Application of CCR1 antagonist in preparation of medicine for improving anti-tumor curative effect of CAR-T cells

Abstract

The invention provides application of a CCR1 antagonist in preparation of a medicine for improving the anti-tumor curative effect of CAR-T cells. The CCR1 antagonist (BX471) improves the tumor immune microenvironment by inhibiting the CCR1-mediated myeloid cell chemotaxis and related fibrosis signals, which is represented by reducing the inhibitory mononuclear / macrophage enrichment and matrix fibrosis level, so that the infiltration and effector functions of CAR-T on tumors are improved. In a mouse tumor model (including but not limited to B cell lymphoma and melanoma), BX471 is continuously given according to a preset treatment course after CAR-T infusion, and compared with single use of CAR-T, better tumor inhibition related indexes can be obtained; no obvious systemic toxic signal is seen under the tested dosage and treatment course. The invention provides a medication direction and an application basis for a tumor microenvironment facing immunological rejection caused by myelogenous enrichment.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunology and cell therapy technology, and relates to CCR1 antagonists, especially the application of CCR1 antagonists in the preparation of drugs that enhance the anti-tumor efficacy of CAR-T cells. Background Technology

[0002] Chimeric antigen receptor T cells (CAR-T) possess the advantages of high programmability and specific recognition. Although they have demonstrated significant efficacy in some hematologic malignancies, in solid tumors, they often suffer from insufficient homing and infiltration, limited in vivo expansion, and persistence due to the immunosuppressive tumor-associated myeloid cells and stromal barrier in the tumor microenvironment. Previous research in this invention, through spatial transcriptome analysis, revealed that one of the main obstacles is the myeloid-mediated immunosuppression and physical barrier in the tumor microenvironment (TME), characterized by a population of monocytes / macrophages expressing CCR1, often presenting as SPP1. + Tumor-associated macrophage phenotype, and FAP + Cancer-associated fibroblasts (CAFs) are spatially coupled within the tumor margin and fibrotic zone; tumor-associated myeloid cells (TAMCs) secrete chemokines such as CCL3 / CCL5 / CCL7, which drive the targeted enrichment of myeloid cells via CCR1. The enriched CCR1... + Tumor-associated fibroblasts (TAMCs) disrupt normal endothelial cells by secreting TGFβ, leading to endothelial-mesenchymal transition (EMT) and forming a physical barrier with tumor-associated fibroblasts (CAFs). Within this barrier, the secreted TGFβs also induce CAR-T cell exhaustion, and previous public data analyses have found this structure is associated with worse survival outcomes. Drug intervention targeting the CCR1 axis holds promise for alleviating the problem of insufficient CAR-T infiltration. However, current technology does not disclose a systemic efficacy enhancement strategy for combining BX471 and CAR-T cells at specific times and dosages in animal models.

[0003] BX471 (molecular formula C21H24ClFN4O3, CAS number 217645-70-0) is a non-peptide, selective CCR1 antagonist, first reported and systematically characterized by the Berlex / Bayer team. It inhibits CCR1-dependent Ca2+ kinase activity by competitively blocking CCR1 receptor-mediated chemokine signaling (typical ligands include CCL3 / MIP-1α, CCL5 / RANTES, etc.). 2+Downstream effects include mobilization, receptor internalization, and chemotaxis. CCR1 is expressed on various myeloid cells (monocytes / macrophages, neutrophils, etc.). The aforementioned blocking effects can reduce the recruitment and polarization of inflammatory myeloid cells, thus exhibiting anti-inflammatory and anti-fibrotic effects in models of multi-organ fibrosis, transplant rejection, and infection / inflammation. Pharmacological studies show that BX471 has high selectivity and functional antagonistic activity against CCR1. In mice, the commonly used dosage window is 2–20 mg / kg (qd or bid) via oral or intravenous administration (depending on the model and pharmacokinetic profile). Preclinical and early clinical assessments indicate good electrocardiographic safety (no significant QTc prolongation). In conjunction with the CAR-T combination strategy involved in this invention, BX471 can be used to inhibit CCR1 in the TME. + The recruitment and barrier construction of myeloid cells significantly enhance CAR-T tumor infiltration and efficacy, consistent with its validated mechanism of action and cross-model translational ability. Summary of the Invention

[0004] The purpose of this invention is to provide the application of a CCR1 antagonist in the preparation of drugs that enhance the anti-tumor efficacy of CAR-T cells. This non-peptide small molecule antagonist (CCR1) improves CAR-T cell infiltration and anti-tumor efficacy by modifying the tumor microenvironment. The tumor microenvironment is characterized by an abundance of myeloid and fibroblast cells and activation of the CCR1 axis.

[0005] The CCR1 antagonist (BX471) improves the tumor immune microenvironment by inhibiting CCR1-mediated myeloid cell chemotaxis and related fibrosis signals, thereby reducing the enrichment of inhibitory monocytes / macrophages and the level of stromal fibrosis, thus enhancing the infiltration and effector function of CAR-T cells on tumors.

[0006] The small molecule antagonist is a non-peptide CCR1 antagonist, BX471. The antagonist is a solid. The powdered solid can be stored at -20°C for 3 years and at 4°C for 2 years. If solution storage is required, the storage solution can be prepared according to the general commercial instructions. It can be stored at -80°C for 2 years and at -20°C it must be used within 1 year.

[0007] The antagonist is generally a solid, and a working solution needs to be prepared before in vivo injection in mice: Take 2.5 μL of DMSO stock solution, add 10 μL of PEG300 / PEG400, mix thoroughly until clear, then add 1.25 μL of Tween 80, mix thoroughly until clear, and then add 11.25 μL of physiological saline. The working solution preparation method is calculated based on an average mouse weight of 20g, an injection dose of BX471 of 5mg / kg, and a dosage of 5uL per mouse. The actual preparation can be adjusted according to the specific weight changes of the mice, following the general working solution preparation ratio in the commercial instructions.

[0008] The tumor cells were Ka539 B-cell lymphocytes and the CD19-overexpressing melanoma cell line B16. Their in vitro culture was conducted as follows: Ka539 cells: 45% DMEM and 45% IMDM were mixed in a volume ratio as the basal medium, followed by the addition of 10% FBS, 1× penicillin-streptomycin, and 50 µM β-mercaptoethanol. The cells were then cultured in suspension, maintaining a cell density of approximately 2×10⁶ cells / year. 5 ~8×10 5 Cells were passaged at a density of [number] cells / mL via fluid replenishment or 1:2 to 1:4 dilution. CD19-B16 cells were preferably cultured in high-glucose DMEM (4.5 g / L glucose) as the basal medium, with the addition of 10% fetal bovine serum (FBS), 1× penicillin-streptomycin solution, 1× non-essential amino acids, 1 mM sodium pyruvate, and 2 mM L-glutamine. Cells were cultured at 37 °C and 5% CO2. When cells reached 70%–90% confluence, they were passaged by digestion with 0.05% Trypsin-EDTA. Both types of tumor cell lines were cultured in vitro at 37 °C and 5% CO2.

[0009] The CAR-T cells include murine CAR-T cells and human CAR-T cells. Murine CAR-T cells are derived from T cells isolated from mouse spleens, and the scFv-CD19 lentivirus infection efficiency must be above 30%. Human CAR-T cells are derived from peripheral blood T cells from volunteers, and the scFv-CD19 infection efficiency in human CAR-T cells must be above 50%. For ease of tracking in in vivo experiments, an mCherry fluorescent tag structure is added to the CAR structure.

[0010] The mice used were C57BL / 6 (for the CD19-CAR-T therapy model) and immunodeficient mice (for the GD2-CAR-T therapy model). The subcutaneous tumor-bearing model was established by subcutaneous injection of Ka539 B-cell lymphoma / CD19-B16 melanoma into the right abdomen of the mice. The in situ tumor-bearing model was established by intramuscular injection of 143B osteosarcoma cells into the right leg of the mice; detailed implementation methods are provided in Examples 3-5.

[0011] The BX471 injection dose was 5 mg / kg. The first administration was completed within 24 hours of CAR-T cell injection, followed by two consecutive tail vein administrations of the same dose every other day, for a total of 3-5 administrations. After one week (7 days) of treatment, the tumor volume, weight, and survival of lymphoma / melanoma mice were recorded. Flow cytometry was used to detect the proportion of intratumoral CAR-T cells and key microenvironment immune components. The integrity of the myeloid immune barrier structure was observed using immunofluorescence imaging.

[0012] Furthermore, this invention conducted a short-term biosafety evaluation of this small molecule antagonist in mice. In subcutaneous xenograft models and tumor-free control mice, the CCR1 antagonist BX471 was administered via tail vein injection according to the aforementioned dosing regimen, and peripheral blood counts and the proportion of bone marrow immune cell populations were measured. Compared with mice not treated with BX471, no abnormal changes in blood counts were observed in the treated group, and the proportion of major immune cell subsets in the bone marrow was also not significantly different, suggesting that this small molecule antagonist has good biosafety under short-term dosing conditions.

[0013] This invention demonstrates that BX471, by inhibiting CCR1-mediated myeloid cell chemotaxis and related fibrosis signals, can be used to prepare drugs that improve the tumor immune microenvironment. This is manifested in reducing the enrichment of suppressor monocytes / macrophages and the level of stromal fibrosis, thereby enhancing the tumor infiltration and effector function of CAR-T cells. In mouse tumor models (including but not limited to B-cell lymphoma and melanoma), continuous administration of BX471 after CAR-T infusion for a predetermined course of treatment yielded superior tumor-suppressive indicators compared to CAR-T monotherapy; no significant systemic toxicity was observed at the tested doses and treatment durations. This invention provides a basis for combination therapy and its applications in tumor microenvironments characterized by myeloid cell enrichment leading to immune rejection.

[0014] This invention employs the CCR1 antagonist BX471 to block CCR1-mediated myeloid chemotaxis and accumulation, thereby weakening CCR1 at its source. + Tumor-associated myeloid cells (TAMCs) — FAP + The tumor immune barrier formed by the CAF axis enhances the infiltration and killing effect of CAR-T cells (chimeric antigen receptor T cells) on solid tumors. This invention provides... Attached Figure Description

[0015] Figure 1 To evaluate the efficacy of BX471 in a subcutaneous mouse model of Ka539 lymphoma treated with CAR-T therapy. (A) Schematic diagram of the combined treatment model; (B) Representative photographs of tumor tissue changes after combined treatment; (C) Immunohistochemical images of the tumor after combined treatment; (D) Survival differences among the four groups of mice; (E) Changes in tumor weight in mice after combined treatment; (FJ) Flow cytometry analysis of tumor cells, CAR-T cells, M2 macrophages, and CCR1 cells in the tumor tissue of mice after combined treatment. + Tumor-associated myeloid cells and FAP + Changes in the proportion of fibroblasts.

[0016] Figure 2Immunofluorescence staining images of tumor structural changes after BX471 combined with CAR-T therapy for Ka539 lymphoma. (A) Immunofluorescence staining protocol and calculation method for cell fluorescence intensity characterization within the field of view; (B) Immunofluorescence staining results of tumor tissues in the CAR-T monotherapy group and the combination therapy group, showing the spatial changes of the fibrous tumor barrier in both groups and the changes in its spatial relationship with infiltrating CAR-T cells; (C) Immunofluorescence spatial changes of the fibrous tumor barrier structure in the combination therapy group and the CAR-T monotherapy group (CCR1). + (D) Spatial relationship changes of tumor-associated myeloid cells and their infiltrating CAR-T cells; (E) Spatial relationship between the fluorescence intensity score of CCR1+ myeloid immune barrier and the fluorescence intensity score of CAR-T cells in tumor tissue after CAR-T therapy in the group without BX471 combined treatment; (X) Spatial relationship between the fluorescence intensity score of CCR1+ myeloid immune barrier and CAR-T cells in tumor tissue after combined BX471 and CAR-T therapy. + Spatial relationship between fluorescence intensity score of myeloid immune barrier and fluorescence intensity score of CAR-T cells; (F) represents the change in CCR1 with the degree of CAR-T infiltration. + Spatial dynamic changes in fluorescence intensity of the myeloid immune barrier.

[0017] Figure 3 This study evaluates the efficacy of BX471 in a CD19-CAR-T therapy in a mouse model of CD19-B16 melanoma with subcutaneous tumors. (A) Schematic diagram of the combined treatment model; (B) Representative photographs of tumor tissue changes after combined treatment; (C) Survival differences among the four groups of mice; (D) Changes in tumor weight in mice after combined treatment; (E) Flow cytometry analysis of tumor cells, CAR-T cells, M2 macrophages, and CCR1 cells in the tumor tissue of mice after combined treatment. + Tumor-associated myeloid cells and FAP + Changes in the proportion of fibroblasts.

[0018] Figure 4 To evaluate the efficacy of BX471 in GD2-CAR-T therapy in an immunodeficient mouse model of 143B osteosarcoma. (A) Schematic diagram of the combined treatment model; (B) Changes in tumor volume in mice after combined treatment; (C) Serial imaging results of tumors in 4 groups of mice.

[0019] Figure 5 Blood test results for short-term in vivo safety evaluation of BX471.

[0020] Figure 6 Bone marrow flow cytometry results for short-term in vivo safety evaluation of BX471. Detailed Implementation

[0021] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0022] This invention discloses the application of a non-peptide small molecule antagonist in improving the tumor microenvironment, enhancing CAR-T cell infiltration, and increasing anti-tumor efficacy. This invention primarily addresses the difficulty of immune infiltration of CD19 CAR-T cells in solid tumors. Therefore, those skilled in the art can refer to the content of this document to expand the application of different CAR-T cells in different solid tumors. The methods and applications of this invention have been described based on favorable examples, all of which are included within the scope of this invention. Those skilled in the art can make appropriate modifications to the methods and applications without departing from the content and scope of this invention to implement and apply them.

[0023] Example 1: Virus Preparation The complete culture medium described in this invention comprises 90% DMEM (high glucose) medium, 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin. PlatoE cells are cultured in this medium in 10 cm culture dishes until the density reaches 60-70%, at which point additional fluid is added.

[0024] Prepare a common plasmid system, which mainly includes 1 μg of the target plasmid (CD28z or 4-1BB), 1 μg of the encapsulation vector Ecoli, and 1 μg of the packaging plasmid PEI. Add these to a 1.5 ml centrifuge tube in the order described above, mix, and let stand for 20 min.

[0025] After settling, change the medium and add 5-6 ml of FBS-free DMEM, adding 400 μL of the prepared medium to each dish. Place the cells with the added virus system in a 37°C, 5% CO2 incubator. After 6-8 hours of infection, remove the supernatant and add 10 ml of complete culture medium. Continue culturing under the aforementioned conditions. Harvest the virus twice, at 48 hours and 72 hours, and store the harvested virus at 4°C.

[0026] The aforementioned virus was centrifuged at 300g for 10 min to remove cell debris, then filtered through a 0.45μm yellow filter membrane and centrifuged at 25000rpm for 3 h at 4°C.

[0027] Remove the supernatant, add RPMI 1640 culture medium and concentrate 100-200 times, let stand at 4℃ for 12h, dispense into EP tubes, 500μl / tube, and store at -80℃.

[0028] Example 2: Preparation of CAR-T cells Prepare C57BL / 6 mice, anesthetic agents, and sterilize surgical instruments. Based on the number of mice, prepare 50 ml of Buffer 1 (PBS + 2% FBS + 2xPS) per mouse, pre-cool on ice, and aliquot into three 15 ml culture dishes. Prepare T cell culture medium, mainly consisting of IMDM (43 ml) + 10% NZ-FBS (5 ml) + 1xSodium Pyruvate (500 μl) + 1xGlutamine + 1xNEA (500 μl) + 1xβ-Me (25 μl) + 2xPS (1000 μl) + 100 U / ml mIL-2 (100 μl).

[0029] The spleen was removed under aseptic conditions, and the connective tissue was separated with surgical scissors. It was then placed in the first culture dish containing Buffer1, washed, transferred to another culture dish containing Buffer1, and then transferred to a culture dish containing Buffer1 in the laminar flow hood.

[0030] In a clean bench, after blowing cells from the spleen using a 5ml syringe, place the spleen into a 40μm filter, gently grind with the syringe plunger, rinse with 5ml Buffer 1, and gently agitate to prevent cell clumping. After thorough grinding, filter the single-cell suspension through a 40μm filter into a 50ml centrifuge tube, centrifuge at 500g for 5 minutes, discard the supernatant, resuspend in 2ml of T-cell culture medium, and slowly add it to a 15ml centrifuge tube above 4ml of mouse lymphocyte separation medium EZ-Sep Mouse 1*, taking care to maintain liquid separation and avoid mixing.

[0031] Set the centrifugation mechanism to 3 up, 0 down, and centrifuge at 800g at room temperature for 30 min. Aspirate the lymphocyte layer, gently mix with 10 ml of 1640 basal medium, and perform cell counting. Centrifuge at 400g at room temperature for 10 min, discard the supernatant, and resuspend the cells in MACS buffer (DPBS + 0.5% BSA + 2mM EDTA) to a concentration of 1×10⁶ cells / mL. 8 / ml.

[0032] Purify T cells according to the standard procedure of the Stem Cell mouse T cell negative selection kit: transfer cells to flow cytometry tubes; add 50 μl / ml Rat Serum and 50 μl / ml Cocktail sequentially and mix well. Incubate at room temperature for 10 min.

[0033] Vortex the magnetic beads for 30 seconds according to the Stem Cell instructions. Add the magnetic beads to the flow cytometer at a rate of 75 μl of magnetic beads / ml of sample, mix well, and incubate at room temperature for 2.5 min.

[0034] Add MACS to bring the total volume of the liquid to 2 ml. Gently pipette and mix 2-3 times. Then place the flow cytometry tube on a magnetic rack and incubate at room temperature for 2 min. After that, transfer the cell suspension in the flow cytometry tube to a new flow cytometry tube with the magnetic rack attached. Add 2 ml of MACS to the original flow cytometry tube again. Repeat this step once.

[0035] After counting the purified cell suspension, centrifuge and then resuspend the cells in 1-2 ml of TCM.

[0036] Activate T cells according to the Gibco #11456D Dynabeads mouse T-activator CD3 / CD28 kit instructions. Add magnetic beads at a 1:1 volume ratio to T cells, as per the instructions.

[0037] Resuspend the magnetic bead in a vortex for 30 seconds, then repeat at 1×10... 6 For T cells, extract the required volume of magnetic beads and add it to a 15ml centrifuge tube. Add 1ml of 1640 basal culture medium and vortex for 5 seconds to mix. Place on a magnetic rack for 1 minute, gently discard the supernatant, and repeat this step twice.

[0038] Resuspend the magnetic beads in the initial volume of T cell suspension, then transfer the cell-magnetic bead mixture to a T25 glass container and mix thoroughly on a shaker for 25 min. Resuspend the magnetic beads in the initial volume of T cell suspension, then transfer the cell-magnetic bead mixture to a T25 glass container and mix thoroughly on a shaker for 30 min.

[0039] After culturing at 37℃ and 5% CO2 for 24 hours, T cells were observed to be larger in volume under an optical microscope, with no obvious cell clusters. The cells were then coated with 0.3 ml of Retronectin and seeded into 12-well plates, and stored overnight at 4℃.

[0040] After adding 1% BSA, incubate at room temperature for 30 minutes.

[0041] According to 2×10 per hole 6 T cells / magnetic beads were seeded into 12-well plates to prepare a 400 μl T cell infection system, which included the concentrated virus prepared in Example 1, polybrene transfection agent, and basal culture medium.

[0042] Resuspend T cells in basal medium, using a viral volume that is three times the volume of T cells. After standing for 30 minutes, centrifuge at 1200g for 2 hours at room temperature, then replace with fresh medium.

[0043] Continue culturing and expanding to the number of cells required for subsequent cases; some CAR-T cells can be cryopreserved at -80℃ for future use.

[0044] Example 3: BX471 improves CAR-T cell infiltration and therapeutic effect in a mouse model of Ka539 B-cell lymphoma. like Figure 1 The BX471 process enhances CAR-T cell infiltration and therapeutic efficacy in a mouse model of B-cell lymphoma, while simultaneously improving CCR1 in the suppressive tumor microenvironment. + Myeloid cell enrichment and degree of fibrosis.

[0045] Twenty 6-week-old C57BL / 6 mice were used, and each mouse was subcutaneously injected with 2×10⁻⁶ mmol / L of the drug via the right lower abdomen. 6 The mice were then randomly divided into four groups: an untreated control group (hereinafter referred to as group 1). The BX471 monotherapy group (hereinafter referred to as Group 2), the CAR-T cell monotherapy group (hereinafter referred to as Group 3), and the BX471 and CAR-T combination therapy group (hereinafter referred to as Group 4) each consisted of 10 animals.

[0046] Ten days after simultaneously injecting tumor cells into four groups of mice, mice in groups 3 and 4 were injected via the tail vein with 2×10⁻⁶ tumor cells. 6 CAR-T cells tagged with mCherry were injected with BX471 via tail vein into groups 2 and 4 on the same day after CAR-T cell injection. The injection dose was 5 mg / kg, administered every 48 hours for two consecutive times, for a total of three administrations. Five animals were sacrificed 21 days later. Tumor tissues from each group were collected to determine tumor size and weight, and tumor sections were prepared for immunohistochemical staining and immunofluorescence experiments. Flow cytometry was used to detect tumor cells, CAR-T cells, and CCR1. + Myeloid cells, FAP + The number of fibroblasts was counted, and statistical differences were calculated. The remaining 5 mice that were not sacrificed were used for survival statistical comparison.

[0047] like Figure 2 Immunofluorescence was performed on tumor tissue sections after treatment in groups 3 and 4. Two consecutive tumor sections were cut and mounted on the left and right sides of a glass slide, respectively. After overnight fixation with 4% PFA, anti-SPP1 and anti-FAP were added to the left side to label the fibroblast barrier, and anti-CCR1 and anti-CD11b were added to the right side to label CCR1. + Tumor-associated myeloid cells, which are generated through processes such as Figure 2 The antibody fluorescence intensity product method shown in Figure A is used to calculate the fluorescence intensity of different cell types, characterizing the average intensity of a certain cell type in the same region. The calculation formula is as follows: Fluorescence Score Indicates cell fluorescence intensity score / field of view. N This refers to the number of fluorescent channels that characterize this cell type. Ik The first in that field of vision k The fluorescence intensity of a single fluorescence channel per pixel. (x,y) This refers to the coordinates of the pixel within the target's field of view.

[0048] Example 4: BX471 improves CAR-T infiltration and efficacy in a CD19-B16 melanoma mouse model like Figure 3 The procedure for establishing a mouse model of CD19-B16 melanoma enhanced by BX471 was implemented. Except that the subcutaneous tumor cells were replaced with CD19-B16 melanoma cell lines, the rest of the implementation plan was the same as in Example 3.

[0049] Example 5: BX471 improves CAR-T therapy efficacy in a mouse model of 143B osteosarcoma. like Figure 4 The procedure for in situ modeling of 143B osteosarcoma in mice by BX471-enhanced GD2 CAR-T therapy was implemented. Twenty immunodeficient mice aged 4-6 weeks were used, and each mouse was injected intramuscularly with 1×10⁻⁶ BX471 in the right leg. 7 Three days after simultaneously injecting tumor cells into the four groups of mice, mice in groups 3 and 4 were injected via the tail vein with 2×10¹² GFP-tagged 143B osteosarcoma cells, grouped according to the same grouping scheme as in Example 3. 6CAR-T cells labeled with mCherry were injected with BX471 via the tail vein into groups 2 and 4 on the same day as the CAR-T cell injection. The injection dose was 5 mg / kg, administered every 48 hours for two consecutive times, for a total of 5 administrations. In the above embodiment, mouse tumor imaging was performed at fixed time points D0, D8, and D22. D0 was the baseline imaging before the treatment regimen was implemented, D8 was 1 week after the aforementioned treatment regimen was implemented, and D20 was 3 weeks after the treatment regimen was implemented. Imaging was performed using an IVIS Spectrum (or equivalent). Animals were anesthetized and kept warm by inhalation with 2–3% isoflurane induction and 1.5–2.0% maintenance. In bioluminescence mode, D-luciferin potassium salt 150 mg / kg was injected intraperitoneally, and acquisition began 10 min after administration, with exposure times of 1–60 s in an automatic range, F / Stop=1, and medium binning. In fluorescence mode, appropriate excitation / emission filters were selected according to the labeled molecules (e.g., mCherry: Ex 570–580 nm / Em 610–620 nm), and the excitation intensity and exposure time were kept within the unsaturated range. The posture, viewing distance, and field of view of each animal were kept consistent at all time points, and dark current and flat field correction were enabled. In Living Image software, the ROI covering the tumor was delineated with the same threshold, and the total photon flux (photons / s) or irradiance was recorded, and normalized to the individual D0 value for longitudinal comparison. The background signal was subtracted from the ipsilateral uninoculated ROI. Data acquisition was repeated on days 8 and 22 under the same conditions. Imaging was stopped and ethical termination criteria followed if ulceration, >20% weight loss, or anesthesia intolerance occurred. All raw images and quantitative data were retained, numbered, and archived, and can be used for statistical analysis in conjunction with caliper volume and survival outcomes. Tumor volume was recorded on day 22.

[0050] Example 6: Short-term safety evaluation in BX471 mice Figure 5 and Figure 6 The short-term safety evaluation experiment of BX471 in mice was conducted using the following groups: a blank control group, a drug treatment group (tumor-free, administered only), and a drug therapy group (administered according to the treatment regimen described in Example 3 after subcutaneous tumor formation), with 5 mice in each group. The administration time, dosage, and tumor inoculation method were consistent with those in Example 3 for each group. Mice were continuously administered the drug and followed up until day 21 after the start of administration, at which point they were sacrificed. Peripheral blood was collected for routine blood tests, and bone marrow samples were taken for flow cytometry analysis. The results showed that, compared with the blank control group, there were no significant abnormalities or statistically significant differences in blood cell counts and the proportion of major immune cell populations in the bone marrow of the drug treatment and drug therapy groups, indicating that BX471 has good short-term in vivo biocompatibility under the above administration conditions.

Claims

1. The application of a CCR1 antagonist in the preparation of drugs that enhance the anti-tumor efficacy of CAR-T cells, characterized in that, The CCR1 antagonist improves the tumor microenvironment and enhances the in vivo anti-tumor efficacy of CAR-T cells. The CCR1 antagonist is BX471.

2. The application according to claim 1, characterized in that, The CAR-T cells include murine CAR-T cells and human CAR-T cells.

3. The application according to claim 1, characterized in that, The tumor cells were B16, a melanoma cell line that overexpressed CD19.

4. The application according to claim 1, characterized in that, The CAR-T includes CD19 CAR-T and GD2 CAR-T.

5. The application according to claim 1, characterized in that, The tumor microenvironment is a tumor microenvironment enriched with myeloid cells and fibroblasts and with activated CCR1 axis.

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