阅读说明：本技术 抗lag-3抗体与shp-2抑制剂联合治疗肿瘤的用途 (Use of anti-LAG-3 antibodies in combination with SHP-2 inhibitors for the treatment of tumors ) 是由 马一明 李家鹏 于 2020-05-08 设计创作，主要内容包括：提供了SHP-2抑制剂与LAG-3抗体联合治疗肿瘤,尤其是非小细胞肺癌的用途。(Uses of SHP-2 inhibitors in combination with LAG-3 antibodies for the treatment of tumors, particularly non-small cell lung cancer, are provided.)

1. The present invention relates to the use of LAG-3 antibodies and SHP-2 inhibitors in the treatment of tumors.

2. Use according to claim 1, characterized in that: the LAG-3 antibody Relatimab is an antibody which blocks the binding of LAG-3 to its ligand MHCII, and the SHP-2 inhibitor SHP-099 is a small molecule compound which inhibits the activity of SHP-2.

3. The two drugs show synergistic inhibitory activity on tumors.

4. The method of claim 1, wherein LAG-3 (CD 223) is a transmembrane protein, and wherein full-length LAG-3 is expressed on the cell membrane, wherein the protein is highly expressed in activated T cells and NK cells, and wherein the protein negatively regulates the immune activity of the T cells.

5. The method of claim 1, wherein the SHP-2 is a tyrosine phosphatase, which is highly expressed in multiple tumors and is closely related to the development of tumors.

6. The invention according to claim 1 establishes the use of LAG-3 antibodies with SHP-2 inhibitors in the treatment of non-small cell lung cancer.

Technical Field

The present invention relates to the use of LAG-3 antibodies and SHP-2 inhibitors in the treatment of tumors.

Background

Lymphocyte activation gene 3 (1 ymphocyte activation gene-3, LAG-3, CD 223) belongs to immunoglobulin superfamily (IgSF) members, and is an immune negative regulator. It is highly expressed mainly on activated T cells and NK cells. Studies have shown that the intracellular segment of LAG-3 is involved in down-regulating the CD3/TCR activation pathway (Iouzalen et al, Eur. J. Immunol. 2001, 2885-2891) and/or in the process of LAG-3 transport to the T cell plasma membrane (Bae et al, J. Immunol. 2014, 193: 3101-3112) and/or in the regulation of STAT5 downstream signaling in activated T cells (Durham et al, Plos One 2014, 9: e 109080). Presently discovered ligands for LAG-3 include MHCII (Workman et al, Eur. J. Immunol., 2003, 33: 970-. LAG-3 negatively regulates the immune activity of T cells and NK cells through interaction with related ligands. In vitro studies have shown that LAG-3 inhibits antigen-dependent T cell proliferation, activation and homeostasis, which plays an important role in the development of immune-related diseases such as tumors, viral infections and autoimmune diseases. LAG-3 is highly expressed on tolerized Tumor Infiltrating Lymphocytes (TILs), mediating the tumor's inhibitory effect on TIL cellular immune activity. Blocking the binding of LAG-3 to MHCII with anti-LAG-3 antibodies has improved T cell proliferation, activation and secretion of Th1 cytokines, contributing to the restoration of T cell immune function, from which therapeutic benefits may be derived (Grosso et al, J Clin Invest 2007, 117: 3383-2292; Gandhi et al, Blood 2006, 108: 2280-2289).

Relatlimab is an antibody developed by BMS corporation that targets the human LAG-3 protein. The Relatimab can block the combination of LAG-3 and ligand MHCII thereof, and further block the activation of the immune negative regulation pathway of the over-expressed LAG-3 on the surface of the tumor cell to the human T cell, thereby enhancing the immune activity of the human T cell and playing a role in killing the tumor. Currently, Relatlimab is already in phase II/III clinical stage, and the major indications include melanoma, non-small cell lung cancer, gastric cancer, colorectal cancer, head and neck squamous cell carcinoma, and the like.

The protein tyrosine phosphatase 2 (Src homology domain 2 binding tyrosine phosphatase-2, SHP-2) containing Src homology region is an evolutionarily conserved non-receptor type Protein Tyrosine Phosphatase (PTP) encoded by PTPN11 gene, mainly comprises two SH2 domains (N-SH 2, C-SH 2) and a PTP catalytic domain, is widely expressed in various tissues of human, and plays an important role in maintaining tissue development, cell homeostasis and the like. SHP-2 is a proto-oncoprotein and is closely related to the development of various cancers. In some human cancers, such as lung, liver, breast, leukemia and stomach cancer, SHP-2 is activated by RTKs, such as overexpressed Endothelial Growth Factor Receptor (EGFR) or human epidermal growth factor receptor 2 (HER 2), which in turn promotes tumor growth.

SHP099 is a small molecule inhibitor of Norwalk pharmaceutical development targeting SHP-2. SHP099 can keep SHP-2 in closed conformation and active site in self-inhibiting state, thereby inhibiting the growth promoting effect on tumor cells. Studies demonstrated that SHP099 was able to selectively strongly inhibit SHP-2 (IC 50 = 0.071 μ M). In animal experiments, SHP099 can kill cancer cells in transplanted tumor mice by oral administration. (Chen et al, Nature 2016, 535: 148-.

Clinical data show that Relatlimab has a low remission rate when administered alone (NCT 02460224), whereas Relatlimab in combination with the PD-1 antibody O drug is able to overcome PD-1 resistance and reduce tumor volume. Therefore, Relatlimab is often administered in combination with other drugs to treat tumors. Therefore, the search for a new antitumor drug which can be co-administered with Relatlimab is of great significance for drug development and clinical treatment of tumors.

Disclosure of Invention

The invention provides, in a first aspect, a method of treating a tumor using a LAG-3 antibody in combination with an SHP-2 inhibitor.

LAG-3: the receptor encoded thereby.

SHP-2: a Src homology sequence-containing tyrosine phosphatase encoded by PTPN 11.

Further, the LAG-3 antibody Relatimab is an agent that blocks the binding of LAG-3 to MHCII.

Further, the SHP2 inhibitor SHP099 is a drug for reducing the activity of SHP2 enzyme.

Further, the tumor is lung cancer, the lung cancer is non-small cell lung cancer, and preferably EGFR activated non-small cell lung cancer.

The experimental result shows that the Relatlimab effectively binds to the LAG-3 antigen protein.

Experimental results show that Relatimab effectively blocks the binding of LAG-3 to MHCII.

Experimental results indicate that Relatlimab is able to activate the TCR signaling pathway.

The experimental result shows that the Relatlimab can promote the primary T cells to secrete IFN gamma.

The results of biochemical experiments show that the SHP099 effectively inhibits the activity of SHP-2.

The results of in vitro pharmacodynamic experiments show that SHP099 effectively inhibits the cell proliferation of NSCLC cell strain NCI-H3255.

The results of in vivo efficacy experiments show that SHP099 effectively inhibits the growth of NSCLC cell NCI-H3255 transplanted tumor.

The results of in vivo efficacy experiments show that SHP099 synergistically inhibits the growth of 3LL tumor.

In some embodiments, the present invention provides methods for inhibiting tumor growth. Examples of cancer include, but are not limited to, primary and/or recurrent cancers, including melanoma, skin cancer, breast cancer, mesothelioma, brain cancer (e.g., glioblastoma multiforme), head and neck squamous cell carcinoma, thyroid cancer, oral cancer, esophageal cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, kidney cancer, membrane adenocarcinoma, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, prostate cancer, intestinal cancer, bladder cancer, bone cancer, and hematological malignancies (e.g., lymphoma, myeloma, or leukemia).

Drawings

FIG. 1 LAG-3 antibody, Relatimab, activates human T cell immune activity. (A) Relatimab binds human LAG-3; (B) the Raji cell expresses MHCII class molecule HLA-DR; (C) human LAG-3 binds to Raji cells (MHC class II molecules); (D) relatimab blocks human LAG-3 binding to Raji cells (MHC class II molecules); (E) relatlimab activates the NFAT signaling pathway in T cells; (F) MLR experiments confirmed that Relatlimab promotes secretion of IFN γ by T cells.

FIG. 2 SHP-2 inhibitor SHP099 inhibited the growth of NSCLC cell line NCI-H3255. (A) SHP099 inhibits SHP-2 enzyme activity; (B) SHP099 inhibited NCI-H3255 cell proliferation (in vitro); (C) SHP099 inhibits NCI-H3255 graft tumor growth (in vivo); (D) SHP099 did not affect mouse body weight (in vivo).

FIG. 3 shows that SHP-2 inhibitor SHP099 cannot inhibit the proliferation of lung cancer cell 3LL as a result of in vitro drug effect evaluation.

FIG. 4 shows in vivo efficacy results showing that SHP-2 inhibitor and LAG-3 antibody synergistically inhibit tumor growth. (A) Tumor volume growth curve of negative control group; (B) tumor volume growth curve of SHP099 alone dosed group; (C) tumor volume growth curves for the mLAG-3 antibody alone group; (D) tumor volume growth curves for the SHP099 and mLAG-3 antibody combination group.

Detailed Description

Example evaluation of the growth inhibitory Activity of an SHP-2 inhibitor on the non-Small cell Lung cancer NCI-H3255 cell line

The protein tyrosine phosphatase SHP-2 is expressed in a recombinant mode. Briefly, the SHP-2 cDNA sequence was cloned into pGEX-4T-2 plasmid, followed by transformation of BL21 (DE 3) pLyss competent cells, plating, and incubation at 37 ℃ overnight. The next day, colonies were picked, shaken at 37 ℃ and 250rpm until OD600 was between 0.6 and 0.8, added with 1mM IPTG, and induced overnight at 37 ℃ and 180 rpm. The next day the cells were harvested by centrifugation, sonicated and subsequently centrifuged at 4 ℃ and 20000rpm for 60 minutes. Subsequently, the supernatant was collected and passed through a Ni NTA affinity column. After washing the hetero-protein, the protein of interest was eluted with 250mM imidazole buffer. Further, the buffer is replaced by a desalting column, and the recombinant SHP-2 protein is further purified by a Q column and a molecular sieve.

Evaluation of the protein tyrosine phosphatase SHP-2 inhibitory Activity by SHP 099. Further, the activity of SHP-2 inhibitor SHP099 was evaluated using the purified SHP-2 protein. Briefly, phosphatase biochemical reactions were performed at room temperature using 96-well black flat-bottom plates in a total volume of 100ul in 50mM HEPES, pH 7.2, 100mM NaCl, 0.5mM EDTA, 0.05% P-20, 1mM DTT. 0.2nM SHP2 was incubated with 0.5uM polypeptide (H2N-LN (pY) IDLDLV (dPEG8) LST (pY) ASINFQK-amide) for 30 min at 25 ℃. The substrate DiFMUP (Invitrogen, cat # D6567) was then added to the above reaction system and read using a microplate reader for dynamic mode readings at excitation and emission wavelengths of 340nm and 450nm, respectively. IC50 is further calculated. The inhibitory activity of SHP099 on SHP2 is shown in figure 1A.

Evaluation of the growth inhibitory activity of the non-small cell lung cancer NCI-H3255 cell line by SHP 099. Non-small cell lung cancer NCI-H3255 cells were plated at a density of 3000 cells per well in 96-well cell culture plates and cultured in medium (DMEM/10% FBS/1% streptomycin). After 24 hours, a concentration gradient of SHP-2 inhibitor SHP099 (10 uM starting concentration, 1: 4 fold dilution, 7 concentrations in total) was added and returned to the incubator for further culture. After 5 days of drug treatment, 10ul of CCK8 was added to each well, mixed well, incubated at 37 ℃ for 3 hours, and absorbance was measured at 450nm with an enzyme-linked immunosorbent assay. The growth inhibitory activity of SHP099 on non-small cell lung carcinoma NCI-H3255 is shown in FIG. 1B.

SHP099 evaluation of growth inhibition of non-small cell lung carcinoma NCI-H3255 transplanted tumor. Non-small cell lung carcinoma NCI-H3255 was inoculated into 6-8 week old C57BL/6J mice. After tumor formation, SHP099 injection was initiated and mice were periodically tested for weight change and tumor volume change. The results show that SHP099 effectively inhibited tumor growth (fig. 1C and fig. 1D).

Example two LAG-3 antibody, Relatimab, enhances T cell immune activity

Relatlimab binds to human LAG-3 recombinant protein. Human LAG-3 protein was recombinantly expressed and the binding of Relatimab to human LAG-3 was further assessed using ELISA. 10ug/mL of human LAG-3 recombinant protein was plated overnight at 4 ℃. The following day, after washing the plate 3 times with 1 XPBST, add 5% BSA/1 XPBST to block for 3 hours, wash the plate 3 times with 1 XPBST, then add the concentration gradient Relatlimab, incubate for 2 hours at room temperature; washing the plate with 1 XPBST for 3 times, adding anti-human IgG secondary antibody marked with HRP, incubating at room temperature for 2 hours, washing the plate with 1 XPBST for 3 times, adding 50ul TMB into each hole, reacting for 10 minutes, stopping the reaction with 2N sulfuric acid, and reading by an enzyme-labeling instrument. The results showed that the LAG-3 antibody, Relatimab, binds to LAG-3 (FIG. 2A).

Ligand blocking experiments. Relatimab was incubated with biotin-labeled human LAG-3 recombinant protein at room temperature for 30 minutes, and then the above mixed system was further incubated with Raji cells at room temperature for 30 minutes, and after washing 3 times with FACS buffer, PE-labeled Streptavidin (1: 1000) was added, at room temperature for 30 minutes, and after washing 3 times with FACS buffer, detection was performed by flow cytometry. The results showed that Relatlimab blocked the binding of LAG-3 to HLA-DR (FIG. 2B).

The NFAT fluorescein reporter evaluates the T cell activation by Relatimab. To evaluate the activation of T cells by Relatlimab, a concentration gradient of Relatlimab was added to the mixed cell system of Jurkat/LAG-3/NFAT-luc and Raji, after 12 hours, SteadyGlo substrate was added, and after 10 minutes of shaking at room temperature, chemiluminescence was detected by a microplate reader. The results showed that Relatlimab activates the NFAT signaling pathway of human T cell line Jurkat (fig. 2C).

Example in a three-in-vitro drug efficacy test, SHP-2 inhibitor did not inhibit proliferation of lung cancer cell line 3LL cells

Evaluation of growth inhibitory Activity of SHP099 on non-Small cell Lung cancer 3LL cell line. Non-small cell lung cancer 3LL cells were seeded at a density of 3000 cells per well in 96-well cell culture plates and cultured in medium (DMEM/10% FBS/1% streptomycin). After 24 hours, a concentration gradient of SHP-2 inhibitor SHP099 (10 uM starting concentration, 1: 4 fold dilution, 7 concentrations in total) was added and returned to the incubator for further culture. After 5 days of drug treatment, 10ul of CCK8 was added to each well, mixed well, incubated at 37 ℃ for 3 hours, and absorbance was measured at 450nm with an enzyme-linked immunosorbent assay. The growth inhibitory activity of SHP099 on non-small cell lung cancer 3LL is shown in fig. 3, and SHP099 did not inhibit the proliferation of lung cancer cell line 3LL cells.

Example SHP-2 inhibitor and LAG-3 antibody synergistically inhibit 3LL Lung cancer transplantable tumor growth in a four-body drug Effect experiment

SHP-2 inhibitor and LAG-3 antibody synergistically inhibit 3LL lung cancer transplanted tumor growth in vivo efficacy experiment. Non-small cell lung carcinoma 3LL cells were inoculated into 6-8 week old C57BL/6J mice. After tumor formation, SHP099 injection was initiated and mice were periodically tested for weight change and tumor volume change. The results show (figure 4) that either the SHP-2 inhibitor or the LAG-3 antibody alone showed a weak inhibition of tumor growth (figures 4B and 4C) and that the combined administration of both showed a significant increase in tumor growth inhibition (figure 4D) compared to the control (figure 4A), indicating that both synergistically inhibit tumor growth, possibly mediated by activation of immune activity in the mouse's body by the SHP-2 inhibitor and the LAG-3 antibody.