Application of composition containing CDK2 inhibitor and anti-PD-1 antibody in preparation of antitumor drugs

Abstract

The invention discloses application of a composition containing a CDK2 inhibitor and an anti-PD-1 antibody in preparation of antitumor drugs, and relates to the technical field of biological medicines, and the composition comprises the CDK2 inhibitor, the anti-PD-1 antibody and an anthracycline chemotherapeutic drug. According to the application of the composition containing the CDK2 inhibitor and the anti-PD-1 antibody in preparation of anti-malignant tumor drugs, the CDK2 inhibitor enhances ICD induced by anthracycline drugs and CDK2 inhibition of up-regulation interferon stimulating genes ISGs and promotes infiltration of T cells and dendritic cells (DCs) by inhibiting activity of tumor cells CDK2 and promoting apoptosis stress, calreticulin (CRT) exposure, HMGB1 release and ATP secretion.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to the use of a composition comprising a CDK2 inhibitor and an anti-PD-1 antibody in the preparation of an anti-tumor drug. Background Art

[0002] In the immunobiological treatment of malignant tumors, although anti-PD-1 / PD-L1 antibodies have significant therapeutic effects, some patients are difficult to respond due to immunosuppression in the tumor microenvironment or insufficient antigen presentation. Immunogenic cell death (ICD) inducers (such as anthracyclines) can activate the immune system by releasing damage-associated molecular patterns (DAMPs), but their efficacy is limited. CDK2, as an oncogenic kinase, is involved in cell cycle regulation and the formation of an immunosuppressive microenvironment. Existing studies have shown that CDK inhibitors (such as Dinaciclib) can enhance the efficacy of chemotherapy or immunotherapy, but the mechanism of action of CDK2-specific inhibition in ICD has not yet been clarified. The present invention has confirmed through experiments that CDK2 inhibitors produce synergistic anti-tumor effects with anti-PD-1 antibodies and anthracyclines by enhancing type I interferon responses and immune cell infiltration. Summary of the Invention

[0003] To achieve the above objectives, the present invention is implemented through the following technical solution: a composition comprising a CDK2 inhibitor and an anti-PD-1 antibody, comprising the following components:

[0004] CDK2 inhibitors selected from small molecule inhibitors (Dinaciclib), RNA interference agents (siRNA, gRNA, or shRNA targeting CDK2), or PROTAC degraders;

[0005] Anti-PD-1 antibodies, including nivolumab, pembrolizumab, or their biosimilars;

[0006] Chemotherapeutic drugs, wherein the chemotherapeutic drugs are specifically anthracyclines.

[0007] The use of a composition comprising a CDK2 inhibitor and an anti-PD-1 antibody in the preparation of an anti-tumor drug is particularly suitable for solid tumors with high CDK2 expression and tumors that are resistant or recurrent to PD-1 monotherapy.

[0008] The present invention provides the use of a composition comprising a CDK2 inhibitor and an anti-PD-1 antibody in the preparation of an anti-tumor drug. The composition has the following beneficial effects:

[0009] (1) Use of the composition comprising a CDK2 inhibitor and an anti-PD-1 antibody in the preparation of an anti-tumor drug, wherein the CDK2 inhibitor inhibits CDK2 activity in tumor cells, promotes apoptotic stress, calreticulin (CRT) exposure, HMGB1 release and ATP secretion, and enhances anthracycline-induced ICD.

[0010] (2) Use of the composition comprising a CDK2 inhibitor and an anti-PD-1 antibody in the preparation of an anti-tumor drug; CDK2 inhibition upregulates interferon-stimulated genes (ISGs), promoting the infiltration of T cells and dendritic cells (DCs).

[0011] (3) Use of the composition comprising a CDK2 inhibitor and an anti-PD-1 antibody in the preparation of an anti-tumor drug. The CDK2 inhibitor reduces the dependence of tumor cells on PD-L1 expression and enhances the immune checkpoint blocking effect of the anti-PD-1 antibody.

[0012] (IV) The use of the composition comprising a CDK2 inhibitor and an anti-PD-1 antibody in the preparation of an anti-tumor drug. In a tumor-bearing mouse model, the combined treatment significantly inhibited tumor progression and induced long-term immune memory. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of the growth inhibition and immune dependence verification of CDK2 knockout (Cdk2- / -) tumors after MTX treatment in the present invention;

[0014] in, Figure 1 A specifically shows the expression of related proteins in WT and Cdk2- / - MCA205 cells by Western blot;

[0015] Figure 1 B Specifically, immunocompetent mice loaded with WT or Cdk2- / - MCA205 tumor cells were intraperitoneally injected with PBS or MTX;

[0016] Figure 1 C is a graph showing the growth curve of WT or Cdk2- / - MCA205 tumor cells in athymic nude mice (nu / nu);

[0017] Figure 1 D specifically shows the size of WT and Cdk2- / - tumors in the MTX-treated and untreated groups on day 29;

[0018] Figure 2 Schematic diagram of the present invention showing that CDK2 deficiency and MTX treatment promote the infiltration of CD8+ T cells and DCs;

[0019] in, Figure 2A is a fluorescence image of infiltrating immune cells in the tumor microenvironment, with tumor-infiltrating CD8+ cells (left) and CD11c+ cells (right) quantified as the percentage of all nucleated (Hoechst+) cells in the same field of view;

[0020] Figure 2 B shows the proportion of CD8+ (left) and CD11c+ (right) occupying nuclear cells (Hoechst+);

[0021] Figure 2 C, D, E, F, G, and H show the enrichment of T cells (C), CD8+ T cells (D), dendritic cells (E), NK cells (F), macrophages (G), and neutrophils (H) in sarcoma patients with high and low CDK2 expression, respectively;

[0022] Figure 3 This is a schematic diagram showing the sensitivity of Cdk2- / - tumor cells to MTX-induced apoptosis;

[0023] in, Figure 3 A is a typical flow cytometric graph of apoptosis after 24 hours of MTX treatment;

[0024] Figure 3 B is a schematic diagram showing the percentage of early and late apoptotic cells;

[0025] Figure 3 C and 3D are western blot images of apoptosis-related proteins in WT and Cdk2- / - cells after treatment with or without MTX (C) or TNFα (D) for 24 h, respectively;

[0026] In the figure, CC3 is cleaved Caspase-3; C3 is Caspase-3; CC8 is cleaved Caspase-8; C8 is Caspase-8; FLIP is FLICE / caspase 8 inhibitory protein; CDK2 is cyclin-dependent kinase 2; GAPDH is glyceraldehyde-3-phosphate dehydrogenase;

[0027] Figure 4 Schematic diagram of the effect of Cdk2 deficiency on cell immunogenic death markers of the present invention;

[0028] in, Figure 4 A and Figure 4 B. WT and Cdk2- / - MCA205 cells were treated with MTX in vitro. Cells were collected at the indicated time points and surface calreticulin exposure was assessed using a rat monoclonal anti-calreticulin antibody. Representative flow cytometry plots (A) and quantitative data (B) are shown.

[0029] Figure 4C Specifically, WT and Cdk2- / - MCA205 cells were treated with MTX in vitro for 24 h, and the HMGB1 protein levels in the cell supernatant were measured by ELISA;

[0030] Figure 4 D is a graph showing the tumor growth curves of WT or Cdk2- / - MCA205 tumor cells implanted subcutaneously into C57 mice or TLR4- / - mice, respectively;

[0031] Figure 4 E Specifically, after WT and Cdk2- / - MCA205 cells were treated with MTX in vitro at different time points, the supernatants were collected and ATP release in the supernatants was measured using a luciferase bioluminescence assay;

[0032] Figure 4 F is a graph showing the tumor growth curves of WT, Cdk2- / -, and CD39-overexpressing Cdk2- / - tumor cells after subcutaneous implantation into C57 mice;

[0033] Figure 4 G: Quantitative RT-PCR measurement of type I IFN-related gene expression in WT and Cdk2- / - tumor cells after 16 hours of in vitro MTX treatment. Fold changes were calculated using the 2^-ddCT method compared with untreated WT MCA205 cells.

[0034] Figure 4 H and I are schematic diagrams of WT (H) or Cdk2- / - (I) MCA205 tumor cells inoculated into immunocompetent mice and intraperitoneally injected with PBS, MTX, or neutralizing IFNAR1 antibody;

[0035] Arrows indicate the time of MTX injection. All groups contained 5 mice, and the experiment was repeated twice. For gene expression data, an unpaired t-test was used. Tumor growth differences were assessed using an unpaired Mann-Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

[0036] Figure 5 Schematic diagram showing that the introduction of wild-type, but not mutant, Cdk2 in the present invention eliminates the therapeutic advantage of Cdk2- / - tumors;

[0037] in Figure 5AD specifically shows that wild-type or mutant (T160A) Cdk2 was stably transfected into Cdk2- / - MCA205 cells and subcutaneously implanted into immunocompetent mice. The responses of Cdk2- / - cells complemented with wild-type (C) or Cdk2- / - cells complemented with T160A (D) to chemotherapy are shown in the figure, along with the responses of wild-type (A) and Cdk2- / - (B) tumors to chemotherapy. Arrows indicate the time of MTX administration.

[0038] Figure 5 E is a schematic diagram of western blot validation of Cdk2 expression after complementation of wild-type or mutant Cdk2. All groups contained at least five mice, and the results shown are representative of one experiment from two experiments. *p < 0.01, ns, not significant (unpaired t-test);

[0039] Figure 6 Schematic diagram of the present invention showing the combination of Cdk2- / - tumor and anti-PD-1 therapy;

[0040] in, Figure 6 A specifically shows the CDK2 expression in melanoma patients who benefited and did not benefit from nivolumab treatment. The data are from GSE91061.

[0041] Figure 6 B is a schematic diagram showing the reduction of CDK2 expression in people who respond to anti-PD-1 therapy. Data from GSE91061.

[0042] Figure 6 C specifically compares the responsiveness of WT and Cdk2- / - tumors to anti-PD-1 antibodies (intravenous injection) in immunocompetent mice. The arrows indicate the time of anti-PD-1 antibody administration.

[0043] Figure 6 D shows the comparison of WT and Cdk2- / - tumor size on day 26 in the anti-PD-1 antibody-treated group and the untreated group. Each group included at least five mice. Mann-Whitney and Wilcoxon rank-sum tests were used to analyze CDK2 gene expression. Unpaired Mann-Whitney U tests were used to compare tumor growth. Unpaired Student's t-tests were used to analyze differences in tumor size. Response categories included CR / PR / SD, with PD defined as no response. *p < 0.05, **p < 0.01, ns, not significant. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0045] For example 1, please refer to Figure 1-6 :

[0046] in vivo antitumor assay;

[0047] To establish the model, wild-type (WT) or Cdk2- / - MCA205 fibrosarcoma cells were inoculated subcutaneously into C57BL / 6 mice;

[0048] Dosage regimen:

[0049] Group 1: PBS control;

[0050] Group 2: MTX (1 mM, intraperitoneal injection on days 5, 7, and 10);

[0051] Group 3: anti-PD-1 antibody (200 μg, intravenous injection on days 7, 9, and 11);

[0052] Results: Cdk2- / - tumors grew slowly in the treatment group. For details, see the appendix of the instructions. Figure 1 B and 6D.

[0053] For example 2, please refer to Figure 1-6 :

[0054] Type I interferon pathway blockade experiment;

[0055] The tumor-bearing mice were treated with IFNAR1 neutralizing antibodies, and the results showed that the combined treatment effect was significantly inhibited. Figure 4 HI, confirmed that type I interferon is the key mechanism.

[0056] For example three, please refer to Figure 6 :

[0057] Clinical relevance analysis;

[0058] Analysis of melanoma patient data from a public database (GSE91061) revealed that patients with low CDK2 expression had a higher response rate to anti-PD-1 therapy. Figure 6 AB, supports the clinical translational potential of this invention.

[0059] Technical conclusions;

[0060] The experimental results showed that CDK2 inhibitors can synergize with anthracyclines and have anti-tumor effects in preclinical models, thus revealing the potential mechanism of combined therapy. According to the results, no enhanced chemotherapy response of Cdk2- / -MCA205 cells was observed in T cell-deficient nude mice ( Figure 2 DE), suggesting that the enhanced antitumor immune response induced by CDK2 inhibition in the setting of MTX is dependent on host T lymphocytes. Furthermore, immune cell infiltration in the tumor microenvironment is increased. In vitro experiments have shown that CDK2 inhibition promotes immunogenic cell death. Type I interferon responses play a key role in the efficacy of combined CDK2 inhibition and MTX therapy, and the antitumor effect of MTX-induced CDK2 loss is related to its kinase activity. Furthermore, CDK2 inhibition enhances the efficacy of anti-PD-1 therapy.

[0061] Although CDK inhibitors have been reported to promote immune cell death, whether Cdk2 deficiency amplifies anthracycline-induced ICD remains to be elucidated. However, our results suggest that Cdk2-knockout tumor cells can enhance anthracycline-induced ICD. The observation that anthracycline-based chemotherapy is more effective against tumors when combined with Cdk2 deficiency suggests a synergistic effect between Cdk2 inhibition and anthracycline chemotherapy. This synergistic effect was observed in immunocompetent experimental mice but not in nu / nu mice. These results highlight the importance of the immune system in mediating the therapeutic interaction between Cdk2 inhibition and anthracycline chemotherapy.

[0062] Furthermore, MTX-treated tumors were infiltrated with more immune cells than those not treated with MTX, particularly in Cdk2-knockout tumors. The enhanced infiltration of dendritic cells and cytotoxic T lymphocytes in Cdk2-knockout tumors after chemotherapy suggests a robust immune response, which may be attributable to the reduced growth of these tumors. This reduced tumor growth may reflect enhanced immune effector activity within the tumor microenvironment, potentially rendering the tumor more susceptible to effective immune attack. The data suggest that Cdk2 deficiency does not directly enhance the cytotoxic effects of anthracyclines but rather promotes the secretion and expression of molecules involved in immune cell death, such as ATP, HMGB1, and CRT, as well as the production of immunostimulatory CXCR3 ligands such as Cxcl10, suggesting that the observed synergistic effect is immune-mediated rather than due to increased direct cytotoxicity. This finding is consistent with the literature suggesting that the immune system plays a key role in the efficacy of chemotherapy.

[0063] Despite increased expression of ICD-associated molecules observed in vitro, we found that in Tlr4- / - mice, tumor growth was similar to that observed in C57 mice when HMGB1 release from Cdk2- / - cells was increased. Cdk2- / - tumor cells overexpressing CD39 exhibited tumor growth comparable to that of Cdk2- / - tumor cells. However, application of an interferon receptor-blocking antibody reversed this effect. Neutralization of the common type I interferon receptor abolished the favorable interaction between CDK2 inhibition and anthracycline-based chemotherapy, establishing a causal link between the exacerbated type I interferon response and the efficacy of the combination therapy. These results suggest that a type I interferon response plays a key antitumor role when CDK2 inhibition is combined with chemotherapy. Subcutaneous inoculation of Cdk2- / - cancer cells with either WT CDK2 or Cdk2T160A complemented Cdk2- / - cells into C57 mice revealed that Cdk2T160A cells, in combination with methotrexate (MTX), were more effective than complemented Cdk2- / - cells. These results suggest that CDK2 kinase activity is involved in the improved response to chemotherapy in Cdk2- / - tumors. Furthermore, mice inoculated with Cdk2- / - cells showed superior responses to PD-1 combination therapy compared to mice loaded with WT cells. Data analysis also revealed decreased CDK2 expression in patients who responded favorably to PD-1 therapy. These observations can be attributed to enhanced sensitivity of tumor cells to immunogenic cell death, increased ISG expression, and increased PD-L1 expression following CDK2 inhibition.

[0064] In summary, these studies reveal that CDK2 inhibition enhances the antitumor effects of anthracycline-based chemotherapy and induces ICD. Combining CDK2 inhibition with anthracycline or anti-PD-1 therapy may provide a new strategy for cancer treatment.

[0065] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a..." does not exclude the presence of additional identical elements in the process, method, article, or device comprising the element.

[0066] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A composition comprising a CDK2 inhibitor and an anti-PD-1 antibody, characterized in that: These include CDK2 inhibitors, anti-PD-1 antibodies, and anthracycline chemotherapy drugs; Wherein, the CDK2 inhibitor is specifically composed of any one of the small molecule inhibitor Dinaciclib, RNA interference agent or PROTAC degrader; The RNA interference agent is specifically composed of any one of siRNA, gRNA or shRNA targeting CDK2; The anti-PD-1 antibody is specifically composed of any one of Nivolumab and Pembrolizumab.

2. Use of the composition comprising a CDK2 inhibitor and an anti-PD-1 antibody according to claim 1 in the preparation of an anti-tumor drug, characterized in that: It is specifically suitable for solid tumors with high expression of CDK2 and tumors that are resistant or recurrent to PD-1 monotherapy.

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