Recombinant oncolytic adenovirus and application thereof
By constructing a recombinant oncolytic adenovirus with a trispecific T-cell adaptor, highly efficient and low-toxicity immunotherapy was achieved in tumor cells. This solved the problem of insufficient T-cell activation capacity in the solid tumor microenvironment of existing oncolytic virus therapies, improved anti-tumor efficacy, and reduced systemic toxicity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LIUZHOU LIUTIE CENT HOSPITAL
- Filing Date
- 2026-02-13
- Publication Date
- 2026-05-15
AI Technical Summary
Existing oncolytic virus therapies have difficulty effectively activating T cells in the solid tumor microenvironment. The BiTE structure lacks co-stimulatory signals, resulting in limited anti-tumor effects and high systemic toxicity. A novel technology platform that can integrate T cell activation signals, tumor targeting signals, and co-stimulatory signals is needed.
A trispecific T-cell adaptor (TriTE) was constructed, which includes a recombinant oncolytic adenovirus that targets CD3 on the surface of T cells, the tumor antigen EpCAM, and provides the co-stimulatory signal 4-1BB. TriTE was delivered locally by the virus to achieve expression and immune activation in tumor cells.
It enhances the lysis effect and immune response of tumor cells, promotes the proliferation, survival and cytokine secretion of T cells, reduces systemic toxicity, and significantly improves the anti-tumor effect.
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Figure CN122038318A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a recombinant oncolytic adenovirus and its applications. Background Technology
[0002] Cancer immunotherapy has become the fourth major cancer treatment method after surgery, radiotherapy, and chemotherapy. In this field, oncolytic virus therapy, as an innovative strategy, can specifically replicate and lyse tumor cells, thereby activating the body to produce an anti-tumor immune response. Among various oncolytic viruses, oncolytic adenoviruses, with their advantages of high infectivity, low pathogenicity, and good genetic stability, have become one of the most promising vectors for clinical application. To enhance the efficacy of oncolytic viruses, researchers have attempted to "arm" them with genetic engineering techniques to express immunomodulatory factors. For example, oncolytic viruses expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) have shown potential in preclinical and early clinical studies. However, the immunosuppressive microenvironment unique to solid tumors often limits the full effectiveness of this type of therapy.
[0003] Bi-specific T cell engagers (BiTEs) are engineered antibodies that simultaneously recognize CD3 molecules and tumor-associated antigens on the surface of T cells, effectively redirecting T cells to the vicinity of tumor cells and activating their cytotoxic function. Epithelial cell adhesion molecule (EpCAM), a transmembrane glycoprotein highly expressed in various epithelial-derived malignancies (such as colorectal cancer, gastric cancer, and pancreatic cancer), is considered an ideal target for developing BiTE drugs. Although the bispecific antibody Catumaxomab, targeting EpCAM and CD3, was approved for the treatment of malignant ascites, it has been largely withdrawn from the market globally due to its limited efficacy and the risk of severe toxic side effects from systemic administration. In recent years, the combined use of BiTEs with oncolytic viruses, or the delivery of BiTEs via oncolytic viruses, has become an important research direction in tumor immunotherapy. However, existing BiTE structures lack co-stimulatory signals, limiting their ability to activate T cells in the solid tumor microenvironment. Even when combined with oncolytic viruses, it remains difficult to achieve ideal and durable anti-tumor effects.
[0004] Co-stimulatory signals are crucial for T cell activation and long-term survival. 4-1BB (CD137) is a key co-stimulatory receptor expressed on T cells, and its activation can effectively promote T cell proliferation, survival, cytokine secretion, and anti-apoptotic capabilities. Although direct use of anti-4-1BB agonist antibodies can enhance T cell function, it is often accompanied by systemic side effects such as hepatotoxicity, limiting its clinical application.
[0005] Therefore, there is an urgent need in the field for a novel technology platform that can organically integrate T-cell activation signals, tumor-targeting signals, and co-stimulatory signals to achieve local tumor delivery, in order to improve efficacy while reducing systemic toxicity. Based on this need, there is an urgent need to develop a new strategy for highly efficient and low-toxicity precision immunotherapy to provide a new solution for breaking through existing technological bottlenecks. Summary of the Invention
[0006] The purpose of this invention is to address the aforementioned problems by providing a recombinant oncolytic adenovirus and its application. It innovatively constructs a tri-specific T cell engager (TriTE) capable of simultaneously targeting T cell activation signals (CD3) and tumor antigens (EpCAM), while providing co-stimulatory signals (4-1BB). Furthermore, it utilizes oncolytic adenovirus to locally deliver TriTE, representing a novel, highly efficient, and low-toxicity strategy for precise immunotherapy, offering a completely new solution to overcome existing technological bottlenecks.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] A recombinant oncolytic adenovirus, wherein the genome of the recombinant oncolytic adenovirus contains a nucleic acid molecule encoding a trispecific T cell adaptor; the trispecific T cell adaptor comprises three functional domains, namely a first binding domain that specifically binds to CD3ε on the surface of T cells, a second binding domain that specifically binds to the tumor-associated antigen EpCAM, and a costimulatory domain that specifically binds to and activates the 4-1BB costimulatory signaling pathway.
[0009] Specifically, the trispecific T-cell adaptor consists of, from N-terminus to C-terminus, a single-chain antibody that specifically binds to CD3ε, the extracellular region of the 4-1BB ligand or a functional fragment thereof that activates the 4-1BB signaling pathway, and a single-chain antibody that specifically binds to epithelial cell adhesion molecules; the amino acid sequence of the trispecific T-cell adaptor is shown in SEQ ID NO: 1 or SEQ ID NO: 3.
[0010] Furthermore, the single-chain antibody that specifically binds to CD3ε is linked to the extracellular region of the 4-1BB ligand or a functional fragment thereof that activates the 4-1BB signaling pathway via a first linker peptide; the extracellular region of the 4-1BB ligand or a functional fragment thereof that activates the 4-1BB signaling pathway is linked to the single-chain antibody that specifically binds to epithelial cell adhesion molecules via a second linker peptide; wherein, the amino acid sequence of the first linker peptide is GGGGS, and the amino acid sequence of the second linker peptide is EASGGPE.
[0011] Furthermore, the nucleotide sequence encoding the amino acid sequence SEQ ID NO: 1 is shown in SEQ ID NO: 2; and the nucleotide sequence encoding the amino acid sequence SEQ ID NO: 3 is shown in SEQ ID NO: 4.
[0012] Furthermore, the expression of the three specific T cell adaptors is regulated by viral promoters or constitutive strong promoters.
[0013] Preferably, the expression of the trispecific T cell adaptor is regulated by the EF1α promoter.
[0014] Furthermore, the recombinant oncolytic adenovirus uses adenovirus type 5 as a vector, with its E1B and E3 regions deleted, and the expression of the E1A gene regulated by the cytomegalovirus promoter, thereby endowing the virus with oncolytic specificity and enhancing its safety.
[0015] This invention also provides a method for preparing the recombinant oncolytic adenovirus, which mainly includes the following steps:
[0016] 1) Gene synthesis and cloning: The gene fragment encoding the three-specific T cell adaptor is artificially synthesized and subcloned into the adenovirus shuttle plasmid to obtain the recombinant shuttle plasmid;
[0017] 2) Viral genome recombination: The above-mentioned recombinant shuttle plasmid and adenovirus backbone plasmid are combined through homologous recombination or site-specific recombination to obtain recombinant adenovirus genome plasmid;
[0018] 3) Virus rescue and amplification: After linearizing the recombinant adenovirus genome plasmid, it was transfected into packaging cells, and virus rescue was performed through cell culture to finally obtain the recombinant oncolytic adenovirus.
[0019] In addition, the recombinant oncolytic adenovirus provided by this invention can be used to prepare drugs for the treatment or prevention of EpCAM-positive tumors.
[0020] In summary, due to the adoption of the above technical solution, the present invention has the following beneficial effects:
[0021] This invention effectively combines the direct oncolytic ability of oncolytic viruses with TriTE-mediated trispecific T cell immune responses to prepare a recombinant oncolytic adenovirus that expresses a trispecific T cell adaptor. This oncolytic adenovirus can selectively replicate and express / secrete the TriTE within tumor cells, thereby combining the direct cell lysis effect of oncolytic viruses with the TriTE-mediated immune activation.
[0022] The trispecific T-cell adaptor TriTE can simultaneously bridge T cells (via CD3ε) and tumor cells (via EpCAM) and provide a crucial co-stimulatory signal (via 4-1BB), effectively activating T cells and synergistically enhancing the anti-tumor immune response. In other words, the recombinant oncolytic adenovirus can not only simultaneously target T-cell activation signals (CD3) and tumor antigens (EpCAM), but also provide a co-stimulatory signal (4-1BB), enhancing the targeting specificity of the oncolytic adenovirus and effectively promoting T-cell proliferation, survival, cytokine secretion, and anti-apoptotic ability, while reducing the toxic side effects of 4-1BB. Furthermore, through the activation of the immune microenvironment by TriTE, the killing effect of the oncolytic adenovirus on solid tumors is increased.
[0023] The oncolytic adenovirus provided by this invention can not only replicate in tumor cells and exert an oncolytic effect, but also significantly promote the infiltration of various immune cells in tumor tissues. It can infect tumor cells and induce them to express a trispecific T-cell adaptor. This trispecific T-cell adaptor can simultaneously provide T-cell activation signals and key co-stimulatory signals, and significantly enhance the activation level, proliferation capacity, and effector function of T cells. Furthermore, this invention, through local viral delivery of TriTE, can improve anti-tumor efficacy while reducing systemic toxicity. Attached Figure Description
[0024] Figure 1 This is a schematic diagram of the structure of recombinant adenovirus type 5 encoding TriTE and its control virus.
[0025] Figure 2 To detect the expression of TriTE (His tag) in the supernatant of oncolytic adenovirus-infected tumor cells using Western blotting.
[0026] Figure 3 To detect oncolytic adenovirus replication using Incucyte live-cell dynamic imaging.
[0027] Figure 4 To detect the oncolytic effect of viruses using crystal violet staining.
[0028] Figure 5 Ad5-TriTE mediates the aggregation of T cells to EpCAM-positive tumor cells and enhances their killing effect on target cells by expressing TriTE molecules.
[0029] Figure 6 To analyze the expression of CD8+ T cell activation markers and effector molecules in a co-culture system of lymphocytes and tumor cells infected with oncolytic adenovirus using flow cytometry.
[0030] Figure 7To investigate the antitumor effects of Ad5-TriTE in mouse subcutaneous xenograft models of colorectal cancer CT26-EpCAM and MC38-EpCAM.
[0031] Figure 8 Ad5-TriTE enhances immune cell infiltration in the tumor microenvironment.
[0032] Figure 9 The antitumor effect of humanized TriTE oncolytic adenovirus Ad5-hTriTE in a humanized mouse colorectal cancer model. Detailed Implementation
[0033] To more clearly illustrate the present invention, the present invention will be further described in detail below with reference to embodiments and comparative examples, but the scope of protection of the present invention is not limited to the following embodiments.
[0034] Example 1: Construction and preparation of recombinant oncolytic adenoviruses Ad5-TriTE and Ad5-hTriTE expressing TriTE molecules.
[0035] Specifically, the following steps are included:
[0036] 1) Gene synthesis and cloning:
[0037] First, gene fragments encoding mouse TriTE and human TriTE (denoted as hTriTE) were artificially synthesized; the amino acid sequence of TriTE is shown in SEQ ID NO: 1, and the corresponding nucleotide sequence is shown in SEQ ID NO. 2; the amino acid sequence of hTriTE is shown in SEQ ID NO: 2, and the corresponding nucleotide sequence is shown in SEQ ID NO. 4.
[0038] Subsequently, the synthesized TriTE or hTriTE gene fragments were double-digested with AgeI (recognition sequence ACCGGT) and EcoRI (recognition sequence GAATTC) restriction endonucleases at 37°C. Simultaneously, the shuttle plasmid pAdS-EF1a (nucleotide sequence shown in SEQ ID NO. 5) was digested with the same endonucleases to generate corresponding sticky ends. The digestion products were separated and purified by 1% agarose gel electrophoresis, and then ligated to the linearized plasmid using T4 DNA ligase. The ligation products were transformed into competent *Escherichia coli* DH5α strain, plated on LB agar plates containing kanamycin, and incubated overnight at 37°C. Single clones were picked for colony PCR and restriction enzyme digestion verification. Positive clones were expanded, and plasmids were extracted for sequencing verification, ultimately yielding the recombinant shuttle plasmids pAdS-TriTE and pAdS-hTriTE, respectively.
[0039] 2) Viral genome recombination:
[0040] The verified recombinant shuttle plasmids pAdS-TriTE or pAdS-hTriTE (containing the attL site and the target gene) were recombined with the adenovirus backbone plasmid pAd / PL-DEST (containing the attR site, the ccdB lethal gene, and the chloramphenicol resistance gene CmR) via LR recombination reaction. The fragment between the attL and attR sites was excised, and the target gene was integrated into the adenovirus backbone. During this process, the ccdB-CmR fragment on the pAd / PL-DEST plasmid was effectively removed.
[0041] The specific procedure is as follows: The shuttle plasmid pAdS-TriTE or pAdS-hTriTE is mixed with the adenovirus backbone plasmid pAd / PL-DEST at a molar ratio of 5:1, and LR Clonase™ II Enzyme Mix is added. The mixture is incubated at 25°C for 1 hour. After the reaction is complete, Proteinase K solution is added to terminate the reaction. The reaction product is transformed into competent E. coli DH5α and plated on LB agar plates containing ampicillin for selection. Single clones are picked and verified by PCR to obtain the correct recombinant adenovirus genomic plasmid pAd-TriTE or pAd-hTriTE.
[0042] 3) Virus rescue and amplification:
[0043] The recombinant adenovirus genome plasmids pAd-TriTE or pAd-hTriTE, which were verified to be correct by sequencing, were digested with PacI restriction endonuclease to remove the ampicillin resistance gene and the origin of replication, thus specifically linearizing them within the inverted terminal repeat (ITR) region of the adenovirus to expose the viral genome ends. After the enzyme digestion reaction, the linearization efficiency was verified by 0.8% agarose gel electrophoresis, and the linearized plasmids were recovered using a DNA purification kit.
[0044] Linearized adenovirus genome plasmids pAd-TriTE or pAd-hTriTE were mixed with jetPRIME® transfection reagent and added to HEK-293A cell culture medium grown in 10 cm cell culture dishes with a confluence of 50-70%. Cells were cultured in a 37°C, 5% CO2 incubator. When cell confluence exceeded 95%, cells were passaged. Since the recombinant adenovirus genome contains a green fluorescent protein (EGFP) reporter gene, observations were made every 24 hours starting 24 hours post-transfection using a fluorescence microscope. 24-72 hours post-transfection, strong diffuse EGFP fluorescence was observed in HEK-293A cells, indicating successful initial transfection and the initiation of target gene expression. Approximately 4-7 days post-transfection, the green fluorescence gradually decreased until a strong fluorescent signal resembling a "comet tail" appeared, signifying successful rescue of the recombinant adenovirus. Simultaneously, typical adenovirus cytopathic effects (CPE) were observed under a bright-field microscope, highly overlapping with the lesion areas observed by fluorescence. When approximately 80% of the cells showed CPE, the cells and their supernatant were collected. The cells were lysed by repeated freeze-thaw cycles to release primary virus particles, thus obtaining the first-generation virus stock solution (P0).
[0045] Freshly plated HEK-293A cells were infected with P0 generation virus solution at an appropriate multiple of infection (MOI) and cultured until extensive CPE occurred. The cells were then collected, frozen, and thawed again to obtain a second-generation virus stock solution (P1) with a higher titer. This amplification process can be repeated to obtain high-titer recombinant oncolytic adenovirus Ad5-TriTE and Ad5-hTriTE.
[0046] 4) Virus titer determination:
[0047] Using a 50% tissue culture infection dose (TCID) reported by green fluorescent protein (EGFP) 50 Virus titration was performed using the endpoint dilution method and converted to plaque-forming units (pfu / mL).
[0048] HEK-293A cells were planted at a density of 1 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator until the cell confluence reached more than 90%.
[0049] Take the stock solution of the virus to be tested and perform 10-fold serial dilutions using serum-free cell culture medium. Discard the old culture medium in the 96-well plate, add the virus solution of each dilution, set up 5 replicates for each dilution, and add 100 μL to each well. At the same time, set up cell control wells with only culture medium added. Place the 96-well plate in an incubator and continue to incubate, and observe the expression of EGFP in the lowest dilution wells using an inverted fluorescence microscope.
[0050] Viral titer (TCID) 50 ( / mL) according to the formula: TCID 50 / mL = 10^[2 + (S / N - 0.5)], where S is the total number of cumulative fluorescent positive wells and N is the number of replicate wells for each dilution. pfu / mL ≈ TCID 50 / mL × 0.7.
[0051] 5) Virus purification:
[0052] Viral particles were purified using an iodixanol gradient ultracentrifugation method. The harvested viral supernatant was centrifuged at 3000 x g for 15 minutes at 4°C to remove cell debris. In an ultracentrifuge tube, iodixanol solutions of different concentrations were slowly layered from bottom to top to form a discontinuous density gradient. Preferably, the bottom layer of the iodixanol solution had a concentration of 54% (w / v), followed by concentrations of 40%, 25%, and 15%. The pretreated viral sample was carefully added to the top layer of the gradient. The mixture was centrifuged at 155,000 x g for 1 hour at 4°C. After centrifugation, a milky-white viral band was observed concentrated in the 40% iodixanol layer. The target band was collected using a tube bottom puncture method, aliquoted, and stored at -80°C.
[0053] Comparative Example 1: Preparation of recombinant oncolytic adenoviruses Ad5-BiTE and Ad5-hBiTE expressing BiTE molecules
[0054] This comparative example aims to prepare a bispecific T-cell adaptor molecule (BiTE) virus for comparison. The construction process is basically the same as in Example 1, with the key difference being the target gene expressed. Specifically, it includes:
[0055] 1) Gene synthesis and cloning:
[0056] Except for replacing the synthesized gene fragment with a BiTE molecule encoding EpCAM and CD3ε (its amino acid sequence is shown in SEQ ID NO: 6, and the corresponding nucleotide sequence is shown in SEQ ID NO: 7) and its humanized version hBiTE (its amino acid sequence is shown in SEQ ID NO: 8, and the corresponding nucleotide sequence is shown in SEQ ID NO: 9), the remaining steps, including enzyme digestion, ligation, transformation, screening and verification, were performed according to the method described in step 1) of Example 1, and the recombinant shuttle plasmids pAdS-BiTE and pAdS-hBiTE were finally obtained.
[0057] 2) Viral genome recombination
[0058] This step is exactly the same as the method described in step 2) of Example 1. The shuttle plasmid pAdS-BiTE or pAdS-hBiTE obtained above is subjected to LR recombination reaction with the adenovirus backbone plasmid pAd / PL-DEST. After transformation, screening and verification, the recombinant adenovirus genome plasmid pAd-BiTE or pAd-hBiTE is obtained.
[0059] 3) Virus rescue and amplification
[0060] This step is performed in exactly the same way as step 3) in Example 1. The linearized recombinant adenovirus genomic plasmid pAd-BiTE or pAd-hBiTE was transfected into HEK-293A cells. EGFP fluorescence and CPE were observed. After virus rescue and amplification, recombinant oncolytic adenoviruses Ad5-BiTE and Ad5-hBiTE were finally obtained.
[0061] Comparative Example 2: Preparation of empty vector control recombinant oncolytic adenovirus Ad5-Control
[0062] This comparative example aims to prepare a blank control virus that does not carry any exogenous therapeutic genes. Details are as follows:
[0063] 1) Viral genome recombination
[0064] This step is exactly the same as the method described in step 2) of Example 1. The shuttle plasmid pAdS-EF1a (as shown in SEQ ID NO.5) without any inserted foreign genes is subjected to LR recombination reaction with the adenovirus backbone plasmid pAd / PL-DEST. After transformation, screening and verification, the empty recombinant adenovirus genome plasmid pAd-Control is obtained.
[0065] 2) Virus rescue and amplification
[0066] This step is exactly the same as step 3) in Example 1. The linearized recombinant adenovirus genome plasmid pAd-Control was transfected into HEK-293A cells. After virus rescue and amplification, the empty vector control recombinant oncolytic adenovirus Ad5-Control was finally obtained.
[0067] The structural schematic diagrams of the recombinant type 5 adenovirus encoding TriTE prepared in Example 1 and its control virus (viruses prepared in Comparative Examples 1 and 2) are shown below. Figure 1 As shown.
[0068] Example 2: In vitro functional verification of recombinant adenovirus
[0069] 1) Protein expression
[0070] To verify the expression of BiTE and TriTE proteins mediated by recombinant adenovirus, mouse colon cancer cell lines C26, CT26, and MC38 were infected with Ad5-Control, Ad5-BiTE, or Ad5-TriTE viruses at a multiplicity of infection (MOI) of 20, respectively. Negative controls were uninfected cells. After 72 hours of infection, the culture supernatant of each group of cells was collected, and proteins were separated by SDS-PAGE. Western blot analysis was then performed using a specific antibody against the His tag carried by the recombinant proteins. Results are as follows: Figure 2 As shown.
[0071] The results showed that in the C26, CT26, and MC38 cell lines, only the Ad5-BiTE and Ad5-TriTE virus-infected groups showed strong specific protein bands in the supernatant. Specifically, in the three Ad5-TriTE-infected cell lines, a clear band was observed at approximately 110 kDa, while in the three Ad5-BiTE-infected cell lines, a clear band was observed at approximately 65 kDa, both molecular weights consistent with expectations. No obvious bands were observed at the corresponding positions in the negative control and Ad5-Control-infected cell lines. This indicates that the Ad5-BiTE and Ad5-TriTE viruses constructed in this patent can effectively express and secrete target proteins of the correct size in tumor cells.
[0072] 2) Virus replication ability
[0073] Mouse colon cancer cell lines CT26 and MC38 were administered at 1×10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 12-well plates. After cell attachment, the cells were infected with recombinant adenoviruses Ad5-Control, Ad5-BiTE, or Ad5-TriTE at a multiplicity of infection (MOI) of 20. The process was continuously monitored within 72 hours post-infection using the IncuCyte ZOOM live-cell dynamic imaging analysis system (Essen Bioscience). Whole-well images were automatically acquired every 8 hours, and viral replication was quantified by quantitative analysis of the area (μm² / field of view) of green fluorescent protein (EGFP) positive regions in the images. Results are as follows: Figure 3 As shown.
[0074] The results showed that in the CT26 and MC38 colon cancer cell lines, the EGFP-positive region induced by Ad5-BiTE, Ad5-TriTE, and Ad5-Control virus infection groups exhibited similar trends at different monitoring time points, and the fluorescence signal intensity among the groups remained at a similar level. Statistical analysis indicated no significant difference (ns) in the replication levels of the three viruses at each time point.
[0075] 3) Viral oncolytic ability
[0076] To evaluate the in vitro oncolytic activity of recombinant adenovirus, crystal violet staining was used for verification. Mouse colon cancer cell lines CT26 and MC38 were stained with crystal violet at 5 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and cultured overnight to allow for full cell adhesion. Subsequently, the cells were infected with Ad5-Control, Ad5-BiTE, or Ad5-TriTE viruses at different multiplicity of infection (MOIs) (0, 5, 25, 125, and 625), and cultured for another 72 hours. After culturing, the culture medium was discarded, and the cells were gently washed with PBS. Crystal violet staining solution was then added to stain the surviving adherent cells to visually demonstrate the oncolytic effect of the virus. Results are as follows: Figure 4 As shown.
[0077] The results showed that all three viruses exhibited significant oncolytic activity in both colon cancer cell lines, and this activity was clearly MOI-dependent. As the MOI increased to 125 and 625, cell clones in each group decreased significantly or even disappeared completely, indicating that the viruses had a strong killing effect on tumor cells.
[0078] Example 3: Ad5-TriTE mediates T cell recruitment and activation through the expression of TriTE molecules.
[0079] 1) Construction and validation of EpCAM overexpression cell lines
[0080] The sequence encoding the mouse EpCAM gene was cloned into a lentiviral expression plasmid, which was then co-transfected with the packaging plasmids PMD2.G and psPAX2 into HEK-293T cells to produce lentivirus. Viral supernatant was collected to infect parental CT26 and MC38 cells, followed by selection for resistance using puromycin to obtain stably transduced EpCAM cells CT26-EpCAM and MC38-EpCAM. Parental CT26 and MC38 cells in logarithmic growth phase, as well as transduced CT26-EpCAM and MC38-EpCAM cells, were washed with PBS and incubated with anti-mouse EpCAM antibody conjugated to allophycocyanin (APC) at 4°C in the dark for 20 minutes. After incubation, the cells were washed twice with PBS to remove unbound antibodies, resuspended, and immediately analyzed by flow cytometry. Results are as follows: Figure 5As shown in A, Figure 5 A showed that, compared with the parental cells, the APC fluorescence signal intensity of the CT26-EpCAM and MC38-EpCAM cell populations was significantly shifted to the right, indicating that the EpCAM-overexpressing tumor cell lines were successfully constructed.
[0081] 2) Analysis of T cell recruitment and target cell killing
[0082] Primary lymphocytes were isolated from the spleen of C57BL / 6 mice and co-cultured with CFSE-labeled MC38-EpCAM target cells at an effector-to-target ratio (E:T ratio) of 10:1. The co-culture system was supplemented with Ad5-Control, Ad5-BiTE, or Ad5-TriTE (MOI=20), respectively. The co-culture process was dynamically monitored using the Incucyte live-cell imaging system. Results are shown below. Figure 5 B, Figure 5 Figure B shows that in the Ad5-TriTE treatment group, significant lymphocyte aggregation was observed around CFSE-labeled MC38-EpCAM cells. Lymphocyte aggregation was also observed in the Ad5-BiTE group, but to a lesser extent than in the Ad5-TriTE group, while no significant aggregation was observed in the Ad5-Control group. After co-culturing for 72 hours, crystal violet staining was performed to assess target cell viability (see Figure B). Figure 5 (C) The results showed that the number of residual cell clones in the Ad5-TriTE treatment group was significantly less than that in other groups, indicating that it induced the strongest target cell killing effect.
[0083] 3) T cell activation
[0084] To assess the functional status of T cells, primary lymphocytes were isolated from the spleen of C57BL / 6 mice and co-cultured with MC38-EpCAM target cells at an effector-to-target ratio (E:T ratio) of 10:1. The co-culture system was supplemented with Ad5-Control, Ad5-BiTE, or Ad5-TriTE (MOI=20). After 24 hours of co-culture, cells were collected for multicolor flow cytometry analysis. First, Fc receptors were blocked using anti-mouse CD16 / CD32 antibodies. Then, the following antibody combinations were used to stain cell surface markers and intracellular cytokines: T cell subsets were first distinguished using APC-Cy7-labeled anti-mouse CD45 antibody (clone 30-F11), BV421-labeled anti-mouse CD3 antibody (clone 145-2C11), and APC-labeled anti-mouse CD8 antibody (clone 53-6.7); then, early activation was detected using PE-Cy7-labeled anti-mouse CD69 antibody, degranulation was detected using PE-labeled anti-mouse CD107a antibody, and effector cytokine expression was assessed using BV786-labeled anti-mouse IFN-γ antibody and BV650-labeled anti-mouse TNF-α antibody.
[0085] Flow cytometry analysis results showed (see) Figure 6 Compared with the Ad5-Control group and the Ad5-BiTE group, Ad5-TriTE treatment significantly increased the proportion of positive cells for CD69, IFN-γ, TNF-α and CD107a in T cells, indicating that Ad5-TriTE can effectively promote T cell activation and cytotoxicity.
[0086] Example 4: Antitumor efficacy of Ad5-TriTE in a mouse model of colorectal cancer
[0087] Male C57BL / 6J and BALB / c mice aged 6-8 weeks (purchased from Nanjing Jicui Pharmaceutical Biotechnology Co., Ltd.) were subcutaneously inoculated with 1×10⁻⁶ mice in the right axilla. 6 MC38-EpCAM or CT26-EpCAM cells were used. Once the tumor volume reached 100 mm³, the tumor-bearing mice were randomly divided into four groups (n=8 per group), receiving intratumoral injection of PBS, 5×10⁻⁶ MC38-EpCAM cells, or CT26-EpCAM cells. 8 PFUs, including Ad5-Control, Ad5-BiTE, or Ad5-TriTE, were administered every two days for a total of four injections. Tumor volume was measured and growth curves were plotted every two days starting with the first dose. Survival was recorded as the endpoint when tumor volume exceeded 2000 mm³ or the animal died. Body weight was monitored every two days to assess systemic toxicity. Results are shown below. Figure 7 .
[0088] Figure 7The results showed that in the MC38-EpCAM or CT26-EpCAM models, both Ad5-BiTE and Ad5-TriTE treatments significantly inhibited tumor growth, with the Ad5-TriTE group exhibiting the most significant tumor-suppressing effect. Survival analysis indicated that Ad5-TriTE treatment significantly prolonged the survival of tumor-bearing mice, showing superior efficacy compared to the Ad5-BiTE group. There was no significant difference in body weight between the virus treatment groups and the PBS control group, indicating good safety for intratumoral injection. In conclusion, Ad5-TriTE effectively inhibits tumor growth and prolongs survival without causing significant systemic toxicity, demonstrating good therapeutic potential.
[0089] Example 5: Effects of Ad5-TriTE on Immune Cell Infiltration in the Tumor Microenvironment
[0090] C57BL / 6 mice were subcutaneously inoculated with 1×10 5 MC38-EpCAM cells were inoculated. After tumor formation was achieved 7 days later, mice were randomly divided into 4 groups, and injected intratumorally with PBS, 5×10⁶ MC38-EpCAM cells ... and 5×10⁶ MC38-EpCAM cells, respectively. 8 PFU (Ad5-Control, Ad5-BiTE, or Ad5-TriTE) was injected every 2 days for a total of 3 injections. After the last treatment, tumor tissue was excised and a single-cell suspension was prepared. Red blood cells were removed using erythrocyte lysis buffer. After cell counting, Fc receptors were blocked with anti-mouse CD16 / CD32 antibodies, followed by staining with a multicolor fluorescent antibody combination at room temperature for 30 minutes. This combination included: APC-Cy7-labeled anti-mouse CD45 antibody (clone 30-F11), BV421-labeled anti-mouse CD3 antibody (clone 145-2C11), FITC-labeled anti-mouse CD4 antibody (clone GK1.5), and APC-labeled anti-mouse CD8 antibody (clone 53-6.7). Finally, the infiltration ratios of various immune cells in the tumor microenvironment were detected by flow cytometry. Results are shown below. Figure 8 .
[0091] Figure 8 The results showed that Ad5-TriTE significantly promoted the infiltration of various immune cells into tumor tissue. Compared with the PBS control group, the Ad5-TriTE treatment group showed significantly higher levels of total lymphocytes and CD45+ in tumor tissue. + White blood cells, CD3 + T cells, CD4 + T cells and CD8 + The proportion of T cell infiltration was significantly increased in all cases (p < 0.0001). Notably, Ad5-TriTE was significantly more effective than the Ad5-Control and Ad5-BiTE groups in increasing the infiltration levels of all the aforementioned cells, especially CD8. +The recruitment effect of cytotoxic T cells was particularly prominent (p < 0.001 vs Ad5-Control or Ad5-BiTE).
[0092] Example 6: The therapeutic effect of human Ad5-hTriTE in a humanized mouse model
[0093] First, the human colorectal cancer cell line HCT116, which highly expresses EpCAM, was selected as the target cell line, and its EpCAM expression level was verified by flow cytometry (see [link to relevant documentation]). Figure 9 A). Inject 1×10 into the tail vein of NCG mice. 6 Human peripheral blood mononuclear cells (PBMCs) were analyzed by flow cytometry after 7 days to detect human CD45 in peripheral blood. + Cell ratio confirms successful implantation into the human immune system (see [reference]). Figure 9 B). Subsequently, humanized NCG mice were subcutaneously inoculated with 5 × 10⁶ cells / mL. 6 HCT116 cells were used until the tumor volume reached approximately 100 mm. 3 Subsequently, tumor-bearing mice were randomly divided into groups and received intratumoral injections of PBS, Ad5-Control, Ad5-hBiTE, or Ad5-hTriTE at a dose of 5 × 10⁻⁶. 8 PFU / dose, once every 3 days, for a total of 3 doses (see below) Figure 9 C). Tumor volume was measured regularly and mouse weight was monitored during treatment.
[0094] The results show (see Figure 9 (D) Compared with the PBS group and the Ad5-Control group, both Ad5-hBiTE and Ad5-hTriTE significantly inhibited the growth of HCT116 tumors. Among them, the Ad5-hTriTE treatment group showed the most significant tumor growth inhibition and the smallest tumor volume, indicating that it mediated the strongest anti-tumor immune response in this model. Throughout the observation period, the body weight of mice in each treatment group remained stable without significant decrease (see [reference]). Figure 9 E), indicating that intratumoral injection of Ad5-hTriTE has good safety.
[0095] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.
Claims
1. A recombinant oncolytic adenovirus, characterized in that, The recombinant oncolytic adenovirus comprises a nucleic acid molecule encoding a trispecific T-cell engager in the genome of the recombinant oncolytic adenovirus; the trispecific T-cell engager comprises three functional domains, which are a first binding domain specifically binding to the surface CD3 epsilon of a T cell, a second binding domain specifically binding to a tumor-associated antigen EpCAM, and a costimulatory domain specifically binding to and activating a 4-1BB costimulatory signaling pathway; The trispecific T-cell engager comprises, from N terminus to C terminus, a single-chain antibody specifically binding to CD3 epsilon, an extracellular region of a 4-1BB ligand or a functional fragment thereof activating the 4-1BB signaling pathway, and a single-chain antibody specifically binding to an epithelial cell adhesion molecule; the amino acid sequence of the trispecific T-cell engager is shown in SEQ ID NO: 1 or SEQ ID NO:
3.
2. The recombinant oncolytic adenovirus according to claim 1, characterized in that, The single-chain antibody specifically binding to CD3 epsilon is connected to the extracellular region of the 4-1BB ligand or the functional fragment thereof activating the 4-1BB signaling pathway through a first connecting peptide; the extracellular region of the 4-1BB ligand or the functional fragment thereof activating the 4-1BB signaling pathway is connected to the single-chain antibody specifically binding to the epithelial cell adhesion molecule through a second connecting peptide; the amino acid sequence of the first connecting peptide is GGGGS, and the amino acid sequence of the second connecting peptide is EASGGPE.
3. The recombinant oncolytic adenovirus of claim 1, wherein, The nucleotide sequence encoding the amino acid sequence SEQ ID NO: 1 is shown in SEQ ID NO: 2; the nucleotide sequence encoding the amino acid sequence SEQ ID NO: 3 is shown in SEQ ID NO:
4.
4. The recombinant oncolytic adenovirus of claim 1, wherein, The expression of the trispecific T-cell engager is regulated by a viral promoter or a constitutive strong promoter.
5. The recombinant oncolytic adenovirus according to claim 4, characterized in that, The expression of the trispecific T-cell engager is regulated by an EF1 alpha promoter.
6. The recombinant oncolytic adenovirus of claim 1, wherein, The recombinant oncolytic adenovirus takes type 5 adenovirus as a carrier, and the E1B region and the E3 region genes are deleted, and the expression of the E1A gene is regulated by a cytomegalovirus promoter.
7. The method of producing a recombinant oncolytic adenovirus according to claim 1, wherein, The method mainly comprises the following steps: 1) Gene synthesis and cloning: artificially synthesizing a gene fragment encoding the trispecific T-cell engager, and subcloning the gene fragment into an adenovirus shuttle plasmid to obtain a recombinant shuttle plasmid; 2) Virus genome recombination: performing homologous recombination or site-specific recombination reaction on the recombinant shuttle plasmid and an adenovirus backbone plasmid to obtain a recombinant adenovirus genome plasmid; 3) Virus rescue and amplification: linearizing the recombinant adenovirus genome plasmid, transfecting into packaging cells, performing virus rescue through cell culture, and finally obtaining the recombinant oncolytic adenovirus.
8. Use of the recombinant oncolytic adenovirus of any one of claims 1-7 in the preparation of a drug for treating or preventing EpCAM-positive tumors.