Blocking IL-34 signal as immunotherapy for p53 mutant liver cancer

Abstract

The invention relates to blocking of IL-34 signal as an immunotherapy for p53 mutant liver cancer. Specifically, the invention relates to application of an IL-34 inhibitor in preparation of a medicine or a kit for treating p53 mutation liver cancer. On the other hand, the invention further relates to application of the IL-34 inhibitor and the anti-PD-1 antibody to preparation of a medicine or a kit for treating the p53 mutation liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of treatment for p53-mutant liver cancer. Background Technology

[0002] Primary liver cancer (PLC) remains a global health challenge, with an estimated incidence exceeding one million cases by 2025 (Llovet et al., 2021). Hepatocellular carcinoma (HCC) accounts for 75% to 85% of PLC cases, while intrahepatic cholangiocarcinoma (ICC) accounts for 10% to 15%. Treatment options for liver cancer are very limited; for advanced HCC, only a few multi-kinase inhibitors offer very limited survival benefits (Ray and Sanoff, 2017). The tumor microenvironment (TME) plays a crucial role in tumor progression and response to treatment, and immunotherapy, represented by immune checkpoint inhibitors (ICIs), has shown potent antitumor activity in some liver cancer patients. However, due to the high heterogeneity of the tumor microenvironment among different liver cancer patients, ICIs are not beneficial to all patients (Sangro et al., 2021).

[0003] Tumor-associated macrophages (TAMs) are the main immunosuppressive cells in the tumor microenvironment. Macrophages can be divided into two subtypes: classically activated macrophages (M1) and alternately activated macrophages (M2). TAMs primarily exhibit an M2 polarized phenotype, enabling them to regulate immune responses, promote angiogenesis, and accelerate tumor progression, invasion, and metastasis. M1 and M2 macrophages have different metabolic phenotypes; M1 macrophages rely on aerobic glycolysis, while M2 macrophages use fatty acid oxidation to promote mitochondrial oxidative phosphorylation. TAM recruitment and polarization are regulated by numerous chemokines and cytokines, such as CCL2 and CSF1 (Mantovani et al., 2022). IL-34, described as a “twin” cytokine of CSF-1, also acts on CSF1R, promoting the growth, survival, and differentiation of monocytes into macrophages (Baghdadi et al., 2018). IL-34 is highly expressed in a variety of cancers and promotes tumor progression (Franzè et al., 2020). (et al., 2021).

[0004] TP53 (encoding the p53 protein, gene ID: 7157) is the most commonly mutated gene in human cancers. Most TP53 mutations occur in the DNA-binding domain, leading to inactivation of its transcriptional function. Loss of p53 protein function is considered a driver of various tumors (Hassin and Oren, 2022). As a transcription factor, p53 is a sequence-specific DNA-binding protein that can directly activate the transcription of many genes, including CDKN1A and MDM2, and can also directly inhibit the expression of many genes, such as CPS1 and NANOG. p53 is known as the "guardian of the genome" because it can induce cell cycle arrest and apoptosis in cells (Hassin and Oren, 2022). In mouse models, reactivation of p53 has proven to be an effective cancer therapy, leading to regression in various tumor types. Notably, reactivation of p53 to induce tumor regression requires the participation of the immune system (Hassin and Oren, 2022). Summary of the Invention

[0005] This invention relates to novel immunotherapeutic targets for p53-mutant liver cancer. We discovered that IL-34 is a specific target for p53-mutant liver cancer, and blocking this target can effectively inhibit the growth of p53-mutant liver cancer. Combined with PD-1 antibodies, this can achieve complete tumor eradication.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] 1. Use of IL-34 inhibitors in the preparation of drugs or kits for the treatment of p53-mutant liver cancer.

[0008] 2. According to the use described in Project 1, wherein the IL-34 inhibitor is an anti-IL-34 antibody or an oligonucleotide for knocking out the IL34 gene; preferably, the sequence of the oligonucleotide for knocking out the IL34 gene is shown in SEQ ID NO:3.

[0009] 3. As described in Project 1, wherein the kit further comprises an anti-PD-1 antibody.

[0010] 4. As described in Project 1, wherein the IL-34 inhibitor is in a form suitable for subcutaneous or intravenous injection.

[0011] 5. Use of IL-34 inhibitors and anti-PD-1 antibodies in the preparation of drugs or kits for the treatment of p53-mutant hepatocellular carcinoma.

[0012] 6. As described in Item 5, wherein the IL-34 inhibitor is an anti-IL-34 antibody or an oligonucleotide for knocking out the IL34 gene.

[0013] 7. As described in Item 5, wherein the IL-34 inhibitor and the anti-PD-1 antibody are in a form suitable for subcutaneous or intravenous injection.

[0014] 8. A kit comprising an IL-34 inhibitor and an anti-PD-1 antibody.

[0015] 9. The kit according to Project 8, wherein the IL-34 inhibitor is an anti-IL-34 antibody or an oligonucleotide for knocking out the IL34 gene.

[0016] In summary, p53 mutations are believed to promote tumorigenesis and play a crucial role in immune surveillance of tumor progression. Our study, using a mouse model of spontaneous hepatocellular carcinoma with different gene mutations, revealed that IL-34 is an indispensable cytokine in p53-mediated immune escape from hepatocellular carcinoma. Mechanistically, IL-34 is a target gene negatively regulated by p53. P53-mutant hepatocellular carcinoma cells secrete IL-34, which acts on tumor-associated macrophages, causing them to metabolically reprogram into immunosuppressive macrophages, thereby promoting tumor immune escape. Blocking IL-34 with antibodies can effectively inhibit the development and progression of p53-mutant hepatocellular carcinoma. Combining the blocking of IL-34 signaling and PD-1 signaling can achieve even better therapeutic effects.

[0017] In this article, TP53 refers to the human p53 gene, Trp53 refers to the mouse p53 gene, and p53 refers to the protein encoded by TP53.

[0018] In this article, "p53-mutant liver cancer" refers to liver cancer caused by missense mutations, nonsense mutations, frameshift mutations, deletion mutations, insertion mutations, point mutations, gain-of-function mutations, and compound mutations in the p53 gene, resulting in the loss of the original tumor-suppressive function of the p53 protein, thereby leading to hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and mixed-type liver cancer. In the prior art, commonly available commercially available kits (such as p53 gene detection kits, such as those from Synotech) can be used to detect whether liver cancer is p53-mutant.

[0019] In this document, the anti-IL-34 antibody refers to conventional anti-IL-34 antibodies in the prior art, such as the IL-34 antibody (Cat#MAB5195) produced by R&D Company. Attached Figure Description

[0020] Figure 1 The diagram shows the construction of a p53-mutant liver cancer model and a non-p53-mutant liver cancer model; (A) is a schematic diagram of the liver cancer model construction; (B) is a physical image of a liver cancer cell.

[0021] Figure 2 A schematic diagram of the construction of primary liver cancer cell lines is shown;

[0022] Figure 3 RNA sequencing showed that Il34 is the most significantly upregulated cytokine in p53-mutant liver cancer;

[0023] Figure 4 The results showed that IL-34 levels were significantly upregulated in p53-mutant hepatocellular carcinoma as demonstrated by qPCR and ELISA assays.

[0024] Figure 5 Data from the TCGA database showing hepatocellular carcinoma (HCC) revealed that the level of IL-34 in p53-mutant HCC was significantly higher than that in p53-normal HCC.

[0025] Figure 6 The results showed that hepatocellular carcinoma tissues with p53 mutations highly expressed IL-34, while hepatocellular carcinoma tissues with normal p53 did not express IL-34; A shows the identification of TP53 gene mutations in human hepatocellular carcinoma tissues by gene sequencing; B shows that Western blot analysis showed that hepatocellular carcinoma tissues with TP53 mutations highly expressed IL-34, while hepatocellular carcinoma tissues with normal TP53 did not express IL-34.

[0026] Figure 7 Showing the Il34 + / - and Il34 - / - Image of a mouse liver after spontaneous hepatocellular carcinoma induced by high-pressure injection of the CRISPR Cas9 plasmid sgTrp53+sgPten into the tail vein. Tumor formation in the mouse liver is reflected by the liver weight / body weight ratio. The sgGFP plasmid is the control plasmid.

[0027] Figure 8 Showing the Il34 + / - and Il34 - / - Survival rate of mice with spontaneous liver cancer induced by high-pressure injection of sgTrp53+sgPten plasmid into the tail vein; sgGFP plasmid was used as the control plasmid.

[0028] Figure 9 The results show that IL-34 was significantly upregulated at both the transcriptional and protein levels after knocking out the Trp53 gene; (A) shows that the expression of the downstream protein p21 of p53 was not induced by the p53 activator nutlin3, proving that the p53 gene was successfully knocked out in CT cells; (B) shows the expression of IL-34 transcriptional level after knocking out the p53 gene in CT cells by quantitative PCR; (C) shows the expression of IL-34 protein level after knocking out the p53 gene in CT cells by Western blot analysis.

[0029] Figure 10 The study showed that IL-34 antibody treatment can significantly inhibit the growth of p53-mutant liver cancer;

[0030] Figure 11 The study showed that IL-34 antibody treatment could significantly reduce macrophage infiltration in p53-mutant tumors;

[0031] Figure 12 The combined gene knockout of IL-34 and antibody blockade of PD-1 showed that p53-mutant tumors could be completely eliminated;

[0032] Figure 13 The combined gene knockout of IL-34 and antibody blockade of PD-1 showed that p53-mutant liver cancer could be completely eliminated;

[0033] Figure 14 The study showed that combined antibody blocking of IL-34 and antibody blocking of PD-1 could completely eliminate p53-mutant tumors. IgG represents the control group, and αIL34+αPD-1 represents the experimental group, which received combined injections of anti-IL34 antibody and anti-PD1 antibody.

[0034] Materials and methods

[0035] mouse

[0036] Five-week-old female C57BL / 6 mice were purchased from Slack, and Il34+ / - mice (C57BL / 6 background) were purchased from the Southern Species and crossbred to obtain Il34+ / - and Il34- / - mice. Genotyping was performed using standard PCR protocols. All experiments used littermate and cage-mate controls. All animals were kept under specific pathogen-free conditions. All experimental procedures involving mice were performed in accordance with the National Animal Research Guidelines (China).

[0037] Cell lines and in vitro culture

[0038] Primary mouse hepatocellular carcinoma (HCC) cell lines were generated as follows: Ctnnb1+Pten (CT) and Trp53+Pten (PT) HCC cells were collected in ice-cold PBS and, following the manufacturer's instructions, were ground and digested using a mouse tumor dissociation kit (Miltenyi Biotec, 130-096-730) and a gentleMACS Dissociator (Miltenyi, 130-093-235). The suspension was then dispersed through a 40 μm cell filter. Cells were initially cultured in medium containing 10% FCS, 100 IU ml-1 penicillin, 100 mg ml-1 streptomycin, insulin, and epidermal growth factor to establish cell lines. After establishment, mouse cell lines were cultured in DMEM medium supplemented with 10% FCS, 100 IU ml-1 penicillin, 100 mg ml-1 streptomycin, and 2 mM L-glutamine. To ensure relevance to parental GEMM tumors, all experiments used low-passage polyclonal cells.

[0039] 293T cells were purchased from the Shanghai Cell Bank (Shanghai, Chinese Academy of Sciences). All cell lines were mycoplasma negative. 293T cells were cultured in DMEM containing 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, and 292 μg / ml L-glutamine.

[0040] Vector construction and cell transfection

[0041] The CRISPR Cas9 plasmids targeting the mutant Trp53 gene (also referred to as "sgTrp53 CRISPR Cas9 plasmids" in this paper), the CRISPR Cas9 plasmids targeting the Pten gene (also referred to as "sgPten CRISPR Cas9 plasmids" in this paper), and the CRISPR Cas9 plasmids targeting Il34 (also referred to as "sgIl34 CRISPR Cas9 plasmids" in this paper) were constructed using conventional methods in the prior art. The blank vectors used were PX330 vectors or LentiCRISPRv2GFP vectors (both purchased from Addgene). The sgRNA sequences are shown in Table 1.

[0042] Table 1, sgRNA sequences

[0043]

[0044] Construction of spontaneous liver cancer model

[0045] Chronic hepatitis was induced in 5-week-old female C57BL / 6 mice, IL34+ / - mice, and IL34- / - mice by intraperitoneal injection of CCL4 for 3 weeks. Following this, hydrodynamic liver injection of the corresponding plasmids to mutate tumor suppressor and oncogenes in the mouse liver was performed. Specifically, plasmid DNA suspended in 2 ml of saline was injected via the tail vein into 8-week-old female C57BL / 6 mice over 5–7 seconds. The injected DNA consisted of 40 μg of transposon plasmid and 20 μg of PB transposase plasmid (see Tang et al., 2022) or 60 μg of pX330 plasmid encoding Cas9 and sgRNA. Animals were monitored for up to one year. Animals were euthanized at specific time points after injection or when signs of tumor development, such as significant abdominal enlargement, appeared. The liver was photographed and weighed, and then fixed or frozen for further processing.

[0046] Immunoblotting

[0047] Cells were lysed using RIPA buffer (ThermoFisher) containing a cocktail of 1 mM PMSF (Beyotime) and a phosphatase inhibitor (APEXBIO, 100×). Lysis was performed on ice for 30 minutes, followed by centrifugation at 12000g for 10 minutes, and the supernatant was collected. After electrophoresis, the separated proteins were electrophoresed onto a PVDF membrane and blocked with 5% w / v milk for 30 minutes at room temperature. The membrane was then incubated overnight at 4°C with a primary antibody (1:1000 dilution). Protein levels were then detected using an HRP-bound antibody (Sangon) as a secondary antibody and an enhanced chemiluminescent substrate (ThermoFisher). The antibodies used were: IL-34 antibody (abcam, ab75723) and GAPDH antibody (Sangon, D110016).

[0048] q-PCR

[0049] RNA was extracted from cells or tissues using an RNA extraction kit (omega). The extracted RNA was then reverse transcribed into cDNA using a reverse transcription kit (Akerui). The cDNA was used as a template for qPCR using SYBR Green (Akerui). The primers for qPCR are listed in Table 2. Each sample was repeated three times. Relative mRNA expression was calculated using the ΔΔCT method.

[0050] Table 2, PCR primers

[0051]

[0052] ELISA

[0053] Collect the ground tissue supernatant and follow the instructions of the ELISA kit manufacturer. The ELISA kit used is as follows: IL34 ELISA kit (R&D).

[0054] Preparation of Trp53 knockout CT cells and Il34 knockout PT cells

[0055] Lentiviral vectors targeting sgTrp53 and sgIl34 (purchased from Addgene), ps.ppAX2 vector (purchased from Addgene), and pMD.2G vector (purchased from Addgene) were transfected into 293T cells (Shanghai, Chinese Academy of Sciences). Supernatants were collected at 48 and 72 hours and used to infect CT and PT cells, respectively. Successful knockout of Trp53 in CT cells and successful knockout of IL34 in PT cells were verified by qPCR and Western blot.

[0056] Tumor implantation and treatment in tumor-bearing mice

[0057] For the treatment of subcutaneous tumors with antibodies, tumor-bearing mice were subcutaneously inoculated with 2×10⁻⁶ antibodies. 5 PT cells or IL34 knockout PT cells were injected intratumorally with anti-IL34 antibody (20 μg each time; BioXcell; clone number: 29F.1A12) and anti-PD-1 antibody (50 μg each time, BioXcell), or a combination of IL-34 antibody and PD-1 antibody, 5 days after inoculation, once every 3 days, for a total of four treatments; the tumor was measured and calculated every 3-4 days, and the tumor volume was calculated as (length × width 2) / 2.

[0058] For the treatment of intrahepatic tumors, tumor-bearing mice were injected intrahepatically with 2×10⁻⁶ mg of [a specific drug]. 5 PT cells or IL34 knockout PT cells were injected intraperitoneally with anti-PD-1 antibody (200 μg BioXcell each time) 5 days after inoculation, once every 3 days, for a total of 4 treatments. Detailed Implementation

[0059] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0060] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents.

[0061] Example 1: Specific upregulation of IL-34 expression in p53-mutant hepatocellular carcinoma

[0062] In this embodiment, the inventors first constructed a liver cancer model, in which they selected the proto-oncogenes Ctnnb1 and Kras, which have the highest mutation frequency in hepatocellular carcinoma in the prior art. G12D A liver cancer model was constructed using the gene (i.e., the most classic oncogenic pathway), the tumor suppressor gene Pten, and the p53 mutation (corresponding to the mouse gene Trp53) to be studied in this invention. In order to accelerate tumor formation, a strategy of combining the above genes in pairs was also chosen.

[0063] In constructing the liver cancer model, we mutated the Trp53 gene (encoding mouse p53 protein) and the Pten tumor suppressor gene (Xue et al., 2014) by high-pressure tail vein injection of CRISPR cas9 plasmid (the construction methods of the CRISPR cas9 plasmid mentioned above are all conventional methods of existing technology, for example, see the "Vector Construction and Cell Transfection" section above), and used transposon plasmids to overexpress the activated forms of Ctnnb1 and Kras. G12DThe gene (the manipulation method and plasmid are known in existing technologies, see Tang et al., 2022 for example). Subsequently, referring to existing information on human liver cancer genes (it was found that Trp53 and Ctnnb1 are mutually exclusive in human liver cancer), the above four mutations were combined in pairs to accelerate the formation of liver cancer, and two p53 mutation-driven (Trp53+Pten, Trp53+Kras) and two non-p53 mutation-driven (Ctnnb1+Pten, Kras+Pten) spontaneous liver cancer models were constructed respectively. Figure 1 ).

[0064] Furthermore, based on the aforementioned liver cancer models, the inventors also constructed two mouse primary liver cancer cell lines derived from the Ctnnb1+Pten and Trp53+Pten liver cancer models, named CT cells and PT cells, respectively. Figure 2 ).

[0065] By analyzing the p53 mutation-driven hepatocellular carcinoma group (Trp53 knockout: Trp53) KO This refers to knocking out the Trp53 gene, i.e., the Trp53+Pten model and the Trp53+Kras model mentioned above, and the non-p53 mutation-driven liver cancer group (Trp53 wildtype: Trp53). WT RNA-seq was performed on liver cancer tissues carrying the wild-type Trp53 gene (i.e., the Ctnnb1+Pten and Kras+Pten models mentioned above). We found that IL-34 was the most significantly upregulated cytokine in both the Trp53+Pten and Trp53+Kras models. Figure 3 q-PCR and ELISA assays both demonstrated that IL-34 expression was significantly upregulated in p53-mutant hepatocellular carcinoma tissue compared to normal liver tissue and non-p53-mutant hepatocellular carcinoma tissue. Figure 4 ).

[0066] Analysis of data from hepatocellular carcinoma (HCC) patients in the TCGA database (currently known to the public) showed that IL-34 levels were significantly upregulated in HCC patients with p53 mutations compared to those with normal p53 levels. Figure 5 The results were consistent with those of the q-PCR and ELISA tests described above. Furthermore, analysis of liver cancer samples collected from the First Affiliated Hospital of Anhui Medical University showed that p53-mutated liver cancer tissue highly expressed IL-34, while p53-normal liver cancer tissue did not express IL-34. Figure 6 ).

[0067] In summary, the above results indicate that IL-34 is highly expressed in p53-mutant hepatocellular carcinoma, therefore, IL-34 may serve as a target for inhibiting p53-mutant hepatocellular carcinoma.

[0068] Example 2: Knocking out IL-34 inhibits the development and progression of p53-mutant liver cancer.

[0069] In this embodiment, the effect of IL-34 on p53-mutant hepatocellular carcinoma was verified by constructing IL-34 knockout mice (i.e., Il34+ / - and Il34- / - mice, where Il34+ / - mice are those with partial IL-34 knockout but still able to express IL-34; and Il34- / - mice are those with complete IL-34 knockout and no IL-34 expression) (purchased from Southern Models). Specifically, Il34+ / - and Il34- / - mice were injected intravenously with saline containing a mixture of CRISPR Cas9 plasmids targeting the mutant Trp53 gene and CRISPR Cas9 plasmids targeting the Pten gene (the construction method of the CRISPR Cas9 plasmids is described in the "Vector Construction and Cell Transfection" section above), and tumor development was assessed at 8-10 months. We observed that tumors had clearly formed in Il34+ / - mice, while tumors had not yet formed in Il34- / - mice, and the survival rate of Il34- / - mice was significantly higher than that of Il34+ / - mice. Figure 7 and Figure 8 This indicates that IL34 plays an important role in the development of p53-mutant liver cancer.

[0070] Example 3: Knockout of Trp53 mediates IL-34 upregulation

[0071] In this embodiment, by directly knocking out the Trp53 gene in primary hepatocellular carcinoma cell line CT cells (obtained in Example 1) that normally express the Trp53 gene, and by inducing the expression of its downstream protein p21 using a p53 activator, it was found that CT cells could not upregulate p21 expression after knocking out the Trp53 gene, demonstrating the successful knockout of the Trp53 gene. After knocking out the Trp53 gene, IL-34 was significantly upregulated at both the transcriptional and protein levels. Figure 9 This demonstrates that the p53 protein regulates the expression of IL-34.

[0072] Example 4: Antibody blocking IL-34 effectively inhibits the growth of p53-mutant liver cancer.

[0073] We subcutaneously inoculated p53-mutant primary hepatocellular carcinoma cell line PT cell (i.e., the mouse primary hepatocellular carcinoma cell line from the Trp53+Pten hepatocellular carcinoma model obtained in Example 1) into C57BL / 6 mice (purchased from Silex Corporation), and then injected the tumors of the mice with anti-IL-34 antibody (purchased from R&D, Cat#MAB5195) as the experimental group, and the group without anti-IL-34 antibody injection as the control group, and examined tumor growth. We observed that anti-IL-34 antibody blockade can effectively inhibit the growth of p53-mutant hepatocellular carcinoma. Figure 10 It also significantly reduced the proportion of infiltrating macrophages in the tumor. Figure 11 ).

[0074] Example 5: Combined blocking of IL-34 and PD-1 can completely eliminate p53-mutant tumors.

[0075] By subcutaneously inoculating mice with p53-mutant primary hepatocellular carcinoma (PT) cells and IL-34 knockout PT cells, followed by intratumoral injection of anti-PD-1 antibody, we observed that blocking PD-1 alone had no therapeutic effect on p53-mutant hepatocellular carcinoma, while blocking IL-34 alone effectively inhibited the growth of p53-mutant hepatocellular carcinoma. Combined blocking of IL-34 and PD-1 achieved complete tumor eradication. Figure 12 ).

[0076] Furthermore, we also inoculated p53-mutant primary hepatocellular carcinoma cell line PT cells and IL34 knockout PT cells into mouse livers, followed by intraperitoneal injection of anti-PD-1 antibodies into the mice. We also observed that blocking PD-1 alone had no therapeutic effect on p53-mutant hepatocellular carcinoma, while blocking IL-34 alone effectively inhibited the growth of p53-mutant hepatocellular carcinoma. Combined blocking of IL-34 and PD-1 achieved complete tumor eradication. Figure 13 ).

[0077] It is worth noting that the combined use of antibodies to block IL-34 and PD-1 can also achieve the effect of completely eliminating p53-mutant tumors. Figure 14 ).

[0078] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0079] References

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[0084] Mantovani,A.,Allavena,P.,Marchesi,F.,and Garlanda,C.(2022).Macrophages as tools and targets in cancer therapy.Nature Reviews DrugDiscovery 21,799-820.

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[0086] Ray,E.M.,and Sanoff,H.K.(2017).Optimal therapy for patients withhepatocellular carcinoma and resistance or intolerance to sorafenib:challenges and solutions.Journal of hepatocellular carcinoma 4,131-138.

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Claims

1. Use of IL-34 inhibitors in the preparation of drugs or kits for the treatment of p53-mutant liver cancer.

2. The use according to claim 1, wherein, The IL-34 inhibitor is an anti-IL-34 antibody or an oligonucleotide for knocking out the IL34 gene; preferably, the sequence of the oligonucleotide for knocking out the IL34 gene is shown in SEQ ID NO:

3.

3. The use according to claim 1, wherein, The kit also contains an anti-PD-1 antibody.

4. The use according to claim 1, wherein, The IL-34 inhibitor is in a form suitable for subcutaneous or intravenous injection.

5. Use of IL-34 inhibitors and anti-PD-1 antibodies in the preparation of drugs or kits for the treatment of p53-mutant hepatocellular carcinoma.

6. The use according to claim 5, wherein the IL-34 inhibitor is an anti-IL-34 antibody or an oligonucleotide for knocking out the IL34 gene.

7. The use according to claim 5, wherein the IL-34 inhibitor and the anti-PD-1 antibody are in a form suitable for subcutaneous or intravenous injection.

8. A kit comprising an IL-34 inhibitor and an anti-PD-1 antibody.

9. The kit according to claim 8, wherein the IL-34 inhibitor is an anti-IL-34 antibody or an oligonucleotide for knocking out the IL34 gene.

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