ANTXR1 as a biomarker for immunosuppressive fibroblast populations and its use to predict response to immunotherapy

Abstract

The present invention provides an in vitro method for detecting immunosuppressive fibroblasts, particularly cancer-associated fibroblasts (CAFs), in a cancer sample from a subject suffering from cancer, the method comprising: detecting ANTXR1 in the cancer sample from the patient; + The present invention also relates to an in vitro method for predicting the response of a subject suffering from cancer to an immunotherapy treatment, an immunotherapy treatment for use in treating cancer in a patient, the use of ANTXR1 as a biomarker for identifying immunosuppressive fibroblasts, particularly immunosuppressive CAFs, and a method for detecting ANTXR1 for use in treating cancer in a patient. + Fibroblast-targeting drugs, a) ANTXR1 + and b) an immunotherapy treatment.

Description

[Technical Field]

[0001] This invention relates to the field of medicine, particularly to the field of oncology. It provides a novel marker for immunosuppressive cell populations and its use. [Background technology]

[0002] In 2018, 9.6 million people died from cancer, making it the second leading cause of death worldwide. With over 15 million new cases diagnosed annually, the prevalence of cancer is extremely high, and the number of new cases is projected to increase by approximately 70% over the next 20 years.

[0003] Currently, there are many treatment options for cancer, including surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy, and palliative care. The best treatment choice for a patient depends on the type, location, and stage of cancer, as well as the patient's health condition and preferences.

[0004] Over the past few decades, immunotherapy has become a crucial part of cancer treatment strategies. Cancer immunotherapy relies on using the immune system to treat cancer. Among the diverse range of immunotherapies developed over time, immune checkpoint inhibitor therapy is particularly promising. However, despite promising results, many patients with advanced cancer do not respond to immune checkpoint inhibitors, and little is known about the mechanisms of primary resistance. In particular, some cancers develop an immunosuppressive microenvironment that contributes to the development of resistance to immunotherapy.

[0005] Therefore, identifying biomarkers that can reliably distinguish between responder and non-responder patients before initiating treatment is necessary to select patients who are likely to benefit from immuno-oncological drugs.

[0006] Cancer-associated fibroblasts (CAFs) are abundant components of cancer and play a significant pro-tumorigenic role. CAFs are heterogeneous, and it is now recognized that different CAF subsets can be defined based on the expression of specific markers. To date, four CAF subsets, called CAF-S1 to CAF-S4, have been identified in human breast and ovarian cancer (Costa A et al., Cancer Cell 2018;33(3):463-79 e10). CAF-S2 and CAF-S3 fibroblasts are also detected in healthy tissue and may resemble normal fibroblasts, while CAF-S1 and CAF-S4 myofibroblasts are limited to cancer and metastatic lymph nodes. Both CAF-S1 and CAF-S4 promote metastasis through complementary mechanisms. In contrast, CAF-S1 promotes immunosuppression in human cancer, while CAF-S4 does not. Because the subpopulation of CAFs remains heterogeneous, it is necessary to identify CAFs that play a specific role in primary resistance to immunotherapy in cancer patients, and in particular, to identify novel markers for detecting immunosuppressive CAFs in cancer.

[0007] Furthermore, there is a strong need to develop new strategies to overcome the immunosuppressive environment, thereby making immunotherapy, particularly immune checkpoint inhibitor therapy, more effective and accessible to all patients. This invention aims to meet these and other needs. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2019 / 020728 [Patent Document 2] U.S. Patent No. 5,618,703 [Patent Document 3] U.S. Patent Application No. 2005 / 0048542 [Patent Document 4] International Publication No. 2014 / 194302 [Patent Document 5] International Publication No. 2017 / 040790 [Patent Document 6] International Publication No. 2017 / 19846 [Patent Document 7] International Publication No. 2017 / 024465 [Patent Document 8] International Publication No. 2017 / 025016 [Patent Document 9] International Publication No. 2017 / 132825 [Patent Document 10] International Publication No. 2017 / 133540 [Patent Document 11] International Publication No. 2006 / 121168 [Patent Document 12] International Publication No. 19232484 [Patent Document 13] International Publication No. 16028656 [Patent Document 14] International Publication No. 16106302 [Patent Document 15] International Publication No. 16191643 [Patent Document 16] International Publication No. 17030823 [Patent Document 17] International Publication No. 17037707 [Patent Document 18] International Publication No. 17053748 [Patent Document 19] International Publication No. 17152088 [Patent Document 20] International Publication No. 18033798 [Patent Document 21] International Publication No. 18102536 [Patent Document 22] International Publication No. 18102746 [Patent Document 23] International Publication No. 18160704 [Patent Document 24] International Publication No. 18200430 [Patent Document 25] International Publication No. 18204363 [Patent Document 26] International Publication No. 19023504 [Patent Document 27] International Publication No. 19062832 [Patent Document 28] International Publication No. 19129221 [Patent Document 29] International Publication No. 19129261 [Patent Document 30] International Publication No. 19137548 [Patent Document 31] International Publication No. 19152574 [Patent Document 32] International Publication No. 19154415 [Patent Document 33] International Publication No. 19168382 [Patent Document 34] International Publication No. 19215728 [Patent Document 35] International Publication No. 20081522 [Patent Document 36] International Publication No. 20132661 [Patent Document 37] International Publication No. 20245173 [Patent Document 38] International Publication No. 21005125 [Patent Document 39] International Publication No. 21005131 [Patent Document 40] U.S. Patent No. 2020246383 [Patent Document 41] International Publication No. 19154859 [Patent Document 42] International Publication No. 19083990 [Patent Document 43] International Publication No. 19118932 [Patent Document 44] International Publication No. 18111989 [Patent Document 45] International Publication No. 17189569 [Patent Document 46] International Publication No. 13107820 [Patent Document 47] International Publication No. 08116054 [Patent Document 48] International Publication No. 07085895 [Patent Document 49] Korean Patent No. 1020190013612 [Patent Document 50] U.S. Patent No. 2016264662A [Patent Document 51] U.S. Patent No. 2017114133A [Patent Document 52] Chinese Patent No. 108707199A [Non-patent literature]

[0009] [Non-Patent Document 1] Costa A et al. Cancer Cell 2018;33(3):463-79 e10 [Non-Patent Document 2] Ohlund D et al., J Exp Med 2017;214(3):579-96 [Non-Patent Document 3] Dominguez et al. Cancer Discov 2020;10(2):232-53 [Non-Patent Document 4] Rooney et al., Cell 2015;160(1-2):48-61 [Non-Patent Document 5] Si-Yang Liu et al., J. Hematol.Oncol.10:136(2017) [Non-Patent Document 6] Jansen et al., ACS Med Chem Lett 2013, May 9;4(5):491-496 [Non-Patent Document 7] Mueller et al., J Biol Chem 1999;274:24947-52 [Non-Patent Document 8] Lindner et al., EJNMMI Radiopharmacy and Chemistry (2019) 4:16 [Non-Patent Document 9] Lidner, Journal of Nuclear Medicine, April 6, 2018

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[0010] Cancer is a systemic disease that involves multiple components, including both tumor cells themselves and host stromal cells. It is now clear that stromal cells in the tumor microenvironment play a crucial role in cancer development. The cancer stroma contains fibroblasts, particularly cancer-associated fibroblasts (CAFs), vascular endothelial cells, immune cells, and extracellular matrix. CAFs are the most abundant component of the tumor stroma, making up the majority of it and influencing the tumor microenvironment to promote cancer development, angiogenesis, invasion, and metastasis. [Means for solving the problem]

[0011] The inventors discovered a novel subpopulation of CAFs that plays a crucial role in establishing the immunosuppressive microenvironment in tumor sites. Using scRNA-seq, the inventors addressed the heterogeneity of the CAF-S1 immunosuppressive subpopulation and identified eight distinct CAF-S1 clusters. Of these, three CAF-S1 clusters (1, 2, 5) belonged to the inflammatory ("iCAF") subgroup, and five CAF-S1 clusters (0, 3, 4, 6, 7) belonged to the myofibroblast ("myCAF") subgroup. The iCAF and myCAF subgroups have been previously described in pancreatic cancer (Ohlund D et al., J Exp Med 2017;214(3):579-96).

[0012] The eight CAF-S1 clusters identified by the inventors are characterized by the high expression of genes encoding extracellular matrix (ECM) proteins (cluster 0), detoxification pathways (cluster 1), interleukin signaling (cluster 2), transforming growth factor β (TGFβ) signaling pathways (cluster 3), wound healing (cluster 4), interferon γ (cluster 5), interferon αβ (cluster 6), and actomyosin pathways (cluster 7). Accordingly, the inventors have annotated them as ecm-myCAF (cluster 0), detox-iCAF (cluster 1), IL-iCAF (cluster 2), TGFβ-myCAF (cluster 3), wound-myCAF (cluster 4), IFNγ-iCAF (cluster 5), IFNαβ-myCAF (cluster 6), and act-myCAF (cluster 7).

[0013] The presence of these CAF-S1 clusters was validated in head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) by analyzing publicly available scRNA-seq data, demonstrating the association of these CAF-S1 clusters across different cancer types.

[0014] Furthermore, the inventors revealed that the abundance of two CAF-S1 clusters within the myCAF subgroup, namely ecm-myCAF (cluster 0) and TGFβ-myCAF (cluster 3), was significantly correlated with the immunosuppressive environment, whereas the content of detox-iCAF and IL-iCAF was not.

[0015] In fact, the ecm-myCAF (cluster 0) and TGFβ-myCAF (cluster 3) clusters exhibited high levels of PD-1 + CTLA-4 + and TIGIT + CD4 + T lymphocytes (which are themselves rich in Treg cells) and CD8 + Tumors with a low T lymphocyte fraction are abundant. Interestingly, the ecm-myCAF (cluster 0) specific signature includes the LRRC15 gene, which was recently identified as a determinant of patient response to immunotherapy in pancreatic cancer (Dominguez et al., Cancer Discov 2020;10(2):232-53).

[0016] In contrast to ecm-myCAF (cluster 0) and TGFβ-myCAF (cluster 3), wound-myCAF (cluster 4) is not associated with an immunosuppressive environment but is correlated with high global infiltration by T lymphocytes. Therefore, because the wound-myCAF cluster (cluster 4) is abundant in tumors of patients who do not respond to immunotherapy, it may serve as a novel surrogate marker for primary resistance to immunotherapy in highly invasive tumors that are usually sensitive to this type of treatment. Thus, assessing the content of specific CAF-S1 clusters in tumors at diagnosis provides an additive value for predicting primary resistance to immune checkpoint inhibitors.

[0017] The inventors specifically demonstrate that, at the time of diagnosis, these particular CAF-S1 clusters are associated with primary resistance to immunotherapy in both melanoma and NSCLC patients. In particular, ecm-myCAF (cluster 0) is associated with FOXP3 highIncrease the fraction of T cells and CD4 + CD25 + Stimulate both PD-1 and CTLA-4 protein levels on the surface of T lymphocytes, which in turn increases the proportion of TGFβ-myCAF (cluster 3). This reveals a positive feedback loop between the immunosuppressive ecm-myCAF (cluster 0) and the CAF-S1 cluster of TGFβ-myCAF (cluster 3), and Tregs that promote immunosuppression and are involved in resistance to immunotherapy. Therefore, these data support the development of a strategy that combines PD-1 and / or CTLA-4 blockade with a therapy targeting specific CAF-S1 cluster components to overcome primary resistance to immune checkpoint blockade.

[0018] Therefore, the inventors' data support the identification of a new subset of patients with ANTXR1 + FAP + CAF, who exhibit immunosuppression and have resistance or low response to immunotherapy. Therefore, in this specific subset of patients, an agent targeting ANTXR1 + FAP + CAF to suppress or reduce its immunosuppressive effect may bring a therapeutic benefit. Furthermore, the combination of this agent and an immunotherapy agent can prevent the development of resistance to the immunotherapy agent or restore the response. Furthermore, even if FAP is expressed by all CAF-S1 cells, ANTXR1 + FAP + FAP + CAF is immunosuppressive and associated with immunotherapy resistance, whereas ANTXR1 + FAP - CAF is not. Therefore, patients with ANTXR1 + FAP + have a greater therapeutic benefit from treatment with a FAP inhibitor. In this regard, the benefit / risk balance is favorable for this treatment in this specific subset of patients, especially in combination with immunotherapy.

[0019] Finally, the inventors identified ANTXR1 as a specific marker for CAF-S1 clusters 0, 3, and 4, enabling the identification of CAFs associated with immunosuppressive effects.

[0020] In a first aspect, the present invention relates to an in vitro method for detecting immunosuppressive fibroblasts in cancer samples from subjects suffering from cancer, the method comprising detecting anthrax toxin receptor 1 (ANTXR1) positive fibroblasts in cancer samples from patients, and ANTXR1 + The presence of fibroblasts is an indicator of immunosuppressive CAF.

[0021] In a second aspect, the present invention relates to an in vitro method for predicting the response of a cancer-affected subject to an immunotherapy agent, wherein the method involves ANTXR1 in a cancer sample from the patient. + This includes detecting fibroblasts and ANTXR1 in cancer samples. + Fibroblasts predict a patient's response to immunotherapy drugs.

[0022] In a third aspect, the present invention relates to an immunotherapy agent for use in the treatment of cancer in a patient, wherein the patient (a) has a low number or percentage of ANTXR1 + Present a cancer sample containing fibroblasts, or (b) ANTXR1 + We present cancer samples that do not contain fibroblasts.

[0023] In particular, methods for predicting the response to the target or immunotherapy agent include ANTXR1 + This further includes determining the proportion of fibroblasts and ANTXR1 + The proportion of fibroblasts is the total number of fibroblasts in the cancer sample or the ANTXR1 ratio relative to the total number of cells in the cancer sample. + This is the number of fibroblasts.

[0024] In particular, ANTXR1 + Fibroblasts are FAP + ANTXR1 + These are fibroblasts. Preferably, ANTXR1 + Fibroblasts are ANTXR1 +Cancer-associated fibroblasts (CAFs), preferably FAPs + ANTXR1 + This is CAF.

[0025] Even more comfortable, ANTXR1 + Fibroblasts are FAP + ANTXR1 + LAMP5 - SDC1 + Fibroblasts, preferably CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - Fibroblasts, preferably CAF; ANTXR1 + SDC1 - LAMP5 - CD9 + Selected from the group consisting of fibroblasts, preferably CAFs.

[0026] In a fourth aspect, the present invention relates to the use of ANTXR1 as a biomarker for the identification of immunosuppressive fibroblasts, preferably immunosuppressive CAFs. The present invention also relates to the use of ANTXR1 as a biomarker for predicting the response of a subject to immunotherapy, as a target tumor in a fibroblast population, preferably a CAF population, that is affected by cancer.

[0027] In a fifth aspect, the present invention relates to ANTXR1 for use in the treatment of cancer in patients. + Regarding drugs that target fibroblasts, patients have ANTXR1 in tumor samples from patients. + FAP + The drug has fibroblasts, preferably CAFs, and ANTXR1 +The immunosuppressive effect on fibroblasts is suppressed or reduced, and preferably the agent is selected from the group consisting of (i) an anti-ANTXR1 antibody which may be conjugated with a cytotoxic drug, a multispecific molecule containing an anti-ANTXR1 moiety, an anti-ANTXR1 T cell receptor (TCR) and an anti-ANTXR1 chimeric antigen receptor (CAR), and (ii) immune cells expressing an anti-ANTXR1 TCR or anti-ANTXR1 CAR, preferably T cells or natural killer cells or any combination thereof.

[0028] In particular, ANTXR1 + Drugs that target fibroblasts are intended to be used in combination with immunotherapy agents.

[0029] In a sixth aspect, the present invention relates to an FAP inhibitor for use in the treatment of cancer in patients, wherein the patient has ANTXR1 in tumor samples from the patient. + FAP + The cells have fibroblasts, preferably CAFs. In certain embodiments, the FAP inhibitor is intended for use in combination with an immunotherapy agent.

[0030] Preferably, the FAP inhibitor is tarabostat or PT-100 (CAS No. 149682-77-9, [(2R)-1-[(2S)-2-amino-3-methylbutanoyl]pyrrolidine-2-yl]boronic acid), linagliptin (Tradjenta) (CAS No. 668270-12-0; 8-[(3R)-3-aminopiperidine-1-yl]-7-buta-2-inyl-3-methyl- FAP inhibitor having a 1-[(4-methylquinazolin-2-yl)methyl]purine-2,6-dione) and N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold, FAPI-02 (CAS number 2370952-98-8, (S)-2,2',2''-(10-(2-(4-(3-((4-((2-(2-cyanopyrrolidine-1-yl)-2-oxoethyl)cal Bamoyl)quinoline-6-yl)oxy)propyl)piperazine-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid), FAPI-04 (CAS No. 2374782-02-0, (S)-2,2',2''-(10-(2-(4-(3-((4-((2-(2-cyano-4,4-difluoropyrrolidine-1-yl)-2 The group consists of peptide target radionuclides such as -oxoethyl)carbamoyl)quinoline-6-yl)oxy)propyl)piperazine-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate) and FAPI-46 (CAS number: 2374782-04-2), FAP-2286, and any combination thereof.

[0031] In a seventh aspect, the present invention relates to a combination preparation for simultaneous, separate, or continuous use in the treatment of a patient's cancer, a) ANTXR1 + b) Products or kits containing drugs that target fibroblasts and immunotherapeutic agents.

[0032] In particular, immunotherapeutic agents are selected from the group consisting of therapeutic procedures that stimulate the patient's immune system to attack malignant tumor cells, immunization of the patient with tumor antigens, molecules that stimulate the immune system such as cytokines, therapeutic antibodies, adoptive T-cell therapy, CAR cell therapy, immune checkpoint inhibitors, and any combination thereof, preferably immune checkpoint inhibitors.

[0033] Preferably, the checkpoint inhibitor is selected from the group consisting of antibodies against cytotoxic T lymphocyte-associated protein (CTLA-4), programmed cell death protein 1 (PD-1), programmed cell death ligand (PD-L1), T cell immune receptor having Ig and ITIM domains (TIGIT), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain-containing T-3 (TIM-3), B- and T lymphocyte attenuator (BLTA), IDO1, or any combination thereof. Preferably, the checkpoint inhibitor is selected from the group consisting of antibodies against CTLA-4, PD-1, PD-L1 and TIGIT, or any combination thereof, more preferably from the group consisting of antibodies against PD-1 or CTLA-4 and combinations thereof.

[0034] In particular, immunotherapeutic agents are selected from the group consisting of ipilimumab, nivolumab, BGB-A317, pembrolizumab, atezolizumab, avelumab or durvalumab, BMS-986016, and epacadostat, or any combination thereof.

[0035] In particular, cancer is selected from the group consisting of prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, stomach cancer, kidney cancer, ovarian cancer, hepatocellular carcinoma, osteosarcoma, melanoma, hypopharyngeal cancer, esophageal cancer, endometrial cancer, cervical cancer, pancreatic cancer, liver cancer, colon or colorectal cancer, adenocarcinoma, sarcoma, non-squamous cell carcinoma, squamous cell carcinoma, neuroendocrine tumors, muscle cancer, adrenal cancer, thyroid cancer, uterine cancer, skin cancer, melanoma, metastatic melanoma, bladder cancer, and head and neck cancer. Preferably, cancer is selected from the group consisting of head and neck cancer, breast cancer, ovarian cancer, NSCLC, melanoma, and metastatic melanoma. More preferably, cancer is selected from the group consisting of head and neck cancer, NSCLC, melanoma, and metastatic melanoma. [Brief explanation of the drawing]

[0036] [Figure 1] This figure shows the identification of different cell clusters of CAF-S1 fibroblasts. Uniform Manifold Approximation and Projection (UMAP) of 18,296 CAF-S1 fibroblasts from 7 BC patients allows for the visualization of 8 CAF-S1 clusters (0-7). The colors indicate different CAF-S1 clusters defined by a graph-based clustering method applied to a space defined by 30 first principal components. [Figure 2] This figure shows the validation of the five most abundant CAF-S1 clusters across different cancer types. The percentage of different clusters among CAF-S1 fibroblasts based on FACS data is shown. Each bar represents one patient (N=44). [Figure 3]This figure shows the detection of CAF-S1 cell clusters in lung cancer and head and neck cancer. (A) UMAP plot combining 18,296 CAF-S1 fibroblasts from BC (red, upper left panel) and FAP+ fibroblasts from HNSCC (data from (30), n=603 FAP+ cells, blue, upper left panel). Scores calculated as the mean z-scores of the genes constituting the specific signature of each CAF-S1 cluster are applied. (B) Same as (A) for NSCLC (data from (31), n=959 FAP+ cells, blue, upper left panel). [Figure 4] This figure shows the correlation between CAF-S1 clusters in breast cancer and immune cells. (A) Detailed correlation curve between the two variables as shown. Each dot represents one tumor (N=37). P-value from Pearson correlation test. (B) Correlation curve between CAF-S1 cluster signature and FOXP3 in the TCGA cohort. Each dot represents one tumor (N=1221). P-value from Pearson correlation test. (C) Same as (B) between CAF-S1 cluster signature and cytolysis index, as specified in Rooney et al., Cell 2015;160(1-2):48-61. [Figure 5]This figure shows the interaction between CAF-S1 clusters and Treg cells. (A) Representative histograms (left) of FOXP3-specific mean fluorescence intensity (speMFI) in the presence of FOXP3 alone (green), iCAF (orange), or ecm-myCAF (red). After 24 hours (h) of co-culture at a 10:1 ratio (T:CAF-S1), the proportion of FOXP3+ cells (center) in CD4+ CD25+ T cells (right) and FOXP3 protein levels were evaluated. P-values ​​from Welch's t-test (N=7 CAF-S1 primary cell lines per condition; n=3 independent experiments). (BG) For the immune checkpoints PD-1 (B), CTLA-4 (C), TIGIT (D), TIM3 (E), and LAG3 (F) in FOXP3+ CD4+ CD25+ Treg cells, the results are the same as in (A). P-values ​​from Welch's t-test. (N=7 CAF-S1 cell lines per condition; n=3 independent experiments). (G) Effect of CD4+ CD25+ T lymphocytes on CAF-S1 cluster identity. Dot plot showing protein levels of CAF-S1 cluster markers on the surface of primary CAF-S1 cell lines. For each marker, the surface protein level is expressed as a specific MFI calculated as follows: Specific MFI = MFI from specific antibody - MFI from isotype control, in absence (-) or presence (+) of CD4+ CD25+ T cells (N=7 CAF-S1 cell lines per condition; n=3 independent experiments). P-values ​​from Mann-Whitney test. [Figure 6]This figure shows the effect of CAF-S1 clusters on resistance to immunotherapy. (A) Gene set enrichment analysis (GSEA) applied to RNA-Seq data from 28 melanoma tumors prior to anti-PD-1 treatment was applied using specific signatures from each CAF-S1 cluster and shows significant enrichment of CAF-S1 gene signatures (top 100 genes) in non-responders (N=13) compared to responsive patients (N=15). (33) Cohort. Below, normal fibroblast signatures (same as above). GSEA analysis shows that clusters 0 (ecm-myCAF), 3 (TGFβ-myCAF), and 4 (wound-myCAF) were significantly associated with non-responders (above), while clusters 1 (detox-iCAF), 2 (IL-iCAF), and 5 (IFN-iCAF) were not significantly associated (below). (B) Expression is assessed by the mean z-score of each CAF-S1 cluster signature in responder and non-responder melanoma patients. (C, D) Same as (B) using normal fibroblast signatures and cytolysis index. (E) Responders and non-responders stratified by low CAF-S1 cluster expression and high CAF-S1 cluster expression (based on the third quartile of the CAF cluster z-score). (F, G) Same as (E) using normal fibroblast signatures and cytolysis index. (H) Same as (A) analyzing the NSCLC cohort of patients. [Figure 7] This figure shows the identification of CAF-S1 clusters. (A) A representative FACS plot showing the gating strategy used to isolate CAF-S1. Cells are first gated to DAPI-EPCAM-CD45-CD31-CD235a- to exclude dead cells, epithelial cells, hematopoietic cells, endothelial cells, and erythrocytes, respectively. Next, FAPHigh CD29Med cells are selected as CAF-S1 fibroblasts. (B) A UMAP plot of 18,296 CAF-S1 fibroblasts (as in Figure 1) shows the mean z-scores of specific gene signatures for the five most abundant CAF-S1 clusters. [Figure 8]This figure shows the identification of CAF-S1 cluster markers for FACS analysis and the selection of CAF-S1 from HNSCC and NSCLC scRNA-seq data. A violin plot shows the distribution of expression (RNA levels from scRNA-seq of 18,296 CAF-S1 fibroblasts from 7 BC patients) of six genes encoding surface markers specific to each of the five most abundant CAF-S1 clusters. [Figure 9] This figure shows the correlation between CAF-S1 clusters and immune cells. Detailed correlation plots between CAF clusters and immune cells (N=37 BCs) are shown. P-values ​​from Pearson correlation tests are also shown. [Figure 10] This figure shows that fibroblasts isolated from paratumors acquire CAF-S1 characteristics by being spread and maintained on a plastic dish. (A) Dot plots show the intracellular specific mean fluorescence intensity (speMFI) FOXP3, PD-1, CTLA-4, and TIGIT of CD4+ CD25+ T cells cultured alone (left) or in the presence of ecm-myCAF (right). P values ​​from Mann-Whitney test (N=4 CAF-S1 primary cell lines). (B) Box plots showing FOXP3, CTLA-4, and TIGIT mRNA levels of CD4+ CD25+ T cells cultured alone (right) or in the presence of ecm-myCAF (left). P values ​​from DESeq2 analysis (N=8). (C) Same as (B) for STAT and NFAT family members. [Figure 11A] This figure shows that a UMAP plot of 18,296 CAF-S1 fibroblasts (as shown in Figure 1) allows for the visualization of eight CAF-S1 clusters (0-7). The three predictive clusters for immunotherapy response, namely cluster 0 = ECM-myCAF, cluster 3 = TGFβ-myCAF, and cluster 4 = wound-myCAF, are indicated by arrows. [Figure 11B1]This figure shows violin plots (left) and UMAP plots (right) illustrating some of the most distinctly expressed genes that define the CAF-S1 signature. The expression of these genes in each CAF-S1 cluster is shown using violin plots and UMAP representations. None of these top representative CAF-S1 genes are specifically expressed in clusters 0, 3, and 4, and therefore predict the immunotherapy response. [Figure 11B2] This is a continuation of Figure 11B1. [Modes for carrying out the invention]

[0037] Detailed description of the invention definition As used herein, the terms “cancer” or “tumor” refer to the presence of cells that have characteristics typical of cancerous cells, such as uncontrolled growth and / or immortality and / or metastatic ability and / or rapid growth and / or proliferation rate and / or specific characteristic morphological features. The term refers to all types of malignant tumors (primary or metastatic) in any type of subject. It may also refer to solid tumors and hematopoietic tumors.

[0038] The term "cancer sample," as used herein, means any sample containing tumor cells and cancer stromal cells derived from the subject, particularly fibroblasts such as cancer-associated fibroblasts (CAFs). Cancer tissue consists, in particular, of cancer cells and their surrounding cancer stromal cells, including cancer-associated fibroblasts (CAFs), vascular endothelial cells, and immune cells, in addition to the extracellular matrix. Preferably, cancer samples contain nucleic acids and / or proteins. Samples may be treated before use.

[0039] As used herein, the terms “cancer-associated fibroblasts” or “CAF” refer to fibroblasts present in the stroma of cancer. They are one of the most abundant stromal components, having a morphology similar to myofibroblasts. CAFs are CD45 - EpCAM - CD31 - CD29 +These are stromal cells.

[0040] As used herein, the term “immunosuppressive fibroblasts” refers to fibroblasts responsible for and / or contributing to immunosuppression in tumors, i.e., partial or complete inhibition or suppression of the individual’s immune response to cancer cells. Depending on the circumstances, fibroblasts may be malignant or normal, particularly in sarcomas.

[0041] As used herein, the terms “immunosuppressive cancer-associated fibroblasts” or “immunosuppressive CAFs” refer to CAFs that are responsible for and / or contribute to the immunosuppression or immunosuppressive microenvironment of a tumor, i.e., the partial or complete inhibition or suppression of the individual’s immune response to cancer cells. The “tumor microenvironment” or “TME” is the environment surrounding a tumor, including perivascular tissue, immune cells, fibroblasts, signaling molecules, and the extracellular matrix (ECM).

[0042] The term "immune response" refers to the action of lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, resulting in the selective damage, destruction, or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues from the human body.

[0043] As used herein, the terms “Anthrax Toxin Receptor 1,” “ANTXR Cell Adhesion Molecule 1,” “ANTXR,” “ANTXR1,” “Tumor Endothelial Marker 8,” “TEM8,” “ATR,” or “GAPO” are equivalent and refer to the product of the human ANTXR1 gene, as described, for example, under the references of gene ID: 84168 and UniProt Q9H6X2. The ANTXR1 protein plays a role in cell adhesion and migration.

[0044] As used herein, the terms “fibroblast-activating protein,” “FAP,” “prolyl endopeptidase FAP,” “dipeptidyl peptidase FAP,” “surface-expressed protease,” “seplus,” “serine membrane endogenous protease,” “SIMP,” “membrane endogenous serine protease,” and “post-proline cleavage enzyme” are used interchangeably and refer to the products of the FAP human gene, such as those listed under references for gene ID: 2191 and UniProt Q12884. FAP is a cell surface glycoprotein serine protease involved in the degradation of the extracellular matrix and is involved in many cellular processes, including tissue remodeling, fibrosis, wound healing, inflammation, and tumor growth.

[0045] As used herein, the terms “Lysosome-associated membrane glycoprotein 5,” “LAMP5,” “Brain and dendritic cell-associated LAMP,” and “Brain-associated LAMP-like protein” are equivalent and refer to the products of the human LAMP5 gene, as described, for example, under GeneID 24141 and UniProt Q9UJQ1 references.

[0046] As used herein, the terms “Syndecan-1,” “SDC1,” “SYND1,” and “CD138” are used interchangeably herein and refer to the product of the human SDC1 gene, as described below, for example, under reference GeneID:6382 and UniProt P18827. SDC1 is a cell surface proteoglycan that possesses both heparan sulfate and chondroitin sulfate and binds the cytoskeleton to the interstitial matrix.

[0047] As used herein, the terms “CD9,” “5H9 antigen,” “cell proliferation suppressor gene 2 protein,” “leukocyte antigen MIC3,” “motility-related protein,” “MRP-1,” “tetraspanin-29,” “Tspan-29,” and “p24” are used interchangeably herein and refer to the products of the human CD9 gene, as described, for example, under the references GeneID:928 and UniProt P21926. CD9 is an integrin-related membrane-bound protein that regulates different processes.

[0048] The "+" indicates cells that express the marker. For example, ANTXR1 + This refers to cells that express ANTXR1. Alternatively, "-" refers to cells that do not express the marker. For example, ANTXR1 - This refers to cells that do not express ANTXR1.

[0049] As used herein, the terms “subject,” “individual,” or “patient” are interchangeable and refer to animals, preferably mammals, and more preferably humans. However, the term “subject” may also refer to non-human animals, particularly mammals, such as dogs, cats, horses, cattle, pigs, sheep, and non-human primates.

[0050] As used herein, the terms “marker” or “biomarker” refer to measurable biological parameters that are useful in predicting the development of cancer, the effectiveness of cancer treatment, or the presence of immunosuppressive cells.

[0051] As used herein, the term “diagnosis” refers to a determination of whether a subject is likely to develop cancer. Those skilled in the art often make diagnoses based on one or more diagnostic markers, the presence, absence, or quantity of which indicate the presence or absence of cancer. “Diagnosis” is also intended to refer to the provision of information useful for diagnosis.

[0052] As used herein, the terms “treatment,” “to treat,” or “to treat” refer to any action intended to improve a patient’s health condition, such as treating, preventing, or delaying a disease. In certain embodiments, such terms refer to the improvement or elimination of a disease or symptoms associated with a disease. In other embodiments, the terms refer to minimizing the progression or worsening of a disease resulting from the administration of one or more therapeutic agents to a subject having such a disease.

[0053] As used herein, the terms “immunotherapy,” “immunotherapy agent,” or “immunotherapy treatment” refer to cancer treatments that utilize an immune system to reject cancer. Therapeutic treatments stimulate the patient’s immune system to attack malignant tumor cells. These treatments include immunization of the patient with tumor antigens (e.g., by administering a cancer vaccine), in which the patient’s own immune system is trained to recognize tumor cells as targets to be destroyed, or administration of immune-stimulating molecules such as cytokines, or administration of therapeutic antibodies as drugs, in which the patient’s immune system is mobilized by the therapeutic antibodies to destroy tumor cells. In particular, antibodies are directed against specific antigens, such as abnormal antigens presented on the surface of tumors.

[0054] A crucial part of the immune system is its ability to distinguish between the body's normal cells and cells considered "foreign," particularly cancer cells. This allows the immune system to attack cancer cells while leaving normal cells intact. To do this, the immune system uses "checkpoints," which are molecules on specific immune cells that need to be activated (or inactivated) to initiate an immune response. Cancer cells may find ways to use these checkpoints to evade attack by the immune system. As used herein, the term "immune checkpoint inhibitor therapy" refers to immunotherapy that targets these checkpoints to enable or enhance the immune system's attack on cancer cells.

[0055] The terms “percentage,” “quantity,” “number,” “amount,” and “level” are used interchangeably herein and may refer to the absolute quantification of molecules or cells in a sample, or the relative quantification of molecules or cells in a sample, i.e., relative to another value, such as a reference value taught herein.

[0056] As used herein, “pharmaceutical composition” refers to a preparation of one or more activators with any other chemical components such as physiologically appropriate carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of an activator to a living organism. The compositions of the present invention may be any conventional route of administration or a form suitable for use. In one embodiment, “composition” is typically intended to be a combination of an activator, e.g., a compound or composition, and a naturally derived or non-naturally derived carrier, and includes inert (e.g., detectable drug or label) or active, e.g., adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, etc., and pharmaceutically acceptable carriers. As used herein, “acceptable vehicle” or “acceptable carrier” is any well-known compound or combination of compounds that is well known to those skilled in the art as being useful in formulating pharmaceutical compositions.

[0057] As used herein, the terms “active ingredient,” “active component,” “pharmaceutical active ingredient,” “therapeutic agent,” “antitemogenic compound,” and “antitemogenic agent” are equivalent and refer to components that have therapeutic effects.

[0058] As used herein, the term “therapeutic effect” means an effect induced by the active ingredient or pharmaceutical composition according to the present invention that can prevent or delay the appearance or development of cancer, or cure or reduce the effects of cancer.

[0059] As used herein, “effective dose” or “therapeutic effective dose” refers to the amount of an activator, either alone or in combination with one or more other activators, required to impart a therapeutic effect to a subject, for example, the amount of an activator required to treat a target disease or disorder, or to produce a desired effect. The “effective dose” varies depending on the drug, the disease and its severity, the characteristics of the subject being treated, including age, physical condition, size, sex, and weight, the duration of treatment, the nature of the combination therapy (if any), the specific route of administration, and factors within the scope of the knowledge and expertise of the healthcare professional. These factors are well known to those skilled in the art and can be addressed through routine experimentation. It is generally preferable to use the maximum dose of individual components or combinations thereof, i.e., the highest safe dose determined by sound medical judgment.

[0060] As used herein, the terms “kit,” “product,” or “combination preparation” specifically define a “kit of components” in the sense that the combination partners (a) and (b) provided in this application can be administered independently or by the use of different fixed combinations of combination partners (a) and (b) in distinct amounts, i.e., simultaneously or at different times. The components of the kit of components can then be administered simultaneously or staggered in time, i.e., at different times for any part of the kit of components. The ratio of the total amounts of combination partner (a) and combination partner (b) administered in the combination preparation can be varied. Combination partners (a) and (b) can be administered by the same route or by different routes.

[0061] As used herein, the term “simultaneous” refers to a pharmaceutical composition, kit, product, or combination preparation according to the present invention in which the active ingredients are used or administered simultaneously, i.e., at the same time.

[0062] As used herein, the term “sequential” refers to a pharmaceutical composition, kit, product, or combination preparation according to the present invention in which the active ingredients are used or administered sequentially, i.e., one after the other. Preferably, when the administration is sequential, all active ingredients are administered in less than about one hour, preferably less than about ten minutes, and more preferably less than one minute.

[0063] As used herein, the term “separate” refers to a pharmaceutical composition, kit, product, or combination preparation according to the present invention in which the active ingredient is used or administered at separate times of day. Preferably, when the administration is separate, the active ingredient is administered at intervals of about 1 hour to about 24 hours, preferably at intervals of about 1 hour and 15 hours, more preferably at intervals of about 1 hour and 8 hours, and even more preferably at intervals of about 1 hour and 4 hours.

[0064] As used herein, the terms "and / or" should be interpreted as specific disclosures of each of two designated features or components, with or without the other. For example, "A and / or B" should be interpreted as specific disclosures of each of (i) A, (ii) B, and (iii) A and B, as if each were described separately.

[0065] The terms "a" or "an" can refer to one or more of the elements they modify unless the context makes it clear whether one or more of the elements being referred to are being described (for example, "reagent" can mean one or more reagents).

[0066] As used herein in relation to any and all values ​​(including the lower and upper limits of numerical ranges), the term "about" means any value having a maximum tolerance deviation range of + / -10% (e.g., + / -0.5%, + / -1%, + / -1.5%, + / -2%, + / -2.5%, + / -3%, + / -3.5%, + / -4%, + / -4.5%, + / -5%, + / -5.5%, + / -6%, + / -6.5%, + / -7%, + / -7.5%, + / -8%, + / -8.5%, + / -9%, + / -9.5%). Using the term "about" at the beginning of a string of values ​​modifies each of the values ​​(i.e., "about 1, 2, and 3" refers to about 1, about 2, and about 3). Furthermore, if a list of values ​​is provided herein (for example, about 50%, 60%, 70%, 80%, 85%, or 86%), this list includes all of their intermediate and fractional values ​​(for example, 54%, 85.4%).

[0067] The methods of the present invention disclosed below may be in vivo, ex vivo, or in vitro methods, preferably in vitro or ex vivo methods.

[0068] Detection of novel fibroblast subpopulations, particularly CAF subpopulations. In a first aspect, the present invention relates to an in vitro method for detecting immunosuppressive fibroblasts, particularly cancer-associated fibroblasts (CAFs), in cancer samples from subjects with cancer, wherein the method involves ANTXR1 + Fibroblasts, especially ANTXR1 + This includes detecting CAF, and ANTXR1 + Fibroblasts, especially ANTXR1 + The presence of CAFs indicates immunosuppressive fibroblasts, particularly immunosuppressive CAFs. Next, the present invention envisions the use of ANTXR1 as a biomarker for identifying immunosuppressive fibroblasts, particularly immunosuppressive CAFs, in cancer samples from subjects affected by cancer. The present invention also envisions ANTXR1 and immunosuppressive ANTXR1 as novel biomarkers in cancer therapy. + Fibroblasts, especially ANTXR1 +This invention relates to the use of CAF populations. In particular, the present invention relates to immunosuppressive ANTXR1 as a novel biomarker of response to immunotherapy. + Fibroblasts, especially ANTXR1 + The invention is intended for use in CAF populations. The present invention also relates to the use of ANTXR1 as a biomarker for tumors of cancerous subjects in fibroblasts, preferably CAF populations, to predict the subject's response to immunotherapy, and in particular to predict whether the subject is likely to respond to immunotherapy.

[0069] In one embodiment, the present invention relates to an in vitro method for detecting immunosuppressive fibroblasts, particularly cancer-associated fibroblasts (CAFs), in cancer samples from subjects suffering from cancer, wherein the method involves detecting ANTXR1 in cancer samples from patients. + Fibroblasts, especially ANTXR1 + This includes detecting CAF.

[0070] Depending on the circumstances, this method may further include steps prior to providing a cancer sample from the patient.

[0071] Preferably, the CAF according to the present invention belongs to the CAF-S1 subgroup, as described, for example, Costa et al., 2018, Cancer Cell, Vol. 33, No. 3, pp. 463-479, e10, or International Publication 2019 / 020728, the disclosure of which is incorporated herein by reference. The CAF-S1 population expresses, in particular, one or more biomarkers selected from the group consisting of CD29, FAP, αSMA, PDGFRβ, and FSP1. In particular, the CAF-S1 according to the present invention expresses FAP + That is the case.

[0072] When used herein, "FAP" + The term "fibroblasts" refers to a subpopulation of fibroblasts that express FAP. Where used herein, "FAP" is used to mean "fibroblasts." + CAF or FAP +The term "cancer-associated fibroblasts" refers to a subpopulation of CAFs that express FAP. Not all CAFs express FAP. Therefore, in preferred embodiments, FAP + CAF detection relies on the detection of CAFs expressing FAP, preferably FAP mRNA and / or protein. + CAF can also be detected using other markers specific to CAF.

[0073] When used herein, "ANTXR1" + The term "fibroblast" refers to a subpopulation of fibroblasts that express ANTXR1. Where used herein, "ANTXR1" is used to mean "fibroblasts that express ANTXR1." + CAF or ANTXR1 + The term "cancer-associated fibroblasts" refers to a subpopulation of CAFs that express ANTXR1. Not all fibroblasts or CAFs express ANTXR1. Therefore, in preferred embodiments, ANTXR1 + The detection of fibroblasts, particularly CAFs, depends on the detection of ANTXR1-expressing fibroblasts, especially CAFs, preferably ANTXR1 mRNA and / or protein. In a preferred embodiment, ANTXR1 detection is performed at the protein level, particularly using an anti-ANTXR1 antibody.

[0074] In one aspect, the present invention relates to an in vitro method for detecting immunosuppressive fibroblasts, particularly cancer-associated fibroblasts (CAFs), in cancer samples from subjects suffering from cancer, wherein the method involves detecting FAP in cancer samples from patients. + ANTXR1 + This includes detecting fibroblasts, particularly CAFs.

[0075] However, ANTXR1 + They can also be detected using other markers specific to fibroblasts, particularly CAFs.

[0076] As used herein, the terms "ecm-myCAF", "CAF-S1 cluster 0", and "ANTXR1" are used. + SDC1+ LAMP5 - "CAF" and "FAP" + ANTXR1 + SDC1 + LAMP5 - "CAF" is used interchangeably and refers to a subpopulation of CAFs, particularly the cluster of the CAF subpopulation CAF-S1. Such a population expresses FAP, ANTXR1, and SDC1 but does not express LAMP5. Thus, in a preferred embodiment, ANTXR1 + SDC1 + LAMP5 - The detection of CAF depends on the detection of CAFs that express ANTXR1 and SDC1 but do not express LAMP5. The detection of ANTXR1, LAMP5, and SCD1 can be performed at the mRNA and / or protein level, preferably at the protein level, particularly by antibodies.

[0077] As used herein, the terms "TGFb-myCAF", "CAF-S1 cluster 3", "ANTXR1 + LAMP5 + SDC1 + / - " and "FAP + ANTXR1 + LAMP5 + SDC1 + / - " are used interchangeably and refer to a subpopulation of CAFs, particularly the cluster of the CAF subpopulation CAF-S1. Such a population expresses FAP, ANTXR1, and LAMP5 and optionally expresses SDC1. Thus, in a preferred embodiment, ANTXR1 + LAMP5 + SDC1 + / - The detection of CAF depends on the detection of CAFs that express ANTXR1, LAMP5, and optionally SDC1. The detection of ANTXR1, LAMP5, and SCD1 can be performed at the mRNA and / or protein level, preferably at the protein level, particularly by antibodies.

[0078] As used herein, the terms "wound-myCAF", "CAF-S1 cluster 4", "ANTXR1 + SDC1 -LAMP5 - CD9 + " and "FAP + ANTXR1 + SDC1 - LAMP5 - CD9 + The term is used interchangeably and refers to a subpopulation of CAF, particularly the CAF subpopulation CAF-S1 cluster. Such populations express FAP, ANTXR1, and CD9, but do not express LAMP5 and SDC1. Therefore, in a preferred embodiment, ANTXR1 + SDC1 - LAMP5 - CD9 + Detection of CAFs relies on detecting CAFs that express ANTXR1 and CD9 but not SDC1 and LAMP5. Detection of ANTXR1, LAMP5, CD9, and SDC1 can be performed at the mRNA and / or protein level, preferably at the protein level, particularly by antibody.

[0079] In one embodiment, the present invention relates to an in vitro method for detecting immunosuppressive fibroblasts, particularly cancer-associated fibroblasts (CAFs), in cancer samples from subjects suffering from cancer, wherein the method involves detecting ecm-myCAF(ANTXR1) in cancer samples from patients. + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and / or wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - This includes detecting CAF. In some cases, the method involves detecting TGFb-myCAF(ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / -This includes detecting CAF. Depending on the case, the method may involve the patient's cancer sample being wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves detecting TGFb-myCAF(ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF.

[0080] In one embodiment, fibroblasts, particularly CAFs, are considered to express FAP, ANTXR1, LAMP5, SDC1, and / or CD9 if their mRNA levels and / or protein levels are significantly different from the corresponding background noise levels, for example, levels measured under the same conditions but without cells, and / or levels corresponding to control conditions, or levels measured in reference RNA or protein, for example, levels measured under the same conditions in cells known not to express FAP, ANTXR1, LAMP5, SDC1, and / or CD9, respectively.

[0081] The measurement of FAP, ANTXR1, LAMP5, SDC1, and / or CD9 expression levels in fibroblasts, particularly CAFs, can be carried out by various techniques well known to those skilled in the art. In particular, the measurement of FAP, ANTXR1, LAMP5, SDC1, and / or CD9 expression levels in CAFs may depend on the detection of FAP, ANTXR1, LAMP5, SDC1, and / or CD9 mRNA (messenger RNA) or protein.

[0082] In one embodiment, the expression of FAP, ANTXR1, LAMP5, SDC1, and / or CD9 in fibroblasts, particularly CAFs, is determined by measuring the expression of their mRNA. Methods for determining the amount of mRNA in cells are well known to those skilled in the art. mRNA can be detected by hybridization (e.g., Northern blotting), particularly by nanostringing and / or amplification (e.g., RT-PCR), particularly by quantitative or semi-quantitative RT-PCR. Other amplification methods include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand substitution amplification (SDA), and nucleic acid sequence-based amplification (NASBA).

[0083] Real-time quantification or semi-quantitative RT-PCR is particularly advantageous. A Taqman probe specific to the protein of the transcript of interest may be used. In a preferred embodiment, the expression levels of FAP, ANTXR1, LAMP5, SDC1 and / or CD9 and any other protein of interest are determined by measuring the amount of their mRNA, preferably by quantitative or semi-quantitative RT-PCR, or by real-time quantitative or semi-quantitative RT-PCR.

[0084] As used herein, the terms “quantitative RT-PCR,” “qRT-PCR,” “real-time RT-PCR,” and “quantitative real-time RT-PCR” are equivalent and interchangeable. Any of the published quantitative RT-PCR protocols may be used in this method (and may be modified as necessary). Suitable quantitative RT-PCR procedures include, but are not limited to, those presented in U.S. Patent No. 5,618,703 and U.S. Patent Application No. 2005 / 0048542, incorporated herein by reference.

[0085] Depending on the circumstances, ANTXR1, FAP, LAMP5, SDC1, and / or CD9 can be determined on tumor samples or on a subset of tumors containing fibroblasts, particularly CAFs. Therefore, the tumor can be pre-treated to isolate fibroblasts, particularly CAFs, from other cells of the tumor, particularly tumor cells, and then ANTXR1, FAP, LAMP5, SDC1, and / or CD9 can be detected. Accordingly, the present invention relates to a method for detecting immunosuppressive fibroblasts, particularly CAFs, in cancer samples of a subject affected by cancer, the method comprising isolating fibroblasts, particularly CAFs, from the cancer sample of the subject, particularly tumor cells, and detecting ANTXR1 of the isolated fibroblasts, particularly CAFs. + This includes detecting fibroblasts, particularly CAFs.

[0086] In another embodiment, the expression levels of ANTXR1, FAP, LAMP5, SDC1, and / or CD9 in fibroblasts, particularly CAFs, are determined by measuring the expression of their respective proteins.

[0087] The amount of protein can be measured by any method well known to those skilled in the art. These methods typically involve contacting a sample with a binding partner capable of selectively interacting with the protein present in the sample. The binding partner is generally a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. Such antibodies can be produced by methods well known to those skilled in the art. These antibodies include those produced by hybridomas, and those produced by genetic engineering using host cells transformed with recombinant expression vectors containing the antibody-coding gene. Hybridomas producing monoclonal antibodies can be obtained as follows: the protein or its immunogenic fragment is used as an antigen for immunity according to conventional immunization methods. The resulting immune cells are fused with known parental cells according to conventional cell fusion methods, and antibody-producing cells are therefore screened from the fused cells using conventional screening methods.

[0088] The antibody according to the present invention can be labeled and / or fused to a detection entity. Preferably, the antibody according to the present invention is labeled or fused to a detection entity.

[0089] In preferred embodiments, the antibody is labeled. The antibody can be labeled with a label selected from the group consisting of radiolabeling, enzymatic labeling, fluorescent labeling, biotin-avidin labeling, chemiluminescence labeling, and the like. The antibody according to the present invention can be labeled by standard labeling techniques well known to those skilled in the art, and the labeled antibody can be visualized using well known methods. In particular, the label generally provides a signal that can be detected by fluorescence, chemiluminescence, radioactivity, colorimetric, mass spectrometry, X-ray diffraction or absorption, magnetism, enzyme activity, and the like.

[0090] Preferably, the detectable label may be a luminescent label. For example, fluorescent labels, bioluminescent labels, chemiluminescent labels, and colorimetric labels may be used in the implementation of the present invention, and more preferably fluorescent labels. Preferably, the label is ligated to the C-terminal end of the antibody.

[0091] In another preferred embodiment, the antibody described above can be fused to a detection entity. The detection entity can be selected from the group consisting of a tag, an enzyme, or a fluorescent protein. The detection entity is preferably located at the C-terminal end of the antibody.

[0092] The amounts of ANTXR1, FAP, LAMP5, SDC1, and / or CD9 can be measured by semi-quantitative Western blotting, enzyme-labeled and mediated immunoassays such as ELISA, biotin / avidin type assays, radioimmunoassays, immunohistochemistry, immunoelectrophoresis or immunoprecipitation, protein or antibody arrays, or flow cytometry such as fluorescence-activated cell sorting (FACS). Reactions generally involve identifying labels such as fluorescence, chemiluminescence, radioactivity, enzyme labeling, or dye molecules, or other methods for detecting the formation of complexes between antigens and antibodies or antibodies that have reacted with them. Preferably, protein expression levels are evaluated by FACS or immunohistochemistry.

[0093] Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. FACS provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell. The cell suspension is placed in the center of a narrow, rapidly flowing liquid stream. The stream is positioned so that the cells are far apart relative to their diameter. An oscillating mechanism causes the cell stream to split into individual droplets. The system is tuned so that the probability of multiple cells being present in each droplet is low. Immediately before the stream splits into droplets, it passes through a fluorescence measurement station, where the fluorescence characteristics of each cell of interest are measured. A charging ring is positioned at the point where the stream splits into droplets. A charge is placed on the ring immediately before the fluorescence intensity is measured, and the opposite charge is trapped in the droplet when it is disconnected from the stream. The charged droplets pass through an electrostatic deflection system that redirects the droplets into containers based on their charge.

[0094] Immunohistochemistry (IHC) is a process that selectively images antigens (proteins, etc.) within cells of tissue sections using the principle of antibodies that specifically bind to antigens in living tissues. Visualization of antibody-antigen interactions can be achieved by several methods well known to those skilled in the art. In the most common example, antibodies are conjugated to enzymes such as peroxidase that can catalyze a color reaction, or tagged with fluorescent dyes such as fluorescein or rhodamine. Immunohistochemistry can be divided into two phases: sample preparation and sample labeling.

[0095] Sample preparation is crucial for preserving cell morphology, tissue structure, and the antigenicity of the target epitope. This requires proper tissue collection, fixation, and sectioning. Paraformaldehyde solutions are often used to fix tissue, but other methods may be employed. The tissue can then be used in slices or whole, depending on the purpose of the experiment or the tissue itself. Before sectioning, tissue samples may be embedded in a medium such as paraffin wax or cryomedia. Sections can be sliced ​​using various instruments, most commonly microtomes, cryostats, or compression tome tissue slicers. Specimens are typically sliced ​​in the range of 3 μm to 50 μm. The slices are then mounted on slides, dehydrated using increasing concentration alcohol washes (e.g., 50%, 75%, 90%, 95%, 100%), cleared using a detergent such as xylene, and then imaged under a microscope. Depending on the fixation and tissue preservation method, the sample may require additional steps, including deparaffinization and antigen retrieval, to make the epitope available for antibody binding. Formalin-fixed, paraffin-embedded tissues often require antigen retrieval, which involves pretreatment of sections with heat or protease. These steps may result in differences between targeted antigen staining and unstained tissue. Depending on the tissue type and antigen detection method, it may be necessary to block or quench endogenous biotin or enzymes, respectively, before antibody staining. Antibodies exhibit preferential binding activity to specific epitopes, but may partially or weakly bind to nonspecific protein sites (also called reaction sites) that resemble homologous binding sites on the target antigen. To reduce background staining in IHC, samples are incubated with a buffer that blocks reaction sites to which primary or secondary antibodies may bind. Common blocking buffers include ordinary serum, skim milk powder, BSA, or gelatin. Methods for removing background staining include diluting primary or secondary antibodies, changing incubation time or temperature, and using different detection systems or different primary antibodies.Quality control should include, at a minimum, a positive control of tissue known to express the antigen, a negative control of tissue known not to express the antigen, and a test tissue probed in the same manner using the omission (or better, absorption) of the primary antibody.

[0096] In immunohistochemical detection strategies, antibodies are classified as primary or secondary reagents as needed. Primary antibodies are produced against the target antigen and are typically unconjugated (i.e., unlabeled), while secondary antibodies are produced against the immunoglobulin of the primary antibody species. Secondary antibodies are usually labeled and / or fused to the detection entity as described above.

[0097] The direct method is a one-step staining method that includes a labeled antibody that directly reacts with the antigen in the tissue section. Because this method uses only one antibody, it is simple and rapid, but in contrast to indirect approaches, it has low sensitivity due to minimal signal amplification.

[0098] The indirect method involves an unlabeled primary antibody (layer 1) that binds to the target antigen in the tissue, and a labeled secondary antibody (layer 2) that reacts with the primary antibody. The secondary antibody must be produced against the IgG of the animal species from which the primary antibody was produced. This method is more sensitive than direct detection strategies due to signal amplification caused by the binding of several secondary antibodies to each primary antibody when the secondary antibody is conjugated to a fluorescent or enzyme reporter. Further amplification can be achieved when the secondary antibody is conjugated to multiple biotin molecules, and complexes of avidin, streptavidin, or neutraavidin protein-conjugating enzymes can be recruited.

[0099] Preferably, the presence of immunosuppressive CAFs is determined by evaluating protein expression levels using FACS, or by immunohistochemistry as described in the experimental section.

[0100] Antibodies that can be used to measure FAP expression levels in CAF by FACS or immunohistochemistry include, for example, the anti-human FAP antibody (R&D Systems, Inc.) reference #MAB3715, ab53066 (abcam, Inc.), ABIN560844, and Vitatex-MABS1001.

[0101] Antibodies that can be used to measure ANTXR1 expression levels in CAF by FACS or immunohistochemistry include, for example, the anti-ANTXR1-AF405 antibody (reference #NB-100-56585, Novus Biological), human TEM8 / ANTXR1 antibody MAB3886 (R&Dsystem), ABIN252539, ANTXR1 antibody (15091-1-AP, Thermo Fischer), NB-100-56585 (Novus), MA1-91702 (Thermo Fischer), ab21270 (Abcam), LS-B13896 (Lifespan Bioscience), rb158588 (Biorbyt), bs-5210R (Bioss), or any of these antibodies with alternative labeling, particularly fluorescent labeling.

[0102] An antibody that can be used to measure LAMP5 expression levels in CAF by FACS or immunohistochemistry is, for example, the anti-LAMP5-PE antibody reference #130-109-156 (Miltenyi Biotech).

[0103] An antibody that can be used to measure the SDC1 expression level of CAF by FACS or immunohistochemistry is, for example, the anti-SDC1-BUV737 antibody, reference #BD-564393 (BD ​​Biosciences), ABIN5680139.

[0104] Antibodies that can be used to measure the CD9 expression level of CAF by FACS or immunohistochemistry include, for example, the anti-CD9-BV711 antibody reference #BD-743050 (BD Biosciences), LS-B5962 (LSBio), or AB_2075893 (BioLegend).

[0105] In a preferred embodiment, ANTXR1 is detected by FACS in patient cancer samples. + CAF detection may include the following steps: - For example, CD45 + cells, EpCAM + Cells and CD31 + By removing the cells, CD45 - EpCAM - CD31 - Exclusion of non-CAF cells by cell selection, and / or - Selection of CAF cells, e.g., CD29 + Cells and / or PDGFRb + Cell selection, preferably CD29 + Cell selection, - Depending on the case, selection of CAF-S1 cells, e.g., CD29 + FAP + , αSMA + , PDGFRβ + and / or FSP1 + Cells, preferably FAP + Cell selection, - In some cases, dead cells can be excluded by using intracellular dyes such as violet LIVE / DEAD dye or DAPI, and by excluding stained cells. - ANTXR1 in cells obtained in the previous step + CAF detection, - Detection of LAMP5, SDC1, and / or CD9-positive or negative cells, depending on the case.

[0106] In another preferred embodiment, immunohistochemistry of ANTXR1 in patient cancer samples is performed. + CAF detection may include the following steps: - For example, identification of CAF in cancer samples based on morphological criteria, - Preferably, ANTXR1 antibody-based CAF based on immunohistochemical staining. + CAF detection, - In some cases, preferably based on FAP antibody immunostaining, FAP is selected from among CAFs. + CAF detection, - Detection of LAMP5, SDC1, and / or CD9-positive or negative cells, depending on the case.

[0107] In one embodiment, the presence of immunosuppressive fibroblasts, particularly CAFs, is associated with the amount of immunosuppressive fibroblasts, particularly CAFs, in the tumor sample or any fraction thereof, and the ANTXR1 ratio of the total number of cells in the sample or any fraction thereof. + Fibroblasts, especially ANTXR1 + This refers to the ratio or proportion of cell numbers in CAF, and the ANTXR1 ratio relative to the total number of stromal cells in the sample or any fraction thereof. + Fibroblasts, especially ANTXR1 + The ratio or percentage of CAF cell numbers, and ANTXR1 relative to the total number of fibroblasts in the sample. + Ratio or percentage of fibroblast cell numbers, ANTXR1 relative to the total number of fibroblasts in the sample + The ratio or percentage of fibroblast cell numbers, or the ratio of ANTXR1 to the total number of CAF cells in the sample or any fraction thereof. + The ratio or percentage of CAF cell numbers, or ANTXR1 relative to the total number of CAF-S1 cells in the sample or any fraction thereof. + This refers to the ratio or percentage of CAF cells in a given area of ​​function (CAF).

[0108] Preferably, "ANTXR1 in tumors" + The term "percentage of fibroblasts" refers to ANTXR1 + This can refer to any ratio having the number of fibroblasts as the numerator (nominator) and the number of reference cells as the denominator. Preferably, "ANTXR1 in tumors" + The term "CAF percentage" refers to ANTXR1 +This can refer to any ratio having the number of CAF cells as the numerator (nominator) and the number of reference cells as the denominator. In cancer samples, such reference cells can be selected from the group consisting of all cells in the cancer sample, cancer cells, stromal cells, CAF cells, and CAF-S1 cells.

[0109] "A low percentage of ANTXR1 + Fibroblasts or low percentage of ANTXR1 + Fibroblasts correspond to less than 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1%.

[0110] "A high percentage of ANTXR1 + "Fibroblasts" or "High percentage of ANTXR1 + Fibroblasts account for 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more than 90%.

[0111] In the first embodiment, ANTXR1 in the patient's cancer sample + The presence of fibroblasts, particularly CAFs, is associated with ANTXR1 in relation to the total number of cells in cancer samples. + Represents the number of fibroblasts, particularly CAF cells. Preferably, ANTXR1 + The presence of fibroblasts, particularly CAFs, corresponds to a proportion of at least 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20%, or 30%.

[0112] In the second embodiment, ANTXR1 in the patient's cancer sample + The presence of fibroblasts, particularly CAFs, is associated with ANTXR1 in relation to the total number of stromal cells in cancer samples. + Represents the number of fibroblasts, particularly CAF cells. Preferably, ATXR1 + The presence of fibroblasts, particularly CAFs, corresponds to a proportion of at least 0.001%, 0.1%, 1%, 5%, 10%, 20%, or 30%.

[0113] In the third aspect, ANTXR1 in the patient's cancer sample + The presence of fibroblasts, particularly CAFs, is associated with the total number of CAF cells in cancer samples, and the ANTXR1+ Represents the number of fibroblasts, particularly CAF cells. Preferably, ATXR1 + The presence of fibroblasts, particularly CAFs, corresponds to at least 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, or 50%.

[0114] In the fourth aspect, ANTXR1 in the patient's cancer sample + The presence of fibroblasts, particularly CAFs, is associated with the total number of CAF-S1 cells in cancer samples, and ANTXR1 + Represents the number of fibroblasts, particularly CAF cells. Preferably, ATXR1 + The presence of fibroblasts, particularly CAFs, corresponds to at least 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, or 50%.

[0115] In one embodiment, ANTXR1 in cancer samples + The presence of fibroblasts, particularly CAFs, for example, ANTXR1 in cancer samples. + The proportion of fibroblasts, particularly CAFs, is inversely proportional to the patient's response to immunotherapy, and in some cases, the method is suitable for immunotherapy as ANTXR1 in cancer samples. + Selecting patients who have no or low proportion of fibroblasts, particularly CAF, and / or ANTXR1 + ANTXR1 in cancer samples for alternative therapies such as immunotherapy combined with treatments that reduce fibroblast-induced immunosuppression, particularly CAF, or anticancer therapy that excludes immunotherapy. + This further includes selecting patients with a moderate or high percentage of fibroblasts, particularly CAFs.

[0116] "Inverse proportion" refers to ANTXR1 in cancer samples. + A higher number of fibroblasts, particularly CAFs, indicates that the patient with cancer is less likely to respond to immunotherapy. These terms do not necessarily represent direct, measurable correlations.

[0117] In one aspect, the present invention relates to an in vitro method for predicting the response of a cancer patient to immunotherapy, wherein the method involves ANTXR1 in a patient's cancer sample. + This includes detecting fibroblasts, particularly CAFs, and ANTXR1 in cancer samples. + Fibroblasts, particularly CAFs, indicate or predict patient non-responsiveness to immunotherapy. In particular, ANTXR1 detected in cancer samples. + The more fibroblasts, especially CAFs, a patient has, the less susceptible they are to responding to immunotherapy.

[0118] Depending on the case, ANTXR1 + Fibroblasts are ANTXR1 + It is CAF. In some cases, ANTXR1 + Fibroblasts are FAP + These are fibroblasts. In some cases, ANTXR1 + CAF is FAP + ANTXR1 + This is CAF.

[0119] Indeed, the present invention relates to the use of ANTXR1, particularly CAF, in a fibroblast population as a biomarker for tumors of a target affected by cancer, in order to predict the target's response to immunotherapy.

[0120] In some cases, the present invention relates to an in vitro method for predicting the response of a cancer patient to immunotherapy, the method comprising (a) ANTXR1 in a patient's cancer sample + Fibroblasts, especially ANTXR1 + This includes detecting CAF in cancer samples, and ANTXR1 + (b) Fibroblasts, particularly CAFs, may indicate or predict patient nonresponsiveness to immunotherapy, and in some cases, ANTXR1 may be suitable for immunotherapy. + Fibroblasts, especially ANTXR1 + Select patients who do not have CAF, and / or ANTXR1 + Fibroblasts, especially ANTXR1 +For alternative therapies such as immunotherapy combined with treatments that reduce CAF-induced immune expression, or anticancer therapy excluding immunotherapy, ANTXR1 + Fibroblasts, especially ANTXR1 + This includes selecting patients who have CAF fibroblasts.

[0121] In some cases, the method involves FAP of the patient's cancer sample. + ANTXR1 + This includes detecting fibroblasts or CAFs. Depending on the case, the method may involve ecm-myCAF(ANTXR1) from the patient's cancer sample. + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and / or wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - This includes detecting CAF. In some cases, the method involves detecting TGFb-myCAF(ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / - This includes detecting CAF. Depending on the case, the method may involve the patient's cancer sample being wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / -This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves detecting TGFb-myCAF(ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF.

[0122] In one embodiment, CAF subpopulations are detected by specific gene signatures. A “gene signature” or “gene expression signature” is a single gene or group of genes in a cell, particularly a fibroblast, that has a unique and characteristic pattern of gene expression. In particular, gene signatures correspond to the deregulation of specific genes, especially gene overexpression.

[0123] In one embodiment, the ecm-myCAF gene signature includes the following genes: ASPN, COL3A1, THY1, SFRP2, COL10A1, COL6A3, LRRC17, CILP, GRP, ITGBL1, COL8A1, COL14A1, ADAM12, OLFML2B, ELN, PLPP4, CREB3L1, FBN1, LOXL1, MATN3, LRRC15, COMP, ISLR, P3H1, COL11A1, SEPT11, NBL1, SPON1, SULF1, FNDC1, CNN1, MIAT, MMP23B, CPXM1, FIBIN, P4HA3, GXYLT2, CILP2, P3H4, and CCDC80. In certain embodiments, the ecm-myCAF gene signature further includes the following genes: FAP and / or ANTXR1.

[0124] In one embodiment, the TGFβ-myCAF gene signature includes the following genes: CST1, LAMP5, LOXL1, EDNRA, TGFB1, TGFB3, TNN, CST2, HES4, COL10A1, ELN, THBS4, NKD2, OLFM2, COL6A3, LRRC17, COL3A1, THY1, HTRA3, TMEM204, SEPT11, COMP, TNFAIP6, ID4, GGT5, INAFM1, CILP, and OLFML2B. In certain embodiments, the TGFβ-myCAF gene signature further includes the following genes: FAP and / or ANTXR1.

[0125] In one embodiment, the wound-myCAF gene signature includes the following genes: SFRP4, CCDC80, OGN, DCN, PTGER3, SFRP2, PDGFRL, SMOC2, MMP23B, CPXM2, COL14A1, ITGBL1, WISP2, CILP2, COL8A1, GAS1, COL3A1, OMD, COL11A1, CILP, NEXN, ASPN, RARRES2, FIBIN, TMEM119, KERA, ID4, GRP, COMP, DPT, ELN, FBLN2, IGF1, and IGF2. In certain embodiments, the wound-myCAF gene signature further includes the following genes: FAP and / or ANTXR1.

[0126] Accordingly, in the methods of this disclosure, ecm-myCAF, TGFβ-myCAF, and / or wound-myCAF are detected by determining the expression of genes containing gene signatures specific to ecm-myCAF, TGFβ-myCAF, and / or wound-myCAF, and their overexpression indicates the presence of these CAF subpopulations. The gene signature may include additional genes, but may include no more than 50 genes, particularly no more than 40 genes.

[0127] The present invention further relates to the use of these gene signatures to detect ecm-myCAF, TGFβ-myCAF, and / or wound-myCAF subpopulations, particularly to detect the presence of immunosuppressive CAFs, and / or to predict the response to immunotherapy.

[0128] In some cases, the sample may be treated to remove non-CAF cells before determining the expression of the gene signature.

[0129] "Overexpressed" or "overexpressed" refers to the expression level measured at the nucleic acid level, particularly at the mRNA level. Expression levels can be measured by any method well known to those skilled in the art. A gene is overexpressed if its expression increases by at least log2 compared to a reference level. The reference level may be the expression of a gene in control cells or cells, where the control is, for example, a population of fibroblasts, particularly CAFs, preferably FAPs. + This is a population of CAFs. In a particular embodiment, the control cells are clusters of CAFs that are not ecm-myCAF (cluster 0), TGFβ-myCAF (cluster 3), and / or wound-myCAF (4), such as detox-iCAF (cluster 1), IL-iCAF (cluster 2), IFNγ-iCAF (cluster 5), IFNαβ-myCAF (cluster 6), and acto-myCAF (cluster 7), or any combination thereof.

[0130] These gene signatures, specific to the ecm-myCAF, TGFβ-myCAF, and / or wound-myCAF subpopulations, can be used in combination with other gene signatures well known to those skilled in the art, particularly those related to response prediction or cancer diagnosis.

[0131] In another embodiment, the present invention relates to an in vitro method for predicting the response of a cancer patient to immunotherapy, the method being: (a) ANTXR1 in patient cancer samples + Fibroblasts, especially ANTXR1 + Detecting CAF, (b) ANTXR1 in cancer samples + Fibroblasts, especially ANTXR1 + The proportion of CAFs is determined, and the patient's response to immunotherapy is determined by ANTXR1 in cancer samples. + Fibroblasts, especially ANTXR1 + The ratio is inversely proportional to the CAF ratio. (c) In some cases, ANTXR1 suitable for immunotherapy treatment + Fibroblasts, especially ANTXR1 + In certain patients without CAF, a low percentage of ANTXR1 + Fibroblasts, especially ANTXR1 + Select patients with CAF and ANTXR1 + Fibroblasts, especially ANTXR1 + Regarding alternative therapies such as immunotherapy combined with treatments that reduce CAF-induced immunosuppression, or anticancer therapy without immunotherapy, a moderate or high percentage of ANTXR1 + Fibroblasts, especially ANTXR1 + Select patients with CAF. Includes.

[0132] In some cases, the method involves FAP of the patient's cancer sample. + ANTXR1 + This includes detecting fibroblasts or CAFs. Depending on the case, the method may involve ecm-myCAF(ANTXR1) from the patient's cancer sample.+ SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and / or wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - This includes detecting CAF. In some cases, the method involves detecting TGFb-myCAF(ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / - This includes detecting CAF. Depending on the case, the method may involve the patient's cancer sample being wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves detecting TGFb-myCAF(ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1- LAMP5 - CD9 + This includes detecting CAF. In some cases, the method involves ecm-myCAF(ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + ) including detecting CAF. Optionally, the method may further include the step of providing a cancer sample from this patient prior to step (a).

[0133] In some cases, the method involves a low percentage of ANTXR1 + The method may further include administering immunotherapy to patients with fibroblasts or CAF. Alternatively, the method may involve a high percentage of ANTXR1 + The process may further include administering an alternative anti-cancer therapy to a patient with fibroblasts or CAF, wherein the alternative therapy is ANTXR1 + This is an anti-cancer treatment that combines immunotherapy with treatment to reduce immunosuppression induced by fibroblasts or CAFs, or an anti-cancer treatment that does not involve immunotherapy. The treatment administered may be selected from a group consisting of surgery, chemotherapy, radiotherapy, hormone therapy, targeted therapy and palliative care, or any combination thereof. Alternatively or additionally, the method may involve ANTXR1 + The procedure may further include administering an immunotherapy treatment in combination with a treatment that reduces immunosuppression induced by fibroblasts or CAFs.

[0134] In another particular aspect, the present invention also relates to a method for selecting patients with tumors for immunotherapy treatment or for determining whether patients with tumors are likely to benefit from immunotherapy treatment, the method comprising ANTXR1 in the patient's cancer sample + To determine the presence of fibroblasts or CAFs, and, if necessary, for immunotherapy treatment, ANTXR1 +Patients without fibroblasts or CAF, or with a low percentage of ANTXR1 + This includes selecting patients with fibroblasts or CAF.

[0135] In some cases, the method further includes a preliminary step of providing a cancer sample from this patient.

[0136] In yet another specific embodiment, the present invention also applies to ANTXR1 + Regarding methods for selecting patients with tumors for alternative therapies such as immunotherapy combined with treatments that reduce immunosuppression induced by fibroblasts or CAFs, or anticancer therapies other than immunotherapy, or regarding patients with tumors who have ANTXR1 + The method relates to a method for determining whether a patient is likely to benefit from an alternative treatment such as immunotherapy combined with a treatment that reduces immunosuppression induced by fibroblasts or CAFs, or from an anticancer treatment other than immunotherapy, wherein the patient's cancer sample is subjected to ANTXR1 + This includes determining the presence of fibroblasts or CAFs, and, if applicable, regarding such alternative therapies, ANTXR1 + Patients with CAF or a high percentage of ANTXR1 + This includes selecting patients with fibroblasts or CAF.

[0137] Depending on the case, ANTXR1 + Fibroblasts or CAFs are FAP + ANTXR1 + Fibroblasts or CAFs. Preferably, ANTXR1 + CAF is FAP + ANTXR1 + CAF, FAP + ANTXR1 + LAMP5 - SDC1 + CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF and ANTXR1 + SDC1 - LAMP5 - CD9+ Selected from the group consisting of FAP + ANTXR1 + LAMP5 - SDC1 + CAF and / or FAP + ANTXR1 + LAMP5 + SDC1 + / - This is CAF.

[0138] Depending on the case, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - )CAF. In some cases, ANTXR1 + CAF is TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - )CAF. In some cases, ANTXR1 + CAF is wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ) and TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - )CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1 + CAF is TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / -) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF.

[0139] In yet another embodiment, the present invention relates to a method for selecting patients for immunotherapy, wherein the method is (a) ANTXR1 in patient cancer samples + To detect fibroblasts or CAFs, (b) ANTXR1 in cancer samples + Determine the proportion of fibroblasts or CAFs. Includes ANTXR1 + A CAF rate of less than 25%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, even more preferably less than 1%, and even more preferably less than 0.5% predicts the patient's response to immunotherapy treatment.

[0140] This method may further include step c) administering an immunotherapy, preferably an immune checkpoint inhibitor.

[0141] In some cases, the method further includes a preliminary step of providing a cancer sample from this patient.

[0142] In yet another embodiment, the present invention relates to a method for selecting patients for alternative anti-cancer treatment, particularly excluding immunotherapy, or the method is (a) ANTXR1 in patient cancer samples+ To detect fibroblasts or CAFs, (b) ANTXR1 in cancer samples + Determine the proportion of fibroblasts or CAFs. Includes ANTXR1 + A CAF rate of more than 1%, preferably more than 5%, more preferably more than 10%, even more preferably more than 20%, even more preferably more than 30%, and even more preferably more than 40% indicates or predicts patient nonresponsiveness to immunotherapy treatment.

[0143] The method may further include (c) administering an alternative treatment selected from the group consisting of surgery, chemotherapy, radiotherapy, hormone therapy, targeted therapy and palliative care or any combination thereof. Alternatively or additionally, the method may include ANTXR1 + The procedure may further include administering an immunotherapy treatment in combination with a treatment that reduces immunosuppression induced by fibroblasts or CAFs.

[0144] In some cases, the method further includes a preliminary step of providing a cancer sample from this patient.

[0145] The present invention further relates to immunotherapy for use in treating cancer in patients, wherein the patient (a) has a low number or percentage of ANTXR1 + (b) Having fibroblasts or CAF or ANTXR1 + The present invention presents cancer samples that do not have fibroblasts or CAFs. The present invention also relates to the use of immunotherapy for manufacturing pharmaceuticals for the treatment of cancer in patients, wherein the patient (a) has a low number or percentage of ANTXR1 + (b) Having fibroblasts or CAF or ANTXR1 + The present invention presents cancer samples that do not contain fibroblasts or CAFs. The present invention further relates to a method for treating cancer in a patient, the method comprising (a) a low number or proportion of ANTXR1 + (b) Selection of patients who have fibroblasts or CAFs, or (b) ANTXR1+ patients who do not have fibroblasts or CAFs, and administration of a therapeutically effective dose of immunotherapy.

[0146] Depending on the case, ANTXR1 + Fibroblasts or CAFs are FAP + ANTXR1 + These are fibroblasts or CAFs. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - )CAF. In some cases, ANTXR1 + CAF is TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - )CAF. In some cases, ANTXR1 + CAF is wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ) and TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - )CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1 + CAF is TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1+ CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF.

[0147] immunotherapy treatment The immunosuppressive fibroblasts according to the present invention, particularly immunosuppressive CAFs, can be used to predict or evaluate the response to immunotherapy.

[0148] Preferably, immunotherapy is selected from the group consisting of immunization of the patient with tumor antigens by therapeutic measures that stimulate the patient's immune system to attack malignant tumor cells, such as administration of cancer vaccines, administration of immune system-stimulating molecules such as cytokines, administration of therapeutic antibodies as drugs, preferably monoclonal antibodies, in particular antibodies against antigens specifically presented or overexpressed on the membrane of tumor cells, or antibodies directed against cell receptors that block or prevent tumor growth, adoptive T cell therapy, immune checkpoint inhibitor therapy, and any combination thereof, preferably immune checkpoint inhibitor therapy.

[0149] In preferred embodiments, immunotherapy is immune checkpoint inhibitor therapy, preferably selected from the group consisting of antibodies against anti-CTLA-4 (cytotoxic T lymphocyte-associated protein 4) such as ipilimumab, antibodies against PD-1 (programmed cell death protein 1) such as nivolumab, pembrolizumab, or BGB-A317, antibodies against PDL1 (programmed cell death ligand) such as atezolizumab, avelumab, or durvalumab, antibodies against LAG-3 (lymphocyte activation gene 3) such as BMS-986016, antibodies against TIM-3 (T cell immunoglobulin and mucin domain-3), antibodies against TIGIT (T cell immune receptor having Ig and ITIM domains), antibodies against BLTA (B and T lymphocyte attenuator), IDO1 inhibitors such as epacadostat, or combinations thereof.

[0150] In the most preferred embodiment, the immunotherapy is an immune checkpoint inhibitor therapy, wherein the immune checkpoint is selected from the group consisting of antibodies against cytotoxic T lymphocyte-associated protein (CTLA-4), programmed cell death protein 1 (PD-1), programmed cell death ligand (PD-L1), T cell immune receptor having Ig and ITIM domains (TIGIT), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain-containing TIM-3 (TIM-3), B- and T lymphocyte attenuator (BLTA), IDO1 inhibitor, or any combination thereof, preferably selected from the group consisting of antibodies against CTLA-4, PD-1 and TIGIT, or any combination thereof, more preferably selected from the group consisting of antibodies against PD-1 or CTLA-4 and combinations thereof.

[0151] Some anti-PD-1 antibodies have already been clinically approved, while others are still in clinical development. For example, anti-PD-1 antibodies include pembrolizumab (also known as keytruda / lambrolizumab, MK-3475), nivolumab (Opdivo, MDX-1106, BMS-936558, ONO-4538), pizilizumab (CT-011), semiplimab (ribtayo), camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317 (tisreizumab), and PDR0 01 (Spartalizumab), MK-3477, SCH-900475, PF-06801591, JNJ-63723283, Genolimuzumab (CBT-501), LZM-009, BCD-100, SHR-1201, BAT-1306, AK-103 (HX-008), MEDI-0680 (also known as AMP-514), MEDI0608, JS001 (Si-Yang Liu et al., J. (See Hematol.Oncol.10:136(2017)), BI-754091, CBT-501, INCSHR1210 (also known as SHR-1210), TSR-042 (also known as ANB011), GLS-010 (also known as WBP3055), AM-0001 (ALMO), STI-1110 (see International Publication No. 2014 / 194302), AGEN2034 (see International Publication No. 2017 / 040790), MGA012 ( You can choose from the group consisting of (see International Publication No. 2017 / 19846), or IBI308 (see International Publication Nos. 2017 / 024465, 2017 / 025016, 2017 / 132825, and 2017 / 133540), monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, 5F4, as described in International Publication No. 2006 / 121168. Other bifunctional or bispecific molecules that target PD-1 include RG7769 (Roche), XmAb20717 (Xencor), MEDI5752 (AstraZeneca), FS118 (F-star), SL-279252 (Takeda Pharmaceutical), and XmAb23104 (Xencor).

[0152] Antibodies against CTLA-4 and bifunctional or bispecific molecules that target CTLA-4 are also known, including ipilimumab, tremelimumab, MK-1308, AGEN-1884, XmAb20717 (Xencor), and MEDI5752 (AstraZeneca).

[0153] Antibodies against TIGIT are also well known in the art, for example, as disclosed in International Publication No. 19232484, such as BMS-986207 or AB154, BMS-986207 CPA.9.086, CHA.9.547.18, CPA.9.018, CPA.9.027, CPA.9.049, CPA.9.057, CPA.9.059, CPA.9.083, CPA.9.089, CPA.9.093, CPA.9.101, CPA.9.103, CH A.9.536.1, CHA.9.536.3, CHA.9.536.4, CHA.9.536.5, CHA.9.536.6, CHA.9.536.7, CHA.9.536.8, CHA.9.560.1, CHA.9.560.3, CHA.9.560.4, CHA.9.5 60.5, CHA.9.560.6, CHA.9.560.7, CHA.9.560.8, CHA.9.546.1, CHA.9.547.1, CHA.9.547.2, CHA.9.547.3, CHA.9.547.4, CHA.9.547.6, CHA.9.547.7, CHA.9.547.8, CHA.9.547.9, CHA.9.547.13, CHA.9.541.1, CHA.9.541.3, CHA.9.541.4, CHA.9.541.5, CHA.9.541.6, CHA.9.541.7, and CHA.9.541.8. Anti-TIGIT antibodies are also listed in International Publication Nos. 16028656, 16106302, 16191643, 17030823, 17037707, 17053748, 17152088, 18033798, 18102536, 18102746, and 18160704. It is disclosed in International Publication Nos. 18200430, 18204363, 19023504, 19062832, 19129221, 19129261, 19137548, 19152574, 19154415, 19168382, and 19215728.

[0154] Preferably, the immunotherapy agent is selected from the group consisting of ipilimumab, nivolumab, BGB-A317, pembrolizumab, atezolizumab, avelumab or durvalumab, BMS-986016, and epacadostat, or any combination thereof.

[0155] ANTXR1 + Fibroblast or CAF targeting agent In another aspect, the present invention also relates to ANTXR1 for use in combination with immunotherapy for the treatment of subjects having cancer, or for use in subjects having cancer. + FAP, a drug that targets fibroblasts + The present invention relates to a drug that targets fibroblasts, or a pharmaceutical composition containing the same, and the subject is particularly a tumor sample of the subject that contains ANTXR1 + It contains fibroblasts.

[0156] In fact, ANTXR1 + Fibroblasts, especially ANTXR1 + Treatments that reduce immunosuppression induced by CAF include, for example, ANTXR1 + This could be a drug that targets fibroblasts or CAFs, or a FAP inhibitor.

[0157] This invention also relates to ANTXR1 + CAF or FAP + The present invention relates to a drug that targets CAF, or a pharmaceutical composition containing the same, for use in combination with immunotherapy for the treatment of subjects with cancer, or for use on subjects with cancer, wherein the subject is particularly targeted to ANTXR1 in the tumor sample of the subject. + It has CAF. More specifically, when using FAP inhibitors, the target is ANTXR1 + FAP + Fibroblasts, especially ANTXR1 + FAP + It has CAF.

[0158] The present invention also relates to the manufacture of pharmaceuticals for the treatment of subjects with cancer, optionally combined with ANTXR1 immunotherapy. +CAF or FAP + Regarding the use of CAF-targeting drugs, in particular, the target tumor sample contains ANTXR1 + It has fibroblasts or CAF. In a further embodiment, the present invention particularly relates to a target tumor sample, a target having cancer, particularly ANTXR1 + ANTXR1 for the treatment of subjects with fibroblasts or CAF + CAF or FAP + This invention relates to the use of a combination of drugs that target CAF. The present invention relates to a method for treating a subject with cancer, wherein the method involves ANTXR1 + Select patients with fibroblasts or CAF, ANTXR1 + CAF or FAP + The method includes administering a therapeutically effective dose of a CAF-targeting agent. The method may further include administering a therapeutically effective dose of immunotherapy. The present invention also relates to patients, particularly those with ANTXR1 + a) ANTXR1 + CAF or FAP + b) Products or kits containing agents that target CAFs, and immunotherapeutic agents. In particular, the subjects are cancer samples containing a moderate or high proportion of ANTXR1 + The tumor sample contains fibroblasts or CAFs.

[0159] This invention also relates to ANTXR1 + CAF or FAP + A method for selecting patients with cancer for treatment with CAF-targeting agents, the method comprising detecting immunosuppressive fibroblasts or CAFs as disclosed above, and the patient having ANTXR1 in the patient's cancer sample. + This includes selecting patients who have fibroblasts or CAF.

[0160] In particular, the drug ANTXR1 + It suppresses or reduces the immunosuppressive effect of fibroblasts or CAFs.

[0161] Preferably, the drug is selected from the group consisting of ANTXR1 inhibitors, anti-ANTXR1 antibodies which may be conjugated with a cytotoxic drug, multispecific molecules containing an anti-ANTXR1 moiety, anti-ANTXR1 T cell receptors (TCRs), anti-ANTXR1 chimeric antigen receptors (CARs), and immune cells expressing anti-ANTXR1 TCRs or anti-ANTXR1 CARs, preferably T cells, or any combination thereof.

[0162] Additionally or alternatively, the drug is selected from the group consisting of FAP inhibitors, anti-FAP antibodies which may be conjugated with cytotoxic drugs, multispecific molecules containing an anti-FAP moiety, anti-FAP T cell receptors (TCRs), anti-FAP chimeric antigen receptors (CARs), and immune cells expressing anti-FAP TCRs or anti-FAP CARs, preferably T cells, or any combination thereof.

[0163] In particular, ANTXR1 inhibitors may be, for example, ebselen or phenylmercury acetate.

[0164] FAP inhibitors (FAPIs) may be selected from the group consisting of tarabostat, PT-100, linagliptin (Tradjenta), FAP inhibitors having an N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) skeleton as disclosed in Jansen et al., ACS Med Chem Lett 2013, May 9;4(5):491-496, MIP-1232 as described in Mueller et al., J Biol Chem 1999;274:24947-52, and FAPI-02 and FAPI-04 as described in Lindner et al., EJNMMI Radiopharmacy and Chemistry (2019) 4:16 and Lidner et al., Journal of Nuclear Medicine, published April 6, 2018. For example, FAP inhibitors are described in International Publication Nos. 20081522, 20132661, 20245173, 21005125, 21005131, U.S. Publication No. 2020246383, 19154859, 19083990, 19118932, 18111989, 17189569, 13107820, 08116054, or 07085895, which are incorporated herein by reference. FAP inhibitors can also be FAP-binding peptides linked to radionuclides (e.g., lutetium-177), such as the FAP inhibitor FAP-2286 developed by Clovis Oncology.

[0165] In certain embodiments, the present invention relates to the use of FAP inhibitors for the treatment of subjects having cancer or for use in the therapy of subjects having cancer, wherein the subjects are particularly present in tumor samples of the subjects or in the tumor microenvironment. + It contains fibroblasts or CAFs.

[0166] In one embodiment, the drug is an anti-ANTXR1 antibody or an anti-FAP antibody. The antibody according to the present invention can be any type of antibody. In particular, the antibody may consist of, or essentially consist of, a classical Y-shaped antibody having two heavy and light chains or a fragment thereof. Preferably, the fragment includes the antigen-binding region or variable region of the antibody. This fragment can be selected from the group consisting of, but are not limited to, Fv, Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv, single-chain Fv(scFv), di-scFv or sc(Fv)2, dsFv, Fd, dAb, CDR, VH, VL, VHH, V-NAR, nanobody, minibody, diabody, and multispecific antibodies formed from antibody fragments.

[0167] The antibody according to the present invention may be a monomeric antibody or a polymeric antibody. In particular, the antibody according to the present invention is a monomeric antibody.

[0168] The antibody according to the present invention may be monoclonal or polyclonal. Preferably, the antibody according to the present invention is monoclonal.

[0169] In another preferred embodiment, the targeting agent is an anti-ANTXR1 or anti-FAP antibody, or peptide-bound ANTXR1 or FAP, conjugated to a drug, and preferably a cytotoxic drug. The anti-ANTXR1 antibody or anti-FAP antibody may optionally have inhibitory or antagonistic activity.

[0170] The drugs according to the present invention are preferably cytotoxic drugs. As used herein, the term “cytotoxic drug” refers to a molecule that, upon contact with a cell and entering the cell, ultimately alters cellular function in a harmful manner (e.g., cell proliferation and / or differentiation and / or metabolism such as protein and / or DNA synthesis) or causes cell death. As used herein, the term “cytotoxic drug” includes toxins, particularly cytotoxics.

[0171] The cytotoxic drugs according to the present invention include drastatin, e.g., drastatin 10, drastatin 15, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), and 5-benzoylvalerate-AE ester (AEVB), meitansine, e.g., ansamitosine, meltansine (also called emtansine or DM1) and labtansine (also called solabtansine or DM4), daunorubicin, and Anthracyclines such as pyrubicin, pirarubicin, idarubicin, zorubicin, serubicin, acrubicin, adliblastin, doxorubicin, mitoxantrone, daunoxom, nemorubicin, and PNU-159682; calicheamicin such as calicheamicin β1Br, calicheamicin gamma1Br, calicheamicin α2I, calicheamicin α3I, calicheamicin β1I, calicheamicin gamma1L, calicheamicin delta1I; esperamycin such as ozogamicin and esperamycin A1; neocartinostatin; bleoma Icin, CC-1065 and duocalmycin A, etc., duocalmycin, anthramycin, abeymycin, tikamicin, DC-81, mazetramycin, neotramycin A and B, etc., pyrrolobenzodiazepines, protracalcin, cibanomycin (DC-102), cibilomycin and tomamycin, pyrrolobenzodiazepine dimers (or PBD), indolino-benzodiazepines, indolino-benzodiazepine dimers, α-amanitin, abraxane, actinomycin, aldesleukin, altretamine, alitretino In, amsacrin, anastrozole, arsenic, asparaginase, azacitidine, azathioprine, bexarotene, bendamustine, bicalutamide, bortezomib, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, chloramphenicol, cyclosporine, cidofovir, coal tar-containing products, colchicine, dacarbazine, dactinomycin, danazol, dasatinib, diethylstilbestrol, dinoprostone, ditranol,Dutasteride, dexrazoxane, docetaxel, doxifluridine, erlotinib, estramustine, etoposide, exemestane, finasteride, flutamide, phloxuridine, flucytosine, fludarabine, fluorouracil, ganciclovir, gefitinib, gemcitabine, goserelin, hydroxyurea, hydroxycarbamide, ifosfamide, irinotecan, imatinib, lenalidomide, leflunomide, letrozole, leuprorelin acetate, lomustine, mechloretamine, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, menotropin, mifepristone, nafarelin, nelarabine, nitrogen mustard, nitrosourea, oxaliplatin, ozogamicin, paclitaxel , podophyllin, pegasparaginase, pemetrexed, pentamidine, pentostatin, procarbazine, raloxifene, ribabalin, larcitrexed, rituximab, romidepsin, sorafenib, streptozosin, sunitinib, sirolimus, streptozosin, temozolomide, temsirolimus, teniposide, thalidomide, thioguanine, thiotepa, topotecan, tacrolimus, taxotere, tafluposide, toremifene, tretinoin, trifluridine, triptorelin, valganciclovir, barrubicin, vinblastine, vidaradin, vincristine, vindesine, vinorelbine, vemurafenib, bismodegib, vorinostat, zidovudine, vedotin, derivatives, and combinations thereof may be selected from the group. Cytotoxic drugs can also be radionuclides such as lutetium-177, iodine-131, samarium-153, and yttrium-90 or astatine-211, bismuth-212, lead-212, bismuth-213, actinium-225, radium-223, and thorium-227.

[0172] In preferred embodiments, the antibody-drug conjugate of the present invention includes a linker between the antibody and the drug. The linker according to the present invention may be cleavable or non-cleavable, and preferably the linker is cleavable. Examples of cleavable linkers according to the present invention include, but are not limited to, disulfides, hydrazones, and peptides. Examples of non-cleavable linkers according to the present invention include, but are not limited to, thioethers.

[0173] In certain embodiments, the drug is linked to a cysteine ​​or lysine residue of the antibody. Preferably, the drug or antigen is linked to a non-natural amino acid incorporated into the antibody.

[0174] Methods for preparing antibody-drug conjugates are well known to those skilled in the art.

[0175] In one embodiment, the agent is an anti-ANTXR1 antibody or anti-FAP CAR cells. As used herein, the terms “chimeric antigen receptor” (CAR), “engineered cell receptor,” “chimeric cell receptor,” or “chimeric immune receptor” (ICR) refer to an engineered receptor that has been implanted with antigen-binding specificity (e.g., an antibody) into immune cells (e.g., T cells or NK cells), and thus combines the antigen-binding properties of the antigen-binding domain with the immunogenic activity of the immune cells, such as the lytic ability and self-regeneration of the immune cells. CAR cells against ANTXR1 are available to those skilled in the art. For example, anti-ANTXR1 CAR T cells have been described in patent applications, Korean Patent No. 1020190013612, U.S. Patent No. 2016264662A, U.S. Patent No. 2017114133A, and Chinese Patent No. 108707199A, as well as in publications such as Byrd et al. (Cancer Res. 2018;78(2):489-500. 10.1158 / 0008-5472.CAN-16-1911).

[0176] The present invention also relates to a pharmaceutical composition comprising an ANTXR1 and / or FAP CAF targeting agent, preferably an FAP inhibitor, an ANTXR1 inhibitor, an anti-FAP antibody, an anti-ANTXR1 antibody, or any combination thereof, wherein the antibody is optionally conjugated with a drug, preferably a cytotoxic drug as described above, or a combination thereof. In particular, such a pharmaceutical composition comprises at least one pharmaceutically acceptable excipient. For this formulation, conventional excipients can be used in accordance with the art well known to those skilled in the art.

[0177] The formulations may be sterilized and, if desired, mixed with pharmaceutically acceptable adjuvants such as carriers, excipients, salts, antioxidants, and / or stabilizers that do not adversely interact with ANTXR1 and / or FAP CAF targeters.

[0178] Depending on the circumstances, the pharmaceutical composition may further include additional therapeutic agents, particularly immunotherapeutic agents as described above.

[0179] Those skilled in the art will understand that the formulations of the present invention may be isotonic with human blood and have essentially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure of about 250 mOSm to about 350 mOSm. Isotonicity can be measured, for example, by vapor pressure or an ice-freezing osmometer.

[0180] ANTXR1 according to the present invention is administered + CAF and / or FAP + The amount of CAF targeting agent or the pharmaceutical composition according to the present invention can be determined by standard procedures well known to those skilled in the art. The patient's physiological data (e.g., age, size, and weight) and route of administration must be taken into consideration to determine an appropriate dose so that a therapeutically effective dose is administered to the patient.

[0181] When the combination preparations, kits, or products for use according to the present invention are administered separately or sequentially, in particular when administered separately, ANTXR1 according to the present invention + CAF and / or FAP +Treatment with CAF-targeting agents is preferably performed before immunotherapy. The treatment method is ANTXR1 according to the present invention. + CAF and / or FAP + After administration of CAF-targeting agents, preferably before administration of immunotherapy, ANTXR1 + CAF and / or FAP + The immunotherapy treatment may further include a step to determine the proportion of CAFs, and the immunotherapy treatment involves ANTXR1 + If the CAF ratio is low, or if ANTXR1 + It is administered only if CAF is not present.

[0182] Use of immunotherapy treatments and therapeutic methods In certain embodiments, the present invention also relates to a patient who has a cancer sample, (a) Low levels or low percentage of ANTXR1 + Fibroblasts, especially ANTXR1 + Present CAF, or (b) ANTXR1 + Fibroblasts, especially ANTXR1 + CAF is not presented. This relates to immunotherapy, preferably immune checkpoint inhibitor therapy, for use in treating cancer in patients.

[0183] This invention also involves a patient using a cancer sample. (a) Low levels or low percentage of ANTXR1 + Presents fibroblasts or CAFs, or (b) ANTXR1 + Fibroblasts or CAFs that do not present This relates to the use of immunotherapy, preferably immune checkpoint inhibitor therapy, for the manufacture of pharmaceuticals for the treatment of cancer.

[0184] The present invention also relates to a method for treating a patient suffering from cancer, wherein the patient, in a sample of the patient's cancer, (a) ANTXR1 in tumor + Low percentage of fibroblasts or CAFs (b) ANTXR1 in the tumor +Absence of fibroblasts or CAFs Selected in the case of The method includes the step of administering an immunotherapy treatment, preferably an immune checkpoint inhibitor treatment, to a patient.

[0185] ANTXR1 + The proportion of fibroblasts or CAFs is as described above. ANTXR1 + Fibroblasts or CAFs are defined as above, cancer is defined as below, and immunotherapy treatment is defined as above.

[0186] Preferably, ANTXR1 + CAF is FAP + ANTXR1 + LAMP5 - SDC1 + CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF and ANTXR1 + SDC1 - LAMP5 - CD9 + Selected from the group consisting of FAP + ANTXR1 + LAMP5 - SDC1 + CAF and / or FAP + ANTXR1 + LAMP5 + SDC1 + / - This is CAF.

[0187] Depending on the case, ANTXR1 + CAF is FAP + ANTXR1 + It is CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - )CAF. In some cases, ANTXR1 + CAF is TGFb-myCAF(ANTXR1 + LAMP5 + SDC1+ / - )CAF. In some cases, ANTXR1 + CAF is wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ) and TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - )CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1 + CAF is TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF.

[0188] Preferably, the immunotherapy is an immune checkpoint inhibitor immunotherapy, preferably selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, and anti-TIGIT antibody or any combination thereof, and more preferably anti-CTLA-4 antibody and / or anti-PD-1 antibody.

[0189] The present invention further relates to a method for treating a target cancer, and a therapeutically effective amount of ANTXR1 according to the present invention. + CAF and / or FAP + A CAF targeting agent, or a pharmaceutical composition containing such an agent, is administered to a subject with cancer, and the patient's cancer or patient's cancer sample is ANTXR1 + CAF is presented, preferably FAP. + ANTXR1 + LAMP5 - SDC1 + CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF and / or ANTXR1 + SDC1 - LAMP5 - CD9 + Preferably FAP + ANTXR1 + LAMP5 - SDC1 + CAF and / or FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF. In particular, the ANTXR1 according to the present invention is administered. + CAF and / or FAP + The amount of CAF targeting agent or the pharmaceutical composition according to the present invention can be determined by standard procedures well known to those skilled in the art. The patient's physiological data (e.g., age, size, and weight) and route of administration must be taken into consideration to determine an appropriate dose so that a therapeutically effective dose is administered to the patient.

[0190] In some embodiments, immunotherapy treatment involves ANTXR1 + FAP, a drug that targets fibroblasts+ A drug targeting fibroblasts and / or a pharmaceutical composition according to the present invention is administered in combination with additional cancer treatments. In particular, immunotherapy, ANTXR1 + FAP, a drug that targets fibroblasts + A drug that targets fibroblasts and / or a pharmaceutical composition according to the present invention can be administered in combination with other targeted therapies, other immunotherapies, chemotherapy and / or radiotherapy.

[0191] In some embodiments, immunotherapy treatment, ANTXR1 + FAP, a drug that targets fibroblasts +A drug targeting fibroblasts and / or a pharmaceutical composition according to the present invention is administered to the patient in combination with chemotherapy. As used herein, the term "chemotherapy" has its general meaning in the art and refers to a treatment consisting of administering chemotherapeutic agents to a patient. Chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocon, metsuredopa and uredopa; altretamine; ethyleneimines and methylamelamamines including triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamamine; acetogenins (especially bratacin and bratacin) Non-, camptothecin (including synthetic analog topotecan), bryostatin, callistin, CC-1065 (including its synthetic analogs adzelesin, karzelesin and bizelesin), cryptophycin (especially cryptophycin 1 and cryptophycin 8), dorastatin, duocalmycin (including synthetic analogs KW-2189 and CB1-TM1), eryuterobin, pancratistatin, sarcodictin, spongistatin, chlorambucil, chlornafadin, cholophosphamide Antibiotics such as estramustine, ifosfamide, mechloretamine, oxidized mechloretamine hydrochloride, melphalan, nobenbitin, phenesterine, prednimustine, trophosphamide, uracil mustard and other nitrogen mustards, carmustine, chlorozotosine, fotemustine, lomustine, nimustine and ranimustine, nitrothreas, antibiotics, for example, engine antibiotics (e.g., calicheamicin, especially calicheamicin gamma and calicheamicin omega Bisphosphonates such as chlordronate, dynemycin, dynemycin A, esperamycin, neocardinostatin chromophore and related chromogenic protein enegyoin antibiotic chromophore, acrasinomycin, actinomycin, autoralnicin, azaserin, bleomycin, kactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,Antimetabolites such as doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins like mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin, methotrexate, and 5-fluorouracil (5-FU). ; Folic acid analogs such as denopterin, methotrexate, pteropterin, trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; androgens such as aminoglutethimide, mitotane, trilostane; anti-adrenal agents such as frolinic acid. Folic acid supplements such as acid, acegraton, aldofamide glycosides, aminolevulinic acid, enyluracil, amsacrin, bestrabusil, bisanthren, edatraxate, defofamin, demecolsin, diazicon, elformitin, eriptinium acetate, epotilon; etoglucide; gallium nitrate; hydroxyurea; lentinan; ronidynin; meitansinoids such as meitansin and ansamitosin; mitogwazone; mitoxantrone ; Mopidammol; Nitraeline; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine; PSK polysaccharide complex); Lazoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2"-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, Beraclin A,Loridine A and Angiidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel and Doxetaxel; Chlorambucil; Gemcitabine; 6-Thiogunine; Mercaptopurine; Methotrexate; Platinum-coordinated complexes such as cisplatin, oxaliplatin and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (e.g., CPT-1); Topoisomerase inhibitor RFS Examples include: difluoromethylromitine (DMFO); retinoids such as retinoic acid; capecitabine; anthracyclines, nitrosourea, antimetabolites, enzymes such as epipodophyllotoxin and L-asparaginase; hormones and antagonists including anthracendione, prednisone and equivalents, corticosteroid antagonists such as dexamethasone and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinylestradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and nonsteroidal antiandrogens such as flutamide; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above. ,

[0192] In some embodiments, immunotherapy treatment, ANTXR1 + FAP, a drug that targets fibroblasts +A drug targeting fibroblasts and / or a pharmaceutical composition according to the present invention is administered to the patient in combination with radiotherapy. Preferred examples of radiotherapy include, but are not limited to, external beam radiotherapy (e.g., superficial X-ray therapy, orthovoltage X-ray therapy, megavoltage X-ray therapy, radiosurgery, stereotactic radiotherapy, fractional stereotactic radiotherapy, cobalt therapy, electron therapy, fast neutron therapy, neutron capture therapy, proton therapy, intensity-modulated radiation therapy (IMRT), three-dimensional conformal radiation therapy (3D-CRT), etc.), close-range radiotherapy, unsealed source radiotherapy, tomotherapy, etc. Gamma rays are another form of photon used in radiotherapy. Gamma rays are spontaneously produced when certain elements (such as radium, uranium, and cobalt-60) decompose or decay and emit radiation. In some embodiments, radiotherapy may be proton therapy or proton minibeam radiotherapy.Proton therapy is a super-precise radiotherapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Control in RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard Proton Therapy. Int J Radiat Oncol Biol Phys. June 1, 2019;104(2):266-271. doi:10.1016 / j.ijrobp.2019.01.080; Prezado Y, Jouvion G, Patriarca A, Nauraye C, Guardiola C, Juchaux M, Lamirault C, Labiod D, Jourdain L, Sebrie C, Dendale R, Gonzalez W, Pouzoulet F. Proton minibeam radiation therapy widens the therapeutic index for high-grade gliomas. Sci Rep. November 7, 2018;8(1):16479. doi:10.1038 / s41598-018-34796-8). The radiotherapy may also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation.FLASH radiotherapy involves delivering radiotherapy at extremely high dose rates, several orders of magnitude higher than currently routine clinical practice (very high dose rates) (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015; 31:121-123. DOI:10.1051 / medsci / 20153102002); Patriarca A., Fouillade CM, Martin F., Pouzoulet F., Nauraye C. et al. Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi:10.1016 / j.ijrobp.2018.06.403.Epub July 11, 2018).

[0193] Patient, regimen and administration The patient is an animal, preferably a mammal, and more preferably a human. However, the patient may also be a non-human animal, particularly a mammal, such as, among other things, a dog, cat, horse, cow, pig, sheep, donkey, rabbit, ferret, gerbil, hamster, chinchilla, rat, mouse, guinea pig, and non-human primate in need of treatment.

[0194] The human patient according to the present invention may be a human in the prenatal stage, a neonatal, child, infant, adolescent or adult, particularly an adult at least 30 years old or at least 40 years old, preferably at least 50 years old, more preferably at least 60 years old, and even more preferably at least 70 years old.

[0195] In one embodiment, the patient is an active smoker or a former smoker.

[0196] Preferably, the patient has been diagnosed with cancer. In another specific embodiment, the patient has metastatic cancer or advanced-stage cancer. In one embodiment, the patient has been diagnosed with stage III or IV cancer.

[0197] In one embodiment, a patient suffering from cancer has metastases, particularly to the brain, liver, bones, and / or kidneys.

[0198] In certain embodiments, the patient has already received at least one line of treatment, particularly one line, two lines, or three or more lines, preferably several lines. Alternatively, the patient has not received any treatment. In particular, the patient has already received nivolumab, pembrolizumab, ipilumumab, or any combination thereof.

[0199] For cancer treatment, immunotherapy, particularly that containing anti-ANTXR1 or anti-FAP agents, can be administered via any conventional route of administration, such as local, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular routes.

[0200] In particular, cancer treatment may include immunotherapy, surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy, palliative care, anti-ANTXR1 or anti-FAP agents, and any combination thereof.

[0201] Preferably, cancer treatment involves ANTXR1 + Fibroblasts, especially ANTXR1 + It is initiated within one month, preferably within one week, of the determination of the presence of CAF.

[0202] Cancer treatment can be administered as a single dose or in multiple doses.

[0203] Preferably, cancer treatment is administered regularly, preferably daily and monthly, more preferably daily and every two weeks, and even more preferably daily and weekly.

[0204] The treatment period preferably includes a period of 1 to 24 weeks, more preferably a period of 1 to 10 weeks, and even more preferably a period of 1 to 4 weeks. In certain embodiments, the treatment is continued for as long as the cancer persists.

[0205] The treatment of cancer, particularly immunotherapy involving anti-ANTXR1 or anti-FAP agents, or the dosage of the treatment, is determined by standard procedures well known to those skilled in the art. The patient's physiological data (e.g., age, size, weight, and health status) and route of administration are considered to determine the appropriate dosage so that an effective therapeutic dose is administered to the patient.

[0206] In relation to the combination of active ingredients, the active ingredients may be administered to the target by the same or different routes of administration. The route of administration usually depends on the pharmaceutical composition used.

[0207] cancer The method of the present invention aims to select and / or treat patients suffering from tumors.

[0208] In one embodiment, tumors include leukemia, seminoma, melanoma, teratoma, lymphoma, non-Hodgkin lymphoma, neuroblastoma, glioma, adenocarcinoma, mesothelioma (including pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and end-stage mesothelioma), rectal cancer, endometrial cancer, thyroid cancer (including papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, anaplastic thyroid carcinoma, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid carcinoma, pheochromocytoma, and paraganglioma), skin cancer (malignant melanoma, basal cell carcinoma, squamous cell carcinoma, carposis sarcoma, keratinized epidermis, mole, dysplastic nevus, lipoma). , including hemangiomas and dermatofibromas), nervous system cancers, brain tumors (including astrocytoma, medulloblastoma, glioma, low-grade glioma, ependymoma, germ blastoma (pineal gland), glioblastoma pleomorphism, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors, spinal nerve fibromas, gliomas or sarcomas), skull cancers (including osteomas, hemangiomas, granulomas, xanthomas or degenerative osteitis), meningeal cancers (including meningiomas, meningiosarcomas or gliomas), head and neck cancers (including squamous cell carcinomas of the head and neck and oral cancers (e.g., cheek cancer, lip cancer, tongue cancer, oral cancer or pharyngeal cancer, etc.)), lymph node cancers, gastrointestinal cancers, liver cancers Cancer (including hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma and hemangioma), colorectal cancer, gastric cancer or gastric cancer, esophageal cancer (including squamous cell carcinoma, laryngeal cancer, adenocarcinoma, leiomyosarcoma or lymphoma), colorectal cancer, intestinal cancer, small intestine or small intestinal cancer (e.g., adenocarcinoma lymphoma, carcinoid tumor, carposis sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma or fibroma), colorectal cancer or colorectal cancer (e.g., adenocarcinoma, tubular adenoma, chorioadenoma, hamartoma or leiomyoma, etc.), pancreatic cancer (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor or bile duct cancer), Breast cancer (including pyoma), ear, nose and throat (ENT) cancer, breast cancer (including HER2-rich breast cancer, luminal A breast cancer, luminal B breast cancer and triple-negative breast cancer), uterine cancer (including endometrial cancer, endometrial stromal sarcoma, malignant mixed Müllerian duct tumor, uterine sarcoma, leiomyosarcoma, gestational trophoblastic disease and other endometrial cancers), ovarian cancer (including germ cell tumor, granulosa cell tumor, Sertli-Leydig cell tumor), cervical cancer, vaginal cancer (including squamous cell vaginal cancer, vaginal adenocarcinoma, clear cell vaginal adenocarcinoma, vaginal germ cell tumor, vaginal sarcoma botryoides and vaginal melanoma), vulvar cancer (squamous cell vulvar cancer, verrucous vulvar cancer,Vulvar melanoma, basal cell vulvar carcinoma, Bartholin's gland carcinoma, vulvar adenocarcinoma, and Queyrat's erythropoiesis), urogenital cancers, kidney cancers (including clear renal cell carcinoma, chromogenic renal cell carcinoma, papillary renal cell carcinoma, adenocarcinoma, Wilms' tumor, nephroblastoma, lymphoma, or leukemia), adrenal cancers, bladder cancers, urethral cancers (e.g., squamous cell carcinoma, transitional cell carcinoma, or adenocarcinoma), prostate cancers (e.g., adenocarcinoma or sarcoma), and testicular cancers (e.g., seminoma, teratoma, embryonic carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor). (or lipoma, etc.), lung cancer (including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC) including squamous cell lung cancer, lung adenocarcinoma (LUAD), and large cell lung cancer, bronchogenic carcinoma, alveolar carcinoma, bronchiololar carcinoma, bronchial adenoma, pulmonary sarcoma, chondromatous hamartoma, and pleural mesothelioma), sarcoma (including Askin tumor, sarcoma botryoides, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma), soft tissue sarcoma (including alveolar soft tissue sarcoma, angiosarcoma, and bladder sarcoma), phyllodes, dermatofibrosarcoma elevations, desmoid tumors, desmoplastic tumors Small round cell tumors, epithelioid sarcomas, exoskeletal chondrosarcomas, exoskeletal osteosarcomas, fibrosarcomas, gastrointestinal stromal tumors (GIST), periangiomas, angiosarcomas, Kaposi's sarcomas, leiomyosarcomas, liposarcomas, lymphangiosarcomas, lymphosarcomas, malignant peripheral nerve sheath tumors (MPNSTs), neurofibrosarcomas, reticular histiocytic tumors, rhabdomyosarcomas, synovial sarcomas and undifferentiated pleomorphic sarcomas, cardiac cancers (including sarcomas such as angiosarcoma, fibrosarcoma, rhabdomyosarcoma or liposarcoma, myxoma, rhabdomyomas, fibromas, lipomas and teratomas), bone cancers (osteogenic sarcomas, osteosarcomas, fibrosarcomas, malignant fibrous histiocytic tumors), Cancers selected from the group consisting of tumors, chondrosarcomas, Ewing's sarcoma, malignant lymphoma and reticular cell sarcoma, multiple myeloma, malignant giant cell tumor, chordoma, bone carcinoma, osteochondroexostosis, benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumor), hematological and lymphoid cancers, hematological cancers (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma and myelodysplastic syndromes), Hodgkin's disease, non-Hodgkin's lymphoma, and hair cell and lymphatic system disorders, as well as their metastases.

[0209] Preferably, the tumor is a cancer selected from the group consisting of prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, stomach cancer, kidney cancer, ovarian cancer, hepatocellular carcinoma, osteosarcoma, melanoma, hypopharyngeal cancer, esophageal cancer, endometrial cancer, cervical cancer, pancreatic cancer, liver cancer, colon or colorectal cancer, neuroendocrine tumor, muscle cancer, adrenal cancer, adenocarcinoma, sarcoma, non-squamous cell carcinoma, squamous cell carcinoma, thyroid cancer, uterine cancer, skin cancer, melanoma, metastatic melanoma, bladder cancer, and head and neck cancer. More preferably, the cancer is selected from the group consisting of ovarian cancer, breast cancer, adenocarcinoma, sarcoma, non-squamous cell carcinoma, squamous cell carcinoma, lung cancer, NSCLC, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, and metastatic melanoma. In a very specific embodiment, the cancer is selected from the group consisting of head and neck cancer, NSCLC, melanoma, and metastatic melanoma.

[0210] In one embodiment, the cancer is ovarian cancer, preferably mesenchymal ovarian cancer, particularly serous high-grade ovarian cancer, or breast cancer, preferably invasive breast cancer and / or its metastases, particularly axillary metastases.

[0211] In another embodiment, the cancer is NSCLC or head and neck cancer.

[0212] In one embodiment, the cancer is a sarcoma, particularly a fibroblastic sarcoma.

[0213] In another embodiment, the cancer is selected from the group consisting of adenocarcinoma, non-squamous cell carcinoma, and squamous cell carcinoma.

[0214] In particular, the microenvironment of a patient's cancer or patient's cancer sample or tumor contains immunosuppressive fibroblasts, especially immunosuppressive CAFs, and especially ANTXR1 + CAF or ANTXR1 + FAP + Present CAF.

[0215] Preferably, the patient's cancer or patient's cancer sample is ANTXR1 + CAF, preferably FAP + ANTXR1 + LAMP5 - SDC1+ CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF and / or ANTXR1 + SDC1 - LAMP5 - CD9 + Preferably FAP + ANTXR1 + LAMP5 - SDC1 + CAF and / or FAP + ANTXR1 + LAMP5 + SDC1 + / - Present CAF.

[0216] Depending on the case, ANTXR1 + CAF is FAP + ANTXR1 + It is CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - )CAF. In some cases, ANTXR1 + CAF is TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - )CAF. In some cases, ANTXR1 + CAF is wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ) and TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - )CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1+ LAMP5 - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1 + CAF is TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + )CAF.

[0217] kit Furthermore, the present invention provides a kit useful for carrying out the methods disclosed herein, the kit being ANTXR1 + Fibroblasts, especially ANTXR1 + CAF, preferably FAP + ANTXR1 + CAF, and even better, FAP + ANTXR1 + LAMP5 - SDC1 + CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF and / or ANTXR1 + SDC1 - LAMP5 - CD9 +Includes means capable of detecting CAF.

[0218] For example, the kit is ANTXR1 + CAF, preferably FAP + ANTXR1 + CAF, and even better, FAP + ANTXR1 + LAMP5 - SDC1 + CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF and / or ANTXR1 + SDC1 - LAMP5 - CD9 + The means may include those necessary for the detection of CAF. Those skilled in the art will know the means required to specifically determine the presence of ANTXR1, preferably FAP and ANTXR1, more preferably FAP, ANTXR1, LAMP5 and SDC1, FAP, ANTXR1, LAMP5 and SDC1, and / or ANTXR1, SDC1, LAMP5 and CD9. The presence can be detected at the nucleic acid level, particularly at the mRNA or protein level. For example, such means may include ANTXR1 + CAF, preferably FAP + ANTXR1 + CAF, and even better, FAP + ANTXR1 + LAMP5 - SDC1 + CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF and / or ANTXR1 + SDC1 - LAMP5 - CD9 +Specific probes, primers, antibodies, and / or aptamers may be used to detect CAF. In particular, such means are antibodies against ANTXR1, FAP, LAMP5, and SDC1 and / or CD9 as disclosed herein. Alternatively, such means may be probes and / or primers specific to ANTXR1, FAP, LAMP5, and SDC1 and / or CD9.

[0219] Additionally or alternatively, the kit includes means, particularly nucleic acids, probes, or primers, for targeting at least one gene of the gene signatures of ecm-myCAF, TGFβ-myCAF, and / or wound-myCAF as described above. More specifically, it includes means for detecting all genes of the gene signatures ecm-myCAF, TGFβ-myCAF, and / or wound-myCAF. In particular, the means are primers and / or probes.

[0220] The aforementioned kit may be a diagnostic kit. The components of the kit may be packaged in either an aqueous medium or a lyophilized form. The kit's container means generally includes at least one vial, test tube, flask, bottle, syringe, or other container means in which the components may be placed, preferably appropriately dispensed. If the kit has multiple components, the kit usually also includes a second, third, or other additional container in which additional components may be placed separately. However, various combinations of components may be contained in the vial.

[0221] In some embodiments, means for collecting samples from individuals and / or means for analyzing samples may be provided. The kit may also include means for containing sterile, pharmaceutically acceptable buffers and / or other diluents. Optionally, a leaflet for guidelines on using such kits may be provided.

[0222] In another aspect, the present invention also relates to the use of the kit disclosed above with respect to the following: - To detect immunosuppressive cancer-associated fibroblasts (CAFs) in cancer samples from individuals affected by cancer. - Predicting the response of cancer patients to immunotherapy treatment. - Selecting or not selecting patients with cancer for immunotherapy treatment. - ANTXR1 + To select or not select patients with cancer for treatment with CAF-targeting drugs. - ANTXR1 + Selecting or not selecting patients with cancer for treatment with CAF-targeting drugs and immunotherapy.

[0223] Further aspects and advantages of the present invention are described in the following examples, which should be considered illustrative and not limiting. [Examples]

[0224] Within the immunosuppressive CAF-S1 subset, a single-cell approach identifies distinct cell clusters. The inventors investigated the heterogeneity of cells within the CAF-S1 immunosuppressive subset using single-cell RNA sequencing (scRNA-seq). CAF-S1 fibroblasts were isolated from human BCs by FACS as described above (see description of prospective cohort 1 in Table 1) (Costa A et al., Cancer Cell 2018;33(3):463-79 e10). In short, from newly excised tumors, the inventors first extracted fragments, dead cells, doublets, and epithelium (EPCAM). + ), hematopoiesis (CD45 + ), endothelium (CD31 + ) and red blood cells (CD235a + ) cells were excluded (Figure 7A). The inventors are EPCAM - CD45 - CD31 - CD235a -This is considered a fraction of cells rich in fibroblasts, and then stained with FAP and CD29 (Figure 7A), and CAF-S1 (FAP) is identified, as previously established by Costa A et al., Cancer Cell 2018;33(3):463-79 e10. High CD29 Med-High ) to other CAF subgroups (CAF-S2:FAP) Neg CD29 Low CAF-S3:FAP Neg CD29 Med CAF-S4:FAP Neg CD29 High This allowed for differentiation from [another gene]. The inventors then performed scRNA-seq on 18,805 CAF-S1 fibroblasts from seven BC patients before any treatment. After quality control, 18,296 CAF-S1 fibroblasts with a median of 2,428 genes detected per cell were saved for further analysis. Unsupervised graph-based clustering identified eight CAF-S1 clusters, which were visualized using the Uniform Manifold Approximation and Projection (UMAP) algorithm (Figure 1). All clusters were found in many patients, albeit at varying levels. Individual clusters were not associated with any particular stage of the cell cycle or high proliferation, as indicated by the G1 / S and G2 / M gene signatures (Tirosh I et al. Science 2016;352(6282)). The inventors confirmed the detection of these different CAF-S1 cell clusters using the label transfer algorithm described in Stuart T et al. Cell 2019;177(7):1888-902 e21. In fact, this algorithm successfully transferred all eight cluster labels from a newly generated, independent CAF-S1 scRNA-seq dataset from the eighth BC patient with high predictive scores.

[0225] Differential gene expression analysis revealed that each cluster is characterized by a specific transcriptional profile. Importantly, the CAF-S1 signatures published in Costa A et al., Cancer Cell 2018;33(3):463-79e10, Givel et al., Nat Commun 2018;9(1):1056, and Kieffer et al., Cancer Discov. 2020;10(9):1330-1351, include genes that are not limited to a specific CAF-S1 cluster (Figure 11). Cluster 0 is associated with ECM remodeling, cell-matrix adhesion, and collagen formation; Cluster 1 has detoxification and inflammatory responses; Cluster 2 is responsive to growth factors, TNF signaling, and interleukin pathways; Cluster 3 has the TGFβ signaling pathway and matrixosomes; Cluster 4 has collagen fiber assembly and wound healing; Cluster 5 is responsive to interferon-γ (IFNγ and cytokine-mediated signaling pathways); Cluster 6 has IFNβα signaling; and Cluster 7 has actomyosin complexes. For example, the inventors have identified the following markers in clusters: LRRC15 (leucine-rich repeat-containing protein 15) and GBJ2 (gap junction protein β2), which were recently identified in pancreatic cancer-derived CAFs, in cluster 0; ADH1B (alcohol dehydrogenase 1) and GPX3 (glutathione peroxidase 3) in cluster 1; RGMA (repulsion-inducing molecule BMP coreceptor) and SCARA5 (scavenger receptor class A member 5) in cluster 2; CST1 (cystatin) and TGFβ1 in cluster 3; and SEMA3C (semaphorin 3C) and SFRP4 (Secreted Frizzled Related High expression of Protein 4) was found in cluster 5, CCL19 and CCL5 (CC motif chemokine ligands 19 and 5), in cluster 6, IFIT3 (interferon-inducing protein with tetratricopeptide repeat 3) and IRF7 (interferon regulator 7), and in cluster 7, GGH (γ-glutamyl hydrolase) and PLP2 (proteolipid protein 2). Interestingly, in human BC, the inventors had previously observed high expression of FAP in pancreatic cancer. +We were able to distinguish between myofibroblasts ("myCAF") and inflammatory ("iCAF") fibroblast subgroups identified within fibroblasts (Elyada et al., Cancer Discov 2019;9(8):1102-23; Ohlund D et al., J Exp Med 2017;214(3):579-96). CAF-S1 clusters 1, 2, and 5 were identified as iCAF, while clusters 0, 3, 4, 6, and 7 were identified as myCAF. Consistent with pancreatic cancer data, iCAF showed high expression of chemokines and pro-inflammatory molecules such as CXCL12 (CXC motif chemokine ligand 12) and SOD2 (superoxide dismutase 2), while myCAF expressed myofibroblast markers including COL1A2 (collagen type 1 alpha 2 chain) and TAGLN (transgerin). Furthermore, the inventors observed that iCAF cluster 5 expresses high levels of CD74, encoding the major histocompatibility complex (MHC) II invariant. CD74 has recently been shown to be specifically expressed in antigen-presenting CAFs ("apCAFs") in pancreatic cancer, suggesting that CAF-S1 cluster 5 may be reminiscent of such apCAFs. In summary, the inventors identified eight distinct CAF-S1 clusters in BC. Clusters 1, 2, and 5 belong to the iCAF subgroup, with cluster 5 potentially corresponding to the apCAF cluster, while clusters 0, 3, 4, 6, and 7 belong to the myCAF subgroup. Furthermore, the iCAF clusters are characterized by detoxification (cluster 1), stimulus response (cluster 2), IFNγ and cytokines (cluster 5), ECM-mediated myCAF clusters (cluster 0), TGFβ (cluster 3), wound healing (cluster 4), IFNαβ (cluster 6), and actomyosin (cluster 7). Therefore, the inventors have identified these different FAPs. High The following nomenclature was proposed for CAF-S1 clusters: cluster 0 = ecm-myCAF, cluster 1 = detox-iCAF, cluster 2 = IL-iCAF, cluster 3 = TGFβ-myCAF, cluster 4 = wound-myCAF, cluster 5 = IFNγ-iCAF, cluster 6 = IFNαβ-myCAF, and cluster 7 = acto-myCAF.

[0226] Finally, the inventors were interested in whether these CAF-S1 clusters accumulated differentially across different BC subtypes. Because the inventors performed analyses on patients before any treatment, the fresh samples collected for scRNA-seq were mostly from luminal (Lum) BC patients, while HER2 and TN BC patients were preferentially treated in the neoadjuvant setting. As a result, prospective cohort 1 contained no HER2 patients and only two TN BC patients (Table 1). Nevertheless, in this dataset, we were able to detect that TN BC patients showed a higher proportion of iCAF clusters and accumulated more myCAF clusters than LumA BC patients (LumA: iCAF=43.4%, myCAF=56.6%, TN: iCAF=57.1%, myCAF=42.9%; P-value = 1.29e-64 from Fisher's exact test). Due to the small number of TN BCs in the dataset, this question was resolved by utilizing the TCGA database, which contains RNA-Seq data from a large number of LumA and TN BC patients. For this purpose, specific signatures of the five most abundant CAF-S1 clusters (accounting for up to 91% of sequenced CAF-S1 cells) were defined by identifying genes that were differentiated in each cluster compared to other clusters (Figure 7B). Since these signatures were also used to detect these clusters in melanoma, NSCLC, and HNSCC data (see Figures 3 and 6 below), the inventors then discarded any genes in these signatures that were also expressed by melanoma, NSCLC, and HNSCC cancer cells to avoid any signals from cancer cells and ensure strictly specific signals for CAF-S1 clusters (Figure 7B regarding the specificity of CAF-S1 cluster signatures). We evaluated the differences in CAF-S1 cluster-specific signature expression between LumA and TN BC subtypes from the TCGA RNA-seq database (https: / / portal.gdc.cancer.gov / ). This confirmed the accumulation of iCAF clusters in TN and myCAF clusters in LumA BC.Specifically, detox-iCAF and IL-iCAF showed higher expression in TN compared to Lum BCs, while ecm-myCAF, TGFβ-myCAF, and wound-myCAF expression was higher in LumA BCs. This increase in iCAF content in TN BCs is consistent with the reported presence of numerous TILs in some TN BCs. In summary, numerous FAPs isolated from BCs... + Using scRNA-seq from CAF-S1 fibroblasts, eight clusters were detected that exhibited a distinct signature and were differentiated in accumulation across BC subtypes.

[0227] CAF-S1 cell clusters are validated by multicolor flow cytometry of BC. Next, the inventors aimed to validate CAF-S1 clusters using multicolor flow cytometry (FACS) on fresh BC samples. By analyzing the proportion of each cluster in CAF-S1 as defined by scRNA-seq, it was initially observed that five initial clusters accounted for up to 91% of the total sequenced cells. Therefore, the inventors decided to focus the FACS analysis on these five most abundant clusters and attempted to identify the surface markers for each cluster. Using pairwise comparison of CAF-S1 cluster expression profiles, six surface markers were identified using commercially available antibodies, and a gating strategy was designed to identify the five most abundant clusters (Figure 8). The inventors attempted to validate the specificity of these six markers in an independent CAF-S1 dataset. To do so, the inventors investigated CAF-S1 scRNA-seq data corresponding to an eighth patient whose cluster labels were successfully transferred by a label transfer algorithm. In fact, a gating strategy based on these six markers and seven BC patients efficiently depicted the five most abundant CAF-S1 clusters in independent datasets. Thus, it was confirmed that these markers were specific to each CAF-S1 cluster. Therefore, fresh BC samples were analyzed by FACS applying the following gating strategy: CAF-S1 fibroblasts (CD45 - EPCAM - CD31 - CD235a - FAP High CD29 Med (Isolated as BC myCAF(ANTXR1) + ) to iCAF(ANTXR1 - They were first isolated based on ANTXR1 protein levels, which distinguished them from fibroblasts. + (myCAF)CAF-S1 clusters 0 (ecm-myCAF), 3 (TGFβ-myCAF), and 4 (wound-myCAF) were distinguished according to SDC1, LAMP5, and CD9 protein levels. ANTXR1 + SDC1+ LAMP5 - Cluster 0 (ECM-myCAF), ANTXR1 + LAMP5 + SDC1 + / - This is defined as cluster 3 (TGFβ-myCAF), and ANTXR1 + SDC1 - LAMP5 - CD9+ was defined as cluster 4 (wound-myCAF). ANTXR1 - (iCAF)CAF-S1 clusters 1 (detox-iCAF) and 2 (IL-iCAF) were separated using the GPC3 and DLK1 markers. ANTXR1 - GPC3 + DLK1 + / - It is defined as cluster 1 (detox-iCAF) and ANTXR1 - GPC3 - DLK1 + It was defined as cluster 2 (IL-iCAF). LAMP5, SDC1 and CD9 and ANTXR1 - GPC3 - DLK1 - ANTXR1 was negative for the cells. +CAF-S1 cells were pooled and labeled as "other clusters." By applying this gating strategy to 44 fresh samples (prospective cohort 2, Table S1), the presence of these five most abundant clusters was verified in BC (Figure 2). The proportion of each cluster within CAF-S1 cells, as defined by FACS, confirmed single-cell results, including clear heterogeneity between CAF-S1 fibroblasts and ecm-myCAF, as the most abundant population in the majority of patients (Figure 2). Next, the inventors analyzed whether there was a correlation between the proportions of each of these five CAF-S1 clusters among patients. There was a correlation between the relative abundances of ecm-myCAF and TGFβ-myCAF (both myCAF), and between the relative abundances of detox-iCAF and IL-iCAF (both iCAF). Conversely, the proportions of ecm-myCAF and TGFβ-myCAF were inversely correlated with the proportions of detox-iCAF and IL-iCAF. Furthermore, wound-myCAF is negatively correlated with detox-iCAF, IL-iCAF, and ecm-myCAF, suggesting that these different CAF-S1 clusters may accumulate in BC, but in a coordinated and differential manner.

[0228] CAF-S1 cell clusters have been identified across different cancer types. The inventors then attempted to test for the presence of CAF-S1 cell clusters in other types of cancer. To this end, they analyzed publicly available scRNA-seq data from head and neck squamous cell carcinoma (HNSCC) (Puram SV et al. Cell 2017;171(7):1611-24 e24) and non-small cell lung cancer (NSCLC) (Lambrechts D et al. Nat Med 2018;24(8):1277-89) because these two studies isolated enough CAFs to investigate the clusters. These published studies included 18 HNSCC patients, five of whom had matched primary tumor and lymph node metastases, and a total of 5,902 cells were analyzed (Puram SV et al. Cell 2017;171(7):1611-24 e24). Furthermore, in the NSCLC cohort, more than 52,000 whole cells were collected from five different patients (Lambrechts D et al. Nat Med 2018;24(8):1277-89). In these two studies, 1,422 cells and 1,465 cells were annotated as CAF in the HNSCC and NSCLC cohorts, respectively. To rigorously analyze CAF-S1 fibroblasts, CAF-S1 was designated as CAF-S1(FAP). High MCAM Low ) and CAF-S4 (FAP Low MCAM HighCAF-S4 cells were distinguished from other CAF-S1 cells based on the expression of two markers, FAP and MCAM, which are regulated at the RNA level, respectively. As a result, 603 CAF-S1 cells from HNSCC and 959 cells from NSCLC were further analyzed. Similarities between CAF-S1 cells from different cancer types were compared by mixing reference (BC) and target (HNSCC or NSCLC) datasets. Data integration was performed using "anchor" correspondences between single cells from different datasets based on the similarity of their expression profiles, as described in Stuart T et al. Cell 2019;177(7):1888-902 e21 (Figure 3). The inventors used CAF-S1 cluster-specific signatures defined by the differentially expressed genes of each cluster compared to other clusters (Figure 7B). Surprisingly, systematic correspondences were found between CAF-S1 clusters from BC and CAF-S1 clusters from either HNSCC (Figure 3A) or NSCLC (Figure 3B). Visualization of clusters using specific signatures confirmed the detection of the five most abundant clusters in HNSCC and NSCLC (Figures 3A and 3B). Therefore, the inventors confirmed the presence of the five most abundant CAF-S1 clusters in HNSCC and NSCLC and highlighted their relevance in other cancers.

[0229] The immunosuppressive environment correlates with specific CAF-S1 clusters. Having demonstrated that CAF-S1 fibroblasts exert immunosuppressive effects in breast and ovarian cancer, the inventors then investigated whether this function is exerted by all CAF-S1 clusters or is limited to specific clusters (Figure 4). The inventors first tested whether a correlation could be detected between CAF-S1 clusters and the content of immune cells. For this purpose, CD4 + CD8 +Fresh BC samples (prospective cohort 2, Table 1) containing T lymphocytes and natural killer (NK) cells were characterized in terms of both CAF-S1 cluster content and immune cell infiltration. The inventors examined the association between stromal cells and immune cells and analyzed variables showing at least one significant correlation with another variable. The correlation matrix obtained by unsupervised hierarchical clustering highlighted that ecm-myCAF and TGFβ-myCAF clusters were clustered together on one side, while detox-iCAF and IL-iCAF clusters were clustered on the other, while wound-myCAF clusters were quite isolated, suggesting that these different clusters interact differentially with T cells. Interestingly, ecm-myCAF, TGFβ-myCAF, and wound-myCAF were found to show a specific association with T lymphocytes. In fact, the proportion of ecm-myCAF in CAF-S1 fibroblasts was found to be in relation to CD45 + A significant correlation with hematopoietic cell infiltration was observed for the first time (Figure 9). More specifically, the amount of ecm-myCAF was found to be related to PD-1 + CTLA-4 + and TIGIT + CD4 + It correlated with T lymphocyte infiltration, but CD8 + It showed an inverse correlation with T lymphocytes. Similarly, the content of TGFβ-myCAF was inversely correlated with CD45 + Although it did not show an overall association with hematopoietic cells, its abundance was CTLA-4 + CD4 + There is a positive correlation with T lymphocyte infiltration, and CD8 + There was a negative correlation with T lymphocytes. Therefore, these data indicate that the abundance of ecm-myCAF and TGFβ-myCAF is associated with a Treg-rich immunosuppressive environment. In contrast to the ecm-myCAF and TGFβ-myCAF clusters, the abundance of detox-iCAF and IL-iCAF was associated with CD8 + It correlated with T cell infiltration. The wound-myCAF cluster was CD45 + It correlates with T lymphocytes overall within the cell (Figure 9), and CTLA-4 + , TIGIT+ , PD-1 + and NKG2A + CD4 + It was inversely correlated with T lymphocytes. Enrichment of wound-myCAF was linked to CTLA-4, a marker of depletion. + CD8 + , TIGIT CD8 + CD244 + CD8 + CD244 + It is also inversely correlated with NK, suggesting an overall association between this cluster and high T lymphocyte infiltration and the immune defense environment. Importantly, the CAF-S1 signatures published in Costa A et al., Cancer Cell 2018;33(3):463-79e10, Givel et al., Nat Commun 2018;9(1):1056, and Kieffer et al., Cancer Discov. 2020;10(9):1330-1351 cannot specifically identify CAF-S1 immunosuppressive clusters associated with immunotherapy responses (i.e., ECM-myCAF, TGFβ-myCAF, and wound-myCAF). These clusters are associated with ANTXR1 + In contrast, the most distinctive CAF-S1 gene was not specifically expressed in these clusters (Figure 11), suggesting that identifying ANTXR1 as a marker for these clusters among the top CAF-S1 genes was not possible by chance.

[0230] To validate these data from an independent, large cohort of BC patients, we next examined the association between CAF-S1 clusters and T cell signatures in the publicly available TCGA database. RNA-seq data from the TCGA database confirmed that the expression of ecm-myCAF and TGFβ-myCAF clusters was positively correlated with the expression of FOXP3, one of the major Treg markers (Figure 4B). Furthermore, wound-myCAF showed no substantial association with FOXP3, while the detox-iCAF and IL-iCAF clusters showed a negative correlation with FOXP3 (Figure 4B). Consistent with these data, a positive correlation was observed between the T cell lysis index and the detox-iCAF and IL-iCAF clusters, but not with ecm-myCAF, TGFβ-myCAF, or wound-myCAF (Figure 4C). In summary, detox-iCAF and IL-iCAF correlate with an immunocompromised environment, while ecm-myCAF and TGFβ-myCAF are both associated with an immunosuppressive environment, and CD8 + CD4 cells are deficient in T lymphocytes and express high levels of immune checkpoints, including PD-1 and CTLA-4. + It is rich in T lymphocytes.

[0231] PD-1 + and CTLA-4 + Positive feedback loop between ecm-myCAF and TGFβ-myCAF with Treg As mentioned above, the inventors determined that the abundance of ecm-myCAF and TGFβ-myCAF, rather than detox-iCAF and IL-iCAF, is the basis for BC's PD-1 + and / or CTLA-4 + The inventors observed a correlation with the abundance of CD4+ T lymphocytes. + CD25 +The role of CAF-S1 clusters in generating an immunosuppressive environment rich in CAF-S1 was investigated. Therefore, the inventors established primary cultures of CAF-S1 clusters to perform in vitro functional assays. Although it was not possible to establish all CAF-S1 clusters in culture, two different methods were used: (1) spreading CAF-S1 fibroblasts directly from BC samples seeded in plastic dishes, and (2) FAP High CD29 Med By sorting cells using FACS and growing them in culture medium on plastic dishes, we successfully isolated ecm-myCAF clusters and iCAF clusters. After several weeks of growth, we performed CD4 assays to determine the identity, i.e., the function, of these different cells under the same culture conditions. + CD25 + We compared cells suitable for co-culture with T lymphocytes (see below). It was observed that CAF-S1 cells obtained by spreading expressed high levels of the myCAF gene, while CAF-S1 cells isolated by selection showed high expression of the iCAF gene. Therefore, the inventors applied cluster-specific signatures established from scRNA-seq data (Figure 7B) and discovered that spread CAF-S1 fibroblasts were rich in ecm-myCAF, while selected CAF-S1 cells were rich in detox-iCAF, IL-iCAF, and IFN-iCAF clusters (see the section on #RNA sequencing methods for primary CAF-S1 cell lines isolated from BC). The inventors also found that the global CAF-S1 subpopulation is CD4 + CD25 - It does not directly affect T cells, but CD4 + CD25 - FOXP3 T lymphocytes +We previously demonstrated that it increases the proportion of Tregs. Since the content of ecm-myCAF and TGFβ-myCAF in BC was associated with a CD4+-rich immunosuppressive microenvironment, but iCAF clusters were not, the inventors used a functional assay as previously performed by Costa A et al., Cancer Cell 2018;33(3):463-79 e10 and Givel et al., Nat Commun 2018;9(1):1056 doi 10.1038 / s41467-018-03348-z.) to test CD4 + CD25 + We compared the functions of these myCAF and iCAF clusters on T cells.

[0232] First, CD4 + CD25 + FOXP3 + The effects of myCAF and iCAF clusters on T lymphocyte content were tested in vitro (Figure 5A). ecm-myCAF is CD4 + CD25 + FOXP3 in a population + The proportion of T cells was increased, and the FOXP3 protein levels of these T cells were enhanced (Figure 5A). In contrast, the iCAF cluster showed CD4 + CD25 + FOXP3 + It did not affect either the percentage of T lymphocytes or FOXP3 protein levels (Figure 5A). The inventors also tested the effects of culture on the identity and immunosuppressive activity of normal fibroblasts. Fibroblasts isolated by spreading from healthy tissue were FAP Neg-Low Initially, it lacked immunosuppressive activity, but in subsequent passages, FAP was developed. Pos-High Furthermore, it was found to be immunosuppressive, suggesting that long-term maintenance of CAF on a plastic plate may activate fibroblasts. Next, ecm-myCAF is CD4 + CD25 + FOXP3 +Based on the ability to increase T lymphocytes, the inventors then considered both the proportion of positive cells and the surface protein levels of these immune checkpoints, and CD4 + CD25 + FOXP3 + PD-1 on T lymphocytes + , CTLA-4 + , TIGIT + , TIM3 + and LAG3 + to compare the ability of the CAF-S1 cluster to regulate their proportions (Figures 5B - 5F). ecm-myCAF significantly increased both the proportion of PD-1 + and CTLA-4 + CD4 + CD25 + FOXP3 + T lymphocytes and their immune checkpoint levels on the surface (Figures 5B, 5C). In contrast to ecm-myCAF, the iCAF cluster did not affect the proportion of PD-1 + and CTLA-4 + T cells or the CTLA-4 protein level (Figures 5B, 5C). Furthermore, the iCAF cluster increased the PD-1 protein level, but this effect was less efficient than that of ecm-myCAF. Both the myCAF cluster and the iCAF cluster increased the proportion of CD4 + CD25 + FOXP3 + TIGIT + cells (Figure 5D), but had no effect on TIM3 + and LAG3 + T cells (Figures 5E, 5F). Therefore, consistent with the correlation between ecm-myCAF and PD-1 + and CTLA-4 + CD4 + T lymphocytes observed in BC, CAF-S1 derived from ecm-myCAF had CD4 + CD25 + FOXP3 +It is shown here that while iCAF clusters directly function against Tregs by enhancing PD-1 and CTLA-4 immune checkpoint levels on the surface of T lymphocytes, they have little to no effect on these cells. Finally, when co-cultured with ecm-myCAF, CD4 + CD25 + Upregulation of immune checkpoints on the surface of T cells was observed to be detected intracellularly (Figure 10A), suggesting that ecm-myCAF increases the total protein level of T cells. Furthermore, the inventors found that CD4 + CD25 + The expression of FOXP3, CTLA-4, and TIGIT in T cells was also upregulated at the mRNA level after co-culturing with ecm-myCAF (Figure 10B), and PD-1 RNA was found to be almost undetectable in T cells in vitro. Furthermore, the inventors found that the mRNA levels of NFAT and STAT family members were also upregulated at the CD4 level during co-culturing with ecm-myCAF. + CD25 + We observed an increase in T lymphocytes (Figure 10C). Since NFAT and STAT are well known transcriptional regulators of immune checkpoints on T cells, these data suggest that ecm-myCAF is elevated in CD4 + CD25 + This study demonstrates that it promotes immune checkpoint upregulation at the RNA level of T lymphocytes, potentially through activation of the NFAT / STAT signaling pathway.

[0233] Considering the influence of CAF-S1 clusters on Tregs, the inventors then became interested in whether T lymphocytes could modulate the identity of CAF-S1 clusters. Therefore, the inventors considered CD4 + CD25 + We evaluated whether co-culturing T lymphocytes had any effect on marker cluster levels on the surface of CAF-S1 fibroblasts. To avoid any contamination by T cells during co-culturing, CAF-S1 was isolated by FACS, and the cluster markers expressed on their surface were analyzed. CD4+ CD25 + Co-culturing of T cells was observed to significantly increase the expression of the TGFβ-myCAF-specific marker LAMP5 on the surface of ecm-myCAF fibroblasts (Figure 5G), thereby increasing the TGFβ-myCAF content in CD4 + CD25 + This suggests an increase in co-culture with T lymphocytes. This effect was detected only in myCAF cells and not in iCAF fibroblasts (Figure 5G), which was expected considering that TGFβ-myCAF and ecm-myCAF CAF-S1 fibroblasts belong to the myCAF subgroup. Consistent with this observation, ANTXR1 protein levels were increased in CD4 + CD25 + When co-cultured with T cells, ecm-myCAF fibroblasts also showed a tendency to increase (though not statistically significant), but iCAF cells did not change significantly and remained low (Figure 5G). Quite surprisingly, DLK1, a marker for IL-iCAF, also increased during co-culture, suggesting potential plasticity between ecm-myCAF and IL-iCAF. In contrast, other markers did not show significant changes during co-culture and remained either high (SDC1) or low (GPC3 and CD9), as expected based on their respective cluster identities (Figure 5G). These observations suggest that CD4 + CD25 + T lymphocytes ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - CD9 + / - ) TGFβ-myCAF(ANTXR1 + LAMP5 + SDC1 + / - CD9 + / - This suggests that ecm-myCAF may facilitate conversion to FOXP3 +We discovered that Tregs can directly influence the levels of PD-1 and CTLA-4 proteins on the surface of T lymphocytes. Conversely, Tregs can promote the conversion of ecm-myCAF to TGFβ-myCAF, thereby affecting these two clusters and CD4+ CD25 + PD-1 + or CTLA-4 + This can underlie the positive feedback loop with T cells and explain the positive correlation observed in BC.

[0234] ecm-myCAF and TGFβ-myCAF are associated with primary resistance to immunotherapy. Considering the direct impact of specific CAF-S1 clusters on Treg PD-1 and CTLA-4 protein levels, the inventors then became interested in whether several CAF-S1 clusters might be associated with immunotherapy resistance. Due to the lack of access to data from BCs treated with immunotherapy, they utilized publicly available data from metastatic melanoma patients treated with anti-PD-1 (pembrolizumab) therapy (Hugo W et al. Cell 2016;165(1):35-44), which revolutionized melanoma treatment. As defined in the aforementioned study, the inventors considered patients "non-responders" to anti-PD-1 if they exhibited progressive disease, and "responders" if they showed a complete or partial response. By performing gene set enrichment analysis, they first observed that, at diagnosis, CAF-S1 specific gene expression was significantly enriched in tumors derived from non-responder patients, although not in normal fibroblast content. The inventors observed, using CAF-S1 cluster-specific signatures, that ecm-myCAF, TGFβ-myCAF, and wound-myCAF gene expression was enriched in non-responders compared to responders, while detox-iCAF, IL-iCAF, and IFN-iCAF clusters were not enriched (Figure 6A). Next, the content of each CAF-S1 cluster was compared between responders and non-responders. The results showed that the expression of ecm-myCAF, TGFβ-myCAF, and wound-myCAF was significantly higher in non-responders than in responders, while the expression of detox-iCAF and IFN-iCAF was similar between the two subgroups of patients (Figure 6B). Furthermore, neither the normal fibroblast content nor the cell lysis index were defined in Rooney et al. Cell 2015;160(1-2):48-61, and they differed between responders and non-responders (Figure 6C, Figure 6D).Consistent with these observations, cross-analysis (i.e., determining the number of responders and non-responders according to low or high CAF-S1 cluster expression) confirmed that the number of non-responder patients was significantly associated with tumors showing high expression of ecm-myCAF, TGFβ-myCAF, or wound-myCAF at diagnosis, while other CAF-S1 clusters, general CAF content, or cytolysis index did not provide useful information regarding patients' response to immunotherapy (Figures 6E-6G). In summary, these data indicate that three specific CAF-S1 clusters (ecm-myCAF, TGFβ-myCAF, and wound-myCAF) are indicative of the diagnosis of anti-PD-1 response in metastatic melanoma patients, while other CAF-S1 clusters (detox-iCAF, IL-iCAF, and IFN-iCAF), total CAF content, or cytolysis index are not. Finally, the inventors sought to investigate the impact of CAF-S1 clusters, particularly ecm-myCAF, TGFβ-myCAF, and wound-myCAF, on first-line immunotherapy resistance in a set of patients with metastatic NSCLC, another recently established clinical indication for immunotherapy (treated with nivolumab in a second-line or third-line setting; see Table 2 for a detailed description of NSCLC Cohort 4). Similar to melanoma, the inventors verified that the CAF-S1 signature, as assessed in tumor specimens sampled at diagnosis, was significantly enriched in non-responder patients. In contrast, the content of normal fibroblasts was higher in responders than in non-responders, suggesting that CAF-S1 was significantly enriched in non-responders. Importantly, the inventors confirmed that ecm-myCAF, TGFβ-myCAF, and wound-myCAF were associated with non-responder NSCLC patients, in contrast to the detox-iCAF, IL-iCAF, and IFNγ-iCAF clusters (Figure 6I). In conclusion, in contrast to the detox-iCAF, IL-iCAF, and IFNγ-iCAF clusters, the abundance of ecm-myCAF and TGFβ-myCAF at diagnosis was associated with resistance to immunotherapy in both melanoma and NSCLC, and with Treg PD-1.+ and CTLA-4 + This is consistent with the ability to increase protein levels.

[0235] Materials and methods Patient cohort Patients with BC: The research developed here is based on samples taken from surgical remnants that are available after histopathological analysis and not necessary for diagnosis. There is no interference with clinical practice. Analysis of primary tumor samples was carried out in accordance with relevant national laws concerning the protection of persons participating in biomedical research. All patients admitted to the Curie Institute (BC patients) received a welcome booklet explaining that samples may be used for research purposes. Thus, all patients included in the study were informed by their referring oncologist that biological samples collected through standard clinical practice may be used for research purposes and that they could object to such use if necessary. If a patient refused, either verbally or in writing, the residual tumor sample was not included in the study. The human experimental procedure for tumor microenvironment analysis by the laboratory of F. Mechta-Grigoriou was approved by the Institutional Review Board and Ethics Committee of the Curie Institute Hospital Group (approved February 12, 2014) and the CNIL (Commission Nationale de l'informatique et des Libertes) (N oApproved by: 1674356 (distributed March 30, 2013). The "Biological Resource Center" (BRC) is part of the Department of Pathology, Department of Diagnostic and Therapeutic Medicine, led by Dr. A. Vincent-Salomon. The BRC is authorized to store and manage human biological specimens in accordance with French law. The BRC has declared the retrieval of defined specimens, which will be continuously incremented upon obtaining patient consent (Declaration No. #DC-2008-57). The BRC adheres to all currently required national and international ethical rules, including the Declaration of Helsinki. The BRC is also certified with the AFNOR NFS-96-900 quality label (renewed and now valid until 2021). Luminous (Lum) tumors were defined by positive immunohistochemical staining for ER (estrogen receptor) and / or PR (progesterone receptor). The cutoff used to define hormone receptor positivity was 10% of stained cells. The Ki67 (proliferation) score further distinguishes Lum A and Lum B tumors (less than 15%: Lum A: Lum B above). HER2-amplified cancers are defined according to ERBB2 immunostaining using ASCO guidelines. The TN immunophenotype was defined as follows: ER with expression of at least one of the following markers. - PR - ERBB2 - :KRT5 / 6 + or EGF-R + .

[0236] NSCLC patients: NSCLC samples were from routine diagnostic samples stored in the Department of Pathology at Bichat Hospital and originated from patients treated with immuno-oncological drugs in the Department of Thoracic Oncology at Bichat Hospital, led by MD Pr.G. Zalcman. The anonymized clinical data are part of the thoracic oncology database of lung cancer patients at the CIC-1425 / CLIP2 Clinical Research Center at Bichat Hospital (co-led by Pr.G. Zalcman), (Approval #17-1381 from the Regional Health Authority) and comply with French regulatory rules for observational clinical studies. Patients were administered checkpoint inhibitors after progressing on chemotherapy-based first or second line treatment, according to the immuno-oncological drug registry. During the current study period, anti-PD-1 nivolumab monoclonal antibody represented the most frequently used drug in such settings. The efficacy of immuno-oncology therapy was assessed every 8–12 weeks by whole-body CT scans, and objective responders (OR), patients with stable disease (SD), and patients showing tumor progression (Progr) with a 20% increase in tumor volume without clinical benefit were defined by a weekly interdisciplinary oncology committee including thoracic radiologists, thoracic oncologists, and pulmonologists, according to RECIST v.1.1 criteria. The best response status observed at 4 months was used for the current study. SD patients who received immuno-oncology drugs for more than 6 months for clinical benefit (n=3) at the 4-month assessment were included in the responder patient group for the current study without any criteria for progressive disease (then called long-lasting SD). The date of progression assessed by CT scan was recorded. Some patients showed early clinical progression requiring early (8 weeks prior) CT scan assessment. These series followed the criteria described above. There were 22 responder patients and 48 progressive patients, with treatment durations of less than 4 months. The progression date was retained as the date of the CT scan indicating RECIST progression. The date and cause of death, or the date of the last report of the patient's life status, were systematically recorded. Secondary or tertiary treatments after progression were registered. There were no imbalances in treatment after progression.PD-L1 staining was performed and interpreted by AG on 4 μm paraffin-embedded sections of diagnostic, pre-treatment biopsy specimens containing at least 200 tumor cells, using the commercially available clone Cell Signaling Technology E1L3N on the Leica Bond platform. All but five patients (those with fewer than 200 tumor cells in the remaining pathological blocks) underwent PD-L1 immunohistochemical analysis.

[0237] CAF-S1 RNA sequencing at the single-cell level Isolation of CAF-S1 from BCs: CAF-S1 fibroblasts were isolated from a total of eight primary BCs (surgical residues before any treatment) (see Table S1 for details of the prospective cohort). Seven BCs were initially studied. Additionally, another BC sample was added to validate CAF-S1 clusters using the Seurat R package label transfer algorithm. CAF-S1 fibroblasts were isolated from BCs using a BDFACS ARIA III® sorter (BD Biosciences). Fresh human primary BC tumors were collected directly from the operating room after macroscopic examination of surgical specimens and selection of target areas by a pathologist. Samples were then cut into small pieces (approximately 1 mm). 3 The cells were cut into 50 μl portions and digested in CO2-independent medium (Gibco #18045-054) supplemented with 150 μg / ml liberase (Roche #05401020001) and DNase I (Roche #11284932001) at 37°C for 40 minutes with shaking (180 rpm). Next, the cells were filtered through a 40 μm cell strainer (Fisher Scientific #223635447) and 5 × 10¹⁴ PBS+ solution (PBS, Gibco #14190; EDTA 2 mM, Gibco #15575; human serum 1%, BioWest #S4190-100) in 50 μl. 5 ~10 6 The cells were resuspended at their final concentration. To isolate CAF-S1 fibroblasts, the inventors first isolated them from the epithelium (EPCAM). + ), hematopoiesis (CD45 + ), endothelium (CD31+ ) and CD235a + (Red blood cell) cells, and then use the CAF-S1 markers (FAP and CD29). To do so, the cells in the suspension were stained with an antibody mix containing anti-EpCAM-BV605 (BioLegend, #324224) for flow cytometry cell sorting, anti-CD31-PECy7 (BioLegend, #303118), anti-CD45-APC-Cy7 (BD Biosciences, #BD-557833), anti-CD235a-PerCP / Cy5.5 (Biolegend, #349109), anti-CD29-Alexa Fluor 700 (BioLegend, #303020), anti-FAP-APC (primary antibody, R&D Systems, #MAB3715), and single cell RNA sequencing was performed. All antibodies except anti-FAP were purchased already conjugated to a fluorochrome. The anti-FAP antibody was conjugated using the Zenon APC mouse IgG1 labeling kit (ThermoFisher Scientific, #Z25051). The isotype control antibodies for each CAF marker used were iso-anti-CD29 (BioLegend, #400144) and iso-anti-FAP (primary antibody, R&D Systems, #MAB002).

[0238] The cell suspension was stained with the antibody mix in PBS+ solution for 15 minutes at room temperature immediately after dissociating the BC tumor sample. 2.5 μg / ml DAPI (ThermoFisher scientific, #D1306) was added immediately prior to flow cytometry sorting. Signals were acquired with a BDFACS ARIA III (trademark) sorter (BD biosciences) for cell sorting. At least 5×10 5The events were recorded. Compensation was performed using single staining for each antibody against anti-mouse IgG and negative control beads (BD Biosciences, #552843). Data analysis was performed using FlowJo version X 10.0.7r2. Cells were first gated based on forward scattering (FSC-A) and lateral scattering (SSC-A) (measuring cell size and granularity, respectively) to eliminate debris. Dead cells were excluded based on positive staining for DAPI. Next, single cells were selected based on SSC-A vs SSC-W parameters. For gating, epithelial cells (EPCAM) were used. + ), hematopoietic cells (CD45 + ), endothelial cells (CD31 + ), red blood cells (CD235a + ) in order to remove EPCAM - CD45 - CD31 - CD235a - It contained cells.

[0239] CAF-S1 RNA sequencing at the single-cell level: Upon isolation, CAF-S1 cells were collected directly into RNase-free tubes (ThermoFisher Scientific, #AM12450) pre-coated with DMEM (GE Life Sciences, #SH30243.01) supplemented with 10% FBS (Biosera, #1003 / 500). At least 6,000 cells were collected per sample. Under these conditions, cell concentration was checked against a control sample and was 200,000 cells / ml. Single-cell capture, lysis, and cDNA library construction were performed using 10X Genomics' Chromium® system with the following kits: Chromium® Single Cell 3' Library & Gel Bead Kit v2 (10X Genomics, #120237) and Chromium® Single Cell A Chip Kit (10X Genomics, #1000009). Gel bead generation in emulsion (GEM), barcoding, post-GEM-RT cleanup (reverse transcription), and cDNA amplification were performed according to the manufacturer's instructions. Target cell retrieval was 3,000 cells per sample to ensure sufficient cell collection while maintaining a low multiplexing rate. Cells were loaded into chromium single-cell A chips accordingly and run for 12 cycles for cDNA amplification. cDNA quality and quantity were checked using an Agilent 2100 bioanalyzer with the Agilent High Sensitivity DNA Kit (Agilent, #5067-4626), and library construction was performed according to the 10X Genomics protocol. Libraries were then run on Illumina HiSeq (for patients P5-7) and NovaSeq (for patients P1-4) at a sequencing depth of 50,000 reads per cell. The processing of raw data, including demultiplexing of raw base call (BCL) files into FASTQ files, alignment, filtering, barcodes, and Unique Molecular Identifiers (UMI) counts, was performed using the 10X Cell Ranger pipeline version 2.1.1.The reads were aligned to the Homo sapiens (human) genome assembly GRCh38 (hg38).

[0240] scRNA-seq data processing scRNAseq: Raw data preprocessing was initially performed using the Cell Ranger software pipeline (version 2.1.1). This process included demultiplexing of raw base call (BCL) files into FASTQ files, reading the alignment of the human genome assembly GRCh38 using STAR, and counting unique molecular identifiers (UMIs). The first set of 18,805 CAF-S1 cells from 7 BC patients (7 sequencing runs, corresponding to patients 1-7) was analyzed using the Seurat R package (version 3.0.0) (Butler et al. Nat Biotechnol 2018;36(5):411-2). A second set of 1,646 CAF-S1 cells from one BC patient (patient 8) was used for validation (see #label transfer) and analyzed using the same methodology.

[0241] Quality Control: As a quality control process, low-quality cells, empty droplets, and multiplet captures were first filtered out based on the distribution (non-zero count) of unique genes detected in each cell for each patient. Cells with fewer than 200 or more than 6,000 detected genes (for patient 1), more than 5,000 detected genes (for patients 3, 5, and 6), more than 4,500 detected genes (for patients 2, 7, and 8), or more than 4,000 detected genes (for patient 4) were excluded. The distribution of cells based on the percentage of expressed mitochondrial genes was also plotted. Cells with a mitochondrial gene percentage exceeding 5% were discarded to eliminate dying cells or low-quality cells with widespread mitochondrial contamination. For each patient, the mitochondrial percentage was calculated using Seurat's PercentageFeatureSet function with argument pattern = "^MT-". Following these QC standards, 18,296 CAF-S1 cells (Patient 1 = 1,825 cells, Patient 2 = 3,300 cells, Patient 3 = 2,810 cells, Patient 4 = 3,153 cells, Patient 5 = 2,486 cells, Patient 6 = 3,179 cells, Patient 7 = 1,543 cells) and 1,582 CAF-S1 cells (Patient 8) were ultimately saved to the first and second datasets, respectively, for downstream analysis.

[0242] Normalization and Data Integration: The integration of scRNA-Seq data from seven BCs from the first dataset was performed using the Seurat functions FindIntegrationAnchors and IntegrateData, after normalizing the library size of each cell using the NormalizeData function with default parameters. 30 dimensions were used for canonical correlation analysis (CCA), and 30 principal components (PCs) were used in the weighting step of the IntegrateData function. The data was scaled using the ScaleData function, and the variables "nUMI" and "percent.mt" were used for regression. The same parameters were used for normalizing the second dataset.

[0243] Clustering and Data Visualization: PCA dimensionality reduction was performed using default parameters. The number of components (PCs) to be included was evaluated using the JackStraw procedure implemented in the JackStraw and ScoreJackStraw functions. 30 PCs were used. Using a graph-based clustering approach, cells in the initial dataset were clustered using the FindNeighbours (k=20) and FindClusters (res=0.35) functions. Ten CAF-S1 clusters were obtained at this resolution. For data visualization, the nonlinear dimensionality reduction method UMAP was applied using Seurat's RunUMAP function.

[0244] Analysis of Differential Gene Expression and Signaling Pathways: Genes specifically upregulated in each of the 10 clusters of the initial dataset were identified using pairwise difference analysis. The median number of genes differentiated in each pairwise combination was 126, but the number of genes differentiated in two combinations was very limited, with only 9 genes between clusters 0 and 5, and 22 genes between clusters 3 and 6. The biological significance of each cluster was determined using the Metascape tool (http: / / metascape.org) with all genes significantly upregulated in each of the 10 initial clusters (one cluster versus all other clusters, function FindAllMarkers, parameters: logfc.threshold=0.25, test=Wilcox rank-sum test). Consistent with pairwise analysis, the biological pathways identified in clusters 0 / 5 on one hand and 3 / 6 on the other were redundant and therefore combined. Next, we defined clusters 0 and 5 as cluster 0 / ecm-myCAF, and clusters 3 and 6 as cluster 3 / TGFβ-myCAF, ultimately identifying eight biologically distinct CAF-S1 clusters.

[0245] Genetic Signatures of CAF-S1, CAF-S1 Clusters, and Normal Fibroblasts: Specific gene signatures for CAF-S1 clusters 0-5 were defined by performing difference analysis (Wilcoxon rank-sum test) between clusters 0-5. Genes that were differentiated in expression between clusters (one cluster versus all other clusters) with an adjusted P-value < 0.05 were selected. Since these signatures were used to detect CAF-S1 clusters in RNA-seq data from single cells and bulk data from different cancer types, including melanoma, NSCLC, and HNSCC, the inventors excluded genes expressed in tumor cells using scRNA-seq data from tumor cells of melanoma (27), NSCLC (31), and HNSCC (30). The inventors defined genes expressed in tumor cells (and therefore excluded from the CAF-S1 cluster signature) if more than 10% of tumor cells showed an expression level greater than 1 in any of the aforementioned scRNA-seq data. The CAF-S1 global signature was first published in Costa A et al., Cancer Cell 2018;33(3):463-79 e10, and subjected to the same type of analysis, excluding genes detected in tumor cells and adapted for bulk analysis. The first 100 most important genes were considered for the CAF-S1 specific signature. The normal fibroblast signature was compared with normal fibroblasts isolated from healthy paratumor tissue (FAP) compared with CAF-S1 fibroblasts isolated from BC. Neg CD29 Med SMA Neg The cytolysis index was defined by genes that were significantly upregulated. Genes expressed in tumor cells were excluded from the signature following the same strategy as above. The cytolysis index was defined as the geometric mean of granzyme A (GZMA) and perforin (PRF1) gene expression, as described in Rooney et al. Cell 2015;160(1-2):48-61.

[0246] Label transfer To validate the CAF-S1 clusters identified in the initial dataset, another dataset corresponding to 1582 CAF-S1 fibroblasts after quality control was collected from an additional BC sample and analyzed using the Seurat pipeline. The label transfer algorithm described in Stuart T et al. Cell 2019;177(7):1888-902 e21 was implemented in the Seurat V3.0 R package and applied using the functions FindTransferAnchors and TransferData. The initial dataset of 18,296 CAF-S1 cells was used as the reference, and the second dataset of 1,582 CAF-S1 cells was used as the query. Dimensionality reduction was performed when finding anchors by projecting PCA from the reference to the query. 30 dimensions were used.

[0247] Integration of single-cell data from BC, HNSCC, and NSCLC Integration between BC and HNSCC or BC and NSCLC single-cell data was performed using the method described in Stuart T et al. Cell 2019;177(7):1888-902 e21 and implemented in the Seurat V3.0 R package. In short, the datasets can be transformed into a shared space by identifying cell-pairwise correspondences (called "anchors") between single cells in the datasets. Dimensionality reduction of both datasets was performed using diagonalized canonical correspondence analysis (CCA), with L2 normalization applied to the canonical correlation vectors before anchor identification. Default parameters were used for the FindIntegrationAnchors and IntegrateData functions in the Seurat V3.0 package.

[0248] Flow cytometry analysis of the five most abundant CAF-S1 clusters and immune cells 44 BCs were cut into small fragments and digested in CO2-independent medium (Gibco, #18045-054) with 5% fetal bovine serum (FBS, PAA, #A11-151), 2 mg / ml collagenase I (Sigma-Aldrich, #C0130), 2 mg / ml hyaluronidase (Sigma-Aldrich, #H3506), and 25 mg / ml DNase I (Roche, #11284932001) at 37°C for 45 minutes with shaking (180 rpm). After tissue digestion, the cells were filtered using a cell strainer (40 mm, Fischer Scientific, #223635447) and washed with PBS solution (Gibco, #14190) supplemented with 2 mM EDTA (Gibco, #15575) and 1% human serum (BioWest, #S4190-100). The cells were then divided into two groups for analysis of CAF-S1 cluster panels and immunocellular cells, respectively.

[0249] CAF-S1 cluster panel: Cells were stained with Live Dead NIR (1:1000, BD Bioscience #565388) in PBS for 20 minutes. Next, the cells were washed and treated with anti-CD235a-APC-Cy7 (1:20, BioLegend, #349115), anti-EpCAM-BV605 (1:25, BioLegend, #324224), anti-CD31-PECy7 (1:50, BioLegend, #303118), anti-CD45-BUV395 (1:25, BD Biosciences, #BD-563792), anti-CD29-Alexa Fluor 700 (1:50, BioLegend, #303020), anti-FAP (1:100, R&D Systems, #MAB3715) linked using the Zenon APC mouse IgG1 labeling kit (Thermo Fisher Scientific, #Z-25051), and anti-ANTXR1-AF405 (1:33, Novus The samples were stained for 45 minutes with an antibody cocktail containing Biological (#NB-100-56585), anti-LAMP5-PE (1:10, Miltenyi Biotech, #130-109-156), anti-SDC1-BUV737 (1:25, BD Biosciences, #BD-564393), anti-GPC3-AF594 (1:20, RnD, #FAB2119T, 100UG), anti-DLK1-AF488 (1:25, RnD, #FAB1144G-100), and anti-CD9-BV711 (1:200, BD Biosciences, #BD-743050).The isotype control antibodies used for each CAF cluster marker were: mouse IgG1 isotype control - BV711 (1:200, BD Bioscience, #563044), mouse IgG1 isotype control - AF405 (1:3, Novus Biological, #IC002V), mouse IgG1 isotype control - BUV737 (1:25, BD Bioscience, #564299), mouse IgG2B isotype control - AF488 (1:12, 5, RnD, #IC0041G), mouse IgG2A isotype control - AF594 (1:5, RnD, #IC003T), and REA control - PE (1:10, Miltenyi Biotech, #130-113-462). Subsequently, the cells were washed and acquired on the same day using an LSR FORTESSA analyzer (BD Biosciences), or fixed with 4% paraformaldehyde (PFA, Electron Microscopy Sciences, #15710) for 20 minutes, washed, and held overnight in PBS+ solution for acquisition the following day. At least 5 × 10⁶ cells. 5 The events were recorded. Compensation was performed using anti-mouse IgG and negative control beads (BD Biosciences, #552843) for each antibody, as well as single staining of cells for Live / Dead staining. Data analysis was performed using FlowJo version 10.4.2 (LLC, USA). Cells were first gated based on forward scattering (FSC-A) and lateral scattering (SSC-A) (measuring cell size and granularity, respectively) to eliminate debris. Dead cells and erythrocytes were excluded based on positive staining for Live / Dead NIR and CD235a, respectively. Next, single cells were selected based on SSC-H vs. SSC-A parameters. Then, epithelial cells (EpCAM) were selected. + ), hematopoietic cells (CD45 + ), endothelial cells (CD31 + To remove the cells, EpCAM - CD45 - CD31 - Gating was performed on the cells. DAPI - EPCAM- CD45 - CD31 - The cells were divided into four subsets (CAF-S1 to CAF-S4) according to FAP and CD29. The CAF-S1 subset was first gated with ANTXR1. + Cells were gated according to SDC1 and LAMP5. ANTXR1 + SDC1 + LAMP5 - Cluster 0 / ecm-myCAF, ANTXR1 + SDC1 - LAMP5 + It was defined as cluster 3 / TGFβ-myCAF. ANTXR1 + SDC1 - LAMP5 - It is gated to CD9 and ANTXR1 + SDC1 - LAMP5 - CD9 + It was defined as cluster 4 / wound-myCAF. ANTXR1 - / Low Cells were gated on DLK1 and GPC3. Cluster 1 (detox-iCAF) was ANTXR1 - GPC3 + DLK1 - / + It is defined as, and cluster 2 is ANTXR1 - GPC3 - DLK1 + Defined as ANTXR1 - GPC3 - DLK1 - and ANTXR1 + SDC1 - LAMP5 - CD9 - It was designated as another cluster.

[0250] Immunotherapy panel: Of the 44 BC samples analyzed for the CAF-S1 cluster, 37 were characterized among them for their immunoconcentration. Cell types were analyzed in the live-dead-negative fraction, and hematopoietic cells (CD45) were identified. + ), CD4+ / CD8 + T lymphocytes, B lymphocytes (CD45 + CD14 - CD3 - CD19 + ), NK(CD45 + CD14 - CD3 - CD56 + ), cytotoxic NK (CD56 + CD16 + ) and non-cytotoxic NK (CD56 + CD16 - ), (CD45 + CD14 - CD3 + CD4 + / CD8 + ) and myeloid cells (CD45 + CD14 +It was defined as follows. For each identified population, the percentage of cells positive for the following checkpoints was also evaluated: PD-1, CTLA-4, NKG2A, TIGIT, CD244, CD158K, CD69, and CD161. Cells were stained with Live Dead (1:1000, Thermo Fisher Scientific, #L34955) in PBS for 20 minutes. Next, the cells were washed and stained with an antibody cocktail for 45 minutes. The antibody cocktail consisted of anti-CD45-APC-cy7 (1:20, BD Biosciences, #BD-557833), anti-CD14-BV510 (1:50, BD Biosciences, #563079), anti-CD56-BUV395 (1:25, BD Biosciences, #563554), anti-CD16-BV650 (1:25, BD Biosciences, #563692), anti-PD-1-BUV737 (1:20, BD Biosciences, #565299), anti-CD3-AF700 (1:25, BD Biosciences, #557943), and anti-NKG2A-BV786 (1:20, BD Biosciences, #747917), anti-TIGIT-BV605 (1:20, BD Biosciences, #747841), anti-CD158K-Pe (1:10, Miltenyi Biotec, #130-095-205), anti-CD244-FITC (1:10, BD Biosciences, #550815), anti-CTLA-4-PE-cy5 (1:10, BD Biosciences, #555854), anti-CD19-Percp-cy5.5 (1:20, BD Biosciences, #561295), anti-CD4 APC (1:25, Miltenyi Biotec, #130-092-374), anti-CD8-PE-TexasRred (1:100, Life It contained anti-CD69-BV710 (1:25, BD Bioscience, #563836) and anti-CD161-PE-VIO770 (1:100, Miltenyi, #130-113-597).Subsequently, the cells were washed and acquired on the same day using an LSR FORTESSA analyzer (BD Biosciences), or fixed with 4% paraformaldehyde (PFA, Electron Microscopy Sciences, #15710) for 20 minutes, washed, and held overnight in PBS+ solution for acquisition the following day. At least 5 × 10⁶ cells. 5 The events were recorded. Compensation was performed for each antibody using anti-mouse IgG and negative control beads (BD Biosciences, #552843) and single staining of cells for Live / Dead staining. Data analysis was performed using FlowJo version 10.4.2 (LLC, USA).

[0251] RNA sequencing of CAF-S1 primary cell line isolated from BC RNA was extracted from CAF-S1 fibroblasts using the Qiagen miRNeasy kit (Qiagen, #217004) according to the manufacturer's instructions. Of the seven CAF-S1 primary cell lines studied here, three were isolated by sorting and four by spreading. These seven CAF-S1 primary cell lines were generated from seven different BC patients. RNA integrity and quality were analyzed using the Agilent RNA 6000 Pico kit (Agilent Technologies, #5067-1513). cDNA libraries were prepared using the TruSeq strand mRNA kit (Illumina, #20020594) and subsequently sequenced using NovaSeq (Illumina). Reads were mapped to the human reference genome (hg38; Gencode release 26) and quantified using STAR (version 2.5.3a) with the parameters "outFilterMultimapNmax=20;alignSJoverhangMin=8;alignSJDBoverhangMin=1;outFilterMismatchNmax=999;outFilterMismatchNoverLmax=0.04;alignIntronMin=20;alignIntronMax=1000000;alignMatesGapMax=1000000;outMultimapperOrder=Random". Only genes with at least one read in 5% of all samples were retained for further analysis. Normalization was performed using the DESeq2 R package, and the raw read matrix was log2 transformed. To identify the identity of primary CAF-S1 cell lines, a score was calculated for each CAF-S1 cluster signature based on the average expression of the genes constituting the iCAF / myCAF signature (as defined in Ohlund et al. J Exp Med 2017;214(3):579-96). The p-values ​​were derived from DESeq2 analysis.

[0252] Functional assay CD4 + CD25 + Isolation of T lymphocytes: CD4 +CD25 + T lymphocytes were isolated from healthy donor peripheral blood obtained from the "Établiment Français du Cin" at the Saint-Antoine-Crosatier Blood Bank in Paris, France, through an agreement with the Curie Institute (Paris, France). Specifically, peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Stemcell, #07861), as previously described by Costa A et al. in Cancer Cell 2018;33(3):463-79 e10. CD4 + CD25+ is human CD4 according to the manufacturer's instructions. + CD25 + Using magnetic cell separation (MACS) with a Treg isolation kit (Miltenyi Biotec, #130-091-301), 5 × 10 8 Refined from PBMC. CD4 + CD25 + The purity of T lymphocytes was determined by flow cytometry, as described by Costa A et al., Cancer Cell 2018;33(3):463-79 e10.

[0253] Isolation of CAF-S1 clusters in culture: To isolate different CAF-S1 clusters, the inventors initially began by sorting cells according to specific markers, but were unable to keep the cells viable with different identities. Next, they tested two different isolation methods by spreading and sorting. In the "spreading" method, tumors were cut into small pieces and incubated in a humidified 1.5% O2 and 5% CO2 incubator in plastic dishes (Falcon, #353003) with 10% FBS (Biosera, #FB-1003 / 500), streptomycin (100 μg / ml), and penicillin (100 U / ml) (Gibco, #15140-122), spreading and expanding fibroblasts at 37°C for at least 2-3 weeks. For the isolation of fibroblasts by the "selection" method, tumor cells were digested using the enzyme cocktail described in (#Isolation of CAF-S1 from BC), and then selected for 2 hours in 48-well plastic dishes (TPP plates, #192048) pre-coated with FBS using BDFACS ARIA III® with the gating strategy detailed in (#CAF-S1 RNA sequencing in single cells). Next, the cells selected for CAF-S1 were grown for 3-4 weeks at 37°C in plastic dishes (TPP plates, #192048) in pericyte medium (ScienCell, #1201) supplemented with 2% FBS (ScienCell, #0010) humidified in a 1.5% O2 and 5% CO2 incubator. To compare the cellular identity of sorted and spread CAF-S1 fibroblasts under the exact same conditions used in the functional assay, both types of fibroblasts (spreaded and sorted) were transferred to plastic dishes (Falcon, #353047) in 20% O2 DMEM medium (HyClone, #SH30243.01), and these medium conditions are suitable for co-culture with CD4+ CD25+ T lymphocytes applied in the in vitro functional assay.Using these protocols, seven distinct CAF-S1 cell lines were isolated from seven different patients, three by sorting and four by spreading. To avoid any in vitro activity, these primary CAF-S1 cell lines isolated by sorting and spreading were used up to passage 5. Furthermore, in each experiment, the properties of spread-and-sorted cells were compared at the same passage.

[0254] Treg-CAF-S1 cluster function assay: 5 × 10 4 Individual CAF-S1 cells (spreading and sorting) were placed in a 24-well plate (Falcon, #353047) in DMEM (HyClone, #SH30243.01) with 10% FBS (Biosera, #FB-1003 / 500) and 1.5% O 2 Sowed overnight to allow to fully adhere. Next, remove the culture medium and 5 × 10 5 CD4 + CD25 + T lymphocytes were added to 500 μl of DMEM 1% FBS (ratio 1-10) and incubated at 37°C and 20% O2 overnight (for RNA analysis) or for 24 hours (for FACS). For FACS analysis, non-adherent cells (CD4) were selected. + CD25 +The samples were collected, washed, and stained at room temperature for 30 minutes using the following markers: Live Dead (1:1000, BD Bioscience, #562247), anti-CD45-BUV395 (1:50, BD Bioscience, #BD-563792), anti-CD4-APC (1:50, Miltenyi Biotec, #130-092-374), anti-CD25 PE-cy7 (1:33, BD Bioscience, #557741), anti-FOXP3-FITC (1:33, ebioscience, #53-4776-42), anti-CTLA-4-Pe-cy5 (1:20, BD Bioscience, #555854), anti-PD-1-BUV737 (BD Bioscience, # Anti-TIGIT-BV605 (1:50, BD Bioscience, #747841), anti-LAG3-BV510 (1:50, BD Bioscience, #744985), anti-TIM3-BV711 (1:50, BD Bioscience, #565566). Adherent cells were trypsin-treated, washed, and stained with Live Dead NIR in addition to CAF-S1 cluster markers (ANTXR1, CD9, SDC1, LAMP5, GPC3, DLK1) to remove dead cells. CD45 was added to remove remaining Treg cells. Cells (Treg panel and CAF-S1 panel) were acquired using a ZE5 cell analyzer (Bio-Rad) and analyzed with Flowjo v10.4.2. Non-adherent cells (CD4) were used for RNA analysis. + CD25 +The RNA was collected by pipetting and spun down. Next, RNA was extracted using a single-cell RNA purification kit (Norgen Biotek, #51800) according to the manufacturer's recommendations. RNA integrity and quality were analyzed using the Agilent RNA 6000 Pico kit (Agilent Technologies, #5067-1513). The cDNA library was prepared using the TruSeq RNA Exome kit (Illumina, #20020189) and subsequently sequenced using NovaSeq (Illumina). Reads were mapped to the human reference genome (hg38; Gencode release 29) and quantified using STAR (version 2.6.1a) with the parameters "outFilterMultimapNmax=20;alignSJoverhangMin=8;alignSJDBoverhangMin=1;outFilterMismatchNmax=999;outFilterMismatchNoverLmax=0.04;alignIntronMin=20;alignIntronMax=1000000;alignMatesGapMax=1000000;outMultimapperOrder=Random". Only genes with at least one read in 5% of all samples were retained for further analysis. Normalization was performed using the DESeq2 R package.

[0255] Treg-CAF-S1 cluster intracellular staining: 5 × 10 4 One CAF-S1 cell (spread) was placed in a 24-well plate (Falcon, #353047) in DMEM (HyClone, #SH30243.01) with 10% FBS (Biosera, #FB-1003 / 500) and 1.5% O 2 Sowed overnight to allow to fully adhere. Next, remove the culture medium and 5 × 10 5 CD4 + CD25 + T lymphocytes were added to 500 μl of DMEM 1% FBS (ratio 1-10) and incubated overnight at 37°C and 20% O2. Next, non-adherent cells (CD4) were analyzed. + CD25+ The cells were collected, washed, and stained with Live Dead (1:1000, BD Bioscience, #562247) at room temperature for 30 minutes. After washing, the cells were divided into two groups (one fixed and permeabilized, the other held without fixation / permeabilization except for FOXP3 staining), and stained with the following markers: anti-CD45-BUV395 (1:50, BD Bioscience, #BD-563792), anti-CD4-APC (1:50, Miltenyi Biotec, #130-092-374), anti-CD25 PE-cy7 (1:33, BD Bioscience, #557741), anti-CTLA-4-Pe-cy5 (1:20, BD Bioscience, #555854), anti-PD-1-BUV737 (BD Bioscience, #565299), anti-TIGIT-BV605 (1:50, BD Bioscience, #747841), anti-LAG3-BV510 (1:50, BD Bioscience, #744985), anti-TIM3-BV711 (1:50, BD Bioscience, #565566) anti-FOXP3-FITC (1:33, ebioscience, #53-4776-42).

[0256] Comparison of fibroblasts derived from normal healthy tissue and CAF-S1 derived from BC. Primary fibroblasts were collected from paratumor tissue, i.e., tissue defined as healthy by the referring pathologist, using the spreading method (see above). The paratumor tissue was cut into small pieces, placed in plastic dishes (Falcon, #353003), and cultured at 37°C for 2-3 weeks in DMEM (HyClone, #SH30243.01) supplemented with 10% FBS (Biosera, #FB-1003 / 500), streptomycin (100 μg / ml), and penicillin (100 U / ml) (Gibco, #15140122). Next, spread fibroblasts were analyzed at early and late passages (passages 2 and 5, respectively) to verify the expression of the CAF-S1 marker. Primary cells were trypsin-treated, resuspended in PBS, stained with LIVE / DEAD® Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific, #L34957), diluted in PBS at room temperature for 20 minutes, and fixed with 4% PFA at room temperature for 20 minutes. After rapid washing with PBS+, cells were stained with anti-FAP antibody (1:100, R&D Systems, #MAB3715) or isotype control (1:100, R&D Systems, #MAB002) in PBS+ at room temperature for 40 minutes. Both the antibody and isotype control were coupled using the Xenon APC mouse IgG1 labeling kit (Thermo Fisher Scientific, #Z-25051). Cells were acquired using an LSR FORTESSA analyzer (BD biosciences). 50,000 events were recorded per sample.

[0257] RNA sequencing from NSCLC samples Formalin-fixed, paraffin-embedded (FFPE) biopsies (N=120) from NSCLC naives from any treatment were processed for RNA extraction using the High FFPET RNA Isolation Kit (Roche, #06650775001) according to the manufacturer's instructions. RNA integrity and quality were analyzed using the Agilent RNA 6000 Pico Kit (Agilent Technologies, #5067-1513). Samples with a DV200 greater than 40% were selected for RNA sequencing (N=70). cDNA libraries were prepared using the Nextera XT Sample Preparation Kit (Illumina, #FC-131-10) and subsequently sequenced using NovaSeq (Illumina). Reads were mapped to the human reference genome (release hg19 / GRCh37) and quantified using STAR (version 2.5.3a) with the parameters "outFilterMultimapNmax=20;alignSJoverhangMin=8;alignSJDBoverhangMin=1;outFilterMismatchNmax=999;outFilterMismatchNoverLmax=0.04;alignIntronMin=20;alignIntronMax=1000000;alignMatesGapMax=1000000;outMultimapperOrder=Random". Only genes with at least one read in 5% of all samples were retained for further analysis. Normalization, unsupervised analysis (PCA), and difference analysis between responder and non-responder patients were performed using the DESeq2R package.

[0258] statistical analysis All statistical analyses and graphical representations of the data were performed using the R environment (https: / / cran.r-project.org, version 3.5.3) or GraphPad Prism software (version 8.1.1). The statistical tests used are consistent with the data distribution. Normality was first checked using the Shapiro-Wilk test, and parametric or nonparametric two-tailed tests were applied according to normality, as shown in the legend of each figure. The scRNA-seq data shown in Figures 1, 2, 3, 7, and 8 were analyzed using the Seurat R package (version 3.0). The correlation matrices shown in Figures 2 and 4 were calculated using the cor function from the stats R package with method="pearson" and use="pairwise.complete.obs". The Corrplot R function was used for clustering and visualization of the correlation matrices with the following parameters: order="hclust" and hclust.method="ward.D2". Quantifications from the FACS analysis shown in Figure 5 are shown using mean ± sem. Figure 10 shows CD4 cultured alone or in the presence of ecm-myCAF. + CD25 + Differential analysis between T cells was performed using the DESeq2 R package. Gene Set Enrichment Analysis (GSEA) software version 3.0 (Broad Institute) was used as shown in Figure 6. For melanoma RNA-seq data, the following parameters were applied: enrichment statistics = "weighted", metric for ranking genes = "Signal2Noise". For NSCLC RNA-seq data, GSEAPreranked was used with log2x change from DESeq2 differential analysis and enrichment score in "classic" mode as the metric for ranking genes.

[0259] [Table 1A]

[0260] [Table 1B]

[0261] NSCLC samples were obtained from routine diagnostic samples from patients treated with immunotumor drugs, stored in the Department of Pathology at Bichat Hospital. All patients received the first cycle of second- or third-line immunotherapy between July 28, 2015, and February 20, 2018, according to drug registration and ongoing clinical trials during that period. The efficacy of immunotumor therapy was assessed every 8–12 weeks by whole-body CT scans according to RECIST v.1.1 criteria. All clinical data were obtained from patients' electronic files. Histological subtypes of non-small cell lung cancer samples were determined from formalin-fixed, paraffin-embedded tissue sections stained with hematoxylin, from bronchoscopy or CT-guided transthoracic biopsy samples, according to the current World Health Organization 2015 classification. Diagnosis of squamous cell carcinoma was supported by p40-positive and TTF-1 (thyroid transcription factor 1)-negative immunohistochemistry. Non-squamous cell lung cancer included both adenocarcinomas showing Alcian blue-positive staining (for mucinous tumor cell content) and / or nuclear TTF-1-positive immunohistochemistry, and large cell carcinomas lacking mucin secretion with p40 and TTF-1-negative immunohistochemistry. The two samples showed mixed features of squamous cell carcinoma and adenocarcinoma differentiation. According to the 8th TNM staging system, the two stage IIIB patients had unresectable lung tumors and contraindications to radiotherapy. * PD-L1 staining was performed and interpreted by AG on 4 μm paraffin-embedded sections from diagnostic, pre-treatment biopsy specimens containing at least 200 tumor cells, using a commercially available Cell Signaling Technology E1L3N clone on the Leica Bond platform. ** The performance status of the EOCG (Eastern Cooperative Oncology Group) was assessed at the time of the first immunotherapy cycle, according to Oken et al., Am J Clin Oncol 1982;5(6):649-55.

[0262] Table 2

Claims

1. This method includes detecting anthrax toxin receptor 1 (ANTXR1) and fibroblast-activating protein (FAP)-positive fibroblasts in cancer samples from subjects suffering from cancer, and ANTXR1 + The presence of FAP+ fibroblasts is an indicator of immunosuppressive cancer-associated fibroblasts (CAFs), and this is an in vitro method for detecting immunosuppressive cancer-associated fibroblasts (CAFs) in cancer samples from the subject.

2. An in vitro method for predicting a subject's response to immunotherapy, comprising detecting ANTXR1+ FAP+ CAF in a cancer sample from a subject suffering from cancer, wherein the ANTXR1+ FAP+ CAF in the cancer sample predicts the subject's response to an immunotherapy agent.

3. An immunotherapy agent for use in the treatment of cancer in a patient, wherein the patient presents (a) a cancer sample having a low number or percentage of ANTXR1+ FAP+ CAF, or (b) a cancer sample not having ANTXR1+ FAP+ CAF.

4. A method for predicting a target response according to claim 2, comprising determining the proportion of ANTXR1+ FAP+ CAF, wherein the proportion of ANTXR1+ FAP+ CAF is the number of ANTXR1+ FAP+ CAF relative to the total number of fibroblasts in a cancer sample or the total number of cells in a cancer sample, or an immunotherapy agent for use according to claim 3.

5. ANTXR1 + FAP + CAF is FAP + ANTXR1 + LAMP5 - SDC1+ CAF, FAP + ANTXR1 + LAMP5 + SDC1+ / - CAF, and FAP + ANTXR1 + SDC1 - LAMP5 - An immunotherapeutic agent for the method according to any one of claims 1, 2, and 4 or the use according to claim 3 or 4, selected from the group consisting of CD9+ CAF.

6. Use of ANTXR1 and FAP+ as biomarkers for identifying immunosuppressive cancer-associated fibroblasts.

7. Use of ANTXR1 and FAP+ in cancer-associated fibroblast populations as biomarkers for the tumor of a target subject to predict the response of the target subject to immunotherapy agents.

8. A drug that targets ANTXR1+ FAP+ CAF for use in the treatment of cancer in patients, wherein the patient has ANTXR1 in tumor samples from the patient. + The drug has FAP+ CAF, and the drug suppresses or reduces the immunosuppressive effect of ANTXR1+ FAP+ CAF, and the drug is selected from the group consisting of (i) an anti-ANTXR1 antibody which may be conjugated with a cytotoxic drug, a multispecific molecule containing an anti-ANTXR1 moiety, an anti-ANTXR1 T cell receptor (TCR) and an anti-ANTXR1 chimeric antigen receptor (CAR), and (ii) immune cells expressing an anti-ANTXR1 TCR or an anti-ANTXR1 CAR, or any combination thereof.

9. The agent according to claim 8, wherein the immune cells are T cells or natural killer cells.

10. A drug targeting ANTXR1+ FAP+ CAF for use according to claim 8 or 9, for use in combination with an immunotherapy agent.

11. The patient had ANTXR1 in tumor samples from the patient. + FAP inhibitors for use in the treatment of cancer in patients with FAP+ CAF.

12. An FAP inhibitor for use according to claim 11, for use in combination with an immunotherapy agent.

13. The FAP inhibitor for use according to claim 11 or 12 is selected from the group consisting of tarabostat, PT-100, linagliptin (Tradjenta), FAPI-02, FAPI-04 and FAPI-46, FAP inhibitors having an N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold, peptide-targeted radionuclides such as FAP-2286, and any combination thereof.

14. A combination preparation for simultaneous, separate, or sequential use in the treatment of a patient's cancer comprises a) an ANTXR1+ FAP+ CAF-targeting agent as described in claim 8 and b) an immunotherapy agent, wherein the patient has ANTXR1 in a tumor sample from the patient. + Products or kits that contain FAP+CAF.

15. The immunotherapy agent is selected from the group consisting of therapeutic agents that stimulate the patient's immune system to attack malignant tumor cells, immunization of the patient having a tumor antigen, molecules that stimulate the immune system such as cytokines, therapeutic antibodies, adoptive T cell therapy, CAR cell therapy, immune checkpoint inhibitors, and any combination thereof, according to the method of any one of claims 2, 4, and 5, the immunotherapy agent for use according to any one of claims 3 to 5, the use according to claim 7, the agent for use according to claim 9, the FAP inhibitor for use according to claim 12 or 13, or the product or kit according to claim 14.

16. The method according to claim 15, or an immunotherapy agent for use according to claim 15, the use according to claim 15, a drug for use according to claim 15, or a product or kit according to claim 15. The method according to claim 15, or an immunotherapy agent for use according to claim 15, the use according to claim 15, a drug for use according to claim 15, a FAP inhibitor for use according to claim 15, or a product or kit according to claim 15.

17. The method according to claim 15, wherein the immunotherapy agent is selected from the group consisting of ipilimumab, nivolumab, BGB-A317, pembrolizumab, atezolizumab, avelumab, or durvalumab, BMS-986016, and epacadostat, or any combination thereof; or the immunotherapy agent for use according to claim 15; the use according to claim 15; the agent for use according to claim 15; the FAP inhibitor for use according to claim 15; or the product or kit according to claim 15.

18. The cancer is selected from the group consisting of prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, stomach cancer, kidney cancer, ovarian cancer, hepatocellular carcinoma, osteosarcoma, melanoma, hypopharyngeal cancer, esophageal cancer, endometrial cancer, cervical cancer, pancreatic cancer, liver cancer, colon or colorectal cancer, adenocarcinoma, sarcoma, non-squamous cell carcinoma, squamous cell carcinoma, neuroendocrine tumor, muscle cancer, adrenal cancer, thyroid cancer, uterine cancer, skin cancer, melanoma, metastatic melanoma, bladder cancer, and head and neck cancer, claims 1, 2 , the method according to any one of claims 4, 5 and 15 to 17, an immunotherapy agent for use according to any one of claims 3 to 5 and 15 to 17, the use according to any one of claims 7 and 15 to 17, a drug for use according to any one of claims 8 to 10 and 15 to 17, an FAP inhibitor for use according to any one of claims 11 to 13 and 15 to 17, or a product or kit according to any one of claims 14 to 17.

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