A probiotic formulation and its use in combination with cart cell therapy

Abstract

The application discloses a genetically engineered probiotic preparation and its use in tumor immunotherapy. The genetically engineered probiotic contains an expression cassette regulated by an inflammation-responsive promoter, which can encode a novel trifunctional fusion protein. The fusion protein contains a secretion signal peptide, a human IL-1 receptor antagonist, a flexible linker, an anti-human GM-CSF nanobody, a second flexible linker, and an anti-human PD-L1 nanobody in sequence. In vitro experiments confirm that the fusion protein has complete biological activity, can simultaneously neutralize GM-CSF, block PD-1 / PD-L1 binding, and inhibit the IL-1 signaling pathway. Animal experiments show that oral administration of the probiotic can significantly enhance the anti-tumor efficacy of CAR-T cells, effectively prevent and alleviate cytokine release syndrome, and provide a safe and effective adjuvant therapy strategy for CAR-T therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a probiotic preparation and its use in combination with CAR-T cell therapy. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy is considered a significant milestone in the field of tumor immunotherapy, achieving remarkable complete remission rates in relapsed or refractory B-cell hematologic malignancies. However, despite its outstanding efficacy, the further promotion of this therapy is still limited by two key issues.

[0003] First, severe immune-related toxicities remain a major obstacle to the clinical application of CAR-T therapy. The most common complication is cytokine release syndrome (CRS). When CAR-T cells recognize and eliminate tumor cells, they proliferate in large numbers and release effector molecules, subsequently activating innate immune cells such as macrophages and monocytes, leading to a cascade release of various pro-inflammatory cytokines (such as IL-6, IL-1, and GM-CSF). This excessive inflammatory response can cause high fever, hypotension, respiratory distress, and even multiple organ failure. Clinically, the IL-6 receptor antagonist tocilizumab is currently the main intervention, but this method is a post-treatment and has limited effectiveness for some patients. Studies show that IL-1 and GM-CSF play upstream driving roles in the early stages of CRS, and currently there are no effective drugs that can simultaneously block these two key signaling pathways.

[0004] Secondly, the efficacy of CAR-T therapy in solid tumors is significantly lower than that in hematologic malignancies. The core reason is that the immunosuppressive tumor microenvironment (TME) hinders the infiltration and sustained activity of CAR-T cells. Highly expressed immune checkpoint molecules (such as PD-L1) in the TME can bind to PD-1 on the surface of CAR-T cells, leading to T cell dysfunction. Although the combined use of PD-1 / PD-L1 blocking antibodies has shown synergistic effects in some studies, the risk of systemic immunosuppression increases, and large-molecule antibodies have difficulty effectively penetrating the core tumor tissue, thus limiting their efficacy.

[0005] To overcome these problems, researchers have attempted to modify CAR-T cells themselves using synthetic biology techniques, such as introducing IL-1 receptor antagonist genes or causing CAR-T cells to secrete single-chain antibodies against PD-L1. While these strategies have shown promise in vitro, they are complex and costly, and the continuous secretion of exogenous proteins may disrupt the body's immune homeostasis, accelerating CAR-T cell depletion and aging.

[0006] To address the aforementioned challenges, this invention proposes an in-situ biotherapy strategy utilizing engineered probiotics. These probiotics can sense changes in inflammatory signals within the body, becoming activated upon the occurrence of CRS or TME, initiating target gene expression on demand, and locally synthesizing and releasing a triadic fusion protein that simultaneously neutralizes IL-1, GM-CSF, and PD-L1. Through this "spatiotemporally precise regulation" model, dynamic suppression of toxic side effects and sustained enhancement of anti-tumor activity can be achieved during CAR-T therapy, thus providing a new approach to the safety and clinical application of CAR-T therapy. Summary of the Invention

[0007] To address the above technical problems, this invention provides a probiotic preparation and its use in conjunction with CAR-T cell therapy.

[0008] Therefore, this invention provides an innovative biotherapy strategy that utilizes genetically engineered probiotics as bioreactors to achieve synergistic enhancement with CAR-T cell therapy. The core of this approach lies in the design of a three-functional fusion protein, TriAb (its amino acid sequence is shown in SEQ ID NO:6), whose structure has been carefully optimized. From the N-terminus to the C-terminus, it is arranged as follows: a secretory signal peptide to ensure effective protein transport to the extracellular space; human IL-1Ra to directly block the IL-1 signaling pathway; a flexible linker peptide to provide spatial freedom between the domains; an anti-human GM-CSF nanobody to specifically neutralize GM-CSF; a second flexible linker peptide; and an anti-human PD-L1 nanobody to effectively block immune checkpoints. This fusion protein innovatively omits the Fc fragment of traditional antibodies, reducing immunogenicity while enhancing tissue penetration.

[0009] Furthermore, the gene encoding this fusion protein was placed under the regulation of an inflammatory-responsive promoter and stably integrated into the probiotic genome, resulting in the engineered strain EcN-TriAb. When this strain is delivered orally, it can colonize the gastrointestinal tract and sense the inflammatory environment induced by CAR-T therapy. Upon activation by inflammatory signals such as NF-κB, the engineered bacteria initiate the expression and secretion of the TriAb protein, achieving in-situ production and precise delivery of the therapeutic protein at the lesion site.

[0010] This invention achieves effective synergy with CAR-T cell therapy through innovative molecular design and delivery system. EcN-TriAb can respond to inflammatory signals in vivo, intelligently secreting a novel fusion protein, TriAb, with triple functions, simultaneously addressing the two key challenges of CRS and TME inhibition. In vitro experiments have confirmed that TriAb possesses complete biological activity, effectively neutralizing GM-CSF, blocking PD-1 / PD-L1 interaction, and inhibiting the IL-1 signaling pathway. Animal experiments show that this formulation can significantly enhance the anti-tumor effect of CAR-T cells, increasing the tumor inhibition rate to 94.5%, while reducing the levels of key CRS-related inflammatory factors by more than 82%, significantly improving the survival status of experimental animals. This oral administration method offers advantages such as convenient use, low production cost, and high safety, providing a new technical approach to address the current problems of toxic side effects and insufficient efficacy in CAR-T therapy, demonstrating significant clinical application value and development prospects. Attached Figure Description

[0011] Figure 1 The results of Western blotting of the fusion protein TriAb are shown in the following diagrams: lane 1 is the marker, lane 2 is the supernatant of EcN-Vector (TNF-α induced), lane 3 is the supernatant of EcN-TriAb (uninduced), and lane 4 is the supernatant of EcN-TriAb (TNF-α induced).

[0012] Figure 2 IL-1Ra standard curve. Detailed Implementation

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0014] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0015] Example 1: Design, preparation and in vitro functional verification of the genetically engineered probiotic EcN-TriAb

[0016] 1. Design and codon optimization of fusion proteins

[0017] 1.1 Design Concept: To synergistically address CRS and tumor immune microenvironment suppression in CAR-T therapy, we designed a novel trifunctional fusion protein, TriAb. This design integrates three key targets: an IL-1 receptor antagonist (IL-1Ra) to block upstream inflammatory signals; an anti-GM-CSF nanobody to neutralize key amplifiers of macrophage activation; and an anti-PD-L1 nanobody to relieve T cell immune checkpoint inhibition. These targets are tandemly linked by a flexible linker peptide to form a single polypeptide chain, ensuring synergistic delivery of all three at the lesion site, achieving a "three birds with one stone" therapeutic effect.

[0018] 1.2 Structural design and sequence: The fusion protein TriAb, from N-terminus to C-terminus, is as follows:

[0019] (1) Secretion signal peptide: PelB signal peptide (specific amino acid sequence as shown in SEQ ID NO:1) is introduced at the N-terminus. This signal peptide will be cleaved after transport, thereby causing the recombinant protein to fold extracellularly.

[0020] (2) Human IL-1 receptor antagonist: The full sequence of the naturally mature human IL-1Ra protein is used to ensure its biological activity and low immunogenicity. The specific amino acid sequence is shown in SEQ ID NO:2.

[0021] (3) First flexible linker peptide: composed of 3 repeating (GGGGS) units, providing sufficient spatial flexibility for IL-1Ra and subsequent nanobody domains to avoid mutual interference. The specific amino acid sequence is shown in SEQ ID NO:3.

[0022] (4) Anti-human GM-CSF nanobody: The high-affinity and high-specificity anti-human GM-CSF nanobody obtained in this study by phage display technology combined with humanization technology (its Kd=0.68±0.045 nM, the control monoclonal antibody (ab316862) Kd=2.32±0.22 nM) is shown in SEQ ID NO:4;

[0023] (5) Second flexible linker peptide: Same as (3), to ensure the independence between GM-CSF nanobody and PD-L1 nanobody, the specific amino acid sequence is shown in SEQ ID NO:3;

[0024] (6) Anti-human PD-L1 nanobody: The high-affinity and high-specificity anti-human PD-L1 nanobody obtained in this study by phage display technology combined with humanization technology (its Kd=0.64±0.036 nM, the control monoclonal antibody (ab213524) Kd=2.26±0.34 nM) is shown in SEQ ID NO:5;

[0025] (7) The amino acid sequence of the fusion protein TriAb designed in this way is shown in SEQ ID NO:6.

[0026] 1.3 Codon Optimization and Gene Synthesis: The amino acid sequence shown in SEQ ID NO:6 was reverse translated, and GenSmart was used to perform the translation. TM The Codon Optimization tool was used to optimize the DNA sequence to suit the E. coli expression system. The codon fitness index of the optimized gene (SEQ ID NO: 7) increased from 0.72 to 0.84, and the GC content was adjusted to 55.24%. The entire gene was synthesized by Genscript Biotech Co., Ltd. and cloned into the pUC57 vector, named pUC57-TriAb-Syn.

[0027] 2. Preparation of genetically engineered probiotic EcN-TriAb

[0028] 2.1 Construction of expression plasmids

[0029] (1) Construction of pUC57-Tn7-pNFκB: First, the commonly used cloning vector pUC57 was used as the backbone. DNA fragments containing Tn7L and Tn7R terminal inverted repeat sequences and intermediate multiple cloning sites were obtained from plasmids containing Tn7 transposon elements (e.g., Addgene, Choi, KH., Gaynor, J., White, K. et al. A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2, 443–448 (2005)) by PCR or enzyme digestion. Simultaneously, the pNFκB promoter fragment was obtained from a commercially available NF-κB reporter gene plasmid (e.g., Beyotime-D2207-1μg-pNFκB-TA-luc or similar products) by enzyme digestion PCR. Appropriate restriction endonucleases (e.g., NdeI and XhoI) were used to digest the pUC57 vector, Tn7 fragment, and pNFκB promoter fragment, respectively. Subsequently, the three linearized fragments were ligated using T4 DNA ligase. The ligation product was transformed into competent *E. coli* cells (e.g., DH5α) and screened on spectinomycin-containing antibiotic plates. Finally, positive clones were selected, and the correctness of the recombinant plasmid was verified by colony PCR, restriction enzyme digestion, and DNA sequencing. Using the above methods, pUC57-Tn7-pNFκB was successfully constructed in this study. After amplification and extraction, it was stored at -80℃ for later use. It should be noted that pUC57-Tn7-pNFκB can also be synthesized and constructed directly by a third-party company, such as Genscript Biotech.

[0030] (2) Preparation of vector and insert fragment: First, the integrative plasmid pUC57-Tn7-pNFκB (backbone, spectinomycin resistant, constructed in our laboratory) containing the NF-κB response promoter and the pUC57-TriAb-Syn plasmid containing the target gene were digested with restriction enzymes. The restriction endonucleases NdeI and XhoI were used, and the reaction was carried out at 37°C for 2 hours, with each reaction system consisting of 30 μL. After digestion, the products were separated by 1% agarose gel electrophoresis. Subsequently, approximately 4.7 kb of the linearized vector fragment and approximately 1.4 kb of the TriAb gene fragment were excised and recovered using a gel recovery kit for subsequent ligation reactions. The concentration and purity of the recovered DNA were determined using Nanodrop 2000: the vector fragment was 45.2 ng / μL (A260 / 280=1.88), and the insert fragment was 32.5 ng / μL (A260 / 280=1.85).

[0031] (3) Ligation and Transformation: The ligation reaction was set up with 50 ng of linearized vector, 30 ng of insert fragment (molar ratio approximately 1:5), 1 μL of T4 DNA ligase (NEB), 1 μL of 10× buffer, and water to a final volume of 10 μL. Ligation was carried out at 16°C for 16 hours. 5 μL of the ligation product was added to 50 μL of DH5α chemocompetent cells, incubated on ice for 30 minutes, heat-shocked at 42°C for 45 seconds, incubated on ice for 2 minutes, and then 500 μL of SOC medium was added. The cells were then incubated at 37°C and 220 rpm for 1 hour. 200 μL of the bacterial culture was spread onto LB agar plates containing 100 μg / mL spectinomycin and incubated at 37°C for 16 hours.

[0032] (4) Identification of positive clones: Eight single colonies were randomly selected for colony PCR. Reaction program: 94°C for 5 min; (94°C for 30 s, 58°C for 30 s, 72°C for 90 s) × 30 cycles; 72°C for 5 min. The results showed that 6 out of the 8 clones could amplify the target band of about 1.4 kb. These 6 positive clones were inoculated with culture medium, and the plasmids were extracted and verified by NdeI / XhoI double digestion. All of them could release a fragment of about 1.4 kb. The plasmid of one of the clones was sent for sequencing. The results showed that the sequence was 100% correct and named pUC57-Tn7-pNFκB-TriAb.

[0033] 2.2 Chromosome integration and strain validation

[0034] (1) Preparation of electrocompetent cells: Wild-type EcN was inoculated into 5 mL of SOB medium and cultured overnight at 37°C. The cells were then transferred to 50 mL of fresh SOB at a ratio of 1:100 and cultured until OD600 = 0.8. The bacterial culture was incubated on ice for 30 minutes, and the cells were collected by centrifugation at 4°C and 5000 × g for 5 minutes. The cells were washed three times with pre-cooled 10% glycerol and finally resuspended in 200 μL of 10% glycerol.

[0035] (2) Electroporation and screening: Take 50 μL of competent cells, mix with 5 ng pUC57-Tn7-pNFκB-TriAb plasmid and 50 ng pTn7 helper plasmid, and transfer to a pre-chilled 2 mm electroporation cuvette. Electroporation was performed using a Bio-Rad electroporator with the following parameters: voltage 2.5 kV, capacitance 25 μF, resistance 200 Ω. Immediately after electroporation, add 1 mL of pre-chilled SOC medium and incubate at 37°C for 3 hours. Spread 200 μL of the culture onto LB agar plates containing 100 μg / mL spectinomycin and incubate at 30°C for 48 hours.

[0036] (3) Genotype verification: Twelve randomly selected single colonies were subjected to colony PCR detection. Nine of them amplified a specific band of approximately 1.6 kb (containing some flanking sequences), while wild-type EcN showed no band. The PCR product was sequenced to confirm that the integrated expression cassette sequence was correct. The correctly integrated strain was named EcN-TriAb and stored at -80°C in 30% glycerol. An empty vector control strain, EcN-Vector, was constructed using the same method.

[0037] 3. Testing of genetically engineered probiotics

[0038] 3.1 Validation of Induced Expression and Protein Secretion

[0039] (1) Bacterial culture and induction: EcN-TriAb and EcN-Vector were cultured overnight in 5 mL LB (containing spectinomycin). The next day, they were transferred to 50 mL fresh medium (250 mL shake flask) at a ratio of 1:100 and cultured at 37°C and 220 rpm until OD600 ≈ 0.6. The OD values ​​at this time were accurately recorded: EcN-TriAb was 0.61 and EcN-Vector was 0.59. The cultures of each bacterium were divided into two equal parts. One part was added with 10 ng / mL human TNF-α, and the other part was added with an equal volume of PBS. Induction was continued for 6 hours.

[0040] (2) Sample preparation: Take 1 mL of bacterial culture and centrifuge at 4°C and 12,000 × g for 10 minutes. Filter the supernatant through a 0.22 μm PVDF filter membrane. Resuspend the bacterial pellet in 1×PBS to the same volume and sonicate (on ice, 30% power, 3s sonication, 5s interval, total time 5min). After centrifugation, take the supernatant as the lysis buffer.

[0041] (3) Western Blot: Take 20 μL of the concentrated supernatant sample after filtration, mix with 5× Loading Buffer, and boil for 10 minutes. Perform 12% SDS-PAGE at a constant voltage of 120V for 90 minutes. Transfer to a PVDF membrane after semi-drying (constant current 1.5 mA / cm², 30 minutes). After blocking with 5% skim milk for 1 hour, incubate overnight at 4°C with rabbit anti-human IL-1Ra monoclonal primary antibody (1:2000, ab124962). Wash 3 times with TBST for 10 minutes each time, and then incubate at room temperature for 1 hour with HRP-labeled goat anti-rabbit secondary antibody (1:5000). After ECL luminescence, develop on an imaging system. The results are as follows. Figure 1 As shown, a clear, single immunoreactive band was detected at approximately 47 kDa only in lane 4 (the supernatant of EcN-TriAb induced by TNF-α), the location of which is consistent with the theoretical molecular weight of the TriAb fusion protein. This band was not observed in any of the other lanes.

[0042] (4) ELISA detection: Human IL-1Ra ELISA Kit was used. The filtered supernatant was processed according to the kit instructions, and the absorbance was read at 450 nm and 540 nm using a BioTek microplate reader. The concentration was calculated based on the standard curve. The results are shown in Table 1 and... Figure 2 As shown, after TNF-α induction, the concentration of IL-1Ra in the EcN-TriAb supernatant reached approximately 1.87 μg / mL, which was 10.6 times that of the uninduced group and significantly higher than that of the EcN-Vector control group (p < 0.001). This indicates that the NF-κB promoter was effectively activated, driving high-level expression and secretion of TriAb protein.

[0043] Table 1. Determination of IL-1Ra concentration in bacterial supernatant (n=3, `X ± SD`)

[0044]

[0045] 3.2 GM-CSF neutralizing activity assay (TF-1 cell proliferation inhibition assay)

[0046] (1) Cell preparation: TF-1 cells were cultured in RPMI-1640 complete medium containing 2 ng / mL human GM-CSF. Before the experiment, the cells were washed three times with GM-CSF-free medium and resuspended to 2×10^5 cells / mL.

[0047] (2) Sample pretreatment: The cell-free supernatant of TNF-α-induced EcN-TriAb and EcN-Vector was mixed with an equal volume of culture medium containing 4 ng / mL human GM-CSF and pre-incubated at 37°C for 1 hour.

[0048] (3) Cell plating and treatment: 50 μL of cell suspension (1×10^4 cells) was added to each well of a 96-well plate. Then 50 μL of pre-mixed "supernatant-GM-CSF" solution was added to make the final volume of each well 100 μL and the final concentration of GM-CSF 2 ng / mL. The experimental groups and settings were as follows: Group A: Blank control (50 μL cells + 50 μL medium without GM-CSF); Group B: GM-CSF control group (50 μL cells + 50 μL medium containing 2 ng / mL GM-CSF); Group C: EcN-Vector supernatant group (50 μL cells + 50 μL pre-incubation mixture of EcN-Vector supernatant and GM-CSF); Group D: EcN-TriAb supernatant group (50 μL cells + 50 μL pre-incubation mixture of EcN-TriAb supernatant and GM-CSF); Group E: Positive control group (50 μL cells + 50 μL medium containing 2 ng / mL GM-CSF and 1 μg / mL anti-human GM-CSF neutralizing antibody). Each group had 5 replicates. The cell culture plates were incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0049] (4) CCK-8 assay: Add 10 μL of CCK-8 solution to each well and continue culturing for 3 hours. Measure the absorbance at 450 nm using a microplate reader.

[0050] (5) Experimental Results: The OD450 value (0.381) of the EcN-TriAb supernatant treatment group (Group D) was significantly lower than that of the GM-CSF control group (Group B, 1.251) and the EcN-Vector supernatant group (Group C, 1.217) (p < 0.001). Its cell proliferation inhibition rate was as high as 69.0%, which was comparable to that of the commercial neutralizing antibody (Group E, 74.8%). This proves that the fusion protein secreted by EcN-TriAb can effectively neutralize the biological activity of GM-CSF. See Table 2 for details.

[0051] Table 2 Raw data and results of GM-CSF neutralizing activity assay (OD450, n=5)

[0052]

[0053] Note: Inhibition rate (%) = [1 - (OD sample group - OD blank) / (ODGM-CSF control group - OD blank)] ×100%.

[0054] 3.3 Detection of PD-L1 binding blocking activity (competitive ELISA)

[0055] (1) Coating and blocking: Dilute recombinant human PD-L1-hFc protein to 2 μg / mL with carbonate coating buffer, add 100 μL to each well of a 96-well high-adsorption microplate, and incubate overnight at 4°C. Discard the coating buffer and wash the plate 3 times with PBST. Add 250 μL of 3% BSA-PBS solution to each well and block at 37°C for 2 hours.

[0056] (2) Competitive incubation: Biotinylated human PD-1-hFc protein was diluted to a working concentration of 2 μg / mL. The supernatants of TNF-α-induced EcN-TriAb and EcN-Vector were serially diluted (stock solution, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128). An equal volume of the diluted supernatant was mixed with an equal volume of biotinylated PD-1 working solution. After washing, 100 μL of the above mixture was added to each well. A maximum signal well (biotinylated PD-1 + dilution only) and a background well (without biotinylated PD-1) were also set up. Commercially available Avelumab (starting at 1 μg / mL, serially diluted 3-fold) was used as a positive control.

[0057] (3) Incubation and detection: Incubate at 7°C for 1.5 hours. Wash the plate 5 times. Add 100 μL of HRP-labeled streptavidin (1:5000 dilution) to each well and incubate at 37°C for 30 minutes. Wash the plate 5 times. Add 100 μL of TMB substrate solution to each well and develop color in the dark for 15 minutes. Add 50 μL of 2 M H2SO4 to each well to stop the reaction. Read the absorbance immediately at 450 nm.

[0058] (4) Results Analysis: Using GraphPad Prism 9 software, a four-parameter logistic curve was fitted with the inhibition rate (% = [1 - (OD sample - OD background) / (OD maximum signal - OD background)] × 100%) as the Y-axis and the logarithm of the sample dilution factor (or the logarithm of the Avelumab concentration) as the X-axis. Competitive ELISA results showed that EcN-TriAb supernatant can dose-dependently block the binding of PD-1 to PD-L1, and its IC50... 50 A 1:12.3 dilution (IC50 of Avelumab)50 The concentration was 18.5 nM, demonstrating that the secreted anti-PD-L1 nanobody has significant biological function. Specific data are shown in Table 3.

[0059] Table 3. Results of PD-L1 binding blocking competitive ELISA (OD450)

[0060]

[0061] Example 2: Evaluation of the enhancing effect of EcN-TriAb on CAR-T therapy in a tumor-bearing mouse model

[0062] 1. Materials and Methods

[0063] 1.1 Animals and Cell Lines

[0064] (1) Experimental animals: 6-8 week old female (NSG) mice, weighing 18-22 g, were housed in an SPF-grade barrier environment (temperature 22±1℃, humidity 50±5%, 12h light-dark cycle) and were allowed free access to sterile feed and sterile water.

[0065] (2) Tumor cell line: The human B-cell lymphoma cell line Raji-luc (stable expression of firefly luciferase) was transduced with a lentiviral vector (pLVX-PD-L1-IRES-Puro) to achieve high expression of human PD-L1 (transduction efficiency verified by flow cytometry >90%). Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (containing 100 U / mL penicillin and 100 μg / mL streptomycin), passaged at 37℃ in a 5% CO2 incubator, and tested for mycoplasma every 3 days to ensure a negative result.

[0066] (3) CAR-T cell preparation: Mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers (with informed consent), and CD3 cells were positively sorted using CD3 magnetic beads. + T cells, purity >95% (flow cytometry verification). Second-generation CAR lentiviral vector (pCDH-CD19scFv-CD28-CD3ζ-IRES-GFP) was used to transduce T cells at MOI=5. Transduction efficiency was assessed 48 h post-transduction by GFP expression rate (>70%). After transduction, T cells were expanded for 7 days in X-VIVO 15 medium containing IL-2 (300 IU / mL). Cell viability (trypan blue rejection >90%) and phenotype (CD4+) were assessed after expansion. + / CD8 + (The ratio is approximately 1:1).

[0067] 1.2 Experimental Grouping and Model Establishment

[0068] (1) Grouping: Based on the preliminary experimental results (n=3 / group), each group was determined to have n=8, as follows: Group 1: PBS control group (model control): PBS was administered by gavage + PBS was injected via the tail vein; Group 2: CAR-T group (positive treatment control): PBS was administered by gavage + CAR-T cells were injected via the tail vein; Group 3: CAR-T + EcN-Vector group (empty vector control): EcN-Vector was administered by gavage + CAR-T cells were injected via the tail vein; Group 4: CAR-T + EcN-TriAb group (experimental group): EcN-TriAb was administered by gavage + CAR-T cells were injected via the tail vein.

[0069] (2) Tumor model establishment: Day 0: All mice were injected with 5×10 via the tail vein. 5 Raji-luc-PD-L1 cells (resuspended in 200 μL PBS) were used to establish a systemic lymphoma model (simulating the hematogenous / bone marrow dissemination characteristics of clinical B-cell lymphoma). Day 7 (model validation): In vivo imaging confirmed successful tumor inoculation in all mice (photon flux > 5 × 10⁻⁶). 8 p / s / cm 2 / sr), mice that failed to model were removed (no mice were removed in this experiment).

[0070] 1.3 Dosing regimen

[0071] (1) Preparation of probiotics: EcN-TriAb and EcN-Vector were prepared in our laboratory (Example 1); the bacterial strains were cultured in LB medium (containing 50 μg / mL kanamycin) at 37°C and 220 rpm with shaking until the logarithmic growth phase (OD). 600 =0.6-0.8), centrifuged at 4℃, 3000×g for 10 min, the precipitate was resuspended three times with sterile PBS, and the concentration was adjusted to 5×10. 9 CFU / mL. (2) Administration time and dosage:

[0072] Probiotic gavage: 200 μL (containing 1×10⁻⁶ probiotics) was administered orally via gavage at the same time (9:00 AM) daily from days 8 to 21. 9 CFU), and the PBS control group were given the same volume of PBS by gavage (to ensure consistent gavage procedures and avoid stress differences).

[0073] CAR-T cell infusion: On day 14, mice in groups 2-4 were injected with 5×10⁻⁶ cells via the tail vein. 6 One CAR-T cell (resuspended in 100 μL PBS) was injected, and the injection site was gently pressed for 30 seconds after injection to avoid leakage; Group 1 was injected with an equal volume of PBS.

[0074] 2. Monitoring Indicators and Methods

[0075] 2.1 Tumor burden monitoring

[0076] (1) In vivo imaging: performed on days 7 (baseline), 14, 21, 28, and 35, as follows: intraperitoneal injection of D-fluorescein potassium salt (150 mg / kg), followed by incubation in the dark for 10 min (to ensure adequate substrate distribution). Bioluminescence images were acquired using an IVIS Spectrum imaging system with an exposure time of 1-5 min (adjusted according to signal intensity).

[0077] (2) Data analysis: The whole-body tumor region (ROI) was delineated using Living Image 4.7.3 software, and the total photon flux (p / s / cm) was quantified. 2 / sr), and the analysis was performed by the same experimenter in a blinded manner to reduce bias.

[0078] (3) Pathological verification of tumor tissue: At the end of the experiment (day 60) or when the mice were dying, the liver, spleen and bone marrow (femur) were dissected, fixed in 4% paraformaldehyde, embedded in paraffin and sectioned, and stained with HE to observe the tumor infiltration (pathological score: 0 points no infiltration, 1 point <30% infiltration, 2 points 30%-70% infiltration, 3 points >70% infiltration).

[0079] 2.2 Monitoring of CRS-related indicators

[0080] (1) Weight and clinical score

[0081] Weight: Weigh yourself at 9:00 AM daily and calculate the percentage change in weight relative to day 14 (CAR-T infusion day) (weight change % = weight on day / weight on day 14 × 100%).

[0082] Clinical scoring: A double-blind method was used (two observers scored independently, and the average score was taken). The detailed criteria were as follows: 0 points: normal activity, quick response, smooth hair, no discharge; 1 point: mild somnolence (slightly slow response to stimuli), slightly fluffy hair, no discharge; 2 points: marked somnolence (delayed response to stimuli), hunched back, fluffy hair, slight discharge around the eyes; 3 points: significantly reduced activity (only occasional movement), tremors, significant discharge around the eyes / nose, weight loss >15%; 4 points: near death (almost no activity), weight loss >20%, meeting the humanitarian endpoint criteria.

[0083] (2) Serum cytokine detection

[0084] Sampling: On day 16 (2 days after CAR-T infusion, early CRS) and day 20 (peak CRS), 200 μL of blood was collected through the posterior orbital venous plexus. The serum was separated by centrifugation at 3000×g for 10 min and stored at -80℃ (avoid repeated freeze-thaw cycles).

[0085] Testing: Using LEGENDplex TMHuman inflammatory factor 13-plex kit, detecting factors including IL-6, IL-1β, GM-CSF, IFN-γ, TNF-α, IL-2, IL-4, IL-10, IL-12p70, IL-17A, MCP-1, CCL3, and CXCL10.

[0086] Instrumentation and Analysis: Flow cytometry was performed using LEGENDplex. TM Data Analysis Software v8.0 analyzes concentrations using a standard curve (R²). 2 Calculate pg / mL using >0.98).

[0087] Temperature monitoring: During the peak of CRS (days 18-20), rectal temperature was monitored daily (electronic thermometer, insertion depth 1cm), and temperature changes were recorded (normal range 36.5-38.5℃, fever is defined as >38.5℃).

[0088] 2.3 Verification of Immune Function and Mechanism

[0089] (1) In vivo distribution and activity of CAR-T cells: On day 21 (7 days after CAR-T infusion), 3 mice from each group were selected, and the number of CAR-T cells (GFP) in peripheral blood, spleen, and tumor tissue was detected by flow cytometry. + ) ratio and CD69 + Expression rate.

[0090] (2) Tumor microenvironment cytokines: Splenic tumor tissue was taken on day 28, homogenized, and the local concentrations of IL-6 and IFN-γ were detected (ELISA kit). The levels were compared with the systemic serum levels to verify the local effect of EcN-TriAb.

[0091] (3) PD-L1 blocking effect: The expression rate of PD-L1 on the surface of tumor cells was detected by flow cytometry (day 28) to evaluate the blocking effect of EcN-TriAb on the PD-1 / PD-L1 pathway.

[0092] 2.4 Survival observation

[0093] The mice were observed until day 60, and their survival status was recorded daily. When a mouse reached a clinical score of 4 or its weight decreased by more than 20%, it was considered to be near death and euthanized. The survival time was recorded.

[0094] 3. Experimental Results and Statistical Analysis

[0095] 3.1 Tumor growth inhibition: On day 14 (before CAR-T infusion), there was no significant difference in tumor burden among the groups (p>0.05), excluding baseline bias. On day 21 (7 days after CAR-T infusion), the tumor signal in the CAR-T + EcN-TriAb group had decreased to 5.2×10⁻⁶.7 The levels were significantly lower than those in the CAR-T group (2.1×10⁻⁶). 9 (p<0.001), indicating early efficacy. On day 28, the tumor inhibition rate in the CAR-T + EcN-TriAb group reached 94.5% (calculated as: (mean of CAR-T group - mean of experimental group) / mean of CAR-T group × 100%), and remained low-load with no rebound trend on day 35. No tumor infiltration was observed in the liver, spleen, and bone marrow of surviving mice on day 60 (score 0), while the residual tumor infiltration score in the CAR-T group mice reached 2-3 points.

[0096] Table 4. Dynamic changes in tumor bioluminescence intensity (unit: p / s / cm² / sr, n=8, x±SD)

[0097]

[0098] Statistical analysis: One-way ANOVA was used for comparisons among multiple groups, ensuring normality and homogeneity of variance (Shapiro-Wilk test and Levene test, p>0.05). Tukey's method was used for pairwise comparisons between groups. On day 28, CAR-T + EcN-TriAb group vs CAR-T group: p<0.001; on day 35: p<0.001.

[0099] 3.2 Changes in CRS-related indicators

[0100] 3.2.1 Changes in body weight and body temperature

[0101] Temperature changes: On day 20, the average body temperature in the CAR-T group was 39.2±0.5℃ (fever), while that in the CAR-T + EcN-TriAb group was 37.8±0.3℃ (normal range), with a difference between the groups p<0.001.

[0102] Weight loss: During the peak of CRS (day 20), the weight of the CAR-T group dropped to 88.7% of the baseline value, while the weight of the CAR-T+EcN-TriAb group dropped only slightly to 97.3%, close to the normal growth of the PBS group (104.3%), indicating a significant reduction in weight loss.

[0103] Table 5. Percentage change in body weight of mice in each group (relative to baseline body weight on day 14, n=8, x±SD)

[0104]

[0105] 3.2.2 Clinical Scoring

[0106] The experimental group had very mild CRS symptoms: the median score on day 20 (peak period) was only 1 point (mild somnolence), which was much lower than the 3 points in the CAR-T group (significantly reduced activity, tremors, etc.), and no mice reached the near-death criteria (4 points).

[0107] Table 6 Clinical scores of mice in each group (n=8, median [interquartile range])

[0108]

[0109] Statistical analysis: Clinical scores were non-normally distributed, and the Kruskal-Wallis H test was used. Dunn's method was used for intergroup comparisons. On day 20, CAR-T + EcN-TriAb group vs CAR-T group: p<0.001.

[0110] 3.2.3 Serum cytokine levels

[0111] The key CRS drivers (IL-6 and GM-CSF) showed the most significant decrease on day 20 (peak): IL-6 decreased by 87.1% and GM-CSF decreased by 82.4%, and were already significantly reduced by day 16 (early stage) (IL-6 decreased by 85.2%), suggesting that EcN-TriAb can inhibit the initiation and progression of CRS. The effector IFN-γ decreased by only 72.4%, and the local IFN-γ concentration in the tumor (285.6±42.3 pg / mL) was significantly higher than that in serum (189.5±25.3 pg / mL, p<0.05), suggesting that CAR-T effector function was preserved locally.

[0112] Table 7 Serum cytokine levels on day 16 and day 20 (pg / mL, n=8, x±SD)

[0113]

[0114] Note: ***p<0.001, compared with the CAR-T group (ANOVA+Tukey method).

[0115] 3.3 Verification of the immune mechanism

[0116] CAR-T cell distribution: On day 21, the proportion of CAR-T cells in the spleen of the CAR-T + EcN-TriAb group (18.5±3.2%) was significantly higher than that of the CAR-T group (9.2±2.1%, p<0.01), and CD69... + The activation rate was 78.3±5.6% (52.1±4.8% in the CAR-T group, p<0.01).

[0117] PD-L1 blockade: On day 28, the PD-L1 expression rate of tumor cells in the CAR-T + EcN-TriAb group (12.3±2.5%) was significantly lower than that in the CAR-T group (68.5±7.3%, p<0.001).

[0118] 3.4 Survival Analysis

[0119] The CAR-T+EcN-TriAb group showed significant survival benefits: median survival > 60 days (not reaching the endpoint), with a 60-day survival rate of 62.5%; while the CAR-T group had a 60-day survival rate of 0 and a median survival of only 46 days, with a statistically significant difference (p<0.001).

[0120] Table 8 Survival status of mice in each group

[0121]

[0122] Statistical analysis: Survival curves were plotted using the Kaplan-Meier method. Log-rank test showed a significant difference in survival between the CAR-T + EcN-TriAb group and the CAR-T group (χ²). 2 =18.6, p<0.001), median survival extended by >30%.

[0123] 4. In summary, this embodiment, through multi-dimensional verification, clarifies the synergistic optimization effect of EcN-TriAb on CAR-T therapy: 4.1 Mechanism of enhanced efficacy: Targeted blocking of PD-L1 in the tumor microenvironment (expression rate decreased by 82%), relieving immunosuppression of CAR-T cells; promoting the enrichment and activation of CAR-T cells in the tumor site (the proportion of CAR-T cells in the spleen increased by 101%), and enhancing the local anti-tumor effect.

[0124] 4.2 CRS relief mechanism: specifically neutralizes CRS driving factors (IL-6, GM-CSF), inhibiting the systemic inflammatory cascade response from the source; locally delivers antibodies (secretion of EcN from intestinal colonization), reducing systemic interference with CAR-T cell effector function (local retention of IFN-γ).

[0125] 4.3 Clinical translational value: The 60-day survival rate reaches 62.5%, solving the problem of recurrence after CAR-T therapy; it significantly reduces the severity of CRS (clinical score ≤1 point), eliminating the need for additional anti-inflammatory drugs and reducing treatment costs and side effects.

[0126] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A fusion protein TriAb, characterized in that, The amino acid sequence of the fusion protein TriAb is shown in SEQ ID NO:

6. From the N-terminus to the C-terminus, the fusion protein TriAb consists of a secretion signal peptide, a human IL-1 receptor antagonist, a first flexible linker peptide, an anti-human GM-CSF nanobody, a second flexible linker peptide, and an anti-human PD-L1 nanobody.

2. The fusion protein TriAb according to claim 1, characterized in that, The amino acid sequence of the secretion signal peptide is shown in SEQ ID NO:

1.

3. The fusion protein TriAb according to claim 1, characterized in that, The amino acid sequence of the human IL-1 receptor antagonist is shown in SEQ ID NO:

2.

4. The fusion protein TriAb according to claim 1, characterized in that, The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:

3.

5. The fusion protein TriAb according to claim 1, characterized in that, The amino acid sequence of the anti-human GM-CSF nanobody is shown in SEQ ID NO:

4.

6. The fusion protein TriAb according to claim 1, characterized in that, The amino acid sequence of the anti-human PD-L1 nanobody is shown in SEQ ID NO:

5.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein TriAb as described in claim 1, and its nucleotide sequence is shown in SEQ ID NO:

7.

8. A genetically engineered probiotic, characterized in that, The probiotic is Escherichia coli Nissle 1917, whose genome integrates an expression cassette containing the nucleic acid molecule described in claim 7.

9. A pharmaceutical composition, characterized in that, The composition comprises the genetically engineered probiotic of claim 8 and a pharmaceutically acceptable carrier.

10. Use of the genetically engineered probiotic of claim 8 in the preparation of a medicament for enhancing the efficacy of CAR-T cell therapy.

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