Novel immune checkpoint inhibitors

Abstract

The present invention relates to a prognostic biomarker for identifying fibrinogen-like protein 1 (FGL1) as reactivity of a cancer patient to an immune checkpoint inhibitor. More specifically, the present invention relates to a method of treating cancer in a human patient comprising administering an immune checkpoint inhibiting antibody that specifically inhibits the interaction of fibrinogen-like protein 1 (FGL1) with lymphocyte activation gene 3 protein (LAG3) wherein the cancer is a cancer expressing FGL1. Furthermore, the present invention relates to a method for assessing the sensitivity to or predicting the responsiveness to a treatment with an immune checkpoint-inhibiting antibody that specifically inhibits the interaction of FGL1 with LAG3 in a cancer patient. The immune checkpoint inhibiting antibody that specifically inhibits the interaction between FGL1 and LAG3 may be an antibody against human FGL1 or an antibody against human LAG3, optionally in combination with other immune checkpoint inhibiting antibodies such as an anti-PD1 or anti-PD-L1 antibody.

Description

[0001] This invention application is a divisional application based on the invention patent application with application date of June 10, 2019, application number 201980038211.5 and name “Novel Immune Checkpoint Inhibitor”. Technical Field

[0002] The present invention relates to the identification of fibrinogen-like protein 1 (FGL1) as a target for treating cancer patients and prognostic biomarkers for predicting the responsiveness of such cancer patients to immune checkpoint inhibitors. More specifically, the present invention relates to a method for treating cancer in a human patient, the method comprising administering an antibody that specifically inhibits the interaction of fibrinogen-like protein 1 (FGL1) with lymphocyte activation gene 3 protein (LAG3), and / or an antibody that specifically binds to FGL1, wherein the cancer is preferably a cancer that expresses FGL1. In addition, the present invention relates to a method for assessing sensitivity to or predicting responsiveness to treatment with an immune checkpoint inhibitory antibody in a cancer patient, the method comprising detecting FGL1 expression in a sample from the patient. The present invention also relates to a kit for selecting cancer patients who will benefit from immune checkpoint inhibitory antibodies. The immune checkpoint inhibitory antibody can be an antibody against human FGL1 or an antibody against human LAG3, optionally in combination with other immune checkpoint inhibitory antibodies such as anti-PD1 antibodies or anti-PD-L1 antibodies. Background Art

[0003] Upregulation of inhibitory receptor (IR) expression is crucial for balancing co-stimulatory receptor activity and limiting T cell activation, thereby preventing autoimmunity, autoinflammation, and tissue damage. However, tumors can hijack these so-called immune checkpoint mechanisms to defend against anti-tumor immune responses initiated by CD4+ and CD8+ T cells. Recent cancer immunotherapy approaches aim to reverse this depletion by targeting inhibitory receptors to overcome immune tolerance, thereby allowing reactivation of cytotoxic T cell responses against tumors.

[0004] Many immunomodulatory agents, primarily antibodies targeting immune system checkpoints such as cytotoxic T lymphocyte antigen 4 (CTLA-4) or programmed death 1 (PD-1) and its ligand (PD-L1), have received regulatory approval for the treatment of a variety of cancers, including malignant melanoma, non-small cell lung cancer, renal cell carcinoma, classical Hodgkin lymphoma, and recurrent or metastatic squamous cell carcinoma of the head and neck. However, the majority of patients treated with checkpoint inhibitors experience little or no benefit, and these treatments are expensive and may have associated toxicities. Therefore, there is a need for additional checkpoint inhibitors, new combinations of checkpoint inhibitors, and / or biomarkers for predicting therapeutic response to immune checkpoint inhibitory antibodies in cancer therapy to optimize patient selection.

[0005] Another immune checkpoint receptor is lymphocyte activation gene 3 (LAG3, CD223). Upregulation of LAG3 is required to control overactivation of the immune system and prevent autoimmunity. Due to its negative regulatory effect on T cells, it is a potential target for cancer immunotherapy. LAG3 is a cell surface T cell inhibitory protein and is thought to work by interacting with major histocompatibility complex class II (MHC-II). Several immunotherapeutics that modulate LAG3 are currently in various stages of clinical and preclinical development, such as the LAG3-Fc fusion protein IMP321 and the anti-LAG3 antibody BMS-986016. Existing anti-LAG3 therapies target interactions with its only known ligand, class II MHC. However, in various experimental settings, interaction with MHC-II does not appear to be required for LAG3-induced inhibitory function, allowing speculation about additional potential functional LAG3 ligands. Summary of the Invention

[0006] The inventors identified the liver-enriched soluble protein fibrinogen-like protein 1 (FGL1) with hepatocyte mitogenic activity and metabolic function as a LAG3 binding partner of great significance for cancer immunotherapy. Given the high expression of FGL1 in patients with primary resistance to anti-PD1 / PD-L1 therapy, the FGL1 / LAG3 pathway may be another potential immune escape mechanism. Therefore, FGL1 and its interaction with LAG3 are potential targets for therapeutic agents, and FGL1 expression is a promising prognostic biomarker for predicting the response to treatment with immune checkpoint inhibitors (such as antibodies that inhibit the interaction between FGL1 and LAG3). Since FGL1 / LAG3 checkpoint inhibition appears to at least complement PD-1 / PD-L1 checkpoint inhibition, FGL1 may also be a negative prognostic marker for other immune checkpoint inhibitory antibodies (such as anti-PD-1 or anti-PD-L1 antibodies).

[0007] On the one hand, a method for treating cancer in a human patient is provided, the method comprising administering to the patient an effective amount of an immune checkpoint inhibitory antibody that specifically inhibits the interaction of fibrinogen-like protein 1 (FGL1) with lymphocyte activation gene 3 protein (LAG3). Also provided is a method for treating cancer in a human patient, the method comprising administering to the patient an effective amount of an antibody that specifically binds to human FGL1. In a specific embodiment, the cancer patient has increased FGL1 expression, preferably, the cancer patient has been determined to have increased FGL1 expression. More specifically, it has been determined that a sample from the patient has increased FGL1 expression, preferably, it has been determined that the sample from the patient has increased FGL1 expression compared to a control. The method may include determining FGL1 expression in a sample from the patient, and administering to the patient an effective amount of an anti-FGL1 antibody or an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3. In one embodiment, the method comprises determining whether FGL1 expression is increased in a sample from the patient, and if FGL1 expression is increased, administering to the patient an effective amount of an anti-FGL1 antibody or an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3.

[0008] Also provided is an antibody that specifically inhibits the interaction of FGL1 and LAG3 (considered an "immune checkpoint inhibitory antibody" due to its interaction with the immune checkpoint pathway) for use in treating cancer in human patients. Further provided is an antibody that specifically binds to human FGL1 for use in treating cancer in human patients. In a specific embodiment, the cancer patient has increased FGL1 expression, preferably, the cancer patient has been determined to have increased FGL1 expression. More specifically, it has been determined that a sample from the patient has increased FGL1 expression, preferably, it has been determined that a sample obtained from the patient has increased FGL1 expression compared to a control. Preferably, it has been determined in vitro that the sample from the patient has increased FGL1 expression. The antibody for use may include determining FGL1 expression in a sample from the patient, followed by administering to the patient an anti-FGL1 antibody or an antibody that specifically inhibits the interaction of FGL1 with LAG3. In one embodiment, the use comprises determining whether FGL1 expression is increased in a sample from the patient, and if FGL1 expression is increased, administering to the patient an anti-FGL1 antibody or an antibody that specifically inhibits the interaction of FGL1 with LAG3.

[0009] The present invention further relates to a method of treating cancer in a human patient or an antibody for use, comprising a) providing a sample from the patient, b) measuring the level of FGL1 expression in the sample, c) comparing FGL1 expression to a control, d) identifying the patient as likely to respond to treatment with an anti-FGL1 antibody or an antibody that specifically inhibits the interaction of FGL1 with LAG3 when the level of FGL1 expression has been measured as increased compared to the control, and e) administering to the patient an effective amount of the anti-FGL1 antibody or the antibody that specifically inhibits the interaction of FGL1 with LAG3.

[0010] The present invention further relates to a method for selecting a therapy for a cancer patient, the therapy comprising an anti-FGL1 antibody or an antibody that specifically inhibits the interaction of FGL1 with LAG3, the method comprising determining the level of FGL1 expression in a sample from the patient, and selecting the therapy for the patient when the level of FGL1 expression is increased.

[0011] Preferably, the FGL1 expression of the antibody according to the method or use of the present invention is increased compared to a control. The control can be a reference value or reference sample of baseline expression from at least one subject without cancer or a non-cancerous tissue sample, wherein the non-cancerous tissue sample is preferably derived from the same organ as the cancer, and more preferably from a cancer patient. The sample from the patient as used in the method or use according to the present invention is preferably a tumor sample and / or a blood sample.

[0012] The antibody that specifically inhibits the interaction of FGL1 and LAG3 used in the method or therapy of the present invention can be an anti-FGL1 antibody, an anti-LAG3 antibody, or a combination thereof. The immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 and LAG3 can be used alone, or in combination with at least one other immune checkpoint inhibitory antibody selected from anti-PD-1 antibodies, anti-PD-L1 (anti-B7-H1) antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-GITR antibodies, anti-VISTA antibodies, anti-BTLA antibodies, and anti-OX40 antibodies. In a specific embodiment, the at least one other immune checkpoint inhibitory antibody is an anti-PD-1 antibody and an anti-PD-L1 antibody. In certain embodiments, the patient may have primary or acquired resistance to at least one immune checkpoint inhibitory antibody selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-GITR antibodies, anti-VISTA antibodies, anti-BTLA antibodies, and anti-OX40 antibodies. More specifically, the patient may have primary or acquired resistance to PD-1 / PD-L1 checkpoint inhibition.

[0013] The cancer as described above can be, but is not limited to, brain cancer, colorectal cancer, melanoma, prostate cancer or lung cancer. Preferably, the cancer is lung cancer or prostate cancer. More preferably, the lung cancer is non-small cell lung cancer (NSCLC). The treatment can be further accompanied by chemotherapy or radiotherapy.

[0014] In another aspect, a method for detecting increased FGL1 expression in a human cancer patient is provided, the method comprising providing a sample from the patient; and detecting FGL1 expression in the sample, wherein the FGL1 expression detected in the sample is preferably compared to a control. The method may optionally further comprise: when FGL1 expression is determined to be increased compared to the control, identifying the patient as potentially susceptible to treatment with an anti-FGL1 antibody or an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3.

[0015] Also provided herein is a method for assessing sensitivity to treatment with an anti-FGL1 antibody or an antibody that specifically inhibits the interaction of FGL1 and LAG3 in a cancer patient, the method comprising: a) optionally providing a sample from the patient, b) detecting FGL1 expression in the sample, c) comparing the FGL1 expression determined in step (b) with a control, wherein the FGL1 expression in the sample is increased compared to the control; and optionally further comprising: when it is determined that FGL1 expression is increased compared to the control, identifying the patient as potentially sensitive to treatment with an anti-FGL1 antibody or an antibody that specifically inhibits the interaction of FGL1 and LAG3.

[0016] Also provided is a method for classifying a tumor in a patient, the method comprising: (a) optionally providing a sample from the patient, (b) detecting FGL1 expression in the sample, and (c) identifying the tumor as a candidate for treatment with an anti-FGL1 antibody or an immune checkpoint inhibitory antibody, preferably an antibody that specifically inhibits the interaction of FGL1 with LAG3. Optionally, the method further comprises the step of comparing the FGL1 expression determined in step (b) with a control, wherein increased FGL1 expression in the sample compared to the control identifies the tumor as a candidate for treatment with an anti-FGL1 antibody or an immune checkpoint inhibitory antibody, preferably an antibody that specifically inhibits the interaction of FGL1 with LAG3.

[0017] Also provided is a method of predicting responsiveness in a human cancer patient, the method comprising: (a) optionally providing a sample from the patient, (b) determining the level of FGL1 expression in the sample, (c) comparing FGL1 expression to a control, and (d) identifying the patient as likely to respond to treatment with an anti-FGL1 antibody or an antibody that specifically inhibits the interaction of FGL1 with LAG3 when the level of FGL1 expression is measured as increased compared to the control, or identifying the patient as unlikely to respond when FGL1 expression is measured as not increased compared to the control.

[0018] Also provided is a method for predicting responsiveness in a human cancer patient, the method comprising: (a) optionally providing a sample from a patient being or previously treated with at least one immune checkpoint inhibitory antibody, (b) determining the level of FGL1 expression in the sample, (c) comparing FGL1 expression to a control, and (d) when it is determined that the level of FGL1 expression is increased compared to the control, identifying the patient as less likely to respond to treatment with the at least one immune checkpoint inhibitory antibody.

[0019] The methods for detecting increased FGL1 expression, methods for assessing sensitivity, methods for classification and methods for predicting responsiveness according to the present invention are generally and preferably in vitro methods. In a specific embodiment, the patient providing the sample may have primary or acquired resistance to at least one checkpoint inhibitory antibody, wherein the at least one checkpoint inhibitory antibody is selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-GITR antibodies, anti-VISTA antibodies, anti-BTLA antibodies and anti-OX40 antibodies. More specifically, the patient may have primary or acquired resistance to PD-1 / PD-L1 checkpoint inhibition. These methods may further include or be followed by a step of administering an anti-FGL1 antibody or an antibody that specifically inhibits the interaction of FGL1 with LAG3 to the cancer patient. The sample used in any of the methods is preferably a tumor or blood sample. FGL1 expression can be detected in a sample by (a) contacting the sample with an anti-FGL1 antibody and detecting binding between FGL1 (FGL1 protein) and the antibody, or (b) detecting the amount of mRNA encoding FGL1 in the sample. In certain embodiments, the control is a reference value or reference sample of baseline expression from at least one subject without cancer or a non-cancerous tissue sample, wherein the non-cancerous tissue sample is preferably derived from the same organ as the cancer, and more preferably from a cancer patient. In certain embodiments, the immune checkpoint inhibitory antibody that inhibits the interaction of FGL1 with LAG3 is an anti-LAG3 antibody or an anti-FGL1 antibody.

[0020] Also provided is a method for treating cancer in a human patient, comprising administering to the patient an effective amount of an anti-FGL1 antibody or an antibody that specifically inhibits the interaction of FGL1 and LAG3, wherein the cancer patient has increased FGL1 expression as determined using any one of the diagnostic methods described herein.

[0021] In another aspect, a kit for selecting a cancer patient who will benefit from an immune checkpoint inhibitory antibody, preferably an anti-FGL1 antibody, or an antibody that specifically inhibits the interaction of FGL1 with LAG3 is provided, wherein the kit comprises means for quantitatively detecting FGL1 expression in a sample from a cancer patient, and a control. The control can be selected from: (a) a reference sample for detecting enhanced FGL1 expression, (b) a reference sample for detecting baseline FGL1 expression, (c) an indication comprising a predetermined reference level of FGL1 expression, the predetermined reference level being associated with sensitivity to an immune checkpoint inhibitory antibody, preferably an anti-FGL1 antibody, or an antibody that specifically inhibits the interaction of FGL1 with LAG3, (d) an indication comprising a predetermined reference level of FGL1 expression, the predetermined reference level being associated with insensitivity to an immune checkpoint inhibitory antibody, preferably an anti-FGL1 antibody, or an antibody that specifically inhibits the interaction of FGL1 with LAG3, and / or a combination of (a), (b), (c), or (d). In one embodiment, the means for quantitatively detecting FGL1 expression in a sample from a cancer patient is (a) an antibody specific for FGL1, (b) a primer pair specific for FGL1 and optionally a probe that hybridizes to the amplified product, and / or (c) a probe that hybridizes to the FGL1 sequence.

[0022] In another aspect, a method for screening immune checkpoint inhibitory antibodies is provided, the method comprising

[0023] (a) providing an FGL1 protein, wherein the FGL1 protein preferably comprises at least the fibrinogen domain of human FGL1;

[0024] (b) providing a LAG3 protein, preferably comprising at least domains 1 and 2 of human LAG3;

[0025] (c) contacting the FGL1 protein with the LAG3 protein in the presence of an antibody to be screened;

[0026] (d) determining the binding of the FGL1 protein to the LAG3 protein; and

[0027] (e) if the binding of the FGL1 protein to the LAG3 protein is reduced compared to the binding of the FGL1 protein to the LAG3 protein in the absence of the antibody to be screened, the antibody to be screened is identified as an immune checkpoint inhibitory antibody that specifically inhibits the interaction between the FGL1 protein and the LAG3 protein. Preferably, the antibody is a human or humanized antibody.

[0028] In another aspect, a method for screening a small molecule that inhibits the binding of (human) FGL1 to (human) LAG3 is provided, the method comprising

[0029] (a) providing an FGL1 protein, wherein the FGL1 protein preferably comprises at least the fibrinogen domain of human FGL1;

[0030] (b) providing a LAG3 protein, preferably comprising at least domains 1 and 2 of human LAG3;

[0031] (c) contacting said FGL1 protein with said LAG3 protein in the presence of a small molecule, e.g., from a compound library, e.g., in a high throughput screening format,

[0032] (d) determining the binding of the FGL1 protein to the LAG3 protein in the presence and absence of the small molecule; and

[0033] (e) identifying small molecules that specifically inhibit the interaction between the FGL1 protein and the LAG3 protein as FGL1-LAG3 checkpoint inhibitor molecules, which are suitable for developing drugs for treating cancer diseases as mentioned above.

[0034] The present invention should also encompass small molecules identified as inhibitors of the binding of (human) FGL1 to (human) LAG3 by the methods described above, and such small molecules can therefore be used as drugs, or can be developed as drugs, which act on this immune checkpoint pathway and can therefore be used in (immuno)therapy of cancer diseases as mentioned above. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Identification of FGL1 as a novel LAG3 binding partner

[0036] (a) Schematic diagram of genome-scale receptor array screening in the following steps: (1) cDNA libraries encoding plasma membrane genes and soluble genes are collected. Selected soluble genes are fused to artificial transmembrane domains to facilitate membrane expression and array detection. (2) Receptor arrays are prepared robotically in 1536-well plates. (3) The gene library in the receptor array is reverse transfected by an ultra-high-speed orbital shaker-based program. The assay is performed using 293T.2A, an engineered 293T cell line that overexpresses several immune-relevant adaptors. (4) Receptor array screening is performed for LAG3-Ig, and array results are collected and analyzed. Human Fc receptors are used as internal positive controls for the screened fusion proteins within each plate. (b) Representative flow cytometric dot plots of FGL1-Ig binding to 293T.2A (1) or mock cells stably infected with mouse LAG3 (2). Binding of control Ig to mouse LAG3-expressing cells is shown (3). All samples were stained with anti-LAG3. (c) Representative Octet sensorgrams of mouse FGL1 binding to immobilized mouse LAG3 fusion protein. (d) Interaction between FGL1 and LAG3. 293T.2A cells were transfected with full-length human or mouse FGL1-TM (FIBCD1(1-57) fused to FGL1(23-312)) (d) or LAG3 (e), as indicated on the y-axis. Fusion proteins were added to the cultures for evaluation of binding to the transfectants using the Cellular Detection System (CDS), as indicated on the x-axis. Screenshots of individual wells captured using the CDS analysis software are shown. (f) Octet analysis of the human FGL1-LAG3 interaction. As shown, the Octet Protein A sensor was loaded with human LAG3-Fc and tested for binding kinetics with human FLAG-FGL1. Kd values ​​were calculated using a 1:1 binding ratio using Octet software. (g) Binding of FGL1-Ig to mouse LAG3-transfected 293T.2A cells in the presence of a control antibody (control) or anti-LAG3 (C9B7W) was tested by FACS. Binding of a control Fc fusion protein was used as a negative control (hatched line).

[0037] Figure 2 Characterization of the FGL1-LAG3 interaction

[0038] (a) Binding of mouse FGL1-Ig, FGL1-Ig with a coiled-coil domain (CCD), or FGL1-Ig with a fibrinogen domain (FD) to 293T.2A cells transfected with mouse LAG3 was measured using a cell-based assay system. (b) Binding of FGL1-Ig to 293T.2A cells transfected with full-length mouse LAG3 or LAG3 with various extracellular domain deletions. The full-length LAG3 protein consists of four extracellular Ig domains (D1 to D4), a transmembrane domain (TM), and an intracellular domain (IC). Binding intensity is indicated as "+++" (strong), "++" (moderate), "+" (weak), and "-" (no binding). (c) 293T.2A cells were transfected with full-length mouse LAG3. FGL1-Ig with various domain deletions or a control Fc fusion protein were added to the culture, and binding to the transfectants was measured using CDS, as indicated. (d) Quantification of FGL1 binding to LAG3 domain deletion / mutation forms as in (c) by CDS software.

[0039] Figure 3 FGL1 exhibits LAG3-dependent inhibitory activity on T cells

[0040] (a) Splenic T cells from WT or LAG3-KO mice were activated for 3 days by suboptimal concentrations of immobilized anti-CD3 antibody in the presence of soluble FGL1-Ig or control-Ig (5 μg / ml), followed by addition of 3 H-dTR. Proliferating T cells were analyzed for thymidine incorporation after 16 hours. (b) 3A9-LAG3 mouse T cell hybridoma cells were cocultured with the LK35.2 B cell line in CellGro serum-free medium in the presence of HEL peptide and varying concentrations of recombinant FGL1 (0, 5, and 50 ng / ml). Shown are the normalized percentages of inhibition of IL-2 levels in the supernatant at 24 hours (no FGL1 as 0% inhibition). (c) Binding of mouse FLAG-FGL1 (100 ng / ml) to mouse LAG3-transfected 293T.2A cells was quantified by CDS in the presence of anti-FGL1, anti-LAG3, or control antibodies at varying antibody:FGL1 ratios. (d) 3A9-LAG3 mouse T cell hybridoma cells were cocultured with the LK35.2 B cell line as in (c) in the presence of HEL peptide and recombinant FGL1 (50 ng / ml), anti-FGL1, and anti-LAG3 antibodies (1 μg / ml). IL-2 levels in the supernatant at 24 hours are shown. *, p < 0.05; **, p < 0.01; NS, not significant.

[0041] Figure 4 Anti-FGL1 enhances antigen-specific T cell responses in vivo

[0042] (a) Schematic diagram of the experimental design. 2x10e6 splenocytes from OT-1 / Rag2KO mice were intravenously transferred into C57 / BL6 mice, followed by treatment with ovalbumin peptide (500 μg per mouse) one day later. Control, anti-FGL1, or anti-LAG3 antibodies were injected on days 1 and 2. (b) Plasma cytokine levels were measured by CBA on day 4. *, p < 0.05; NS, not significant.

[0043] Figure 5 FGL1-KO mice confer a pro-inflammatory phenotype

[0044] (ab) FGL1 expression values ​​in different mouse tissues and immune cell subsets, as indicated in the BioGPS microarray database.

[0045] Figure 6 Enhanced immune response in the blood of FGL1-KO mice

[0046] (a) Frequency of the indicated immune lineages in the CD45 compartment of peripheral blood from WT and FGL1 KO mice. (b) Quantification of the indicated immune subsets in peripheral blood from WT (solid) and FGL1 KO (striped) mice. *, p<0.05.

[0047] Figure 7 FGL1 deficiency or blockade delays tumor growth

[0048] (ab) FGL1-KO, LAG3-KO, or WT littermates (WT) were inoculated with MC38 tumor cells (0.5 x 106 cells per mouse) on day 0. The mice were monitored for mean tumor diameter (a) and survival (b) at the indicated days after tumor inoculation. (cf) C57 / BL6 mice were inoculated with MC38 tumor cells (0.5 x 106 cells per mouse) on day 0 and treated with anti-FGL1, anti-LAG3, or control mAbs every four days from day 6 to day 18. Tumor responses (c) and the absolute number of CD45 (left panel), CD8 (center panel), and CD4 (right panel) cells per mg of tumor are depicted (d). Furthermore, the bar graph (e) shows the functional phenotype of CD8 TILs in tumors from FGL-1KO and anti-FGL1-treated mice. (f) WT or FGL1-KO mice were inoculated with MC38 tumor cells and treated with anti-LAG3 or control antibodies as in (c). Mean tumor diameters of these mice are shown. (g) LAG3-KO mice were inoculated with MC38 tumor cells and treated with anti-FGL1 or control antibodies as in (c). Mean tumor diameters of these mice are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.005; ****, p < 0.001; NS, not significant.

[0049] Figure 8 FGL1 expression is upregulated in solid cancers

[0050] (a) FGL1 expression values ​​in different human tissues, as shown in the BioGPS microarray database (mRNA). (b) Frequency of studies demonstrating upregulation or downregulation of the FGL1 gene in cancer lesions compared with corresponding normal tissues (P value < 0.05, fold change > 3 or < 0.3 as cutoffs) in major solid cancers analyzed using the R program based on the Oncomine database. (c) FGL1 expression values ​​(mRNA) in lung adenocarcinoma and other solid cancers (left-hand panel) compared with corresponding normal tissues (right-hand panel) analyzed using the TCGA cancer database.

[0051] Figure 9 FGL1 is upregulated in human cancers and has prognostic value

[0052] (a) Distribution of FGL1 expression as shown by quantitative immunofluorescence (QIF) staining in a YTMA250 NSCLC tissue array stained for FGL1, DAPI nuclear, and cytokeratin. The median expression value from normal lung tissue was used as the cutoff for FGL1 upregulation. (b) Association of tumor FGL1 expression quantified by quantitative immunofluorescence with overall patient survival in YTMA250 lung cancers. (c) Plasma FGL1 levels in a Spanish cohort of NSCLC cancer patients and healthy donors were tested by ELISA. (d) Survival analysis of NSCLC (d) or melanoma (e) patients with high or low FGL1 after anti-PD1 therapy.

[0053] Figure 10 Synergistic effects of FGL1 targeting and anti-PD therapy

[0054] (a) C57 / BL6 mice were inoculated with MC38 tumor cells on day 0 (0.5x10e6 per mouse) and subsequently treated with anti-FGL1, anti-LAG3, or control antibodies every four days from day 6 to day 18. In some groups, mice were also treated with a single dose of anti-B7-H1 (10B5) on day 6. Survival of mice in these groups is shown. *, p<0.05; **, p<0.01. (b) C57 / BL6 mice were inoculated with MC38 tumor cells on day 0 (0.5x10e6 per mouse) and then treated with anti-FGL1, anti-LAG3, or control antibodies every four days from day 6 to day 18. In some groups, mice were also treated with a single dose of anti-B7-H1 (10B5) on day 6. Survival of mice in these groups is shown. *, p<0.05; **, p<0.01. 6 ), followed by treatment with anti-FGL1, anti-LAG3, or control antibodies every four days from day 6 to day 18. In some groups, mice were also treated with a single dose of anti-B7-H1 (10B5) on day 6. The mean tumor diameters in the indicated groups of mice at day 22 are shown. DETAILED DESCRIPTION

[0055] The general embodiment "comprising" or "comprised" encompasses the more specific embodiment "consisting of." Furthermore, the singular and plural forms are not used in a limiting manner. As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless expressly stated to be singular only.

[0056] As used herein, the term "protein" is used interchangeably with "amino acid residue sequence" or "polypeptide" and refers to a polymer of amino acids of any length. These terms also include proteins that are post-translationally modified by reactions including, but not limited to, glycosylation, acetylation, phosphorylation, glycation, or protein processing. Modifications and changes can be made in the structure of a polypeptide, such as fusion to other proteins, amino acid sequence substitutions, deletions, or insertions, while the molecule retains its biological functional activity. For example, certain amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence, and a protein with the same properties can be obtained. The term "polypeptide" generally refers to a sequence having more than 10 amino acids, and the term "peptide" means a sequence having a length of up to 10 amino acids. However, these terms can be used interchangeably.

[0057] As used herein, the term "gene" refers to a DNA locus of a heritable genomic sequence that affects the properties of an organism by being expressed as a functional product or by regulating gene expression. Genes and polynucleotides may comprise introns and exons, as in genomic sequences, or only comprise coding sequences, as in cDNA, such as open reading frames (ORFs), which comprise a start codon (methionine codon) and a translation stop codon. Genes and polynucleotides may also comprise regions that regulate their expression, such as transcription initiation, translation, and transcription termination. Therefore, regulatory elements such as promoters are also included.

[0058] The terms "enhanced," "increased," and "increased" are used interchangeably herein and refer to an increase of at least about 10% compared to a control, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300%, or a decrease of any integer between 10-300% compared to a control. As used herein, a "control" is a reference value or reference sample of baseline expression from at least one subject without cancer or a non-cancerous tissue sample, wherein the non-cancerous tissue sample is preferably derived from the same organ as the cancer, and more preferably from a cancer patient.

[0059] The cut-off value for establishing a marker such as FGL1 and the method and sample type for measuring the FGL1 level in the sample obtained from the individual or patient to be analyzed are matched or identical to each other. A cut-off value can be obtained in a control group, i.e., a value above which increased expression or overexpression (e.g., increased FGL1 expression compared to a control) is confirmed. The control group on which the cut-off value is based is selected to match the individual / patient group being studied.

[0060] A "vector" is a nucleic acid that can be used to introduce a heterologous polynucleotide into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), wherein additional DNA or RNA segments can be introduced into the viral genome.

[0061] The terms "encode" and "codes for" broadly refer to any process whereby the information in a polymer macromolecule is used to guide the production of a second molecule that is different from the first molecule. In other aspects, a DNA molecule can encode an RNA molecule (e.g., by using a transcription process of a DNA-dependent RNA polymerase). Moreover, as in the translation process, an RNA molecule can encode a polypeptide. When used to describe the translation process, the term "encode" is also extended to the triplet codons encoding amino acids. In some aspects, an RNA molecule can encode a DNA molecule, such as by incorporating a reverse transcription process of an RNA-dependent DNA polymerase. On the other hand, a DNA molecule can encode a polypeptide, wherein it should be understood that the "encode" used in this case incorporates both transcription and translation processes.

[0062] The term "allele" refers to any of the different forms of a gene, genetic target region, or generally a DNA sequence at a single locus (i.e., chromosomal location). This includes coding sequences, non-coding sequences, and regulatory sequences. Different alleles within a genome are not necessarily identical in nucleotide sequence.

[0063] The term "antibody" refers to a protein consisting of one or more polypeptides that are substantially encoded by immunoglobulin genes. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes and countless immunoglobulin variable region genes. The terms "antibody" and "immunoglobulin" are used interchangeably herein. The term "antibody" is used in its broadest sense herein and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, and multispecific antibodies (e.g., bispecific antibodies). The term "antibody" also encompasses antibody fragments, such as, for example, Fv, Fab, Fab', F(ab)2, or other antigen-binding subsequences of antibodies, i.e., antigen-binding fragments of original or "full-length" antibodies. The term "antibody" also encompasses engineered molecules, such as nanobodies (R), i.e., humanized VHH domains derived from camelid antibodies, and "domain antibodies" or "single domain antibodies," i.e., immunoglobulins that can bind to their antigens through a single immunoglobulin domain (rather than through two paired VH / VL domains). The term "antibody" further encompasses antibody conjugates. Classical 4-chain full-length "antibodies" or "immunoglobulins" are generally heterotetrameric glycoproteins of about 150 kDa, consisting of two identical light chains and two identical heavy chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy chain and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has an amino-terminal variable domain (VH) and subsequently three carboxyl-terminal constant domains (CH). Each light chain has an N-terminal variable domain (VL) and a single C-terminal constant domain (CL). The term "antibody" further refers to a class of antibodies, which includes a variety of individual antibodies with the same specificity (variable domains) and the same constant domains.

[0064] The term "immune checkpoint inhibitory antibody" refers to an antibody as defined above that binds to an inhibitory receptor (IR) or more broadly interferes with the interaction between an inhibitory receptor and its ligand, wherein the inhibitory receptor is crucial for balancing co-stimulatory receptor activity and limiting T cell activation. Therefore, this antibody targets immune system checkpoints, such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death factor 1 (PD-1) or its ligand (PD-L1). Examples of immune checkpoint inhibitory antibodies suitable for use in the present invention are anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-GITR antibodies, anti-OX40 antibodies or anti-LAG3 antibodies, but are not limited thereto. In addition, anti-FGL1 antibodies can be considered as immune checkpoint inhibitors. In most cases, the immune checkpoint inhibitory antibody is an antagonist or blocking antibody, i.e., a blocking antibody selected from the group consisting of anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-LAG3 antibodies, anti-VISTA antibodies, and anti-BTLA antibodies. However, the immune checkpoint inhibitory antibody may also be an agonist antibody, such as an anti-GITR or anti-OX40 antibody. With regard to anti-LAG3 antibodies, the antibody may be an antibody that selectively inhibits the interaction of LAG3 with MHC class II molecules, or an antibody that selectively inhibits the interaction of LAG3 with FGL1, or an antibody that inhibits the interaction of LAG3 with FGL1 and MHC class II molecules. Preferably, the immune checkpoint inhibitory antibody is an IgG antibody, more preferably a human or humanized IgG antibody, and even more preferably a human or humanized IgG1 or IgG4 antibody.

[0065] A "fusion protein" is defined as a protein that contains the complete sequence or any portion of the sequence of two or more originally separate natural or modified heterologous proteins, or a combination of the complete sequence or any portion of the sequence of two or more originally separate natural or modified heterologous proteins. Fusion proteins can be constructed by genetic engineering methods by fusing two or more genes or portions thereof that originally encoded two or more originally separate natural or heterologous proteins or portions thereof. This produces a fusion protein that has functional properties derived from each of the original proteins. Fusion proteins include, but are not limited to, Fc fusion proteins. As used herein, a fusion protein can also be a Flag fusion protein. Fusion proteins such as FGL1 fusion proteins or LAG3 fusion proteins can also be used in therapeutic methods or for treating cancer according to the present invention in place of or in addition to immunosuppressive antibodies. Preferably, the fusion protein is an FGL1-Fc (FGL1-Ig) or LAG3-Fc (LAG3-Ig) protein.

[0066] The term "cytokine" refers to a small protein that is released by cells and acts as an intercellular mediator, for example, affecting the behavior of cells surrounding the secreting cell. Cytokines can be secreted by immune cells such as T cells, B cells, NK cells and macrophages or other cells. Cytokines can participate in intercellular signal transduction events such as autocrine signal transduction, paracrine signal transduction and endocrine signal transduction. They can mediate a series of biological processes, including but not limited to immunity, inflammation and hematopoiesis. Cytokines can be chemokines, interferons, interleukins, lymphokines or tumor necrosis factors.

[0067] As used herein, the term "expression" or "expressing" refers to the transcription and / or translation of a nucleic acid sequence in a host cell. The expression level of a target gene product in a cancer or cancer patient can be determined based on the amount of the corresponding mRNA present in the sample provided or by the amount of a protein encoded by a selected gene, particularly encoding FGL1. For example, RNA transcribed from genes such as the FGL1 gene can be quantitatively assayed by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization with cellular RNA, or by PCR (such as qPCR). Proteins encoded by genes such as the FGL1 gene can be quantitatively assayed by various methods, for example, by ELISA, by Western blotting, by radioimmunoassay, by immunoprecipitation, by immunohistochemistry, by testing the biological activity of the protein, by immunostaining of the protein and subsequent FACS analysis, or by homogeneous time-resolved fluorescence (HTRF) assay.

[0068] As used herein, the term "cancer patient has increased FGL1 expression" refers to a patient who has a cancer that expresses FGL1, particularly a cancer that has increased FGL1 expression, more particularly a cancer that has increased FGL1 expression compared to a control. FGL1 expression can be detected or determined in a cancer sample, such as a biopsy of cancerous tissue. However, FGL1 expression can also be detected or determined in a blood sample of a cancer patient, such as a plasma or serum sample. FGL1 can be present in a bound and / or free (soluble) form. Thus, the term "cancer patient has increased FGL1 expression" encompasses that the patient has a cancer that has increased FGL1 expression and / or that the patient has an increased level of FGL1 in their blood. Thus, in one embodiment, the patient has a cancer that has increased FGL1 expression, particularly compared to a control, such as a non-cancerous reference sample of the same tissue of the patient, or in a reference sample of the same tissue of at least one reference subject, preferably a reference sample of the same tissue of more than one reference subject, or a reference value. Reference value can be based on the reference sample of the same tissue of at least one reference subject, preferably more than the reference sample of the same tissue of one reference subject.Described at least one reference subject is preferably a subject not suffering from cancer.Enhanced expression can be detected as the percentage (tumor proportion score (TPS)) of the signal that rises in tissue sample or the cell that rises that is positive for FGL1.In another embodiment or in addition to previous embodiment, the patient has the FGL1 expression of increase in blood such as in serum and / or plasma, particularly the FGL1 level of increase, more particularly the FGL1 protein level of increase.Preferably, FGL1 expression is increased compared with control, described control is such as in non-cancerous reference blood sample, such as serum or plasma sample of at least one reference subject, preferably more than the reference blood sample of one reference subject, or reference value.Reference value can be based on the reference blood sample of at least one reference subject, preferably more than the reference blood sample of one reference subject.Described at least one reference subject is preferably a subject not suffering from cancer.Preferably, reference subject does not have further liver damage.In one embodiment, patient suffers from the cancer that expresses FGL1, wherein said cancer is not liver cancer. Preferably, the patient has a cancer with increased expression of FGL1, wherein the cancer is not liver cancer, more particularly a cancer with increased expression of FGL1 compared to a control, wherein the cancer is not liver cancer.

[0069] The term "gene product" refers to RNA polynucleotides and polypeptides encoded by a gene or DNA polynucleotide.

[0070] As used herein, the term "sample" refers to a tissue sample or a body fluid sample such as a blood sample. A tissue sample is a slice of a body organ or tissue that typically includes several cell types, optionally with a cytoskeleton structure that holds the cells together. A tissue sample can be obtained by biopsy, which, for example, includes cutting, slicing, or punching. It involves extracting sample cells or tissue for inspection. However, the terms "providing a sample," "sample from a patient," or "sample obtained from a patient" do not include the active steps of extracting tissue or blood, but rather refer to providing a sample that has been extracted, i.e., providing an ex vivo sample from a patient. In addition, a tissue sample can be a tumor sample or a corresponding control sample, which is preferably from the same tissue. Preferably, a tissue sample is used as a formalin-fixed, paraffin-embedded (FFPE) sample. A blood sample can be a serum or plasma sample, and is preferably a plasma sample. However, a blood sample can also include a cell fraction, for example, for complete blood cell count and hematology, such as a peripheral blood smear. In addition to blood, other body fluid samples include, but are not limited to, mucus, semen, saliva, sputum, bronchial lavage fluid, breast milk, bile, and urine. The sample may also be a bone marrow biopsy or aspirate or cerebrospinal fluid. The sample may be analyzed, for example, but not limited to, by cytochemistry (chemical stains (dyes) that react with certain substances found in or on cells of different types), flow cytometry, and immunohistochemistry (IHC), such as by using antibody staining, fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR), or ELISA. As used herein, the term "sample obtained from a patient" refers to a sample obtained in vitro (i.e., after collection), and is synonymous with "providing a sample from a patient" or "providing an in vitro sample from a patient."

[0071] As used herein, the terms "detecting FGL1 expression" and "determining FGL1 expression" refer to measuring the level of FGL1 expression by any means or method known to those skilled in the art, wherein FGL1 expression can be determined at the protein or mRNA level. Preferably, the means or method for measuring FGL1 expression is capable of quantitatively detecting or determining FGL1 expression, preferably human FGL1 expression. Quantitatively detecting or determining FGL1 expression includes detecting or determining relative amounts or absolute amounts. Suitable methods for quantitatively detecting and determining FGL1 expression include contacting a sample with an anti-FGL1 antibody and detecting the binding between FGL1 and the antibody, such as by ELISA or by IHC, or detecting the amount of mRNA encoding the FGL1 protein in the sample, such as by PCR. In a specific embodiment, detecting or determining FGL1 expression or increased FGL1 expression involves administering an anti-FGL1 antibody to a patient and performing in vivo imaging, particularly if the antibody is labeled, such as by radioactivity, fluorescence or other means.

[0072] The term "fibrinogen-like protein 1," abbreviated as FGL1 (synonyms LFIRE-1, HFREPI, Hepassocin, HP-041), refers to a protein of the fibrinogen family, which also includes fibrinogen-like protein 2 and coagulation factors V, VIII, and XIII. However, unless otherwise specified, the term "FGL1 expression" refers to both protein and mRNA expression in humans. FGL1 is encoded by the FGL1 gene. FGL-1 contains a fibrinogen-related domain in its C-terminal portion, which contains four conserved cysteines shared by all members of the fibrinogen family. However, FGL-1 lacks the platelet binding site, crosslinking region, and thrombin-sensitive site that allow other members of the fibrinogen family to assist in the formation of fibrin clots. FGL1 is upregulated in regenerating livers and is fully associated with the fibrin matrix after clot formation. Although most FGL1 is found in plasma, approximately 20% of FGL1 remains in the serum after blood clotting. The human FGL1 precursor has an amino acid sequence as provided in the NCBI Reference Sequence Database under ID No. NP_004458.3, wherein amino acids 1 to 22 correspond to the signal peptide and amino acids 23 to 312 correspond to the mature protein. The C-terminal globular domain of fibrinogen corresponds to amino acids 78 to 304.

[0073] As used herein, the term "lymphocyte activation gene 3 protein," abbreviated as LAG3, refers to an inhibitory molecule involved in the regulation of T cell activation, proliferation, and homeostasis. It is also known as CD223. LAG3 is a cell surface molecule that is activated and induced by CD4-related activation that acts as an immune checkpoint receptor. LAG3 is expressed on activated T cells, natural killer cells (NK cells), B cells, and plasmacytoid dendritic cells (pDCs). The protein negatively regulates proliferation, activation, and homeostasis in a manner similar to CTLA-4 and PD-1. It is reported that it plays a role in Treg suppressive function and helps maintain CD8+ T cells in a tolerogenic state. LAG3 is structurally related to CD4 and binds to MHC class II molecules with high affinity. A single lysine residue (K468) within the conserved "KIEELE" motif is crucial for interaction with downstream signal transduction molecules. It has four extracellular Ig-like domains with conserved structural motifs between the D1 and D3 domains and between the D2 and D4 domains. The first domain is an Ig-like V-type domain, and the next three domains are Ig-like C2-type domains. Human LAG3 shares approximately 70% homology with murine LAG3. A unique distinguishing feature of LAG3 is a 30-aa proline-rich loop between the C and C' beta strands of the D1 domain. Although both human and murine LAG-3 possess this loop, their homology is lowest in this region. The amino acid sequence of human LAG3 is provided by the NCBI GenBank database as ID number AAH52589.

[0074] The term "immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 and LAG3" refers to an antibody as defined above that binds to human FGL1 or human LAG3, thereby blocking the binding of FGL1 to LAG3. This blocking or antagonistic activity of the antibody may be due to binding to the binding pocket (or binding interface) of FGL1 for LAG3 or alternatively LAG3 for FGL1, or due to any other spatial interaction with the binding of FGL1 to LAG3, but is not limited thereto. With respect to anti-LAG3 antibodies, the antibody may be an antagonistic antibody that selectively inhibits the interaction of LAG3 with FGL1, or an antagonistic antibody that inhibits the interaction of LAG3 with FGL1 and MHC II. Preferably, the immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3 is an IgG antibody, more preferably a human or humanized IgG antibody, and even more preferably a human or humanized IgG1 antibody.

[0075] As used herein, the term "programmed death factor 1 (PD-1)" refers to an immune checkpoint that limits excessive immune responses to antigens and prevents autoimmunity. PD-1 can be expressed on a variety of immune cells, including T cells, B cells, natural killer T (NKT) cells, activated monocytes, and dendritic cells (DCs). It is expressed on the surface of activated T cells, but not resting T cells, and has two known ligands, PD-L1 and PD-L2. PD-L1 is expressed on activated T cells, DCs, monocytes, and myeloid cells, and outside the hematopoietic system, PD-L1 is expressed in the lungs, vascular endothelium, liver non-parenchymal cells, placental syncytiotrophoblasts, and keratinocytes. Many agents targeting PD-1 or PD-L1 interactions are in clinical development at various stages. Nivolumab and pembrolizumab are humanized PD-1 blocking antibodies that have been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of unresectable or advanced malignant melanoma (MM), non-small cell lung cancer (NSCLC) that has progressed on or after platinum-based chemotherapy, and recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN) that has progressed on or after platinum-based chemotherapy. Nivolumab is further approved for the treatment of advanced renal cell carcinoma (RCC) that has progressed after prior therapy and classical Hodgkin lymphoma that has relapsed or progressed after autologous hematopoietic stem cell transplantation (HSCT). Pembrolizumab has been further approved for the first-line treatment of patients with 50% or greater PD-L1 expression (TPS ≥ 50%). Atezolizumab is a fully humanized PD-L1 blocking antibody that is approved by the FDA and EMA for patients with locally advanced or metastatic urothelial carcinoma (UC) after prior platinum-containing chemotherapy or who are considered ineligible for cisplatin, and for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) after prior chemotherapy. Other anti-PD-L1 antibodies are durvalumab and avelumab.

[0076] Uses of immune checkpoint inhibitory antibodies

[0077] In the present invention, a method for treating cancer in a human patient is provided, comprising administering to the patient an effective amount of an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 and LAG3, and / or an anti-FGL1 antibody (in the latter case, regardless of whether direct inhibition of the interaction of FGL1 and LAG3 can be observed).

[0078] Also provided is a method for treating cancer in a human patient, comprising administering to the patient an effective amount of an immune checkpoint inhibitory antibody, preferably an antibody that specifically inhibits the interaction of FGL1 with LAG3, and / or an anti-FGL1 antibody, wherein the cancer patient has increased FGL1 expression, preferably wherein the cancer patient has been determined to have increased FGL1 expression. In specific embodiments, a sample obtained from the patient has been determined to have increased FGL1 expression. The increased FGL1 expression is typically increased compared to a control. The method may include determining FGL1 expression in a sample obtained from the patient and administering to the patient an effective amount of an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3, or an anti-FGL1 antibody. The method may further include determining whether FGL1 expression is increased in the sample obtained from the patient, and if FGL1 expression is increased, administering to the patient an effective amount of an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3, or an effective amount of an anti-FGL1 antibody.

[0079] Also provided is an immune checkpoint inhibitory antibody, or anti-FGL1 antibody, that specifically inhibits the interaction between FGL1 and LAG3, for use in treating cancer in a human patient. In certain embodiments, the cancer patient has increased FGL1 expression, preferably, the cancer patient has been determined to have increased FGL1 expression. In specific embodiments, a sample from the patient has been determined to have increased FGL1 expression. The increased FGL1 expression is typically increased compared to a control. Preferably, increased FGL1 expression has been determined in vitro in the sample from the patient. Administration of the antibody may include determining FGL1 expression in the sample from the patient, followed by administration of the immune checkpoint inhibitory antibody, or anti-FGL1 antibody, that specifically inhibits the interaction between FGL1 and LAG3 to the patient. Administration of the antibody may further include determining whether FGL1 expression is increased in the sample from the patient, and if FGL1 expression is increased, administering the immune checkpoint inhibitory antibody, or anti-FGL1 antibody, that specifically inhibits the interaction between FGL1 and LAG3 to the patient.

[0080] In another aspect, a method for treating cancer in a human patient is provided, comprising administering to the patient an effective amount of an antibody that binds to human FGL1. Also provided is an antibody that binds to human FGL1 for use in treating cancer in a human patient. In certain embodiments, the cancer patient has increased FGL1 expression, preferably, the cancer patient has been determined to have increased FGL1 expression. In specific embodiments, a sample from the patient has been determined to have increased FGL1 expression. The increased FGL1 expression is typically increased compared to a control. Preferably, the FGL1 antibody inhibits the interaction between FGL1 and LAG3.

[0081] The method or the antibody for use may further comprise a) identifying a patient in need of cancer treatment, b) determining that the patient has increased FGL1 expression, and c) administering to the patient an effective amount of an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3, or an anti-FGL1 antibody. The method or the antibody for use may further comprise determining increased FGL1 expression in a sample from the patient. The method or the antibody for use may further comprise providing a sample from the patient to determine FGL1 expression.

[0082] The present invention further relates to a method for treating cancer in a human patient or an antibody for use, comprising a) optionally providing a sample from the patient, b) measuring the level of FGL1 expression in the sample, c) comparing FGL1 expression to a control, d) identifying the patient as likely to respond to treatment with an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3 or with an anti-FGL1 antibody when the level of FGL1 expression has been measured as increased compared to the control, and e) administering to the patient an effective amount of the immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3, or the anti-FGL1 antibody.

[0083] The present invention further relates to a method for selecting a therapy for a cancer patient, wherein the therapy comprises an immune checkpoint inhibitory antibody that specifically inhibits the interaction between FGL1 and LAG3, or an anti-FGL1 antibody, the method comprising measuring the level of FGL1 expression in a sample obtained from the patient, and selecting the therapy for the patient when the FGL1 expression level is increased.

[0084] Preferably, the FGL1 expression of the method according to the present invention or the antibody for use is increased compared to a control. The control can be a reference value or a reference sample of baseline expression from at least one subject without cancer or a noncancerous tissue sample, wherein the noncancerous tissue sample is preferably derived from the same organ as the cancer, and more preferably from a cancer patient. The sample from the patient as used in the method according to the present invention or the purposes is preferably a tissue sample and / or a blood sample, preferably a tumor or a blood sample. Therefore, in one embodiment, the patient suffers from a cancer with increased FGL1 expression, particularly compared to a control, the control being a noncancerous reference sample of the same tissue of the patient, or in a reference sample of the same tissue of at least one reference subject, preferably more than a reference sample of the same tissue of a reference subject, or a reference value. The reference value can be based on a reference sample of the same tissue of at least one reference subject, preferably more than a reference sample of the same tissue of a reference subject. The at least one reference subject is preferably a subject not suffering from cancer. Enhanced expression can be detected as an elevated signal or the percentage (tumor proportion score (TPS)) of cells that are positive for FGL1. In another embodiment or in addition to the previous embodiment, the patient has increased FGL1 expression in blood such as serum or plasma, particularly increased FGL1 levels, more particularly increased FGL1 protein levels. Preferably, FGL1 expression is increased compared to a control, such as in a reference blood sample, such as a serum or plasma sample of at least one reference subject, preferably a reference blood sample of more than one reference subject, or a reference value. The reference value can be based on a reference blood sample of at least one reference subject, preferably a reference blood sample of more than one reference subject. The at least one reference subject is preferably a subject not suffering from cancer. Preferably, the reference subject does not have further liver damage. In one embodiment, the patient suffers from a cancer that expresses FGL1, wherein the cancer is not liver cancer. Preferably, the patient suffers from a cancer that has increased FGL1 expression, wherein the cancer is not liver cancer, more particularly a cancer that has increased FGL1 expression compared to a control, wherein the cancer is not liver cancer. Tissue or tumor samples can be used as formalin-fixed, paraffin-embedded samples for FGL1 detection.

[0085] The immune checkpoint inhibitory antibody that specifically inhibits the interaction between FGL1 and LAG3 can be an anti-LAG3 antibody, an anti-FGL1 antibody, or a combination thereof. In a specific embodiment, the immune checkpoint inhibitory antibody is an anti-LAG3 antibody, wherein the anti-LAG3 antibody inhibits the interaction between FGL1 and LAG3. In another specific embodiment, the immune checkpoint inhibitory antibody is an anti-FGL1 antibody. In yet another embodiment, the immune checkpoint inhibitory antibody is an anti-LAG3 antibody and an anti-FGL1 antibody. Preferably, the anti-LAG3 and anti-FGL1 antibodies inhibit the interaction between FGL1 and LAG3. In a specific embodiment, the anti-FGL1 antibody is used to treat cancer in a human patient.

[0086] The immune checkpoint inhibitory antibodies and / or anti-FGL1 antibodies that specifically inhibit the interaction of FGL1 and LAG3 used in the methods or treatments according to the present invention may also be used in addition to at least one other immune checkpoint inhibitory antibody (the term still includes its antigen-binding fragment), wherein the other immune checkpoint inhibitory antibodies can be any checkpoint inhibitory antibodies other than the immune checkpoint inhibitory antibodies or anti-FGL1 antibodies that specifically inhibit the interaction of FGL1 and LAG3. Preferably, the at least one other immune checkpoint inhibitory antibody is selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-BTLA antibodies, anti-VISTA antibodies, anti-GITR antibodies, anti-OX40 antibodies and anti-LAG3 antibodies. In particular, the anti-LAG3 antibody selectively inhibits the interaction of LAG3 with MHC class II molecules. More preferably, the at least one immune checkpoint inhibitory antibody is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody, and even more preferably, the at least one immune checkpoint inhibitory antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0087] The immune checkpoint inhibitory antibody and the at least one other immune checkpoint inhibitory antibody can be administered to the patient systemically (e.g., orally, parenterally, subcutaneously, intravenously, rectally, intramuscularly, intraperitoneally, intranasally, transdermally, or by inhalation or an intracavitary device), topically, or by application to a mucous membrane (such as the nose, throat, and bronchi). Preferably, the immune checkpoint inhibitory antibody and the at least one other immune checkpoint inhibitory antibody are administered intravenously or subcutaneously. When more than one immune checkpoint inhibitory antibody is used, each immune checkpoint inhibitory antibody can be independently administered by a suitable administration route (preferably by intravenous or subcutaneous means). The immune checkpoint inhibitory antibody and the at least one other immune checkpoint inhibitory antibody can be administered simultaneously or separately from each other. Typically, they are provided in a separate formulation or dosage form, which can be combined or administered separately before administration.

[0088] In certain embodiments, cancers treated with immune checkpoint inhibitory antibodies that specifically inhibit the interaction of FGL1 and LAG3, or with anti-FGL1 antibodies, include but are not limited to solid tumors, blood cancers (e.g., leukemias, lymphomas, myelomas, e.g., multiple myeloma), and metastatic lesions. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignancies, such as sarcomas and cancers, such as adenocarcinomas of various organ systems, such as those affecting the lungs, breasts, ovaries, lymphomas, gastrointestinal tract (e.g., colon), anus, genitalia, and urogenital tract (e.g., kidney, urothelium, bladder cells, prostate), pharynx, CNS (e.g., brain, nerves, or glial cells), head and neck, skin (e.g., melanoma), and pancreas, as well as adenocarcinomas including malignancies, such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, and esophageal cancer. Preferably, the cancer is brain cancer, colorectal cancer, melanoma, prostate cancer, or lung cancer. More preferably, the cancer is lung cancer or prostate cancer, and even more preferably, the lung cancer is non-small cell lung cancer (NSCLC). The treatment may be further accompanied by chemotherapy or radiotherapy. The cancer may be an early, mid-stage, late-stage or metastatic cancer. In a specific embodiment, the cancer is not liver cancer, such as hepatocarcinoma, with or without a viral infection, such as chronic viral hepatitis.

[0089] In one embodiment, the cancer is selected from lung cancer (e.g., non-small cell lung cancer (NSCLC) (e.g., NSCLC with squamous and / or non-squamous histology, or NSCLC adenocarcinoma)), melanoma (e.g., advanced melanoma), kidney cancer (e.g., renal cell carcinoma), myeloma (e.g., multiple myeloma), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two, or all of estrogen receptor, progesterone receptor, or Her2 / neu, such as triple-negative breast cancer), colorectal cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), anal cancer, gastroesophageal cancer, thyroid cancer, cervical cancer, lymphoproliferative disorder (e.g., post-transplant lymphoproliferative disorder), brain cancer or blood cancer, T-cell lymphoma, B-cell lymphoma, non-Hodgkin lymphoma, or leukemia (e.g., myeloid leukemia or lymphoid leukemia).

[0090] In another embodiment, the cancer is selected from cancer (e.g., advanced or metastatic cancer), melanoma, or lung cancer (e.g., non-small cell lung cancer). In one embodiment, the cancer is lung cancer, e.g., non-small cell lung cancer or small cell lung cancer, preferably non-small cell lung cancer. In one embodiment, the cancer is melanoma, e.g., advanced melanoma. In one embodiment, the cancer is an advanced or unresectable melanoma that is unresponsive to other therapies. In another embodiment, the cancer is prostate cancer, e.g., advanced prostate cancer. In yet another embodiment, the cancer is a myeloma, e.g., multiple myeloma. In yet another embodiment, the cancer is kidney cancer, e.g., renal cell carcinoma (RCC) (e.g., metastatic RCC or clear cell renal cell carcinoma (CCRCC)).

[0091] In certain embodiments, the patient to be treated for cancer may have primary or acquired resistance to at least one immune checkpoint inhibitory antibody, wherein the at least one immune checkpoint inhibitory antibody is selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-BTLA antibodies, anti-VISTA antibodies, anti-GITR antibodies, and anti-OX40 antibodies. In some embodiments, the patient has primary or acquired resistance to PD-1 / PD-L1 checkpoint inhibition. Patients with primary (innate) resistance to checkpoint inhibitors may also be referred to as non-responders. However, late recurrences have been observed in some cases, indicating acquired (secondary) resistance to treatment.

[0092] Preferably, the cancer patient has or is identified as having increased FGL1 expression. In certain embodiments, the cancer patient has or is identified as having a tumor with increased FGL1 expression. In some embodiments, the methods described herein further comprise identifying a cancer patient suitable for treatment with an immune checkpoint inhibitory antibody based on having increased FGL1 expression or having a tumor with increased FGL1 expression. In some embodiments, the cancer patient has or is identified as having a high percentage of cancer cells that are positive for FGL1.

[0093] In some embodiments, the methods described herein further comprise identifying a cancer patient based on: having increased FGL1 expression; or having a high percentage of FGL1-positive cancer cells; and having lung cancer (e.g., squamous cell lung cancer or lung adenocarcinoma), head and neck cancer; squamous cell cervical cancer; gastric cancer; esophageal cancer; thyroid cancer; melanoma, brain cancer, colorectal cancer, prostate cancer, and / or nasopharyngeal carcinoma (NPC), preferably brain cancer, colorectal cancer, melanoma, prostate cancer, or lung cancer. The methods and antibodies disclosed herein can be used to treat metastatic lesions associated with increased FGL1 expression and the above-mentioned cancers.

[0094] Also provided is a method for treating cancer in a human patient, comprising administering to the patient an effective amount of an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 and LAG3, and / or an anti-FGL1 antibody, wherein the cancer patient has increased FGL1 expression as determined according to any one of the diagnostic methods described herein.

[0095] If the antibodies of the present invention bind to FGL1, preferably human FGL1, the antibodies may or may not directly inhibit the binding of FGL1 to (human) LAG3. The antibodies may or may not compete with human LAG3 for binding to FGL1. Although the molecular mechanism by which such non-competitive antibodies exert their immunomodulatory effects is not fully understood, such antibodies may have the same therapeutic effects as anti-FGL1 antibodies (which compete with LAG3 for binding to FGL1).

[0096] In yet another aspect, an antibody that binds to human LAG3 (anti-LAG3) is provided, wherein the antibody inhibits the interaction of human LAG3 with human FGL1.

[0097] Preferably, the anti-FGL1 antibody and / or anti-LAG3 antibody according to the present invention is a human or humanized antibody, more preferably a human or humanized IgG1 or IgG4 antibody.

[0098] Diagnostic methods

[0099] Provided herein is a method for detecting increased FGL1 expression in a human cancer patient, the method comprising providing a sample from the patient; and detecting FGL1 expression in the sample, wherein the quantitatively detected FGL1 expression in the sample is preferably compared to a control, wherein the method may optionally further comprise: when FGL1 expression has been determined to be increased compared to a control, identifying the patient as potentially sensitive to treatment with an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3, or with an anti-FGL1 antibody.

[0100] Also provided herein is a method for assessing sensitivity to treatment with an immune checkpoint inhibitory antibody in a cancer patient, the method comprising: a) providing a sample from the patient, b) detecting FGL1 expression in the sample, c) comparing the FGL1 expression determined in step (b) with a control, wherein the FGL1 expression in the sample is increased compared to the control; and optionally further comprising: when FGL1 expression has been determined to be increased compared to the control, identifying the patient as potentially sensitive to treatment with an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3 or with an anti-FGL1 antibody.

[0101] Also provided is a method for classifying a patient's tumor, the method comprising (a) providing a sample from the patient, (b) detecting FGL1 expression in the sample, and (c) identifying the tumor as a candidate for treatment with an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3, or with an anti-FGL1 antibody, wherein the method optionally further comprises a step of comparing the FGL1 expression determined in (b) with a control, wherein increased FGL1 expression in the sample compared to the control identifies the tumor as a candidate for such treatment. At the same time, increased FGL1 expression levels in the sample can also indicate that the tumor has escaped treatment with another immune checkpoint inhibitory antibody, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-IDO antibody, an anti-CTLA-4 antibody, an anti-Tim-3 antibody, an anti-BTLA antibody, an anti-VISTA antibody, an anti-GITR antibody, and an anti-OX40 antibody. In this case, the combined use of such other immune checkpoint inhibitory antibodies together with immune checkpoint inhibitory antibodies that specifically inhibit the interaction of FGL1 with LAG3, or together with anti-FGL1 antibodies, can overcome primary or acquired resistance to such other immune checkpoint inhibitory antibodies and, therefore, can overcome the poor prognosis associated with increased FGL1 expression.

[0102] Also provided is a method for predicting responsiveness in a human cancer patient, the method comprising: (a) providing a sample from the patient, (b) measuring the level of FGL1 expression in the sample, (c) comparing FGL1 expression to a control, and (d) identifying the patient as likely to respond to treatment with an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3, or with an anti-FGL1 antibody, when it is determined that the level of FGL1 expression is increased compared to the control, or identifying the patient as unlikely to respond when it is determined that FGL1 expression is not increased compared to the control.

[0103] Also provided is a method for predicting responsiveness in a human cancer patient, the method comprising: (a) providing a sample from a patient who is or was previously treated with at least one immune checkpoint inhibitory antibody, (b) determining the level of FGL1 expression in the sample, (c) comparing FGL1 expression to a control, and (d) identifying the patient as less likely to respond to treatment with the at least one immune checkpoint inhibitory antibody when the level of FGL1 expression is determined to be increased compared to the control. The at least one immune checkpoint inhibitory antibody is an immune checkpoint inhibitory antibody other than an immune checkpoint inhibitory antibody that specifically inhibits the interaction of FGL1 with LAG3 and other than an anti-FGL1 antibody, but is preferably selected from the group consisting of anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-GITR antibodies, and anti-OX40 antibodies. More preferably, the antibody is selected from the group consisting of anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies, and more preferably, the antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. The poor prognosis associated with increased FGL1 expression can be overcome by add-on therapy with immune checkpoint inhibitory antibodies or anti-FGL1 antibodies according to the present invention that specifically inhibit the interaction of FGL1 with LAG 3.

[0104] Taking into account the various interactions between immune checkpoint pathways, enhanced or increased FGL1 expression may also indicate that treatment of this patient with any immune checkpoint inhibitor may achieve a therapeutic effect in the treatment of cancer diseases, including (antagonistic) anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG3 antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-GITR antibodies, anti-OX40 antibodies or anti-VISTA antibodies. Therefore, according to another aspect of the present invention, there are provided methods of treatment, methods of predicting the responsiveness of human cancer patients, methods of assessing the sensitivity of tumors or cancer diseases, methods of classifying tumors in patients, etc., wherein enhanced or increased FGL1 expression is used as an indicator indicating that this patient, tumor or cancer disease can be treated by an immune checkpoint inhibitor as mentioned above, such as, for example, (antagonistic) anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG3 antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-VISTA antibodies, anti-GITR antibodies, anti-OX40 antibodies or anti-VISTA antibodies.

[0105] The methods for detecting increased FGL1 expression, methods for assessing sensitivity, methods for classification, and methods for predicting responsiveness according to the present invention are preferably in vitro methods. In addition, the step of detecting FGL1 expression particularly involves quantitatively detecting FGL1 expression or FGL1 expression levels, preferably in a sample from a patient. The method may further include or be followed by the step of administering to the cancer patient an immune checkpoint inhibitory antibody or an anti-FGL1 antibody that specifically inhibits the interaction between FGL1 and LAG3. The sample is preferably a tissue and / or body fluid sample, preferably a tissue and / or blood sample, more preferably a tumor and / or blood sample. For example, FGL1 expression can be quantitatively detected in a sample by: (a) contacting the sample with an anti-FGL1 antibody and detecting binding between FGL1 (FGL1 protein) and the antibody, or (b) detecting the amount of mRNA encoding FGL1 in the sample. In addition, an increase in FGL1 copy number can be determined. In one embodiment, the control is a reference value of baseline expression or a reference sample from at least one reference subject without cancer or a non-cancerous tissue sample, wherein the non-cancerous tissue sample is preferably derived from the same organ as the cancer, and more preferably from a cancer patient.

[0106] The FGL1 expression as detected according to the method is preferably increased compared to a control. The control can be a reference value or reference sample of baseline expression from at least one reference subject without cancer or a non-cancerous tissue sample, wherein the non-cancerous tissue sample is preferably derived from the same organ as the cancer, and more preferably from a cancer patient. The sample from the patient as used in the method according to the invention is preferably a tissue sample and / or a blood sample, preferably a tumor and / or a blood sample.

[0107] Therefore, in one embodiment, the patient suffers from a cancer with increased FGL1 expression, particularly compared to a control, such as a non-cancerous reference sample of the same tissue of the patient, or in a reference sample of the same tissue of at least one reference subject, preferably more than a reference sample of the same tissue of a reference subject, or a reference value. The reference value may also be referred to as a cutoff value. The reference value may be based on a reference sample of the same tissue of at least one reference subject, preferably more than a reference sample of the same tissue of a reference subject. The at least one reference subject is preferably a subject not suffering from cancer. The enhanced expression may be detected as an elevated signal or elevated percentage of cells that are positive for FGL1 (tumor proportion score (TPS)).

[0108] In another embodiment or in addition to previous embodiment, the patient has the FGL1 expression of increase in blood such as serum or plasma, particularly the FGL1 level of increase, more particularly the FGL1 protein level of increase. Preferably, FGL1 expression increases compared to a control, the control being as in a reference blood sample, such as the serum or plasma sample of at least one reference subject, preferably more than a reference blood sample of a reference subject, or a reference value. The reference value can be based on the reference blood sample of at least one reference subject, preferably more than a reference blood sample of a reference subject. The at least one reference subject is preferably a subject not suffering from cancer. Preferably, the reference subject does not have further liver damage.

[0109] In one embodiment, the patient has a cancer that expresses FGL1, wherein the cancer is not liver cancer. Preferably, the patient has a cancer with increased FGL1 expression, wherein the cancer is not liver cancer, more particularly a cancer with increased FGL1 expression compared to a control, wherein the cancer is not liver cancer.

[0110] In specific embodiments, FGL1 expression is detected in a sample from a patient with primary or acquired resistance to at least one checkpoint inhibitory antibody, or from a patient with primary or acquired resistance to PD-1 / PD-L1 checkpoint inhibition, wherein the at least one checkpoint inhibitory antibody is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-IDO antibody, an anti-CTLA-4 antibody, an anti-Tim-3 antibody, an anti-GITR antibody, and an anti-OX40 antibody.

[0111] The cancer patient with increased FGL1 expression can be a patient suffering from a cancer expressing FGL1, a cancer particularly with increased FGL1 expression, a cancer more particularly with increased FGL1 expression compared to a control. Can be in a cancer sample, such as in a biopsy of cancerous tissue, detect or determine that FGL1 is expressed. However, can also be in a blood sample of a cancer patient, such as in a blood plasma or serum sample, detect or determine that FGL1 is expressed. FGL1 can exist in a combined and / or free (soluble) form.

[0112] In one embodiment, the patient has a cancer with increased FGL1 expression, particularly compared to a control, such as a non-cancerous reference sample of the same tissue of the patient, or in a reference sample of the same tissue of at least one reference subject, preferably a reference sample of the same tissue of more than one reference subject, or a reference value. The reference value can be based on a reference sample of the same tissue of at least one reference subject, preferably a reference sample of the same tissue of more than one reference subject. The at least one reference subject is preferably a subject without cancer. Enhanced expression in the tumor sample can be detected as an increased signal (staining intensity) and / or an increased percentage of cells positive for FGL1 (tumor proportion score (TPS)). Typically, the sample is in the form of a formalin-fixed, paraffin-embedded tissue sample, for example, as a tissue microarray (TMA) section. The percentage of tumor cells with FGL1 staining used to assess the patient's TPS is between 0% and 100%. TPS is the percentage of viable tumor cells that show partial or complete staining relative to all viable tumor cells present in the sample (positive and negative). Infiltrating immune cells, normal cells, necrotic cells, and debris must be excluded from the score. At least 100 viable tumor cells are required to determine FGL1 expression. The FGL1 expression level of the sample can be interpreted as "no FGL1 expression: TPS < 1%", "FGL1 expression: TPS ≥ 1%", "high FGL1 expression: TPS ≥ 50%". Other relevant factors may be the expression level (staining intensity). Staining can be performed using methods known in the art, using in situ hybridization, preferably fluorescence in situ hybridization (FISH), or preferably immunohistochemistry (IHC). For IHC, sections of formalin-fixed, paraffin-embedded tissue samples are generally deparaffinized and rehydrated. After antigen retrieval, endogenous peroxidases should be quenched, for example with hydrogen peroxide, before reacting with anti-FGL1 antibodies or LAG3 fusion proteins. Other relevant factors may also be an increase in gene copy number (an additional gene copy in the genome), such as can be identified using FISH (Inoue, Y. et al., Clinical significance of PD-L1 and PD-L2 copy number gains in non-small-cell lung cancer, Oncotarget, 7 (22): 32113-32128). In addition, circulating free tumor DNA from plasma, such as EDTA-anticoagulated peripheral whole blood, can be quantitatively detected. In addition to primary tumor samples, FGL1 expression can also be determined in metastatic regional lymph node samples. Lymph nodes can also be analyzed in patients with lymphoma or leukemia.

[0113] Nucleic acid hybridization only involves contacting a probe (e.g., an oligonucleotide or a larger polynucleotide) with a target nucleic acid under conditions where the probe and its complementary target can form a stable heteroduplex by complementary base pairing. As used herein, hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed in, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Labs Press (incorporated by reference in its entirety, specifically referring to pages 9.31-9.62). In addition, formulas for calculating appropriate hybridization and washing conditions to achieve hybridization allowing varying degrees of nucleotide mispairing are disclosed in, for example, Meinkoth et al. (1984) Anal. Biochem. 138, 267-284. Nucleic acids that do not form heteroduplexes are washed off from hybrid nucleic acids, and then hybrid nucleic acids can be detected, typically by detecting attached detectable labels. Nucleic acid denaturation is achieved by increasing the temperature or reducing the salt concentration of the buffer containing nucleic acids. Under low stringency conditions (e.g., low temperature or high salt or both), hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even when the annealed sequences are not completely complementary. Thus, the specificity of hybridization decreases at lower stringencies. Conversely, at higher stringencies (e.g., higher temperatures or lower salts), successful hybridization requires fewer mismatches.

[0114] In another embodiment or in addition to the tissue sample, the patient has increased FGL1 expression in blood such as serum and / or plasma, particularly increased FGL1 levels, more particularly increased FGL1 protein levels. Preferably, FGL1 expression is increased compared to a control, such as in a non-cancerous reference blood sample, such as a serum or plasma sample of at least one reference subject, preferably more than a reference blood sample of a reference subject, or a reference value. The reference value can be based on a reference blood sample of at least one reference subject, preferably more than a reference blood sample of a reference subject. The at least one reference subject is preferably a subject not suffering from cancer. Preferably, the reference subject does not have further liver damage. FGL1 expression in serum and / or plasma can be detected, for example, using enzyme-linked immunosorbent assay (ELISA) as a detection agent using anti-FGL1 antibodies or LAG3 fusion proteins, and is known in the art. The concentration of FGL1 in serum is within the range of ng / ml.

[0115] Hybridized nucleic acids or antibodies are detected by detecting one or more labels attached to the sample nucleic acid or antibody. Labels can be incorporated by any of a number of means well known to those skilled in the art. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the present invention include fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, Alexa fluors, Spectrum dyes, etc.), quantum dots, radioactive labels (e.g., 3 H. 125 I. 35 S. 14 C. or 32 Radiolabeled antibodies can be detected by fluorescence in situ hybridization (FISH) or colorimetric labeling. The methods for detecting such labels are well known to those skilled in the art. Therefore, for example, radiolabeled antibodies can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector, a fluorescence microscope, or flow cytometry that detects the emitted light. Colorimetric labels can be detected simply by visualizing the colored labels. Preferably, hybrid nucleic acids are detected by fluorescent labeling and most preferably, in the case of fluorescence in situ hybridization (FISH) assays. FISH assays are well known in the art. Antibodies are preferably detected by fluorescent labeling or colorimetric labeling.

[0116] In one embodiment, the patient has a cancer that expresses FGL1, wherein the cancer is not liver cancer. Preferably, the patient has a cancer with increased FGL1 expression, wherein the cancer is not liver cancer, more particularly a cancer with increased FGL1 expression compared to a control, wherein the cancer is not liver cancer. Cancer patients suitable for the methods of the present invention have a cancer as specified for therapeutic use of immune checkpoint inhibitory antibodies.

[0117] Screening methods

[0118] The present invention also provides a method for screening immune checkpoint inhibitory antibodies, the method comprising

[0119] (a) providing an FGL1 protein, wherein the FGL1 protein preferably comprises at least the fibrinogen domain of human FGL1;

[0120] (b) providing a LAG3 protein, preferably comprising at least domains 1 and 2 of human LAG3;

[0121] (c) contacting the FGL1 protein with the LAG3 protein in the presence of an antibody to be screened;

[0122] (d) determining the binding of FGL1 protein to LAG3 protein; and

[0123] (e) if the binding of the FGL1 protein to the LAG3 protein is reduced compared to the binding of the FGL1 protein to the LAG3 protein in the absence of the antibody to be screened, the antibody to be screened is identified as an immune checkpoint inhibitory antibody that specifically inhibits the interaction between the FGL1 protein and the LAG3 protein. Preferably, the antibody is a human or humanized antibody.

[0124] The present invention further provides a method for screening small molecules that inhibit the binding of (human) FGL1 to (human) LAG3, the method comprising

[0125] (a) providing an FGL1 protein, wherein the FGL1 protein preferably comprises at least the fibrinogen domain of human FGL1;

[0126] (b) providing a LAG3 protein, preferably comprising at least domains 1 and 2 of human LAG3;

[0127] (c) contacting said FGL1 protein with said LAG3 protein in the presence of a small molecule, e.g., from a compound library, e.g., in a high throughput screening format,

[0128] (d) determining the binding of the FGL1 protein to the LAG3 protein in the presence and absence of the small molecule; and

[0129] (e) identifying a small molecule that specifically inhibits the binding of the FGL1 protein to the LAG3 protein as an FGL1-LAG3 checkpoint inhibitor molecule, which is suitable for developing a drug for treating the cancer diseases mentioned above.

[0130] In addition, the present invention encompasses small molecules identified as inhibitors of the binding of (human) FGL1 to (human) LAG3 by the methods described above, and such small molecules can therefore be used as drugs, or can be developed as drugs, which act on this immune checkpoint pathway and can therefore be used in (immuno)therapy of cancer diseases as mentioned above.

[0131] In certain embodiments, the binding of FGL1 (e.g., full-length human FGL1 or tagged FGL1, such as 3xFLAG-tagged FGL1 protein) to a soluble protein of LAG3 (e.g., a fusion protein of human LAG3, such as a LAG3-Fc fusion protein) is measured. The binding or interaction can be measured, for example, using a biosensor such as an Octet instrument.

[0132] In other embodiments, at least one protein comprises a transmembrane domain to promote membrane expression, and the other binding partner is a soluble protein. Human LAG3 is a transmembrane protein and therefore contains a transmembrane domain. FGL1 is a soluble protein that can be fused with an artificial transmembrane domain (ATM), such as from the FIBCD1 gene (ATM inserted into the N-terminus) or the B7-H6 gene (ATM inserted into the C-terminus), to promote membrane expression. Preferably, FGL1 fused with the artificial transmembrane domain is transfected into cells that overexpress several immune-related adaptors that promote membrane expression (such as DAP10, DAP12, FcRγ, and CD3ε), for example, into 293T.2A cells. The soluble protein can be FGL1, optionally labeled with a detectable marker (such as a tag (e.g., 3xFLAG tag), an Fc domain, or a fluorescent marker), or can be detected using a secondary antibody. Alternatively, the soluble protein can be a soluble fusion protein of LAG3, such as a LAG3-Fc fusion protein, and can be directly labeled or detected using a secondary antibody or any other secondary reagent known in the art. In a preferred embodiment, the FGL1 protein comprises amino acids 78-304 or amino acids 23-312 of the amino acid sequence provided in the NCBI Reference Sequence Database as NP_004458.3. Methods for determining protein-protein interactions are known in the art and may include, but are not limited to, the use of a cellular detection system (CDS), fluorescence microscopy, and flow cytometry.

[0133] Reagent test kit

[0134] In another aspect, a kit for selecting cancer patients who will benefit from immune checkpoint inhibitory antibodies is provided, wherein the kit comprises a means for quantitatively detecting FGL1 expression in a sample from a cancer patient, and a control. The control can be selected from: (a) a reference sample for detecting enhanced FGL1 expression, (b) a reference sample for detecting baseline FGL1 expression, (c) an indication of a predetermined reference level of FGL1 expression, the predetermined reference level having been associated with sensitivity to immune checkpoint inhibitory antibodies, (d) an indication of a predetermined reference level of FGL1 expression, the predetermined reference level having been associated with insensitivity to immune checkpoint inhibitory antibodies, and / or a combination of (a), (b), (c) or (d). In one embodiment, the means for quantitatively detecting FGL1 expression in a sample from a cancer patient is (a) an antibody specific for FGL1, (b) a primer pair specific for FGL1 and, optionally, a probe that hybridizes to an amplified product, and / or a probe that hybridizes to an FGL1 target sequence, preferably an FGL1 mRNA target sequence.

[0135] Example

[0136] method

[0137] animal

[0138] C57BL / 6 (B6) mice of 6 to 8 week old are purchased from Charles River (Wilmington, Massachusetts).FGL1 conditional knockout mice are obtained from European mutant mouse resource bank (EMMA), and in genetically engineered C57 / BL6 ES cells (Agouti JM8A), this mouse strain has been carried out homology targeting.By hybridizing with CMV-Cre mice (Jackson Lab), FGL1 is completely knocked out (whole body knockout) mice (Fgl1- / -, referred to as FGL1-KO in this article).All mouse protocols all comply with NIH guidelines and have obtained the approval of Yale University School of Medicine Animal Care and Use Committee.

[0139] Plasmids, fusion proteins, and antibodies

[0140] Human cDNA libraries encoding approximately 6,500 full-length plasma membrane genes and approximately 1,300 soluble genes were collected from Genecopeia (Rockville, MD) and Open Biosystems (Huntsville, AL) or cloned individually. All genes used in the ultra-high-throughput receptor array described below were cloned into mammalian expression vectors. Human and mouse fusion proteins were generated by tagging the relevant genes encoding the extracellular domain with human Fc or 3xFLAG, expressed in 293T cells, and purified by affinity columns. Domain deletion plasmids for FGL1 and LAG3 were generated by PCR or GeneArt synthesis (Thermo Fisher, Waltham, MA). Antibodies used in flow cytometry and in vitro studies were purchased from BD Biosciences (San Jose, CA), BioLegend (San Diego, CA), or ThermoFisher (Waltham, MA). The hybridoma used for the anti-LAG3 monoclonal antibody (C9B7W; directed against mouse LAG3) was from Dr. Dario Vignali (University of Pittsburgh). Rat mAbs against mouse FGL1 and mouse mAbs against human FGL1 were generated by immunizing animals with FGL1 fusion protein.

[0141] Ultra-high-throughput receptor array technology

[0142] In addition to plasma membrane genes, soluble genes were selected based on expression profiles, homology with known immune-related molecules (bioinformatics analysis was performed using BioGPS, Immgen, HPM, UCSC genome browser, and NCBI databases), and then fused with an artificial transmembrane domain (ATM) from the FIBCD1 gene (ATM inserted at the N-terminus) or the B7-H6 gene (ATM inserted at the C-terminus) to promote membrane expression. To construct each receptor array plate, the cDNA library plasmid was diluted individually in OptiMEM (4ug / ml) and then transferred to a 384-deep-well plate (VWR) with 200ul of plasmid solution in each well. Two microliters (ul) of plasmid solution were transferred from each well of four 384 plates to a 1536-well plate (Greiner) using a robotic liquid handling system (PlateMate, Thermo Fisher). The entire human receptor array contains five 1536 plates. Unless otherwise specified, these 1536 plates were further foiled and stored at -80°C. For receptor array screening, the array plate was taken out from -80 ° C and placed in an incubator until the solution was completely melted. The array plate was spun down (600g, 2min) and dispensed with 2ul of OptiMEM (7ul lipofectamine / ml) containing lipofectamine 3000 per well using a Multidrop Combi robotic dispenser (Thermo Fisher) and rapidly shaken for 1min on an ultra-high-speed orbital shaker. All plates were stored at room temperature for 20min, followed by addition of 2x10e3 293T.2A cells (293T cells overexpressing several immune-related adapters (DAP10, DAP12, FcRγ, and CD3ε) that promote membrane expression) in 4ul DMEM culture medium per well by Multidrop Combi. The plate was further spun down (1000g, 4min) to remove air bubbles from the inside of each well and then incubated at 37 ° C. Eighteen hours after transfection, 2 ng of LAG3-Fc fusion protein (human or mouse) and 3 ng of anti-Fc secondary antibody were added to each well. Six to eight hours later, the plates were read using the Applied Biosystems 8200 Cellular Detection System (CDS) and analyzed using CDS 8200 software. Human Fc receptors were included in each plate as an internal positive control for the fusion proteins being screened.

[0143] Protein-protein interaction analysis

[0144] Protein interactions were measured and analyzed on an Octet Red instrument (PALL, New York, NY) at room temperature. Octet Protein A sensors were immersed in a solution containing mouse or human LAG3-Fc fusion protein (10 μg / ml) and then loaded with varying concentrations of 3xFLAG-tagged FGL1 protein (two-fold serial dilutions starting at 10 μg / ml). Protein association and dissociation were monitored and analyzed using Octet software.

[0145] In vitro T cell function assay

[0146] Anti-mouse CD3 mAb (eBiosciences) was pre-coated in 96-well plates at the indicated concentrations. Mouse splenocytes from C57 / BL6 mice or LAG3-KO mice were added to each well at 3x10e5 / well in the presence of 5ug / ml of FGL1-Fc fusion protein or isotype-matched control Fc and cultured for 3 days. 3 H-TdR, and thymidine incorporation was counted using a MicroBeta Trilux liquid scintillation counter (PerkinElmer, Waltham, MA).

[0147] For antigen-specific T cell experiments, 3A9 T cell hybridomas or 3A9 cells overexpressing the full-length mouse LAG3 gene were incubated with the LK35.2 cell line in the presence of HEL peptide (1.5 μg / ml). The indicated concentrations of FGL1 fusion protein or controls were also included to test in vitro function. In some groups, anti-FGL1 or anti-LAG3 antibodies (5 μg / ml) were used to monitor antagonistic effects. 24 hours after cell co-culture, IL-2 levels in the supernatant were analyzed using a CBA kit (BD Pharmingen).

[0148] In vivo tumor experiments

[0149] In vivo tumor experiments were performed using 8- to 10-week-old LAG3-KO, FGL1-KO mice, or control littermates. On day 0, tumors were inoculated by a single subcutaneous injection of MC38 or Hepa1-6 cells (5 × 10 5 cells and for Hepa1-6, 1x10 6cells), and mice were treated intraperitoneally with 100 μg of anti-FGL1 or anti-LAG3 (C9B7W) antibodies on days 6, 10, 14, and 18, with saline or rat Ig as controls. For the anti-PD-L1 (B7-H1) combination therapy, mice were further treated with a hamster mAb against PD-L1 (clone 10B5; 100 μg) on ​​day 6. Tumor growth was monitored twice a week by electronic calipers and the average tumor diameter was measured as (length + width) / 2.

[0150] Bioinformatics

[0151] Microarray data on solid cancer lesions and corresponding normal tissues were collected from Oncomine (Thermo Fisher) (a total of 254 datasets covering 14 types of human cancer). The frequency of FGL1 upregulation or downregulation in each cancer dataset was further analyzed using the R program (fold change >3 for upregulation or <0.3 for downregulation, with a P value <0.05 as a cutoff). In addition, FGL1 expression in several cancer individuals was compared with corresponding normal tissues using the TCGA cancer database. The raw microarray data were normalized using the Cancer Browser (https: / / genome-cancer.ucsc.edu / ) and then analyzed using R.

[0152] Histology and immunohistochemistry

[0153] Tissues were removed from naive or tumor-bearing mice, embedded in paraffin, and cut into 5-μm-thick sections for H&E or immunohistochemical staining. For FGL1 staining, tissues were deparaffinized and rehydrated before antigen retrieval. Tissue sections were then stained with an anti-FGL1 antibody (177R4) and subsequently incubated with an amplification system (DakoCytomation, Glostrup Municipality, Denmark).

[0154] Statistical analysis

[0155] Statistical analysis was performed using Student's t-test and two-way anova, and P values ​​reflect comparisons within the control sample. P values ​​less than 0.05 were considered statistically significant. Error bars in the figures represent standard error of the mean (SEM).

[0156] Example 1

[0157] Lymphocyte activation gene 3 (LAG3) is a cell-surface T-cell inhibitory protein thought to function through interaction with major histocompatibility complex class II (MHC-II). However, in various experimental settings, LAG3-induced inhibitory function does not require this interaction, suggesting the existence of other functional LAG3 ligands. Using a genome-scale receptor array system, we identified fibrinogen-like protein 1 (FGL1), a liver-enriched soluble protein with hepatocyte mitogenic activity and metabolic functions, as a LAG3 binding partner.

[0158] Using immunoglobulin (Ig) Fc-tagged LAG3 extracellular domain fusion proteins, a genome-scale receptor array platform (RAP) was used to search for new LAG3 binding proteins ( Figure 1 Panel a). RAP is a semi-automated large-scale gene expression system in which individual human cDNAs encoding transmembrane and soluble proteins can be transiently overexpressed on the surface of 293T cells (Yao, S. et al. B7-h2 is acostimulatory ligand for CD28 in human. Immunity 34, 729-740, (2011)). The current form of RAP contains more than 90% of all annotated genes encoding human transmembrane (about 6,500) and soluble (about 1,300) proteins. To promote membrane expression, several immune adaptor genes (DAP10, DAP12, FcRγ and CD3ε) were engineered into 293T cells, and genes encoding soluble proteins were fused to artificial transmembrane domains so that they could be expressed on the cell surface ( Figure 1 Panel a). Furthermore, to improve efficiency, the original 384-well format was modified to a 1,536-well format. In a RAP screen, the secreted protein FGL1 was shown to be the primary binding protein for LAG3-Fc.

[0159] Example 2

[0160] The interaction appears to be conserved between human and mouse species ( Figure 1 Small picture d and Figure 1 Panel e). FGL1 / LAG3 interaction can be analyzed by flow cytometry ( Figure 2 Panel b) and Octet biolayer interferometry with approximately 1.5 nM Kd ( Figure 1 Panel d) was validated, indicating a high affinity interaction. The slow off-rates in both mouse and human binding suggest the possible stability of this interaction ( Figure 1 Small picture c and Figure 1Panel f). Other FGL1 homologs, including FGL2, and several angiopoietin family members involved in regulating Treg function did not show interaction with LAG3 (data not shown), indicating that the FGL1 / LAG3 interaction is highly specific.

[0161] We sought to identify the domains of FGL1 that bind to LAG3 and whether this influences MHC-II interactions. FGL1 is composed of a helix-helix domain (CCD) and a fibrinogen-like domain (FD) (Yamamoto, T. et al. Molecular cloning and initial characterization of a novel fibrinogen-related gene, HFREP-1. Biochem Biophys Res Commun 193, 681-687, (1993)). Through domain deletion experiments, we demonstrated that the FD, rather than the CCD, is responsible for LAG3 binding. Figure 2 Small picture a and Figure 2 Panel c). LAG3 protein consists of four Ig-like domains (D1-D4), a transmembrane domain (TM) and an intracellular domain (IC) (Huard, B. et al. Characterization of the major histocompatibility complex class II binding site on LAG-3 protein. Proc Natl Acad Sci USA 94, 5744-5749, (1997); Triebel, F. et al. LAG-3, a novel lymphocyte activation gene closely related to CD4. J Exp Med 171, 1393-1405, (1990)) Figure 2 Panel b). Deletion of the D3-D4 domain of LAG3 did not affect FGL1 binding, whereas the D1 or D2 moieties alone reduced binding ( Figure 2 Small picture b and Figure 2Panel d), indicating that both D1 and D2 contribute to the FGL1 / LAG3 interaction. Previous studies have demonstrated that a single point mutation (Y73F) in the C' chain of the D1 domain of mouse LAG3 (corresponding to Y77F in human LAG3) disrupts MHC class II binding (Huard, B. et al. Characterization of the major histocompatibility complex class II binding site on LAG-3 protein. Proc Natl Acad Sci USA 94, 5744-5749, (1997); Workman, CJ, Dugger, KJ & Vignali, D. A., Couting edge: molecular analysis of the negative regulatory function of lymphocyte activation gene-3. J Immunol 169, 5392-5395, (2002)). Interestingly, we found that FGL1-Fc bound to the LAG3 Y73F mutant as strongly as to LAG3 WT ( Figure 2 Small picture b and Figure 2Panel d). In addition, we utilized an anti-LAG3 mAb (clone C9B7W), which has been reported to bind to the D2 domain of mouse LAG3 without disrupting the LAG3 / MHC class II interaction (Andrews, LP, Marciscano, AE, Drake, CG & Vignali, DALAG3 (CD223) as a cancer immunotherapy target. Immunol Rev 276, 80-96, (2017); Workman, CJ, Rice, DS, Dugger, KJ, Kurschner, C. & Vignali, DAPhenotypic analysis of themurine CD4-related glycoprotein, CD223 (LAG-3). Eur J Immunol 32, 2255-2263, (2002); Cemerski, S., Zhao, S., Chenard, M., Laskey, J., Cui, L., Shukla, R., Haines, B. ,Hsieh,E.,Beaumont,M.,Mattson,J.,Blumenschein,W.,Hirsch,H.,Fayadat-Dilman,L.,Liang,L.,De Waal Malefyt, R., T cell activation and anti-tumor efficacy of anti-LAG-3antibodies is independent of LAG-3–MHCII blocking capacity. Journal for Immunotherapy of Cancer 3(Suppl 2), 183, (2015)). In the 293T LAG3 + After preincubation of cells with C9B7W, we found that FGL1 / LAG3 binding was completely abolished ( Figure 1 Panel g). These data indicate that FGL1 interacts with LAG3 in a large region involving both D1 and D2.

[0162] To directly test whether FGL1 and MHC class II compete for LAG3 binding, we cultured LAG3+ cells with MHC class II I-Ab fusion proteins in combination with high concentrations of FGL1 (up to 100-fold increase), and no significant competition was observed (data not shown). These data suggest that FGL1 interacts with LAG3 through the fibrinogen C-terminal region in a large region involving both D1 and D2, which is not redundant for MHC class II binding.

[0163] Example 3

[0164] In a competition assay, we further determined whether existing monoclonal anti-LAG3 antibodies blocked the FGL1-LAG3 interaction. Human LAG3-expressing CHO cells or primary T cells activated with anti-CD3 antibodies to induce LAG3 expression were stained with fluorescently labeled FGL1 (1 ug / ml FGL-A647) in the presence or absence of anti-LAG3 antibodies (10 ug / ml anti-LAG3-A488 antibody; R&D Systems) to determine whether they competed with FGL1 for binding to LAG3. In the presence of anti-LAG3 antibodies, FGL1 binding was not reduced (data not shown), thus appearing to not interfere with the FGL1:LAG3 interaction.

[0165] Example 4

[0166] Next, we analyzed the effects of the FGL1 / LAG3 interaction on T cells in vitro. After activation, LAG3 is highly expressed on T cells. By flow cytometry analysis, the FGL1-Fc fusion protein bound to activated T cells from WT mice but not from LAG3KO mice (data not shown). FGL1-Fc inhibited splenic T cell proliferation after anti-CD3 stimulation, but this inhibitory activity was lost in LAG3-KO splenocytes ( Figure 3 Panel a). Similarly, FGL1 fusion protein was able to inhibit antigen-specific activation of the murine 3A9-LAG3+ T cell line in a dose-dependent manner ( Figure 3 Panel b). Thus, FGL1 inhibits T cell proliferation and function in vitro. To further demonstrate the dependence of the FGL1 / LAG3 pathway on T cell suppression, we generated a specific anti-FGL1 mAb (clone 177R4), which blocked the binding of the FGL1 fusion protein to LAG3-expressing 293T cells in a manner similar to anti-LAG3 ( Figure 3 Panel c). Both antibodies abolished the inhibitory effect of FGL1 on IL-2 production in 3A9-LAG3 cells ( Figure 2 Panel d) and promoted antigen-specific T cell activation in vivo, as determined by elevated plasma levels of IFN-γ and TNF-α ( Figure 4 Small picture a and Figure 4 Panel b). Our results support that FGL1 and LAG3 function as a receptor / ligand pair that negatively regulates T cell responses.

[0167] Similar results were obtained in activated human primary T cells. When stimulated with anti-CD3 antibodies at or above 1 ug / ml within 24 hours, CD4 T cells and CD8 T cells showed enhanced LAG3 expression. After anti-CD3 stimulation with 1 or 3 ug / ml, LAG3 expression was maintained for at least 72 hours, as analyzed by flow cytometry (data not shown). On day 4 after activation with anti-CD3 / anti-CD28 coated beads, LAG3 expression on activated primary T cells was further detected by FGL1-Fc staining (10 μg / ml) using T cells isolated from human PBMCs (data not shown).

[0168] To evaluate the potential role of FGL-1 in regulating the immune system, a mouse embryonic stem cell line (JM8) modified by wild-type phenotypic genes was used to generate FGL1 knockout (FGL1-KO) in a C57BL / 6 background (Pettitt, SJ et al. Agouti C57BL / 6N embryonic stem cells for mouse genetic resources. Nat Methods 6, 493-495, (2009)). In wild-type (WT) mice, FGL1 is selectively expressed in the liver but not by the immune system ( Figure 5Panel a) (Yan, J. et al. Cloning and characterization of a mouse liver-specific gene mfrep-1, up-regulated in liver regeneration. Cell Res 12, 353-361, (2002); Li, CY et al. Recombinant human hepassocin stimulates proliferation of hepatocytes in vivo and improves survival in rats with fulminant hepatic failure. Gut 59, 817-826, (2010); Liu, Z. & Ukomadu, C. Fibrinogen-like protein 1, a hepatocyte derived protein is an acute phase reactant. Biochem Biophys Res Commun 365, 729-734, (2008)). The absence of hepatic FGL-1 expression was confirmed by Western blotting and plasma FGL-1 levels were confirmed by ELISA in FGL1-KO mice compared to WT littermates (data not shown). FGL1-KO mice have an overall normal appearance, organ size and pups, indicating that FGL1 does not affect the development and growth of mice as a whole. However, FGL1-KO has a high incidence of dermatitis with obvious lymphocytes. Eight of 20 FGL1 KO mice (12 to 16 months old) developed dermatitis, while only one of 15 WT littermates developed dermatitis. Five of eight 12 to 16 month old females (rather than males) had increased anti-double-stranded DNA autoantibodies in serum (data not shown). Peripheral blood mononuclear cell analysis (Bendall, SC et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687-696, (2011)) showed a trend of higher frequencies of CD8 T cells rather than CD4 T cells or other immune subsets in FGL1 KO mice compared with FGL-1WT ( Figure 6 Small picture a and Figure 6Panel b). In contrast, T cell subsets in peripheral tissues such as the spleen or liver showed no significant differences (data not shown). These data suggest that internalized FGL1 does not affect development and growth, but may be involved in regulating physiological immune responses.

[0169] Example 5

[0170] Given the promising role of LAG3 as a target in cancer immunotherapy and the newly identified role of the FGL1 / LAG3 pathway in suppressing T cell immunity, we tested FGL1 deficiency or blockade in tumor immunity. Using the immunogenic MC38 mouse colon cancer model, we found that mice deficient in FGL1 or LAG3 were resistant to transplanted tumors in the majority of KO mice (P < 0.05) at day 35, compared to tumor growth in all wild-type (WT) mice. Figure 7 Panel a). While all WT mice reached the endpoint (mean tumor diameter of 15 mm) within 60 days, approximately 50% of FGL1-KO or LAG3-KO mice were tumor-free after 150 days ( Figure 7 Panel b). Anti-FGL1 and anti-LAG3 (clone C9B7W) mAbs also significantly inhibited the growth of MC38 murine colon carcinoma subcutaneously implanted in syngeneic C57BL / 6 mice ( Figure 7 Panel c) and Hepa1-6 murine hepatocarcinoma cell lines (data not shown). In the MC38 model, depletion of CD8+ or CD4+ T cells by specific mAbs abolished the antitumor effects of both FGL1 and LAG3 mAbs (data not shown), indicating that the antitumor effects were dependent on T cells. By analysis of tumor-infiltrating leukocytes (TILs) by mass cytometry, we observed that the absolute numbers of leukocytes (CD45+ cells), CD8 and CD4 T cells per mg of tumor were significantly increased in treated (anti-FGL1 or anti-LAG3) mice compared to controls ( Figure 7 Panel d). Moreover, a significant increase in the effector memory T cell population was observed in the tumors of treated mice compared to control mice, but not in the tumor-draining lymph nodes and spleen (data not shown). When characterizing the functional changes in TILs following FGL1 deficiency or FGL1 blockade, significant increases in functional markers (granzyme B, CD44, Ly6C, FAS) and a decrease in LAG-3 were observed in both FGL-1KO-treated and anti-FGL1-treated mice compared to controls ( Figure 7 Small figure e).

[0171] To rigorously exclude the role of one or more other LAG3 ligands, we tested the antitumor effect of C9B7W in FGL1 KO mice. Although anti-LAG3 mAb (C9B7W) significantly slowed MC38 tumor growth in WT mice, this effect was abolished in FGL1 KO mice ( Figure 7 Panel f). Thus, the antitumor effect of the anti-LAG3 mAb C9B7W is mediated by blocking FGL1 rather than MHC class II or other LAG3 ligands. In addition, we found that the effect of anti-FGL1 is also dependent on LAG3, as this antibody fails to confer tumor resistance in LAG3-KO mice ( Figure 7 Panel g). Thus, our findings support a critical role for FGL1 / LAG3 as a functional and interdependent pair in the control of anti-tumor immunity.

[0172] Example 6

[0173] Given the relevance of FGL1 / LAG3 in mouse cancer models, we explored the potential of FGL1 as a novel anti-tumor target in the clinical setting. Figure 8 Panel a) and a recently reported human proteome analysis (Kim, MS et al. A draft map of the human proteome. Nature 509, 575-581, (2014)), FGL1 expression in healthy individuals is largely restricted to the liver. Comprehensive analysis of the Oncomine database revealed that FGL1 mRNA was upregulated in several solid tumors, with the highest expression in the lung cancer dataset (8 / 23 or 35%) ( Figure 8 Panel b). In addition, FGL1 was confirmed to be one of the most upregulated genes in the TCGA lung adenocarcinoma database and in many other cancers except liver cancer ( Figure 8 Panel c). To reliably measure FGL1, we validated the immunofluorescence assay using 293T cells transfected with FGL1 and tumor and normal lung tissue for establishing threshold expression cutpoints. FGL1 expression was then analyzed by multiplex quantitative immunofluorescence (QIF) in 275 non-small cell lung cancer (NSCLC) cases, 30 of which were paired with normal lung tissue. FGL1 expression was restricted to the tumor cell compartment, with minimal expression in the stroma and negative staining in normal paired lung tissue (data not shown). In all lung cancer cases, we found positive staining in approximately 15% of the specimens ( Figure 9 Panel a), and tumors with high FGL1 expression in tumor biopsies were associated with a poorer prognosis ( Figure 9 In addition, we also conducted two independent groups (Group 1, from the University of Navarra ( Figure 9Panel c), and cohort 2, from Fujian Medical University (data not shown), found that plasma FGL1 levels in NSCLC patients were higher than those in healthy donors, as analyzed by ELISA. However, there was no difference in plasma FGL1 levels between patients with or without liver damage (ALT ≥ 40 U / L; ALT < 40 U / L) and between patients with or without metastasis (data not shown).

[0174] Example 7

[0175] We further tested whether the FGL1 / LAG-3 pathway could be an alternative inhibitory pathway acting independently or in synergy with the B7-H1 (PD-L1) / PD-1 pathway in the tumor microenvironment. Therefore, we analyzed the correlation between baseline plasma FGL1 levels and the efficacy of anti-PD-1 therapy in patients with metastatic NSCLC (cohort 1). In patients treated with anti-PD-1 mAb, higher FGL1 expression was associated with poor overall survival ( Figure 9 Panel d). Similar results were observed in an independent cohort of patients with metastatic melanoma treated with anti-PD-1 mAb (cohort 3, from Yale University) ( Figure 9 Small figure e).

[0176] We further tested the effects of anti-FGL1 / anti-LAG3 in the context of anti-B7-H1 blockade using the MC38 tumor model. Single-agent anti-B7-H1 mAb slowed tumor growth and improved survival, with death occurring within 90 days ( Figure 10 Panel a). Similarly, single-agent anti-FGL1 or anti-LAG3 mAb treatment failed to prolong survival beyond 3 months. Surprisingly, the combination of anti-B7-H1 mAb with anti-FGL1 or anti-LAG3 mAb significantly improved survival compared to single-agent therapy ( Figure 10 Panel a) and tumor burden ( Figure 10 In addition, more than 30% of the mice remained tumor-free for more than 150 days ( Figure 10 Panel a). Together, these data support a role for the FGL1 / LAG-3 pathway as a primary resistance mechanism in cancer patients treated with anti-PD1 / PDL1 therapy. Thus, anti-FGL1 or anti-LAG3 blocking antibodies enhance T cell-mediated tumor immunity and inhibit tumor growth in several mouse models in a receptor-ligand interdependent manner and synergize with B7-H1 (PD-L1) / PD-1 blockade therapy.

[0177] Summarize

[0178] Known anti-LAG3 therapies target the interaction with its only known ligand, class II MHC. The inventors have proposed an alternative, independent LAG3 pathway involving FGL1 that is potentially important for cancer immunotherapy. FGL1 inhibits T cell responses, and (antagonistic) anti-FGL1 or anti-LAG3 blocking antibodies impair tumor growth. Although FGL1 expression is limited in normal tissues, it is highly expressed in several human solid cancers, including lung cancer and melanoma, and has important prognostic value. Given the high expression of FGL1 in patients with primary resistance to anti-PD1 / PD-L1 therapy, the FGL1 / LAG3 pathway may be a potential immune escape mechanism. In addition, the data suggest that FGL1 expression has prognostic value as a biomarker for successful treatment with drugs that interfere with the FGL1 / LAG3 immune checkpoint pathway, as well as as a biomarker for predicting anti-PD-1 response, predicting poor prognosis for anti-PD1 / PD-L1 therapy and potentially other checkpoint inhibitory molecules.

[0179] In summary, the present invention includes but is not limited to the following:

[0180] 1. A method for treating cancer in a human patient, comprising administering to the patient an effective amount of an antibody that specifically inhibits the interaction between fibrinogen-like protein 1 (FGL1) and lymphocyte activation gene 3 protein (LAG3).

[0181] 2. A method for treating cancer in a human patient according to claim 1, comprising administering to the patient an effective amount of the antibody that specifically inhibits the interaction of FGL1 and LAG3, wherein the cancer patient has increased FGL1 expression.

[0182] 3. The method according to item 1, wherein the cancer patient has been determined to have increased FGL1 expression.

[0183] 4. The method of claim 3, wherein the sample from the patient has been determined to have increased FGL1

[0184] Express.

[0185] 5. The method according to claim 1, comprising

[0186] a) determining whether FGL1 expression is increased in a sample from said patient, and

[0187] b) if FGL1 expression is increased, administering to said patient an effective amount of said antibody that specifically inhibits the interaction of FGL1 with LAG3.

[0188] 6. The method according to claim 1, comprising

[0189] a) identifying patients in need of cancer treatment, and

[0190] b) determining that the patient has increased FGL1 expression, thereafter

[0191] c) administering to the patient an effective amount of the antibody that specifically inhibits the interaction between FGL1 and LAG3.

[0192] 7. A method for selecting a therapy for a cancer patient, the therapy comprising an antibody that specifically inhibits the interaction of FGL1 and LAG3, the method comprising determining the level of FGL1 expression in a sample from the patient, and selecting the therapy for the patient when the level of FGL1 expression is increased.

[0193] 8. The method of any one of items 2 to 7, wherein the FGL1 expression is increased compared to a control.

[0194] 9. A method of treating cancer in a human patient, the method comprising

[0195] a) providing a sample from said patient,

[0196] b) measuring the expression level of FGL1 in the sample,

[0197] c) comparing FGL1 expression to controls,

[0198] d) identifying said patient as likely to respond to treatment with at least one antibody that specifically inhibits the interaction of FGL1 with LAG3 when the level of FGL1 expression has been measured as increased compared to said control, and

[0199] e) administering to the patient an effective amount of the antibody that specifically inhibits the interaction between FGL1 and LAG3.

[0200] 10. The method according to any one of items 1 to 9, wherein the antibody that specifically inhibits the interaction of FGL1 and LAG3 is an anti-FGL1 antibody or an anti-LAG3 antibody.

[0201] 11. A method for treating cancer in a human patient, comprising administering to the patient an effective amount of an antibody that binds to human FGL1.

[0202] 12. The method according to any one of items 1 to 11, wherein the method comprises administering an anti-PD-1

[0203] At least one additional immune checkpoint inhibitory antibody selected from the group consisting of anti-PD-L1 antibody, anti-IDO antibody, anti-CTLA-4 antibody, anti-Tim-3 antibody, anti-GITR antibody, anti-VISTA antibody, anti-BTLA antibody, and anti-OX40 antibody.

[0204] 13. The method according to any one of items 1 to 12, wherein the patient has

[0205] (i) primary or acquired resistance to at least one immune checkpoint inhibitory antibody selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-IDO antibody, anti-CTLA-4 antibody, anti-Tim-3 antibody, anti-GITR antibody, anti-VISTA antibody, anti-BTLA antibody and anti-OX40 antibody, or

[0206] (ii) wherein the patient has primary or acquired resistance to PD-1 / PD-L1 checkpoint inhibition.

[0207] 14. The method according to any one of items 1 to 13, wherein the cancer is brain cancer, colorectal cancer, melanoma, prostate cancer or lung cancer, preferably non-small cell lung cancer (NSCLC).

[0208] 15. The method of any one of items 1 to 14, wherein the treatment is further accompanied by chemotherapy or radiotherapy.

[0209] 16. A method for evaluating cancer patients for treatment with immune checkpoint inhibitory antibodies, preferably specifically inhibiting FGL1

[0210] A method for treating a cell with an antibody that interacts with LAG3, the method comprising

[0211] (a) providing a sample from said patient,

[0212] (b) detecting FGL1 expression in the sample, and

[0213] (c) comparing the FGL1 expression determined in step (b) with a control, wherein the FGL1 expression in the sample is increased compared to the control.

[0214] 17. A method for classifying a tumor in a patient, the method comprising

[0215] (a) providing a sample from said patient,

[0216] (b) detecting FGL1 expression in the sample, and

[0217] (c) identifying the tumor as a candidate for treatment with an immune checkpoint inhibitory antibody, preferably an antibody that specifically inhibits the interaction of FGL1 and LAG3.

[0218] 18. The method of claim 16 or 17, wherein the method is an in vitro method.

[0219] 19. A method for predicting responsiveness in a human cancer patient, the method comprising

[0220] (a) providing a sample from said patient,

[0221] (b) determining the expression level of FGL1 in the sample,

[0222] (c) FGL1 expression was compared with the control,

[0223] (d) identifying the patient as likely to respond to treatment with an immune checkpoint inhibitory antibody, preferably an antibody that specifically inhibits the interaction of FGL1 and LAG3, when it has been determined that the level of FGL1 expression is increased compared to the control, or identifying the patient as unlikely to respond when it has been determined that FGL1 expression is not increased compared to the control; and optionally

[0224] (e) further comprising the step of administering to the cancer patient an immune checkpoint inhibitory antibody, preferably an antibody that specifically inhibits the interaction between FGL1 and LAG3.

[0225] 20. A method of treating cancer in a human patient, comprising administering to the patient an effective amount of at least one immune checkpoint inhibitory antibody, wherein the cancer patient has increased

[0226] FGL1 expression.

[0227] 21. A method for screening immune checkpoint inhibitory antibodies, the method comprising

[0228] (a) providing an FGL1 protein, preferably comprising at least the fibrinogen of human FGL1

[0229] domain;

[0230] (b) providing a LAG3 protein, preferably comprising at least domains 1 and 2 of human LAG3;

[0231] (c) contacting the FGL1 protein with the LAG3 protein in the presence of an antibody to be screened;

[0233] (d) determining the binding of the FGL1 protein to the LAG3 protein; and

[0234] (e) if the binding of the FGL1 protein to the LAG3 protein is reduced as compared to the binding of the FGL1 protein to the LAG3 protein in the absence of the antibody to be screened, the antibody to be screened is identified as an antibody that specifically inhibits the interaction between the FGL1 protein and the LAG3 protein.

[0235] 22. A method for screening small molecules that inhibit the binding of FGL1 to LAG3, the method comprising: a) providing an FGL1 protein, wherein the FGL1 protein preferably comprises at least the fibrinogen of human FGL1;

[0236] domain;

[0237] (b) providing a LAG3 protein, preferably comprising at least domains 1 and 2 of human LAG3;

[0238] (c) contacting said FGL1 protein with said LAG3 protein in the presence of a small molecule, e.g., from a compound library, in a format, e.g., for high throughput screening,

[0239] (d) determining the binding of the FGL1 protein to the LAG3 protein in the presence and absence of the small molecule; and

[0240] (e) identifying a small molecule that specifically inhibits the binding of the FGL1 protein to the LAG3 protein as an FGL1-LAG3 checkpoint inhibitor molecule.

[0241] 23. A kit for selecting cancer patients who will benefit from immune checkpoint inhibitory antibodies, preferably antibodies that specifically inhibit the interaction between FGL1 and LAG3, wherein the kit

[0242] Include

[0243] (a) means for quantitatively detecting FGL1 expression in a sample from a cancer patient, and

[0244] (b) Control.

[0245] 24. The kit according to item 23, wherein the control is selected from:

[0246] (a) Reference samples for detecting enhanced FGL1 expression,

[0247] (b) Reference samples for detecting baseline FGL1 expression,

[0248] (c) comprises a description of a predetermined reference level of FGL1 expression, said predetermined reference level having been correlated with sensitivity to an antibody that specifically inhibits the interaction of FGL1 with LAG3,

[0249] (d) comprises a description of a predetermined reference level of FGL1 expression, said predetermined reference level having been correlated with insensitivity to an antibody that specifically inhibits the interaction of FGL1 with LAG3, and / or

[0250] (e) A combination of (a), (b), (c) or (d).

[0251] 25. The kit according to item 23, wherein the means for quantitatively detecting FGL1 expression in a sample from a cancer patient is

[0252] (a) an antibody specific for FGL1,

[0253] (b) a primer pair specific for FGL1 and optionally a probe that hybridizes to the amplified product, and / or

[0254] (c) Probe hybridized to the FGL1 target sequence.

[0255] 26. An antibody that binds to human FGL1, wherein the antibody inhibits the binding of human FGL1 to human LAG3.

[0256] 27. An antibody that binds to human LAG3, wherein the antibody inhibits binding of human LAG3 to human FGL1.

[0257] 28. The antibody of item 27, wherein the antibody further inhibits the interaction of human LAG3 with human MHC class II molecules.

[0258] 29. An anti-FGL1 antibody for use in the treatment of cancer.

[0259] 30. An anti-FGL1 antibody for use in the treatment of cancer according to claim 29, wherein the cancer disease is selected from solid tumors, blood cancers and metastatic lesions, more specifically selected from leukemias, lymphomas (including Hodgkin's lymphoma), myelomas (including multiple myeloma), sarcomas and carcinomas, including adenocarcinomas, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, brain cancer, colorectal cancer, melanoma, prostate cancer, head and neck cancer, recurrent or metastatic head and neck squamous cell carcinoma, squamous cell lung cancer, lung adenocarcinoma, squamous cell cervical cancer, gastric cancer, esophageal cancer and thyroid cancer.

[0260] 31. The anti-FGL1 antibody of item 29 or item 30, wherein the antibody is a human or humanized antibody.

[0261] 32. The anti-FGL1 antibody according to item 29 or item 30, wherein the cancer patient to be treated with the anti-FGL1 antibody has increased FGL1 expression.

[0262] 33. The anti-FGL1 antibody according to item 32, wherein the cancer patient has been determined to have increased

[0263] FGL1 expression.

[0264] 34. The anti-FGL1 antibody according to item 33, wherein the sample from the cancer patient has been determined to have increased FGL1 expression.

[0265] 35. The anti-FGL1 antibody according to item 34, wherein the treatment comprises

[0266] (a) determining whether FGL1 expression is increased in a sample from the patient, and

[0267] (b) if FGL1 expression is increased, administering an effective amount of the anti-FGL1 antibody to the patient.

[0268] 36. A method of treating cancer in a human patient, the method comprising

[0269] a) providing a sample from said patient,

[0270] b) measuring the expression level of FGL1 in the sample,

[0271] c) comparing FGL1 expression to controls,

[0272] d) identifying said patient as likely to respond to treatment with an anti-FGL1 antibody when it has been determined that the level of FGL1 expression is increased compared to said control, and

[0273] e) administering to the patient an effective amount of an anti-FGL1 antibody.

Claims

1. A method for treating cancer in a human patient, the method comprising administering to the patient an effective amount of an antibody that specifically inhibits the interaction between fibrinogen-like protein 1 (FGL1) and lymphocyte activation gene 3 protein (LAG3).

2. A method for treating cancer in a human patient according to claim 1, comprising administering to the patient an effective amount of the antibody that specifically inhibits the interaction of FGL1 and LAG3, wherein the cancer patient has increased FGL1 expression.

3. The method of claim 1, wherein the cancer patient has been determined to have increased FGL1 expression. The method of claim 3 , wherein the sample from the patient has been determined to have increased expression of FGL1.

5. The method according to claim 1, comprising: a) determining whether FGL1 expression is increased in a sample from said patient, and b) if FGL1 expression is increased, administering to said patient an effective amount of said antibody that specifically inhibits the interaction of FGL1 and LAG3.

6. The method according to claim 1, comprising: a) identifying patients in need of cancer treatment, and b) determining that the patient has increased FGL1 expression, thereafter c) administering to the patient an effective amount of the antibody that specifically inhibits the interaction between FGL1 and LAG3.

7. A method for selecting a therapy for a cancer patient, the therapy comprising an antibody that specifically inhibits the interaction of FGL1 and LAG3, the method comprising determining the level of FGL1 expression in a sample from the patient, and selecting the therapy for the patient when the level of FGL1 expression is increased.

8. The method according to any one of claims 2 to 7, wherein the FGL1 expression is increased compared to a control.

9. A method of treating cancer in a human patient, the method comprising a) providing a sample from said patient, b) measuring the expression level of FGL1 in the sample, c) Comparison of FGL1 expression with controls, d) identifying said patient as likely to respond to treatment with at least one antibody that specifically inhibits the interaction of FGL1 with LAG3 when the level of FGL1 expression has been measured as increased compared to said control, and e) administering to the patient an effective amount of the antibody that specifically inhibits the interaction between FGL1 and LAG3. 10 . The method according to claim 1 , wherein the antibody that specifically inhibits the interaction of FGL1 and LAG3 is an anti-FGL1 antibody or an anti-LAG3 antibody.

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