Use of SRG-1 compound in the preparation of a drug for treating KRAS-G12C inhibitor resistance related tumor

Abstract

The present application relates to the use of SRG-1 compound or its pharmaceutically acceptable salt in the preparation of a medicament for treating KRAS-G12C inhibitor drug resistance related tumors, especially to the treatment of acquired drug resistance solid tumors carrying KRAS-G12C-Y96C double mutation. Experiments show that SRG-1 can directly bind to KRAS-G12C-Y96C double mutant protein, inhibit the proliferation of drug-resistant tumor cells and induce apoptosis, inhibit the growth of drug-resistant tumors in vivo and prolong the survival of tumor-bearing animals. SRG-1 can also reduce the level of regulatory T cells in the tumor microenvironment, promote NK cell, CD8+ effector T cell infiltration, and increase the expression of Granzyme B and IFN-gamma and other anti-tumor effector molecules. The present application also provides SRG-1 combined with anti-CD25 antibody or its antigen binding fragment and the corresponding pharmaceutical composition, which can further relieve immune suppression and enhance the anti-tumor effect, and provide a new treatment strategy for KRAS-G12C inhibitor drug resistance tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the use of SRG-1 compound in the preparation of drugs for treating tumors associated with resistance to KRAS-G12C inhibitors. Background Technology

[0002] In recent years, covalent inhibitors targeting KRAS G12C mutations, such as Sotorasib and Adagrasib, have been introduced into clinical applications, providing new treatment options for the precision treatment of KRAS-mutant tumors.

[0003] However, KRAS-G12C inhibitors still face significant resistance issues in clinical application. Some patients show poor efficacy in the early stages of treatment, while others experience disease progression after the initial response. Studies have shown that KRAS-G12C inhibitor resistance can be mediated by multiple mechanisms, including secondary KRAS mutations, reactivation of the upstream receptor tyrosine kinase pathway, compensatory activation of bypass signaling pathways, downstream pathway reactivation, and tumor microenvironment immunosuppression. Among these, secondary mutations in KRAS Y96C can affect the binding between KRAS-G12C inhibitors and target proteins, weakening the inhibitory effect of existing inhibitors on KRAS Y96C mutant proteins, and are one of the important mechanisms of acquired resistance.

[0004] Current KRAS-G12C inhibitors are primarily designed around the single mutation target of KRAS G12C. When tumor cells further develop secondary resistance mutations such as Y96C, the interaction between the drug and the KRAS protein switch-II pocket may be affected, leading to reduced drug binding capacity and decreased inhibitory effect, thereby resulting in tumor recurrence or progression. Therefore, developing novel therapeutic drugs that can effectively target drug-resistant mutations such as KRAS-G12C-Y96C is a pressing technical problem that needs to be solved in the treatment of KRAS-G12C inhibitor-resistant tumors.

[0005] On the other hand, KRAS-mutant tumors not only possess advantages in autonomous cell proliferation and survival, but can also promote immune escape by regulating the tumor immune microenvironment. Regulatory T cells, or Treg cells, are an important immunosuppressive cell population in the tumor microenvironment, which can inhibit the activation and killing functions of anti-tumor immune effector cells such as CD8+ T cells and natural killer cells. Increased Treg cell levels, decreased CD8 / Treg ratio, and insufficient expression of effector molecules such as Granzyme B and IFN-γ may all lead to a reduced response of tumors to targeted therapy or immunotherapy.

[0006] Therefore, a novel therapeutic strategy is needed in the field to treat KRAS-G12C inhibitor-resistant tumors, particularly acquired resistant solid tumors carrying the KRAS-G12C-Y96C double mutation. This therapeutic strategy preferably possesses the combined effects of directly inhibiting the proliferation of resistant tumor cells, inducing tumor cell apoptosis, reducing Treg immunosuppressive levels, promoting the infiltration of anti-tumor immune effector cells, and synergistically enhancing efficacy with Treg clearance strategies. This would provide new drug uses and combination therapy options for KRAS-G12C inhibitor-resistant tumors. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides the use of the SRG-1 compound in the preparation of drugs for treating tumors associated with resistance to KRAS-G12C inhibitors.

[0008] In accordance with a first aspect of the invention, use is provided of an SRG-1 compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors associated with resistance to KRAS-G12C inhibitors.

[0009] Preferably, the KRAS-G12C inhibitor resistance-related tumors are tumors that are insensitive to Sotorasib and / or Adagrasib treatment, or are primary or acquiredly resistant to the drug.

[0010] Preferably, the KRAS-G12C inhibitor resistance-associated tumor is a solid tumor carrying a KRAS G12C mutation.

[0011] Preferably, the KRAS-G12C inhibitor resistance-associated tumor further carries a secondary KRAS Y96C mutation.

[0012] Preferably, the drug is used to inhibit tumor cell proliferation, induce tumor cell apoptosis, reduce regulatory T cell levels, and / or promote the infiltration of anti-tumor immune effector cells.

[0013] According to a second aspect of the invention, the use of an SRG-1 compound or a pharmaceutically acceptable salt thereof in combination with an antiCD25 antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating tumors associated with resistance to KRAS-G12C inhibitors is provided.

[0014] Preferably, the anti-CD25 antibody or its antigen-binding fragment is used to reduce the level of regulatory T cells (Tregs) in the tumor microenvironment.

[0015] According to a third aspect of the invention, there is provided a pharmaceutical composition comprising: (1) an SRG-1 compound or a pharmaceutically acceptable salt thereof; (2) an antiCD25 antibody or an antigen-binding fragment thereof; and (3) a pharmaceutically acceptable excipient and / or carrier.

[0016] Preferably, the pharmaceutical composition is used to treat tumors associated with resistance to KRAS-G12C inhibitors.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] First, this invention discovers that the SRG-1 compound or a pharmaceutically acceptable salt thereof can be used to treat tumors associated with KRAS-G12C inhibitor resistance, particularly those with acquired drug-resistant solid tumors carrying the KRAS-G12C-Y96C double mutation. Existing KRAS-G12C inhibitors, such as Sotorasib / AMG510, exhibit significantly reduced binding affinity and antitumor activity to target proteins after the development of secondary Y96C mutations. In contrast, SRG-1 can still directly bind to the KRAS-G12C-Y96C double-mutated protein, thus providing a new therapeutic option for overcoming this type of secondary resistance.

[0019] Second, this invention demonstrates that SRG-1 still exhibits a clear inhibitory effect on the proliferation of KRAS-G12C-Y96C resistant tumor cells. Experimental results show that SRG-1 can not only inhibit the proliferation of KRAS-G12C inhibitor-sensitive cells, but also inhibit the proliferation of tumor cells carrying secondary resistance mutations in Y96C, indicating that it can continue to exert its anti-tumor effect even when the efficacy of traditional KRAS-G12C inhibitors declines.

[0020] Third, this invention demonstrates that SRG-1 can induce apoptosis in KRAS-G12C-Y96C drug-resistant tumor cells. After SRG-1 treatment, the proportion of apoptotic drug-resistant tumor cells increased with increasing drug concentration, indicating that its anti-tumor effect is not only reflected in inhibiting tumor cell proliferation, but also in promoting the death of drug-resistant tumor cells, thereby helping to improve the therapeutic effect on drug-resistant tumors.

[0021] Fourth, the SRG-1 of this invention did not exhibit significant cytotoxicity in non-malignant cells. Compared to broad-spectrum cytotoxic drugs, SRG-1 did not show significant toxicity to normal bronchial epithelial cells, normal lung fibroblasts, and mouse embryonic fibroblasts, suggesting that it has good selectivity and a potential safe therapeutic window, which is beneficial for subsequent drug development and clinical translation.

[0022] Fifth, this invention demonstrates that SRG-1 has in vivo anti-tumor effects in the KRAS-G12C-Y96C drug-resistant tumor animal model. SRG-1 can inhibit the growth of drug-resistant tumors, reduce tumor burden, and prolong the survival of tumor-bearing animals, while no significant weight loss or systemic toxicity was observed, indicating that it not only has in vitro anti-drug resistance activity but also in vivo therapeutic effects.

[0023] Sixth, this invention reveals that SRG-1 can improve the immunosuppressive microenvironment of KRAS-mutant tumors. SRG-1 can reduce the level of CD4+Foxp3+Treg cells in peripheral blood, spleen, and tumor microenvironment, and promote the infiltration of NK1.1+ natural killer cells, while increasing the expression levels of anti-tumor effector molecules such as Granzyme B and IFN-γ, thereby helping to relieve tumor immunosuppression and enhance anti-tumor immune responses.

[0024] Seventh, this invention demonstrates that SRG-1 can block the process of PBMC polarization induced by KRAS-mutant tumor cells to Treg, and enhance the killing effect of PBMCs on KRAS-mutant and KRAS-G12C-Y96C drug-resistant tumor cells. This result indicates that SRG-1 can not only directly act on drug-resistant tumor cells, but also improve the host immune system's ability to clear drug-resistant tumors by regulating the interaction between tumor cells and immune cells.

[0025] Eighth, this invention provides a novel treatment regimen for KRAS-G12C-Y96C drug-resistant tumors using a combination of SRG-1 and anti-CD25 antibody. The anti-CD25 antibody can further reduce Treg levels, and when combined with SRG-1, it can enhance tumor suppression and improve the anti-tumor effects of CD8+ effector T cells, NK cells, and cytotoxic effector molecules. This combination regimen can synergistically exert therapeutic effects from two levels: "directly inhibiting drug-resistant tumor cells" and "relieving immunosuppression."

[0026] Ninth, this invention also provides a method for screening suitable patients or suitable tumor models based on indicators such as KRAS-G12C-Y96C mutation, elevated Treg levels, decreased CD8 / Treg ratio, and changes in Granzyme B or IFN-γ expression. This is beneficial for achieving precise treatment of KRAS-G12C inhibitor-resistant tumors and improving the rationality of drug selection and treatment response rate.

[0027] In summary, this invention addresses KRAS-G12C inhibitor resistance, particularly secondary resistance tumors caused by KRAS-G12C-Y96C, by providing SRG-1 monotherapy and its combination with anti-CD25 antibodies. This regimen exhibits multiple effects, including directly inhibiting drug-resistant tumor cells, inducing tumor cell apoptosis, reducing Treg immunosuppression, and promoting the infiltration of anti-tumor immune effector cells. It provides a novel pharmaceutical use and combination therapy strategy for the treatment of KRAS-G12C inhibitor-resistant tumors. Attached Figure Description

[0028] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0029] Figure 1 The diagram shows the structure of the SRG-1 compound and the detection results of its binding ability with the KRAS-G12C-Y96C double mutant protein according to embodiments of the present invention; wherein, Figure 1 A is the structural formula of the SRG-1 compound. Figure 1 B represents the binding / dissociation curves of SRG-1 and KRAS-G12C-Y96C protein. Figure 1 C represents the binding / dissociation curves of AMG510 and KRAS-G12C-Y96C proteins.

[0030] Figure 2 This is a diagram showing the in vitro efficacy evaluation results of SRG-1 against KRAS-G12C inhibitor-sensitive and drug-resistant tumor cells according to embodiments of the present invention; wherein, Figure 2 A and Figure 2 B represents the proliferation inhibition results of AMG510 on NCI-H358 parental cells and NCI-H358-Y96C resistant cells. Figure 2 C and Figure 2 D represents the inhibition of SRG-1 on the proliferation of NCI-H358-Y96C resistant cells and NCI-H358 parental cells. Figure 2 E represents the inhibition of cell proliferation by SRG-1 in a KRAS-G12C-Y96C double mutant cell model. Figure 2 F to Figure 2 H represents the toxicity evaluation results of SRG-1 against non-malignant cells. Figure 2 I arrive Figure 2 J represents the flow cytometry results of SRG-1-induced apoptosis in NCI-H358-Y96C drug-resistant cells.

[0031] Figure 3 This is a diagram illustrating the in vivo antitumor effect and immune microenvironment regulation results of SRG-1 in a KRAS-G12C-Y96C drug-resistant tumor animal model according to an embodiment of the present invention; wherein, Figure 3 A represents representative images of tumors in mice from each group at the experimental endpoint. Figure 3 B represents the tumor growth curve. Figure 3 C represents the curve of mouse body weight change. Figure 3 D represents the mouse survival curve. Figure 3 E to Figure 3 H represents the tumor growth trajectory of a single mouse in each group. Figure 3 I arrive Figure 3 K represents the proportion of Treg cells detected in peripheral blood, spleen, and the tumor microenvironment. Figure 3 L and Figure 3 M represents the expression results of Granzyme B and IFN-γ in tumor tissue. Figure 3N represents the detection result of NK1.1 positive natural killer cell infiltration in tumor tissue.

[0032] Figure 4 The image shows the detection results of KRAS-mutant tumor cells inducing PBMC polarization towards Treg, as provided in the embodiments of the present invention. It shows the changes in the proportion of CD4+Foxp3+Treg cells after PBMCs were co-cultured with H23, H23-Y96C, H2122, and H2122-Y96C cells, respectively.

[0033] Figure 5 The figure shows the detection results of SRG-1 enhancing PBMC-mediated tumor cell killing provided by the embodiments of the present invention. It shows the changes in relative luciferase activity of target cells such as H23, H23-Y96C, H2122, and H2122-Y96C that stably express luciferase and PBMC after co-culturing with SRG-1 treatment.

[0034] Figure 6 This is a graph showing the experimental results of combining SRG-1 with anti-CD25 antibody for the treatment of KRAS-G12C-Y96C resistant tumors according to an embodiment of the present invention; wherein, Figure 6 A represents representative images of tumors in mice from each group at the experimental endpoint. Figure 6 B represents the statistical results of the weight of the tumor in vitro. Figure 6 C represents the result of detecting the proportion of CD8+ Granzyme B+ cells in the tumor microenvironment. Figure 6 D represents the result of the CD8+IFN-γ+ cell proportion assay. Figure 6 E represents the result of CD8+Ki67+ cell proportion detection. Figure 6 G represents the result of the CD4+Foxp3+Treg cell proportion assay. Figure 6 H represents the result of the NK1.1 positive cell proportion detection. Detailed Implementation

[0035] The present invention will be further described below with reference to embodiments. It should be understood that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Any equivalent substitutions, conventional adjustments or combinations made based on the disclosure of the present invention without departing from the essential concept of the present invention should fall within the scope of protection of the present invention.

[0036] The SRG-1 compound described in this invention is the compound shown in Formula I, and its chemical name is Ethyl(1R,7S,8R)-8-(4-chlorobenzyl)-1,7-dimethyl-2,3,5,6-tetraoxa-11-azaspiro[bicyclo[5.3.1]undecane-4,1'-cycloheptane]-8-carboxylate.

[0037] In this invention, the SRG-1 compound may be used in its free form or in the form of a pharmaceutically acceptable salt thereof. The pharmaceutically acceptable salt includes, but is not limited to, hydrochloride, sulfate, phosphate, methanesulfonate, p-toluenesulfonate, maleate, fumarate, citrate, tartrate, acetate, or any combination thereof.

[0038] Unless otherwise stated, the cell culture conditions in the following examples are 37°C, Experimental data are expressed as mean ± SD; statistical significance is expressed as... express.

[0039] The technical contribution of this invention lies in the discovery that SRG-1 can be used to treat KRAS-G12C inhibitor-related tumors, especially acquired drug-resistant tumors carrying KRAS-G12C-Y96C double mutations, and can achieve the treatment of drug-resistant tumors by reducing Treg levels, promoting the infiltration of anti-tumor immune effector cells, and synergizing with anti-CD25 antibodies.

[0040] Example 1 Preparation of SRG-1 compound

[0041] This embodiment is for illustration. Figure 1 The SRG-1 compound shown in A can be prepared by a one-pot three-component reaction, thereby ensuring the availability of the active ingredient described in this invention.

[0042] 1. Experimental materials

[0043] The experimental materials included ethyl 2-acetyl-2-(4-chlorobenzyl)-5-oxohexanoate, 1,1-dihydroperoxycycloheptane, ammonium acetate, ethanol, and an ethanol / water mixture.

[0044] 2. Preparation method

[0045] Ethyl 2-acetyl-2-(4-chlorobenzyl)-5-oxohexanoate was dissolved in ethanol at 20°C to 25°C to obtain a reaction substrate solution. Ammonium acetate and 1,1-dihydroperoxycycloheptane were added sequentially to the reaction substrate solution, wherein the amount of ammonium acetate was 3 molar equivalents and the amount of 1,1-dihydroperoxycycloheptane was 1.5 molar equivalents. After the addition was complete, the reaction mixture was stirred at 20°C to 25°C for 24 hours. After the reaction was completed, the reaction system was placed at approximately -22°C overnight to allow the target product to crystallize. The precipitated crystals were collected by filtration and washed with a cold ethanol / water mixture (volume ratio 40:60). After washing and drying, the SRG-1 compound was obtained.

[0046] 3. Structural Confirmation

[0047] The obtained SRG-1 compound can be structurally confirmed by liquid chromatography-mass spectrometry, proton nuclear magnetic resonance (HMR) spectroscopy, carbon nuclear magnetic resonance (CMR) spectroscopy, high-performance liquid chromatography (HPLC), and / or single-crystal X-ray diffraction (SCD). In one specific embodiment, the obtained SRG-1 compound is analyzed by HPLC and has a purity of not less than 95%, preferably not less than 98%. In a further embodiment, the structural confirmation data of SRG-1 includes LC-MS, 1H NMR, 13C NMR, HPLC purity, and, if necessary, single-crystal X-ray diffraction data.

[0048] Figure 1 The SRG-1 compound shown in Figure A can be prepared via a one-pot, three-component reaction, exhibiting availability and reproducibility. The resulting SRG-1 compound can serve as the active ingredient for treating KRAS-G12C inhibitor-resistant tumors according to this invention.

[0049] Example 2 Preparation of pharmaceutically acceptable salt of SRG-1

[0050] This example illustrates that the SRG-1 compound can be further prepared into a pharmaceutically acceptable salt.

[0051] 1. Preparation of SRG-1 hydrochloride

[0052] The SRG-1 compound obtained in Example 1 was added to an appropriate amount of anhydrous ethanol, methanol, ethyl acetate, dichloromethane, or a mixture thereof, and stirred until dissolved. Under ice bath or room temperature conditions, a hydrochloric acid-ether solution or a hydrochloric acid-ethanol solution was added dropwise to the above solution to form a salt with hydrochloric acid. After the reaction was complete, the solution was concentrated under reduced pressure or an antisolvent was added to precipitate the salt. After filtration, washing, and drying, SRG-1 hydrochloride was obtained.

[0053] 2. Preparation of SRG-1 methanesulfonate

[0054] The SRG-1 compound obtained in Example 1 was dissolved in a suitable organic solvent, and an equimolar or slightly excess amount of methanesulfonic acid was added. The mixture was stirred at room temperature. After the reaction was complete, the mixture was concentrated under reduced pressure, crystallized, filtered, washed, and dried to obtain SRG-1 methanesulfonate.

[0055] 3. Preparation of SRG-1 fumarate

[0056] The SRG-1 compound obtained in Example 1 was dissolved in ethanol or an ethanol / water mixture, and fumaric acid was added. The mixture was heated and stirred to allow it to react completely. Subsequently, the mixture was cooled to crystallize, filtered, and dried to obtain SRG-1 fumarate.

[0057] The SRG-1 compound can be prepared as a hydrochloride, methanesulfonate, fumarate, or other pharmaceutically acceptable salt. These salts can be used to prepare medicaments for treating tumors associated with resistance to KRAS-G12C inhibitors.

[0058] Example 3: KRAS-G12C Inhibitor-Resistant Tumor Model and Applicable Subjects

[0059] 1. Tumors associated with resistance to KRAS-G12C inhibitors

[0060] The KRAS-G12C inhibitor resistance-related tumors described in this invention refer to solid tumors carrying the KRAS-G12C mutation and exhibiting primary or acquired resistance to KRAS-G12C inhibitor treatment. In a preferred embodiment, the KRAS-G12C inhibitor is Sotorasib or Adagrasib.

[0061] In a more preferred embodiment, the KRAS-G12C inhibitor resistance-associated tumor is a solid tumor that has progressed after treatment with Sotorasib and / or Adagrasib and further carries a secondary KRAS Y96C mutation.

[0062] The solid tumor is a solid tumor carrying the KRAS G12C mutation. More preferably, the solid tumor is non-small cell lung cancer or colorectal cancer carrying the KRAS-G12C-Y96C double mutation.

[0063] 2. Methods for determining drug resistance

[0064] In one specific implementation, resistance to KRAS-G12C inhibitors can be confirmed by one or more of the following methods: detection of KRAS G12C mutations in tumor tissue or circulating tumor DNA; disease progression in patients after treatment with KRAS-G12C inhibitors; further detection of secondary KRAS Y96C mutations in tumor tissue or circulating tumor DNA; and decreased sensitivity of tumor cell models to Sotorasib, AMG510, or Adagrasib.

[0065] The preferred therapeutic target of the present invention is acquired drug-resistant solid tumors of KRAS-G12C inhibitors carrying KRAS-G12C-Y96C double mutations, especially non-small cell lung cancer or colorectal cancer.

[0066] Example 4: Direct binding of SRG-1 to the KRAS-G12C-Y96C double mutant protein

[0067] This example illustrates that SRG-1 can directly bind to the KRAS-G12C-Y96C double mutant protein, supporting its use at the molecular level for the treatment of tumors resistant to KRAS-G12C inhibitors. Experimental results are shown below. Figure 1 B and Figure 1 C.

[0068] 1. Experimental Materials

[0069] The experimental materials included SRG-1 compound, recombinant KRAS-G12C-Y96C double mutant protein, AMG510, and the sensors and buffers required for biomembrane interferometry detection.

[0070] 2. Experimental Methods

[0071] The binding ability of SRG-1 to the KRAS-G12C-Y96C double mutant protein was detected using biomembrane interference technology.

[0072] SRG-1 was prepared into a series of concentration solutions ranging from 4.6875 μM to 150 μM and diluted in a 2-fold gradient.

[0073] The recombinant KRAS-G12C-Y96C double mutant protein was immobilized on the sensor surface, and baseline equilibration, binding detection, and dissociation detection were performed sequentially. The binding phase detection time was approximately 120 seconds, after which the sensor was transferred to a compound-free buffer for dissociation detection. The binding affinity between AMG510 and the KRAS-G12C-Y96C double mutant protein was detected using the same method. Steady-state affinity was fitted based on binding / dissociation curves at different concentrations, and the equilibrium dissociation constant KD was calculated.

[0074] 3. Experimental Results

[0075] like Figure 1 As shown in Figure B, SRG-1 exhibited a dose-dependent binding response to the KRAS-G12C-Y96C double mutant protein within a concentration range of 4.6875 μM to 150 μM. Steady-state affinity fitting revealed that the KD of SRG-1 for the KRAS-G12C-Y96C double mutant protein was 6.64 × 10⁻⁶. -5 M. For example Figure 1 As shown in C, the KD of AMG510 against the KRAS-G12C-Y96C double mutant protein, used as a control, is 3.94 × 10⁻⁶. -3 M. Compared to AMG510, SRG-1 exhibits a stronger binding affinity to the KRAS-G12C-Y96C double mutant protein.

[0076] At two-fold dilutions from 4.6875 μM to 150 μM, the compound exhibited a clear dose-dependent binding response. Its binding kinetics curves displayed a typical "fast binding, fast dissociation" characteristic. In the binding phase (<120 seconds), the molecule rapidly occupied the target site and reached thermodynamic steady state. In the elution phase (to the right of the red dashed line), the binding signal experienced a steep exponential decay, rapidly returning to baseline, indicating that the complex has an extremely short half-life. Steady-state affinity fitting yielded an equilibrium dissociation constant (KD) of 6.64 × 10⁻⁶ for the KRAS-G12C-Y96C protein. -5 The AMG510, however, only measures 3.94×10. -3 This indicates an off-target state. This micromolar-level weak affinity directly confirms at the biophysical level that the key Switch-II pocket residue mutation in the KRAS-G12C-Y96C mutant protein may disrupt the core hydrogen bond network between the drug and protein, causing severe steric hindrance or conformational instability, thereby significantly weakening the AMG510 molecule's anchoring ability to the target protein, thus leading to drug resistance. Meanwhile, the SRG-1 compound still exhibits high affinity for the Y96C mutant protein. This provides direct molecular-level evidence for future clinical application of SRG-1 compound-driven targeted therapy for secondary mutation resistance in Y96C.

[0077] Example 5: Inhibitory effect of SRG-1 on the proliferation of KRAS-G12C inhibitor-sensitive and drug-resistant tumor cells

[0078] This example demonstrates that SRG-1 can inhibit the proliferation of KRAS-G12C inhibitor-sensitive tumor cells and KRAS-G12C-Y96C resistant tumor cells. Experimental results are shown below. Figure 2 A to Figure 2 H and Figure 2 J.

[0079] 1. Experimental Materials

[0080] The experimental cells included parental NCI-H358 lung cancer cells, NCI-H358-Y96C drug-resistant cells carrying the KRAS-G12C-Y96C double mutation, engineered BEAS-2B cells stably overexpressing the KRAS-G12C-Y96C double mutation, as well as human normal bronchial epithelial cells BEAS-2B, human normal lung fibroblasts CCD19Lu, and mouse embryonic fibroblasts NIH3T3. The test compound was SRG-1, and the control drug was AMG510.

[0081] 2. Experimental Methods

[0082] Cell proliferation activity was assessed using the MTT assay. Cells were seeded in 96-well plates, and after cell attachment, different concentrations of SRG-1 or AMG510 were added. After 72 hours of treatment, MTT reagent was added for further incubation. The supernatant was then discarded, and DMSO was added to dissolve formazan crystals. Absorbance values ​​were measured using a microplate reader. Cell viability was calculated for each treatment group, using the untreated group or the excipient group as controls, and the half-maximal inhibitory concentration (IC50) was calculated based on the dose-response curve.

[0083] 3. Experimental Results

[0084] like Figure 2 As shown in Figure A, AMG510 exhibited a strong inhibitory effect on the proliferation of KRAS G12C single-mutant NCI-H358 parental cells, with an IC50 of 23.62 nM. Figure 2 As shown in Figure B, the proliferation inhibitory activity of AMG510 was significantly reduced in NCI-H358-Y96C drug-resistant cells carrying the KRAS-G12C-Y96C double mutation.

[0085] like Figure 2 D and Figure 2 As shown in Figure C, SRG-1 exhibited inhibitory effects on the proliferation of both NCI-H358 parental cells and NCI-H358-Y96C resistant cells. Specifically, the IC50 of SRG-1 against NCI-H358 parental cells was 14.99 μM, and the IC50 against NCI-H358-Y96C resistant cells was 17.02 μM.

[0086] like Figure 2 As shown in E, in engineered BEAS-2B cells that stably overexpress the KRAS-G12C-Y96C double mutant, SRG-1 still exhibited a significant inhibitory effect on proliferation, with an IC50 of 10.44 μM.

[0087] like Figure 2 F, Figure 2 H and Figure 2As shown in J, in parallel toxicology tests, SRG-1 did not show significant cytotoxicity against non-malignant cells such as BEAS-2B, CCD19Lu, and NIH3T3.

[0088] These results indicate that SRG-1 can inhibit not only the proliferation of KRAS-G12C inhibitor-sensitive tumor cells, but also the proliferation of drug-resistant tumor cells carrying the KRAS-G12C-Y96C double mutation. Unlike AMG510, which shows reduced activity in KRAS-G12C-Y96C-resistant cells, SRG-1 maintains its inhibitory effect on KRAS-G12C-Y96C-resistant cells. Furthermore, SRG-1 did not exhibit significant cytotoxicity against non-malignant cells, suggesting its potential for treating tumors associated with KRAS-G12C inhibitor resistance.

[0089] Example 6: SRG-1 induces apoptosis in KRAS-G12C-Y96C resistant tumor cells

[0090] This example demonstrates that SRG-1 can induce apoptosis in KRAS-G12C-Y96C drug-resistant tumor cells. Experimental results are shown below. Figure 2 I.

[0091] 1. Experimental Materials

[0092] The experimental cells were NCI-H358-Y96C drug-resistant cells. The test compound was SRG-1. The apoptosis detection reagent was the Annexin V / PI apoptosis detection kit or other equivalent flow cytometry apoptosis detection reagent.

[0093] 2. Experimental Methods

[0094] NCI-H358-Y96C cells were seeded in culture plates. After cell adhesion and entry into the logarithmic growth phase, they were treated with 8 μM, 16 μM, and 36 μM SRG-1 for 24 hours, respectively. A control group without treatment was set up. After treatment, cells were collected and stained according to the apoptosis detection kit instructions. Flow cytometry was then used to detect the proportions of early and late apoptotic cells. Each group was performed in triplicate.

[0095] 3. Experimental Results

[0096] like Figure 2 As shown in Figure I, the basal spontaneous apoptosis rate of the untreated control group NCI-H358-Y96C cells was 14.85%. After SRG-1 treatment, as the SRG-1 concentration increased from 8 μM to 16 μM and 36 μM, the apoptosis rate gradually increased and showed a statistically significant difference compared with the control group.

[0097] These results demonstrate that SRG-1 can induce apoptosis in KRAS-G12C-Y96C resistant tumor cells in a concentration-dependent manner. This supports the potential use of SRG-1 in the development of drugs for treating tumors resistant to KRAS-G12C inhibitors and supports the possibility that its mechanism of action includes inducing tumor cell apoptosis.

[0098] Example 7: Antitumor effect of SRG-1 in a KRAS-G12C-Y96C drug-resistant tumor animal model

[0099] This example demonstrates that SRG-1 can inhibit the growth of KRAS-G12C-Y96C resistant tumors and prolong the survival of tumor-bearing animals in vivo. Experimental results are shown below. Figure 3 A to Figure 3 H.

[0100] 1. Experimental Materials

[0101] The experimental animals were immunocompetent C57BL / 6 mice. The tumor cells were MC38-Y96C cells carrying the KRAS-G12C-Y96C double mutation. The test compound was SRG-1. The control group used the excipient Vehicle, and an AMG510 control group was also included.

[0102] 2. Modeling and Grouping

[0103] Log-phase MC38-Y96C cells were digested, counted, and resuspended in suitable buffer or culture medium. The cells were then subcutaneously seeded into C57BL / 6 mice to establish an MC38-Y96C syngeneic subcutaneous xenograft model. Once the tumor volume reached the drug administration criteria, the mice were randomly divided into a Vehicle control group, an AMG510 group, a low-dose SRG-1 group, and a high-dose SRG-1 group.

[0104] In one specific implementation, the low-dose and high-dose groups of SRG-1 were administered different doses of SRG-1 to evaluate its dose-dependent antitumor effect. The specific dosage, route of administration, and frequency of administration of SRG-1 and AMG510 can be determined according to the experimental protocol or preclinical study requirements.

[0105] 3. Detection Indicators

[0106] During drug administration, the long and short diameters of the tumor were measured periodically, and the tumor volume was calculated using the formula: Tumor Volume = 1 / 2 × Long Diameter × Short Diameter². The mouse weight and general condition were also recorded. At the experimental or humanitarian endpoint, the animals were sacrificed, the tumor tissue was dissected, photographed, and weighed. Survival curves were plotted based on the survival status of each group of animals.

[0107] 4. Experimental Results

[0108] like Figure 3 A and Figure 3 As shown in Figure B, compared with the Vehicle control group, the SRG-1 treatment group showed significant in vivo antitumor effects. Both the low-dose and high-dose SRG-1 groups were able to inhibit the progression of MC38-Y96C resistant tumors, showing a dose-dependent trend.

[0109] like Figure 3 E to Figure 3 As shown in H, individualized tumor growth curves indicate that tumor progression in the SRG-1 treatment group was consistently suppressed, with no significant early rebound observed. Figure 3 As shown in Figure D, the survival analysis results showed that, compared with the Vehicle control group, the overall survival of tumor-bearing mice in the SRG-1 treatment group was prolonged, with the high-dose SRG-1 group maintaining a high survival rate during the 30-day observation period.

[0110] like Figure 3 As shown in Figure C, the weight monitoring results showed that the weight of mice in the SRG-1 treatment group did not decrease significantly compared with the Vehicle control group, and no obvious drug-related systemic toxicity was observed.

[0111] SRG-1 was able to inhibit tumor growth and prolong the survival of tumor-bearing animals in a KRAS-G12C-Y96C-resistant tumor animal model, without observing significant weight loss or systemic toxicity. These results support the use of SRG-1 or a pharmaceutically acceptable salt thereof in the preparation of drugs for treating KRAS-G12C inhibitor-resistant tumors.

[0112] Example 8: SRG-1 reduces Treg levels and promotes infiltration of anti-tumor immune effector cells.

[0113] This example illustrates that SRG-1 can reduce regulatory T cell levels and promote the infiltration of anti-tumor immune effector cells, thereby reshaping the immune microenvironment of KRAS-G12C-Y96C resistant tumors. Experimental results are shown below. Figure 3 I arrive Figure 3 N.

[0114] 1. Experimental Materials

[0115] Peripheral blood, spleen, and tumor tissue were collected from mice at the experimental endpoint in Example 7. Detection antibodies included flow cytometry antibodies such as CD45, CD4, Foxp3, NK1.1, Granzyme B, and IFN-γ, or other equivalent antibodies.

[0116] 2. Experimental Methods

[0117] At the experimental endpoint, peripheral blood, spleen, and tumor tissue were collected from mice. Tumor tissue was mechanically minced and enzymatically digested to prepare a single-cell suspension; spleen was ground, filtered, and lysed with erythrocytes to prepare a single-cell suspension; peripheral blood was lysed with erythrocytes to obtain a leukocyte suspension. Flow cytometry was used to detect the proportion of CD4+Foxp3+Treg cells in different tissues, and the infiltration level of NK1.1+ natural killer cells and the expression levels of Granzyme B and IFN-γ in tumor tissue were further examined.

[0118] 3. Experimental Results

[0119] like Figure 3 I arrive Figure 3 As shown in K, compared with the Vehicle control group, SRG-1 treatment can reduce the proportion of CD4+Foxp3+Treg cells in the peripheral blood, spleen and tumor microenvironment of tumor-bearing mice, with the Treg level in the tumor microenvironment showing a dose-dependent decreasing trend.

[0120] like Figure 3 As shown in N, SRG-1 treatment can increase the infiltration level of NK1.1+ natural killer cells in tumor tissue. Figure 3 L and Figure 3 As shown in M, the expression levels of antitumor effector molecules such as Granzyme B and IFN-γ increased in tumor tissue after SRG-1 treatment.

[0121] These results indicate that SRG-1 can not only directly inhibit the growth of KRAS-G12C-Y96C resistant tumors, but also improve the tumor immune microenvironment by reducing Treg levels, promoting NK cell infiltration, and enhancing Granzyme B and IFN-γ expression. These results support the use of SRG-1 to reduce regulatory T cell levels and promote the infiltration of anti-tumor immune effector cells.

[0122] Example 9: KRAS-mutant tumor cells induce PBMC polarization towards Treg and block SRG-1.

[0123] This example illustrates that KRAS-mutant tumor cells can induce peripheral blood monocytes to polarize towards the Treg axis, and that SRG-1 can block this immunosuppressive process. Experimental results are shown below. Figure 4 .

[0124] 1. Experimental Materials

[0125] Tumor cells included non-small cell lung cancer cells H23 and H2122 carrying the KRAS G12C mutation, as well as the corresponding H23-Y96C and H2122-Y96C cells. Control cells were BEAS-2B cells. Immune cells were peripheral blood mononuclear cells (PBMCs) derived from healthy donors. The test compound was SRG-1.

[0126] 2. Experimental Methods

[0127] PBMCs were co-cultured with BEAS-2B, H23, H23-Y96C, H2122, or H2122-Y96C cells, respectively. After co-culture, cells were collected and single-cell suspensions were prepared. The proportion of CD4+Foxp3+ Treg cells was detected by flow cytometry. In the SRG-1 intervention experiment, different concentrations of SRG-1 were added to the co-culture system, with the Vehicle treatment group as a control, to detect the effect of SRG-1 on Treg polarization.

[0128] 3. Experimental Results

[0129] like Figure 4 As shown, compared with the control group co-cultured with PBMCs and BEAS-2B cells, the proportion of CD4+Foxp3+ Treg cells increased after PBMCs were co-cultured with KRAS-mutant tumor cells H23, H23-Y96C, H2122, or H2122-Y96C. This result suggests that KRAS-mutant tumor cells can promote the polarization of PBMCs towards an immunosuppressive Treg phenotype. The addition of SRG-1 inhibited this Treg polarization trend, indicating that SRG-1 can block the immunosuppressive regulation induced by KRAS-mutant tumor cells.

[0130] This embodiment demonstrates that KRAS-mutant tumor cells can promote Treg amplification or polarization, while SRG-1 can inhibit this process. These results further support the use of SRG-1 to treat KRAS-G12C inhibitor-resistant tumors by reducing Treg levels and relieving immunosuppression.

[0131] Example 10: SRG-1 enhances PBMC-mediated killing of KRAS-G12C-Y96C resistant tumor cells

[0132] This example illustrates that SRG-1 can enhance the immunocytotoxic effect of PBMCs against KRAS-mutant and KRAS-G12C-Y96C resistant tumor cells. Experimental results are shown below. Figure 5 A to Figure 5 F.

[0133] 1. Experimental Materials

[0134] Target cells were H23, H23-Y96C, H2122, and H2122-Y96C tumor cells stably expressing luciferase. Effector cells were healthy donor-derived PBMCs. The assay compound was SRG-1. The assay substrate was a luciferase substrate.

[0135] 2. Experimental Methods

[0136] Stable luciferase-expressing target tumor cells were seeded into culture plates and then co-cultured with PBMCs. The effector-to-target ratio (E:T) was set to 5:1. Different concentrations of SRG-1 were added to the co-culture system, with the Vehicle group serving as a control. After co-culture, luciferase substrate was added, and the luminescence intensity of each well was measured. The luminescence intensity was positively correlated with the number of surviving target cells. The killing effect of PBMCs on target tumor cells was evaluated based on the decrease in luminescence intensity.

[0137] 3. Experimental Results

[0138] like Figure 5 A to Figure 5 As shown in Figure F, compared with the Vehicle control group, the relative luciferase activities of target cells H23, H23-Y96C, H2122, and H2122-Y96C were significantly decreased in the SRG-1 treatment group, showing a dose-dependent trend. This result suggests that SRG-1 can enhance the killing effect of PBMCs on KRAS-mutant tumor cells, especially those carrying secondary drug-resistant Y96C mutations.

[0139] SRG-1 enhances the immunogenicity of PBMCs against KRAS-G12C-Y96C resistant tumor cells. This result supports the use of SRG-1 in the development of drugs for treating KRAS-G12C inhibitor-resistant tumors and demonstrates its ability to promote anti-tumor immune responses.

[0140] Example 11: Combined treatment of KRAS-G12C-Y96C resistant tumors with SRG-1 and anti-CD25 antibody

[0141] This example illustrates that the combined use of SRG-1 and anti-CD25 antibody can produce an enhanced anti-tumor effect. Experimental results are shown below. Figure 6 A to Figure 6 H.

[0142] 1. Experimental Materials

[0143] The experimental animals were immunocompetent C57BL / 6 mice. The tumor cells were MC38-Y96C cells. The test compound was SRG-1. The combined drug component was an anti-CD25 antibody or its antigen-binding fragment, preferably an anti-CD25 monoclonal antibody with Treg depletion activity. The control group used an isotype control antibody or a vehicle.

[0144] 2. Modeling and Grouping

[0145] A subcutaneous xenograft model of MC38-Y96C was established according to the method described in Example 7. After the tumor volume reached the drug administration standard, the tumor-bearing mice were randomly divided into Vehicle or isotype control group, anti-CD25 antibody monotherapy group, SRG-1 monotherapy group, and SRG-1 and anti-CD25 antibody combined drug therapy group.

[0146] In one embodiment, SRG-1 and anti-CD25 antibody are administered simultaneously. In another embodiment, SRG-1 and anti-CD25 antibody are administered sequentially. In yet another embodiment, SRG-1 is administered first to inhibit KRAS-G12C-Y96C resistant tumor cells and reduce Treg induction signaling, followed by administration of anti-CD25 antibody to further eliminate Tregs.

[0147] 3. Detection of tumor growth and immune microenvironment

[0148] Tumor volume was measured periodically during drug administration. At the experimental endpoint, tumor tissue was dissected, photographed, and weighed. Tumor volume and weight were compared among the groups to evaluate the antitumor efficacy of single-drug and combination therapy. At the experimental endpoint, single-cell suspensions were prepared from tumor tissues of each group, and flow cytometry was used to detect the proportions of CD4+Foxp3+Treg cells, CD8+Granzym B+ cells, CD8+IFN-γ+ cells, CD8+Ki67+ cells, and NK1.1+ cells.

[0149] 4. Experimental Results

[0150] like Figure 6 A and Figure 6 As shown in Figure B, both anti-CD25 antibody monotherapy and SRG-1 monotherapy were able to inhibit the growth of MC38-Y96C tumors. Compared with either monotherapy group, the combination therapy of SRG-1 and anti-CD25 antibody showed stronger tumor inhibition, further reduction in tumor weight, and significant reduction in tumor burden in some animals.

[0151] like Figure 6 As shown in G, anti-CD25 antibody can reduce CD4+Foxp3+ Treg levels in the tumor microenvironment, and SRG-1 monotherapy can also reduce Treg levels. Treg levels were further reduced in the combination therapy group.

[0152] like Figure 6 C to Figure 6 F and Figure 6 As shown in Figure H, the proportions of CD8+GranzymeB+ cells, CD8+IFN-γ+ cells, CD8+Ki67+ cells, and NK1.1+ cells were increased in the combined treatment group, suggesting that the combined treatment can enhance the local cytotoxic immune response in the tumor.

[0153] The combined use of SRG-1 and anti-CD25 antibodies enhances the therapeutic effect against KRAS-G12C-Y96C resistant tumors. This enhancement is associated with reduced Treg levels, enhanced CD8+ effector T cell function, promoted NK cell infiltration, and increased expression of cytotoxic effector molecules. These results support the use of SRG-1 or a pharmaceutically acceptable salt thereof in combination with anti-CD25 antibodies or their antigen-binding fragments in the preparation of drugs for treating KRAS-G12C inhibitor-resistant tumors.

[0154] Example 12: Biomarker combinations for screening suitable patients or suitable tumor models

[0155] This embodiment is used to illustrate the preferred application of SRG-1 monotherapy or SRG-1 combined with anti-CD25 antibody in this invention.

[0156] 1. Test Sample

[0157] The test samples can be tumor tissue, blood samples, circulating tumor DNA, peripheral blood mononuclear cells, tumor-infiltrating immune cells, or a combination thereof.

[0158] 2. Detection Indicators

[0159] In one specific implementation, suitable recipients of SRG-1 therapy possess one or more of the following characteristics: presence of a KRAS G12C mutation; disease progression following treatment with a KRAS-G12C inhibitor; further, presence of a secondary KRAS Y96C mutation; decreased sensitivity of tumor cells to Sotorasib, AMG510, or Adagrasib; increased proportion of CD4+Foxp3+Treg cells in the tumor microenvironment; decreased CD8 / Treg ratio in the tumor microenvironment; insufficient infiltration of NK1.1+ cells in the tumor; and low expression levels of Granzyme B or IFN-γ.

[0160] 3. Preferred Targets

[0161] In a preferred embodiment, patients suitable for receiving combination therapy with SRG-1 and anti-CD25 antibody are those who have developed acquired resistance after treatment with KRAS-G12C inhibitors; have been diagnosed with KRAS-G12C-Y96C double mutations; have elevated Treg levels in the tumor microenvironment; or have a decreased CD8 / Treg ratio in solid tumors.

[0162] By combining the above biomarkers, we can screen for patient groups that are more suitable for receiving SRG-1 monotherapy or SRG-1 combined with anti-CD25 antibody therapy, thereby improving the treatment efficacy of tumors associated with KRAS-G12C inhibitor resistance.

[0163] Example 13 Pharmaceutical composition containing SRG-1 and anti-CD25 antibody

[0164] This embodiment illustrates a pharmaceutical composition for treating tumors associated with resistance to KRAS-G12C inhibitors.

[0165] 1. Composition of the composition

[0166] A pharmaceutical composition comprising an SRG-1 compound or a pharmaceutically acceptable salt thereof, an anti-CD25 antibody or an antigen-binding fragment thereof, and pharmaceutically acceptable excipients and / or a carrier. The SRG-1 compound or a pharmaceutically acceptable salt thereof can be formulated as an oral formulation, an injectable formulation, a lyophilized formulation, a suspension, a liposome formulation, a nanoparticle formulation, or other pharmaceutical dosage forms suitable for cancer treatment. The anti-CD25 antibody or its antigen-binding fragment can be formulated as an injection solution or a lyophilized powder for injection.

[0167] 2. Exemplary Formulation

[0168] In one embodiment, the SRG-1 compound or a pharmaceutically acceptable salt thereof is mixed with pharmaceutically acceptable excipients to prepare an oral tablet or capsule. The excipients include fillers, disintegrants, binders, lubricants, solubilizers, or stabilizers. In another embodiment, the SRG-1 compound or a pharmaceutically acceptable salt thereof is prepared as an injectable formulation. The injectable formulation includes one or more of water for injection, physiological saline, buffered saline solution, solubilizer, stabilizer, isotonic regulator, and pH adjuster. In another embodiment, an anti-CD25 antibody or its antigen-binding fragment is formulated with a buffer, stabilizer, surfactant, and isotonic regulator to form an antibody injection solution. In yet another embodiment, the SRG-1 formulation and the anti-CD25 antibody formulation are prepared separately and placed together in the same kit. In use, the SRG-1 and anti-CD25 antibody can be administered simultaneously, separately, or sequentially.

[0169] 3. Applications

[0170] The pharmaceutical composition is used to treat KRAS-G12C inhibitor-resistant tumors. In a preferred embodiment, the KRAS-G12C inhibitor-resistant tumor is a solid tumor that has acquired resistance after treatment with sotorasib. In a further embodiment, the KRAS-G12C inhibitor-resistant tumor is a solid tumor that has acquired resistance after treatment with adagrasib. In a more preferred embodiment, the KRAS-G12C inhibitor-resistant tumor is a solid tumor carrying a KRAS-G12C-Y96C double mutation. The solid tumor includes non-small cell lung cancer, colorectal cancer, pancreatic cancer, biliary tract cancer, ovarian cancer, or other solid tumors carrying KRAS G12C mutations.

[0171] SRG-1 compounds or pharmaceutically acceptable salts thereof may be combined with anti-CD25 antibodies or their antigen-binding fragments to form a pharmaceutical composition for the treatment of tumors associated with KRAS-G12C inhibitor resistance. This pharmaceutical composition exerts its therapeutic effects by inhibiting tumor cell proliferation, inducing tumor cell apoptosis, reducing Treg levels, and promoting the infiltration of anti-tumor immune effector cells.

[0172] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of SRG-1 compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of tumors associated with resistance to KRAS-G12C inhibitors.

2. The use according to claim 1, wherein, The KRAS-G12C inhibitor resistance-related tumors are tumors that are insensitive to Sotorasib and / or Adagrasib treatment, or that are primary or acquiredly resistant to the drug.

3. The use according to claim 1 or 2, wherein, The KRAS-G12C inhibitor resistance-related tumors are solid tumors carrying KRAS G12C mutations.

4. The use according to claim 3, wherein, The KRAS-G12C inhibitor resistance-associated tumors further carry secondary KRAS Y96C mutations.

5. The use according to any one of claims 1 to 4, wherein, The drug is used to inhibit tumor cell proliferation, induce tumor cell apoptosis, reduce regulatory T cell levels, and / or promote the infiltration of anti-tumor immune effector cells.

6. Use of an SRG-1 compound or a pharmaceutically acceptable salt thereof in combination with an antiCD25 antibody or an antigen-binding fragment thereof in the preparation of a medicament for the treatment of tumors associated with resistance to KRAS-G12C inhibitors.

7. The use according to claim 6, wherein, The anti-CD25 antibody or its antigen-binding fragment is used to reduce the level of regulatory T cells (Tregs) in the tumor microenvironment.

8. A pharmaceutical composition comprising: (1) SRG-1 compound or a pharmaceutically acceptable salt thereof; (2) Anti-CD25 antibody or its antigen-binding fragment; and (3) Pharmaceutically acceptable excipients and / or carriers.

9. The pharmaceutical composition according to claim 8, wherein, The pharmaceutical composition is used to treat tumors associated with resistance to KRAS-G12C inhibitors.

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