An artificial antigen-loaded red blood cell immune product and its application in treatment of solid tumors

Abstract

The present application relates to a kind of artificial antigen loaded red blood cell immunization product and its application in solid tumor treatment, belong to biological medicine technical field.The present application constructs the RBC (RBC-Vac) of surface carrying artificial antigen FITC.At the same time, design and construct the amplification antigen receptor BAR capable of specifically recognizing FITC, and it is respectively introduced into tumor antigen specificity TCR-T cell and CAR-T cell, obtain BAR-TCR-T cell and BAR-CAR-T cell.The present application is activated and amplified by targeting spleen delivery artificial antigen BAR-TCR-T or BAR-CAR-T cell in vitro, to enhance the curative effect of ACT in solid tumor.The method can solve the problem of limited curative effect of ACT in solid tumor, provide new strategy and application value for ACT to treat solid tumor.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a red blood cell immunotherapy product loaded with artificial antigens and its application in the treatment of solid tumors. Background Technology

[0002] The application of adoptive T-cell therapy (ACT), such as chimeric antigen receptor T-cell (CAR-T cell) and T-cell receptor T-cell (TCR-T cell) therapy, in solid tumors remains limited, primarily due to factors such as the low number of ACT cells within the tumor and poor functional persistence. Most systemically injected ACT cells accumulate in secondary lymphoid organs, such as the spleen and lymph nodes, after circulating in the body. The spleen and non-draining lymph nodes lack tumor antigens, and ACT cells accumulated in these sites cannot effectively maintain their activated state, resulting in reduced efficacy and numbers. Activating and expanding ACT cells accumulated in secondary lymphoid organs, thereby increasing their recirculation and tumor infiltration rates, is becoming a new strategy to improve the efficacy of ACT in solid tumors.

[0003] Therapeutic vaccines work by delivering tumor antigens and adjuvants to antigen-presenting cells (APCs), such as dendritic cells (DCs), which then promote the presentation of antigens to TCRs using MHC molecules. This activates and expands these tumor antigen-specific endogenous cytotoxic T cells (CTLs) to inhibit tumor growth. Genetically modified TCR-T cells that recognize specific tumor or viral antigens and express CARs can yield bispecific TCR-CAR-T cells. After the vaccine delivers the target antigen to the TCR, the APCs present the antigen, stimulating the TCR to activate these bispecific CAR-T cells. However, TCR-dependent activation is limited by MHC molecules, significantly narrowing its application scope. Furthermore, current technologies for delivering CAR target antigens have weak spleen targeting and difficulty in controlling the expression cells and levels of CAR target antigens, easily leading to CAR-T cells accidentally damaging normal spleen cells or insufficient CAR-T cell activation. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problems of low number of ACT cells in vivo, functional decline and insufficient activation in the prior art.

[0005] To address the aforementioned technical problems, this invention provides a red blood cell immunotherapy product loaded with artificial antigens and its application in the treatment of solid tumors. This invention constructs an RBC-based amplified vaccine utilizing DSPE-PEG, which has membrane intercalation capabilities. 2000The artificial antigen FITC is modified onto the surface of RBCs to prepare RBCs carrying FITC on their surface (RBC-Vac). Simultaneously, a boosting antigen receptor (BAR) capable of specifically recognizing FITC is designed and constructed, and introduced into tumor antigen-specific TCR-T cells and CAR-T cells, respectively, to obtain BAR-TCR-T cells and BAR-CAR-T cells. By targeting the spleen with the artificial antigen, BAR-TCR-T or BAR-CAR-T cells in the spleen are activated and expanded, increasing tumor infiltration and thus enhancing the efficacy of ACT in solid tumors. This method addresses the limited efficacy of ACT in solid tumors, providing a new strategy and application value for ACT treatment of solid tumors.

[0006] The first objective of this invention is to provide a red blood cell immune product loaded with FITC antigen, which is prepared by modifying the surface of red blood cells with FITC antigen.

[0007] Furthermore, the erythrocyte immune product is prepared by co-incubating erythrocytes with phospholipids terminally modified with FITC-polyethylene glycol-FITC.

[0008] Furthermore, the ratio of phospholipid-polyethylene glycol-FITC to erythrocytes is (0.5-10) μg: 1×10 6 One red blood cell.

[0009] A second objective of the present invention is to provide an immune composition targeting the spleen, the immune composition comprising the above-described erythrocyte immune product and cells containing an amplified antigen receptor that specifically recognizes the FITC antigen.

[0010] Furthermore, the cells include T cells, which include chimeric antigen receptor T cells and / or T cell receptor T cells.

[0011] Furthermore, the amplified antigen receptor includes a single-chain variable domain that recognizes FITC, a hinge region and transmembrane segment of CD28, an intracellular 4-1BB co-stimulatory domain and a CD3ζ signaling domain, a P2A cleavage peptide, and a reporter protein tNGFR.

[0012] A third objective of this invention is to provide an application of the above-described immune composition in the preparation of tumor treatment products.

[0013] Furthermore, the tumor treatment product targets the spleen and activates and expands T cells within the spleen.

[0014] Further, the effector-to-target ratio of the erythrocyte immune product to cells containing amplified antigen receptors that specifically recognize FITC antigens is (5-100):1. Preferably, the effector-to-target ratio of the erythrocyte immune product to cells containing amplified antigen receptors that specifically recognize FITC antigens is (10-100):1.

[0015] A fourth object of the present invention is to provide a product for the treatment of solid tumors, the product comprising the above-described immune composition.

[0016] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0017] The RBC-Vac of this invention targets and enriches spleen and tumors. The artificial antigen FITC on the surface of the RBC-Vac can be recognized by engineered T cells expressing BAR, thereby achieving specific activation and expansion of BAR-TCR-T cells and BAR-CAR-T cells in vivo, increasing their chances of re-infiltrating the circulation and infiltrating tumors, and enhancing their therapeutic effect on tumors. By delivering the artificial antigen in vivo and constructing a synthetic receptor that can specifically recognize the antigen, specific activation of different adoptive T cells such as TCR-T and CAR-T cells can be achieved, exhibiting broad applicability. This invention provides a new strategy and experimental evidence for enhancing ACT (Active Therapy for Solid Tumors) such as CAR-T cells. Attached Figure Description

[0018] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0019] Figure 1 A is a flowchart of the preparation of RBC-Vac and BAR-T cells; B is a diagram showing the effect of RBC-Vac activating BAR-T cells in the spleen, enhancing their ability to expand, recirculate and infiltrate tumors, thereby enhancing their anti-tumor efficacy.

[0020] Figure 2This section presents the modification, characterization, and in vitro safety evaluation of RBC-Vac; A and B are representative flow cytometry plots and average fluorescence intensity of FITC fluorescence on the RBC-Vac surface; C is the drug loading of DP-FITC on the erythrocyte surface; D and E are flow cytometry histograms and average fluorescence intensity of Annexin expression on the erythrocyte surface after RBC-Vac preparation with different concentrations of DSPE-PEG-FITC; F is the permeability and fragility curve of RBC-Vac at different NaCl concentrations; G and H are the hemolysis experiments and hemolysis rates of different RBC-Vacs; I and J are representative flow cytometry histograms and average fluorescence intensity of FITC fluorescence on the surface of RBC-Vac at 0, 24, 48, and 72 h after preparation; K is the FITC fluorescence distribution observed by confocal microscopy.

[0021] Figure 3 This document describes the construction and functional study of BAR-TCR-T cells. A is a schematic diagram of the BAR gene sequence; B is a schematic diagram of the BAR-TCR-T cell structure; C is a flow cytometry histogram of tNGFR expression of BAR reporter protein on the surface of transfected T cells; D and E are flow cytometry histograms of CD69 expression and the average fluorescence intensity of E after activating BAR-TCR-T cells with different RBC-Vac ratios; F is an experimental flowchart; G shows the effect of RBC-Vac on the tumor-killing ability of BAR-TCR-T cells at different effector-to-target ratios (0.5, 1, 5, 10); H is the concentration of IFN-γ in the culture supernatant at an effector-to-target ratio of 10:1.

[0022] Figure 4 This study investigates the biodistribution of RBC-Vac in mice; A is a schematic diagram of the experimental procedure; B shows fluorescence imaging of various organs after treatment with different administration methods; C shows the average fluorescence percentage of each organ; D is a schematic diagram of the experimental procedure; E shows fluorescence imaging of organs after treatment with different concentrations of RBC-Vac; FH represents the proportion of fluorescence signal per unit mass in the spleen, lymph nodes, and tumor tissue relative to the total organ signal.

[0023] Figure 5 A is the activation of BAR-TCR-T cells in mice; B is the experimental flowchart; C is the line graph of the proportion of adoptive T cells (ACT) in PBMCs; D is the schematic diagram of the experimental procedure; E is the representative flow cytometry plot of the proportion of ACT in mouse spleen and lymph nodes; F is the average proportion and number of ACT in mouse spleen; GH is the average proportion and number of ACT in mouse lymph nodes.

[0024] Figure 6The diagram shows how RBC-Vac promotes the proliferation of BAR-TCR-T cells in the spleen, lymph nodes, and tumors in a tumor model. A is the experimental flowchart; B is a representative flow cytometry plot of the proportion of ACT in the spleen, lymph nodes, and tumors of mice; C is the average proportion of ACT in the spleen of mice; D is the average proportion of ACT in the lymph nodes of mice; E is the average proportion of ACT in the tumors of mice; F is the statistical count of ACT per milligram of spleen; G is the statistical count of ACT per milligram of lymph nodes; H is the statistical count of ACT per milligram of tumor.

[0025] Figure 7 The diagram shows how different dosing intervals of RBC-Vac promote the proliferation of BAR-TCR-T cells in tumors, spleen, lymph nodes, and peripheral blood in a tumor model; A is the experimental flowchart. BC are representative flow cytometry plots showing the percentage of ACT in mouse tumor and spleen cells after single and double doses of RBC-Vac injection, respectively; DF is the average percentage of ACT in mouse tumors, spleen, and lymph nodes; GH is the number of ACTs in mouse tumors and spleen.

[0026] Figure 8 RBC-Vac significantly enhances the therapeutic effect of BAR-TCR-T cells on subcutaneous solid tumors in mice; A and E are experimental flowcharts; B and F are tumor growth curves; C and G are survival curves; D and H are changes in body weight.

[0027] Figure 9 This section describes the construction and functional study of BAR-CAR-T cells; A is a schematic diagram of the CAR gene sequence; B is a schematic diagram of the BAR-CAR-T cell structure; C is a flow cytometry zebra plot showing the expression of reporter proteins tNGFR and Thy1.1 on the surface of transfected T cells; DE are flow cytometry histograms and mean fluorescence intensities of CD69 expression after BAR-CAR-T cells are activated by different RBC-Vac ratios; F is an experimental flowchart; G shows the effect of RBC-Vac on the tumor-killing ability of BAR-CAR-T cells at different effector-target ratios (0.5, 1, 5, 10); H is the concentration of IFN-γ in the culture supernatant.

[0028] Figure 10 The diagram shows the effects of RBC-Vac on the proliferation of BAR-CAR-T cells in tumors, spleen, lymph nodes, and peripheral blood cells in a tumor model. A is the experimental flowchart; B and D are representative flow cytometry plots showing the percentage of ACT in mouse tumors and spleen after single and double doses of RBC-Vac injection, respectively; C and E are the average percentage of ACT in mouse tumors and spleen; F and G are representative flow cytometry plots and average percentage of ACT in mouse tumors; J and K are the number of ACT cells in mouse tumors and spleen; I is the number of IFN-γ+ ACT cells in mouse tumors.

[0029] Figure 11 RBC-Vac significantly enhances the therapeutic effect of BAR-CAR-T cells on subcutaneous solid tumors in mice; A is the experimental flowchart; B is the tumor growth curve; C is the survival curve; D is the weight change. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0031] The detection methods involved are:

[0032] 1. Materials

[0033] DSPE-PEG 2000 -FITC (brand name: LZ-DS-YW0076), DSPE-PEG 2000 Cy5 was purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd.; mouse IL-2 and IL-7 were purchased from Peprotech; mouse anti-CD28 and anti-CD3 antibodies were purchased from Bio X Cell; fetal bovine serum, non-essential amino acids, sodium pyruvate, and HEPES were purchased from Gibco; and HieffTrans liposome nucleic acid transfection reagent was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0034] 2. Experimental cells and animals

[0035] HEK293T, B16-OVA, and B16-F10 cells were purchased from ATCC. B16-hCD19, B16-OVA-Luc, and B16-hCD19-Luc cells were constructed in the laboratory. C57BL / 6 mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd., and all mice were 6-8 weeks old. OT-1 mice were purchased from Shanghai Southern Model Biotechnology Co., Ltd., and Pmel-1 mice were purchased from Jackson Laboratory, USA. All mice were bred and housed in an SPF-grade animal experimental center under conditions of 20 ± 2 ℃ humidity, 50 ± 5% temperature, and 12 h of light per day. All animal experimental procedures complied with the relevant requirements of laboratory animal welfare and the laboratory animal ethics guidelines of Soochow University.

[0036] 3. Instruments

[0037] The ultra-high-speed refrigerated centrifuge and the small high-speed centrifuge were purchased from Eppendorf, Germany; the flow cytometer was purchased from BD, USA; and the full-wavelength multi-functional microplate reader was purchased from TECAN, Switzerland.

[0038] Example 1: Preparation and characterization of erythrocyte vaccines anchored to the artificial antigen FITC

[0039] 1. Experimental Methods

[0040] 1.1 Anchoring the artificial antigen FITC on the surface of red blood cells

[0041] Dissolve precisely weighed DSPE-PEG in DMSO 2000 DP-FITC (10 mg / mL stock solution) was prepared for use. One day prior to the experiment, the chest hair of C57BL / 6 mice was shaved. After anesthesia, blood was collected via subxiphoid puncture of the ventricle into heparin sodium anticoagulant tubes. After dilution with PBS, the cells were centrifuged at 3000×g for 5 min at 4°C, the supernatant was discarded, and the cells were washed 4-5 times until the supernatant was clear to obtain purified red blood cells, which were then resuspended in PBS for storage. DP-FITC was added to the red blood cell suspension at concentrations of 0.1, 1, 5, and 10 μg / mL (1×10⁻⁶). 6 ( / mL), incubate at 37℃ for 30 min, add serum-containing medium to terminate the reaction, wash with PBS and centrifuge to obtain FITC-modified erythrocyte vaccine (RBC-Vac) for subsequent flow cytometry analysis.

[0042] 1.2 Detection of drug loading of DP-FITC on the surface of erythrocytes

[0043] After co-incubating RBCs with DP-FITC (final concentrations of 1, 2, 4, 8, 16, and 32 μg / mL), the cells were centrifuged at 3000×g for 5 min to remove free DP-FITC. After counting the red blood cells, an appropriate amount of RIPA was added for lysis, releasing FITC from the membrane surface. Fluorescence intensity was measured using a microplate reader, with excitation / emission wavelengths set according to FITC standards. A fluorescence-concentration curve was established using the DP-FITC standard solution, and the DP-FITC loading on red blood cells in each group was calculated.

[0044] 1.3 Effect of FITC modification on RBC activity

[0045] 1.3.1 Annexin V staining experiment

[0046] Red blood cells were extracted and modified with DP-FITC membranes to prepare RBC-Vacs with different drug loading capacities (final concentrations of 0.5, 1, 2, 5, and 10 μg / mL). Samples were washed twice with pre-cooled PBS and once with 1×Annexin V Binding Buffer, then resuspended in 100 μL buffer, Annexin V antibody and nucleic acid dye were added, and the mixture was incubated at room temperature in the dark for 15 min. Then, 400 μL of buffer was added, and flow cytometry was performed to assess the effect of drug loading concentration on red blood cell membrane state.

[0047] 1.3.2 Penetrating brittleness test

[0048] Sodium chloride solutions of different concentrations were prepared, thoroughly mixed with red blood cells, and incubated at room temperature for 20 min. The supernatant was collected by centrifugation and transferred to 96-well plates. PBS was used as a blank control, and absorbance was measured at 540 nm. The absorbance corresponding to complete hemolysis in the 17.1 mM NaCl treatment group (i.e., 1 mg / mL) was defined as 100%, and the hemolysis rate under each NaCl concentration was calculated to plot the osmotic fragility curve.

[0049] 1.3.3 Hemolysis test

[0050] Different concentrations of DP-FITC (final concentrations of 0.5, 1, 2, 5, and 10 μg / 10) were used. 6 RBC-Vac samples were prepared by co-incubating erythrocytes with 100 cells (3 cells) in each well, with 3 replicates per group. The prepared RBC-Vacs were incubated at room temperature for 4 h, and hemolysis was observed. An equal volume of PBS was used as a negative control (Abs1), and an equal volume of deionized water was used as a positive control (Abs2). After incubation, the samples were centrifuged at 3000×g for 3 min at 4℃, and the erythrocyte sedimentation and hemolysis status of the supernatant were recorded by photograph. 100 μL of the supernatant from each group was transferred to a 96-well plate, and the absorbance at 577 nm was measured using a microplate reader (with 655 nm as the reference wavelength), recorded as Abs3, and the hemolysis rate was calculated.

[0051] 1.4 Retention of DP-FITC on the surface of erythrocytes

[0052] Flow cytometry was used to evaluate the retention capacity of FITC modified with different concentrations of DP-FITC on the surface of erythrocytes. The end of the intercalation reaction was recorded as 0 h, and RBC-Vac samples were collected at 0, 24, 48, and 72 h for flow cytometry analysis. Furthermore, a DP-FITC dosage of 5 μg / 102 was selected as the optimal level. 6 RBC-Vac was prepared for use in cells, and samples were collected at 0, 24, 48, and 72 h. The red blood cells were resuspended in 200 μL PBS, and an appropriate amount of anti-fluorescence quenching mounting medium was added to the cells under light-protected conditions. The cells were then prepared into slides, and the distribution of FITC on the surface of the red blood cells and the fluorescence intensity were observed using a laser confocal microscope.

[0053] 2. Discussion of Results

[0054] After co-incubating FITC-terminated DSPE-PEG (DP-FITC) with erythrocytes, the artificial antigen FITC was anchored on the erythrocyte surface, constructing the FITC-carrying erythrocyte vector vaccine RBC-Vac. Flow cytometry analysis showed that increasing the amount of DP-FITC significantly increased the average fluorescence intensity of FITC on the erythrocyte surface. Figure 2(AB). The FITC loading on the surface of red blood cells increased in a dose-dependent manner with the amount of DP-FITC added, reaching a peak at approximately 16 μg / 10. 6 Approaching saturation at 100 cells ( Figure 2 (C).

[0055] To assess the effect of high drug loading on erythrocyte membrane properties, flow cytometry was used to detect erythrocyte senescence markers ( ). Figure 2 The results showed that, compared with untreated erythrocytes, the Annexin V positive signal in DP-FITC-modified erythrocytes increased with increasing drug loading, suggesting that high concentrations of DP-FITC may promote phosphatidylserine eversion, but no significant disruption to membrane integrity was observed. Further hemolysis and osmotic fragility tests were performed. Figure 2 (FH), found below 5 μg / 10 6 The hemolysis rate of DP-FITC-modified erythrocytes per cell was less than 5%, and the osmotic fragility was not significantly different from that of normal erythrocytes, indicating that RBC-Vac maintained good biocompatibility under these conditions, while high concentrations (e.g., 10 μg / 10 cells) showed better biocompatibility. 6 (Each cell) may cause slight membrane damage.

[0056] Based on a comprehensive consideration of drug loading and biosafety, 5 μg / 10 6 Subsequent experiments were conducted on individual cells with DP-FITC to assess the retention time of FITC loading on the surface of erythrocytes. Figure 2 (IK). Flow cytometry analysis showed that the FITC signal on the RBC-Vac surface could be maintained for more than 72 h. Laser confocal microscopy showed that at 0 h, the FITC fluorescence was mainly distributed in a uniform ring shape, which highly coincided with the outline of the erythrocyte membrane; after 24 h, the fluorescence outline was still intact; and at 72 h, although the fluorescence intensity decreased, a significant FITC signal could still be detected on the erythrocyte surface, indicating that the artificial antigen could be stably anchored on the erythrocyte membrane surface for a long time.

[0057] Example 2: Activation of BAR-TCR-T cells by RBC-Vac

[0058] 1. Experimental Methods

[0059] 1.1 Construction of BAR vector and virus preparation

[0060] In pFucci-CMV-Amp +Double enzyme digestion and cloning were performed on the vector to construct the following BAR sequences: kozak sequence, mCD8αleader (NCBI: 12525), anti-FITC scFv (clone 4m5.3), mCD28 hinge (NCBI: 12487), mCD28TM, 4-1BB (NCBI: 21942), CD3ζ (NCBI: 12503), P2A and tNGFR (NCBI: 4804).

[0061] One day prior to the experiment, HEK293T cells were plated and cultured to a density of 70-80%. A three-plasmid transfection system was constructed (psPAX2:pMD2.G:target plasmid = 2:1:3, total volume 18 μg), with Hieff Lipo transfection reagent used at a volume 2.5-3 times that of the plasmid. The plasmid and transfection reagent were added separately to 800 μL of serum-free medium and mixed thoroughly. The mixture was allowed to stand at room temperature for 20 min to form a complex, which was then slowly added to the cells. Fluorescent protein expression could be observed the day after transfection. Viral supernatant was collected at 48 h and 72 h, centrifuged at 1000×g for 5 min, filtered through a 0.45 μm filter, and stored at 4℃ for short-term storage. After ultracentrifugation for concentration, the supernatant was stored at -80℃ for long-term storage.

[0062] 1.2 Mouse CD8 + Extraction and culture of T cells

[0063] (1) C57BL / 6 mouse CD8 + Extraction and culture of T cells

[0064] One day prior to the experiment, anti-CD3 antibody (1 μg / mL) and anti-CD28 antibody (5 μg / mL) were pre-coated in 6-well plates, 5 mL per well, and incubated overnight at 4°C. T-cell culture medium was prepared pre-: RPMI 1640 as the basal medium, with the addition of 10% FBS, 1% penicillin-dextrin antibody, 1% sodium pyruvate, 1% non-essential amino acids, and 1% HEPES. The following day, spleens from C57BL / 6 mice were harvested and mechanically homogenized using a 40 μm cell filter, followed by centrifugation at 4°C and 750×g for 4 min. After discarding the supernatant, 1 mL of ACK erythrocyte lysis buffer was added, and lysis was performed for 3 min, followed by termination of the reaction with 2 volumes of PBS. The resulting cell suspension was passed through a membrane again and centrifuged at 4°C and 750×g for 4 min to obtain the final spleen cell suspension. CD8+ cells from the spleen cells were isolated using the STEMCELL isolation kit according to the prescribed procedure. + T cells were resuspended in the previously prepared T cell culture medium, and after adding IL-2 and IL-7, they were transferred to coating plates and activated in a 37°C, 5% CO2 incubator for 48 h. After changing the medium, the cells were expanded for another 48 h before being used for subsequent experiments.

[0065] (2) Transgenic mouse CD8 + Activation and culture of T cells

[0066] After isolating the spleens of OT-1 / Pmel-1 mice, the same grinding, cleavage, and centrifugation procedures as described above were performed. Then, 20 mL of solution containing IL-2, IL-7, and OVA was added. 257-264 T cell culture medium containing peptide / gp10025-33 was resuspended, activated in an incubator for 3 days, and then subjected to gradient centrifugation with Ficoll-Pague Plus mononuclear cell separation medium. After purification, OT-1 / Pmel-1 CD8 was obtained. + T cells were used in subsequent experiments.

[0067] 1.3 Validation of RBC-Vac in vitro activation of BAR-TCR-T cells

[0068] 1.3.1 Lentiviral infection of T cells

[0069] The virus concentrate was diluted with serum-free RPMI 1640 and the titer was determined. HEK293T cells in logarithmic growth phase were adjusted to 0.1 × 10⁻⁶ cells / mL. 6 Spread the virus at a concentration of / mL onto a plate and incubate overnight. lentiviruses are serially diluted 10-fold and polybrene (5 μg / mL) is added. The medium is replaced with DMEM complete medium the day after infection, and cultured for another 48-72 h. Viral titers are then calculated.

[0070] T cells were infected with lentivirus at an MOI of 10 (1×10). 6 Cells / 500 μL virus solution), with a final Polybrene concentration of 8 μg / mL. After 0.5 days of infection, double the concentration of IL-2 and IL-7 was added to the culture medium, and the cells were cultured for another 2.5 days. Cells were then collected for flow cytometry analysis to assess infection efficiency.

[0071] 1.3.2 Effect of RBC-Vac on BAR-TCR-T cell activation

[0072] RBC-Vacs were prepared, resuspended in fresh culture medium, and counted. OT-1 mouse T cells were extracted and infected with anti-FITC.4-1BB.tNGFR CAR lentivirus to construct BAR-TCR-T cells. The BAR-TCR-T cell density was adjusted to 0.3 × 10⁻⁶ cells / year. 6Cells / mL were used to set different ratios of RBC-Vac / BAR-TCR-T, with 3 replicates per group. The specific groupings were as follows: G1: BAR-TCR-T; G2: RBC-Vac + BAR-TCR-T (30:1); G3: RBC-Vac + BAR-TCR-T (40:1); G4: RBC-Vac + BAR-TCR-T (50:1); G5: RBC-Vac + BAR-TCR-T (100:1). After co-incubation, the cells were cultured at 37°C for 24 h, followed by flow cytometry staining and analysis.

[0073] 1.4 Verification of the in vitro enhancement of BAR-TCR-T cell tumor-killing ability by RBC-Vac

[0074] BAR-TCR-T cells were prepared using the same method described above. One day in advance, B16-OVA-Luc cells were seeded into 96-well plates with a black transparent bottom, at a density of 1 × 10⁶ cells per well. 4 Cells were cultured overnight. Three replicates were used per group, with the following cell groups: G1: BAR-TCR-T; G2: RBC + BAR-TCR-T (50:1); G3: RBC + BAR-TCR-T (100:1); G4: RBC-Vac + BAR-TCR-T (50:1); G5: RBC-Vac + BAR-TCR-T (100:1). Different effector-to-target ratios (E / T) were set (0.5, 1, 5, 10), and the cells were co-cultured for 2 days at 37℃ and 5% CO2. The IFN-γ content was detected according to the ELISA kit instructions. 100 μL of PBS and 100 μL of Bright-Lite™ assay reagent were added to each well of the original plate, and the Luciferase intensity was detected using a microplate reader after 2 min of reaction.

[0075] 2. Discussion of Results

[0076] This embodiment successfully constructed the BAR molecule, whose sequence is anti-FITC.4-1BB.tNGFR ( Figure 3 (AB). In this embodiment, a three-plasmid lentiviral packaging system was used to package and prepare recombinant lentivirus in HEK293T cells. The concentrated virus was then used to infect T cells, ultimately obtaining BAR-TCR-T cells that stably express BAR. Flow cytometry results showed that the expression rate of the reporter molecule tNGFR reached 90.2% (AB). Figure 3 The C value indicates that BAR-TCR-T cells were successfully constructed.

[0077] To evaluate the activation effect of RBC-Vac on BAR-TCR-T cells, this study first constructed an OT-1CD8 cell expressing BAR. +T cells were incubated in vitro with different ratios of RBC-Vac, and the activation level of BAR-TCR-T cells was assessed by detecting the expression level of CD69, an activation molecule on the surface of BAR-TCR-T cells. The results showed that RBC-Vac increased CD69 expression in BAR-TCR-T cells under all ratio conditions, and the activation effect was further enhanced with increasing RBC-Vac ratio. Figure 3 (DE).

[0078] B16-OVA-Luc cells stably express luciferase, which, upon cleavage, catalyzes the oxidation of the substrate Luciferin to oxidative luciferin. This process releases photons, forming a detectable bioluminescent signal; therefore, the luminescence intensity can reflect the number and survival status of tumor cells. RBC-Vac was mixed with BAR-TCR-T cells at ratios of 50:1 and 100:1, and tumor cell killing experiments were conducted according to different effector-target ratios. Figure 3 The results showed that co-culturing ordinary erythrocytes with BAR-TCR-T cells did not significantly enhance their cytotoxic effect; however, RBC-Vac significantly enhanced the cytotoxic activity of BAR-TCR-T cells against B16-OVA-Luc cells under different effector-to-target ratios. When RBC-Vac:BAR-TCR-T=100:1, the additional killing rate of BAR-TCR-T cells against tumor cells could be increased by approximately 25% (F). Figure 3 Further analysis of IFN-γ levels in the culture supernatant at an effector-to-target ratio of 10:1 revealed that IFN-γ secretion levels increased synchronously with the increase in the RBC-Vac ratio. Figure 3 The above results indicate that RBC-Vac can effectively activate BAR-TCR-T cells in vitro and enhance their anti-tumor effects.

[0079] Example 3: Targeting capability and delivery efficiency of RBC-Vac

[0080] 1. Experimental Methods

[0081] 1.1 Distribution characteristics of RBC-Vac in major tissues of mice

[0082] B16-OVA cells were washed three times with PBS and then counted, with the count adjusted to 1×10⁻⁶. 7 / mL, subcutaneously inoculate each 6-8 week old female C57BL / 6 mouse with 1×10 6 One tumor cell (100 μL) was inoculated, and the day of inoculation was recorded as Day 0. Tumor volume was measured every 3 days. Red blood cells were used at 5 μg / 102 6Cy5-labeled RBCs were constructed by modifying individual cells with DP-Cy5 for in vivo distribution analysis. Each experimental group consisted of four mice, as follows: G1: PBS; G2: Free DP-Cy5 (sc); G3: DP-Cy5@RBC (iv); G4: Cy5-RBC (iv). The time of drug administration was recorded as 0 h. Mice were sacrificed 24 h later, and organs and tumor tissues were isolated for in vitro imaging analysis.

[0083] 1.2 Comparison of RBC-Vac distribution characteristics with different modification concentrations

[0084] B16-OVA tumor cells were inoculated using the method described above, and tumor volume was measured every 3 days. Red blood cells were extracted according to the method in Example 1 and treated with 2, 5, and 10 μg / 10⁻⁶ cells, respectively. 6 DP-Cy5 was modified in individual cells to prepare different amounts of RBC-Vac. Each experimental group consisted of 5 cells, and the groupings were as follows: G1: PBS; G2: DP-Cy5 (2 μg / 10⁻⁶) 6 (5 μg / 10 cells) @RBC; G3:DP-Cy5 ... 6 (10 μg / 10 cells) @RBC; G4:DP-Cy5 ... 6 (1 cell) @RBC. The time of drug administration was recorded as 0 h. Mice were sacrificed 24 h later, and organs and tumor tissues were isolated for small animal in vitro imaging analysis.

[0085] 2. Discussion of Results

[0086] To verify the organ distribution characteristics of RBC-Vac in vivo, this study used Cy5 instead of FITC for tracing to reduce imaging background in small animals. Following the CAR-T therapy dosage of RBC-Vac:BAR-TCR-T = 100:1, DP-Cy5@RBC and FarRed-labeled red blood cells were intravenously injected into C57BL / 6 tumor model mice, with a control group receiving subcutaneous injection of free DP-Cy5. The number of mice in each experimental group was the same. Figure 4 A).

[0087] The results showed that the Cy5 fluorescence signal in the spleen and tumor tissue after intravenous injection of RBC-Vac (G3) was significantly higher than that in the subcutaneous injection of free DP-Cy5 group (G2), while the distribution trend in the Far Red labeled erythrocyte group (G4) was consistent with that in G3. Compared with the G2 group, the fluorescence signal in the spleen of the G3 group increased by approximately 5.1 times, and the signal in the inguinal and axillary lymph nodes increased by approximately 5 times; the proportion of tissue fluorescence per unit mass showed that the spleen signal accounted for 53.4% ​​of the total organ fluorescence, which was approximately 4.1 times higher than that in the G2 group. In the tumor tissue, the signal in the G3 group was approximately 1.9 times higher than that in the G2 group, and the trend in the G4 group was consistent with that in the G3 group. Figure 4The results showed that RBC-Vac has a significant spleen-targeting delivery capability, providing experimental evidence for the in vivo activation of CAR-T cells.

[0088] To further compare the in vivo delivery efficiency of RBC-Vac with different drug loading concentrations, different DP-Cy5-loaded RBC-Vacs were intravenously injected using the same dosing ratio (RBC-Vac:BAR-TCR-T=100:1). Figure 4 The results showed that the distribution patterns of RBC-Vac at various drug loading concentrations were consistent in major organs, with significant enrichment in immune-related organs such as the spleen, suggesting that the delivery system has good organ targeting (D). Figure 4 The fluorescence intensity comparison showed that there was no significant overall difference between different drug loading amounts, indicating that within the experimental range, increasing the drug loading amount did not significantly change the in vivo distribution of RBC-Vac (E). Figure 4 (FH).

[0089] Example 4: Verification of the effect of RBC-Vac on activating BAR-TCR-T in vivo

[0090] 1. Experimental Methods

[0091] 1.1 RBC-Vac amplification of peripheral blood BAR-TCR-T cells

[0092] Five days prior to T-cell infusion, BAR-TCR-T cells were extracted and prepared. One day before infusion, cyclophosphamide was dissolved in PBS and pretreated in mice by intraperitoneal injection at a dose of 100 mg / kg. On the day of T-cell infusion, BAR-TCR-T cells were washed three times with PBS, and 5 × 10⁵ cells were injected into each mouse via the tail vein. 6 Cells. Two days after infusion, administer 5 μg / 10 6 RBC-Vacs were prepared from DP-FITC cells, and each mouse was injected with an equal amount of BAR-TCR-T cells at a dose of 50 times that of RBC-Vacs via the tail vein. The BAR-TCR-T injection diary was designated as Day 0, and the RBC-Vac injection as Day 2. Subsequently, PBMCs were extracted from the eyeballs every 3 days for flow cytometry staining and analysis.

[0093] 1.2 Validation of RBC-Vac's in vivo activation of BAR-TCR-T cells

[0094] Five days prior to T cell infusion, BAR-TCR-T cells were extracted and prepared. One day prior to infusion, mice were intraperitoneally injected with cyclophosphamide. Five mice were divided into groups as follows: G1: PBS; G2: BAR-TCR-T; G3: BAR-TCR-T + sc DP-FITC; G4: BAR-TCR-T + iv RBC-Vac (1:20); G5: BAR-TCR-T + iv RBC-Vac (1:100). On the day of T cell infusion, BAR-TCR-T cells were washed three times with PBS, and 5 × 10⁵ cells were injected into each mouse via the tail vein. 6 BAR-TCR-T cells. Two days after infusion, administer 5 μg / 10 6 RBC-Vac was prepared using DP-FITC per cell and administered according to groupings. Three days after administration of RBC-Vac or DP-FITC, the spleen and lymph nodes of mice were isolated, single-cell suspensions were prepared, and flow cytometry staining and analysis were performed.

[0095] 2. Discussion of Results

[0096] To investigate the kinetics of RBC-Vac-induced BAR-TCR-T cell proliferation in vivo, this study prepared RBC-Vac using DP-FITC-modified erythrocytes and constructed BAR-TCR-T cells using C57BL / 6 mouse-derived T cells. Two days after intravenous infusion of BAR-TCR-T cells, RBC-Vac was administered. Peripheral blood was collected every three days, and PBMCs were isolated and analyzed by flow cytometry. Figure 5 The results showed that 3 days after RBC-Vac administration (Day 5), the ACT ratio increased by approximately 3.2 times compared to Day 2, reaching its highest level, and then gradually decreased, demonstrating the in vivo expansion dynamics of BAR-TCR-T cells after RBC-Vac activation, providing a basis for optimizing dosing time. Figure 5 B).

[0097] To evaluate the effect of RBC-Vac on activating BAR-TCR-T cells in vivo, in healthy mice, BAR-TCR-T cells were intravenously infused for 2 days and then treated with RBC-Vac. After 3 more days of activation, the spleen and lymph nodes were isolated, and single-cell suspensions were prepared for flow cytometry analysis. Figure 5The results showed that, compared with the control group (G2) which received only BAR-TCR-T cells, subcutaneous injection of DP-FITC (G3) and intravenous injection of RBC-Vac (G4, G5) both increased the proportion of BAR-TCR-T cells in the spleen. Subcutaneous injection of DP-FITC increased the proportion by approximately 1.7 times, while 20-fold and 100-fold doses of RBC-Vac increased it by 2.1 times and 5.4 times, respectively. The number of ACTs per milligram of spleen tissue also showed the same trend, suggesting that RBC-Vac has a significant advantage in ACT amplification in the spleen. Figure 5 (EF). In lymph nodes, treatment with different doses of RBC-Vac increased the proportion of BAR-TCR-T cells by approximately 2.1 times and the number of ACTs per milligram of lymph node by approximately 1.8 times. The increase in the number of ACTs in lymph nodes may be due to the activation and redistribution of BAR-TCR-T cells in the spleen. In contrast, the subcutaneous injection of DP-FITC resulted in slightly better local activation of lymph nodes due to the homing effect of DSPE with albumin, leading to an increase in the proportion of ACTs by approximately 3.1 times and the number of ACTs by approximately 2.1 times. Figure 5 (GH).

[0098] Overall, RBC-Vac promotes the expansion of BAR-TCR-T cells in the spleen and partial homing to lymph nodes. Although subcutaneous DP-FITC injection has some advantages in local activation of lymph nodes, RBC-Vac performs better in terms of overall lymphoid organ ACT expansion levels.

[0099] Example 5: The role of RBC-Vac in a subcutaneous tumor model

[0100] 1. Experimental Methods

[0101] The methods for B16-OVA tumor modeling and preparation of BAR-TCR-T cells were the same as described above. Mice were grouped according to body weight and tumor volume, with 5 mice per group. The specific groupings were as follows: G1: PBS; G2: RBC-Vac; G3: BAR-TCR-T; G4: BAR-TCR-T + DP-FITC (sc); G5: BAR-TCR-T + RBC-Vac (iv). On the day of T cell infusion, BAR-TCR-T cells were washed three times. Each mouse in groups G3-G5 received a tail vein injection of 5 × 10⁵ cells. 6 BAR-TCR-T cells. On days 2 and 4 after BAR-TCR-T cell infusion, respectively, at 5 μg / 102 6 RBC-Vac was prepared using DP-FITC per cell and administered according to groupings. Seven days after BAR-TCR-T cell reinfusion, mouse spleen, lymph nodes, and tumor tissue were isolated, weighed, ground, and used to prepare single-cell suspensions for flow cytometry staining and analysis.

[0102] 2. Discussion of Results

[0103] To further evaluate the role of RBC-Vac in a subcutaneous tumor model, RBC-Vac was administered on days 2 and 4 after intravenous infusion of BAR-TCR-T cells. After activation, lymph nodes, spleen, and tumor tissue were collected for flow cytometry analysis. Figure 6 To rule out the potential impact of RBC-Vac itself on the body's immune response, a control group (G2) was established, receiving only RBC-Vac. Results showed that compared to the G3 group, which only received BAR-TCR-T cells, the RBC-Vac treatment group (G5) significantly increased the proportion of BAR-TCR-T cells in the spleen, by 15.4 times. Figure 6 The C), the amount of ACT per milligram of spleen tissue also showed the same trend (C), Figure 6 In contrast, the subcutaneous injection of DP-FITC (G4) only increased the relevant indicators by about 3.7 times, which was significantly weaker than RBC-Vac. In lymph nodes, because DP-FITC in the G4 group can enter the lymphatic system through albumin-mediated activation of local BAR-TCR-T cells, the expansion of BAR-TCR-T cells in the lymph nodes was the most significant. Compared with the G3 group, the proportion of BAR-TCR-T cells in the lymph nodes of the G4 group increased by about 1.8 times, and the number of BAR-TCR-T cells per milligram of lymph node increased by about 1.9 times. Figure 6 The D and G groups are mentioned. RBC-Vac is mainly distributed via the bloodstream and does not directly enter the lymphatic circulation; therefore, its promoting effect on lymph nodes is not as significant as that of DP-FITC. Notably, RBC-Vac has a more significant enrichment and expansion effect on BAR-TCR-T cells in tumor tissue. Compared with the G3 group, RBC-Vac increased the proportion of BAR-TCR-T cells in tumor tissue by approximately 15.4 times and the number of BAR-TCR-T cells per milligram of tumor tissue by approximately 14.7 times. Figure 6 The E and H values ​​were observed. Treatment with the same amount of DP-FITC only increased this indicator by approximately 3.7 times. This indicates that RBC-Vac can more effectively promote the in vivo expansion of BAR-TCR-T cells and tumor invasion in tumor models.

[0104] Example 6: Effect of RBC-Vac dosing interval on in vivo expansion of BAR-TCR-T cells

[0105] 1. Experimental Methods

[0106] The methods for establishing the B16-F10 tumor model and preparing BAR-TCR-T cells were the same as described above. Mice were grouped according to body weight and tumor volume, with 5 mice in each group. The specific groupings were as follows: G1: PBS; G2: BAR-TCR-T; G3: BAR-TCR-T + sc DP-FITC; G4: BAR-TCR-T + iv RBC-Vac (1:20); G5: BAR-TCR-T + iv RBC-Vac (1:50); G6: BAR-TCR-T + iv RBC-Vac (1:100). On the day of T cell infusion, the BAR-TCR-T cells were washed three times, and each mouse in groups G2-G6 received a tail vein injection of 5 × 10⁵ cells. 6 Two BAR-TCR-T cells were infused. On day 2 after BAR-TCR-T cell infusion, RBC-Vac was prepared at a dosage of 5 μg / 10⁶ cells and administered according to the assigned groups. Seven days later, blood was collected from the orbital cavity, and the spleen, lymph nodes, and tumor tissue of the mice were isolated, ground, and analyzed by flow cytometry. On the same day, the remaining mice were again administered the same dose of RBC-Vac or DP-FITC, and flow cytometry staining and analysis were performed. Seven days after the second administration, peripheral blood was collected again, and the spleen, lymph nodes, and tumor tissue were isolated and analyzed by flow cytometry again.

[0107] 2. Discussion of Results

[0108] To assess the effect of prolonged RBC-Vac dosing intervals on in vivo expansion of BAR-TCR-T cells, tumor tissue and spleen of some mice were analyzed 7 days after the first RBC-Vac administration (Day 9); the remaining mice were given the same dose of RBC-Vac again on the same day, and samples were taken again for analysis 7 days later. Figure 7 The results showed that double-needle RBC-Vac treatment significantly increased the number and expansion level of BAR-TCR-T cells in tumor tissue (A). Figure 7 Compared with the unactivated control group (G2), the number of BAR-TCR-T cells in tumors increased by approximately 3.3-fold, 7.7-fold, and 15.4-fold, respectively, in the different dose RBC-Vac groups (G4-G6); the double-dose administration groups at each dose showed a higher number of BAR-TCR-T cells than the corresponding single-dose groups, suggesting that even with prolonged dosing intervals, repeated administration of RBC-Vac can further promote the infiltration and maintenance of BAR-TCR-T cells in tumor tissue. Figure 7 (of D, G).

[0109] In the spleen, double-needle RBC-Vac also significantly promoted the expansion of BAR-TCR-T cells. Compared with the G2 group, the number of BAR-TCR-T cells in the different dose treatment groups increased by approximately 3-fold, 8.1-fold, and 11-fold, respectively. Figure 7(C, E, H). The overall amplification effect of each RBC-Vac group was better than that of the subcutaneous injection group with the same FITC dose (G3), indicating that the erythrocyte carrier is beneficial for antigen enrichment in the spleen and effective activation of BAR-TCR-T cells. The number of BAR-TCR-T cells in the spleen of the multiple-dose group was significantly higher than that of the single-dose group, suggesting that repeated administration of RBC-Vac can further enhance the sustained amplification and activation effect in the spleen.

[0110] Overall, RBC-Vac, delivered to the spleen via erythrocyte carriers, effectively enhances the expansion of BAR-TCR-T cells in circulating immune organs, thereby increasing their invasiveness in tumor tissues. The two-needle administration strategy, even with extended dosing intervals, significantly improves BAR-TCR-T cell expansion and accumulation, providing a basis for optimizing RBC-Vac dosing frequency and enhancing therapeutic efficacy.

[0111] Example 7: RBC-Vac enhances the anti-tumor efficacy of BAR-TCR-T cells

[0112] 1. Experimental Methods

[0113] 1.1 Study on the enhancement of BAR-TCR-T cell antitumor efficacy by RBC-Vac in subcutaneous tumor models

[0114] The methods for B16-OVA tumor modeling and preparation of BAR-TCR-T cells were the same as described above. Five mice were used in each group, as follows: G1: PBS; G2: RBC-Vac; G3: BAR-TCR-T; G4: BAR-TCR-T + sc DP-FITC; G5: BAR-TCR-T + ivRBC-Vac (1:10); G6: BAR-TCR-T + ivRBC-Vac (1:100). Mice in groups G3-G6 received 5×10⁵ PBS via tail vein infusion. 6 100 BAR-TCR-T cells were administered. Drugs were given according to group assignments on days 2 and 4 after T cell infusion. Mouse body weight and tumor volume were measured every 2 days after T cell infusion. Tumor volume was considered decreased when it reached 1500 mm². 3 The time was used as the termination criterion for the experiment, and mouse survival curves were plotted at the same time.

[0115] 1.2 Antitumor efficacy of RBC-Vac enhanced by different dosing frequencies in a subcutaneous tumor model against BAR-TCR-T cells

[0116] The methods for B16-OVA tumor modeling and BAR-TCR-T cell preparation were the same as described above. Five mice were assigned to each group as follows: G1: PBS; G2: BAR-TCR-T; G3: BAR-TCR-T + RBC-Vac (1:50) every 4 days; G4: BAR-TCR-T + RBC-Vac (1:50) every 7 days; G5: BAR-TCR-T + RBC-Vac (1:100) every 7 days. Mice in groups G2-G5 received 5 × 10⁵ BAR-TCR-T cells via tail vein infusion. 6 One BAR-TCR-T cell was infused. Two days after T cell infusion, RBC-Vac was administered via tail vein injection according to the prescribed dosing frequency and dosage. Tumor volume was measured and body weight was recorded every two days after T cell infusion. When the tumor volume reached 1500 mm, the infusion was recorded. 3 The time was taken as the end point of the experiment, and survival curves were plotted.

[0117] 2. Discussion of Results

[0118] To evaluate the antitumor effect of combined RBC-Vac and BAR-TCR-T cell therapy, this study validated this strategy in a B16-OVA subcutaneous tumor model. First, different doses of RBC-Vac were constructed, and BAR-TCR-T cells were constructed using OT-1-derived TCR-T cells. Tumor-bearing mice were then assigned to different treatment groups. Figure 8 (A). Experimental results showed that the tumor volume in the PBS group had not yet reached 1500 mm. 3 On day 14, subcutaneous injection of DP-FITC, compared to infusion of BAR-TCR-T cells alone, further enhanced the anti-tumor effect of BAR-TCR-T cells, increasing the tumor growth inhibition rate to 83%. Figure 8 Based on this, different doses of RBC-Vac significantly enhanced the therapeutic effect of BAR-TCR-T cells. By day 26, the tumor volume in the G6 group was only 16.7% of that in the G5 group, and the survival time of mice was significantly prolonged. Figure 8 (C). Although both subcutaneous injection of DP-FITC and intravenous injection of RBC-Vac enhanced the anti-tumor effect of BAR-TCR-T cells, RBC-Vac showed a more significant advantage in long-term tumor control and prolonging mouse survival. Furthermore, there was no significant difference in body weight change among the treatment groups, suggesting that the above treatment strategies did not cause significant systemic toxicity. Figure 8 D).

[0119] Based on this, this study further optimized the dosage and frequency of RBC-Vac administration. By setting different dosages and dosing intervals, the optimal dosing regimen for RBC-Vac to enhance the therapeutic effect of BAR-TCR-T cells was explored. Tumor-bearing mice were divided into five groups, receiving PBS (G1), BAR-TCR-T cells (G2), BAR-TCR-T cells combined with 50-fold RBC-Vac every 4 days (G3), BAR-TCR-T cells combined with 50-fold RBC-Vac every 7 days (G4), and BAR-TCR-T cells combined with 100-fold RBC-Vac every 7 days (G5), respectively. Figure 8 (E). The results showed that the tumor volume in the PBS group had not yet reached 1500 mm. 3 On day 16, infusion of BAR-TCR-T cells alone inhibited tumor growth by approximately 52%. Figure 8 (F). Different dosing frequencies of RBC-Vac all further enhanced the anti-tumor effect of BAR-TCR-T cells. Because the dosing frequency and dose in group G4 were lower than in group G3 and group G5, the tumor volume in this group showed a brief rebound after day 10, but decreased again after reactivation with RBC-Vac, eventually reaching the cure standard. Groups G3 and G5 also showed similar trends. By day 22, the tumor volume in groups G3, G4, and G5 was only 1.4%, 5.4%, and 0.25% of that in group G2, respectively. Figure 8 The F), and significantly prolonged the survival of mice ( Figure 8 Notably, the survival rates of groups G3 and G5 were 60% and 80%, respectively, due to higher doses or higher dosing frequencies; while group G4, which provided a gentler and more sustained stimulation under lower dose and lower frequency conditions, achieved a 100% survival rate. These results indicate that RBC-Vacs administered at different frequencies can significantly enhance the anti-tumor effect of BAR-TCR-T cells, but a relatively lower dose and lower frequency dosing strategy is more advantageous in long-term tumor control and prolonging survival. Furthermore, no significant changes in body weight were observed in any of the treatment groups, further demonstrating the good safety profile of this treatment regimen. Figure 8 H).

[0120] Example 8: RBC-Vac in vitro activation of BAR-CAR-T cells

[0121] 1. Experimental Methods

[0122] 1.1 Construction of CAR vector

[0123] In pFucci-CMV-Amp +Double enzyme digestion and cloning were performed on the vector to construct CAR sequences: kozak sequence, mCD8αleader, anti-hCD19 scFv (clone FMc36), mCD28 hinge, mCD28 TM, CD28, CD3ζ, P2A and Thy1.1.

[0124] 1.2 Validation of RBC-Vac in vitro activation of BAR-CAR-T cells

[0125] CD8 was extracted from OT-1 mice. + T cells were infected with CD8 using anti-FITC.4-1BB.tNGFR CAR and anti-hCD19.CD28.Thy1.1 viral concentrate. + T cells were used to prepare BAR-CAR-T cells. The density of BAR-CAR-T cells was adjusted to 0.3 × 10⁻⁶. 6 Cells were analyzed at a ratio of 10 cells / mL, with the RBC-Vac / BAR-CAR-T activation ratio set. Each group had three replicates, and the specific groupings were as follows: G1: BAR-CAR-T; G2: RBC-Vac + BAR-CAR-T (10:1); G3: RBC-Vac + BAR-CAR-T (50:1); G4: RBC-Vac + BAR-CAR-T (100:1). After activation, cells were collected and subjected to flow cytometry staining and analysis.

[0126] 1.3 Validation of RBC-Vac's in vitro enhancement of the tumor-killing ability of BAR-CAR-T cells

[0127] BAR-CAR-T cells were prepared using the same method described above. One day in advance, B16-hCD19-Luc cells were seeded into 96-well plates with a black transparent bottom, at a density of 1 × 10⁶ cells per well. 4 Cells were cultured overnight. IFN-γ levels and cytotoxic activity were detected according to the method in Section 2.8. Each group had three replicates, with the following specific groupings: G1: BAR-CAR-T; G2: RBC-Vac + BAR-CAR-T (50:1); G3: RBC-Vac + BAR-CAR-T (100:1).

[0128] 2. Discussion of Results

[0129] This study further constructed the CAR gene sequence, the specific structure of which is as follows: Figure 9As shown in Figures AB. BAR-CAR-T cells were successfully constructed by co-infecting T cells with two lentiviruses, BAR and anti-hCD19.CD28.Thy1. Flow cytometry results showed that the proportion of double-positive cells simultaneously expressing the BAR reporter molecule tNGFR and the CD19-CAR reporter molecule Thy1.1 reached 74.9%. Figure 9 (C).

[0130] To verify the activating effect of RBC-Vac on BAR-CAR-T cells, this study co-incubated RBC-Vac with BAR-CAR-T cells at different ratios and detected CD69 expression by flow cytometry. The results showed that when RBC-Vac:BAR-CAR-T = 10:1, RBC-Vac significantly activated BAR-CAR-T cells, increasing the average fluorescence intensity of CD69 by approximately 3 times. Figure 9 (DE). With further increases in the RBC-Vac ratio, the activation level of BAR-CAR-T cells continued to enhance. Under conditions of 50:1 and 100:1, the CD69 fluorescence intensity increased by approximately 6-fold and 18-fold, respectively. This indicates that RBC-Vac can effectively promote the activation of BAR-CAR-T cells, and this effect is significantly dose-dependent.

[0131] The effect of RBC-Vac on the tumor-killing function of BAR-CAR-T cells was further investigated. The results showed that RBC-Vac significantly enhanced the killing ability of BAR-CAR-T cells against B16-hCD19-Luc cells, increasing the tumor killing rate by 22-45% under different effector-to-target ratios. Figure 9 The results indicate that RBC-Vac can not only promote the activation of BAR-CAR-T cells, but also further enhance their in vitro anti-tumor effect.

[0132] Example 9: Regulatory effect of RBC-Vac on the in vivo function of BAR-CAR-T cells

[0133] 1. Experimental Methods

[0134] The methods for establishing B16-hCD19 tumor models and preparing BAR-CAR-T cells were the same as described above. Mice were randomly divided into groups of 5 mice each, based on tumor volume, as follows: G1: PBS; G2: BAR-CAR-T; G3: BAR-CAR-T + iv RBC-Vac (1:20); G4: BAR-CAR-T + iv RBC-Vac (1:50); G5: BAR-CAR-T + iv RBC-Vac (1:100). Mice in groups G2-G5 received 5 × 10⁵ PBS via tail vein infusion. 6Two days after T cell infusion, RBC-Vacs were prepared and administered via tail vein injection according to the assigned group. Seven days later, peripheral blood was collected, and mice were dissected. Lymph nodes, spleen, and tumor tissue were isolated, weighed, ground, and subjected to flow cytometry staining and analysis. RBC-Vac injections were administered again on the same day, and blood and tissue collection were repeated after another seven days. Flow cytometry analysis was performed using the same method.

[0135] 2. Discussion of Results

[0136] To further evaluate the regulatory effect of RBC-Vac on the in vivo function of BAR-CAR-T cells, this study investigated its impact on the activation and tumor-killing ability of BAR-CAR-T cells in a subcutaneous tumor model. First, CD8+ cells derived from OT-1 mice were used... + BAR-CAR-T cells were constructed based on T cells; subsequently, on days 2 and 4 after intravenous infusion of BAR-CAR-T cells, corresponding doses of RBC-Vac were administered to simulate multiple antigen stimulation conditions. Three days after the last activation, mice were dissected, and lymph nodes, spleen, and tumor tissue were isolated. Flow cytometry was used to analyze the distribution and functional status of BAR-CAR-T cells in different tissues. Figure 10 A).

[0137] The results showed that compared with simple BAR-CAR-T cell infusion, double-needle RBC-Vac treatment significantly increased the accumulation of BAR-CAR-T cells in tumor tissue. Compared with group G2, different doses of RBC-Vac treatment groups (G3-G5) significantly increased the proportion of BAR-CAR-T cells in tumor tissue, increasing by approximately 5-fold, 17.4-fold, and 21.1-fold, respectively. Figure 10 The number of ACT cells per milligram of tissue in tumor tissue also showed a consistent trend (BC); the number of ACT cells per milligram of tissue in tumor tissue also showed a consistent trend (BC). Figure 10 (J). This result indicates that RBC-Vac can effectively promote the expansion of BAR-CAR-T cells in vivo and enhance their recruitment or retention at tumor sites. Combined with the previous finding that RBC-Vac has a strong accumulation capacity in the spleen, this suggests that RBC-Vac may first provide additional stimulatory signals to BAR-CAR-T cells in immune-related organs such as the spleen, thereby promoting their expansion and ultimately increasing the number of effector cells entering tumor tissue.

[0138] Further analysis of the functional status of BAR-CAR-T cells revealed that the number of IFN-γ-positive BAR-CAR-T cells in tumor tissue significantly increased after RBC-Vac treatment, reaching 2.4 times, 6.4 times, and 3.7 times that of the G2 group, respectively. Figure 10The results (FG) indicate that RBC-Vac not only promoted the accumulation of BAR-CAR-T cells in tumors but also enhanced their effector activation state. IFN-γ is an important functional cytokine for T cells to exert anti-tumor effects, and its elevation usually indicates stronger immune activation and cytotoxic potential. Therefore, the above results show that the promoting effect of RBC-Vac on BAR-CAR-T cells is not only reflected in increased numbers but also in enhanced function, that is, it can maintain or enhance the effector activity of cells while increasing tumor infiltration. This is particularly important for CAR-T therapy of solid tumors, because tumor infiltration alone without sustained functional output often fails to translate into a stable anti-tumor effect. It is noteworthy that although the G5 group showed a higher number of infiltrated BAR-CAR-T cells, its activation level was lower than that of the G4 group, suggesting that excessively high doses of continuous stimulation may not necessarily lead to better functional output. This phenomenon suggests that there may be a specific dose window for the regulation of BAR-CAR-T cells by RBC-Vac: moderately enhanced artificial antigen stimulation is beneficial for promoting BAR-CAR-T cell expansion and activation, while excessive or continuous stimulation may induce T cells into a functionally limited state. Combined with existing knowledge in the ACT field, long-term or high-intensity antigen exposure can lead to gradual functional decline in T cells, manifested as decreased cytokine secretion, weakened cytotoxic activity, and upregulation of exhaustion-related phenotypes. Therefore, the results of group G5 suggest that when using RBC-Vac to enhance the therapeutic effect of BAR-CAR-T cells, a higher antigen dose is not necessarily better; rather, a balance needs to be struck between promoting expansion and avoiding excessive stimulation. The above results indicate that repeated administration of RBC-Vac can significantly promote the expansion, infiltration, and functional activation of BAR-CAR-T cells in tumor tissues, thereby enhancing their in vivo anti-tumor potential. Simultaneously, the differences between different dose groups also suggest that rationally optimizing the dosage and frequency of RBC-Vac administration may be an important direction for further improving the therapeutic effect of BAR-CAR-T cells and avoiding functional exhaustion.

[0139] In addition, multiple RBC-Vac treatment also significantly increased the expansion level of BAR-CAR-T cells in the spleen. Figure 10 Compared with the G2 group, the proportion of BAR-CAR-T cells in the spleen increased by 2.8-fold, 11.8-fold, and 10.2-fold, respectively, after treatment with different doses of RBC-Vac. Figure 10 The number of BAR-CAR-T cells per milligram of spleen tissue increased by 3.2-fold, 12.5-fold, and 9.3-fold, respectively. Figure 10The results indicate that RBC-Vac can effectively promote the enrichment and expansion of BAR-CAR-T cells in the spleen. Combined with the aforementioned results, this suggests that the spleen may be an important site for RBC-Vac-mediated in vivo activation of BAR-CAR-T cells. After the delivery of artificial antigens by erythrocyte carriers, continuous stimulation of BAR-CAR-T cells can be provided within this immune organ, thereby enhancing their in vivo expansion capacity.

[0140] Overall, dual-dose RBC-Vac administration can promote the expansion of BAR-CAR-T cells in the spleen and further increase their migration and accumulation in immune organs such as lymph nodes, while the changes in peripheral blood are relatively limited. This suggests that RBC-Vac mainly exerts its anti-tumor effect in vivo by enhancing the expansion and tissue redistribution of BAR-CAR-T cells in immune organs.

[0141] Example 10: Applicability of RBC-Vac in Different Types of ACT Treatment

[0142] 1. Experimental Methods

[0143] The methods for establishing B16-hCD19 tumor models and preparing BAR-CAR-T cells were the same as described above. Mice were randomly divided into groups of 5 mice each, based on tumor volume, as follows: G1: PBS; G2: BAR-CAR-T; G3: BAR-CAR-T + RBC-Vac (1:50) administered every 4 days; G4: BAR-CAR-T + RBC-Vac (1:50) administered every 7 days; G5: BAR-CAR-T + RBC-Vac (1:100) administered every 7 days. Mice in groups G2-G5 received 5 × 10⁵ BAR-CAR-T cells via tail vein infusion. 6 One BAR-CAR-T cell was infused. Two days after T cell infusion, RBC-Vac was administered via tail vein injection according to the dosage and frequency set in the experimental group. Tumor volume was then measured and mouse weight recorded every two days. The tumor volume was recorded when it reached 1500 mm². 3 The experiment was terminated and survival curves were plotted.

[0144] 2. Discussion of Results

[0145] To further evaluate the applicability of RBC-Vac in different types of ACT treatment, this study further validated its impact on the anti-tumor efficacy of BAR-CAR-T cells in a B16-hCD19 tumor model. Different combinations of RBC-Vac dosages and dosing frequencies were used to screen for the optimal dosing strategy. Tumor-bearing mice were divided into five groups, receiving PBS (G1), BAR-CAR-T cells (G2), BAR-CAR-T cells combined with 50-fold RBC-Vac every 4 days (G3), BAR-CAR-T cells combined with 50-fold RBC-Vac every 7 days (G4), and BAR-CAR-T cells combined with 100-fold RBC-Vac every 7 days (G5), respectively. Figure 11 A).

[0146] The results showed that the tumor volume in the PBS group had not yet reached 1500 mm. 3 On day 18, infusion of BAR-CAR-T cells alone inhibited tumor growth by approximately 71.3%. Figure 11 Based on this, RBC-Vac at different dosing frequencies significantly enhanced the anti-tumor efficacy of BAR-CAR-T cells, almost completely inhibiting tumor growth. Although the tumor volume reduction rate in group G4 was slightly slower than in groups G3 and G5 due to the lower dosing frequency and dosage, the tumor gradually shrank and eventually achieved complete cure after subsequent RBC-Vac reactivation. Groups G3 and G5 also achieved complete tumor regression and significantly prolonged the survival of mice (B). Figure 11 (C). In terms of survival rate, the survival rate of mice in group G3 was 80%, while that in groups G4 and G5 both reached 100%. Overall, RBC-Vac at different dosing frequencies significantly enhanced the anti-tumor effect of BAR-CAR-T cells, but reasonable dosage and dosing intervals helped to reduce the potential immune stimulation burden while maintaining efficacy. Furthermore, no significant changes in body weight were observed in any of the treatment groups, suggesting that this combined treatment strategy has good in vivo safety. Figure 11 D).

[0147] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A red blood cell immunotherapy product loaded with FITC antigen, characterized in that, The red blood cell immune product is prepared by modifying the surface of red blood cells with FITC antigen.

2. The red blood cell immune product according to claim 1, characterized in that, The red blood cell immunotherapy product is prepared by co-incubating red blood cells with phospholipids terminally modified with FITC-polyethylene glycol-FITC.

3. The red blood cell immune product according to claim 2, characterized in that, The ratio of phospholipid-polyethylene glycol-FITC to erythrocytes is (0.5-10) μg: 1×10 6 One red blood cell.

4. An immune composition targeting the spleen, characterized in that, The immune composition comprises the red blood cell immune product according to any one of claims 1-3 and cells containing an amplified antigen receptor that specifically recognizes the FITC antigen.

5. The immune composition according to claim 4, characterized in that, The cells include T cells, which include chimeric antigen receptor T cells and / or T cell receptor T cells.

6. The immune composition according to claim 4, characterized in that, The amplified antigen receptor includes a single-stranded variable domain that recognizes FITC, a hinge region and transmembrane segment of CD28, an intracellular 4-1BB co-stimulatory domain and a CD3ζ signaling domain, a P2A cleavage peptide, and a reporter protein tNGFR.

7. The use of the immune composition according to any one of claims 4-6 in the preparation of tumor treatment products.

8. The application according to claim 7, characterized in that, The tumor treatment product targets the spleen and activates and expands T cells within the spleen.

9. The application according to claim 7, characterized in that, The effector-to-target ratio of the erythrocyte immune product to cells containing amplified antigen receptors that specifically recognize FITC antigens is (5-100):

1.

10. A product for the treatment of solid tumors, characterized in that, The product comprises the immune composition according to any one of claims 4-6.

Images