Engineered probiotic bacteria, outer membrane vesicles of a hmgb1 neutralizing single-domain antibody and uses thereof

Abstract

The application provides an engineered probiotic of a single-domain antibody neutralizing HMGB1, an outer membrane vesicle and application thereof, relates to the fields of antibody engineering, artificial intelligence assisted protein design, synthetic biology, microbial engineering and bacterial outer membrane vesicle, and particularly relates to a synthetic single-domain antibody AnHM specifically binding to high mobility group protein B1 (HMGB1), engineered gram-negative bacteria expressing the antibody, outer membrane vesicles of the bacteria and application thereof. The OMV-AnHM expressed by the engineered probiotic can reduce detectable recombinant HMGB1 in a system, and inhibit nuclear factor kappa B signal and inflammatory factor release induced by HMGB1. Oral administration of EcN-AnHM can be distributed in the gastrointestinal tract for a short time, reduce plasma HMGB1 and inflammatory indexes, alleviate organ damage and improve short-term survival in heatstroke mice. The application also relates to corresponding nucleic acids, expression cassettes, pharmaceutical compositions, preparation methods and application thereof in heatstroke prevention or alleviation.

Description

Technical Field

[0001] This invention relates to the fields of antibody engineering, artificial intelligence-assisted protein design, synthetic biology, microbial engineering, and bacterial outer membrane vesicles. Specifically, it relates to a synthetic single-domain antibody (VHH) AnHM targeting high mobility group B1 (HMGB1), a nucleic acid encoding the single-domain antibody, a fusion protein and expression cassette formed by the antibody and an outer membrane vesicle anchoring protein, an engineered Gram-negative probiotic expressing the fusion protein, outer membrane vesicles (OMVs) with AnHM surface generated therefrom, and the application of the above-mentioned engineered bacteria and outer membrane vesicles in the prevention or mitigation of heatstroke. Background Technology

[0002] Heatstroke is a critical illness caused by severe heat stress. Its pathological process involves heat-induced cell damage, release of damage-related molecular patterns, intestinal barrier disruption, endothelial dysfunction, coagulation disorders, amplification of systemic inflammation, and multi-organ damage. Current clinical treatment mainly relies on rapid cooling, fluid resuscitation, correction of coagulation and electrolyte abnormalities, and organ support. Specific biological therapies that can directly inhibit key nodes of inflammation amplification are still lacking.

[0003] High-mobility group box 1 (HMGB1) is a highly conserved nuclear protein. Upon cellular stimulation by heat, necrosis, pyroptosis, or inflammation, HMGB1 can be released extracellularly and act as a damage-related molecular model, activating receptor pathways such as Toll-like receptor 4 / myeloid differentiation protein 2 (TLR4 / MD-2) and receptor for advanced glycation end products (RAGE), promoting nuclear factor κB (NF-κB) signaling and the export of inflammatory factors. Therefore, extracellular HMGB1 is a candidate intervention target for amplifying inflammation in heatstroke.

[0004] Direct injection of anti-HMGB1 antibodies faces limitations in production, storage, transportation, and application scenarios. Engineered probiotics can survive briefly in the gastrointestinal tract after oral administration and produce therapeutic molecules; outer membrane vesicles (OMVs) released by Gram-negative bacteria can serve as nanoscale protein carriers. However, whether exogenous single-domain antibody modules, after being expressed by engineered bacteria and entering OMVs, can maintain surface accessibility and HMGB1 neutralizing activity, and whether oral engineered bacterial systems can effectively intervene in heatstroke, still require specific technical solutions and experimental verification. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide an HMGB1 neutralizing system with a specific synthetic single-domain antibody AnHM sequence as its core, which can be expressed by engineered probiotics and displayed on the surface of outer membrane vesicles, thereby reducing extracellular HMGB1 and its downstream inflammatory output, and for the prevention or mitigation of heatstroke-related systemic inflammation and organ damage.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a synthetic single-domain antibody (VHH) AnHM, the complete amino acid sequence of which is shown in SEQ ID NO: 1, and the heavy chain complementarity-determining regions 1, 2, and 3 (HCDR1, HCDR2, and HCDR3) are shown in SEQ ID NO: 2-4, respectively. AnHM uses the B-box surface of HMGB1 as the computationally designed target region; E108, Y109, K112, M132, T136, D140, P143, Y144, and K147 are used as predicted contact residues and as the basis for artificial intelligence-aided design.

[0007] Secondly, the present invention provides a nucleic acid molecule that encodes the aforementioned synthetic single-domain antibody.

[0008] Thirdly, the present invention provides a fusion protein comprising an outer membrane vesicle anchoring protein and the aforementioned synthetic single-domain antibody. Preferably, the fusion protein comprises, from the N-terminus to the C-terminus, cytolysin A, a flexible linker peptide, an HA epitope tag, and AnHM, and its amino acid sequence is shown in SEQ ID NO: 6.

[0009] Fourthly, the present invention provides a recombinant expression cassette, comprising a promoter and a nucleic acid molecule encoding the aforementioned fusion protein operably linked to the promoter. Preferably, the promoter is an arabinose-inducible araBAD promoter, and the nucleotide sequence of the recombinant expression cassette is shown in SEQ ID NO: 7.

[0010] Fifthly, the present invention provides a recombinant vector comprising the above-described recombinant expression cassette.

[0011] In a sixth aspect, the present invention provides an engineered Gram-negative bacterium comprising the recombinant vector described above. Preferably, the engineered Gram-negative bacterium is a non-pathogenic or probiotic *Escherichia coli*. More preferably, the engineered Gram-negative bacterium expresses the ClyA-HA-AnHM fusion protein after induction, and enriches the fusion protein in the outer membrane and / or outer membrane vesicles.

[0012] In a seventh aspect, the present invention provides an outer membrane vesicle produced by the aforementioned engineered Gram-negative bacteria, and displaying on its outer surface a synthetic single-domain antibody AnHM with an amino acid sequence as shown in SEQ ID NO: 1.

[0013] Eighthly, the present invention provides a pharmaceutical composition comprising the aforementioned engineered Gram-negative bacteria and / or their generated outer membrane vesicles, and a pharmaceutically acceptable carrier, protectant, stabilizer, excipient, or combination thereof. Preferably, the pharmaceutical composition is an oral liquid, suspension, lyophilized bacterial powder, granules, capsules, enteric-coated formulation, or microcapsule formulation; when it contains inducible engineered bacteria, it further comprises an inducer capable of inducing the expression of a fusion protein.

[0014] In a ninth aspect, the present invention provides a drug kit comprising separately packaged component a and component b, wherein component a is the aforementioned engineered Gram-negative bacterium, and component b is an inducer for inducing the engineered Gram-negative bacterium to express ClyA-HA-AnHM.

[0015] In a tenth aspect, the present invention provides a method for preparing the above-mentioned engineered Gram-negative bacteria, comprising the following steps: constructing the recombinant vector as described in claim 8; introducing the recombinant vector into non-pathogenic or probiotic Gram-negative bacteria; screening positive transformants; and verifying the expression of the ClyA-HA-AnHM fusion protein.

[0016] Eleventhly, the present invention provides a method for preparing the above-mentioned outer membrane vesicles, comprising the following steps: culturing the engineered Gram-negative bacteria as described in claim 9; inducing the expression of ClyA-HA-AnHM fusion protein; removing bacterial cells; isolating the outer membrane vesicles from the cell-free culture supernatant; and verifying the presence of AnHM and HMGB1 binding activity in the outer membrane vesicles.

[0017] In a twelfth aspect, the present invention provides the use of the above-described synthetic single-domain antibody, nucleic acid molecule, fusion protein, recombinant expression cassette, recombinant vector, engineered Gram-negative bacteria, outer membrane vesicles, pharmaceutical composition or pharmaceutical kit in the preparation of a medicament for the prevention or reduction of heatstroke-related systemic inflammation and organ damage.

[0018] Beneficial effects: (1) AnHM has a well-defined 123 amino acid synthetic single-domain antibody sequence and complementarity-determining region combination, which can be independently manufactured, expressed and quality controlled, and can be used to define the corresponding technical solutions.

[0019] (2) Artificial intelligence was used to design candidate single-domain antibodies from scratch, and the 23 candidates were prepared as independent monoclonal phages for parallel screening. This can reduce the impact of differences in growth, packaging and amplification during the mixed screening process on the selection of lead molecules.

[0020] (3) The ClyA fusion configuration enables AnHM to be expressed in engineered bacteria and enter the outer membrane vesicles; the results of proteinase K treatment support that AnHM in OMV-AnHM has surface accessibility.

[0021] (4) OMV-AnHM can reduce the detectable recombinant HMGB1 in the system and inhibit HMGB1-induced NF-κB p65 phosphorylation and inflammatory factor release, indicating that AnHM still maintains its functional activity after being displayed by outer membrane vesicles.

[0022] (5) After oral administration of EcN-AnHM, the live bacteria were mainly recovered in the gastrointestinal tract sample, and no significant and continuous growth of live bacteria was observed in the blood and most extraintestinal tissues. In heatstroke mice, EcN-AnHM can reduce plasma HMGB1 and inflammatory markers, alleviate multi-organ damage and improve short-term survival. Attached Figure Description

[0023] Figure 1 A schematic diagram illustrating the role of high-mobility group box 1 (HMGB1) in neutralizing the engineered bacteria-outer membrane vesicle system.

[0024] Figure 2 Figures show the AI-assisted de novo design of AnHM, screening of candidate single-domain antibody phages, AnHM expression and purification, HMGB1 binding characteristics, and in vitro neutralization results. In the figures, (A) the binding screening results of 23 candidate single-domain antibody monoclonal phages to HMGB1, with bovine serum albumin (BSA) as a non-specific binding control. (B) the binding inhibition results of 6 candidate single-domain antibodies under competitive conditions with different concentrations of soluble HMGB1. (C) the expression and purification results of AnHM. (D) the surface plasmon resonance (SPR) detection of the binding kinetics between AnHM and HMGB1. (E) the binding specificity of AnHM to human HMGB1 and its A and B boxes, as well as mouse HMGB1, porcine HMGB1, HMGB2, BSA, and lipopolysaccharide (LPS). (F) the effect of AnHM on the release of interleukin-6 (IL-6) from macrophages after stimulation with recombinant HMGB1. Effect of (G)AnHM on the release of tumor necrosis factor-α (TNF-α) from macrophages after stimulation with recombinant HMGB1.

[0025] Figure 3Figure 1 shows the construction of the ClyA-HA-AnHM expression cassette and engineered E. coli Nissle 1917 (EcN), the fusion protein induction expression, the outer membrane vesicle particle size, and the identification results of the surface accessibility of the fusion protein in OMV-AnHM. In the figure, (A) is a schematic diagram of the construction of the EcN-AnHM and control engineered strain EcN-Ctrl expression cassettes and the production of OMV-AnHM by the engineered strains and its binding to HMGB1. (B) is the polymerase chain reaction (PCR) identification results of the EcN-Ctrl and EcN-AnHM recombinant vectors. (C) is the particle size distribution of wild-type outer membrane vesicles (OMV-WT), control outer membrane vesicles (OMV-Ctrl), and OMV-AnHM. (D) is the immunoblotting detection of L-arabinose-induced expression of the ClyA-HA or ClyA-HA-AnHM fusion protein in different engineered strains. (E) Immunoblotting was used to detect ClyA-HA or ClyA-HA-AnHM in OMV, and the surface accessibility of the fusion protein was evaluated by proteinase K (PK) treatment.

[0026] Figure 4 The figure shows the effect of OMV-AnHM on the detectability of recombinant HMGB1 and its inhibition of HMGB1-induced NF-κB p65 phosphorylation and inflammatory factor release. In the figure, (A) the relative levels of detectable recombinant HMGB1 in the system under different outer membrane vesicle treatments, with polymyxin B (PMB) pretreatment and proteinase K pretreatment groups used to evaluate the effects of outer membrane vesicle-related components and surface proteins. (B) the effect of Western blotting on HMGB1-induced phosphorylation of nuclear factor κB p65 (NF-κBp65). (C) the effect of different doses of OMV-AnHM on HMGB1-induced IL-6 release from macrophages. (D) the effect of different doses of OMV-AnHM on HMGB1-induced TNF-α release from macrophages.

[0027] Figure 5 This image shows the in vitro fluorescence distribution of the gastrointestinal tract and major extraintestinal tissues after oral administration of EcN-WT or EcN-AnHM, the results of viable bacterial culture in different tissues, and the HA-related signal results in liver and kidney tissues. In the image, (A) is the in vitro fluorescence imaging of the gastrointestinal tract and major extraintestinal tissues at different time points after oral administration of EcN-WT or EcN-AnHM. (B) is the quantitative result of the change in fluorescence signal over time in the stomach, jejunum, cecum, and colon after oral administration of EcN-WT or EcN-AnHM. (C) is the viable bacterial culture result of blood, intestinal, and major extraintestinal tissue samples at different time points. (D) is the immunofluorescence detection of HA-related signals in liver and kidney tissues after oral administration of different engineered bacteria.

[0028] Figure 6The figure shows the effects of oral EcN-AnHM on plasma HMGB1, organ NF-κB p65 phosphorylation, and plasma inflammatory factors in heatstroke mice. In the figure, (A) plasma HMGB1 levels in heatstroke mice after different doses of EcN-AnHM and different control treatments; (B) levels of NF-κB p65 and phosphorylated NF-κB p65 in the heart, liver, lung, and kidney tissues of heatstroke mice in different treatment groups as detected by Western blotting; (C) plasma interleukin-1β (IL-1β) levels in heatstroke mice in different treatment groups; (D) plasma IL-6 levels in heatstroke mice in different treatment groups; and (E) plasma TNF-α levels in heatstroke mice in different treatment groups.

[0029] Figure 7 The figure shows the effects of oral administration of EcN-AnHM on blood organ damage indicators, histopathological changes, and 72-hour short-term survival in heatstroke mice. In the figure, (A) plasma alanine aminotransferase (ALT) levels in heatstroke mice under different treatment groups; (B) plasma aspartate aminotransferase (AST) levels; (C) blood urea nitrogen (BUN) levels; (D) creatinine (Cr) levels; (E) creatine kinase isoenzyme MB (CK-MB) levels; (F) lactate dehydrogenase (LDH) levels; (G) hematoxylin-eosin (HE) staining results of heart, liver, lung, and kidney tissues from mice under different treatment groups; and (H) 72-hour survival curves of heatstroke mice under different treatment groups. Detailed Implementation

[0030] The following examples are used to illustrate the technical solutions of the present invention and do not constitute a limitation on the scope of protection of the present invention. Experimental methods without specific conditions can use conventional conditions in the art, conditions recommended by reagent suppliers, or optimized equivalent conditions.

[0031] Table 1: Terms and Definitions.

[0032] the term meaning HMGB1 High-mobility group B1 proteins include full-length proteins or fragments with extracellular pro-inflammatory activity. AnHM A synthetic single-domain antibody (VHH) was obtained by computationally designing the target region using the B-box surface of HMGB1, through artificial intelligence-assisted de novo design and parallel screening of independent monoclonal phages; the complete sequence is shown in SEQ ID NO: 1. EcN-AnHM Engineered Escherichia coli Nissle 1917 (EcN) containing and capable of expressing the ClyA-HA-AnHM fusion protein. EcN-Ctrl Control engineered bacteria expressing ClyA-HA. OMV-AnHM External membrane vesicles (OMVs) generated by EcN-AnHM and exhibiting AnHM on the outer surface. neutralization This includes reducing HMGB1-induced receptor signaling or cytokine output by directly binding to, capturing, or isolating extracellular HMGB1. Example 1: AnHM's AI-assisted de novo design, screening, and validation.

[0033] 1.1 Artificial intelligence-assisted design and candidate generation.

[0034] Using the three-dimensional structure of the B-box of HMGB1 as the design target region, E108, Y109, K112, M132, T136, D140, P143, Y144 and K147 were set as computational design hotspots. Artificial intelligence was used to assist in de novo design to obtain 23 candidate sequences in single-domain antibody (VHH) format.

[0035] 1.2 Parallel screening of independent monoclonal phages.

[0036] Twenty-three candidate single-domain antibodies were cloned into the same pIII phage display vector, and 23 monoclonal phages were prepared under the same host bacteria, helper phages, and packaging conditions. The first round of parallel phage ELISA assays used full-length HMGB1 as the target antigen and bovine serum albumin (BSA) as a non-specific binding control. Candidate molecules were screened based on the difference in binding signals between HMGB1 and BSA.

[0037] The candidates selected in the first round of screening were further subjected to competitive binding detection using different concentrations of soluble HMGB1. The binding specificity of the lead molecule was compared with that of HMGB1 in its B-box, A-box, HMGB2, and related control proteins. Based on the comprehensive screening results, HM_20 was identified as the lead single-domain antibody and named AnHM.

[0038] 1.3 AnHM sequence, expression purification and HMGB1 binding.

[0039] AnHM is a synthetic single-domain antibody consisting of 123 amino acids. Its complete amino acid sequence is: EVQLVESGGGLVQPGGSLRLSCAASGFDFSQHAMGWFRQAPGKGRELVAAIGPGGGVTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAASSGPGMARLENYAFWGQGTQVTVSS. Soluble expression and affinity purification of AnHM were performed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie brilliant blue staining revealed a major protein band at approximately 15 kilodaltons (kDa), consistent with the expected molecular weight. Surface plasmon resonance (SPR) was used to detect the binding of AnHM to the full-length HMGB1. The results showed a concentration-dependent response to different concentrations of AnHM, with the equilibrium dissociation constant KD measured to be approximately 12.5 nanomolars (nM). Further enzyme-linked immunosorbent assay (ELISA) showed that AnHM significantly bound to human HMGB1 and its B box, but had lower binding to the A box, HMGB2, BSA and lipopolysaccharide (LPS), and could recognize mouse and porcine HMGB1.

[0040] 1.4 In vitro neutralization effect of AnHM.

[0041] The effect of AnHM on the inflammatory response induced by recombinant HMGB1 was evaluated using bone marrow-derived macrophages (BMDMs). Control groups, AnHM alone, recombinant HMGB1 alone, and groups combining recombinant HMGB1 with different doses of AnHM were included. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels were measured. Compared with the recombinant HMGB1 alone group, AnHM dose-dependently reduced the release of IL-6 and TNF-α, supporting its functional HMGB1 neutralizing activity.

[0042] Example 2: ClyA-HA-AnHM expression cassette, engineered EcN, and OMV-AnHM.

[0043] 2.1 Construction of expression cassettes and engineered bacteria.

[0044] A ClyA-HA-AnHM fusion expression cassette driven by the araBAD promoter was constructed and introduced into *E. coli* Nissle 1917 (EcN). Simultaneously, a control engineered strain expressing ClyA-HA, EcN-Ctrl, was constructed, and the unmodified EcN was used as EcN-WT. The recombinant vector was identified by polymerase chain reaction (PCR); the primer positions and representative amplification results are shown below. Figure 2 B.

[0045] 2.2 Fusion protein expression and preparation of outer membrane vesicles.

[0046] After adding L-arabinose, immunoblotting was performed using an anti-HA antibody. EcN-Ctrl detected a ClyA-HA band of approximately 35 kDa, and EcN-AnHM detected a ClyA-HA-AnHM band of approximately 48 kDa, while the uninduced group showed weak signals or no obvious bands. OMV was obtained from the culture supernatant after cell removal, 0.22 μm filtration, concentration, and outer membrane vesicle separation. Particle size analysis showed that OMV-WT, OMV-Ctrl, and OMV-AnHM were all distributed in the nanoscale range.

[0047] 2.3 Identification and surface accessibility of fusion proteins in OMV-AnHM.

[0048] Western blotting was used to detect purified outer membrane vesicles. ClyA-HA was detected in OMV-Ctrl, and ClyA-HA-AnHM was detected in OMV-AnHM. OmpA, an outer membrane vesicle-associated protein, was also detected as a reference. Treatment with proteinase K (PK) significantly reduced the signal of the HA-related fusion protein in OMV-AnHM, supporting the fact that the fusion protein in AnHM is exposed on the surface of the outer membrane vesicles and is accessible to proteases.

[0049] Example 3: Effects of OMV-AnHM on the detectability of HMGB1 and in vitro inflammatory response.

[0050] 3.1 The effect of OMV-AnHM on the detectability of recombinant HMGB1.

[0051] Recombinant HMGB1 was incubated with equal or different doses of OMV-WT, OMV-Ctrl, or OMV-AnHM, with the detectability level of recombinant HMGB1 alone used as a reference. Compared to OMV-WT and OMV-Ctrl, OMV-AnHM dose-dependently reduced the detectable recombinant HMGB1 in the system. Pretreatment with polymyxin B (PMB) retained the effect of reducing HMGB1 detectability; this effect was significantly weakened after proteinase K pretreatment, supporting the involvement of the surface protein AnHM in HMGB1 binding or capture.

[0052] 3.2 Effect of OMV-AnHM on nuclear factor κB signaling.

[0053] Macrophages were stimulated with recombinant HMGB1, and the study included a control group, a recombinant HMGB1 alone group, a HMGB1 combined with OMV-WT or OMV-Ctrl group, and a HMGB1 combined with different doses of OMV-AnHM group. Western blotting was used to detect phosphorylated nuclear factor κB p65 (p-NF-κB p65), total NF-κB p65, and β-actin. OMV-AnHM reduced HMGB1-induced NF-κB p65 phosphorylation with increasing dose, while OMV-WT and OMV-Ctrl showed weaker inhibitory effects.

[0054] 3.3 Effects of OMV-AnHM on the release of inflammatory factors.

[0055] IL-6 and TNF-α were detected under the above treatment conditions. OMV-AnHM dose-dependently reduced HMGB1-induced IL-6 and TNF-α release, and the inhibitory effect of high-dose OMV-AnHM was more significant, indicating that AnHM retains its HMGB1-related neutralizing function after being displayed by outer membrane vesicles.

[0056] Example 4: In vivo distribution, viable bacterial recovery and HA-related signals after oral administration of EcN-AnHM.

[0057] 4.1 Short-term distribution of the gastrointestinal tract and major tissues.

[0058] Following oral administration of EcN-WT or EcN-AnHM containing detectable fluorescent signals, in vitro tissue imaging was performed at 0.5, 2, 4, 6, 12, and 24 hours. The fluorescent signals were primarily distributed in the gastrointestinal tract, gradually weakening over time as they shifted from the proximal intestinal segment towards the cecum and colon. Quantification of the radioactivity in the stomach, jejunum, cecum, and colon revealed similar gastrointestinal temporal distribution trends for EcN-WT and EcN-AnHM.

[0059] 4.2 Live bacteria recovery and liver and kidney HA-related signals.

[0060] Selective cultures were performed on blood, heart, jejunum, cecum, colon, liver, kidney, lung, and spleen samples at 1, 3, 6, 12, and 24 hours. Viable bacteria were primarily recovered from jejunum, cecum, and colon samples; no significant persistent colony growth was observed in blood or most extraintestinal tissues. Immunofluorescence assays using anti-HA antibodies revealed varying degrees of HA-related fluorescence signals in the liver and kidneys after oral administration of EcN-AnHM-induced expression. This signal is used to indicate the tissue detectability of ClyA-HA-AnHM-related components and is not used alone to determine whether they originate from intact engineered bacteria or intact outer membrane vesicles.

[0061] Example 5: Effects of oral administration of EcN-AnHM on inflammation and organ damage in heatstroke mice.

[0062] 5.1 Grouping, administration and sampling.

[0063] Mice were orally treated before heat exposure to establish a heatstroke model. The main subgroups included a sham treatment + phosphate-buffered saline (PBPS) group, a heatstroke + PBPS group, an EcN-WT group, an EcN-Ctrl group, different doses of EcN-AnHM, and an EcN-AnHM group without L-arabinose inducer. EcN-AnHM doses were set at 0.25 × 10^11, 0.5 × 10^11, and 1 × 10^11 colony-forming units / mL (CFU / mL), and the induction groups were given L-arabinose. Major inflammatory and organ damage markers were measured 12 hours after heatstroke; survival was observed up to 72 hours.

[0064] 5.2 Plasma HMGB1, inflammatory factors and organ NF-κB signaling.

[0065] Plasma levels of HMGB1, interleukin-1β (IL-1β), IL-6, and TNF-α were measured 12 hours after heatstroke. Western blotting was used to detect p-NF-κB p65 and total NF-κB p65 in heart, liver, lung, and kidney tissues. Compared with the heatstroke + phosphate-buffered saline group, the EcN-WT group, and the EcN-Ctrl group, induced expression of EcN-AnHM decreased plasma HMGB1 and inflammatory cytokine levels with increasing dose, and reduced NF-κB p65 phosphorylation in multiple organs. The improvement was weaker in the EcN-AnHM group without L-arabinose inducer.

[0066] 5.3 Organ injury, histopathology and short-term survival.

[0067] Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (Cr), creatine kinase isoenzyme MB (CK-MB), and lactate dehydrogenase (LDH) were measured 12 hours after heatstroke. Induced EcN-AnHM expression dose-dependently reduced these organ damage markers. Hematoxylin-eosin (HE) staining showed that the EcN-AnHM-induced group had less damage to heart, liver, lung, and kidney tissues compared to the control engineered bacteria group. During the 72-hour survival observation, the short-term survival rate of the highest dose EcN-AnHM combined with L-arabinose group was higher than that of the phosphate-buffered saline, EcN-WT, EcN-Ctrl, and uninduced EcN-AnHM groups.

[0068] Example 6: Oral formulations, combined use and quality control.

[0069] EcN-AnHM can be formulated into fresh bacterial suspensions, lyophilized bacterial powders, enteric-coated capsules, microcapsules, or granules. L-arabinose can be administered with the engineered bacteria at specific times or packaged separately into drug kits. The engineered bacteria or outer membrane vesicle formulations can be used in combination with rapid cooling, fluid resuscitation, and organ support.

[0070] Quality control items may include strain identity, viable cell titer, inducible expression capacity, outer membrane vesicle size, outer membrane protein A (OmpA) and ClyA-HA-AnHM content, HMGB1 binding or neutralization activity in cells, sterility check, bacterial residues, and endotoxin levels.

[0071] Alternative implementations: Without departing from the core concept of this invention, EcN can be replaced with other non-pathogenic or probiotic Gram-negative bacteria; ClyA can be replaced with other anchoring proteins capable of localizing effector proteins to the outer membrane or the surface of outer membrane vesicles; and the araBAD promoter can be replaced with other controllable promoters. The above substitutions maintain the common technical concept of "specific HMGB1 neutralizing single-domain antibody—engineered bacterial expression—outer membrane vesicle surface display—HMGB1 binding and neutralization—heatstroke application".

[0072] Industrial Applicability: The recombinant expression cassette, engineered bacteria, and outer membrane vesicles of this invention can be prepared using conventional molecular cloning, microbial culture, and membrane vesicle isolation processes. Quality control can be achieved through indicators such as particle size, fusion protein expression, and HMGB1 binding or neutralization activity in cells. The product can be used for heatstroke prevention or adjunctive intervention in high-temperature operations, military training, competitive sports, disaster relief, and clinical emergency scenarios.

[0073] Sequence Identification Description: This invention relates to the sequences shown in SEQ ID NO: 1-11, including the complete AnHM single-domain antibody, heavy chain complementarity-determining regions 1-3, AnHM-encoded nucleic acid, ClyA-HA-AnHM fusion protein and expression cassette, ClyA, HA epitope tags and linker peptide sequences, as shown in Table 2 below.

[0074] Table 2: Sequence Identifier Description SEQ ID NO sequence Type and Length 1 AnHM complete single-domain antibody (VHH) amino acid sequence Protein; 123 amino acids 2 AnHM heavy chain complementarity-determining region 1 (HCDR1) Protein; 8 amino acids 3 AnHM heavy chain complementarity-determining region 2 (HCDR2) Protein; 8 amino acids 4 AnHM heavy chain complementarity-determining region 3 (HCDR3) Protein; 16 amino acids 5 AnHM encoded nucleotide sequence Deoxyribonucleic acid (DNA); 369 nucleotides 6 ClyA-HA-AnHM fusion protein Protein; 441 amino acids 7 araBAD-ClyA-HA-AnHM expression kit Deoxyribonucleic acid (DNA); 1764 nucleotides 8 ClyA amino acid sequence Protein; 305 amino acids 9 ClyA encodes nucleotide sequence Deoxyribonucleic acid (DNA); 915 nucleotides 10 HA epitope-tagged amino acid sequence Protein; 9 amino acids 11 Short linker peptide between HA and AnHM Protein; 4 amino acids

Claims

1. A synthetic single-domain antibody that specifically binds to high-mobility group box protein B1, characterized in that, The single-domain antibody is named AnHM, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The synthetic single-domain antibody according to claim 1, characterized in that, According to the international immunogenetic information system numbering system, the single-domain antibody includes heavy chain complementarity-determining region 1 as shown in SEQ ID NO: 2, heavy chain complementarity-determining region 2 as shown in SEQ ID NO: 3, and heavy chain complementarity-determining region 3 as shown in SEQ ID NO:

4.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the synthetic single-domain antibody of claim 1.

4. A fusion protein, characterized in that, The fusion protein includes an outer membrane vesicle anchoring protein and the synthetic single-domain antibody as described in claim 1.

5. The fusion protein according to claim 4, characterized in that, The fusion protein comprises, from N-terminus to C-terminus, cytolysin A, a flexible linker peptide, an HA epitope tag, and AnHM, and its amino acid sequence is shown in SEQ ID NO:

6.

6. A recombinant expression cassette, characterized in that, Includes a promoter and a nucleic acid molecule operatively linked to the promoter that encodes the fusion protein of claim 4 or 5.

7. The recombinant expression cassette according to claim 6, characterized in that, The promoter is an arabadose-inducible araBAD promoter, and the nucleotide sequence of the recombinant expression cassette is shown in SEQ ID NO:

7.

8. A recombinant vector, characterized in that, The recombinant vector comprises the recombinant expression cassette of claim 7.

9. An engineered Gram-negative bacterium, characterized in that, The engineered Gram-negative bacteria comprise the recombinant vector of claim 8.

10. The engineered Gram-negative bacteria according to claim 9, characterized in that, The engineered Gram-negative bacteria are non-pathogenic or probiotic Escherichia coli.

11. The engineered Gram-negative bacteria according to claim 9, characterized in that, The engineered Gram-negative bacteria expressed the ClyA-HA-AnHM fusion protein after induction, and the fusion protein was enriched in the outer membrane and / or outer membrane vesicles.

12. An outer membrane vesicle, characterized in that, The outer membrane vesicles are produced by the engineered Gram-negative bacteria of claim 9, and their outer surface displays the synthetic single-domain antibody AnHM with the amino acid sequence shown in SEQ ID NO:

1.

13. A pharmaceutical composition, characterized in that, Includes the engineered Gram-negative bacteria of claim 9 and / or their generated outer membrane vesicles, as well as pharmaceutically acceptable carriers, protectants, stabilizers, excipients, or combinations thereof.

14. The pharmaceutical composition according to claim 13, characterized in that, The pharmaceutical composition is an oral liquid, suspension, lyophilized bacterial powder, granules, capsules, enteric-coated preparations, or microcapsule preparations; when it contains inducible engineered bacteria, it also includes an inducer capable of inducing the expression of the fusion protein.

15. A medicine kit, characterized in that, It includes separately packaged components a and b, wherein component a is the engineered Gram-negative bacterium of claim 9, and component b is an inducer for inducing the engineered Gram-negative bacterium to express ClyA-HA-AnHM.

16. A method for preparing the engineered Gram-negative bacteria of claim 9, characterized in that, The method includes the following steps: constructing the recombinant vector as described in claim 8; introducing the recombinant vector into non-pathogenic or probiotic Gram-negative bacteria; screening for positive transformants; and verifying the expression of the ClyA-HA-AnHM fusion protein.

17. A method for preparing the outer membrane vesicles of claim 12, characterized in that, Includes the following steps: Cultivation The engineered Gram-negative bacteria of claim 9; induce expression of ClyA-HA-AnHM fusion protein; remove bacterial cells; isolate outer membrane vesicles from cell-free culture supernatant; verify the presence of AnHM and HMGB1 binding activity in the outer membrane vesicles.

18. Use of the synthetic single-domain antibody of claim 1, the nucleic acid molecule of claim 3, the fusion protein of claim 4, the recombinant expression cassette of claim 6, the recombinant vector of claim 8, the engineered Gram-negative bacteria of claim 9, the outer membrane vesicle of claim 12, the pharmaceutical composition of claim 13, or the pharmaceutical kit of claim 15 in the preparation of a medicament for the prevention or relief of heatstroke-related systemic inflammation and organ damage.

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