Application of LOXL2 as tumor immunotherapy drug target

Abstract

The invention belongs to the field of medicines, and particularly relates to improvement of sensitivity of tumors to immunotherapy by targeted inhibition of LOXL2. The invention reveals that inhibition of LOXL2 can promote T cell infiltration, inhibit tumor metastasis and significantly enhance the T cell treatment effect. The invention provides an LOXL2 inhibitor and an immunotherapy pharmaceutical composition, which can effectively improve the curative effect of tumor immunotherapy, especially in the treatment of metastatic liver cancer and lung cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an application of LOXL2 as a drug target for tumor immunotherapy. Background Art

[0002] Currently, the treatment of tumor metastasis and post-transplant recurrence remains a significant challenge. According to statistics, approximately 30%-40% of cancer patients experience metastasis or recurrence after treatment, with metastasis and recurrence being particularly common in malignant tumors such as lung cancer, breast cancer, and liver cancer. Clinical data show that the recurrence rate after chemotherapy remains high at over 50%, and while immunotherapy has shown some effectiveness, the long-term control rate remains below 30%.

[0003] At the same time, with the promotion of targeted drugs, the survival of some patients has been extended, but the overall efficacy is limited and the side effects are severe. Therefore, precise treatment options for metastatic and recurrent tumors are urgently needed to improve patients' survival rate and quality of life.

[0004] Immunotherapy, represented by immune checkpoint inhibitors (ICIs), has greatly improved the clinical efficacy of malignant tumor treatment. Although ICIs have demonstrated clinical activity across a wide range of tumor types, a significant proportion of patients remain unresponsive to ICI therapy. In recent years, the success of CAR-T cell infusion in the treatment of leukemia and lymphoma has demonstrated the importance of T cell therapy in anti-tumor immunity.

[0005] However, T cell therapy has been ineffective in treating solid tumors, a key reason being the suppressive microenvironment of solid tumors. This immunosuppressive microenvironment, including dense stroma and immunosuppressive cells and factors, prevents T cells from effectively activating and infiltrating these “cold tumors.”

[0006] Lysyl oxidase-like protein 2 (LOXL2) belongs to the lysyl oxidase (LOX) family and primarily acts as an extracellular enzyme involved in extracellular matrix (ECM) remodeling. It also has a variety of non-enzyme-dependent functions, including regulation of epithelial-mesenchymal transition (EMT), chromatin modification, and metabolic reprogramming. Clinical studies have shown that LOXL2 overexpression is associated with a variety of cancers. However, clinical trials targeting LOXL2 in combination with chemotherapy drugs have failed in the treatment of various cancers (Adv. Ther. 2020, 3, 1900119; Oncologist 2017, 22, 241; Oncologist 2017, 22, e23.).

[0007] There are studies both domestically and internationally that have reported targeting LOXL2 in combination with PD-1 for tumor treatment (e.g., CN109312408 and US11555029B2), but there is no data on metastasis or post-transplant recurrence. Treatment for these metastases and post-transplant recurrence is a pressing challenge that needs to be addressed. Summary of the Invention

[0008] The present invention aims to solve at least one of the above technical problems. To this end, the present invention has found that targeted inhibition of LOXL2 can promote T cell infiltration, reduce the metastasis of various tumors, and significantly improve the efficacy of T cell therapy.

[0009] The first aspect of the present invention aims to provide a use of targeting LOXL2 in the preparation of a drug for promoting T cell infiltration in the microenvironment of a solid tumor.

[0010] Preferably, the solid tumor is a metastasis, more preferably a metastasis of lung cancer or / and liver cancer, and most preferably lung cancer.

[0011] Preferably, the solid tumor is lung metastasis of breast cancer, lung metastasis of lung cancer and / or liver metastasis of pancreatic cancer.

[0012] The second aspect is to provide a pharmaceutical composition for enhancing the sensitivity of tumors to immunotherapy, comprising a LOXL2 inhibitor and an immunotherapy drug.

[0013] Preferably, the tumor is lung cancer and / or liver cancer, preferably a metastasis, more preferably a metastasis of lung cancer and / or liver cancer, and most preferably lung cancer.

[0014] Preferably, the tumor is a lung metastasis of breast cancer, a lung metastasis of lung cancer and / or a liver metastasis of pancreatic cancer.

[0015] Preferably, immunotherapy includes but is not limited to T cell therapy and / or immune checkpoint therapy.

[0016] More preferably, T cell therapy comprises TCR-T, CAR-T and / or TIL; immune checkpoint therapy comprises anti-PD1, PD-L1 and / or CTLA-4.

[0017] More preferably, the T cell therapy is CAR-T therapy.

[0018] Preferably, the LOXL2 inhibitor is PSX5505.

[0019] The third aspect aims to provide a use of a LOXL2 inhibitor combined with immunotherapy in the preparation of a drug for increasing the sensitivity of tumors to immunotherapy.

[0020] Preferably, the tumor is lung cancer and / or liver cancer, preferably a metastasis, more preferably a metastasis of lung cancer and / or liver cancer, and most preferably lung cancer.

[0021] Preferably, the tumor is a lung metastasis of breast cancer, a lung metastasis of lung cancer and / or a liver metastasis of pancreatic cancer.

[0022] Preferably, immunotherapy includes but is not limited to immune checkpoint therapy and / or T cell therapy.

[0023] More preferably, T cell therapy comprises TCR-T, CAR-T and / or TIL; immune checkpoint therapy comprises anti-PD1, PD-L1 and / or CTLA-4.

[0024] More preferably, the T cell therapy is CAR-T therapy.

[0025] Preferably, the LOXL2 inhibitor is PSX5505. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shown is the relationship between LOXL2 expression and T cell infiltration in patients analyzed from clinical data.

[0027] Figure 2 As shown, KP cells of the control group (Vector), Nf2 knockout (NKO), and Nf2 and LOXL2 double knockout group (NL-DKO) were injected into C57BL / 6 cells via the tail vein to form lung metastases, and intratumoral CD8 and CD11b staining analysis was performed on Day 12.

[0028] Figure 3 It shows that Figure 2 Statistical results of KP cells; the control group is Ctrl, the Nf2 knockout group is NKO, and the Nf2 and LOXL2 double knockout group is NL-DKO. The left panel shows CD11b, and the right panel shows CD8.

[0029] Figure 4 The figure shows the results of proteomic analysis of samples from patients with liver cancer metastasis; patients with liver cancer who received liver transplantation were divided into a LOXL2 protein high expression group (n=19) and a LOXL2 low expression group (n=69).

[0030] Figure 5 The results show that the recurrence of hepatocellular carcinoma after liver transplantation is correlated with the expression of LOXL2 in the tumor; n=17 were high expression of LOXL2 (LOXL2-H), and n=62 were low expression of LOXL2 (LOXL2-L).

[0031] Figure 6The data show that the survival of patients with hepatocellular carcinoma who underwent liver transplantation was correlated with the expression of LOXL2; n=19 were high expression of LOXL2 (LOXL2-H), and n=69 were low expression of LOXL2 (LOXL2-L).

[0032] Figure 7 Clinical data analysis shows the correlation between the level of LOXL2 expression in patients and their survival time after anti-PD-1 treatment.

[0033] Figure 8 The data show that, based on clinical data analysis, high expression of LOXL2 is correlated with immunotherapy responsiveness.

[0034] Figure 9 Shown are the overall survival curves of cancer patients receiving anti-PD-1 treatment from the KMPlot database, The Cancer Genome Atlas (TCGA), divided into LOXL2 high expression group (n=105) and LOXL2 low expression group (n=306).

[0035] Figure 10 、 11 It shows that based on Figure 9 We analyzed data from multiple clinical immune checkpoint immunotherapy datasets to assess the activity of extracellular matrix containing collagen ( Figure 10 ) and LOXL2 plus collagen gene expression signatures ( Figure 11 ) and the response to checkpoint immunotherapy (R, Response; NR, Non-Response).

[0036] Figure 12 Shown are Kaplan-Meier survival curves of mice injected with LOXL2-KO or control cells (Vector) and treated with PBS or OT-I CD8+ T cells (ACT) (n=5) on day 7. The cell line is KP.

[0037] Figure 13 Shown are C57BL / 6 mice that were intravenously injected with vector (E0771-OVA-Vector) or LOXL2 knockout group (LOXL2-KO E0771-OVA) and treated with PBS or OT-I CD8+ T cells on day 18 (n=6); bioluminescent images of lung metastases were generated 2 days after treatment; cell line E0771.

[0038] Figure 14Shown are Kaplan-Meier survival curves of mice injected with LOXL2 knockout (LKO) or control (Vector) E0771 cell lines and treated with PBS or OT-I CD8+ T cells (ACT) 18 days later (PBS-treated LKO group, n=6, other groups n=7).

[0039] Figure 15 Shown are Kaplan-Meier survival curves of NSG mice injected with LOXL2 knockout (LKO) or vector control (WT) A549 cells and treated with PBS or HER2 CAR-T cells (ACT) on day 28.

[0040] Figure 16 Shown are bioluminescent images of lung metastases generated by MDA-MB-231-CD19 (M19) cancer cells in NSG mice 3 days after PBS or CD19-CAR-T (CAR-T) treatment; LOXL2 knockout group (M19-LOXL2-KD), control group (M19-Vector), n=5.

[0041] Figure 17 It shows that Figure 16 Quantization diagram of .

[0042] Figure 18 It is shown that Figure 16 Kaplan-Meier survival curve of experimental mice.

[0043] Figure 19 The figure shows that mice were injected with LOXL2 inhibitor (PXS-5505, LOX-IN-3 dihydrochloride, product number T39986, produced by TargetMol Chemicals Inc) and DMSO (control group), and treated with PBS or CD19-CAR-T (CAR-T) on the 29th day after tumor cell injection. Bioluminescent images of lung metastasis produced by cancer cells were detected 3 days later (DMSO combined with PBS group, 8 mice; PXS-5505 combined with ACT group, 8 mice; DMSO combined with ACT group, 7 mice; PXS-5505 combined with PBS group, 7 mice).

[0044] Figure 20 It is shown that Figure 19 Quantification diagram, D is DMSO group, X is PXS5505 group, PBS is PBS treatment group, ACT is CAR-T treatment group.

[0045] Figure 21 It is shown that Figure 19 Kaplan-Meier survival curve of experimental mice, PXS is PXS5505. DETAILED DESCRIPTION

[0046] the term

[0047] The terms used herein have the meanings commonly understood by persons of ordinary skill in the relevant art. However, to facilitate a better understanding of the present invention, some definitions and explanations of related terms are provided below. It should be noted that the explanations of terms provided herein are intended solely to facilitate a better understanding of the present invention by those skilled in the art and are not intended to limit the present invention.

[0048] The tumor microenvironment (TME) is the environment surrounding a tumor. The TME is composed of tumor cells, tumor stromal cells (including stromal fibroblasts), endothelial cells, and immune cells (such as microglia, macrophages, and lymphocytes), as well as non-cellular components of the extracellular matrix, such as collagen, fibronectin, hyaluronic acid, and laminin.

[0049] Immune checkpoint therapy is one of the most significant breakthroughs in cancer treatment in recent years. It blocks inhibitory signals that tumors use to evade the immune system, reactivating the body's own T cells to attack cancer. Currently, immune checkpoint inhibitors (ICIs) are the most commonly used immunotherapy option. As an immunosuppressive pathway, they are crucial for maintaining autoimmune tolerance and regulating the duration and scope of immune responses in peripheral tissues.

[0050] Currently used immune checkpoint inhibitors include programmed cell death 1 (PD-1) and its ligand (PD-L1), anti-PD1, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0051] T cell therapy is a type of immunotherapy that enhances the body's immune cells to attack tumors. Current T cell infusion therapies include tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor T cells (CAR-T), and T cell receptor-engineered T cells (TCR-T).

[0052] Tumor suppressor genes (TSGs) are a class of key genes that regulate cell growth, proliferation, and apoptosis. The loss or inactivation of their function may lead to uncontrolled cell proliferation and promote tumorigenesis.

[0053] The Hippo-YAP pathway is a highly conserved signaling pathway that primarily regulates organ size, tissue regeneration, stem cell self-renewal, and tumorigenesis. Its core function is to regulate cell proliferation, apoptosis, and differentiation by controlling the activity of the effector proteins YAP / TAZ. In the Hippo-YAP pathway, Nf2, Lats1, Lats2, and Arhgap35 (Lats1, Lats2) are key players in lung metastasis, immune evasion, and resistance to T cell therapy.

[0054] Targeting LOXL2 promotes T cell infiltration in lung metastases and the clinical relevance of LOXL2

[0055] It is hypothesized that LOXL2 promotes the polarization of immunosuppressive macrophages and restricts T cell infiltration by cross-linking collagen and stiffening the extracellular matrix (ECM). Clinical datasets have shown that the proportion of tumor-infiltrating T cells is negatively correlated with LOXL2 expression (Dataset: PRJEB25780, GSE91061, GSE78220; Clinical datasets: hugo2016, kim2018, riaz2017_naive, prat2017). Figure 1 ).

[0056] To test whether inhibition of LOXL2 would abrogate the rejection of T cell therapy in the tumor microenvironment, we performed immunofluorescence staining of lung metastases formed by control (Vector or Ctrl), Nf2 knockout (Nf2-KO), and Nf2LOXL2 double knockout (NL-DKO) KP cells. Lung metastases were formed by tail vein injection into C57BL / 6 mice, and intratumoral CD8 and CD11b staining were analyzed on Day 12.

[0057] Figure 2 It can be seen that compared with the control group, the CD8 of Nf2-KO tumors are mainly distributed in the periphery; while after LOXL2 knockout, the T cells of NL-DKO infiltrate into the tumor. The results showed that LOXL2 knockout significantly reduced the infiltration of CD11b+ myeloid cells and the restored CD8+ T cell infiltration in Nf2 knockout lung metastases ( Figure 3 ).

[0058] To verify the clinical significance of LOXL2 and further analyze the relationship between LOXL2 expression and cancer, this example focuses on the recurrence and postoperative survival rate of patients with hepatocellular carcinoma who have undergone liver transplantation. Based on proteomics, the patients were divided into a LOXL2 high expression group (LOXL2-H, High) and a LOXL2 low expression group (LOXL2-L, Low) according to protein expression. Figure 4 ), where protein expression values greater than 4 were defined as the high expression group, and protein expression values less than 4 were defined as the low expression group.

[0059] The analysis results showed that the LOXL2 high expression group had a higher recurrence rate after liver transplantation and a worse overall survival (OS) than the LOXL2 low expression group. Figure 5 、 Figure 6 ), and after anti-PD-1 treatment, the survival time of patients with high LOXL2 expression was significantly shorter ( Figure 7 ), and were significantly less responsive to immunotherapy ( Figure 8 ). This indicates that increased LOXL2 expression can lead to increased cancer recurrence rate and decreased survival rate in patients after transplantation.

[0060] Supporting analysis of the public cohort (database source: KMPlot, https: / / www.kmplot.com / ) showed that LOXL2 expression was negatively correlated with OS of patients after anti-PD1 treatment ( Figure 9 Further analysis found that the immune checkpoint therapy responder cohort had significantly lower levels of extracellular matrix (ECM) proteins containing collagen, or a combination of collagen and LOXL2 ( Figure 10 、 Figure 11 ), this finding supports that LOXL2-mediated collagen cross-linking has an immunosuppressive role in the setting of metastases and / or tumor recurrence.

[0061] Targeting LOXL2 promotes T cell therapy for lung metastatic cancer

[0062] The inventors investigated the potential of LOXL2 as a common target for enhancing immunotherapy response in lung metastasis. Different types of tumor cells were injected into C57BL / 6 mice ( Figure 12-14 ) or B-NDG( Figures 15-21 -DJ) mice developed lung metastases and were then treated with T cells ( Figure 15-18 ) or LOXL2 inhibitor (PXS-5505) combined with T cell therapy ( Figure 19-21 ) and examined the tumor progression and survival of mice.

[0063] First, LOXL2 was knocked out in a mouse triple-negative breast cancer cell line (E0771-OVA) and a lung cancer cell line (KP) that stably express OVA (ovalbumin). These cells were then injected into C57BL / 6 mice to establish a lung metastasis model of triple-negative breast cancer. The mice were then treated with PBS or HER2 CAR-T cells. The results showed that knocking out LOXL2 significantly reduced lung metastasis of cancer cells and promoted the tumor's response to T cell therapy ( Figure 12 、 13 , 14).

[0064] Secondly, Loxl2 was knocked out and knocked down in human lung cancer cell lines (A549) and triple-negative breast cancer cell lines (MDA-MB-231), respectively, and injected into B-NDG mice through the tail vein to form lung metastases. The formed lung metastases were then treated with CAR-T therapy. Bioluminescence imaging and Kaplan-Meier survival analysis showed that LOXL2 knockdown not only significantly reduced the lung metastasis of M19 cells, but also increased the sensitivity of A549 and M19 lung metastases to CAR-T therapy ( Figure 15-18 ).

[0065] Finally, LOXL2 inhibitor (PXS-5505) and T cell therapy were combined. On day 0, M19-Vec and M19-shLoxl2 were injected into NSG mice; from day 3 to day 31, Loxl2 inhibitor PXS-5505 (20 mg / kg) was continuously injected intraperitoneally; on day 29, CD19 CAR-T adoptive therapy was performed; and tumor bioluminescence imaging was performed on day 32. The results showed that inhibition of LOXL2 significantly improved the efficacy of CAR-T therapy in MDA-MB-231 lung metastatic cancer mice ( Figure 19-21 ).

[0066] These findings suggest that targeted inhibition of Loxl2 reduces lung metastasis of tumor cells and enhances the ability of immunotherapy, especially T cell therapy, to kill lung metastatic cancer.

[0067] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. Application of targeting LOXL2 in the preparation of drugs that promote T cell infiltration in the solid tumor microenvironment.

2. The use according to claim 1, characterized in that The solid tumor is a metastatic lesion.

3. The use according to claim 2, characterized in that The metastatic lesions are lung metastatic lesions and / or liver metastatic lesions.

4. The use according to claim 2, characterized in that The solid tumor is lung metastasis of breast cancer, lung metastasis of lung cancer, or liver metastasis of pancreatic cancer.

5. A pharmaceutical composition for enhancing the sensitivity of tumors to immunotherapy, characterized in that: Contains LOXL2 inhibitors and immunotherapy drugs.

6. The pharmaceutical composition according to claim 5, characterized in that The tumor is a metastatic lesion.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that The metastatic lesions are lung metastatic lesions and / or liver metastatic lesions.

8. The pharmaceutical composition according to claim 5 or 6, characterized in that The tumor is lung cancer and / or liver cancer.

9. The pharmaceutical composition according to claim 5, characterized in that The immunotherapy includes but is not limited to T cell therapy and / or immune checkpoint therapy.

10. The pharmaceutical composition according to claim 9, characterized in that The T cell therapy includes TCR-T, CAR-T and / or TIL.

11. The pharmaceutical composition according to claim 9, characterized in that The immune checkpoint therapy includes anti-PD1, PD-L1 and / or CTLA-4.

12. The pharmaceutical composition according to claim 5, characterized in that The LOXL2 inhibitor is PSX5505.

13. Use of a LOXL2 inhibitor combined with immunotherapy in the preparation of a drug for increasing the sensitivity of tumors to immunotherapy.

14. The use according to claim 13, characterized in that The tumor is a metastatic lesion.

15. The use according to claim 13, characterized in that The immunotherapy includes but is not limited to immune checkpoint therapy and / or T cell therapy.

16. The use according to claim 15, characterized in that The T cell therapy includes TCR-T, CAR-T and / or TIL.

17. The use according to claim 15, characterized in that Immune checkpoint therapy includes anti-PD1, PD-L1 and / or CTLA-4.

Images