Application of TIGIT antibody and TNF alpha blocking agent in preparation of antitumor drugs

Abstract

The invention provides an application of a TIGIT antibody and a TNF alpha blocking agent in preparation of antitumor drugs. In a plurality of animal tumor models, the combined treatment of the two shows a remarkable synergistic anti-tumor effect, the tumor growth is inhibited, the lifetime of mice is prolonged, and meanwhile, the tumor immune microenvironment is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tumor immunotherapy, and in particular, the present application provides the use of TIGIT antibodies and TNFα blockers in the preparation of anti-tumor drugs. BACKGROUND

[0002] Tumor immunotherapy, which activates the body's own immune system to fight tumors, has become an important means of treating malignant tumors in the past decade. Immune checkpoint inhibitors (ICIs) such as PD-1, PD-L1 and CTLA-4 antibodies have shown significant efficacy in melanoma, non-small cell lung cancer and other cancers. However, the objective response rate of existing ICIs alone is still low, only a portion of patients benefit, and there are problems such as high incidence of immune-related adverse events (irAEs). Therefore, it is urgent to develop new immune checkpoint targets and more safe and effective combination therapy strategies.

[0003] TIGIT (T cell immunoreceptor with Ig and ITIM domains) as a new generation of immune checkpoint receptor has received extensive attention in recent years. TIGIT is highly expressed in tumor-infiltrating CD8 + T cells and natural killer cells (NK cells), and is closely related to T cell functional exhaustion. TIGIT inhibits T / NK cell activity by competitively binding to the ligand CD155, and enhances the immune suppressive function of Tregs. A number of preclinical studies have shown that blocking TIGIT signaling can effectively enhance anti-tumor immune responses, and combination therapy with PD-1 / PD-L1 antibodies can significantly improve efficacy. However, the results of clinical studies have not been satisfactory. For example, a number of phase III clinical trials such as SKYSCRAPER-01 and KeyVibe-002 in non-small cell lung cancer patients failed to achieve the primary endpoint, triggering extensive discussions in the academic community on the effectiveness of TIGIT monotherapy or combination therapy strategies.

[0004] Recent studies have shown that the expression of TIGIT is highly microenvironment-dependent, with significantly different expression levels in different tumor types or inflammatory backgrounds, suggesting that its upstream regulatory mechanisms are complex and not fully understood. It has been reported that TIGIT is also highly expressed in peripheral blood T / NK cells of patients with chronic viral infections (such as HBV or HIV), suggesting that persistent antigen stimulation or inflammatory factors may be key inducers.

[0005] Tumor necrosis factor alpha (TNFα) is a classic inflammatory factor that is continuously highly expressed in various tumor microenvironments, both pro-inflammatory and tumor cell-killing, and can mediate immune suppression through activation of signaling pathways such as NF-κB. The team of the present application found that TNFα can specifically activate NF-κB and AP-1 transcription factors through its receptor TNFR2, thereby inducing chromatin opening in the TIGIT expression enhancer region and significantly up-regulating TIGIT expression. Based on this, the present application proposes an immunotherapy strategy of combined blocking of TNFα pathway and TIGIT, in order to improve the therapeutic effect of TIGIT antibody, broaden the benefiting population, and reduce immunotoxicity. SUMMARY

[0006] In one aspect, the present application provides the use of a TIGIT antibody and a TNFα blocker in the preparation of an antitumor drug.

[0007] Further, the TIGIT antibody is Tiragolumab, Vibostolimab or Domvanalimab.

[0008] Further, the TNFα blocker is Infliximab, adalimumab, Sacituzumab or Etanercept.

[0009] Further, the TIGIT antibody is Ab01258-1.1 of VivopureX, and the TNFα neutralizing antibody is BE0058 of BioXcell.

[0010] Further, the antitumor drug is an antitumor drug for mice.

[0011] Both known and under research TIGIT antibodies and TNFα blockers in the art can be used, and those known to a person skilled in the art can block the corresponding pathways to achieve similar effects.

[0012] Further, the tumor is colon cancer or melanoma.

[0013] Further, the tumor is melanoma.

[0014] Further, the tumor is melanoma lung metastasis.

[0015] The TIGIT antibody and TNFα blocker in the drug can be prepared in the form of a composition, can be provided separately and placed in the same package, or can be provided as separate drugs and used in combination according to the instructions.

[0016] Further, the drug further comprises a pharmaceutically acceptable excipient.

[0017] Further, the drug is an injection or an oral agent.

[0018] Depending on the properties of the specific TIGIT antibody and TNFα blocker, other dosage forms besides injections and oral agents can also be used. Those skilled in the art can select excipients for the drug according to the routine in the pharmaceutical field when designing the drug, including but not limited to solvents, cosolvents, osmotic pressure regulators, pH regulators, antioxidants, surfactants, binders, fillers, lubricants, preservatives, taste-masking agents, flavoring agents, sustained-release agents, controlled-release agents, coatings, capsules, etc.

[0019] An anti-tumor drug comprising a TIGIT antibody and a TNFα blocker.

[0020] In another aspect, the present application provides a TIGIT antibody and a TNFα blocker for use in the preparation of a medicament for reducing the proportion of TIGIT + immune cells, enhancing CD8 + T cells and NK cells, enhancing tumor infiltration and functional activity, slowing tumor progression, inhibiting distant metastasis, and prolonging survival.

[0021] The present application also proposes a patient screening method suitable for the combined treatment strategy, which comprises detecting TIGIT high expression and TNFα signal activation state in tumor tissue to predict the response of patients to TIGIT+TNFα antibody combination therapy.

[0022] In the MC38 subcutaneous tumor model and B16F10 lung metastasis model, the combined therapy showed significant synergistic anti-tumor effect, inhibited tumor growth, prolonged the survival of mice, and improved the tumor immune microenvironment. Tissue analysis showed that TIGIT + T / NK cell ratio decreased, accompanied by increased infiltration of effector T cells and NK cells.

[0023] Further mechanism research found that TNFα induced TIGIT expression through TNFR2-mediated NF-κB and AP-1, and enhanced its chromatin accessibility and enhancer activity. TNFα antibody can effectively block this pathway, significantly reduce the transcription level of TIGIT, and break the inflammatory-induced immunosuppression state.

[0024] The present application discloses the key upstream regulation mechanism of TIGIT expression, and based on this, proposes a treatment strategy of combined blocking of TIGIT and TNFα, which provides new theoretical support and clinical transformation path for optimizing TIGIT targeted immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 TNFα stimulation on NK cells and CD8 +Figure showing the results of the impact of TIGIT expression in T cells.

[0026] Figure 2A Figure showing the correlation of TIGIT and TNF in various tumor types.

[0027] Figure 2B Figure showing the correlation of TIGIT and TNF in kidney cancer tissue samples.

[0028] Figure 2C Figure showing the results of the chip immunohistochemical staining study of the correlation of TIGIT and TNF.

[0029] Figure 3A Figure showing the enhancer accessibility of the TIGIT gene neighborhood after TNFα stimulation.

[0030] Figure 3B Figure showing the results of the ChIP-qPCR detection of the binding of epigenetic factors P300, BRD4, and transcription factors NF-κB p65 and c-JUN.

[0031] Figure 4A Figure showing the effect of TNFα neutralizing antibodies and their combination with TIGIT antibodies on NK cells and CD8 + Figure showing the results of the impact of TIGIT expression on the surface of T cells.

[0032] Figure 4B Figure showing the effect of TNFα neutralizing antibodies and their combination with TIGIT antibodies on NK cells and CD8 + Figure showing the results of the immunofluorescence analysis of the tumor infiltration of T cells.

[0033] Figure 5A Figure showing the experimental protocol for the mice of Example 6.

[0034] Figure 5B Figure showing the results of the in vivo imaging of the mice of Example 6 and the statistical graph of the fluorescence intensity.

[0035] Figure 5C Figure showing the statistical graph of the survival of the mice of Example 6.

[0036] Figure 6 Figure showing the regulatory mechanism of TNFα-induced TIGIT expression. DETAILED DESCRIPTION

[0037] The reagents and experimental methods used in the examples are as follows:

[0038] Molecular biology reagents: Antibodies BRD4 (#13440), P300 (#54062), H3K4mel purchased from CST company, Antibodies H3K27ac (#4729), CD161 (#234107), TIGIT (#243903) purchased from abcam company, Antibodies CD8, TNFa (#GB11188) purchased from Servicebio company, HRP labeled secondary antibodies and DAB developing kit purchased from Zhongshanjingqiao company. Recombinant human TNFa, IL-17A, IL-1b, IL-6 and IL-10 purchased from Peprotech company. Flow cytometry antibodies CD27-BV605 (#563365), CD4-BV786 (#563331), CD8-FITC (#553031), NK1.1-BV650 (#564143), NK1.1-APC (#550627), CD16-AF700 (#557920) purchased from BD Pharmingen company; CD45-APC / Cyanine7 (#157204), CD8-BV421 (#301036), CD56-BV605 (#318334), CD3-Pacific Blue (#100214), APC-Mouse IgG1 kappa Isotype Control (#400120) purchased from BioLegend company; aCD8 (#16-0289-85), aCD3 (#16-0037-81), CD11b-AF700 (#56-0112-82), TIGIT-APC (#17-9500-42), TIGIT-PE (#12-9500-42), TIGIT-percp-eFluor710, percp-eFluor710-Rat IgG2a kappa Isotype Control purchased from eBioscience company; CD3-FITC (#35-0038-T100), CD4-percp (#65-0049-T100), Ghost Dye Violet 510 (#13-0870-T100) cell viability dye purchased from Tonbo Biosciences company.

[0039] Example 1

[0040] Take 5 mL of peripheral blood from healthy donors, take 5 mL of Lymphoprep lymphocyte separation medium into a 15 mL centrifuge tube, gently add the peripheral blood on top of the lymphocyte, 2000 rpm, 9 for speed, 2 for speed, 10 minutes after gently sucking the middle white membrane layer, which is the lymphocyte. After washing with PBS, centrifuge at 300 g for 5 minutes, resuspend with 1 mL of FACS buffer (1 mL of fetal bovine serum + 100 μL of 0.5M EDTA, pH: 8.0), and filter with a 40 μm filter. Use EasySep TM Human NK cell isolation kit (Stem Cell Technologies, item number 17955), follow the instructions, the purity of NK cells can reach more than 90%. The isolated NK cells were cultured in vitro in a 24-well plate, the culture medium was RPMI1640 supplemented with 10% fetal bovine serum (FBS), recombinant human IL-2 (100 U / mL) and cytokines TNFα (50 ng / mL), IL-17A (20 ng / mL), IL-1β (20 ng / mL), IL-6 (20 ng / mL) and IL-10 (20 ng / mL) for 24 hours, then collect the cells. Add CD3-FITC, CD56-BV605, CD16-AF700, TIGIT-PE pre-mixed cocktail (1:100, 1 μL / ab / tube). Incubate at 4°C for 30 minutes, then add 200 μL of FACS buffer and mix gently, centrifuge at 400 g for 5 minutes, then fix with 300 μL of 2% paraformaldehyde and detect the data collected by flow cytometry. Human CD8 + T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors by immunomagnetic bead negative selection, using CD8 + T cell isolation kit (item number 130-096-495), follow the manufacturer's instructions. The isolated CD8 + T cells were cultured in vitro in a 24-well plate, the culture medium was RPMI1640 supplemented with 10% fetal bovine serum (FBS) and human recombinant IL-2 (100 U / mL), and stimulated with αCD3 and αCD8 and TNFα (50 ng / mL). Add CD3-FITC, CD8-BV421, TIGIT-PE pre-mixed cocktail (1:100, 1 μL / ab / tube). Incubate at 4°C for 30 minutes, then add 200 μL of FACS buffer and mix gently, centrifuge at 400 g for 5 minutes, then fix with 300 μL of 2% paraformaldehyde and detect the data collected by flow cytometry.

[0041] Figure 1Parts A and B of FIG. 1 show the change of TIGIT expression on peripheral blood NK cells after exposure to multiple inflammatory cytokines (IL-1β, IL-17A, TNFα) and immunosuppressive factors commonly found in tumor microenvironment (IL-6, IL-10). Among them, only TNFα can significantly up-regulate the expression of TIGIT. Further in Part C of FIG. 1, it is shown that TNFα can also enhance the expression level of TIGIT in activated CD8 Figure 1 + T cells.

[0042] The data indicate that TNFα is an important inducer of TIGIT up-regulation.

[0043] Example 2

[0044] We collected a tissue chip containing kidney cancer adjacent and cancer tissues, a total of 58 available cancer tissue samples. The tissue chip was first baked in a 55°C oven for 30 minutes to promote paraffin dissolution, and then dewaxed and dehydrated in turn with xylene and different concentration gradients of ethanol. After dewaxing, the slices were blocked with 5% goat serum at room temperature for 1 hour, and the primary antibody was incubated overnight at 4°C. The next day, the wet box was restored to room temperature, and PBST buffer was used to wash 3 times for 5 minutes each time on a horizontal shaker to remove unbound antibodies. Then incubate the secondary antibody at room temperature for 1 hour, and adjust the DAB color development time according to the expression level of the target protein. Finally, use resin mounting, scan to obtain images and collect experimental data.

[0045] The results of TIMER2.0 database analysis show that TIGIT and TNF are significantly positively correlated in multiple tumor types Figure 2A ). To further verify this result, we analyzed kidney cancer tissue samples and found that they also had a significant positive correlation Figure 2B ), and the chip immunohistochemical staining further supports the stability and consistency of this correlation Figure 2C ).

[0046] The results show that the high expression of TIGIT in the tumor microenvironment is likely driven by TNFα induction.

[0047] Example 3

[0048] ​Chromatin immunoprecipitation sequencing (ChIP-seq) and ChIP-qPCR were performed according to standard protocols with slight modifications. Briefly, cells were cross-linked with 1% formaldehyde for 10 min at room temperature to fix protein-DNA complexes, followed by the addition of 125 mM glycine to stop the reaction for 5 min. After washing with PBS, cells were lysed and chromatin was sonicated to 200-500 bp fragments. 5 μg of anti-H3K4mel or H3K27ac antibody was added, and the target chromatin fragments were enriched by protein A / G magnetic beads. The immunoprecipitated DNA was reverse cross-linked by incubation at 65 °C overnight, followed by DNA extraction using the Qiagen PCR purification kit. Library construction for sequencing was performed using the NEBNext Ultra II DNA Library Prep Kit (NEB), including steps such as end repair, ligation of adapters, screening of fragment length, and index primer amplification. After library quality was detected by Agilent Bioanalyzer, double-end sequencing was performed on the MGI DNBSEQ-T7 platform. In the ChIP-qPCR experiments of P300, BRD4, and transcription factors NF-κB p65 and c-JUN, part of the immunoprecipitated product was used as a template, and the enrichment of specific sites was detected by SYBR Green real-time fluorescent quantitative PCR. The results were expressed as a percentage relative to the input DNA, and are shown as mean ± S.D.

[0049] After TNFα stimulation, the enhancer accessibility in the TIGIT gene neighborhood significantly increased, as evidenced by the increased H3K27ac, H3K4mel ChIP-seq and ATAC-seq signals Figure 3A Further ChIP-qPCR detection of epigenetic factors P300, BRD4, and transcription factors NF-κB p65 and c-JUN showed that TNFα stimulation significantly increased the enrichment of these factors in the enhancer region Figure 3B

[0050] The results showed that TNFα enhanced the chromatin openness of the enhancer near the TIGIT gene, promoted the binding of NF-κB and AP-1, and thus enhanced the transcriptional activity of TIGIT.

[0051] Example 4

[0052] For the colorectal cancer model, 1 x 10 6 ​MC38 mouse colon cancer cells were suspended in 150 μL PBS and injected subcutaneously into the right flank of C57BL / 6J mice. Three days after inoculation, the mice were randomly divided into groups and treatment was initiated. The treatment groups were injected intraperitoneally with TIGIT antibody (200 μg, VivopureX, Ab01258-1.1), TNFα neutralizing antibody (125 μg, BioXcell, BE0058), combination therapy, or isotype control antibody (200 μg, Selleck), respectively, for a total of 5 injections, once every 3 days. The long and short diameters of the tumors were measured every 2-3 days using a vernier caliper, and the tumor volume was calculated using the formula: long x short2x 0.5. The tumor tissue was removed on day 20 after inoculation for subsequent analysis. Half of the tumor was fixed in 4% paraformaldehyde for paraffin sectioning, and the other half was chopped with an ophthalmic scissors and placed in a 2 mg / ml collagenase digestion solution for 30 minutes in a 37°C water bath. The solution was removed every 10 minutes and shaken for 15 seconds on a shaker. After digestion, the same volume of DMEM containing 10% fetal bovine serum buffer was added to terminate the digestion. The filtrate was collected using a 200-mesh filter, centrifuged at 500g for 10 minutes, and resuspended in 3 mL of 40% percoll (3.6 mL percoll + 0.4 mL 10x PBS + 6 mL 1x PBS). The solution was gently added to 70% percoll (6.3 mL percoll + 0.7 mL 10x PBS + 3 mL 1x PBS), centrifuged at 1260g, with an ascending speed of 6 and a descending speed of 2, for 30 minutes. The middle white membrane layer was collected, mixed with 5 mL of 1x PBS, and centrifuged at 500g for 10 minutes. The cells were resuspended in 1 mL of FACS and filtered through a 40-μm filter, centrifuged at 400g for 5 minutes. CD3-BV421, NK1.1-BV650, CD8-FITC, CD4-BV786, and TIGIT-percp-eFluor710 were mixed into a cocktail (1:100, 1 μL / ab / tube) and incubated at 4°C for 30 minutes in the dark. After incubation, 200 μL of FACS buffer was added, mixed gently, and centrifuged at 400g for 5 minutes. The cells were fixed with 300 μL of 2% paraformaldehyde and analyzed by flow cytometry.

[0053] Example 5

[0054] MC38 subcutaneous tumor tissues were fixed with 10% neutral formaldehyde and routinely paraffin-embedded, with a section thickness of 4 μm. The sections were deparaffinated with xylene and rehydrated in different concentrations of ethanol. Then, the sections were subjected to antigen retrieval with citric acid buffer at pH 6.0. After membrane permeabilization with 0.3% Triton X-100, the sections were blocked with 3% BSA for 1 h at room temperature. Then, the sections were incubated with primary antibodies, including CD8a antibody and CD161 antibody, at 4 °C overnight. The next day, the sections were incubated with fluorescently labeled secondary antibodies, and the nuclei were stained with DAPI. The final images were collected by Olympus FV1200 confocal microscope and scanned and analyzed using 3DHISTECH whole slide scanning system.

[0055] Figure 4A It was shown that TNFα neutralizing antibody could significantly reduce the expression of TIGIT on the surface of NK cells and CD8 + T cells in the tumor microenvironment, and this inhibitory effect was further enhanced after combination therapy with TIGIT antibody. Figure 4B For immunofluorescence analysis results, it was shown that the tumor infiltration of NK cells and CD8 + T cells in the combination therapy group was significantly increased, suggesting that this combination strategy could effectively improve the tumor immune microenvironment.

[0056] Example 6

[0057] For the melanoma lung metastasis model, 1.5 × 10 5 B16F10-Luc melanoma cells were suspended in 150 μL of PBS and injected into C57BL / 6J mice via the tail vein. Treatment began 3 days after inoculation, and TIGIT antibody (200 μg), TNFα neutralizing antibody (125 μg), combination therapy group, or isotype control antibody (200 μg) were injected intraperitoneally, a total of 4 times, every 3 days. On day 15 after inoculation, the mice were anesthetized (isoflurane inhalation) 10 minutes after intraperitoneal injection of the fluorescent substrate D-luciferin (20 mg / mL, Gold Biotechnology) at a dose of 150 mg / kg body weight, and then imaged in the AniView100 in vivo multi-modality imaging system (Biolight Biotechnology). The fluorescence intensity was calculated and analyzed by Living Image software.

[0058] The anti-tumor effect of combination therapy in the mouse B16-F10-Luc melanoma lung metastasis model was demonstrated. The results of small animal in vivo imaging Figure 5A and Figure 5B showed that the combination of TNFα neutralizing antibody and TIGIT antibody significantly inhibited the formation of lung melanoma metastases and significantly prolonged the survival of mice.Figure 5C )。

Claims

1. Application of TIGIT antibody and TNFα blocker in the preparation of antitumor drugs.

2. The application according to claim 1, wherein the TIGIT antibody is Tiragolumab, Vibostolimab, or Domvanalimab.

3. In the application according to claim 1 or 2, the TNFα blocker is Infliximab, adalimumab, Sacituzumab, or Etanercept.

4. The application according to claim 1, wherein the tumor is colon cancer or melanoma.

5. The application according to claim 4, wherein the tumor is melanoma.

6. The application according to claim 5, wherein the tumor is a melanoma lung metastasis.

7. The application according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable excipient.

8. The application according to claim 1, wherein the drug is an injection or an oral preparation.

9. An antitumor drug, characterized in that, The anti-tumor drugs include TIGIT antibodies and TNFα blockers.

10. The medicament according to claim 9, wherein the TIGIT antibody is Tiragolumab, Vibostolimab, or Domvanalimab; and the TNFα blocker is Infliximab, adalimumab, Sacituzumab, or Etanercept.

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