A rAAV vector targeting a tumor containing a bispecific t cell engager encoding gene and uses thereof

Abstract

The application discloses a recombinant adeno-associated virus (rAAV) containing a nucleic acid sequence encoding a bispecific T cell engager (BiTE), and discloses construction, preparation and application of the rAAV vector in tumor treatment. The application firstly attempts to introduce the BiTE gene into the AAV, delivers the BiTE gene to the body through the rAAV vector, and continuously synthesizes the BiTE by the cell after rAAV infection, so that the problem of short half-life of the BiTE is fundamentally solved, and the purpose of continuously killing tumors can be achieved by single administration.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the construction and preparation of a recombinant adeno-associated virus expression vector and its application in tumor treatment. Background Technology

[0002] Adeno-associated virus (AAV) vectors have been widely used in gene therapy. Recombinant adeno-associated virus (rAAV) binds to receptors on the surface of target cell membranes, triggering an internalization effect. After entering the cell nucleus, the AAV particle disintegrates, releasing a single-stranded DNA genome. This genome replicates into a double strand within the nucleus, initiating the expression of the target gene, and persists long-term within the target cell. AAV gene therapy can be used for gene replacement (delivering genes expressing normal proteins to compensate for loss-of-function genetic mutations, such as hemophilia and Parkinson's disease), gene silencing (delivering treatments for acquired gene mutations, such as Huntington's disease), delivery of novel genes (using muscle-expressed therapeutic antibodies to treat HIV), gene editing, and other gene therapies. The scientific community currently agrees that AAV does not cause any human disease, and AAV vectors offer advantages such as non-integration, highly efficient long-term gene expression, ease of gene manipulation, and low immune response. Therefore, rAAV has become the primary vector platform for in vivo gene therapy delivery.

[0003] Bispecific T cell engagers (BiTEs) are bispecific antibodies, approximately 50-60 kDa in size, consisting of a flexible linker and two antigen-specific single-chain variable fragments (scFvs). One scFv targets tumor-associated surface antigens, while the other targets CD3 on the surface of T cells. When BiTE molecules bind to both T cells and tumor cells simultaneously, T cells are activated, promoting the direct secretion of perforin and granzymes by CD8+ T cells. CD4+ T cells secrete cytokines to further recruit and activate cytotoxic T cells, thereby killing tumor cells.

[0004] Because BiTE does not contain the Fc domain of traditional antibodies, it has a simple structure, good tissue penetration, and significantly reduced immunogenicity. Currently, various BiTEs targeting different tumor antigens have been reported to have high anti-tumor activity, such as CD19, CD33, EpCAM, CEA, PSMA, and BCMA.

[0005] However, because BiTE does not contain an Fc structure, its half-life is extremely short, generally only 2-4 hours, requiring frequent dosing in clinical practice. For example, Amgen's Blincyto, which contains tandem anti-CD19 scFv and anti-CD3 scFv, is one of the antibody drugs with the highest dosing frequency. One course of treatment requires continuous intravenous infusion every day for 4 weeks, with a maximum of 5 courses. The cost of the drug is about $180,000 per year, and patient compliance is poor.

[0006] To address the short-acting drawback of BiTE, current technologies are limited to improving antibody molecular structure by fusing Fc fragments, designing IgG-like structures, or fusing HAS antibodies to increase serum half-life. For example, Amgen developed a half-life-extending BiTE technology (HLE-BiTE) based on BiTE technology, fusing BiTE with an Fc fragment. However, this approach carries potential problems, including poor tissue penetration due to high molecular weight, complex process control and quality analysis costs due to antibody glycosylation, alterations in molecular structure leading to changes in antibody affinity for target antigens, and the introduction of fusion proteins increasing complex biological effects and affecting activity. Therefore, it is difficult to simultaneously leverage the advantages of BiTE while increasing its half-life. Furthermore, industrial antibody production commonly uses mammalian cells such as Chinese hamster ovary cells and HEK293 cells, with typical fermentation yields reaching 300-1000 mg / L. The high clinical dosage of antibodies places high demands on antibody production.

[0007] Although both BiTE and AAV technologies have wide applications, no one has yet attempted to introduce the BiTE gene into AAV for gene therapy. It remains unknown whether AAV carrying BiTE can be successfully constructed and effectively expressed. Therefore, the applicant conducted the research work for this invention. Summary of the Invention

[0008] This invention is the first attempt to introduce the bispecific T cell engager (BiTE) encoding gene into adeno-associated virus (AAV), and deliver the BiTE gene into the body through a recombinant adeno-associated virus (rAAV) vector. By utilizing the continuous synthesis of BiTE by cells infected with rAAV, the problem of the short half-life of BiTE is fundamentally solved, thereby achieving the purpose of continuous tumor killing.

[0009] This invention provides a recombinant adeno-associated virus (rAAV) vector containing a nucleic acid sequence encoding a bispecific T-cell conjugate.

[0010] Studies have reported on various BiTEs targeting different tumor antigens, such as BiTEs targeting CD19, CD33, EpCAM, CEA, PSMA, and BCMA. In a specific embodiment of this invention, the feasibility of introducing BiTE nucleic acid sequences targeting Her2 and CD26 into AAVs was demonstrated. Based on this, those skilled in the art can reasonably anticipate that introducing BiTEs targeting other than Her2 and CD26 into AAVs can also achieve sustained tumor killing, thus solving the problem of the short half-life of BiTEs.

[0011] The adeno-associated virus (aAV) can be targeted to specific organs based on its bispecific T-cell binder. The key to rAAV therapeutic delivery efficiency lies in the affinity between the AAV capsid and target cells, which is related to different AAV serotypes, capsid types, and target cell receptor proteins. For example, AAV1 has high affinity for muscle, liver, and the nervous system; AAV2 has broad cell affinity and the widest range of research applications; AAV5 has high affinity for the liver, retina, and nervous system; and AAV6 has tissue affinity for immune cells, lungs, heart, and muscle. Therefore, selecting an appropriate highly specific AAV serotype has a significant impact on efficacy when targeting different organs and indications. In a specific embodiment of this application, AAV6 serotype was used to match BiTE targeting Her2, CD26-positive tumors, achieving the expected results. For different targeted tumor sites, a suitable tissue-affinity AAV serotype can be selected from different AAV serotypes.

[0012] When the bispecific T cell binder is CD26-CD3 or Her2-CD3, and the AAV6 serotype is selected, the gene linking sequence in the rAAV vector is as follows: 5' inverted repeat (ITR), CMV promoter, β intron enhancer sequence, signal peptide sequence, bispecific T cell binder gene sequence, polyA termination sequence, and 3' inverted repeat (ITR).

[0013] When BiTE is Her2-CD3 BiTE, the BiTE gene sequence is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2. When BiTE is CD26-CD3 BiTE, the BiTE gene sequence is shown in SEQ ID NO: 3, and the encoded amino acid sequence is shown in SEQ ID NO: 4.

[0014] The 5' inverted repeat sequence is shown in SEQ ID NO: 5, the CMV promoter is shown in SEQ ID NO: 6, the β intron enhancer sequence is shown in SEQ ID NO: 7, the polyA terminator sequence is shown in SEQ ID NO: 8, and the 3' inverted repeat sequence is shown in SEQ ID NO: 9. The signal peptide gene sequence is shown in SEQ ID NO: 14, and the amino acid sequence is shown in SEQ ID NO: 15.

[0015] The present invention also provides host cells comprising the above-described rAAV vector. Host cells may include 293T cells, 293 cells, etc.

[0016] The present invention also provides a composition comprising the above-described rAAV carrier.

[0017] This invention also provides a method for preparing the above-mentioned rAAV vector, comprising:

[0018] 1. Construct an rAAV expression vector containing a nucleic acid sequence encoding a bispecific T cell binder;

[0019] 2. Co-transfect the plasmid constructed in step 1 into the packaging cells; the transfection method adopts conventional methods in the art, such as liposome transfection, electroporation, microinjection, calcium phosphate precipitation, etc.

[0020] 3. Harvest the cells from step 2, and extract and purify rAAV.

[0021] The present invention also provides the use of the above-mentioned rAAV carrier in the preparation of tumor therapeutic drugs.

[0022] This invention is the first to attempt to introduce the bispecific T-cell engager (BiTE) gene into adeno-associated virus (AAV), and deliver the BiTE gene into the body via a recombinant adeno-associated virus (rAAV) vector. The resulting rAAV-infected cells continuously synthesize BiTE. This not only maintains the original structure and advantages of BiTE, but also enables continuous expression and maintenance of a certain drug concentration using the host cell as a cell factory, achieving long-lasting BiTE efficacy. This fundamentally solves the problem of BiTE's short half-life, achieving the goal of sustained tumor killing. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the AAV expression vector backbone of this application, including the plasmid replication origin pUC ori, the Amp resistance gene, the adeno-associated virus 5' inverted repeat sequence (ITR), the CMV promoter, the β intron enhancement sequence, the multiple cloning site, the polyA termination sequence, and the 3' inverted repeat sequence (ITR).

[0024] Figure 2This is a schematic diagram of an AAV expression cassette used for gene delivery, in which the target gene is inserted. Figure 1 The multiple cloning sites in the expression vector backbone are the GFP gene, the 21R21 gene, and the 21R23 gene.

[0025] Figure 3 This is a silver-stained SDS-PAGE gel image of the purified AAV sample. M represents the protein marker. Lane 1 is the GFP AAV sample, lane 2 is the 21R21 AAV sample, and lane 3 is the 21R23 AAV sample. The loading volume is 5-10 × 10e9 vg. Three bands of recombinant AAV capsid proteins VP1, VP2, and VP3 (87 KDal, 72 KDal, and 62 KDal) are visible, and the ratio of the three is approximately 1:1:10 (VP1:VP2:VP3).

[0026] Figure 4 Five 293T cells were infected with 1×10e10vg of AAV sample, and GFP expression was detected by fluorescence microscopy after 3 days.

[0027] Figure 5 This study used flow cytometry to detect the OSRC2 labeling activity in the supernatant of AAV-infected 293T cells.

[0028] Figure 6 This study detected the expression levels of CD26 and Her2 in 786-0-GFP and OSRC-2-GFP stable cells.

[0029] Figure 7 The image shows the growth status of tumor cells under a microscope 48 hours after AAV administration to wells co-cultured 786-0-GFP cells and PBMCs. The arrow indicates the PBMC cell cluster.

[0030] Figure 8 The image shows the growth status of tumor cells under a microscope after 96 hours of AAV administration in wells co-cultured with OSRC-2-GFP cells and PBMCs. The arrow indicates the PBMC cell cluster.

[0031] Figure 9 This refers to the tumor volume changes in NOD / SCID mice carrying OSRC-2 (renal carcinoma) xenografts after AAV delivery of the BiTE gene.

[0032] Figure 10 This refers to the change in body weight in NOD / SCID mice carrying OSRC-2 (renal carcinoma) xenografts after AAV delivery of the BiTE gene.

[0033] Figure 11 This describes the changes in mouse survival in NOD / SCID mice carrying OSRC-2 (renal carcinoma) xenografts after AAV delivery of the BiTE gene.

[0034] Figure 12 The method involves using ELISA to detect changes in the OD value of CD26-CD3 BiTE molecules in mouse muscle tissue.

[0035] Figure 13 The method used is ELISA to detect changes in the OD value of Her2-CD3 BiTE molecules in mouse muscle tissue. Detailed Implementation

[0036] Example 1: Construction of AAV expression vector

[0037] Construction of pAAV-GFP expression vector:

[0038] After amplification of the GFP gene sequence (SEQ ID NO: 10), the multiple cloning site MCS (between the XbaI and BamHI restriction sites) between the "β intron enhancement sequence" and the "polyA termination sequence" of the pAAV-CMV vector was constructed to form the pAAV-GFP expression vector.

[0039] Construction of pAAV-BiTE expression vector:

[0040] When BiTE is Her2-CD3 BiTE, the BiTE gene sequence is shown in SEQ ID NO: 1, and the encoded amino acid sequence is shown in SEQ ID NO: 2. When BiTE is CD26-CD3 BiTE, the BiTE gene sequence is shown in SEQ ID NO: 3, and the encoded amino acid sequence is shown in SEQ ID NO: 4.

[0041] The signal peptide gene SEQ ID NO: 14 was fused with the two BiTE gene sequences mentioned above and amplified to construct the multiple cloning site MCS (between BamHI and EcoRI restriction sites) between the β intron enhancement sequence and the polyA termination sequence of the pAAV-CMV vector, forming AAV expression vectors: pAAV-21R21 (containing Her2-CD3 BiTE) and pAAV-21R23 (containing CD26-CD3 BiTE).

[0042] Example 2: Production and purification of recombinant AAV virus

[0043] 293T cell culture: After resuscitation, 293T cells are cultured adherently in DMEM medium containing 10% FBS and 1% Glutamax. After 2-3 passages, the cells are ready for packaging. 150mm cell culture dishes are coated with 15ml of 0.1% gelatin at room temperature for 30min. After trypsin digestion, 293T cells are resuspended, counted, and the coating medium is discarded. 1.2-1.8×10e7 293T cells are seeded into each 150mm dish, and 293T medium is added to a final volume of 30ml. The cells are incubated overnight at 37℃ with 5% CO2.

[0044] Transfection: On the morning of the second day, when 293T cells reached 70-80% confluence, the medium was replaced with fresh 293T medium (30 ml per dish), and the dishes were returned to the incubator. The transfection mixture was prepared by adding the three constructed AAV expression vector plasmids, pRC6 and pHelper plasmids respectively, to serum-free DMEM medium at a mass ratio of 1:1:1, with a total DNA volume of 30 μg per 15 cm dish. Lipo8000 liposomes (Beyotime Biotechnology, catalog number: C0533) were then gently added and mixed thoroughly. The mixture was incubated at room temperature for 30 min. 1.5 ml of the transfection mixture was added to each dish, gently mixed, and the dishes were returned to the incubator. 24 h after transfection, the medium was replaced with 30 ml of AAV harvest medium (DMEM medium containing 2% FBS, 1% Glutamax, and 1% 1M HEPES), and the dishes were returned to the incubator.

[0045] rAAV collection and purification: 72 hours after infection, add 1 / 80 volume of 0.5M EDTA (pH 8.0) to a culture dish containing 293T cells. Resuspend the cells by pipetting and collect them into a 50ml sterile centrifuge tube. Centrifuge at 2000g for 10 minutes at 4℃, discard the supernatant, and collect the cell pellet after completely removing the supernatant. This pellet contains host 293T cells containing AAV particles. Subsequent steps follow... Purification Kit Maxi (TAKARA catalog number: 6666) is used to purify and concentrate AAV according to the instructions. After aliquoting, store at -80°C.

[0046] Example 3: In vitro expression and activity detection of BiTE gene delivered by recombinant AAV

[0047] Step 1: The purified recombinant AAV sample was subjected to qPCR quantitative titer detection using primer pairs 5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO: 11) and 5'-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO: 12). The detection results indicate that the AAV particles containing the BiTE gene were successfully packaged, with a high titer comparable to that of the GFP control virus (see Table 1).

[0048] Table 1

[0049] GFP 1.69x10e12 21R21 1.03x10e12 21R23 2.87x10e12

[0050] The purity of the AAV purified sample was detected by SDS-PAG electrophoresis. Figure 3 After purification, the three capsid proteins VP1, VP2, and VP3 of AAV were clearly expressed in a ratio close to 1:1:10, indicating that the three capsid proteins of AAV were expressed in a normal proportion, the assembly was correct, there were few impurities, and the purity was high. Furthermore, the theoretical BiTE protein molecule size (approximately 55 kDa) could not be observed in the 21R21 and 21R23 viral lanes, indicating that the purification process essentially removed BiTE molecules that might have been expressed during the packaging process.

[0051] Step 2: 293T cells were digested and passaged into 24-well plates, with 5 × 10⁵ cells per well, and incubated at 37°C for 24 hours. The next day, fresh medium was added to each well, and 1 × 10⁵ vg of recombinant AAV sample was added for infection. Cells were incubated at 37°C. Three days after infection, fluorescence microscopy revealed that the 293T cells infected with the control AAV virus emitted significant GFP fluorescence with a high positive rate, indicating high infectivity of AAV. Cells 21R21 and 21R23 do not contain the GFP gene and therefore showed no fluorescence. Figure 4 The medium for 293T cells was changed, and the supernatant was recovered 24 hours later.

[0052] Step 3: Screening of target cells

[0053] The expression levels of CD26 and Her2 on the surface of renal cell carcinoma cells OSRC-2 and 786-0 were detected using CD26 or Her2 monoclonal antibodies. The results are shown in Table 2. Both cell lines were found to be highly expressive of CD26 and Her2. Therefore, these two cell lines were selected as target cells.

[0054] Table 2

[0055] OSRC-2 Kidney cancer 97.8％ 84.5％ 786-0 Kidney cancer 99.9％ 86.8％

[0056] Step 4: After trypsin digestion of OSRC-2 cells, resuspend them in 1640 mL of medium, centrifuge at 2500 rpm for 5 min, discard the medium, and resuspend the cells in pre-chilled PBS buffer. Repeat the centrifugation and PBS washing steps once to remove residual medium. Resuspend the cells in PBS until the density reaches 1 × 10e6 cells / mL. Aliquot 100 μL of OSRC-2 cells into 8 centrifuge tubes, labeling them 1-8.

[0057] Add 10 μl of the blank culture supernatant from step 2 to cell number 3; add 10 μl of the culture supernatant from the GFPAAV infection post-infection culture in step 2 to cell number 4; add 200 ng of Herceptin monoclonal antibody to cell number 5; add 10 μl of the culture supernatant from the 21R21AAV infection post-infection culture in step 2 to cell number 6; add 200 ng of 19G294BiTE antibody (SEQ ID NO: 13) to cell number 7; add 10 μl of the culture supernatant from the 21R23AAV infection post-infection culture in step 8. Mix well and incubate at 4°C for 30 min. After incubation, centrifuge and discard the supernatant. Wash the cell pellet twice with PBS to remove unbound antibody molecules.

[0058] Cells numbered 2-8 were treated with fluorescein-labeled goat anti-human IgG (H+L)-Alexa647 (Jackson ImmunoResearch catalog number 109-606-003) as a secondary antibody. After mixing, the cells were incubated with the target cells at 4°C in the dark for 30 min. After incubation, the cells were washed three times with PBS, centrifuged, and the supernatant was discarded. The cell pellet was collected and resuspended in 100 μl of PBS. The average fluorescence intensity of the supernatant and antibody molecules labeled with the target cells was analyzed by flow cytometry.

[0059] Identified by flow cytometry, Figure 5 The results showed that the supernatant from the blank culture medium and the supernatant from the GFP AAV infection group (numbered 3 and 4) had no labeling activity against OSRC-2. The positive controls Herceptin and 19G294 BiTE (numbered 5 and 7) both bound to OSRC-2 cells. The supernatant from the 21R21 and 21R23 AAV infection groups (numbered 6 and 8) also showed binding activity against OSRC-2, and the flow cytometry fluorescence intensity was close to that of the positive controls. These data demonstrate that the bispecific antibody recombinant AAV vector constructed in Example 1 has the activity of delivering and inducing the expression of bispecific antibody genes, and that the expressed bispecific antibody has the function of recognizing the target antigen.

[0060] Example 4: Detection of in vitro antitumor activity of recombinant AAV delivering the BiTE gene

[0061] Step 1: Use lentivirus containing the GFP gene to infect tumor cells to construct GFP-stable tumor cells, obtaining OSRC-2-GFP and 786-0-GFP cells. Use CD26 or Her2 monoclonal antibodies to detect the GFP-stable tumor cells. It was found that it did not affect the expression levels of CD26 and Her2, and the GFP fluorescence was easy to observe. Therefore, GFP-carrying tumor cells were used to detect the in vitro antitumor activity. Figure 6The test results showed that the GFP positivity rate of 786-0-GFP cells reached 99.0%, the CD26 positivity rate reached 98.0%, and the Her2 positivity rate reached 87.8%. The GFP positivity rate of OSRC-2-GFP cells reached 94.2%, the CD26 positivity rate reached 98.9%, and the Her2 positivity rate reached 81.8%.

[0062] Step 2: Renal cell carcinoma cells 786-0-GFP and OSRC-2-GFP were digested separately, centrifuged, resuspended, counted, and adjusted to 3×10e5 cells / ml using 1640 medium containing 10% FBS. 100 μl was seeded into each well of a 96-well plate and incubated at 37°C for 2 hours to allow tumor cells to adhere. After tumor cell adhesion, PBMC cells were collected and centrifuged. Cells were resuspended in fresh 1640 medium, counted, and adjusted to 3×10e6 cells / ml. The first well contained only tumor cells without PBMCs. 50 μl of PBMCs was added to each of the second to fifth wells to achieve an effector-to-target ratio (E / T) of 5 / 1. Add 50 μl of culture medium to the second well, add 1×10evg of AAV-GFP sample to the third well, add 1×10evg of AAV-21R21 sample to the fourth well, and add 1×10evg of AAV-21R23 sample to the fifth well, and then add culture medium to each well to a total volume of 200 μl. Each group has two replicates. Incubate at 37℃ for 2-4 days, and observe the status of GFP-positive tumor cells under a fluorescence microscope. It was found that after 48 h, AAV-21R21 and AAV-21R23 significantly mediated the killing of 786-0-GFP by PBMCs. PBMC activation and aggregation were observed under bright field (BF) light, and a significant reduction in GFP-positive cells was observed under GFP field light. Figure 7 After 96 hours, AAV-mediated PBMC killing of OSRC-2 was observed, and PBMC activation and aggregation were observed, with a significant decrease in GFP-positive cells. Figure 8 ).

[0063] Existing studies on the mechanism of action of BiTE bispecific antibodies have shown that after BiTE mediates T cell recognition of tumors, it can stimulate T cell activation and rapidly promote T cell adhesion, aggregation and expansion on the surface of tumor cells. Therefore, T cells can be observed to wrap around tumor cells to form clusters.

[0064] The above data demonstrate that the bispecific antibody recombinant AAV vector constructed in Example 1 has the ability to deliver bispecific antibody genes and induce their expression, and that the bispecific antibody expressed in vitro has the function of mediating PBMC killing tumor cells.

[0065] Example 5: Recombinant AAV delivery of BiTE gene and the efficacy of BiTE in NOD / SCID mice bearing OSRC-2 (renal carcinoma) xenografts.

[0066] Experiment 1: Efficacy of recombinant AAV delivery of bispecific antibody gene and bispecific antibody in NOD / SCID mice bearing OSRC-2 (renal carcinoma) xenograft tumors.

[0067] Methods: NOD-SCID mice weighing 18–22 g and approximately 5–7 weeks old were selected. OSRC-2 cell suspension (4 × 10e7 cells / ml) and PBMC cell suspension (6 × 10e7 cells / ml) were thoroughly mixed at a 1:1 (volume ratio). GFP, 21R21, and 21R23 AAV purified samples were diluted to 1 × 10e11 vg / ml. 0.1 ml of OSRC-2 + PBMC cell suspension was mixed with either PBS or 0.1 ml of AAV sample from the treatment group and subcutaneously injected into each NOD-SCID mouse (0.2 ml per mouse). The groups were: Model group, Group A (GFP group), Group B (21R21 group), and Group D (21R23 group), with 6 animals in each group. Tumor volume and body weight were measured starting on day 5 of modeling, and the survival status of the mice was observed.

[0068] Conclusion: This experiment established a mouse xenograft model of human renal cell carcinoma by subcutaneous injection of a mixture of OSRC-2 and PBMC cells into NOD-SCID mice. Based on this model, each mouse was treated once with 10e10vg of recombinant AAV expressing a bispecific antibody against BiTE. From day 8 of modeling, tumors in the model group and the GFP-A group began to increase significantly. Treatment with AAV significantly inhibited tumor growth in groups B and D, and by day 12, tumors in groups B and D had completely regressed. The results are as follows: Figure 9 As shown in Table 3.

[0069] Table 3

[0070]

[0071] Furthermore, the OSRC-2 human renal cell carcinoma model resulted in animal death during the trial, while animals in the AAV treatment group did not experience weight loss and tolerated the treatment well. Figure 10 , 11 As shown.

[0072] Experiment 2: Efficacy of BiTE in NOD / SCID mice bearing OSRC-2 (renal carcinoma) xenografts

[0073] Methods: NOD-SCID mice weighing 18–22g and approximately 5–7 weeks old were selected, and 3 × 10⁻⁶ mice were used. 6 cells / 0.1ml OS-RC-2 cell suspension and 6×10 6PBMC cell suspension was thoroughly mixed at a 1:1 (volume ratio) and subcutaneously injected into the right side of NOD-SCID mice, with 0.2 ml injected into each mouse. Animals were randomly divided into two groups according to body weight: the Model group and the 19G294 group, with 5 animals in each group. Drug administration began on day 1, with a frequency of once daily for 5 consecutive days per week, followed by a 2-day drug-free period.

[0074] Conclusion: The test sample 19G294 significantly inhibited tumor growth in human renal cell carcinoma model mice at a dose of 30 μg / mouse, as shown in Table 4.

[0075] Table 4

[0076]

[0077] The results of the two experiments show that the BiTE bispecific antibody delivered by recombinant AAV can completely inhibit human renal cell carcinoma model with only one dose, which significantly reduces the number of doses compared to the traditional daily BiTE administration method, and has significant efficacy and good safety.

[0078] Example 6: Detection of BiTE gene and bispecific antibody expression in NOD / SCID mice bearing OSRC-2 (renal carcinoma) xenografts via recombinant AAV delivery

[0079] Step 1: Following the animal experiments of Experiment 1 in Example 5, all animals were euthanized using CO2 and dissected. 20-30 mg of subcutaneous muscle from the right side (tumor-inoculated) and the left side (non-tumor-inoculated) from each mouse was harvested and homogenized using a glass homogenizer. 300 μl of RIPA lysis buffer (Beyotime P0013C) was added to each sample, and the mixture was transferred to centrifuge tubes and centrifuged at 12000 rpm, 4°C for 10 min. Approximately 200 μl of the supernatant was collected into a new centrifuge tube. The left subcutaneous muscle sample from the two animals in the mixed model group was designated Control-L, and the right subcutaneous muscle sample was designated Control-R; ​​the left subcutaneous muscle sample from the two animals in Mixed Group A was designated A-LM, and the right subcutaneous muscle sample was designated A-RM; the left subcutaneous muscle sample from the three animals in Mixed Group B was designated B-LM, and the right subcutaneous muscle sample was designated B-RM; the left subcutaneous muscle sample from the three animals in Mixed Group D was designated D-LM, and the right subcutaneous muscle sample was designated D-RM.

[0080] Step 2: Dilute CD26 protein to 5 μg / ml, coat 96-well microplates with 100 μl / well, and incubate overnight at 4°C. After coating, wash the plate with 1×PBST and block with 2% (w / v) BSA PBST (2 h, 37°C). Muscle tissue protein extraction samples from the model groups Control-LM, Control-RM, Group A (A-LM, A-RM), and Group D (D-LM, D-RM) were diluted 2-fold, 6-fold, and 18-fold with 2% (w / v) BSA PBST. Add 100 μl to each well of the blocked microplate (1 h, 37°C). Wash the plate 3 times and agitate dry. Dilute HRP-labeled CD3 protein (1 mg / ml) 1:8000 and add 100 μl to each well of the microplate (1 h, 37°C). Wash the plate 4 times and agitate dry. Add 100 μl of single-component TMB chromogenic solution I to each well of the microplate (15 min, 37℃). Stop the reaction by adding 50 μl of 2M H₂SO₄ to each well. Measure A450 and A620 using a microplate reader, calculate (sample value - Control) / Control, and analyze the changes in OD values.

[0081] Her 2 protein was diluted to 2 μg / ml, and 100 μl / well was used to coat a 96-well microplate. The plate was incubated overnight at 4°C. The detection method was the same as above, detecting A-LM and A-RM samples from group A, and B-LM and B-RM samples from group B.

[0082] Results: The test revealed the following... Figure 12 The OD signal value of the right muscle tissue in group D mice treated with 21R23 AAV was higher than that in the left muscle, the model group, and group A, indicating that significant BiTE molecular signal was detected in the right muscle of group D, proving the sustained expression of 21R23 AAV in vivo. Figure 13 The OD signal in the right muscle tissue of mice treated with 21R21 AAV was higher than that in the left muscle of group B and the left and right sides of group A, which also indicates the sustained expression of 21R21 AAV in vivo in group B.

Claims

1. A recombinant adeno-associated virus (rAAV) vector containing a nucleic acid sequence encoding a bispecific T-cell engager (BiTE), characterized in that, The BiTE is CD26-CD3 BiTE or Her2-CD3 BiTE, the adeno-associated virus is AAV6 serotype, and the gene linking sequence in the rAAV vector is: 5' inverted repeat (ITR), CMV promoter, β intron enhancer sequence, signal peptide sequence, BiTE gene sequence, polyA termination sequence, and 3' inverted repeat (ITR).

2. The recombinant adeno-associated virus (rAAV) vector of claim 1, wherein BiTE is Her2-CD3 BiTE, and the amino acid sequence encoded by the BiTE gene sequence is shown in SEQ ID NO:

2.

3. The recombinant adeno-associated virus (rAAV) vector of claim 1, wherein BiTE is CD26-CD3 BiTE, and the amino acid sequence encoded by the BiTE gene sequence is shown in SEQ ID NO:

4.

4. The recombinant adeno-associated virus (rAAV) vector of claim 1, wherein the 5' terminal inverted repeat sequence is shown in SEQ ID NO: 5, the CMV promoter is shown in SEQ ID NO: 6, the β intron enhancement sequence is shown in SEQ ID NO: 7, the polyA termination sequence is shown in SEQ ID NO: 8, and the 3' terminal inverted repeat sequence is shown in SEQ ID NO:

9.

5. A host cell comprising the rAAV vector according to any one of claims 1-4.

6. A composition comprising the rAAV carrier according to any one of claims 1-4.

7. A method for preparing the rAAV vector according to any one of claims 1-4, comprising: Step 1: Construct an rAAV expression vector containing a nucleic acid sequence encoding a bispecific T cell binder; Step 2: Transfect the plasmid constructed in Step 1 into packaging cells; Step 3: Harvest the cells from Step 2, and extract and purify rAAV.

8. Use of the rAAV carrier according to any one of claims 1-4 in the preparation of a medicament for treating renal cell carcinoma.

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