Aars2-mediated cd112 lactylation inhibitors and screening methods

Abstract

The present application relates to the field of biological medicine and tumor immunity, and discloses an AARS2-mediated CD112 K412 lactylation inhibitor and a screening method thereof.AARS2 promotes CD112 K412 lactylation, weakens the combination of UBE3C and CD112, inhibits CD112 ubiquitination and lysosome-related degradation, thereby maintaining tumor cell surface CD112 and inhibiting CD8+ T cell and NK cell functions.The screening method sequentially detects AARS2-related functions, CD112 K412 lactylation, UBE3C-CD112 combination, CD112 ubiquitination or degradation, surface CD112 and immune cell killing, and is used to obtain candidates capable of reducing CD112 stability and enhancing anti-tumor immunity.The present application also discloses nucleic acid molecules for reducing AARS2 expression and corresponding pharmaceutical compositions, which can be used for preparing anti-tumor drugs.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and tumor immunotherapy, specifically involving the AARS2-mediated CD112 412 lysine lactylation regulatory axis, in vitro screening methods for candidate antitumor substances targeting this regulatory axis, nucleic acid molecules that reduce AARS2 expression, and related drug uses.

[0002] More specifically, the present invention relates to a technical solution that restores the binding and ubiquitination of UBE3C to CD112 by reducing AARS2-mediated CD112 K412 lactylation, promotes lysosomal-associated degradation of CD112, reduces CD112 on the surface of tumor cells, and enhances the anti-tumor effects of CD8⁺ T cells and NK cells. Background Technology

[0003] Tumor cells often exhibit enhanced glycolysis and lactate production. Besides participating in energy metabolism and tumor microenvironment acidification, lactate can also serve as a metabolic source related to lysine lactylation, altering protein function, interactions, or stability. Establishing verifiable and screenable drug intervention pathways targeting specific non-histone lactylation events remains a challenge in the field of cancer treatment.

[0004] CD112, also known as NECTIN2 or PVRL2, is a cell membrane protein involved in cell adhesion and immune regulation. On the surface of tumor cells, CD112 can associate with immunomodulatory receptors such as TIGIT and CD112R, thereby affecting T cell and NK cell effects. Current strategies primarily focus on blocking receptor-ligand interactions, lacking technical solutions that address the regulation of endogenous protein stability within tumor cells to reduce CD112 surface abundance.

[0005] AARS2 is generally considered to be involved in mitochondrial alanine-tRNA-related functions. The inventors have discovered that AARS2 is also involved in the regulation of CD112 lactylation. AARS2 deficiency enhances the killing of tumor cells by CD8⁺ T cells and NK cells, and inhibits tumor growth in an immune-intact animal tumor model.

[0006] Further research revealed that lysine residue 412 of CD112 is a functionally significant lactylation site. Lactylation of CD112K412 weakens the binding of CD112 to the E3 ubiquitin ligase UBE3C, reducing CD112 ubiquitination and subsequent degradation, thereby maintaining the stability of the CD112 protein and its expression on the cell surface.

[0007] Therefore, it is necessary to provide a screening method that can read out the AARS2-CD112 K412 lactylation-UBE3C axis as a continuous mechanism, and to provide drug use that can reduce this lactylation event, promote CD112 clearance and enhance anti-tumor immunity. Summary of the Invention I. Technical problems to be solved

[0008] One of the technical problems to be solved by this invention is to provide a technical solution that targets the AARS2-CD112 lactylation axis in tumor cells, so that the screening of candidates is not limited to single binding or enzyme activity readout, but can be verified step by step along CD112 K412 lactylation, UBE3C binding, ubiquitination, degradation, membrane surface expression and immune cell killing.

[0009] Another technical problem to be solved by the present invention is to provide a drug use that reduces AARS2 expression or function and thereby reduces CD112 on the surface of tumor cells, and to provide specific nucleic acid target sequences and mechanistic support for this use. II. Technical Solution

[0010] In a first aspect, the present invention provides an in vitro screening method for candidate antitumor substances targeting the AARS2-CD112 lactylation axis. The method includes contacting the candidate with a detection system containing AARS2 and CD112, and detecting the CD112 K412 lactylation level after candidate treatment.

[0011] The detection system can be a cell-free system, a recombinant protein system, a cell lysate system, or a cell system expressing AARS2 and CD112. The cell system is preferably tumor cells or cells suitable for exogenous expression and immunoprecipitation.

[0012] In some implementations, the effect of the candidate on AARS2 binding to lactate, AARS2 binding to CD112, or AARS2-mediated CD112 lactylation is detected before or simultaneously with the detection of CD112 K412 lactylation. AARS2 R110A, W210A, and G269A mutants can be used as controls for functional defect mechanisms.

[0013] CD112 K412 lactylation can be determined by immunoprecipitation combined with lysine lactylation detection, liquid chromatography-tandem mass spectrometry, or specific immunoassay for K412 lactylation. Candidates with reduced CD112 K412 lactylation relative to the vector or untreated control can proceed to the next stage of screening.

[0014] The next stage of screening will assess at least one of the following after candidate treatment: CD112 binding to UBE3C, CD112 ubiquitination, CD112 protein stability, total CD112 protein level, and cell surface CD112 level. Preferred candidates will increase UBE3C-CD112 binding and CD112 ubiquitination, and decrease CD112 stability, total protein, or surface expression.

[0015] CD112 protein stability can be detected by a follow-up assay after protein synthesis inhibition; total CD112 protein can be detected by immunoblotting; cell surface CD112 can be detected by flow cytometry, cell surface biotinylation or other membrane protein quantification methods.

[0016] In a further embodiment, tumor cells treated with the candidate are co-cultured with CD8⁺ T cells and / or NK cells, and tumor cell death, immune cell cytotoxicity, degranulation, cytokine release, or other effector functions are detected. Preferably, the candidate can enhance tumor cell death and strengthen at least one immune effect.

[0017] Mechanism validation can be performed using AARS2-deficient cells, cells expressing the CD112 K412R mutant, or UBE3C-deficient cells. If the candidate's effect is significantly weakened in the above mechanism controls, it supports the view that its action depends on the AARS2-CD112K412 lactylation-UBE3C axis.

[0018] To improve selectivity, the effects of the candidate on at least one of AARS1, mitochondrial protein translation, cellular respiration, mitochondrial function, normal cell viability, and immune cell viability can also be detected. Preferred candidates do not produce unacceptable nonspecific cytotoxicity under effective conditions of reducing CD112 K412 lactylation.

[0019] Candidates can be small molecule compounds, peptides, proteins, antibodies or antibody fragments, nucleic acid molecules, target protein degradation molecules, or molecular gels that promote the binding of UBE3C to CD112.

[0020] In a second aspect, the present invention provides a kit for performing the above-described screening method. The kit comprises at least AARS2 or cells expressing AARS2, CD112 or cells expressing CD112, and a reagent for detecting CD112 lactylation.

[0021] The kit may also contain UBE3C or cells expressing UBE3C, ubiquitination detection reagents, cell surface CD112 detection reagents, CD8⁺ T cell or NK cell function detection reagents, and one or more mechanism controls from wild-type AARS2, AARS2 R110A, W210A or G269A, wild-type CD112, CD112 K412R and UBE3C-deficient cells.

[0022] In a third aspect, the present invention provides the use of AARS2 inhibitors in the preparation of antitumor drugs. The AARS2 inhibitors reduce AARS2-mediated CD112 K412 lactylation, promote UBE3C binding to CD112, CD112 ubiquitination and lysosomal-related degradation, and reduce CD112 on the surface of tumor cells.

[0023] The AARS2 inhibitors exert their effects by reducing AARS2 expression, inhibiting AARS2 binding to lactate, blocking the interaction between AARS2 and CD112, or inhibiting AARS2-mediated CD112 lactylation. To minimize the impact on classical mitochondrial function of AARS2, candidates with high selectivity for regulating non-classical CD112 lactylation of AARS2 are preferred.

[0024] The antitumor drug is applicable to malignant tumors with AARS2-CD112 axis activation. Tumors can be characterized by at least one of the following indicators: AARS2 expression, CD112 K412 lactylation, total CD112 protein, or CD112 on the surface of tumor cells. Representative tumors include breast cancer, melanoma, and pancreatic cancer, preferably triple-negative breast cancer.

[0025] In a fourth aspect, the present invention provides a nucleic acid molecule for reducing AARS2 expression. The nucleic acid molecule targets the AARS2 nucleic acid region comprising the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0026] The nucleic acid molecule can be a small interfering RNA, a short hairpin RNA, an antisense oligonucleotide, or a guide nucleic acid of the CRISPR system. The nucleic acid molecule can be combined with a pharmaceutically acceptable carrier to form a drug composition for the preparation of antitumor drugs.

[0027] This invention reveals that β-alanine can interfere with AARS1 / 2-related lactate binding, reduce CD112 lactylation, and increase CD112 ubiquitination and degradation. Therefore, β-alanine can be used as a pharmacological positive control in screening methods; however, this result does not indicate that β-alanine is a selective inhibitor of AARS2.

[0028] AARS2 status can also influence the adjunctive therapeutic effect of TIGIT×PD-L1 bispecific antibodies in tumor models. This observation supports understanding immune checkpoint therapy response using the AARS2-CD112 axis; the co-administration conditions of AARS2 inhibitors with the aforementioned bispecific antibodies can be determined through further screening.

[0029] CD112 can also participate in HSV-2 entry into host cells. This invention observed a correlation between Aars2 status and the therapeutic outcome of HSV-2 oncolytic virus in the EO771 tumor model. Since reducing AARS2 may simultaneously reduce surface CD112, the direct combination of AARS2 inhibition and HSV-2 oncolytic virus should be determined by considering both viral entry and amplification phases to determine the order of administration. III. Beneficial Effects

[0030] This invention establishes a continuous regulatory relationship between lactate-AARS2-CD112 K412 lactylation-UBE3C-CD112 stability-tumor immunity, providing an intervention level that differs from simply blocking receptor-ligand interactions for reducing CD112 on the surface of tumor cells.

[0031] The screening method of this invention combines upstream AARS2-related functions, midstream CD112 K412 lactylation and UBE3C binding, and downstream CD112 degradation and immune cell function, which can reduce false positive candidates that only change a single biochemical readout and lack functional effects.

[0032] This invention also provides mechanism controls such as AARS2 deletion, AARS2 defective mutants, CD112 K412R and UBE3C deletion, and provides AARS1 and mitochondrial functional screening pathways, which is beneficial to improve the consistency and selectivity of candidates with target mechanisms.

[0033] This invention provides AARS2 nucleic acid target sequences that have been used in experiments, providing a concrete basis for nucleic acid intervention against AARS2 and corresponding drug compositions. Attached Figure Description

[0034] Figure 1 A schematic diagram of the AARS2-CD112 K412 lactylation axis and its intervention mechanism.

[0035] Figure 2 Experimental results demonstrating that Aars2 deficiency enhances CD8⁺ T cell and NK cell-mediated anti-tumor immunity.

[0036] Figure 3 The results represent experimental findings on the regulation of CD112 lactylation, surface expression, and T / NK cell killing by lactate metabolism and AARS2-related functions.

[0037] Figure 4 The results show the experimental findings on CD112 K412 lactylation regulating UBE3C binding, CD112 ubiquitination, and lysosomal-related degradation.

[0038] Figure 5 To investigate the influence of Aars2 status on the antitumor effect of TIGIT×PD-L1 bispecific antibody and the therapeutic results of this bispecific antibody in a mouse tumor model.

[0039] Figure 6 To observe the therapeutic outcomes and safety of Aars2 status and HSV-2 oncolytic virus in the EO771 tumor model. Detailed Implementation Terminology and Testing Principles

[0040] Unless otherwise stated, AARS2 represents alanyl-tRNA synthetase 2; the mouse homolog is denoted as Aars2. CD112, NECTIN2, and PVRL2 refer to the same protein in this application. K412la represents lysine lactylation at position 412 of CD112. UBE3C represents E3 ubiquitin ligase UBE3C.

[0041] In this application, "decrease," "increase," or "enhancement" all refer to a detectable directional change compared to the corresponding untreated, vector, or negative control. Changes can be determined using biological replicates and appropriate statistical methods based on the experimental system. Unless expressly defined in the claims, these terms do not require fixed percentage thresholds.

[0042] In this application, "lysosome-related degradation" refers to the degradation process of CD112 that can be blocked or weakened by lysosomal function inhibition conditions, and is not used to limit specific intracellular transport complexes that have not yet been directly determined experimentally.

[0043] Tumor cell death can be detected by flow cytometry analysis after labeling tumor cells with CellTracker and staining with propidium iodide, or by equivalent cell death or cytotoxicity detection methods available in the art. Cell surface CD112 can be detected by flow cytometry under impermeable conditions. Example 1: Aars2 deficiency enhances CD8⁺ T cell and NK cell-mediated anti-tumor immunity

[0044] B16-F10 and EO771 cells expressing negative control sgRNA, Aars1 sgRNA, or Aars2 sgRNA were established, respectively. Cells from each group were subcutaneously inoculated into C57BL / 6 mice, and tumor volume was monitored. Compared with the control and Aars1-deficient groups, Aars2 deficiency significantly inhibited tumor growth in both B16-F10 and EO771 cells.

[0045] Aars2 cDNA was reintroduced into Aars2-deficient EO771 cells. Compared with empty vector reintroduction, Aars2 reexpression restored tumor growth in vivo, supporting the association between tumor phenotype and Aars2 deficiency.

[0046] Spatial transcriptomic analysis and histological observation were performed on control and Aars2-deficient EO771 tumors. After Aars2 deletion, the spatial distribution of tumor cells, CD8⁺ T cells, NK cells, macrophages and fibroblasts changed.

[0047] In the EO771 model, immune cell depletion was performed using anti-CD8 antibody, anti-NK1.1 antibody, or a combination of both. After depletion of CD8⁺ T cells or NK cells alone, the Aars2-deficient group still showed some tumor suppression; however, after simultaneous depletion of both CD8⁺ T cells and NK cells, the growth difference between the control and Aars2-deficient tumors essentially disappeared. These results indicate that the in vivo tumor-suppressive effect of Aars2 deficiency depends on the combined action of CD8⁺ T cells and NK cells.

[0048] Figure 2 In the figures, a and b represent the B16-F10 inoculation regimen and tumor growth curve, respectively; c and d represent the EO771 inoculation regimen and tumor growth curve, respectively; e and f represent the Aars2 replenishment regimen and tumor growth curve, respectively; g and h represent the spatial transcriptome and histological analysis of EO771 tumors; i represents the immune cell clearance regimen; j represents the validation of CD8⁺ T cell clearance; k and l represent the tumor volume and weight after CD8⁺ T cell clearance; m represents the validation of NK cell clearance; n and o represent the tumor volume and weight after NK cell clearance; p and q represent the tumor volume and weight after simultaneous clearance of CD8⁺ T cells and NK cells. Example 2: Lactic acid metabolism and AARS2 function regulation of CD112 and immune cell killing

[0049] MDA-MB-231 cells treated with 2-deoxyglucose or oxalamide to reduce lactate-related effects, when co-cultured with CD8⁺ T cells or NK cells, showed increased tumor cell death. Treatment with ARC155858 or AZD3965 to promote intracellular lactate accumulation reduced CD8⁺ T cell or NK cell-mediated tumor cell death.

[0050] In AARS2-deficient MDA-MB-231 cells, the effects of the above-mentioned lactate metabolism intervention on CD8⁺ T cell and NK cell killing were significantly weakened, indicating that its functional effect is at least partially dependent on AARS2.

[0051] β-alanine treatment enhances CD8⁺ T cell and NK cell-mediated tumor cell killing. Replenishment of wild-type AARS2 in AARS2-deficient cells restores the immune resistance phenotype, while R110A, W210A, and G269A defective mutants do not achieve the same restorative effect as wild-type AARS2.

[0052] AARS2 deficiency reduced CD112 on the surface of MDA-MB-231 cells. Oxalamyric acid treatment reduced the difference in surface CD112 between control and AARS2-deficient cells. Immunoprecipitation and mass spectrometry results showed that AARS2 is associated with CD112, and that AARS2 and its associated functions (R110, W210, and G269) are involved in the regulation of CD112 lactylation and stability.

[0053] Figure 3 In the diagram, ah shows the effect of lactate production or transport intervention on cell death in control or AARS2-deficient tumor cells under co-culture conditions of CD8⁺ T cells and NK cells; i and j show the role of β-alanine; k and l show the results of AARS2 wild-type and functionally deficient mutant complementation; m and n show the flow cytometry detection and quantification of surface CD112; o shows the effect of AARS2 and lactate on CD112 lactylation; pr shows the CD112 interaction proteomic profile, AARS2 identification, and peptide coverage; s shows the co-immunoprecipitation of AARS2 and CD112; t shows the effect of AARS2 wild-type and mutant on CD112 lactylation; and u shows the CD112 protein level after AARS2 complementation. Example 3: CD112 K412 is a functional lactylation site

[0054] The CD112 lactylated peptide was identified using liquid chromatography-tandem mass spectrometry, and K412 lactylation was detected in the amino acid region from position 388 to 414 of CD112. Substitution of arginine with candidate lysine residues in the intracellular region of CD112 resulted in a significant reduction in CD112 lactylation due to the K412R mutation.

[0055] Sequence alignment of CD112 homologs across different species revealed conservation in the region adjacent to K412. Follow-up experiments following protein synthesis inhibition showed that the K412R mutant degraded faster than wild-type CD112.

[0056] The K412R mutant exhibited a high tendency for ubiquitination in both the presence and absence of AARS2. These results indicate that CD112 K412 lactylation is a functional post-translational modification that enhances the stability of the CD112 protein. Example 4: UBE3C-mediated CD112 ubiquitination and lysosomal-related degradation

[0057] Candidate E3 ubiquitin ligases were screened from CD112 interaction proteomic profiles, yielding candidate proteins such as TRIM4, UBE3C, and SMURF1. Co-immunoprecipitation and expression gradient experiments showed that UBE3C interacts with CD112 and decreases CD112 protein levels with increasing UBE3C levels.

[0058] UBE3C deficiency increases cell surface CD112 and decreases endogenous CD112 ubiquitination; UBE3C overexpression increases CD112 ubiquitination and accelerates CD112 reduction. Experiments using wild-type ubiquitin and K48R or K63R ubiquitin mutants showed that both K48 and K63-related ubiquitin chains are involved in UBE3C-mediated CD112 ubiquitination.

[0059] Degradation pathway intervention showed that NH4Cl could restore the CD112 reduction caused by UBE3C, and Bafilomycin A1 produced a corresponding blocking effect; MG132 and 3-MA did not show the same recovery pattern, supporting that UBE3C promotes lysosomal degradation of CD112.

[0060] NaLac treatment reduces UBE3C-mediated CD112 ubiquitination and CD112-UBE3C binding; reducing lactate or using β-alanine treatment enhances the binding. Complementation experiments with AARS2 wild-type and defective mutants, as well as comparisons of CD112 K412R, further demonstrate that AARS2-mediated K412 lactylation weakens UBE3C recognition of CD112.

[0061] Figure 4 In the above diagrams, ah shows the effects of lactate metabolism and AARS2 function on CD112 lactylation, ubiquitination, and protein levels; im shows the identification, mutation, conservation, stability, and ubiquitination of the K412 site; n shows the screening of candidate E3 ligases; o shows the interaction between candidate E3 and CD112; p and q show the surface CD112 and its quantification after UBE3C deletion; r shows the stability tracking of CD112 under UBE3C conditions; s shows the comparison of inhibitors of different degradation pathways; t and u show the time treatments of NH4Cl and Bafilomycin A1, respectively; v shows the promotion of CD112 ubiquitination by UBE3C; w shows the dose effect of UBE3C; x shows the ubiquitination of CD112 after UBE3C deletion; y shows the detection of K48 and K63-related ubiquitin chains; z shows the effect of NaLac on UBE3C-mediated CD112 ubiquitination; aa-ac shows the effects of lactate-related treatments and AARS2 mutants on CD112-UBE3C binding, respectively; ad shows the effects of CD112... The effect of K412R on the binding of UBE3C. Example 5: Multi-stage screening targeting the AARS2-CD112 lactylation axis

[0062] A detection system expressing AARS2 and CD112 was established. The candidate compound was first contacted with the system, and at least one of the following was detected: AARS2 binding to lactate, AARS2 binding to CD112, or CD112 lactylation. Wild-type AARS2 and the R110A, W210A, and G269A mutants were used as mechanistic controls.

[0063] For initial screening candidates, CD112 K412 lactylation is detected. This can be achieved by first immunoprecipitating CD112, followed by detection with a lysine lactylation antibody; alternatively, mass spectrometry or a K412 lactylation-specific reagent can be used. Candidates with reduced K412 lactylation are selected for the next stage.

[0064] Further analysis was conducted on UBE3C-CD112 binding, CD112 ubiquitination, CD112 protein stability, total CD112 protein, and cell surface CD112 after candidate treatment. The preferred candidate increased UBE3C-CD112 binding and CD112 ubiquitination while decreasing CD112 stability or surface expression.

[0065] Tumor cells treated with the candidate were co-cultured with CD8⁺ T cells or NK cells, and tumor cell death was detected. The presence of AARS2-deficient, CD112 K412R-deficient, or UBE3C-deficient cells was used to determine whether the candidate's effect was dependent on the target regulatory axis.

[0066] Finally, the effects of the candidate on AARS1, mitochondrial protein translation, cellular respiration, and the viability of normal and immune cells were examined. This yielded a candidate with readouts for mechanism, CD112 degradation, and immune function. β-alanine was used as a pharmacological positive control but not as a selectivity standard for AARS2. Example 6: Aars2 status and response to TIGIT×PD-L1 bispecific antibody therapy

[0067] A bispecific antibody containing both anti-TIGIT and anti-PD-L1 binding modules was constructed. In the C57BL / 6 mouse EO771 model, IgG, anti-TIGIT antibody, anti-PD-L1 antibody, and TIGIT×PD-L1 bispecific antibody were compared. The bispecific antibody showed stronger tumor control and survival benefits compared to single-target antibodies.

[0068] The TIGIT×PD-L1 bispecific antibody was constructed using conventional bispecific antibody technology platforms in the field, including but not limited to symmetric IgG-scFv fusion format, Knobs-into-Holes (KiH) heterodimer format, CrossMAb heavy chain exchange format, DVD-Ig dual variable region-Ig format, or VHH / nanobody-based bispecific antibody formats. During construction, binding modules capable of simultaneously recognizing human and mouse TIGIT and PD-L1 were selected to simultaneously meet the drug administration needs of both the mouse C57BL / 6 model and the humanized NCG mouse PDX model; alternatively, two sets of bispecific antibodies, one humanized and one mouse homologous, were constructed for different animal models.

[0069] The anti-TIGIT binding module can be derived from the complementarity-determining region (CDR) sequence of a publicly disclosed clone (such as clones 10A7, 22G2, or homologous antibodies like Ociperlimab, BMS-986207, and Tiragolumab), or from VH / VL pairs obtained through screening of anti-TIGIT hybridomas or phage / yeast display libraries known in the art. The anti-PD-L1 binding module can be derived from the CDR sequence of a publicly disclosed clone (such as Atezolizumab, Avelumab, Durvalumab, or similar antibodies already marketed / in clinical trials in China), or from VH / VL pairs obtained through screening of anti-PD-L1 hybridomas or display libraries known in the art. Preferably, the anti-TIGIT and anti-PD-L1 binding modules use a common light chain or are modified to avoid heavy / light chain mismatches.

[0070] The heavy and light chain coding sequences of the bispecific antibody were cloned into dual-promoter mammalian expression vectors (such as pcDNA3.1 or similar vectors), or co-expressed using IRES or self-cleaving peptides (2A peptides). HEK293F or CHO-S suspension cell lines were used for transient or stable transfection expression. The culture supernatant was initially purified by Protein A affinity chromatography and / or Protein L affinity chromatography, followed by cation / anion exchange chromatography and molecular sieve (SEC) purification. The purified product was identified for aggregation state and purity using non-reducing and reducing SDS-PAGE, SEC-MALS, and HPLC. Its binding affinity to human TIGIT, human PD-L1, mouse Tigit, and mouse Cd274 was determined by surface plasmon resonance (SPR) or biomembrane interference (BLI) techniques. Flow cytometry was used to verify its specific binding to TIGIT and PD-L1 expressing cells. Its biological activity was verified by T cell activation, tumor cell killing, or immune checkpoint blockade reporter gene assays. The bispecific antibody can be modified via Fc segment modification (such as L234A / L235A / N297A or similar mutations) to weaken ADCC and CDC effects, or retain the intact Fc segment to maintain in vivo half-life and effector function.

[0071] The study compared IgG and TIGIT×PD-L1 bispecific antibodies in control and Aars2-deficient EO771 tumors. Aars2 deficiency itself inhibited tumor growth and weakened the additional effect of bispecific antibodies relative to IgG, indicating that Aars2 status is related to the immunosuppressive environment targeted by this treatment.

[0072] Spatial transcriptomic analysis revealed altered spatial neighborhood relationships among tumor cells, CD8⁺ T cells, NK cells, cancer-associated fibroblasts, and tumor-associated macrophages after bispecific antibody treatment, with enrichment of immune effector-related pathways in lymphocytes. The antitumor effect of bispecific antibodies was also demonstrated in a humanized NCG mouse xenograft model derived from triple-negative breast cancer patients.

[0073] Figure 5 In the figures, a shows the structural diagrams of IgG, single-target antibodies, and bispecific antibodies; b shows the dosing regimens for control or Aars2-deficient EO771 tumors receiving IgG or bispecific antibodies; c and d show the corresponding tumor growth and survival curves; e shows the regimens for EO771 tumors receiving IgG, anti-TIGIT, anti-PD-L1, or bispecific antibodies; f and g show the corresponding tumor growth and survival curves; hl shows spatial transcriptomics, cell population, spatial interaction, and pathway enrichment analyses; mo shows the dosing regimen, tumor growth, and survival curves for a humanized NCG mouse triple-negative breast cancer patient-derived xenograft model. The antibody dose shown is 10 mg / kg, administered intravenously every 3 days. Example 7: Aars2 status and HSV-2 oncolytic virus treatment results

[0074] C57BL / 6 mice were subcutaneously inoculated with either control or Aars2-deficient EO771 cells. After tumor establishment, HSV-2 oncolytic virus or corresponding vectors were administered intratumorally as controls, and tumor volume, survival, body weight, peripheral blood routine tests, and histological examination of major organs were monitored.

[0075] HSV-2 inhibited the growth of control EO771 tumors. Aars2-deficient tumors themselves grow slowly, and after HSV-2 administration, the tumor burden remained at a low level and showed improved survival. No significant weight loss was observed during treatment, and no significant abnormalities were found in peripheral blood leukocytes, erythrocytes, hemoglobin, and platelets. Histological examination of the heart, liver, spleen, lungs, and kidneys showed no significant systemic toxicity.

[0076] Figure 6 In the figures, a represents the control or Aars2-deficient EO771 tumors receiving intratumoral administration of the vector or HSV-2; b represents the tumor growth curve; c represents the survival curve; d represents relative body weight; e represents H&E staining of major organs; and f represents peripheral blood routine indicators.

[0077] The above results indicate that Aars2 status is correlated with the outcome of HSV-2 oncolytic virus treatment. If a sequential protocol is implemented, the timing of Aars2 inhibitor administration can be determined based on the time required for the virus to complete initial infection and amplification. Example 8 Nucleic Acid Target Sequence

[0078] The experiments used sgRNA or shRNA target sequences targeting human AARS2, human CD112, and human UBE3C. Sequence identifiers and sequences are listed in the nucleotide or amino acid sequence listing of this application. SEQ ID NO:1-4 targeting human AARS2 can be used to construct nucleic acid molecules that reduce AARS2 expression; SEQ ID NO:5-8 can be used to construct mechanism control cells.

[0079] Those skilled in the art can, based on the expression vector, RNA form, and delivery system used, convert the listed DNA target sequences into corresponding RNA sequences without altering the target region, and configure appropriate promoters, backbones, loop structures, or delivery vectors.

Claims

1. An in vitro screening method for candidate antitumor substances targeting the AARS2-CD112 lactylation axis, characterized in that, include: Contact the candidate with the detection system containing AARS2 and CD112; The lactylation level of lysine at position 412 of CD112 was detected after the candidate was treated. Select candidates that reduce the level of lactylation relative to the control; and The candidate is tested to determine whether it causes at least one of the following changes: increased binding of CD112 to UBE3C, increased CD112 ubiquitination, decreased CD112 protein stability, decreased total CD112 protein level, or decreased CD112 level on the cell surface.

2. The screening method according to claim 1, characterized in that, It also includes at least one of the following: Before detecting the lactylation level of lysine at position 412 of CD112, the inhibitory effect of the candidate on at least one of AARS2 binding to lactate, AARS2 binding to CD112, or AARS2-mediated CD112 lactylation was detected. or The level of lactylation was determined by immunoprecipitation combined with lysine lactylation detection, liquid chromatography-tandem mass spectrometry, or specific immunoassay for lysine lactylation at position 412 of CD112.

3. The screening method according to claim 1, characterized in that, It also includes at least one of the following: Tumor cells treated with the candidate were co-cultured with CD8⁺ T cells and / or NK cells, and candidates that could improve tumor cell death or enhance the effector function of CD8⁺ T cells and / or NK cells were selected. Using AARS2-deficient cells, cells expressing the CD112 K412R mutant, and / or UBE3C-deficient cells, we verified whether the effect of the candidate depends on the AARS2-CD112 K412 lactylation-UBE3C regulatory axis; or The effects of the candidate on at least one of AARS1, mitochondrial protein translation, cellular respiration, normal cell viability, or immune cell viability were examined to rule out non-selective inhibition or non-specific cytotoxicity.

4. The screening method according to claim 1, characterized in that, The candidates are small molecule compounds, peptides, proteins, antibodies or antibody fragments, nucleic acid molecules, target protein degradation molecules, or molecular gels that promote the binding of UBE3C to CD112.

5. An in vitro screening kit for candidate antitumor substances targeting the AARS2-CD112 lactylation axis, characterized in that, The kit contains AARS2 or cells expressing AARS2, CD112 or cells expressing CD112, and a reagent for detecting CD112 lactylation; the kit also contains at least one of UBE3C or cells expressing UBE3C, a ubiquitination detection reagent, a cell surface CD112 detection reagent, a CD8⁺ T cell function detection reagent, or an NK cell function detection reagent.

6. The use of AARS2 inhibitors in the preparation of antitumor drugs, characterized in that, The AARS2 inhibitor reduces AARS2-mediated lactylation of lysine at position 412 of CD112, thereby promoting the binding of UBE3C to CD112, increasing CD112 ubiquitination and lysosomal-associated degradation, and reducing CD112 levels on the surface of tumor cells.

7. The use according to claim 6, characterized in that: The AARS2 inhibitor is a nucleic acid molecule, a small molecule compound, a polypeptide, a protein, an antibody or antibody fragment, or a protein degradation molecule that targets AARS2; The AARS2 inhibitors exert their effects by reducing AARS2 expression, inhibiting AARS2 binding to lactate, blocking AARS2-CD112 interaction, or inhibiting AARS2-mediated CD112 lactylation; and The tumor is breast cancer, melanoma, or pancreatic cancer; preferably, the breast cancer is triple-negative breast cancer.

8. A nucleic acid molecule for reducing AARS2 expression, characterized in that, The nucleic acid molecule targets the AARS2 nucleic acid region containing the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and the nucleic acid molecule is a small interfering RNA, a short hairpin RNA, an antisense oligonucleotide or a guide nucleic acid of the CRISPR system.

9. A pharmaceutical composition, characterized in that, It comprises the nucleic acid molecule of claim 8 and a pharmaceutically acceptable carrier.

10. Use of the nucleic acid molecule of claim 8 in the preparation of antitumor drugs.

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