experiments.

[0034] Ex-Vivo Organoid Culture

[0035] Tumor samples are turned into single cell suspension after enzymatic digestion. And then, 3D culture is performed on membrane matrix-covered surfaces in order to best characterize the tumor structure.

[0036] Primary NSCLC Cells and T Cell Co-Culture PBMCs containing isolated T cells and cancer cells grown in a 3D culture environment are cultured simultaneously in the same environment (Co-Culture). At this stage, culturing tumor cells and PBMCs (T cells) in the same environment best simulates the tumor environment within the body. In this way, the drug response obtained by adding different concentrations of Atezolizumab (anti-PD-L1) to the co-culture medium can predict the therapeutic response with high clinical relevance.

[0037] Cellular Analysis

[0038] Atelozumab are monoclonal antibodies that bind to the PD-L1 ligand on tumor cells and immune cells. In this way, it blocks the PD-1 / PD-L1 interaction and promotes T cell-mediated tumor death through the formation of an immune response. Therefore, after adding Atezolizumab (anti-PD-L1) to the co-culture medium described in Element 4, treatment response can be evaluated as a result of a 4-stage series of analyses.

[0039] 1. T-Cell Mediated Cell Death Analysis

[0040] Since ATP is an indicator of metabolically active cells, the number of living cells can be evaluated based on the amount of ATP present. At this point, the ATP cell viability test is a highly sensitive homogeneous test widely used in cell culture experiments to estimate the number of viable cells in laboratories. The basic operating principle of the method exploits the ATP utilization of the luciferin-luciferase complex and the resulting light production to assess the amount of ATP present in cell cultures.

[0041] T cell-mediated tumor cell death will be determined by ATP cell viability analysis.

[0042] 2. T-Cells-Mediated Apoptosis Analysis

[0043] T cells, through the FAS receptor or various signaling molecules (perforins and granzymes) they secrete, cause caspase activation in cancer cells and consequently induction of apoptotic cell death. However, PD-L1 binding to PD-1 transmits an inhibitory signal. Upregulation of PD-L1 in cancer cells allows cancers to evade the host immune system. In element 4, Atezolizumab (anti- PD-L1) added to primary NSCLC cells and T cell co-culture medium inhibits PD-L1 / PD-1 binding and induces T cell-mediated apoptotic cell death. T umor cells in the culture medium are first lysed and the substrate added to the cell lysate is broken down by caspases and aminoluciferin is released. The amount of luminescence proportional to caspase 3 / 7 activity in the cells is measured as RLU (Relative Light Unit) unit with a luminometer reader. A number of cytokines have also been proposed as factors that induce or sensitize cells to apoptosis. Among these cytokines, IFN-y is produced by many immune cells in the tumor microenvironment. Natural killer cells (NK Cells) and CD8+T lymphocytes are the two main producers of IFN-y in the tumor microenvironment. Additionally, CD4+T lymphocytes secrete IFN- y to tumor sites, which supports the activation of lymphocytes involved in innate and adaptive immunity. IFN-y effects have broad anti-tumor effects. IFN-y exerts direct anticancer activity through inhibition of cell proliferation, for example through upregulation of p21 and p27 molecules to arrest the cell cycle, or through mediation of apoptotic cell death. In addition, interferon gamma released by T-cells stimulates the expression of tumor antigen-presenting MHC molecules to increase the immunogenicity of tumor cells, making them more susceptible to immune recognition and destruction. For this reason, the levels of IFN-6, which is also a marker of T cell-induced cell death, are taken from the co-culture medium described in Element 4 and measured using elisa microplates with added IFN-6 antibodies.

[0044] 4. T Cell Proliferation Levels

[0045] The immune response to cancer develops over many years, and in the case of progressive and metastatic cancer, treatment fails. Numerous factors may contribute to this failure, but one key factor is that the responses of effector T cells may gradually weaken after prolonged antigenic exposure. This phenomenon is called T-cell exhaustion and is characterized by decreased production of IFN-y and reduced cancer cell reactivity. These observations, among others, ultimately led to the development of PD-1 / PD-L1 pathway blockade as a therapeutic anti-cancer strategy. At this stage, CD4+or CD8+T Cells will be marked with CellTrace CFSE (Carboxyfluorescein succinimidyl ester) Cell Proliferation Kit (Excitation / Emission: 492*517) and co-cultured to prevent T cell exhaustion due to Atezolizumab treatment resistance. In this way, the proliferation of T cells in the presence or absence of Atezolizumab will be evaluated simultaneously on the flow cytometry device.

[0046] Statistical Evaluation

[0047] Statistical evaluation is a multi-step evaluation process that includes T cell-mediated cell death, Apoptosis and IFN-6 analyses, followed by CD8+T cell proliferation levels. This evaluation process is carried out with the Co-Culture + Atezolizumab group (D) and Co-culture (C) experimental groups (Figure 1). T-test and Anova approaches are used for the statistical significance of the data obtained as a result of the analyzes performed in Elements 1 , 2 and 3 compared to the control group. The algorithm processor according to statistical significance levels is given in Figure 5. Explanations regarding the analysis results regarding the functionality of the invention are given below.

[0048] 1. T-Cell Mediated Cell Death Analysis

[0049] Cell viability was calculated with RLU (relative luminescence units) values obtained from the luminometer reader using the formula %Cell Viability= [(RLU Sample - RLU Blind) / ( RLU Control - RLU Blind)]*100. Since PD-L1 receptor blockade by atezolizumab makes the cancer cell vulnerable to T cells, determining cell death is important for treatment response. As a result of Atezolizumab application to the co-culture medium, cell viability was measured as <75% in 28 samples. The fact that cell viability was measured as >75% in 18 samples is explained by the high cell viability of this sample group despite the application of Atezolizumab and its resistance to T cell cytotoxicity.

[0050] 2. T-Cells-Mediated Apoptosis Analysis

[0051] Cell viability was calculated with RLU (relative luminescence units) values obtained from the luminometer reader using the formula %Caspase Activity= [(RLU Sample - RLU Blind) / ( RLU Control - RLU Blind)]*100. Apoptosis-induced T cell-induced cytotoxicity results in increased caspase activation parallel to increased cell death. As a result of Atezolizumab application to the Co-culture group, an increase in caspase 3 / 7 activation (p < 0.05) was observed in 30 samples compared to the tumor cells and the Co-culture group. In 5 samples (37, 39, 42, 43, 44), the increase in activation observed in Co-culture decreases in the Co-culture + Atezolizumab group (p < 0.05). This may be an indication that resistance to treatment may develop later in the presence of atezolizumab.

[0052] 3. Determination of IF N-5 Levels

[0053] In 38 samples, it was determined that in the Co-culture + Atezolizumab group, IFN-6 levels (pg / ml) increased compared to tumor cells (p < 0.05). In 20 samples, it was observed that IFN-6 levels increased in the Co-culture + Atezolizumab group compared to the Co-culture group (p < 0.05).

[0054] 4. T Cell Proliferation Levels

[0055] 5. Statistical Evaluation

[0056] Statistical evaluation is a multi-step evaluation process that includes T cell-mediated cell death, Apoptosis and IFN-6 analyses, followed by CD8+T cell proliferation levels. This evaluation process is carried out with the Co-Culture + Atezolizumab group (D) and Co-culture (C) experimental groups (Figure 5). Tumor cells are generally eliminated after sequential interactions of multiple cytotoxic T lymphocytes. Individual cytotoxic T lymphocyte contacts can cause varying levels of cellular damage until apoptosis is induced or target cell recovery is achieved. Additionally, depending on the characteristics of the cell population, immune checkpoint receptor interactions result in T cell cytotoxicity.

[0057] The decrease in cell viability in the Co-Culture group (p<0.05) can be explained in this context and a T cell-mediated cytotoxic effect in the culture medium can be mentioned. T cell-mediated cytotoxicity is expected to increase as atezolizumab disrupts PD-L1 and PD-1 immune checkpoint receptor interactions. Therefore, the increase in cell viability or the observation of statistically insignificant decreases in the Co-Culture + Atezolizumab group (D) compared to the Co-culture (C) group is explained by the lack of Atezolizumab effect and therefore anti-PD-L1 treatment resistance (Figure 6). Examples 3, 13, 15, and 20-45 were examined in this group.

[0058] On the other hand, due to the decrease in T cell death, a decrease or no change in apoptotic activity may be observed in rare cases. This situation brings to mind the activation of different cell death mechanisms other than apoptosis, and the indication that cell death is T cell-derived is explained by IFN-6 levels at this point. Because even if the tumor reactivity of cytotoxic T cells occurs through a different death pathway, changes in the levels of IFN-6 are expected as a result of the reactivity.

[0059] In the Co-Culture + Atezolizumab group (D) compared to the Co-culture (C) group; DSC in T cell-mediated cell death (p<0.05) T cell-mediated apoptosis DSC IFN-6 levels DSC or statistically insignificant increases did not show any T cell markers (increased IFN-6 levels or apoptotic activity) that would correspond to cell death, so these samples are classified in the group with atezolizumab treatment resistance (Figure 7).

[0060] If an increase in IFN-6 levels (D>C, p<0.05) is observed, cytotoxic T cell (CD8+) proliferation levels are decisive. Because the increase in T cell proliferation (D>C, p<0.05) due to increased IFN-6 levels, i.e. the observation of reactivity against the tumor (reactivity in this case is the decrease in Co-Culture cell viability as a result of Atezolizumab administration) results in Atezolizumab response (Figure 8). Within 45 samples, no decrease in apoptotic activity was observed in the case of statistically significant T cell cytotoxicity, so the algorithm described in Figures 7 and 8 was not applied. In another case, the Co-Culture + Atezolizumab group (D) was higher than the Co-culture (C) group;

[0061] > DSC in T cell-mediated cell death (p<0.05)

[0062] > T cell-mediated apoptosis DSC (p>0.05) If IFN-6 levels are DSC or D>C (p>0.05)

[0063] Cytotoxic T cell (CD8+) proliferation levels are decisive. Because an increase in T cell proliferation (DSC, p<0.05) can explain T cell reactivity. And these samples were examined in the group responding to atezolizumab treatment (Figure 9). This scenario was not encountered among 45 examples. Atezolizumab resistance is present when a decrease in CD8+T cell proliferation is observed (D<C) or D>C (p>0.05) (Figure 9). Samples 1 and 2 were examined in this group.

[0064] In another case, the Co-Culture + Atezolizumab group (D) was higher than the Co-culture (C) group;

[0065] > DSC in T cell-mediated cell death (p<0.05)

[0066] > T cell-mediated apoptosis D>C (p>0.05)

[0067] > IFN-6 levels D>C T cell proliferation DSC or D<C (p>0.05)

[0068] There is a response to atezolizumab administration. Because increased cytotoxic CD8+ T cell proliferation is the result of atezolizumab-induced T cell tumor contacts and consequently tumor reactivity due to decreased cell viability proportional to increased IFN-6 levels. Samples 10, 11 and 12 were examined in this group (Figure 10).

[0069] In the same scenario, if T cell proliferation is D<C (p<0.05), there is a response to Atezolizumab application in the sample groups. Because the increase in apoptotic activity and IFN-6 levels parallel to the decrease in cell viability is a result of cytotoxic CD8 + T cell reactivity. In this case, the decrease in CD8+T cell proliferation is explained by the T cell depletion observed as a result of tumor reactivity. Samples 14, 16, 17, 18 and 19 were examined in this group (Figure 10).

[0070] In another case, the Co-Culture + Atezolizumab group (D) was higher than the Co-culture (C) group;

[0071] > D<C in T cell-mediated cell death (p>0.05)

[0072] > T cell-mediated apoptosis D<C (p>0.05)

[0073] > IFN-6 levels D>C (p<0.05)

[0074] Cytotoxic T cell (CD8+) proliferation levels are decisive. In case of T cell proliferation D<C (p<0.05), there is resistance to Atezolizumab administration (Figure 11). Samples 4, 5, 6, 7, 8, 9 are classified in this group. When these samples are examined, although the T cell-mediated cytotoxicity results after Atezolizumab application to the co-culture are statistically significant, there is an average decrease of 8.4% in cell viability. At this point, the decrease in CD8+T cell proliferation confirms the low cytotoxic effect. And it also shows that the selectivity of the algorithm is strong. In case of T cell proliferation D<C (p>0.05) or DSC, there is a response to Atezolizumab administration. The increase in IFN-6 levels (p<0.05) occurs due to increased cytotoxic CD8+ T cell proliferation and the process results in T cell cytotoxicity (Figure 11).

[0075] REFERENCES

[0076] Kim, H. A., Yom, C. K., Moon, B. I., Choe, K. J., Sung, S. H., Han, W. S., ... Oh, S. Y. 2008. "The use of an in vitro adenosine triphosphate-based chemotherapy response assay to predict chemotherapeutic response in breast cancer". Breast, 17(1), 19-26.

[0077] Lehnhardt, M., Muehlberger, T., Kuhnen, C., Brett, D., Steinau, H. U., Jafari, H. J., ... Homann, H. H. 2005. "Feasibility of chemosensitivity testing in soft tissue sarcomas". World Journal of Surgical Oncology, 3. Zhang, J., Li, H. 2015. "Heterogeneity of tumor chemosensitivity in ovarian epithelial cancer revealed using the adenosine triphosphate-tumor chemosensitivity assay". Oncology Letters, 9(5), 2374-2380.

Claims

CLAIMS1. Immunotherapy sensitive test method for preliminary determination of anti-PD-L1 treatment resistance, characterized by comprising following process steps:• Preparing tumor samples for culture• Isolation of PBMC from blood,• Growing cells obtained as a result of PBMC isolation in a 3D culture environment on membrane matrix coated surfaces,• Simultaneous culture (Co-Culture) of cancer cells grown in a 3D culture environment with PBMC cells in same environment,• Evaluation of T cell-mediated response by adding different concentrations of Atezolizumab to the co-culture medium,• Performing cellular analyzes after atezolizumab treatment,• Evaluation of results.

2. The method according to claim 1 , characterized in that said cellular analyses are T-Cell Mediated Cell Death Analysis, T-Cell Mediated Apoptosis Analysis, Determination of IFN- 3 Levels and T-Cell Proliferation Levels analyses.

Images