Antxr1 as a biomarker of immunosuppressive fibroblast population and its use for predicting response to immunotherapies

Abstract

The present invention provides an in vitro method for detecting immuno-suppressive fibroblasts, in particular cancer-associated fibroblasts (CAFs), in a cancerous sample from a subject suffering from a carcinoma, the method comprising detecting ANTXR1 + fibroblasts in a cancerous sample from this patient.SOLUTION: The present invention also relates to an in vitro method for predicting the response of a subject suffering from cancers to an immunotherapeutic treatment, an immunotherapeutic treatment for use in treating cancers in patients, the use of ANTXR1 as a biomarker for the identification of immunosuppressive fibroblasts, in particular immunosuppressive CAFs, an agent targeting ANTXR1 + fibroblasts for use in treating cancers in patients, an article of manufacture or a kit comprising a) an agent targeting ANTXR1 + fibroblasts and b) an immunotherapeutic treatment.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of medicine, particularly to the field of oncology. It provides new markers for immunosuppressive cell populations and uses thereof. [Background technology]

[0002] Cancer is the second leading cause of death worldwide, with 9.6 million deaths in 2018. With over 15 million new cases diagnosed each year, cancer prevalence is also very high, with the number of new cases expected 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. Choosing the best treatment for a patient depends on the type, location, and grade of the cancer, as well as the patient's health and preferences.

[0004] Over the past few decades, immunotherapy has become an important part of cancer treatment strategies. Cancer immunotherapy relies on using the immune system to treat cancer. Among the diverse immunotherapy treatments 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, identification of biomarkers that can reliably distinguish responder from non-responder patients before initiating treatment is necessary to select patients who are likely to benefit from immuno-oncology drugs.

[0006] Cancer-associated fibroblasts (CAFs) are abundant components of cancer and perform important pro-tumorigenic functions. It is now recognized that CAFs are heterogeneous and distinct CAF subsets can be defined based on the expression of specific markers. To date, four CAF subsets, designated CAF-S1-S4, have been identified in human breast and ovarian cancers (Costa A et al., Cancer Cell 2018;33(3):463-79 e10). While CAF-S2 and CAF-S3 fibroblasts are also detected in healthy tissues and may be reminiscent of normal fibroblasts, CAF-S1 and CAF-S4 myofibroblasts are restricted 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 cancers, whereas CAF-S4 does not. Because CAF subpopulations remain heterogeneous, there is a need to identify CAFs that play specific roles in primary resistance to immunotherapy in cancer patients, and there is a need to identify new markers, especially for detecting immunosuppressive CAFs in cancer.

[0007] There is also a persistent need to develop new strategies to overcome the immunosuppressive environment, thereby making immunotherapy, and in particular immune checkpoint inhibitor therapy, more effective and accessible to all patients. The present invention seeks 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, both tumor cells themselves and host stromal cells. It is now clear that stromal cells in the tumor microenvironment play an important role in cancer development. The tumor stroma contains fibroblasts, particularly cancer-associated fibroblasts (CAFs), vascular endothelial cells, immune cells, and the extracellular matrix. CAFs are the most abundant component of the tumor stroma, occupying the majority of the stroma and influencing the tumor microenvironment to promote cancer development, angiogenesis, invasion, and metastasis. [Means for solving the problem]

[0011] We discovered a novel subpopulation of CAFs that plays a key role in establishing an immunosuppressive microenvironment at tumor sites. Using scRNA-seq, we addressed the heterogeneity of the CAF-S1 immunosuppressive subpopulation and identified eight distinct CAF-S1 clusters. Three of these clusters (1, 2, and 5) belong to the inflammatory (iCAF) subgroup, while five of the clusters (0, 3, 4, 6, and 7) belong 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 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 pathways (cluster 4), interferon γ (cluster 5), interferon αβ (cluster 6), and actomyosin pathways (cluster 7). Accordingly, we annotated these clusters as follows: ecm-iCAF (cluster 0), detox-iCAF (cluster 1), IL-iCAF (cluster 2), TGFβ-iCAF (cluster 3), wound-iCAF (cluster 4), IFNγ-iCAF (cluster 5), IFNαβ-iCAF (cluster 6), and act-iCAF (cluster 7).

[0013] The existence 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 relevance of these CAF-S1 clusters across different cancer types.

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

[0015] Indeed, the ecm-myCAF (cluster 0) and TGFβ-myCAF (cluster 3) clusters express high levels of PD-1 + , CTLA-4 + and TIGIT + CD4 + T lymphocytes (which are themselves enriched in Tregs) and CD8 + It is enriched in tumors with low T lymphocyte fractions. 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-myCAFs (cluster 0) and TGFβ-myCAFs (cluster 3), wound-myCAFs (cluster 4) are not associated with an immunosuppressive environment but are correlated with high global infiltration by T lymphocytes. Therefore, the wound-myCAF cluster (cluster 4) is enriched in tumors from patients who do not respond to immunotherapy and may therefore serve as a novel surrogate marker of primary resistance to immunotherapy in highly invasive tumors that are normally sensitive to this type of treatment. Therefore, assessing the content of specific CAF-S1 clusters in tumors at diagnosis provides additional value for predicting primary resistance to immune checkpoint inhibitors.

[0017] In particular, we show 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-myCAFs (cluster 0) express FOXP3 highIncreased the fraction of T cells and CD4 + CD25 + It stimulates both PD-1 and CTLA-4 protein levels on the surface of T lymphocytes, which in turn increases the proportion of TGFβ-myCAFs (cluster 3). This reveals a positive feedback loop between the CAF-S1 cluster of immunosuppressive ECM-myCAFs (cluster 0) and TGFβ-myCAFs (cluster 3) and Tregs, which promote immunosuppression and contribute to resistance to immunotherapy. Thus, these data support the development of strategies that combine PD-1 and / or CTLA-4 blockade with therapies targeting specific CAF-S1 cluster components to overcome primary resistance to immune checkpoint blockade.

[0018] Therefore, our data suggest that ANTXR1 + FAP + Supporting the identification of a new subset of patients with CAF, these patients exhibit immunosuppression and have resistance or poor response to immunotherapy. Thus, in this particular subset of patients, ANTXR1 + FAP + ANTXR1 to inhibit or reduce the immunosuppressive effects of CAFs + Drugs targeting CAFs may offer therapeutic benefits. Furthermore, the combination of these drugs with immunotherapeutic agents can prevent the development of resistance to immunotherapeutic agents or restore response. Furthermore, even when FAP is expressed by all CAF-S1 cells, ANTXR1 + FAP + CAFs are immunosuppressive and associated with immunotherapy resistance, whereas ANTXR1 + FAP - CAF is not, so ANTXR1 + FAP + Patients may derive greater therapeutic benefit from treatment with FAP inhibitors, and in this context, the benefit / risk balance favors this treatment in this particular 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, allowing 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 a cancer sample from a subject suffering from cancer, the method comprising detecting anthrax toxin receptor 1 (ANTXR1)-positive fibroblasts in the cancer sample from the patient, wherein ANTXR1 + The presence of fibroblasts is indicative of immunosuppressive CAFs.

[0021] In a second aspect, the present invention relates to an in vitro method for predicting the response of a subject suffering from cancer to an immunotherapeutic agent, the method comprising measuring ANTXR1 in a cancer sample from the patient. + ANTXR1 in cancer samples, including detecting fibroblasts + Fibroblasts predict patient response to immunotherapeutic agents.

[0022] In a third aspect, the present invention relates to an immunotherapeutic agent for use in treating cancer in a patient, the patient having (a) a low number or percentage of ANTXR1 + (b) providing a cancer sample containing fibroblasts; or (c) providing a sample containing ANTXR1 + A cancer sample without fibroblasts is presented.

[0023] In particular, the method for predicting a subject's or immunotherapeutic agent response includes the step of: + The method further comprises determining the proportion of fibroblasts, + The percentage of fibroblasts is the ratio of ANTXR1 to the total number of fibroblasts in the cancer sample or the total number of cells in the cancer sample. + The number of fibroblasts.

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

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

[0026] In a fourth aspect, the present invention relates to the use of ANTXR1 as a biomarker for identifying immunosuppressive fibroblasts, preferably immunosuppressive CAFs.The present invention also relates to the use of ANTXR1 in fibroblast populations, preferably CAF populations, as a tumor biomarker for predicting the response of subjects suffering from cancer to immunotherapy.

[0027] In a fifth aspect, the present invention provides an ANTXR1 inhibitor for use in treating cancer in a patient. + For agents targeting fibroblasts, the patient may be tested for ANTXR1 in a tumor sample from the patient. + FAP + fibroblasts, preferably CAFs, and the agent is ANTXR1 +The agent inhibits or reduces the immunosuppressive effect of fibroblasts, and preferably the agent is selected from the group consisting of (i) an anti-ANTXR1 antibody optionally conjugated to a cytotoxic drug, a multispecific molecule comprising an anti-ANTXR1 moiety, an anti-ANTXR1 T cell receptor (TCR) and an anti-ANTXR1 chimeric antigen receptor (CAR), and (ii) an immune cell, preferably a T cell or a natural killer cell, expressing an anti-ANTXR1 TCR or an anti-ANTXR1 CAR, or any combination thereof.

[0028] In particular, ANTXR1 + The fibroblast-targeting agent is intended for use in combination with an immunotherapeutic agent.

[0029] In a sixth aspect, the present invention relates to a FAP inhibitor for use in treating cancer in a patient, the patient having ANTXR1 in a tumor sample from the patient. + FAP + fibroblasts, preferably CAFs. In certain embodiments, the FAP inhibitor is for use in combination with an immunotherapeutic agent.

[0030] Preferably, the FAP inhibitor is talabostat or PT-100 (CAS number 149682-77-9, [(2R)-1-[(2S)-2-amino-3-methylbutanoyl]pyrrolidin-2-yl]boronic acid), linagliptin (Tradjenta) (CAS number 668270-12-0; 8-[(3R)-3-aminopiperidin-1-yl]-7-but-2-ynyl-3-methyl- 1-[(4-methylquinazolin-2-yl)methyl]purine-2,6-dione), a FAP inhibitor with an N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold, FAPI-02 (CAS no. 2370952-98-8, (S)-2,2',2''-(10-(2-(4-(3-((4-((2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)carbamate) (S)-2,2',2''-(10-(2-(4-(3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid), FAPI-04 (CAS number 2374782-02-0, (S)-2,2',2''-(10-(2-(4-(3-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid), -oxoethyl)carbamoyl)quinolin-6-yl)oxy)propyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) and peptide-targeted radionuclides such as FAPI-46 (CAS Number: 2374782-04-2), FAP-2286, and any combination thereof.

[0031] In a seventh aspect, the present invention provides a combination preparation for simultaneous, separate or sequential use in the treatment of cancer in a patient, comprising: a) ANTXR1 + The present invention relates to a product or kit comprising a) a fibroblast-targeting agent and b) an immunotherapeutic agent.

[0032] In particular, the immunotherapeutic agent is selected from the group consisting of therapeutic treatments 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 immunoreceptor with Ig and ITIM domains (TIGIT), lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), B- and T-lymphocyte attenuator (BLTA), IDO1, or any combination thereof, preferably antibodies against CTLA-4, PD-1, PD-L1 and TIGIT, or any combination thereof, more preferably antibodies against PD-1 or CTLA-4 and a combination thereof.

[0034] In particular, the immunotherapeutic 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.

[0035] In particular, the cancer is selected from the group consisting of prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, gastric cancer, renal cancer, ovarian cancer, hepatocellular carcinoma, osteosarcoma, melanoma, hypopharyngeal cancer, esophageal cancer, uterine 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 gland cancer, thyroid cancer, uterine cancer, skin cancer, melanoma, metastatic melanoma, bladder cancer and head and neck cancer; preferably, the cancer is selected from the group consisting of head and neck cancer, breast cancer, ovarian cancer, NSCLC, melanoma and metastatic melanoma; more preferably, the cancer is selected from the group consisting of head and neck cancer, NSCLC, melanoma and metastatic melanoma. [Brief explanation of the drawings]

[0036] [Figure 1] Figure 1. Identification of distinct cell clusters of CAF-S1 fibroblasts. Uniform Manifold Approximation and Projection (UMAP) of 18,296 CAF-S1 fibroblasts across seven BC patients allows visualization of eight CAF-S1 clusters (0-7). Colors indicate distinct CAF-S1 clusters defined by a graph-based clustering method applied to the space defined by the 30 first principal components. [Figure 2] Figure 1 shows validation of the five most abundant CAF-S1 clusters in different cancer types. Percentage of distinct clusters among CAF-S1 fibroblasts based on FACS data. Each bar represents one patient (N=44). [Figure 3]Detection of CAF-S1 cell clusters in lung and head and neck cancers. (A) UMAP plot combining 18,296 CAF-S1 fibroblasts from BC (red, top left panel) and FAP+ fibroblasts from HNSCC (data from (30), n=603 FAP+ cells, blue, top left panel). Scores calculated as the average z-score of 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, top left panel). [Figure 4] Correlation between CAF-S1 clusters and immune cells in breast cancer. (A) Detailed correlation curves between two variables as indicated. Each dot represents one tumor (N=37). P-values ​​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-values ​​from Pearson correlation test. (C) Same as (B) between CAF-S1 cluster signature and cytolytic index, as defined in Rooney et al., Cell 2015;160(1-2):48-61. [Figure 5]Figure 1 shows the reciprocal effect of CAF-S1 clusters and Tregs. (A) Representative histograms (left) of FOXP3-specific mean fluorescence intensity (speMFI) in the presence of iCAFs (orange) or ecm-myCAFs (red) alone (green). After 24 hours (h) of coculture at a 10:1 ratio (T:CAF-S1), the percentage of FOXP3 cells (center) and FOXP3 protein levels were assessed among CD4+ CD25+ T cells (right). *P values ​​from Welch's t-test (N = 7 primary CAF-S1 cell lines per condition; n = 3 independent experiments). (B-G) Same as (A) for PD-1 (B), CTLA-4 (C), TIGIT (D), TIM3 (E), and LAG3 (F) immune checkpoints in FOXP3+ CD4+ CD25+ Tregs. *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 CAF-S1 primary cell lines. For each marker, surface protein levels are expressed as specific MFI, calculated as follows: specific MFI = MFI derived from specific antibody - MFI derived from isotype control, in the absence (-) or presence (+) of CD4+ CD25+ T cells (N = 7 CAF-S1 cell lines per condition; n = 3 independent experiments). P values ​​from the Mann-Whitney test. [Figure 6]Figure 1 shows the influence of the CAF-S1 cluster on resistance to immunotherapy. (A) Gene set enrichment analysis (GSEA) applied to RNA-Seq data from 28 melanoma tumors before anti-PD-1 treatment using specific signatures from each CAF-S1 cluster shows significant enrichment of the CAF-S1 gene signature (top 100 genes) in non-responding (N = 13) patients compared with responding (N = 15) patients from the cohort (33). Bottom, same as for the normal fibroblast signature (top). GSEA analysis shows that clusters 0 (ecm-iCAF), 3 (TGFβ-iCAF), and 4 (wound-iCAF) are significantly associated with non-responders (top), while clusters 1 (detox-iCAF), 2 (IL-iCAF), and 5 (IFN-iCAF) are not significantly associated (bottom). (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 the normal fibroblast signature and cytolytic index. (E) Responders and non-responders stratified by low and high CAF-S1 cluster expression (based on the third quartile of CAF cluster z-score). (F, G) Same as (E) using the normal fibroblast signature and cytolytic index. (H) Same as (A) analyzing a cohort of patients with NSCLC. [Figure 7] Figure 1. Identification of CAF-S1 clusters. (A) Representative FACS plot showing the gating strategy used to isolate CAF-S1. Cells were first gated on DAPI-EPCAM-CD45-CD31-CD235a- to exclude dead cells, epithelial cells, hematopoietic cells, endothelial cells, and erythrocytes, respectively. FAPHighCD29Med cells were then selected as CAF-S1 fibroblasts. (B) UMAP plot (as in Figure 1) of 18,296 CAF-S1 fibroblasts shows the average z-scores of the specific gene signatures of the five most abundant CAF-S1 clusters. [Figure 8]Figure 1. Identification of markers of the CAF-S1 cluster for FACS analysis and selection of CAF-S1 from HNSCC and NSCLC scRNA-seq data. Violin plot showing the distribution of expression (at RNA levels from scRNA-seq from 18,296 CAF-S1 fibroblasts from seven BC patients) of six genes encoding surface markers specific for each of the five most abundant CAF-S1 clusters. [Figure 9] Figure 1 shows the correlation between CAF-S1 clusters and immune cells. Detailed correlation plots (N=37 BCs) between CAF clusters and immune cells as indicated. P values ​​from Pearson correlation test. [Figure 10] Fibroblasts isolated from paratumor cells acquire CAF-S1 characteristics upon expansion and maintenance on plastic dishes. (A) Dot plots show the intracellular specific mean fluorescence intensity (speMFI) of FOXP3, PD-1, CTLA-4, and TIGIT in CD4+ CD25+ T cells cultured alone (left) or in the presence of ecm-myCAFs (right). P values ​​from the Mann-Whitney test (N = 4 CAF-S1 primary cell lines). (B) Box plots showing FOXP3, CTLA-4, and TIGIT mRNA levels in CD4+ CD25+ T cells cultured alone (right) or in the presence of ecm-myCAFs (left). P values ​​from DESeq2 analysis (N = 8). (C) Same as (B) for STAT and NFAT family members. [Figure 11A] A UMAP plot (as in Figure 1) of 18,296 CAF-S1 fibroblasts allows visualization of eight CAF-S1 clusters (0–7). Three predictive clusters of immunotherapy response are indicated by arrows: Cluster 0 = ECM-myCAF, Cluster 3 = TGFβ-myCAF, and Cluster 4 = wound-myCAF. [Figure 11B1]Violin plot (left) and UMAP plot (right) depict some of the most differentially expressed genes that define the CAF-S1 signature. Expression of these genes in each CAF-S1 cluster is shown using violin plot and UMAP representation. None of these top representative CAF-S1 genes are specifically expressed in clusters 0, 3, or 4, and therefore predictive of immunotherapy response. [Figure 11B2] This is a continuation of Figure 11B1. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description of the Invention definition As used herein, the term "cancer" or "tumor" refers to the presence of cells that have characteristics typical of cancer-causing cells, such as uncontrolled proliferation, and / or immortality, and / or metastatic potential, and / or rapid growth and / or proliferation rate, and / or certain characteristic morphological features. The term refers to any type of malignant tumor (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, refers to any sample containing tumor cells and cancer stromal cells, particularly fibroblasts such as cancer-associated fibroblasts (CAFs), derived from a subject. Cancer tissue is particularly composed of cancer cells and their surrounding cancer stromal cells, such as cancer-associated fibroblasts (CAFs), vascular endothelial cells, and immune cells, in addition to the extracellular matrix. Preferably, the cancer sample contains nucleic acids and / or proteins. The sample can be processed before use.

[0039] As used herein, the term "cancer-associated fibroblasts" or "cancer-associated fibroblasts" or "CAFs" refers to fibroblasts present in the stroma of cancer. They are one of the most abundant stromal components with a morphology resembling myofibroblasts. CAFs express CD45 - EpCAM - CD31 - CD29 +They are interstitial cells.

[0040] As used herein, the term "immunosuppressive fibroblasts" refers to fibroblasts that are responsible for and / or contribute to immunosuppression in tumors, i.e., the partial or complete inhibition or suppression of an individual's immune response to cancer cells. In some cases, fibroblasts can be malignant, particularly in sarcomas, or normal.

[0041] As used herein, the term "immunosuppressive cancer-associated fibroblasts" or "immunosuppressive CAFs" refers to CAFs that are responsible for and / or contribute to the immunosuppressive or immunosuppressive microenvironment of a tumor, i.e., the partial or complete inhibition or suppression of an individual's immune response to cancer cells. The "tumor microenvironment" or "TME" is the environment surrounding a tumor, including surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix (ECM).

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

[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, e.g., as described under Gene ID: 84168 and reference to UniProt Q9H6X2. The ANTXR1 protein plays a role in cell adhesion and migration.

[0044] As used herein, the terms "fibroblast activation protein," "FAP," "prolyl endopeptidase FAP," "dipeptidyl peptidase FAP," "surface-expressed protease," "Sepras," "serine integral membrane protease," "SIMP," "integral membrane serine protease," and "post-proline cleaving enzyme" are used interchangeably and refer to the product of the FAP human gene, e.g., as described under Gene ID: 2191 and reference to 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, such as tissue remodeling, fibrosis, wound healing, inflammation, and tumor growth.

[0045] As used herein, the terms "lysosome-associated membrane glycoprotein 5", "lysosome-associated membrane protein 5", "LAMP5", "brain- and dendritic cell-associated LAMP" and "brain-associated LAMP-like protein" are equivalent and refer to the product of the human LAMP5 gene as described, for example, under GeneID 24141 and UniProt Q9UJQ1 reference.

[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, e.g., as described under reference to GeneID: 6382 and UniProt P18827. SDC1 is a cell surface proteoglycan that contains both heparan sulfate and chondroitin sulfate and connects the cytoskeleton to the interstitial matrix.

[0047] As used herein, the terms "CD9," "5H9 antigen," "cytostatic gene 2 protein," "leukocyte antigen MIC3," "motility-associated protein," "MRP-1," "tetraspanin-29," "Tspan-29," and "p24" are used interchangeably herein and refer to the product of the human CD9 gene, e.g., as described under reference to GeneID:928 and UniProt P21926. CD9 is an integral membrane protein associated with integrins and regulates different processes.

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

[0049] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to animals, preferably mammals, and even more preferably humans. However, the term "subject" can also refer to non-human animals, particularly mammals, such as dogs, cats, horses, cows, pigs, sheep, and non-human primates.

[0050] As used herein, the term "marker" or "biomarker" refers to a measurable biological parameter that helps predict the development of cancer, the efficacy of cancer treatment, or the presence of immunosuppressive cells.

[0051] As used herein, the term "diagnosis" refers to determining whether a subject is likely to suffer from cancer.Those skilled in the art often make a diagnosis based on one or more diagnostic markers, the presence, absence or amount of which indicates the presence or absence of cancer." Diagnosis" is also intended to refer to providing information useful for diagnosis.

[0052] As used herein, the terms "treatment," "treat," or "treating" refer to any action intended to improve the well-being of a patient, including the treatment, prevention, prophylaxis, and delay of disease. In certain embodiments, such terms refer to the amelioration or eradication of a disease or symptoms associated with a disease. In other embodiments, the terms refer to minimizing the spread or worsening of a disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.

[0053] As used herein, the terms "immunotherapy," "immunotherapeutic agent," or "immunotherapeutic treatment" refer to cancer therapeutic treatments that use the immune system to reject cancer. Therapeutic treatments stimulate the patient's immune system to attack malignant tumor cells. Therapeutic treatments include immunizing 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 administering molecules that stimulate the immune system, such as cytokines, or administering therapeutic antibodies as drugs, in which the patient's immune system is recruited by the therapeutic antibodies to destroy tumor cells. In particular, the antibodies are directed against specific antigens, such as abnormal antigens displayed on the surface of tumors.

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

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

[0056] As used herein, a "pharmaceutical composition" refers to a preparation of one or more active agents and any other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an active agent to an organism. The compositions of the present invention can be in a form suitable for any conventional route of administration or use. In one embodiment, a "composition" typically contemplates a combination of an active agent, e.g., a compound or composition, with a naturally occurring or non-naturally occurring carrier, and includes an inert (e.g., a detectable agent or label) or active agent, e.g., an adjuvant, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc., and a pharmaceutically acceptable carrier. An "acceptable vehicle" or "acceptable carrier" as referred to herein refers to any known compound or combination of compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0057] As used herein, the terms "active ingredient," "active ingredient," "active pharmaceutical ingredient," "therapeutic agent," "antineoplastic compound," and "antineoplastic agent" are equivalent and refer to an ingredient that has a therapeutic effect.

[0058] As used herein, the term "therapeutic effect" refers to an effect induced by an active ingredient or pharmaceutical composition according to the present invention, which is capable of preventing or delaying the appearance or development of cancer, or curing or attenuating the effects of cancer.

[0059] As used herein, "effective amount" or "therapeutically effective amount" refers to the amount of an active agent, alone or in combination with one or more other active agents, required to confer a therapeutic effect on a subject, e.g., the amount of active agent required to treat a target disease or disorder or to produce a desired effect. An "effective amount" will vary 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 concomitant therapy (if any), the particular route of administration, and factors within the knowledge and expertise of the medical practitioner. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is generally preferred that maximum doses of the individual components or combinations thereof be used, i.e., the highest safe doses according to sound medical judgment.

[0060] The terms "kit," "product," or "combined preparation" as used herein specifically define a "kit of parts" in the sense that the combination partners (a) and (b) defined in this application can be administered independently or by using different fixed combinations with distinct amounts of combination partners (a) and (b), i.e., simultaneously or at different times. The parts of the kit of parts can then be administered simultaneously or chronologically staggered, i.e., at different times for any part of the kit of parts. The ratio of the total amounts of combination partner (a) and combination partner (b) administered in the combined preparation can vary. The combination partners (a) and (b) can be administered by the same route or by different routes.

[0061] As used herein, the term "concurrently" refers to a pharmaceutical composition, kit, product or combined preparation according to the invention wherein the active ingredients are used or administered simultaneously, i.e. simultaneously.

[0062] As used herein, the term "sequential" refers to a pharmaceutical composition, kit, product, or combined preparation according to the present invention in which the active ingredients are used or administered sequentially, i.e., one after the other. Preferably, when administration is sequential, all active ingredients are administered within less than about 1 hour, preferably less than about 10 minutes, and even more preferably less than about 1 minute.

[0063] As used herein, the term "separate" refers to a pharmaceutical composition, kit, product, or combined preparation according to the present invention, wherein the active ingredients are used or administered at separate times in a day. Preferably, when administered separately, the active ingredients are administered at intervals of about 1 hour to about 24 hours, preferably at intervals of about 1 and 15 hours, more preferably at intervals of about 1 and 8 hours, and even more preferably at intervals of about 1 and 4 hours.

[0064] The term "and / or" as used herein should be construed as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. For example, "A and / or B" should be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were listed individually.

[0065] The terms "a" or "an" can refer to one or more of the elements that it modifies, unless the context makes it clear whether one element or more than one element is being described (e.g., "a reagent" can mean one or more reagents).

[0066] The term "about" as used herein in connection with any and all values ​​(including the lower and upper limits of a numerical range) means any value having an acceptable deviation range of up to + / -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%). Use of the word "about" at the beginning of a value string modifies each of the values ​​(i.e., "about 1, 2, and 3" refer to about 1, about 2, and about 3). Furthermore, when a list of values ​​is set forth herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), this list includes all intermediate and fractional values ​​thereof (e.g., 54%, 85.4%).

[0067] The methods of the 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 the CAF subpopulation In a first aspect, the present invention relates to an in vitro method for detecting immunosuppressive fibroblasts, in particular 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. + Fibroblasts, especially ANTXR1 + detecting CAFs, and ANTXR1 + Fibroblasts, especially ANTXR1 + The presence of CAFs indicates immunosuppressive fibroblasts, particularly immunosuppressive CAFs. Next, the present invention contemplates the use of ANTXR1 as a biomarker for identifying immunosuppressive fibroblasts, particularly immunosuppressive CAFs, in cancer samples from subjects suffering from cancer. The present invention also provides novel biomarkers for ANTXR1 and immunosuppressive ANTXR1 in cancer treatment. + Fibroblasts, especially ANTXR1 +In particular, the present invention relates to the use of the immunosuppressive ANTXR1 as a novel biomarker for response to immunotherapy. + Fibroblasts, especially ANTXR1 + The present invention also relates to the use of ANTXR1 in fibroblasts, preferably CAF populations, as a tumor biomarker in subjects suffering from cancer to predict the subject's response to immunotherapy, in particular to predict whether the subject will be 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 a cancer sample from a subject suffering from cancer, the method comprising detecting ANTXR1 in the cancer sample from the patient. + Fibroblasts, especially ANTXR1 + This includes detecting CAFs.

[0070] Optionally, the method may further comprise the prior step of providing a cancer sample from the patient.

[0071] Preferably, the CAFs according to the present invention belong to the CAF-S1 subgroup, as described, for example, in Costa et al., 2018, Cancer Cell, Vol. 33, No. 3, pp. 463-479.e10, or in International Publication No. WO 2019 / 020728, the disclosures of which are incorporated herein by reference. The CAF-S1 population expresses 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 is characterized by FAP. + is.

[0072] As used herein, "FAP" + The term "fibroblasts" refers to a subpopulation of fibroblasts that express FAP. + CAF" or "FAP" +The term "cancer-associated fibroblasts" refers to a subpopulation of CAFs that express FAP. Not all CAFs express FAP. Thus, in a preferred embodiment, FAP + The detection of CAFs relies on the detection of CAFs that express FAP, preferably FAP mRNA and / or protein. + Other markers specific for CAFs can also be used to detect them.

[0073] As used herein, "ANTXR1" + The term "fibroblasts" refers to a subpopulation of 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 a preferred embodiment, ANTXR1 + The detection of fibroblasts, particularly CAFs, relies on the detection of fibroblasts, particularly CAFs, that express ANTXR1, preferably the detection of ANTXR1 mRNA and / or protein. In a preferred embodiment, the detection of ANTXR1 is carried out 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 a cancer sample from a subject suffering from cancer, the method comprising detecting FAPs in the cancer sample from the patient. + ANTXR1 + This includes detecting fibroblasts, particularly CAFs.

[0075] However, ANTXR1 + Other markers specific to fibroblasts, and in particular CAFs, can also be used to detect them.

[0076] As used herein, the terms "ecm-myCAF," "CAF-S1 cluster 0," and "ANTXR1" refer to + SDC1+ LAMP5 - CAF and FAP + ANTXR1 + SDC1 + LAMP5 - "CAFs" are used interchangeably and refer to a subpopulation of CAFs, in particular the cluster of 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 CAFs relies on the detection of CAFs that express ANTXR1 and SDC1 but do not express LAMP5. The detection of ANTXR1, LAMP5 and SCD1 can be carried out at the mRNA and / or protein level, preferably at the protein level, in particular by antibodies.

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

[0078] As used herein, the terms "wound-myCAF," "CAF-S1 cluster 4," and "ANTXR1" refer to + SDC1 -LAMP5 - CD9 + " and "FAP + ANTXR1 + SDC1 - LAMP5 - CD9 + " are used interchangeably and refer to a subpopulation of CAFs, in particular the cluster of CAF subpopulation CAF-S1. Such a population expresses FAP, ANTXR1 and CD9, but does not express LAMP5 and SDC1. Thus, in a preferred embodiment, ANTXR1 + SDC1 - LAMP5 - CD9 + The detection of CAFs relies on the detection of CAFs that express ANTXR1 and CD9 but not SDC1 and LAMP5. Detection of ANTXR1, LAMP5, CD9 and SCD1 can be performed at the mRNA and / or protein level, preferably at the protein level, in particular by antibodies.

[0079] In one aspect, the present invention relates to 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 ecm-myCAFs (ANTXR1) in a cancer sample from the patient. + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and / or wound-myCAF (ANTXR1 + SDC1 - LAMP5 - CD9 + Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - Optionally, the method comprises detecting TGFb-myCAF (ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / -Optionally, the method includes detecting wound-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 - LAMP5 - CD9 + Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + Optionally, the method comprises detecting TGFb-myCAF (ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + ) 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 their protein levels are significantly different from the corresponding background noise levels, e.g., levels measured under the same conditions but in the absence of cells, and / or levels corresponding to control conditions, or from reference RNA or protein, e.g., levels measured under the same conditions but in cells known not to express FAP, ANTXR1, LAMP5, SDC1 and / or CD9, respectively.

[0081] Measurement of FAP, ANTXR1, LAMP5, SDC1, and / or CD9 expression levels in fibroblasts, particularly CAFs, can be performed by various techniques well known to those skilled in the art. In particular, measurement of FAP, ANTXR1, LAMP5, SDC1, and / or CD9 expression levels in CAFs may rely on 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 blot analysis), particularly by the NanoString method and / or amplification (e.g., RT-PCR), particularly quantitative or semi-quantitative RT-PCR. Other methods of amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA).

[0083] Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. Taqman probes specific for the proteins of the transcripts of interest may be used. In a preferred embodiment, the expression levels of FAP, ANTXR1, LAMP5, SDC1 and / or CD9 and any other proteins 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 can be used interchangeably. Any published quantitative RT-PCR protocol can be used (and modified, if necessary) for use in the present method. Suitable quantitative RT-PCR procedures include, but are not limited to, those set forth in U.S. Pat. No. 5,618,703 and U.S. Patent Application No. 2005 / 0048542, which are incorporated herein by reference.

[0085] Optionally, ANTXR1, FAP, LAMP5, SDC1 and / or CD9 can be determined on tumor samples or on subsets of tumors containing fibroblasts, particularly CAFs. Thus, tumors 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. Thus, the present invention can relate to a method for detecting immunosuppressive fibroblasts, particularly CAFs, in a cancer sample from a subject suffering from cancer, the method comprising isolating fibroblasts, particularly CAFs, from the subject's cancer sample, particularly from tumor cells, and detecting ANTXR1 in the isolated fibroblasts, particularly CAFs. + This includes detecting fibroblasts, particularly CAFs, respectively.

[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 a protein can be measured by any method known to those skilled in the art. Typically, these methods involve contacting a sample with a binding partner capable of selectively interacting with a protein present in the sample. The binding partner is generally a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Such antibodies can be produced by methods well known to those skilled in the art. These antibodies include, in particular, those produced by hybridomas and those produced by genetic engineering using host cells transformed with a recombinant expression vector carrying a gene encoding the antibody. Hybridomas producing monoclonal antibodies can be obtained as follows: the protein or an immunogenic fragment thereof is used as an antigen for immunization according to conventional immunization methods. The resulting immunocytes are fused with known parent cells according to conventional cell fusion methods, and antibody-producing cells are then screened from the fused cells using conventional screening methods.

[0088] The antibodies according to the invention can be fused to a label and / or a detectable entity. Preferably, the antibodies according to the invention are labeled or fused to a detectable entity.

[0089] In a preferred embodiment, the antibody is labeled. The antibody can be labeled with a label selected from the group consisting of a radioactive label, an enzyme label, a fluorescent label, a biotin-avidin label, a chemiluminescent label, and the like. The antibodies according to the present invention can be labeled by standard labeling techniques well known to those skilled in the art, and the labeled antibodies can be visualized using well-known methods. In particular, the label generally provides a signal that can be detected by fluorescence, chemiluminescence, radioactivity, colorimetry, mass spectrometry, X-ray diffraction or absorption, magnetism, enzymatic 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 practice of the present invention, more preferably fluorescent labels. Preferably, the label is linked to the C-terminal end of the antibody.

[0091] In another preferred embodiment, the above-described antibody 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 at the C-terminal end of the antibody.

[0092] The amount of ANTXR1, FAP, LAMP5, SDC1, and / or CD9 can be measured by semi-quantitative Western blot, 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). The reaction generally involves revealing a label such as fluorescent, chemiluminescent, radioactive, enzyme-labeled, or dye molecule, or other methods for detecting the formation of a complex between an antigen and an antibody or an antibody reacted therewith. Preferably, the protein expression level is assessed 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, one cell at a time, into two or more containers based on the specific light scattering and fluorescence characteristics of each cell. A cell suspension is contained in the center of a narrow, rapidly flowing liquid stream. The stream is positioned so that the cells are separated by a large distance compared to their diameter. A vibrating mechanism breaks the stream of cells into individual droplets. The system is adjusted to reduce the probability of multiple cells per droplet. Just before the stream breaks into droplets, it passes through a fluorescence measurement station, where the fluorescent characteristics of each cell of interest are measured. A charging ring is positioned at the point where the stream breaks into droplets. A charge is placed on the ring just before the fluorescence intensity is measured, trapping the droplets when the opposite charge is disconnected from the stream. The charged droplets then pass through an electrostatic deflection system that diverts droplets to containers based on their charge.

[0094] Immunohistochemistry (IHC) refers to the process of selectively imaging intracellular antigens (e.g., proteins) in tissue sections using the principle of antibodies specifically binding to antigens in biological tissue. 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-producing reaction, or are 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 important to preserve cell morphology, tissue architecture, and the antigenicity of target epitopes. This requires appropriate tissue collection, fixation, and sectioning. Paraformaldehyde solutions are often used to fix tissues, but other methods may also be used. The tissue can then be sliced ​​or used whole, depending on the purpose of the experiment or the tissue itself. Prior to sectioning, tissue samples may be embedded in a medium such as paraffin wax or cryomedia. Sections can be sliced ​​using a variety of instruments, most commonly a microtome, cryostat, or compression tome tissue slicer. Specimens are typically sliced ​​at thicknesses ranging from 3 μm to 50 μm. Slices are then mounted on slides, dehydrated using increasing concentrations of alcohol washes (e.g., 50%, 75%, 90%, 95%, 100%), and cleared using detergents such as xylene before imaging under a microscope. Depending on the fixation and tissue preservation method, samples may require additional steps, including deparaffinization and antigen retrieval, to make epitopes accessible for antibody binding. Formalin-fixed, paraffin-embedded tissues often require antigen retrieval, which involves pretreating sections with heat or proteases. These steps can result in the difference between staining and non-staining of the target antigen. Depending on the tissue type and antigen detection method, blocking or quenching endogenous biotin or enzymes, respectively, may be necessary before antibody staining. Antibodies exhibit preferential binding activity for their specific epitopes but may also bind partially or weakly to sites on nonspecific proteins (also called reactive sites) that resemble their cognate binding site on the target antigen. To reduce background staining in IHC, samples are incubated with a buffer that blocks reactive sites to which primary or secondary antibodies may bind. Common blocking buffers include normal serum, nonfat dry milk, BSA, or gelatin. Methods for eliminating background staining include diluting the primary or secondary antibody, changing the incubation time or temperature, and using a different detection system or a different primary antibody.Quality controls should include, at a minimum, a tissue known to express the antigen as a positive control and a negative control of a tissue known not to express the antigen, as well as test tissues probed in the same manner using omission of the primary antibody (or better, absorption of the primary antibody).

[0096] In immunohistochemical detection strategies, antibodies are classified as primary or secondary reagents, as appropriate. Primary antibodies are raised against the antigen of interest and are typically unconjugated (i.e., unlabeled), while secondary antibodies are raised against the immunoglobulins of the primary antibody species. Secondary antibodies are usually fused to a label and / or detection entity, as described above.

[0097] Direct methods are one-step staining methods that involve a labeled antibody that reacts directly with the antigen in the tissue section. This method is simple and fast because it uses only one antibody, but in contrast to indirect approaches, it is less sensitive because there is little signal amplification.

[0098] Indirect methods involve the use of an unlabeled primary antibody (first layer) that binds to a target antigen in the tissue, followed by a labeled secondary antibody (second layer) that reacts with the primary antibody. The secondary antibody must be raised against the IgG of the animal species in which the primary antibody was raised. This method is more sensitive than direct detection strategies because of signal amplification resulting from the binding of several secondary antibodies to each primary antibody if the secondary antibodies are conjugated to a fluorescent or enzyme reporter. Further amplification can be achieved if the secondary antibody is conjugated to multiple biotin molecules, which can recruit avidin, streptavidin, or neutravidin protein-linked enzyme complexes.

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

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

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

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

[0103] Antibodies that can be used to measure the SDC1 expression level of CAFs by FACS or immunohistochemistry are, 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 CAFs by FACS or immunohistochemistry are, for example, the anti-CD9-BV711 antibodies reference #BD-743050 (BD Biosciences), LS-B5962 (LSBio) or AB_2075893 (BioLegend).

[0105] In a preferred embodiment, ANTXR1 is detected by FACS in a patient cancer sample. + The detection of CAFs may include the following steps: - e.g. CD45 + cells, EpCAM + Cells and CD31 + Exclude cells, thereby CD45 - EpCAM - CD31 - Cell selection to exclude non-CAF cells, and / or - Selection of CAF cells, e.g., CD29 + Cells and / or PDGFRb + Selection of cells, preferably CD29 + Cell selection, Optionally, selection of CAF-S1 cells, e.g., CD29 + , FAP + , αSMA + , PDGFRβ + and / or FSP1 + Cells, preferably FAP + Cell selection, - optionally, exclusion of dead cells by excluding stained cells using intracellular dyes, such as Violet LIVE / DEAD dye or DAPI, and - ANTXR1 in the cells obtained in the previous step + Detection of CAFs, Optionally, detection of LAMP5, SDC1 and / or CD9 positive or negative cells.

[0106] In another preferred embodiment, ANTXR1 was detected by immunohistochemistry in a patient's cancer sample. + The detection of CAFs may include the following steps: - Identification of CAFs in cancer samples, e.g., based on morphological criteria, - Preferably, ANTXR1 among CAFs based on ANTXR1 antibody immunostaining + Detection of CAFs, Optionally, FAP among CAFs, preferably based on FAP antibody immunostaining + Detection of CAFs, Optionally, detection of LAMP5, SDC1 and / or CD9 positive or negative cells.

[0107] In one aspect, the presence of immunosuppressive fibroblasts, particularly CAFs, can be determined by the amount of immunosuppressive fibroblasts, particularly CAFs, in the tumor sample or any fraction thereof, the ratio of ANTXR1 to the total number of cells in the sample or any fraction thereof, + Fibroblasts, especially ANTXR1 + This refers to the ratio or percentage of CAF cell numbers, and the ratio of ANTXR1 to the total number of stromal cells in a sample or any fraction thereof. + Fibroblasts, especially ANTXR1 + Ratio or percentage of CAF cell number, ANTXR1 to the total number of fibroblasts in the sample + The ratio or percentage of fibroblast cell number, ANTXR1 to the total number of fibroblasts in the sample + The ratio or percentage of fibroblast cell numbers, ANTXR1 to the total number of CAF cells in the sample or any fraction thereof + The ratio or percentage of the cell number of CAFs or ANTXR1 to the total number of CAF-S1 cells in the sample or any fraction thereof + This refers to the ratio or percentage of CAF cell numbers.

[0108] Preferably, "ANTXR1 in tumors" + The term "fibroblast fraction" refers to the ANTXR1 + It can refer to any ratio having the number of fibroblasts as the numerator and the number of reference cells as the denominator. + The term "CAF fraction" refers to ANTXR1 +It can refer to any ratio having the number of CAFs as the numerator and the number of reference cells as the denominator. In a cancer sample, such reference cells can be selected from the group consisting of all cells of the cancer sample, cancer cells, stromal cells, CAF cells and CAF-S1 cells, among others.

[0109] "Low percentage of ANTXR1 + fibroblasts" or "low percentage of ANTXR1 + "Fibroblasts" represents less than 20%, 10%, 5%, 2%, 1%, 0.5% or 0.1%.

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

[0111] In a first aspect, ANTXR1 in a patient cancer sample is + The presence of fibroblasts, especially CAFs, significantly increased the ANTXR1 expression level relative to the total number of cells in cancer samples. + It represents the number of fibroblasts, particularly CAF cells. Preferably, ANTXR1 + The presence of fibroblasts, in particular CAFs, represents a proportion of at least 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20% or 30%.

[0112] In a second aspect, ANTXR1 in a patient cancer sample is + The presence of fibroblasts, especially CAFs, significantly increased the ANTXR1 expression relative to the total number of stromal cells in cancer samples. + It represents the number of fibroblasts, particularly CAF cells. Preferably, ATXR1 + The presence of fibroblasts, in particular CAFs, represents a proportion of at least 0.001%, 0.1%, 1%, 5%, 10%, 20% or 30%.

[0113] In a third aspect, ANTXR1 in a patient cancer sample is + The presence of fibroblasts, especially CAFs, significantly increased the ANTXR1 expression relative to the total number of CAF cells in cancer samples.+ It represents the number of fibroblasts, particularly CAF cells. Preferably, ATXR1 + The presence of fibroblasts, in particular CAFs, represents a proportion of at least 0.1%, 1%, 5%, 10%, 20%, 30%, 40% or 50%.

[0114] In a fourth aspect, ANTXR1 in a patient cancer sample is + The presence of fibroblasts, especially CAFs, significantly increased the ANTXR1 expression level relative to the total number of CAF-S1 cells in cancer samples. + It represents the number of fibroblasts, particularly CAF cells. Preferably, ATXR1 + The presence of fibroblasts, in particular CAFs, represents a proportion of at least 0.1%, 1%, 5%, 10%, 20%, 30%, 40% or 50%.

[0115] In one embodiment, ANTXR1 in a cancer sample + The presence of fibroblasts, especially CAFs, e.g., ANTXR1 in cancer samples + The proportion of fibroblasts, particularly CAFs, is inversely proportional to the patient's responsiveness to immunotherapy treatment, and optionally, the method can be used to identify ANTXR1 in a cancer sample as being suitable for immunotherapy treatment. + Selecting patients who have no or a low proportion of fibroblasts, particularly CAFs, and / or ANTXR1 + ANTXR1 in cancer samples for alternative treatments, such as immunotherapy combined with treatments that reduce the immunosuppression induced by fibroblasts, especially CAFs, or anti-cancer treatments that exclude immunotherapy treatments. + It further includes selecting patients with a moderate or high proportion of fibroblasts, particularly CAFs.

[0116] "Inversely proportional" refers to the level of ANTXR1 in cancer samples. + The more fibroblasts, especially CAFs, present, the less likely a cancer patient is to respond to immunotherapy. These terms do not necessarily imply a direct, measurable correlation.

[0117] In one aspect, the present invention relates to an in vitro method for predicting the response of a subject suffering from cancer to immunotherapy treatment, the method comprising detecting ANTXR1 in a cancer sample from the patient. + ANTXR1 in cancer samples, including detecting fibroblasts, particularly CAFs + Fibroblasts, particularly CAFs, are indicative of or predictive of patient non-responsiveness to immunotherapy treatment. In particular, ANTXR1 detected in cancer samples + The more fibroblasts, especially CAFs, there are, the less susceptible the patient is to respond to immunotherapy treatment.

[0118] In some cases, ANTXR1 + Fibroblasts express ANTXR1 + CAF. In some cases, ANTXR1 + Fibroblasts are FAP + fibroblasts. In some cases, ANTXR1 + CAF is FAP + ANTXR1 + CAF.

[0119] Indeed, the present invention relates to the use of ANTXR1, particularly CAFs, in fibroblast populations as a tumor biomarker in subjects suffering from cancer to predict the subject's response to immunotherapy.

[0120] Optionally, the present invention relates to an in vitro method for predicting the response of a subject suffering from cancer to immunotherapy treatment, the method comprising: (a) detecting ANTXR1 in a cancer sample from the patient; + Fibroblasts, especially ANTXR1 + detecting ANTXR1 in a cancer sample, + (b) that fibroblasts, particularly CAFs, are indicative of or predictive of patient non-responsiveness to immunotherapy treatment; and (c) that ANTXR1, optionally amenable to immunotherapy treatment, is + Fibroblasts, especially ANTXR1 + Selecting patients without CAF and / or ANTXR1 + Fibroblasts, especially ANTXR1 +For alternative treatments, such as immunotherapy combined with treatments that reduce CAF-induced immune expression or anti-cancer treatments that exclude immunotherapy, ANTXR1 + Fibroblasts, especially ANTXR1 + This involves selecting patients with CAF fibroblasts.

[0121] Optionally, the method comprises detecting FAP in a patient cancer sample. + ANTXR1 + Optionally, the method comprises detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and / or wound-myCAF (ANTXR1 + SDC1 - LAMP5 - CD9 + Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - Optionally, the method comprises detecting TGFb-myCAF (ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / - Optionally, the method includes detecting wound-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 - LAMP5 - CD9 + Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / -Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + Optionally, the method comprises detecting TGFb-myCAF (ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + ) detecting CAF.

[0122] In one embodiment, CAF subpopulations are detected by specific gene signatures. A "gene signature" or "gene expression signature" is a single or group of genes in cells, particularly fibroblasts, that have a unique, characteristic pattern of gene expression. In particular, a gene signature corresponds to the deregulation of specific genes, particularly the overexpression of genes.

[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 comprises 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 comprises 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 comprises the following genes: FAP and / or ANTXR1.

[0126] Thus, in the methods of the present disclosure, ecm-myCAF, TGFβ-myCAF, and / or wound-myCAF are detected by determining the expression of genes comprising a gene signature specific for ecm-myCAF, TGFβ-myCAF, and / or wound-myCAF, the overexpression of which indicates the presence of these CAF subpopulations. Optionally, 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, in particular to detect the presence of immunosuppressive CAFs, and / or to predict response to immunotherapeutic treatment.

[0128] Optionally, the sample can be treated to remove non-CAF cells before determining the expression of the gene signature.

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

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

[0131] In another embodiment, the present invention relates to an in vitro method for predicting the response of a subject suffering from cancer to immunotherapy treatment, the method comprising: (a) ANTXR1 in patient cancer samples + Fibroblasts, especially ANTXR1 + Detecting CAFs, (b) ANTXR1 in cancer samples + Fibroblasts, especially ANTXR1 + The patient's response to immunotherapy treatment is determined by determining the proportion of CAFs in the cancer sample. + Fibroblasts, especially ANTXR1 + Inversely proportional to the CAF rate, (c) optionally, ANTXR1 suitable for immunotherapy treatment; + Fibroblasts, especially ANTXR1 + Low rates of ANTXR1 in certain patients without CAF + Fibroblasts, especially ANTXR1 + Patients with CAF were selected and ANTXR1 + Fibroblasts, especially ANTXR1 + Alternative therapies, such as immunotherapy combined with treatments that reduce CAF-induced immunosuppression or anti-cancer treatments that exclude immunotherapy, are considered. + Fibroblasts, especially ANTXR1 + Selecting patients with CAF Includes:

[0132] Optionally, the method comprises detecting FAP in a patient cancer sample. + ANTXR1 + Optionally, the method comprises detecting ecm-myCAF (ANTXR1) in the patient's cancer sample.+ SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and / or wound-myCAF (ANTXR1 + SDC1 - LAMP5 - CD9 + Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - Optionally, the method comprises detecting TGFb-myCAF (ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / - Optionally, the method includes detecting wound-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 - LAMP5 - CD9 + Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + Optionally, the method comprises detecting TGFb-myCAF (ANTXR1) in the patient's cancer sample. + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1- LAMP5 - CD9 + Optionally, the method includes detecting ecm-myCAF (ANTXR1) in the patient's cancer sample. + SDC1 + LAMP5 - ), TGFb-myCAF(ANTXR1 + LAMP5 + SDC1 + / - ) and wound-myCAF(ANTXR1 + SDC1 - LAMP5 - CD9 + ) detecting CAFs. Optionally, the method may further comprise the step of providing a cancer sample from the patient prior to step (a).

[0133] Optionally, the method comprises: + The method may further comprise administering an immunotherapy treatment to a patient having fibroblasts or CAFs. + The method may further comprise administering an alternative anti-cancer treatment to the patient with fibroblasts or CAFs, the alternative treatment comprising administering to the patient an alternative anti-cancer treatment that inhibits ANTXR1 + The anti-cancer treatment is immunotherapy or an anti-cancer treatment excluding immunotherapy treatment in combination with a treatment that reduces immunosuppression induced by fibroblasts or CAFs. The administered treatment may be selected from the group consisting of surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy, and palliative care, or any combination thereof. Alternatively or additionally, the method comprises administering to a patient a cancer-related tumor comprising: + The method may further comprise 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 a patient suffering from a tumor for immunotherapy treatment or determining whether a patient suffering from a tumor is likely to benefit from immunotherapy treatment, the method comprising detecting ANTXR1 in a cancer sample from the patient. + Determining the presence of fibroblasts or CAFs, and optionally, for immunotherapy treatment, ANTXR1 +Patients without fibroblasts or CAFs or with a low percentage of ANTXR1 + This involves selecting a patient with fibroblasts or CAFs.

[0135] Optionally, the method further comprises the prior step of providing a cancer sample from said patient.

[0136] In yet another specific embodiment, the present invention also provides ANTXR1 + The present invention relates to a method for selecting a patient suffering from a tumor for an alternative treatment, such as an anti-cancer treatment other than immunotherapy or immunotherapy treatment, in combination with a treatment that reduces the immunosuppression induced by fibroblasts or CAFs, or for selecting a patient suffering from a tumor for an alternative treatment, such as an anti-cancer treatment other than immunotherapy treatment, or for selecting a patient suffering from a tumor for an anti-cancer treatment other than immunotherapy treatment, in combination with a treatment that reduces the immunosuppression induced by fibroblasts or CAFs. + The present invention relates to a method for determining whether a patient is likely to benefit from an alternative treatment, such as immunotherapy or an anti-cancer treatment other than immunotherapy, in combination with a treatment that reduces fibroblast- or CAF-induced immunosuppression, the method comprising: + and optionally determining the presence of ANTXR1 for such alternative treatments. + Patients with CAF or high rates of ANTXR1 + This involves selecting a patient who has fibroblasts or CAFs.

[0137] In some cases, ANTXR1 + Fibroblasts or CAFs are FAP + ANTXR1 + fibroblasts or CAFs. Preferably, ANTXR1 + CAF, FAP + ANTXR1 + CAF, FAP + ANTXR1 + LAMP5 - SDC1 + CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF and ANTXR1 + SDC1 - LAMP5 - CD9+ and preferably FAP. + ANTXR1 + LAMP5 - SDC1 + CAF and / or FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF.

[0138] In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ) CAF. In some cases, ANTXR1 + CAFs express 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 + CAFs express 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 a patient for immunotherapy, the method comprising: (a) ANTXR1 in patient cancer samples + Detecting fibroblasts or CAFs; (b) ANTXR1 in cancer samples + Determining the percentage of fibroblasts or CAFs Contains ANTXR1 + A CAF percentage 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%, even more preferably less than 0.5% is predictive of patient responsiveness to immunotherapy treatment.

[0140] The method may further comprise a step c) of administering an immunotherapy, preferably the immunotherapy is an immune checkpoint inhibitor.

[0141] Optionally, the method further comprises the prior step of providing a cancer sample from said patient.

[0142] In yet another embodiment, the present invention relates to a method for selecting a patient for an alternative anti-cancer treatment, in particular excluding immunotherapy, or the method comprises: (a) ANTXR1 in patient cancer samples+ Detecting fibroblasts or CAFs; (b) ANTXR1 in cancer samples + Determining the percentage of fibroblasts or CAFs Contains ANTXR1 + A percentage of CAFs greater than 1%, preferably greater than 5%, more preferably greater than 10%, even more preferably greater than 20%, even more preferably greater than 30%, even more preferably greater than 40% is indicative of or predictive of patient non-responsiveness to immunotherapy treatment.

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

[0144] Optionally, the method further comprises the prior step of providing a cancer sample from said patient.

[0145] The present invention further relates to an immunotherapy treatment for use in treating cancer in a patient, the patient having (a) a low number or percentage of ANTXR1 + (b) fibroblasts or CAFs, or (b) ANTXR1 + The present invention also relates to the use of an immunotherapy treatment for the manufacture of a medicament for the treatment of cancer in a patient, the patient having (a) a low number or percentage of ANTXR1 + (b) fibroblasts or CAFs, or (b) ANTXR1 + The present invention also provides a cancer sample that does not have fibroblasts or CAFs. The present invention further relates to a method of treating cancer in a patient, the method comprising: (a) detecting a low number or percentage of ANTXR1 + It involves selecting a patient who (a) has fibroblasts or CAFs or (b) does not have ANTXR1+ fibroblasts or CAFs, and administering a therapeutically effective amount of immunotherapeutic treatment.

[0146] In some cases, ANTXR1 + Fibroblasts or CAFs are FAP + ANTXR1 + fibroblasts or CAFs. + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ) CAF. In some cases, ANTXR1 + CAFs express 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 + CAFs express 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, particularly immunosuppressive CAFs, according to the present invention can be used to predict or assess response to immunotherapy.

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

[0149] In a preferred embodiment, the immunotherapy treatment is an immune checkpoint inhibitor treatment, 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 containing-3), antibodies against TIGIT (T-cell immunoreceptor with Ig and ITIM domains), antibodies against BLTA (B and T lymphocyte attenuator), IDO1 inhibitors such as epacadostat, or combinations thereof.

[0150] In a 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 immunoreceptor with Ig and ITIM domains (TIGIT), lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing 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 antibodies against PD-1 or CTLA-4 and a combination 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), pidilizumab (CT-011), cemiplimab (Libtayo), camrelizumab, AUNP12, AMP-224, AGEN-2034, BGB-A317 (tisleizumab), 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. 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 ( and the monoclonal antibodies 5C4, 17D8, 2D3, 4H1, 4A11, 7D3, 5F4, described in WO 2006 / 121168. Other known 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 known in the art, and include, for example, BMS-986207 or AB154, BMS-986207, as disclosed in WO 19232484. 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 may also be used in combination with other antibodies disclosed in WO 16028656, WO 16106302, WO 16191643, WO 17030823, WO 17037707, WO 17053748, WO 17152088, WO 18033798, WO 18102536, WO 18102746, WO 18160704 No. 18200430, No. 18204363, No. 19023504, No. 19062832, No. 19129221, No. 19129261, No. 19137548, No. 19152574, No. 19154415, No. 19168382 and No. 19215728.

[0154] Preferably, the immunotherapeutic 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 agents In another aspect, the present invention also provides an ANTXR1 inhibitor for use in combination with an immunotherapy treatment for the treatment of a subject with cancer, or for use in a subject with cancer. + Fibroblast-targeting drugs, FAP + In relation to a fibroblast-targeting agent or a pharmaceutical composition comprising the same, the subject is particularly + Contains fibroblasts.

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

[0157] The present invention also relates to ANTXR1 + CAF or FAP + The present invention relates to an agent or pharmaceutical composition comprising the same that targets CAF, for use in combination with immunotherapy treatment for the treatment of a subject with cancer, or for use in a subject with cancer, wherein the subject has a tumor sample that specifically contains ANTXR1. + More specifically, when using a FAP inhibitor, the subject has ANTXR1 + FAP + Fibroblasts, especially ANTXR1 + FAP + It has CAF.

[0158] The present invention also provides a method for the treatment of a subject with cancer, comprising administering to a subject a therapeutically effective amount of ANTXR1, optionally in combination with immunotherapy treatment, for the manufacture of a medicament for the treatment of a subject with cancer. +CAF or FAP + Regarding the use of agents that target CAFs, in particular, the subject may have a tumor sample containing ANTXR1 + In a further aspect, the present invention provides a method for detecting ANTXR1 in a subject with cancer, particularly in a tumor sample from the subject, comprising detecting ANTXR1 in a subject with cancer, particularly in a tumor sample from the subject. + ANTXR1 for the treatment of subjects with fibroblasts or CAFs + CAF or FAP + The present invention relates to a method of treating a subject with cancer, the method comprising administering to a subject a therapeutic agent that targets ANTXR1. + Selecting patients with fibroblasts or CAFs, ANTXR1 + CAF or FAP + The method further comprises administering a therapeutically effective amount of an immunotherapeutic treatment to a patient, particularly a patient with ANTXR1. + As a combined preparation for simultaneous, separate or sequential use in the treatment of cancer in patients with fibroblasts or CAFs, a) ANTXR1 + CAF or FAP + The present invention relates to a product or kit comprising a) a drug targeting CAFs, and b) an immunotherapeutic agent. In particular, the subject is a cancer sample containing a medium or high proportion of ANTXR1 + We have tumor samples with fibroblasts or CAFs.

[0159] The present invention also relates to ANTXR1 + CAF or FAP +

[0013] The present invention relates to a method for selecting a patient having cancer for treatment with an agent that targets CAFs, the method comprising detecting immunosuppressive fibroblasts or CAFs as disclosed above, and determining whether the patient has ANTXR1 in the patient's cancer sample. + This includes selecting patients if they have fibroblasts or CAFs.

[0160] In particular, the drug inhibits ANTXR1 + Inhibit or reduce the immunosuppressive effects of fibroblasts or CAFs.

[0161] Preferably, the agent is selected from the group consisting of an ANTXR1 inhibitor, an anti-ANTXR1 antibody optionally conjugated to a cytotoxic drug, a multispecific molecule comprising an anti-ANTXR1 moiety, an anti-ANTXR1 T cell receptor (TCR), an anti-ANTXR1 chimeric antigen receptor (CAR), and an immune cell, preferably a T cell, expressing an anti-ANTXR1 TCR or an anti-ANTXR1 CAR, or any combination thereof.

[0162] Additionally or alternatively, the agent is selected from the group consisting of a FAP inhibitor, an anti-FAP antibody optionally conjugated to a cytotoxic drug, a multispecific molecule comprising an anti-FAP moiety, an anti-FAP T-cell receptor (TCR), an anti-FAP chimeric antigen receptor (CAR), and an immune cell, preferably a T cell, expressing an anti-FAP TCR or an anti-FAP CAR, or any combination thereof.

[0163] In particular, the ANTXR1 inhibitor may be, for example, ebselen or phenylmercuric acetate.

[0164] The FAP inhibitor (FAPI) may be selected from the group consisting of talabostat, PT-100, linagliptin (Tradjenta), FAP inhibitors having an N-(4-quinolinol)-Gly-(2-cyanopyrrolidine) backbone 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 FAP inhibitors such as FAPI-02 and FAPI-04 as described in Lindner et al., EJNMMI Radiopharmacy and Chemistry (2019) 4:16 and Lindner et al., Journal of Nuclear Medicine, April 6, 2018. For example, FAP inhibitors are described in WO20081522, WO20132661, WO20245173, WO21005125, WO21005131, U.S. Patent No. 2020246383, WO19154859, WO19083990, WO19118932, WO18111989, WO17189569, WO13107820, WO08116054 or WO07085895, the disclosures of which are incorporated herein by reference. The FAP inhibitor can also be a FAP-binding peptide linked to a radionuclide (eg, 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 a FAP inhibitor for the treatment of, or for use in the therapy of, a subject having cancer, wherein the subject has ANTXR1, particularly in the subject's tumor sample or in the tumor microenvironment. + It contains fibroblasts or CAFs.

[0166] In one embodiment, the agent 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 can comprise, consist of, or essentially consist of a classic Y-shaped antibody having two heavy and light chains or fragments thereof. Preferably, the fragment comprises the antigen-binding or variable region of the antibody. The fragment can be selected from the group consisting of, but not limited to, Fv, Fab, Fab', F(ab)2, F(ab')2, F(ab)3, Fv, single-chain Fv (scFv), dis-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 invention may be a monomeric antibody or a multimeric antibody. In particular, the antibody according to the invention is a monomeric antibody.

[0168] The antibodies according to the invention may be monoclonal or polyclonal. Preferably, the antibodies according to the invention are monoclonal.

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

[0170] The drug according to the present invention is preferably a cytotoxic drug. As used herein, the term "cytotoxic drug" refers to a molecule that, upon contact with a cell, ultimately upon internalization into the cell, alters cell function in a detrimental way (e.g., cell proliferation and / or growth and / or differentiation and / or metabolism, such as protein and / or DNA synthesis) or causes cell death. As used herein, the term "cytotoxic drug" encompasses toxins, particularly cytotoxins.

[0171] Cytotoxic drugs according to the present invention include dolastatins such as dolastatin 10, dolastatin 15, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), and 5-benzoylvaleric acid-AE ester (AEVB), maytansines such as ansamitocin, mertansine (also called emtansine or DM1) and ravtansine (also called soravtansine or DM4), daunorubicin, ertansine ... Anthracyclines such as pirubicin, pirarubicin, idarubicin, zorubicin, cerubicin, acurubicin, adlibrastin, doxorubicin, mitoxantrone, daunoxomil, nemorubicin, and PNU-159682; calicheamicins such as calicheamicin β1Br, calicheamicin gamma 1Br, calicheamicin α2I, calicheamicin α3I, calicheamicin β1I, calicheamicin gamma 1L, and calicheamicin delta 1I; ozogamicin; esperamicins such as esperamicin A1; neocarzinostatin; bleomycin; Duocarmycins such as Isin, CC-1065 and Duocarmycin A, pyrrolobenzodiazepines such as anthramycin, abeymycin, ticamycin, DC-81, mazethramycin, neothramycin A and B, polothramycin prothracarcin, sivanomycin (DC-102), sibiromycin and tomamycin, pyrrolobenzodiazepine dimers (or PBDs), indolino-benzodiazepines, indolino-benzodiazepine dimers, α-amanitin, Abraxane, actinomycin, aldesleukin, altretamine, alitretinoin In, amsacrine, 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, dithranol,Dutasteride, dexrazoxane, docetaxel, doxifluridine, erlotinib, estramustine, etoposide, exemestane, finasteride, flutamide, floxuridine, flucytosine, fludarabine, fluorouracil, ganciclovir, gefitinib, gemcitabine, goserelin, hydroxyurea, hydroxycarbamide, ifosfamide, irinotecan, imatinib, lenalidomide, leflunomide, letrozole, leuprorelin acetate, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, menotropin, mifepristone, nafarelin, nelarabine, nitrogen mustard, nitrosourea, oxaliplatin, ozogamicin, paclitaxel , podophyllin, pegasparaginase, pemetrexed, pentamidine, pentostatin, procarbazine, raloxifene, ribavarin, raltitrexed, rituximab, romidepsin, sorafenib, streptozocin, sunitinib, sirolimus, streptozocin, temozolomide, temsirolimus, teniposide, thalidomide, thioguanine, thiotepa, topotecan, tacrolimus, taxotere, tafluposide, toremifene, tretinoin, trifluridine, triptorelin, valganciclovir, valrubicin, vinblastine, vidalazine, vincristine, vindesine, vinorelbine, vemurafenib, vismodegib, vorinostat, zidovudine, vedotin, derivatives and combinations thereof. The cytotoxic drug may also be a radionuclide 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 a preferred embodiment, the antibody-drug conjugate of the present invention comprises 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 an unnatural amino acid incorporated into the antibody.

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

[0175] In one embodiment, the agent is an anti-ANTXR1 antibody or an anti-FAP CAR cell. 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 transfers antigen-binding specificity (e.g., an antibody) into an immune cell (e.g., a T cell or NK cell), thus combining the antigen-binding properties of the antigen-binding domain with the immunogenic activities of the immune cell, such as its lytic capacity and self-renewal. 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, and 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 pharmaceutical compositions comprising ANTXR1 and / or FAP CAF targeting agent, preferably FAP inhibitor, ANTXR1 inhibitor, anti-FAP antibody, anti-ANTXR1 antibody, or any combination thereof, and antibody is optionally conjugated with drug, preferably cytotoxic drug as described above, or combination thereof.In particular, such pharmaceutical compositions comprise at least one pharmaceutically acceptable excipient.For this formulation, conventional excipients can be used according to the techniques well known to those skilled in the art.

[0177] The formulations can be sterilized and, if desired, mixed with pharmaceutically acceptable carriers, excipients, salts, antioxidants and / or stabilizers that do not adversely interact with the ANTXR1 and / or FAP CAF targeting agent.

[0178] Optionally, the pharmaceutical composition may further comprise an additional therapeutic agent, particularly an immunotherapeutic agent as described above.

[0179] The formulations of the present invention can be isotonic with human blood, and those skilled in the art will understand that the formulations of the present invention 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 by an ice-freezing osmometer.

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

[0181] When the combined preparations, kits or products for use according to the invention are administered separately or sequentially, in particular when administered separately, the ANTXR1 + CAF and / or FAP +Treatment with the CAF targeting agent is preferably performed prior to immunotherapy treatment. + CAF and / or FAP + After administration of the treatment with the CAF targeting agent, preferably before administration of the immunotherapy treatment, ANTXR1 + CAF and / or FAP + The immunotherapy treatment may further comprise determining the proportion of CAFs, and + If the CAF rate is low or ANTXR1 + It is administered only if CAFs are not present.

[0182] Immunotherapeutic Treatments and Uses of Therapeutic Methods In certain aspects, the present invention also provides a method for a patient to administer to a cancer sample: (a) low levels or proportions of ANTXR1 + Fibroblasts, especially ANTXR1 + Present your CAF, or (b) ANTXR1 + Fibroblasts, especially ANTXR1 + Not presenting CAF The present invention relates to immunotherapy treatments, preferably immune checkpoint inhibitor treatments, for use in treating cancer in a patient.

[0183] The present invention also provides a method for detecting cancer samples containing: (a) low levels or proportions of ANTXR1 + presenting fibroblasts or CAFs, or (b) ANTXR1 + does not present fibroblasts or CAFs, The present invention relates to the use of an immunotherapy treatment, preferably an immune checkpoint inhibitor treatment, for the manufacture of a medicament for the treatment of cancer.

[0184] The present invention also relates to a method for treating a patient suffering from cancer, the method comprising: (a) ANTXR1 in tumors + low percentage of fibroblasts or CAFs, (b) ANTXR1 in tumors +No fibroblasts or CAFs If selected, The method includes administering to the patient an immunotherapy treatment, preferably an immune checkpoint inhibitor treatment.

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

[0186] Preferably, ANTXR1 + CAF, FAP + ANTXR1 + LAMP5 - SDC1 + CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF and ANTXR1 + SDC1 - LAMP5 - CD9 + and preferably FAP + ANTXR1 + LAMP5 - SDC1 + CAF and / or FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF.

[0187] In some cases, ANTXR1 + CAF is FAP + ANTXR1 + CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ) CAF. In some cases, ANTXR1 + CAFs express 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 + CAFs express 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 an anti-CTLA-4 antibody, an anti-PD-1 antibody and an anti-TIGIT antibody or any combination thereof, more preferably an anti-CTLA-4 antibody and / or an anti-PD-1 antibody.

[0189] The present invention further relates to a method of treating cancer in a subject, comprising administering 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, is administered to a subject suffering from cancer, and the patient's cancer or the patient's cancer sample is detected to detect ANTXR1. + Presenting with 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 + / - In particular, the ANTXR1 according to the present invention is administered + CAF and / or FAP + The amount of the CAF targeting agent or pharmaceutical composition according to the present invention can be determined by standard procedures well known to those skilled in the art. The physiological data of the patient (e.g., age, size, and weight) and the route of administration must be taken into account to determine the appropriate dosage so that a therapeutically effective amount is administered to the patient.

[0190] In some embodiments, the immunotherapy treatment is ANTXR1 + Fibroblast-targeting drugs, FAP+ The fibroblast-targeting agent and / or pharmaceutical composition according to the present invention is administered in combination with an additional cancer treatment, particularly an immunotherapy treatment, such as ANTXR1. + Fibroblast-targeting drugs, FAP + The fibroblast-targeting agent and / or pharmaceutical composition according to the present invention may be administered in combination with other targeted therapies, other immunotherapies, chemotherapy and / or radiation therapy.

[0191] In some embodiments, the immunotherapy treatment, ANTXR1 + Fibroblast-targeting drugs, FAP +The fibroblast-targeting agent and / or pharmaceutical composition according to the present invention is administered to a 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 a chemotherapeutic agent to a patient. Chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide, alkylsulfonates such as busulfan, improsulfan, and piposulfan, aziridines such as benzodopa, carboquone, metuledopa, and uredopa, altretamine, ethylenimines and methylameramines, including triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine, acetogenins (particularly bullatacin and bullatacin), and the like. non), camptothecin (including the synthetic analog topotecan), bryostatin, kallistatin, CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs), cryptophycins (especially cryptophycin 1 and cryptophycin 8), dolastatins, duocarmycins (including synthetic analogs KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictin, spongistatin, chlorambucil, chlornaphazine, chorofosfamidis estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, nitrogen mustards such as uracil mustard, nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine, antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma and calicheamicin omega), Bisphosphonates such as clodronate, including dynemicin, dynemicin A, esperamicin, and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autarubicin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, 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, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate and 5-fluorouracil (5-FU) folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calsterone, androgens such as dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; adrenal anti-inflammatory drugs such as aminoglutethimide, mitotane, and trilostane; florinic acid Folic acid supplements such as aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatlaxate, defofamine, demecolcine, diaziconazole, elformitin, elliptinium acetate, epothilones; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone ; Mopidanmol; Nitraeline; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2"-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veracrine A,Roridin A and Anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel and Doxetaxel; Chlorambucil; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum coordination 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 inhibitors RFS 2000; difluoromethylromycin (DMFO); retinoids such as retinoic acid; capecitabine; anthracyclines, nitrosoureas, antimetabolites, epipodophyllotoxins, enzymes such as L-asparaginase; hormones and antagonists including anthracenediones, 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 ethinyl estradiol 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 pharmaceutically acceptable salts, acids, or derivatives of any of the above. ,

[0192] In some embodiments, the immunotherapy treatment, ANTXR1 + Fibroblast-targeting drugs, FAP +The fibroblast-targeting agent and / or pharmaceutical composition according to the present invention is administered to a patient in combination with radiation therapy. Suitable examples of radiation therapy include, but are not limited to, external beam radiation therapy (e.g., superficial X-ray therapy, orthovoltage X-ray therapy, megavoltage X-ray therapy, radiosurgery, stereotactic radiotherapy, fractionated 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.), brachytherapy, unsealed source radiation therapy, tomotherapy, etc. Gamma rays are another form of photons used in radiation therapy. Gamma rays are spontaneously generated when certain elements (such as radium, uranium, and cobalt-60) emit radiation during decomposition or decay. In some embodiments, the radiation therapy may be proton therapy or proton minibeam radiation therapy.Proton therapy is an ultra-precise radiation therapy 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.2019 Jun 1;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. 2018 Nov 7;8(1):16479. doi:10.1038 / s41598-018-34796-8). The radiation therapy can also be FLASH radiotherapy (FLASH-RT) or FLASH proton beam radiation.FLASH radiotherapy involves the ultrafast delivery of radiation therapy at dose rates several orders of magnitude greater than currently routine clinical practice (ultra-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 setup 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] Patients, Regimen and Administration The patient is an animal, preferably a mammal, and even more preferably a human, however, the patient may also be a non-human animal, particularly a mammal, such as, inter alia, 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] A human patient according to the present invention may be a human at the prenatal stage, a newborn, a child, an infant, an adolescent or an adult, in particular an adult at least 30 years of age or at least 40 years of age, preferably an adult at least 50 years of age, more preferably an adult at least 60 years of age, and even more preferably an adult at least 70 years of age.

[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 or advanced cancer. In one embodiment, the patient has been diagnosed with stage III or IV cancer.

[0197] In one embodiment, the patient suffering from cancer has metastases, particularly brain, liver, bone metastases and / or suprarenal metastases.

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

[0199] Cancer treatments, particularly immunotherapy treatments or therapies involving anti-ANTXR1 or anti-FAP agents, can be administered by any conventional route of administration, including topical, enteral, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular routes of administration.

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

[0201] Preferably, the cancer treatment comprises administering to a mammalian subject an ANTXR1 + Fibroblasts, especially ANTXR1 + Treatment is initiated within one month, preferably within one week, after the determination of the presence of CAF.

[0202] The cancer treatment can be administered in a single dose or multiple doses.

[0203] Preferably, the cancer treatment is administered on a regular basis, preferably between daily and monthly, more preferably between daily and every two weeks, and even more preferably between daily and weekly.

[0204] The treatment period preferably comprises between 1 day and 24 weeks, more preferably between 1 day and 10 weeks, and even more preferably between 1 day and 4 weeks. In certain embodiments, treatment continues as long as the cancer persists.

[0205] The amount of cancer treatment, particularly immunotherapy treatment or treatment including anti-ANTXR1 or anti-FAP agents, administered 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 the route of administration are taken into consideration to determine the appropriate dosage so that a therapeutically effective amount is administered to the patient.

[0206] In the context of a combination of active ingredients, the active ingredients may be administered to a subject by the same or different routes of administration, which generally depend on the pharmaceutical composition used.

[0207] cancer The methods of the present invention are aimed at selecting and / or treating patients suffering from tumors.

[0208] In one embodiment, the tumor is selected from the group consisting of leukemia, seminoma, melanoma, teratoma, lymphoma, non-Hodgkin's lymphoma, neuroblastoma, glioma, adenocarcinoma, mesothelioma (including pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, and late-stage mesothelioma), rectal cancer, endometrial cancer, thyroid cancer (including papillary thyroid cancer, follicular thyroid cancer, medullary thyroid cancer, anaplastic thyroid cancer, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, and paraganglioma), skin cancer (including malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, mole, dysplastic nevi, lipoma, and the like). , hemangioma, and dermatofibroma), nervous system cancer, brain tumor (including astrocytoma, medulloblastoma, glioma, low-grade glioma, ependymoma, embryonal tumor (pineal), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor, spinal neurofibroma, glioma, or sarcoma), skull cancer (including osteoma, hemangioma, granuloma, xanthomas, or osteitis deformans), meningeal cancer (including meningiomas, meningeal sarcomas, or gliomatosis), head and neck cancer (including head and neck squamous cell carcinoma and oral cancer (e.g., buccal cavity cancer, lip cancer, tongue cancer, oral cancer, or pharyngeal cancer, etc.)), lymph node cancer, digestive system cancer, liver cancer (including hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma and hemangioma), colon cancer, stomach cancer or gastric cancer, esophageal cancer (including squamous cell carcinoma, laryngeal carcinoma, adenocarcinoma, leiomyosarcoma or lymphoma), colorectal cancer, intestinal cancer, small intestine or small bowel cancer (e.g., adenocarcinoma lymphoma, carcinoid tumor, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma or fibroma), colon or large intestine cancer (e.g., adenocarcinoma, renal tubular adenoma, villous adenoma, hamartoma or leiomyoma, etc.), pancreatic cancer (including ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor or biliary tract cancer), poma), ear, nose and throat (ENT) cancer, breast cancer (including HER2-high breast cancer, luminal A breast cancer, luminal B breast cancer and triple-negative breast cancer), uterine cancer (including endometrial cancer such as endometrial carcinoma, endometrial stromal sarcoma, malignant mixed Müllerian tumor, uterine sarcoma, leiomyosarcoma and gestational trophoblastic disease), ovarian cancer (including germinoma, granulosa cell tumor and Sertoli-Leydig cell tumor), cervical cancer, vaginal cancer (including squamous cell vaginal carcinoma, vaginal adenocarcinoma, clear cell vaginal adenocarcinoma, vaginal germ cell tumor, vaginal sarcoma botryoides and vaginal melanoma), vulvar cancer (squamous cell vulvar carcinoma, warty vulvar carcinoma,vulvar melanoma, basal cell carcinoma of the vulva, Bartholin's gland carcinoma, vulvar adenocarcinoma, and Queyrat's erythropoiesis), genitourinary cancer, kidney cancer (including clear cell renal cell carcinoma, chromogenic renal cell carcinoma, papillary renal cell carcinoma, adenocarcinoma, Wilms' tumor, nephroblastoma, lymphoma, or leukemia), adrenal gland cancer, bladder cancer, urethral cancer (e.g., squamous cell carcinoma, transitional cell carcinoma, or adenocarcinoma), prostate cancer (e.g., adenocarcinoma or sarcoma), and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, etc.) 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, bronchiolar carcinoma, bronchial adenoma, pulmonary sarcoma, chondromatous hamartoma, and pleural mesothelioma), sarcoma (including Askin tumor, sarcoma botryoides, chondrosarcoma, Ewing sarcoma, malignant hemangioendothelioma, malignant neurilemmoma, osteosarcoma, and soft tissue sarcoma), soft tissue sarcoma (including alveolar soft part sarcoma, angiosarcoma, and bladder sarcoma), phyllodes, dermatofibrosarcoma protuberans, desmoid tumor, desmoplasia Stick small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangioperioma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, reticular fibrohistiocytic tumor, rhabdomyosarcoma, synovial sarcoma and undifferentiated pleomorphic sarcoma, cardiac cancer (including sarcomas such as angiosarcoma, fibrosarcoma, rhabdomyosarcoma or liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma), bone cancer (osteogenic sarcoma, osteosarcoma, fibrosarcoma, malignant fibrohistiocytic tumor, the cancer is selected from the group consisting of: rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis (including rheumatoid arthritis, ...

[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, gastric cancer, renal cancer, ovarian cancer, hepatocellular carcinoma, osteosarcoma, melanoma, hypopharyngeal cancer, esophageal cancer, uterine 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 particular 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, in particular high-grade ovarian cancer of the serous type, or breast cancer, preferably invasive breast cancer and / or metastases thereof, in particular 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 the patient's cancer or the patient's cancer sample or tumor is rich in immunosuppressive fibroblasts, particularly immunosuppressive CAFs, particularly ANTXR1 + CAF or ANTXR1 + FAP + Present CAF.

[0215] Preferably, the patient's cancer or patient's cancer sample contains 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] In some cases, ANTXR1 + CAF is FAP + ANTXR1 + CAF. In some cases, ANTXR1 + CAF is ecm-myCAF(ANTXR1 + SDC1 + LAMP5 - ) CAF. In some cases, ANTXR1 + CAFs express 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 + CAFs express 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 The present invention further provides kits useful for carrying out the methods disclosed herein, the kits comprising: + Fibroblasts, especially ANTXR1 + CAF, preferably FAP + ANTXR1 + CAF, and even more preferably FAP + ANTXR1 + LAMP5 - SDC1 + CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF and / or ANTXR1 + SDC1 - LAMP5 - CD9 +It includes a means capable of detecting CAF.

[0218] For example, the kit contains ANTXR1 + CAF, preferably FAP + ANTXR1 + CAF, and even more preferably FAP + ANTXR1 + LAMP5 - SDC1 + CAF, FAP + ANTXR1 + LAMP5 + SDC1 + / - CAF and / or ANTXR1 + SDC1 - LAMP5 - CD9 + The present invention may include means for detecting CAFs. Those skilled in the art are aware of the means required to specifically determine the presence of ANTXR1, preferably the presence of FAP and ANTXR1, and even more preferably the presence of 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 level, or at the protein level. For example, such means can be used to detect ANTXR1. + CAF, preferably FAP + ANTXR1 + CAF, and even more preferably 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 CAFs. In particular, such means are antibodies against ANTXR1, FAP, LAMP5, SDC1, and / or CD9 as disclosed herein. Alternatively, such means may be probes and / or primers specific to ANTXR1, FAP, LAMP5, SDC1, and / or CD9.

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

[0220] The kit may be a diagnostic kit. The components of the kit may be packaged either in aqueous media or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, preferably suitably aliquoted. Where there are multiple components in the kit, the kit will also typically include second, third, or other additional containers into which the additional components may be separately placed. However, various combinations of components may be contained in vials.

[0221] In some embodiments, means for obtaining a sample from an individual and / or means for analyzing the sample may be provided. Kits may also include means for containing sterile, pharmaceutically acceptable buffers and / or other diluents. Optionally, a leaflet with guidelines for using such kits is provided.

[0222] In another aspect, the present invention also relates to the use of the kit disclosed above for: - detecting immunosuppressive cancer-associated fibroblasts (CAFs) in cancer samples from subjects suffering from cancer - predicting the response of a subject suffering from cancer to immunotherapy treatment - Selecting or not selecting patients with cancer for immunotherapy treatment - ANTXR1 + Selecting or not selecting patients with cancer for treatment with CAF-targeted drugs - ANTXR1 + Selecting or not selecting patients with cancer for treatment with CAF-targeted drugs and immunotherapy treatment.

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

[0224] Within the immunosuppressive CAF-S1 subset, a single-cell approach identifies distinct cell clusters. The inventors used single-cell RNA sequencing (scRNA-seq) to investigate cellular heterogeneity within the CAF-S1 immunosuppressive subset. CAF-S1 fibroblasts were isolated from human BC by FACS as previously described (see description of prospective cohort 1 in Table 1) (Costa A et al., Cancer Cell 2018;33(3):463-79 e10). Briefly, from freshly resected tumors, the inventors first isolated debris, dead cells, doublets, and epithelial (EPCAM)-like cells. + ), hematopoiesis (CD45 + ), endothelium (CD31 + ) and erythrocytes (CD235a + ) cells were excluded (Figure 7A). - CD45 - CD31 - CD235a -We considered these as a fibroblast-rich cell fraction and then performed FAP and CD29 staining (Figure 7A). We identified CAF-S1 (FAP) as a fibroblast-rich cell fraction, as previously established in Costa A et al., Cancer Cell 2018;33(3):463-79 e10. High CD29 Med-High ) to another CAF subpopulation (CAF-S2:FAP Neg CD29 Low ;CAF-S3:FAP Neg CD29 Med ;CAF-S4:FAP Neg CD29 High ) could be distinguished from other BC fibroblasts. We 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 retained 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 specific cell cycle phases or high proliferation, as demonstrated using G1 / S and G2 / M gene signatures (Tirosh I et al. Science 2016;352(6282)). The inventors confirmed the detection of these distinct CAF-S1 cell clusters using the label transfer algorithm described in Stuart T et al. Cell 2019;177(7):1888-902 e21. Indeed, this algorithm successfully transferred all eight cluster labels in a newly generated, independent CAF-S1 scRNA-seq dataset from an 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, contain genes that are not restricted to a specific CAF-S1 cluster (Figure 11). Cluster 0 is associated with ECM remodeling, cell-substrate adhesion and collagen formation, cluster 1 has detoxification and inflammatory response, cluster 2 is responsive to growth factors, TNF signaling and interleukin pathways, cluster 3 has TGFβ signaling pathway and matrisome, cluster 4 has collagen fiber assembly and wound healing, cluster 5 is responsive to interferon gamma (IFNγ and cytokine-mediated signaling pathway), cluster 6 has IFNβα signaling, and cluster 7 has the actomyosin complex. As an example, the inventors have identified markers recently identified in CAFs derived from pancreatic cancer, LRRC15 (leucine-rich repeat containing 15) and GBJ2 (gap junction protein β2) in cluster 0, ADH1B (alcohol dehydrogenase 1) and GPX3 (glutathione peroxidase 3) in cluster 1, RGMA (repulsive guidance molecule BMP co-receptor) 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 Protein) in cluster 4. We found high expression of CCL19 and CCL5 (CC motif chemokine ligands 19 and 5) in cluster 5, IFIT3 (interferon-inducible protein with tetratricopeptide repeats 3) and IRF7 (interferon regulatory factor 7) in cluster 6, and GGH (γ-glutamyl hydrolase) and PLP2 (proteolipid protein 2) in cluster 7. Interestingly, in human BC, the inventors have previously reported that FAP in pancreatic cancer is associated with high expression of CCL19 and CCL5 (C-C motif chemokine ligands 19 and 5) in cluster 6, IFIT3 (interferon-inducible protein with tetratricopeptide repeats 3) and IRF7 (interferon regulatory factor 7) in cluster 7. +We were able to distinguish between myofibroblast ("myCAF") and inflammatory ("iCAF") fibroblast subgroups identified among 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 iCAFs, while clusters 0, 3, 4, 6, and 7 were identified as myCAFs. Consistent with data from pancreatic cancer, iCAFs showed high expression of chemokines and proinflammatory molecules such as CXCL12 (C-X-C motif chemokine ligand 12) and SOD2 (superoxide dismutase 2), whereas myCAFs expressed myofibroblast markers, including COL1A2 (collagen type 1 alpha 2 chain) and TAGLN (transgelin). Furthermore, we observed that iCAF cluster 5 expressed high levels of CD74, which encodes the major histocompatibility complex (MHC) II invariant chain. CD74 has recently been shown to be specifically expressed on antigen-presenting CAFs ("apCAFs") in pancreatic cancer, suggesting that CAF-S1 cluster 5 may be reminiscent of such apCAFs. In summary, we identified eight distinct CAF-S1 clusters in BC. Clusters 1, 2, and 5 belong to the iCAF subgroup, and cluster 5 may correspond 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), response to stimuli (cluster 2), IFNγ and cytokines (cluster 5), ECM-mediated myCAF cluster (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 the CAF-S1 cluster: 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, we were interested in whether these CAF-S1 clusters differentially accumulated in different BC subtypes. Because we performed analyses on patients before any treatment, the fresh samples collected for scRNA-seq were mostly from luminal (Lum) patients, whereas HER2 and TN BC patients were preferentially treated in the neoadjuvant setting. Consequently, there were no HER2 patients and only two TN BC patients in prospective cohort 1 (Table 1). Nevertheless, in this dataset, we were able to detect that TN BC patients exhibited 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 our dataset, this question was addressed by utilizing the TCGA database, which contains RNA-Seq data from a large number of LumA and TN BC patients. To this end, specific signatures of the five most abundant CAF-S1 clusters (accounting for up to 91% of sequenced CAF-S1 cells) were defined by identifying differentially expressed genes in each cluster compared to other clusters (Figure 7B). Because these signatures were also used to detect these clusters in melanoma, NSCLC, and HNSCC data (see Figures 3 and 6 below), we next discarded any genes in these signatures that were also expressed by melanoma, NSCLC, and HNSCC cancer cells to avoid any signal from cancer cells and ensure a strictly specific signal for the CAF-S1 cluster (Figure 7B for the specificity of the CAF-S1 cluster signature). We assessed the differential expression of CAF-S1 cluster-specific signatures between LumA and TN BC subtypes from the TCGA RNA-seq database ( https: / / portal.gdc.cancer.gov / ), which 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 with LumA BC, whereas ecm-myCAF, TGFβ-myCAF, and wound-myCAF expression were higher in LumA BC. This increase in iCAF content in TN BC is consistent with the reported presence of numerous TILs in some TN BC. In summary, numerous FAPs were isolated from BC. + Using scRNA-seq from CAF-S1 fibroblasts, eight clusters were detected that showed distinct signatures and were differentially accumulated in BC subtypes.

[0227] CAF-S1 cell clusters are validated by multicolor flow cytometry of BC Next, we sought to validate the CAF-S1 clusters using multicolor flow cytometry (FACS) on fresh BC samples. By analyzing the proportion of each cluster in CAF-S1 cells defined by scRNA-seq, we first observed that the five initial clusters accounted for up to 91% of all sequenced cells. Therefore, we decided to focus our FACS analysis on these five most abundant clusters and attempted to identify surface markers for each cluster. Using pairwise comparisons of CAF-S1 cluster expression profiles, we identified six surface markers using commercially available antibodies and designed a gating strategy to identify the five most abundant clusters (Figure 8). We then sought to validate the specificity of these six markers in an independent CAF-S1 dataset. To do so, we investigated the CAF-S1 scRNA-seq data corresponding to the eighth patient whose cluster labels were successfully transferred by the label transfer algorithm. Indeed, a gating strategy relying on these six markers and based on seven BC patients efficiently delineated the five most abundant CAF-S1 clusters in an independent dataset. Thus, these markers were confirmed to be 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 myCAFs (ANTXR1 + ) to iCAF(ANTXR1 - ) were first separated based on ANTXR1 protein levels in comparison with 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. + SDC1+ LAMP5 - is cluster 0 (ECM-myCAF), ANTXR1 + LAMP5 + SDC1 + / - is defined as cluster 3 (TGFβ-myCAF), and ANTXR1 + SDC1 - LAMP5 - CD9+ was defined as cluster 4 (wound-myCAF). - (iCAF) CAF-S1 clusters 1 (detox-iCAF) and 2 (IL-iCAF) were isolated using the GPC3 and DLK1 markers. - GPC3 + DLK1 + / - is defined as cluster 1 (detox-iCAF), and ANTXR1 - GPC3 - DLK1 + were defined as cluster 2 (IL-iCAFs). LAMP5, SDC1, CD9, and ANTXR1 - GPC3 - DLK1 - ANTXR1 was negative for cells +CAF-S1 cells were pooled and designated "other clusters." 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 defined by FACS confirmed the single-cell results, including clear heterogeneity between CAF-S1 fibroblasts and ecm-myCAFs, as the most abundant population in the majority of patients (Figure 2). Next, we analyzed whether there was a correlation between the proportions of each of these five CAF-S1 clusters across patients. The relative abundance of ecm-myCAFs and TGFβ-myCAFs (both myCAFs) and the relative abundance of detox-iCAFs and IL-iCAFs (both iCAFs) were correlated. Conversely, the proportions of ecm-myCAFs and TGFβ-myCAFs were inversely correlated with the proportions of detox-iCAFs and IL-iCAFs. Furthermore, wound-myCAFs were also negatively correlated with detox-iCAFs, IL-iCAFs, and ecm-myCAFs, suggesting that these distinct CAF-S1 clusters may differentially accumulate in BC, but in a coordinated manner.

[0228] CAF-S1 cell clusters have been identified across cancer types We next sought to examine the presence of CAF-S1 cell clusters in other cancer types. To do so, we 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), as these two studies isolated enough CAFs to investigate clusters. These published studies included 18 HNSCC patients, five of whom had matched pairs of primary tumors and lymph node metastases, and analyzed a total of 5,902 cells (Puram SV et al. Cell 2017;171(7):1611-24 e24). Furthermore, in the NSCLC cohort, over 52,000 total cells were collected from five different patients (Lambrechts D et al. Nat Med 2018;24(8):1277-89). In these two studies, 1,422 and 1,465 cells were annotated as CAFs in the HNSCC and NSCLC cohorts, respectively. To strictly analyze CAF-S1 fibroblasts, CAF-S1 was classified as CAF-S1 (FAP). High MCAM Low ) and CAF-S4(FAP Low MCAM HighCAF-S1 cells were distinguished from CAF-S4 based on the expression of FAP and MCAM, two markers regulated at the RNA level of HNSCC and NSCLC, respectively. Consequently, 603 CAF-S1 cells from HNSCC and 959 CAF-S1 cells from NSCLC were further analyzed. The similarities between CAF-S1 cells from different cancer types were compared by combining 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). We used CAF-S1 cluster-specific signatures defined by differentially expressed genes in each cluster compared to other clusters (Figure 7B). Surprisingly, we found systematic correspondences between CAF-S1 clusters from BC and 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). Thus, we confirm the existence of the five most abundant CAF-S1 clusters in HNSCC and NSCLC and highlight their relevance in other cancers.

[0229] Immunosuppressive environment correlates with specific CAF-S1 clusters Having demonstrated that CAF-S1 fibroblasts exert immunosuppressive effects in breast and ovarian cancer, we next investigated whether this function is exerted by all CAF-S1 clusters or is restricted to specific clusters (Figure 4). We first tested whether we could detect a correlation between CAF-S1 clusters and immune cell content. To this end, we investigated 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. We examined the association between stromal 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 clustered together on one side, while detox-iCAF and IL-iCAF clusters clustered on the other, whereas the wound-myCAF cluster was fairly isolated, suggesting that these distinct clusters differentially interact with T cells. Interestingly, ecm-myCAF, TGFβ-myCAF, and wound-myCAF were found to exhibit specific associations with T lymphocytes. Indeed, the proportion of ecm-myCAF in CAF-S1 fibroblasts was significantly higher than that of CD45 fibroblasts. + We first observed a significant correlation with hematopoietic cell infiltration (Figure 9). More specifically, the abundance of ecm-myCAFs correlated with the expression of PD-1. + , CTLA-4 + and TIGIT + CD4 + Correlated with T lymphocyte infiltration, but CD8 + Similarly, the content of TGFβ-myCAF was negatively correlated with that of CD45 + Although it showed no overall association with hematopoietic cells, its abundance was significantly higher than that of CTLA-4 + CD4 + It is positively correlated with infiltration by T lymphocytes and CD8 + These data therefore indicate that the abundance of ecm-myCAF and TGFβ-myCAF is associated with an immunosuppressive environment rich in Tregs. In contrast to the ecm-myCAF and TGFβ-myCAF clusters, the abundance of detox-iCAF and IL-iCAF was negatively correlated with CD8 + The wound-myCAF clusters were associated with T cell infiltration. + Among cells, there was an overall correlation with T lymphocytes (Figure 9), and CTLA-4 + , TIGIT+ , PD-1 + and NKG2A + CD4 + Enrichment of wound-myCAFs was inversely correlated with T lymphocyte exhaustion, CTLA-4, a marker of T lymphocyte exhaustion. + CD8 + , TIGIT CD8 + , CD244 + CD8 + , CD244 + It is also anti-correlated with NK, suggesting a general association of this cluster with high T lymphocyte infiltration and an immune-protective environment. Importantly, the CAF-S1 signature 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, fails to specifically identify the CAF-S1 immunosuppressive clusters (i.e., ECM-myCAF, TGFβ-myCAF, and wound-myCAF) associated with immunotherapy response. These clusters are associated with ANTXR1. + In contrast, these clusters showed that the most differentially expressed CAF-S1 genes were not differentially expressed (Figure ​(Figure11),11), suggesting that the identification of ANTXR1 among the top CAF-S1 genes as a marker for these clusters was not by chance.

[0230] To validate these data in an independent large cohort of BC patients, we next examined the association of the CAF-S1 cluster with T cell signatures from the publicly available TCGA database. RNA-seq data from the TCGA database confirmed that the expression of the ecm-myCAF and TGFβ-myCAF clusters was positively correlated with the expression of FOXP3, one of the key Treg markers (Figure 4B). Furthermore, wound-myCAF clusters showed no significant association with FOXP3, whereas detox-iCAF and IL-iCAF clusters showed a negative correlation with FOXP3 (Figure 4B). Consistent with these data, we observed a positive correlation between the T cell lytic index and the detox-iCAF and IL-iCAF clusters, but not with ecm-myCAF, TGFβ-myCAF, or wound-myCAF clusters (Figure 4C). In summary, detox-iCAFs and IL-iCAFs correlate with an immunocompetent environment, whereas ecm-myCAFs and TGFβ-myCAFs are both associated with an immunosuppressive environment, and CD8 + CD4 T lymphocytes are poor 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-myCAFs and TGFβ-myCAFs with Tregs As mentioned above, the inventors have demonstrated that the abundance of ecm-myCAF and TGFβ-myCAF, but not detox-iCAF and IL-iCAF, is related to the upregulation of PD-1 in BC. + and / or CTLA-4 + We observed that the abundance of CD4+ T lymphocytes correlated with the abundance of CD4 + CD25 +We investigated the role of CAF-S1 clusters in generating a fibroblast-rich, immunosuppressive environment. Therefore, we established primary cultures of CAF-S1 clusters to perform in vitro functional assays. Although we were unable to establish all CAF-S1 clusters in culture, we used two different methods: (1) direct escape and spreading of CAF-S1 fibroblasts from BC samples seeded on plastic dishes, and (2) FAP. High CD29 Med By sorting the cells by FACS and growing them in culture medium on plastic dishes, we successfully isolated ecm-myCAF and iCAF clusters. After several weeks of growth under the same culture conditions, we were able to identify the identities of these distinct cells, namely CD4 and iCAF, for functional assays. + CD25 + We compared CAF-S1 cells obtained by spreading with those adapted for co-culture with T lymphocytes (see below). We observed that CAF-S1 cells obtained by spreading expressed high levels of myCAF genes, while CAF-S1 cells isolated by sorting showed high expression of iCAF genes. Therefore, we applied the cluster-specific signatures established from the scRNA-seq data (Figure 7B) and found that spread CAF-S1 fibroblasts were enriched in ecm-myCAFs, whereas sorted CAF-S1 cells were enriched in detox-iCAF, IL-iCAF, and IFN-iCAF clusters (see the section on RNA sequencing methods for CAF-S1 primary cell lines isolated from BC). We also found that global CAF-S1 subpopulations were CD4+ / CD ... + CD25 - It does not directly affect T cells, but CD4 + CD25 - FOXP3 in T lymphocytes +We previously demonstrated that ecm-myCAF and TGFβ-myCAF content in BC was associated with a CD4+-rich immunosuppressive microenvironment, but not iCAF clusters, and therefore we used functional assays to assess the CD4+-rich microenvironment, as previously performed in 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. + CD25 + The functions of these myCAF and iCAF clusters on T cells were compared.

[0232] First, CD4 + CD25 + FOXP3 + The effect of myCAF and iCAF clusters on T lymphocyte content was examined in vitro (Figure 5A). + CD25 + FOXP3 in the population + The iCAF clusters increased the proportion of CD4 T cells and enhanced FOXP3 protein levels in these T cells (Figure 5A). + CD25 + FOXP3 + There was no effect on either the proportion of T lymphocytes or FOXP3 protein levels (Figure 5A). We also examined the effect of culture on the identity and immunosuppressive activity of normal fibroblasts. Fibroblasts isolated by spreading from healthy tissues were found to be resistant to FAP. Neg-Low Although it initially lacked immunosuppressive activity, it developed FAP in subsequent passages. Pos-High These results suggest that long-term maintenance of CAFs on plastic dishes may activate fibroblasts. + CD25 + FOXP3 +Based on the ability to increase T lymphocytes, the inventors next considered both the percentage of positive cells for these immune checkpoints and their surface protein levels to determine the CD4 + CD25 + FOXP3 + PD-1 on T lymphocytes + , CTLA-4 + , TIGIT + , TIM3 + and LAG3 + We compared the ability of CAF-S1 clusters to regulate the proportion of PD-1 (Figure 5B-F). + and CTLA-4 + CD4 + CD25 + FOXP3 + In contrast to ecm-myCAFs, iCAF clusters significantly increased both the proportion of T lymphocytes and the levels of immune checkpoints on their surface (Figure 5B, C). + and CTLA-4 + Neither the proportion of T cells nor the CTLA-4 protein level was affected (Figure 5B, Figure 5C). Furthermore, iCAF clusters increased PD-1 protein levels, but this effect was less efficient than that of ecm-myCAFs. Both myCAF and iCAF clusters increased CD4 + CD25 + FOXP3 + TIGIT + Although TIM3 increased the proportion of cells (Fig. 5D), + and LAG3 + There was no effect on T cells (Figure 5E, Figure 5F). + and CTLA-4 + CD4 + Consistent with the correlation with T lymphocytes, ecm-myCAF-derived CAF-S1 expresses CD4 + CD25 + FOXP3 +It is shown here that iCAF clusters have no or minimal effect on Tregs, whereas they directly affect Tregs by enhancing PD-1 and CTLA-4 immune checkpoint levels on the surface of T lymphocytes. Finally, co-culture with ecm-myCAFs results in increased CD4 + CD25 + We observed that the upregulation of immune checkpoints on the surface of T cells was also detected intracellularly (Fig. 10A), suggesting that ecm-myCAF increases the total protein levels of T cells. + CD25 + We found that the expression of FOXP3, CTLA-4, and TIGIT in T cells was also upregulated at the mRNA level after co-culture with ecm-myCAFs (Fig. 10B), and PD-1 RNA was barely detectable in T cells in vitro. Furthermore, we found that the mRNA levels of NFAT and STAT family members were also upregulated in CD4 T cells upon co-culture with ecm-myCAFs. + CD25 + We observed that ecm-myCAFs were elevated in T lymphocytes (Figure 10C). Because NFAT and STAT are well-known transcriptional regulators of immune checkpoints in T cells, these data support the notion that ecm-myCAFs are upregulated by CD4 + CD25 + We show that it promotes immune checkpoint upregulation at the RNA level in T lymphocytes, potentially through activation of the NFAT / STAT signaling pathway.

[0233] Considering the influence of the CAF-S1 cluster on Tregs, we next wondered whether T lymphocytes could regulate the identity of the CAF-S1 cluster. + CD25 + We evaluated whether co-culture with T lymphocytes had any effect on the level of cluster markers on the surface of CAF-S1 fibroblasts. To avoid any contamination with T cells during co-culture, CAF-S1 were isolated by FACS and analyzed for cluster markers expressed on their surface.+ CD25 + We observed that co-culture of T cells significantly increased the expression of the TGFβ-myCAF-specific marker LAMP5 on the surface of ecm-myCAF fibroblasts (Figure 5G), thereby confirming that the content of TGFβ-myCAFs is related to the CD4 + CD25 + This suggests that ANTXR1 protein levels are increased by co-culture with T lymphocytes. This effect was detected only in myCAFs, but not in iCAF fibroblasts (Figure 5G), which was expected given that TGFβ-myCAFs and ecm-myCAFs, as well as CAF-S1 fibroblasts, belong to the myCAF subgroup. Consistent with this observation, ANTXR1 protein levels were significantly increased by CD4 + CD25 + Ecm-myCAF fibroblasts also showed a tendency to increase (although it did not reach significance) upon coculture with T cells, whereas iCAF cells remained strictly unchanged and low (Figure 5G). Quite surprisingly, DLK1, a marker of IL-iCAF, also increased upon coculture, suggesting potential plasticity between ecm-myCAF and IL-iCAF. In contrast, other markers did not show significant fluctuations upon coculture and remained either high (SDC1) or low (GPC3 and CD9), as expected based on their respective cluster identities (Figure 5G). These observations are consistent with the CD4 + CD25 + T lymphocytes express ecm-myCAF (ANTXR1 + SDC1 + LAMP5 - CD9 + / - ) TGFβ-myCAF (ANTXR1 + LAMP5 + SDC1 + / - CD9 + / - These results suggest that ecm-myCAF may promote the conversion of ecm-myCAF to FOXP3. +We found that Tregs can directly affect the levels of PD-1 and CTLA-4 proteins on the surface of T lymphocytes. Conversely, Tregs can promote the conversion of ECM-myCAFs to TGFβ-myCAFs, thereby promoting the differentiation of these two clusters and the CD4+ CD25 + PD-1 + or CTLA-4 + This could be the basis for a positive feedback loop between T cells and BC, explaining the positive correlation observed.

[0234] Ecm-myCAF and TGFβ-myCAF are associated with primary resistance to immunotherapy Given the direct influence of specific CAF-S1 clusters on Treg PD-1 and CTLA-4 protein levels, we next investigated whether some CAF-S1 clusters might be associated with immunotherapy resistance. Because we lacked access to data from BC patients treated with immunotherapy, we utilized published data from metastatic melanoma patients treated with anti-PD-1 (pembrolizumab) therapy (Hugo W et al. Cell 2016;165(1):35-44), which has revolutionized melanoma treatment. As defined in the aforementioned study, we considered patients to be "non-responders" to anti-PD-1 if they showed progressive disease and "responders" if they showed a complete or partial response. By performing gene set enrichment analysis, we first observed that, at the time of diagnosis, the expression of CAF-S1-specific genes was significantly enriched in tumors from non-responder patients, but not in normal fibroblast content. Using the CAF-S1 cluster-specific signature, we observed that gene expression of ecm-myCAF, TGFβ-myCAF, and wound-myCAF clusters was enriched in non-responders compared with responders, whereas detox-iCAF, IL-iCAF, and IFN-iCAF clusters were not (Figure 6A). Next, we compared the abundance of each CAF-S1 cluster 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, whereas 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 cytolytic index, as defined by Rooney et al. Cell 2015;160(1-2):48-61, differed between responders and non-responders (Figure 6C, Figure 6D).Consistent with these observations, reciprocal 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-responders was significantly associated with tumors showing high expression of Ecm-myCAF, TGFβ-myCAF, or wound-myCAF at diagnosis, whereas other CAF-S1 clusters, general CAF content, or cytolytic index did not provide useful information regarding patient response to immunotherapy (Figures 6E-G). Collectively, these data indicate that the three specific CAF-S1 clusters (ecm-myCAF, TGFβ-myCAF, and wound-myCAF) are diagnostic indicators of anti-PD-1 responses in metastatic melanoma patients, whereas other CAF-S1 clusters (detox-iCAF, IL-iCAF, and IFN-iCAF), total CAF content, or cytolytic index are not. Finally, we sought to examine the impact of the CAF-S1 cluster, particularly Ecm-myCAF, TGFβ-myCAF, and wound-myCAF, on first-line immunotherapy resistance in a series of metastatic NSCLC patients (here treated in the second- or third-line setting with nivolumab; see Table 2 for a detailed description of NSCLC Cohort 4). Similar to melanoma, we verified that the CAF-S1 signature, 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, we confirmed that ecm-myCAF, TGFβ-myCAF, and wound-myCAF, in contrast to the detox-iCAF, IL-iCAF, and IFNγ-iCAF clusters, are associated with non-responder NSCLC patients (Figure 6I). In conclusion, the abundance of ecm-myCAF and TGFβ-myCAF at diagnosis, in contrast to the detox-iCAF, IL-iCAF, and IFNγ-iCAF clusters, is associated with resistance to immunotherapy in both melanoma and NSCLC, and with the upregulation of PD-1 in Tregs.+ and CTLA-4 + Consistent with its ability to increase protein levels.

[0235] Materials and Methods Patient cohort BC patients: The study developed here was based on samples collected from surgical residues available after histopathological analysis and not necessary for diagnosis. There was no interference with clinical practice. Analysis of primary tumor samples was performed in accordance with the relevant national legislation regarding the protection of people participating in biomedical research. All patients admitted to Institut Curie (BC patients) received a welcome booklet explaining that their samples could be used for research purposes. Therefore, all patients included in the study were informed by their referring oncologist that biological samples collected through standard clinical practice could be used for research purposes and that they could object to such use if necessary. If the patient refused, either verbally or in writing, the residual tumor samples were not included in the study. The human experimental procedures for tumor microenvironment analysis by F. Mechta-Grigoriou's laboratory were approved by the Institutional Review Board and Ethics Committee of the Institut Curie Hospital Group (approved February 12, 2014) and the CNIL (Commission Nationale de l'Informatique et des Libertes) (NIL). oApproval: 1674356 delivered March 30, 2013. The "Biological Resource Center" (BRC) is part of the Pathology Department of the Department of Diagnostic and Therapeutic Medicine, led by Dr. A. Vincent-Salomon. The BRC is authorized to store and manage human biological samples in accordance with French law. The BRC has declared a defined sample collection that is continuously incremented as patient consent is obtained (Declaration No. DC-2008-57). The BRC adheres to all currently required national and international ethical regulations, including the Declaration of Helsinki. The BRC is also certified with the AFNOR NFS-96-900 quality label (updated and now valid until 2021). Luminal (lumen) tumors were defined by positive immunostaining 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 from Lum B tumors (<15%: Lum A: Lum B as above). HER2-amplified cancers were defined according to ERBB2 immunostaining using ASCO guidelines. TN immunophenotype was defined as follows: ERBB2 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 Pathology Department of Bichat Hospital and derived from patients treated with immuno-oncology drugs at the Thoracic Oncology Department of Bichat Hospital, led by Pr. G. Zalcman, MD. De-identified clinical data were part of the Thoracic Oncology Database of Lung Cancer Patients at the CIC-1425 / CLIP2 Clinical Investigation Center of Bichat Hospital (co-led by Pr. G. Zalcman) (Regional Health Authority Approval #17-1381), in accordance with French regulatory rules for observational clinical research. Patients received checkpoint inhibitors after progression on first- or second-line chemotherapy-based treatments, according to the Registry of Immuno-Oncology Drugs. During the current study period, the anti-PD-1 monoclonal antibody nivolumab represented the most frequent drug used in such settings. The efficacy of immuno-oncology treatment was evaluated every 8–12 weeks by whole-body CT scans. A weekly multidisciplinary tumor committee, including a thoracic radiologist, thoracic oncologist, and pulmonologist, defined objective responders (OR), patients with stable disease (SD), and patients showing tumor progression (progression)—a 20% increase in tumor volume without clinical benefit—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 agents for >6 months with clinical benefit (n=3) at the 4-month evaluation were included in the responder group without any criteria for progressive disease (long-term sustained SD at the time). The date of progression as assessed by CT scan was recorded. Some patients showed early clinical progression requiring earlier (before 8 weeks) CT scan evaluation. In these series, the above criteria were followed. There were 22 responder patients and 48 progressive patients, and the treatment duration was less than 4 months. The progression date was retained as the date of the CT scan showing RECIST progression. The date and cause of death or date of last known vital status were systematically recorded. Second- or third-line treatment after progression was registered. There were no imbalances by treatment after progression.PD-L1 staining was performed and interpreted by AG on 4-μm paraffin-embedded sections from diagnostic, pretreatment biopsy specimens containing at least 200 tumor cells using a commercially available clone, Cell Signaling Technology E1L3N, on a Leica Bond platform. All but five patients (with fewer than 200 tumor cells in the remaining pathology blocks) underwent PD-L1 immunohistochemistry.

[0237] CAF-S1 RNA sequencing at the single-cell level Isolation of CAF-S1 from BC: CAF-S1 fibroblasts were isolated from a total of eight primary BCs (surgical remnants prior to any treatment) (see Table S1 for details of the prospective cohort). Seven BCs were initially studied. Additionally, another BC sample was added to validate the CAF-S1 cluster using the label transfer algorithm in the Seurat R package. CAF-S1 fibroblasts were isolated from BCs using a BD FACS ARIA III™ sorter (BD Biosciences). Fresh human BC primary tumors were collected directly from the operating room after gross inspection of the surgical specimen and selection of the area of ​​interest by a pathologist. Samples were cut into small pieces (approximately 1 mm 3 ) 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 (min) with shaking (180 rpm). Cells were then filtered through a 40 μm cell strainer (Fisher Scientific #223635447) and diluted to 5 × 10 in 50 μl of PBS+ solution (PBS, Gibco #14190; EDTA 2 mM, Gibco #15575; human serum 1%, BioWest #S4190-100). 5 ~10 6 To isolate CAF-S1 fibroblasts, we first isolated epithelial (EPCAM) cells and resuspended them at a final concentration of 0.1%. + ), hematopoiesis (CD45 + ), endothelium (CD31+ ), and CD235a + Selection was applied to exclude (erythroid) cells, followed by CAF-S1 markers (FAP and CD29). Cells in suspension were stained with an antibody mix for flow cytometry cell sorting, including anti-EpCAM-BV605 (BioLegend, #324224), 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), and anti-FAP-APC (primary antibody, R&D Systems, #MAB3715), and then single-cell RNA sequencing was performed. All antibodies, except FAP, were purchased pre-conjugated with fluorescent dyes. Anti-FAP antibodies were conjugated using the Fluorescent Dye Zenon APC Mouse IgG1 Labeling Kit (ThermoFisher Scientific, #Z25051). Isotype control antibodies for each CAF marker were iso-anti-CD29 (BioLegend, #400144) and iso-anti-FAP (primary antibody, R&D Systems, #MAB002).

[0238] Cell suspensions were stained with the antibody mix in PBS+ solution for 15 minutes at room temperature immediately after dissociation of BC tumor samples. 2.5 μg / ml DAPI (ThermoFisher Scientific, #D1306) was added immediately before flow cytometry sorting. Signals were acquired on a BD FACS ARIA III™ sorter (BD Biosciences) for cell sorting. At least 5 × 10 5Events were recorded. Compensation was performed using single staining for anti-mouse IgG and negative control beads (BD biosciences, #552843) for each antibody. Data analysis was performed using FlowJo version X 10.0.7r2. Cells were first gated based on forward scatter (FSC-A) and side scatter (SSC-A) (measures of cell size and granularity, respectively) to exclude debris. Dead cells were excluded based on positive staining for DAPI. Single cells were then selected based on SSC-A vs. SSC-W parameters. Gating included epithelial cells (EPCAM) and endothelial cells (ECMC). + ), hematopoietic cells (CD45 + ), endothelial cells (CD31 + ), erythrocytes (CD235a + ) to remove the EPCAM - , CD45 - , CD31 - , CD235a - contained cells.

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

[0240] scRNA-seq data processing scRNA-seq: Raw data preprocessing was initially performed using the Cell Ranger software pipeline (version 2.1.1). This included demultiplexing raw base call (BCL) files into FASTQ files, aligning reads to the human genome assembly GRCh38 using STAR, and counting unique molecular identifiers (UMIs). A first set of 18,805 CAF-S1 cells from seven BC patients (seven 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 step, we first filtered out low-quality cells, empty droplets, and multiple captures based on the distribution (non-zero counts) of unique genes detected in each cell for each patient. Cells with fewer than 200 and 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 percentage of mitochondrial genes greater than 5% were discarded to eliminate dying cells or low-quality cells with extensive mitochondrial contamination. For each patient, the mitochondrial percentage was calculated using the PercentageFeatureSet function in Seurat with the argument pattern="^MT-". Following these QC criteria, 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 finally saved in the first and second datasets, respectively, for downstream analysis.

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

[0243] Clustering and data visualization: PCA dimensionality reduction was performed using default parameters. The number of included components (PCs) was assessed using the JackStraw procedure implemented in the JackStraw and ScoreJackStraw functions. 30 PCs were used. A graph-based clustering approach was used to cluster cells in the initial dataset using the FindNeighbors (k = 20) and FindClusters functions (res = 0.35). At this resolution, 10 CAF-S1 clusters were obtained. For data visualization, the nonlinear dimensionality reduction method UMAP was applied using the RunUMAP function in Seurat.

[0244] Differential gene expression and signaling pathway analysis: Genes specifically upregulated in each of the 10 clusters in the initial dataset were identified using pairwise differential analysis. While the median number of differentially expressed genes in each pairwise combination was 126, the number of genes differentially expressed between two combinations was very limited: 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) by searching all genes significantly upregulated in each of the 10 initial clusters (one cluster vs. all other clusters, function FindAllMarkers, following parameters: logfc.threshold=0.25, test=Wilcox with Wilcoxon rank sum test). Consistent with the pairwise analysis, 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-myCAFs, and clusters 3 and 6 as cluster 3 / TGFβ-myCAFs, ultimately identifying eight biologically distinct CAF-S1 clusters.

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

[0246] Label Transfer To validate the CAF-S1 clusters identified in the first dataset, another dataset corresponding to 1,582 quality-controlled CAF-S1 fibroblasts 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 and implemented in the Seurat V3.0 R package, was applied using the functions FindTransferAnchors and TransferData. The first 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. When finding anchors, dimensionality reduction was performed 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. Briefly, the datasets can be transformed into a shared space by identifying pairwise correspondences between single cells (called "anchors") between the datasets. Dimensionality reduction of both datasets was performed using diagonalized canonical correspondence analysis (CCA), with L2 normalization applied to the canonical correlation vectors prior to 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 Forty-four BCs were cut into small pieces and digested in CO2-independent medium (Gibco, #18045-054) supplemented 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) for 45 minutes at 37°C with shaking (180 rpm). After tissue digestion, cells were strained using a 40 mm cell strainer (Fischer Scientific, #223635447) and washed with PBS (Gibco, #14190) supplemented with 2 mM EDTA (Gibco, #15575) and 1% human serum (BioWest, #S4190-100). Cells were then divided into two groups for analysis of the CAF-S1 cluster panel and immune cell panel, respectively.

[0249] CAF-S1 cluster panel: Cells were stained with Live Dead NIR (1:1000, BD Bioscience #565388) in PBS for 20 minutes. Cells were then washed and stained 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), anti-ANTXR1-AF405 (1:33, Novus Biosciences, #10 ... The cells were stained for 45 minutes with an antibody cocktail containing 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 Biologicals, #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). Cells were then washed and acquired the same day using an LSR FORTESSA analyzer (BD Biosciences) or fixed with 4% paraformaldehyde (PFA, Electron Microscopy Sciences, #15710) for 20 min, washed, kept in PBS+ solution overnight, and acquired the next day. At least 5 × 10 cells were collected. 5 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 scatter (FSC-A) and side scatter (SSC-A) (measuring cell size and granularity, respectively) to exclude debris. Dead cells and red blood cells were excluded based on positive staining for Live / Dead NIR and CD235a, respectively. Single cells were then selected based on SSC-H vs. SSC-A parameters. Epithelial cells (EpCAM) were then analyzed. + ), hematopoietic cells (CD45 + ), endothelial cells (CD31 + ) to remove EpCAM - , CD45 - , CD31 - Gating was performed on cells. DAPI - ,EPCAM- , CD45 - , CD31 - Cells were divided into four subsets (CAF-S1 to CAF-S4) according to FAP and CD29. The CAF-S1 subset was first gated on ANTXR1. Then, ANTXR1 + Cells were gated according to SDC1 and LAMP5. + SDC1 + LAMP5 - is cluster 0 / ecm-myCAF, ANTXR1 + SDC1 - LAMP5 + was defined as cluster 3 / TGFβ-myCAF. ANTXR1 + SDC1 - LAMP5 - Gated on CD9 and ANTXR1 + SDC1 - LAMP5 - CD9 + was defined as cluster 4 / wound-myCAF. ANTXR1 - / Low Cells were gated on DLK1 and GPC3. Cluster 1 (detox-iCAF) expresses ANTXR1. - GPC3 + DLK1 - / + Cluster 2 is defined as ANTXR1 - GPC3 - DLK1 + Defined as: ANTXR1 - GPC3 - DLK1 - and ANTXR1 + SDC1 - LAMP5 - CD9 - were designated as other clusters.

[0250] Immune panel: Of the 44 BC samples analyzed for the CAF-S1 cluster, 37 were characterized for their immune content in the meantime. Cell types were analyzed in the live / dead negative fraction, and hematopoietic cells (CD45 + ), 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 +For each identified population, the percentage of cells positive for the following checkpoints was also assessed: 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. Cells were then washed and stained for 45 minutes with an antibody cocktail consisting 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-BUV 737 (1:20, BD Biosciences, #565299), anti-CD3-AF700 (1:25, BD Biosciences, #557943), and anti-NKG2A-BV786 (1:20, BD Biosciences, #563692). 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 Technologies, #MHCD0817), anti-CD69-BV710 (1:25, BD Biosciences, #563836), and anti-CD161-PE-VIO770 (1:100, Miltenyi, #130-113-597).Cells were then washed and acquired the same day using an LSR FORTESSA analyzer (BD Biosciences) or fixed with 4% paraformaldehyde (PFA, Electron Microscopy Sciences, #15710) for 20 min, washed, kept in PBS+ solution overnight, and acquired the next day. At least 5 × 10 cells were collected. 5 Events were recorded. Compensation was performed using anti-mouse IgG and negative control beads (BD Biosciences, #552843) for each antibody, 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 on a NovaSeq (Illumina) system. Reads were mapped to the human reference genome (hg38; Gencode release 26) and quantified using STAR (version 2.5.3a) with the following parameters: outFilterMultimapNmax=20; alignSJoverhangMin=8; alignSJDBoverhangMin=1; outFilterMismatchNmax=999; outFilterMismatchNoverLmax=0.04; alignIntronMin=20; alignIntronMax=1000000; alignMatesGapMax=1000000; outMultimapperOrder=Random. Only genes with one read in at least 5% of all samples were retained for further analysis. Normalization was performed with the DESeq2 R package, and the raw read matrix was log2 transformed. To identify the identity of CAF-S1 primary cell lines, a score was calculated for each CAF-S1 cluster signature by the average expression of genes comprising the iCAF / myCAF signature (as defined in Ohlund et al. J Exp Med 2017;214(3):579-96). P-values ​​are from DESeq2 analysis.

[0252] Functional assays CD4 + CD25 + Isolation of T lymphocytes: CD4 +CD25 + T lymphocytes were isolated from peripheral blood of healthy donors obtained from the Saint-Antoine Crozatier blood bank "Établissement Français du Cent" in Paris through a treaty with the Institut Curie (Paris, France). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated using Lymphoprep (Stemcell, #07861) as previously described in Costa A et al., Cancer Cell 2018;33(3):463-79 e10. CD4 + CD25+ was purified from human CD4 + CD25 + 5 × 10 cells were cultured using magnetic cell sorting (MACS) with a Treg isolation kit (Miltenyi Biotec, #130-091-301). 8 Purified from PBMCs of CD4 + CD25 + T lymphocyte purity was determined by flow cytometry as described in Costa A et al., Cancer Cell 2018;33(3):463-79 e10.

[0253] Isolation of CAF-S1 clusters in culture: To isolate distinct CAF-S1 clusters, we first began by sorting cells according to specific markers, but were unable to maintain viability of distinct identities. Next, we tested two different isolation methods: 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 on plastic dishes (Falcon, #353003) in DMEM (HyClone, #SH30243.01) supplemented with 10% FBS (Biosera, #FB-1003 / 500), streptomycin (100 μg / ml), and penicillin (100 U / ml) (Gibco, #15140-122) at 37°C for at least 2–3 weeks to allow fibroblasts to spread and expand. For fibroblast isolation by the "sorting" method, tumors were digested with the enzyme cocktail described in (#Isolation of CAF-S1 from BC) and sorted for 2 hours on FBS-precoated 48-well plastic dishes (TPP plates, #192048) using the gating strategy detailed in (#CAF-S1 RNA Sequencing on Single Cells) using a BDFACS ARIA III™. CAF-S1 sorted cells were then grown on plastic dishes (TPP plates, #192048) in pericyte medium (ScienCell, #1201) supplemented with 2% FBS (ScienCell, #0010) at 37°C for 3–4 weeks in a humidified incubator with 1.5% O and 5% CO. To compare the cellular identity of sorted and spread CAF-S1 fibroblasts under the exact same conditions used in the functional assays, both types of fibroblasts (spreading and sorting) were transferred to plastic dishes (Falcon, #353047) in DMEM medium (HyClone, #SH30243.01) with 20% O. These medium conditions are compatible with the co-culture with CD4+ CD25+ T lymphocytes applied in the in vitro functional assays.Using these protocols, seven different 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 the spreading and sorted cells were compared at the same passage.

[0254] Treg-CAF-S1 cluster functional assay: 5 × 10 4 CAF-S1 cells (spreading and sorting) were plated in 24-well plates (Falcon, #353047) in DMEM (HyClone, #SH30243.01) supplemented with 10% FBS (Biosera, #FB-1003 / 500) and 1.5% O. 2 The medium was then removed 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, 20% O for overnight (for RNA analysis) or 24 h (for FACS). For FACS analysis, non-adherent cells (CD4 + CD25 +) were harvested, washed, and stained for 30 minutes at room temperature with 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, BD Bioscience, #53-4776-42), anti-CTLA-4-Pe-cy5 (1:20, BD Bioscience, #555854), anti-PD-1-BUV737 (BD Bioscience, # Antibodies were used: anti-TIGIT-BV605 (1:50, BD Bioscience, #747841), anti-LAG3-BV510 (1:50, BD Bioscience, #744985), and anti-TIM3-BV711 (1:50, BD Bioscience, #565566). Adherent cells were trypsinized, washed, and stained for Live Dead NIR plus CAF-S1 cluster markers (ANTXR1, CD9, SDC1, LAMP5, GPC3, DLK1) to remove dead cells, and CD45 to remove remaining Tregs. Cells (Treg panel and CAF-S1 panel) were acquired using a ZE5 cell analyzer (Bio-Rad) and analyzed using Flowjo v10.4.2. For RNA analysis, non-adherent cells (CD4 + CD25 +The cells were collected by pipetting and spun down. RNA was then 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). cDNA libraries were prepared using the TruSeq RNA Exome Kit (Illumina, #20020189) and subsequently sequenced on a NovaSeq (Illumina) system. Reads were mapped to the human reference genome (hg38; Gencode release 29) and quantified using STAR (version 2.6.1a) with the following parameters: outFilterMultimapNmax=20; alignSJoverhangMin=8; alignSJDBoverhangMin=1; outFilterMismatchNmax=999; outFilterMismatchNoverLmax=0.04; alignIntronMin=20; alignIntronMax=1000000; alignMatesGapMax=1000000; outMultimapperOrder=Random. Only genes with one read in at least 5% of all samples were retained for further analysis. Normalization was performed with the DESeq2 R package.

[0255] Treg-CAF-S1 cluster intracellular staining: 5 × 10 4 CAF-S1 cells (spread) were plated in a 24-well plate (Falcon, #353047) in DMEM (HyClone, #SH30243.01) supplemented with 10% FBS (Biosera, #FB-1003 / 500) and 1.5% O. 2 The medium was then removed and 5 × 10 5 CD4 + CD25 + T lymphocytes were added to 500 μl of DMEM 1% FBS (ratio 1 to 10) and incubated overnight at 37 °C and 20% O. Next, non-adherent cells (CD4 + CD25+ ) were harvested, 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 kept 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 normal healthy tissue-derived fibroblasts and BC-derived CAF-S1 Primary fibroblasts were collected from paratumor tissue, i.e., tissue defined as healthy by the pathologist in question, 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, we analyzed the spread fibroblasts at early and late passages (passages 2 and 5, respectively) to verify the expression of the CAF-S1 marker. Primary cells were trypsinized, resuspended in PBS, stained with LIVE / DEAD™ Fixable Aqua Dead Cell Stain dye (ThermoFisher Scientific, #L34957), diluted in PBS for 20 minutes at room temperature, and fixed with 4% PFA for 20 minutes at room temperature. After a quick wash 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+ for 40 minutes at room temperature. Both the antibody and isotype control were coupled using the fluorochrome Zenon APC Mouse IgG1 Labeling Kit (ThermoFisher 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-naive patients without any treatment were processed for RNA extraction using the High FFPE 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 on a NovaSeq (Illumina). Reads were mapped to the human reference genome (release hg19 / GRCh37) and quantified using STAR (version 2.5.3a) with the following parameters: outFilterMultimapNmax=20; alignSJoverhangMin=8; alignSJDBoverhangMin=1; outFilterMismatchNmax=999; outFilterMismatchNoverLmax=0.04; alignIntronMin=20; alignIntronMax=1000000; alignMatesGapMax=1000000; outMultimapperOrder=Random. Only genes with one read in at least 5% of all samples were retained for further analysis. Normalization, unsupervised PCA, and differential analysis between responder and non-responder patients were performed with the DESeq2R package.

[0258] statistical analysis All statistical analyses and graphical representations of 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 were consistent with the data distribution. Normality was first checked using the Shapiro-Wilk test, and then a parametric or nonparametric two-tailed test was applied according to normality, as indicated in each figure legend. 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". Quantification from the FACS analysis shown in Figure 5 is shown using mean ± SEM. Figure 10. CD4 cells cultured alone or in the presence of ecm-myCAFs + 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 in Figure 6. For melanoma RNA-seq data, the following parameters were applied: enrichment statistic = "weighted" and metric for ranking genes = "Signal2Noise." For NSCLC RNA-seq data, GSEAPreranked was used with log2 fold change from DESeq2 differential analysis and the "classic" mode of enrichment score as the metric for ranking genes.

[0259] [Table 1A]

[0260] [Table 1B]

[0261] NSCLC samples were collected from routine diagnostic specimens of patients treated with immuno-oncology drugs, stored in the Pathology Department of Bichat Hospital. All patients received their first cycle of second- or third-line immunotherapy between July 28, 2015, and February 20, 2018, according to the drug registration and ongoing clinical trials during that period. The efficacy of immuno-oncology treatment was evaluated every 8–12 weeks by whole-body CT scan according to RECIST v.1.1 criteria. All clinical data were retrieved from the patient's electronic file. The histological subtype of NSCLC samples was determined according to the current World Health Organization 2015 classification on hematoxylin-stained formalin-fixed, paraffin-embedded tissue sections from bronchoscopy or CT-guided transthoracic biopsy specimens. The diagnosis of squamous cell carcinoma was supported by p40-positive and TTF-1 (thyroid transcription factor 1)-negative immunostaining. Non-squamous lung cancers included both adenocarcinomas showing positive Alcian blue staining (for mucin tumor cell content) and / or positive nuclear TTF-1 immunostaining, and large cell carcinomas lacking mucin secretion with negative p40 and TTF-1 immunostaining. Two specimens showed mixed features of squamous cell carcinoma and adenocarcinoma differentiation. According to the 8th TNM staging system, two stage IIIB patients had unresectable lung tumors and contraindications to radiation therapy. * 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 the Cell Signaling Technology E1L3N commercial clone on a Leica Bond platform. ** Eastern Cooperative Oncology Group (EOCG) performance status 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. detecting anthrax toxin receptor 1 (ANTXR1)-positive fibroblasts in a cancer sample from a patient suffering from cancer, + An in vitro method for detecting immunosuppressive fibroblasts in a cancer sample from a subject suffering from cancer, wherein the presence of fibroblasts is indicative of immunosuppressive CAFs.

2. ANTXR1 in cancer samples from patients with cancer + ANTXR1 in cancer samples, including detecting fibroblasts + 1. An in vitro method of predicting the response of a subject suffering from cancer to immunotherapy treatment, wherein fibroblasts are predictive of said patient's response to an immunotherapeutic agent.

3. The patient has (a) a low number or percentage of ANTXR1 + (b) providing a cancer sample containing fibroblasts; or (c) providing a sample containing ANTXR1 + An immunotherapeutic agent for use in treating cancer in a patient presenting with a cancer sample that does not have fibroblasts.

4. ANTXR1 + Determining the proportion of fibroblasts and ANTXR1 + The percentage of fibroblasts is determined by the ratio of ANTXR1 to the total number of fibroblasts in the cancer sample or the total number of cells in the cancer sample. + The method for predicting a subject's response according to claim 2 or the immunotherapeutic agent for use according to claim 3, wherein the number of fibroblasts is determined.

5. ANTXR1 + Fibroblasts are FAP + ANTXR1 + 5. The immunotherapeutic agent for the method of claim 2 or 4 or the use of claim 3 or 4, which is a fibroblast.

6. ANTXR1 + Fibroblasts express ANTXR1 + CAF, preferably FAP + ANTXR1 + 6. The immunotherapeutic agent for the method according to any one of claims 2 and 4 to 5, or the immunotherapeutic agent for the use according to any one of claims 3 and 4 to 5, which is CAF.

7. ANTXR1 + Fibroblasts are FAP + ANTXR1 + LAMP5 - SDC1 + Fibroblasts, preferably CAFs and FAPs + ANTXR1 + LAMP5 + SDC1 + / - Fibroblasts, preferably CAFs, ANTXR1 + SDC1 - LAMP5 - 7. The method according to any one of claims 2 and 4 to 6, wherein the cell is selected from the group consisting of CD9+ fibroblasts, preferably CAFs; 7. An immunotherapeutic agent for use according to any one of claims 3 and 4 to 6.

8. Use of ANTXR1 as a biomarker for the identification of immunosuppressive fibroblasts, preferably immunosuppressive CAFs.

9. The use of ANTXR1 in a fibroblast population, preferably a CAF population, as a biomarker of a subject's tumor to predict the response of a subject suffering from cancer to an immunotherapeutic agent.

10. ANTXR1 for use in treating cancer in a patient + a patient receiving a drug that targets fibroblasts, the drug comprising: + FAP + fibroblasts, preferably CAFs, and the agent is ANTXR1 + An agent that suppresses or reduces the immunosuppressive effect of fibroblasts, preferably the agent is selected from the group consisting of (i) an anti-ANTXR1 antibody optionally conjugated to a cytotoxic drug, a multispecific molecule comprising an anti-ANTXR1 moiety, an anti-ANTXR1 T cell receptor (TCR) and an anti-ANTXR1 chimeric antigen receptor (CAR), and (ii) an immune cell, preferably a T cell or a natural killer cell, expressing an anti-ANTXR1 TCR or an anti-ANTXR1 CAR, or any combination thereof.

11. ANTXR1 for use according to claim 10, for use in combination with immunotherapeutic agents. + Drugs that target fibroblasts.

12. Patients were found to have ANTXR1 in tumor samples from patients. + FAP + 1. A FAP inhibitor for use in treating cancer in a patient having fibroblasts, preferably CAFs.

13. 13. The FAP inhibitor for use according to claim 12, for use in combination with an immunotherapeutic agent.

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

15. a) ANTXR1 according to claim 10 as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a patient + and b) an immunotherapeutic agent that targets fibroblasts, and the patient has a tumor sample from the patient that detects ANTXR1. + FAP + A product or kit comprising fibroblasts, preferably CAFs.

16. The immunotherapeutic 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 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, the method of claim 2 and any one of claims 4 to 7, the immunotherapeutic agent for use according to any one of claims 3 to 7, the use of claim 9, the agent for use according to claim 11, the FAP inhibitor for use according to claim 13 or 14 or the product or kit of claim 15.

17. 17. The method of claim 16, or the immunotherapeutic agent for the use of claim 16, the use of claim 16, the agent for the use of claim 16, the FAP inhibitor for the use of claim 16, or the product or kit of claim 16, wherein the checkpoint inhibitor is selected from the group consisting of antibodies against cytotoxic T-lymphocyte-associated protein (CTLA-4), programmed death protein 1 (PD-1), programmed death-ligand (PD-L1), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), B- and T-lymphocyte attenuator (BLTA), IDO1, or any combination thereof, preferably selected from the group consisting of antibodies against CTLA-4, PD-1, PD-L1 and TIGIT, or any combination thereof, more preferably antibodies against PD-1 or CTLA-4 and a combination thereof.

18. 17. The method of claim 16, or the immunotherapeutic agent for the use of claim 16, the use of claim 16, the agent for the use of claim 16, the FAP inhibitor for the use of claim 16, or the product or kit of claim 16, wherein the immunotherapeutic 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.

19. The cancer is selected from the group consisting of prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, gastric cancer, kidney cancer, ovarian cancer, hepatocellular carcinoma, osteosarcoma, melanoma, hypopharyngeal cancer, esophageal cancer, uterine 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; preferably, the cancer is selected from the group consisting of head and neck cancer, breast cancer, ovarian cancer, NSCLC, melanoma and metastatic melanoma; more preferably, the cancer is 19. The method of any one of claims 2, 4 to 7 and 16 to 18, the immunotherapeutic agent for use of any one of claims 3 to 7 and 16 to 18, the use of any one of claims 9 and 16 to 18, the agent for use of any one of claims 10 to 11 and 16 to 18, the FAP inhibitor for use of any one of claims 12 to 14 and 16 to 18 or the product or kit of any one of claims 15 to 18, wherein the cancer is selected from the group consisting of head and neck cancer, NSCLC, melanoma and metastatic melanoma.

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