Recombinant oncolytic influenza virus for delivering anti-gpA33*CD3 bispecific antibody and application

Abstract

The invention discloses a recombinant oncolytic influenza virus for delivering an anti-gpA33 * CD3 bispecific antibody and application of the recombinant oncolytic influenza virus. Belongs to the technical field of biological medicine. According to the recombinant oncolytic influenza virus provided by the invention, a heavy chain variable region and a light chain variable region of an anti-gpA33 antibody and an anti-CD3 antibody are inserted into an influenza virus genome, the recombinant oncolytic influenza virus is rescued through a reverse genetics technology, and the virus can stably express an anti-gpA33 * CD3 bispecific antibody in colorectal cancer cells; one end of the antibody can be specifically combined with CD3 molecules on the surfaces of the T cells to efficiently activate the T cells, the other end of the antibody can be specifically combined with biomarker molecules gpA33 on the surfaces of the colorectal cancer cells, and under the bridging action of the antibody, the activated T cells are collected to the surfaces of the colorectal cancer cells to induce the T cells to directly generate the effect of targeted killing of the colorectal cancer cells, so that the colorectal cancer cells are efficiently killed. Therefore, the purpose of treating tumors is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to recombinant oncolytic influenza virus delivering anti-gpA33×CD3 bispecific antibodies and its applications. Background Technology

[0002] Colorectal cancer (CRC) is a leading cause of cancer death, seriously threatening people's health and lives. Currently, the main treatments for colorectal cancer include surgery, chemotherapy, radiotherapy, and targeted therapy. Although these treatments have made significant progress, they still have problems such as significant side effects and the development of drug resistance.

[0003] Oncolytic viruses (OVs) therapy has been proven to be an effective tumor immunotherapy. Currently, four oncolytic virus preparations have been approved for marketing in China and abroad. In 2021, the oncolytic virus Teserpaturev (G47Δ, Delytact) was approved for the treatment of malignant glioma, making oncolytic virus therapy a focus of tumor immunotherapy once again. Currently, adenoviruses and herpes simplex viruses are among the most studied oncolytic viruses, but these viruses suffer from problems such as low replication efficiency and easy integration of viral DNA into the host genome.

[0004] Influenza virus is an excellent oncolytic virus vector. As an RNA virus, it lacks reverse transcriptase activity and DNA integration activity, meaning its viral genes do not integrate into the host genome. Furthermore, the N-acetylsialic acid receptor (SAR), the binding receptor for influenza virus, is widely distributed on the surface of various tumor cells, including lung cancer, colorectal cancer, and prostate cancer, allowing influenza virus to infect a wide range of tumor cells. Influenza virus exhibits strong genetic plasticity; using reverse genetics, eight gene fragments of the virus can be individually modified, a simple and easy process. Influenza virus possesses a natural oncolytic ability against colorectal cancer cells, making it the preferred oncolytic virus vector for targeted therapy of colorectal cancer. The surface of colorectal cancer cells not only contains a large number of SARs, but colorectal cancer cells also secrete cysteine ​​protease cathepsin B or L, which helps the influenza virus hemagglutinin (HA) undergo hydrolysis and conformational changes, promoting the recognition and binding of viral HA to SARs on the surface of colorectal cancer cells, thereby increasing the viral infection and replication rates.

[0005] Bispecific antibodies are a class of non-natural antibodies containing two Fab fragments that can simultaneously bind to two different antigenic sites. Bispecific antibodies used in tumor therapy research can specifically bind to TAAs on the surface of tumor cells at one end and to trigger molecules on immune effector cells at the other, acting as a bridge between the two ends. This recruits activated effector cells to the surface of tumor cells, achieving targeted killing of tumor cells. Given that bispecific antibodies can recruit, activate, and mediate T cell-mediated, highly efficient tumor-targeting killing effects, the continuous expression and delivery of bispecific antibodies via influenza virus would significantly improve the oncolytic efficiency of the influenza virus.

[0006] In summary, how to provide a recombinant oncolytic influenza virus expressing bispecific antibodies for the treatment of colorectal cancer is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] The technical problem to be solved by this invention is how to improve the efficacy of immunotherapy for tumors, especially for the treatment of colorectal cancer.

[0008] To address the aforementioned technical problems, this invention provides a recombinant oncolytic influenza virus delivering a bispecific antibody against gpA33×CD3, named rPR8-gpA33-CD3. The recombinant oncolytic influenza virus is ① or ②: ① Influenza virus containing bispecific antibodies against gpA33×CD3; ② Influenza virus expressing bispecific antibodies against gpA33×CD3; The anti-gpA33×CD3 bispecific antibody is a protein of the following type 1), 2), or 3): 1) Contains the peptide segment whose amino acid sequence is shown in SEQ ID No. 1 (heavy chain variable region of anti-gpA33 antibody, VH). gpA33 ), encoded by nucleotides 2410-2760 as shown in SEQ ID No. 5; containing the amino acid sequence of the peptide segment shown in SEQ ID No. 2 (the light chain variable region of the anti-gpA33 antibody, VL gpA33 ), encoded by nucleotides 2287-2610 as shown in SEQ ID No. 6; containing the amino acid sequence of the peptide segment shown in SEQ ID No. 3 (the light chain variable region of the anti-CD3 antibody, VL CD3 ), encoded by nucleotides 2806-3123 as shown in SEQ ID No. 5; containing the amino acid sequence of the peptide segment shown in SEQ ID No. 4 (the heavy chain variable region of the anti-CD3 antibody, VH CD3 ), encoded by nucleotides 2656-3012 as shown in SEQ ID No. 6.

[0009] 2) Proteins derived from 1) having the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4; 3) Recombinant proteins obtained by linking tags and / or other genes to the C-terminus and / or N-terminus of 1) or 2).

[0010] The genome of the aforementioned recombinant oncolytic influenza virus consists of eight single-stranded, segmented negative-sense RNAs. The negative-sense RNA of the recombinant oncolytic influenza virus is transcribed into a set of complementary positive-sense RNAs, including PB2-RNA and PB1-VH. gpA33 -VL CD3 -RNA, HA-RNA, PA-VL gpA33 -VH CD3 -RNA, NP-RNA, NA-RNA, M-RNA and NS-RNA; The PB2-RNA is a positive-sense RNA encoding the PB2 gene in influenza virus strains; The PB1-VH gpA33 -VL CD3 -RNA encodes the recombinant gene PB1-VH gpA33 -VL CD3 Positive-sense RNA; The HA-RNA is a positive-sense RNA encoding the HA gene in the influenza virus strain. The PA-VL gpA33 -VH CD3 -RNA encodes the recombinant gene PA-VL gpA33 -VH CD3 Positive-sense RNA; The NP-RNA is a positive-sense RNA encoding the NP gene in the influenza virus strain; The M-RNA is a positive-sense RNA encoding the M gene in the influenza virus strain; The NA-RNA is a positive-sense RNA encoding the NA gene in the influenza virus strain; The NS-RNA is a positive-sense RNA encoding the NS gene in the influenza virus strain.

[0011] In the aforementioned recombinant oncolytic influenza virus, the complete set of positive-sense RNA may also consist solely of the PB2-RNA and the PB1-VH. gpA33 -VL CD3 -RNA, the HA-RNA, the PA-VL gpA33 -VH CD3It consists of -RNA, the NP-RNA, the M-RNA, the NA-RNA and the NS-RNA.

[0012] In the above-mentioned recombinant oncolytic influenza virus, the PB1-VH gpA33 -VL CD3 -The RNA sequence is the linker T2A gene sequence, nucleotides 2296-2346 of SEQ ID No. 5, inserted sequentially from 5' to 3' before the 3' NCR stop codon of the PB1 gene in the influenza virus strain; the Igκ signal peptide gene sequence, nucleotides 2347-2409 of SEQ ID No. 5; VH gpA33 Gene sequence, nucleotides 2410-2760 of SEQ ID No. 5; flexible linker (G4S)3 gene sequence, nucleotides 2761-2805 of SEQ ID No. 5; VL CD3 The sequence obtained is as follows: gene sequence, nucleotides 2806-3123 of SEQ ID No. 5; short peptide FNRGECC gene sequence of antibody hinge region, nucleotides 3124-3144 of SEQ ID No. 5; hydrolyzable linker P2A gene sequence, nucleotides 3145-3210 of SEQ ID No. 5; PB1 gene 3'NCR end packaging signal sequence gene, nucleotides 3211-3374 of SEQ ID No. 5, with all other nucleotides unchanged.

[0013] The recombinant gene can be a VH gene inserted into the influenza virus gene PB1. gpA33 -VL CD3 It is derived from genes and can also be inserted into other genes in the influenza virus genome. gpA33 -VL CD3 It is inherited from genes.

[0014] In the above-mentioned recombinant oncolytic influenza virus, the PA-VL gpA33 -VH CD3 -The RNA sequence is the linker T2A gene sequence, nucleotides 2173-2223 of SEQ ID No. 6, inserted sequentially from 5' to 3' before the 3' NCR stop codon of the PA gene in the influenza virus strain; the Igκ signal peptide gene sequence, nucleotides 2224-2286 of SEQ ID No. 6; VL gpA33Gene sequence, nucleotides 2287-2610 of SEQ ID No. 6; flexible linker (G4S)3 gene sequence, nucleotides 2611-2655 of SEQ ID No. 6; VH CD3 The sequence obtained was obtained by modifying the gene sequence (nucleotides 2656-3012 of SEQ ID No. 6), the antibody hinge region short peptide VEPKSCC gene sequence (nucleotides 3013-3033 of SEQ ID No. 6), the hydrolyzable linker P2A gene sequence (nucleotides 3034-3099 of SEQ ID No. 6), and the PA gene 3'NCR terminal packaging signal sequence (nucleotides 3100-3283 of SEQ ID No. 6), with all other nucleotides remaining unchanged.

[0015] The recombinant gene can be a VL gene embedded in the influenza virus gene PA. gpA33 -VH CD3 VL can be obtained through genetic modification or embedded in other genes in the influenza virus genome. gpA33 -VH CD3 It is inherited from genes.

[0016] The influenza virus strains include, but are not limited to, any subtype of influenza A or influenza B virus.

[0017] To address the aforementioned technical problems, this invention also provides a method for constructing a recombinant oncolytic influenza virus expressing a bispecific antibody against gpA33×CD3. The method comprises transporting a recombinant vector pHW2000-PB2 containing a DNA molecule encoding the PB2-RNA, and a DNA molecule encoding the PA-VL... gpA33 -VH CD3 -Recombinant vector for RNA-DNA molecules pHW2000-PA-VL gpA33 -VH CD3 The recombinant vectors pHW2000-HA, pHW2000-NP, pHW2000-M, pHW2000-NA, pHW2000-NS, and pHW2000-NS contain DNA molecules encoding the HA-RNA, respectively. gpA33 -VL CD3 -Recombinant vector for RNA-DNA molecules pHW2000-PB1-VH gpA33 -VL CD3The virus was introduced into packaging cells to obtain recombinant oncolytic influenza virus expressing anti-gpA33×CD3 bispecific antibodies. The vector was pHW2000 plasmid, but not limited to pHW2000 plasmid.

[0018] To solve the above-mentioned technical problems, the present invention also provides any of the following products: (1) The genome of the recombinant oncolytic influenza virus; (2) A complete set of vectors for treating tumors, namely, a vector containing the encoding gene of the anti-gpA33×CD3 bispecific antibody; (3) A complete expression cassette for treating tumors, namely, an expression cassette containing the gene encoding the anti-gpA33×CD3 bispecific antibody; (4) A complete set of genes for treating tumors, namely the encoding gene of the anti-gpA33×CD3 bispecific antibody; (5) A set of proteins for treating tumors, namely the anti-gpA33×CD3 bispecific antibody protein.

[0019] To address the aforementioned technical problems, the present invention also provides biomaterials related to the recombinant oncolytic influenza virus, wherein the biomaterials are any of the following products: (1) A set of DNA molecules encoding the set of positive-sense RNA of the recombinant oncolytic influenza virus; the set of DNA molecules consists of a DNA molecule encoding the PB2-RNA and a DNA molecule encoding the PA-VL. gpA33 -VH CD3 -RNA DNA molecules that encode the PB1-VH gpA33 -VL CD3 It consists of a DNA molecule encoding NP-RNA, a DNA molecule encoding NA-RNA, a DNA molecule encoding HA-RNA, a DNA molecule encoding M-RNA, and a DNA molecule encoding NS-RNA; (2) A set of recombinant vectors; the set of recombinant vectors consists of a recombinant vector containing a DNA molecule encoding the PB2-RNA, and a recombinant vector containing a DNA molecule encoding the PA-VL. gpA33 -VH CD3 Recombinant vectors containing DNA molecules encoding HA-RNA, recombinant vectors containing DNA molecules encoding NP-RNA, recombinant vectors containing DNA molecules encoding M-RNA, recombinant vectors containing DNA molecules encoding NA-RNA, recombinant vectors containing DNA molecules encoding NS-RNA, and recombinant vectors containing DNA molecules encoding PB1-VH gpA33 -VL CD3 -The recombinant vector of RNA and DNA molecules; (3) Microorganisms containing the recombinant oncolytic influenza virus; (4) Animal cells containing the recombinant oncolytic influenza virus; (5) Animal tissues containing the recombinant oncolytic influenza virus; (6) Animal organs containing the recombinant oncolytic influenza virus.

[0020] Among the above-mentioned biological materials, the animal cells, animal tissues, and animal organs do not include reproductive materials.

[0021] In the above-mentioned biological materials, the microorganisms, animal cells, animal tissues, and animal organs can all serve as hosts for recombinant oncolytic viruses.

[0022] In one embodiment of the present invention, the recombinant vector containing the DNA molecule encoding the PB2-RNA in (2) is pHW2000-PB2, and the recombinant vector containing the DNA molecule encoding the PA-VL is pHW2000-PB2. gpA33 -VH CD3 The recombinant vector for RNA-DNA molecules is pHW2000-PA-VL. gpA33 -VH CD3 The PB1-VH encoding is included. gpA33 -VL CD3 The recombinant vector for RNA-DNA molecules is pHW2000-PB1-VH. gpA33 -VL CD3 The recombinant vector containing the DNA molecule encoding the NP-RNA is pHW2000-NP, the recombinant vector containing the DNA molecule encoding the NA-RNA is pHW2000-NA, the recombinant vector containing the DNA molecule encoding the HA-RNA is pHW2000-HA, the recombinant vector containing the DNA molecule encoding the M-RNA is pHW2000-M, and the recombinant vector containing the DNA molecule encoding the NS-RNA is pHW2000-NS. pHW2000-PB1-VH gpA33 -VL CD3 It can express the VH shown in SEQ ID No. 1 gpA33 Polypeptides, and VL as shown in SEQ ID No. 3 CD3 Polypeptide. The pHW2000-PA-VL gpA33 -VH CD3 It can express the VL shown in SEQ ID No. 2 gpA33 The polypeptide, and the VH shown in SEQ ID No. 4. CD3 Polypeptide.

[0023] To address the aforementioned technical problems, the present invention also provides a tumor treatment drug, wherein the active ingredient of the drug is the recombinant oncolytic influenza virus.

[0024] The active ingredient of the above-mentioned drug may also be a composition obtained by combining the recombinant oncolytic influenza virus with other antitumor drugs.

[0025] To address the aforementioned technical problems, the present invention also provides any of the following applications: (1) The application of the recombinant oncolytic influenza virus in the preparation of drugs for treating tumors; (2) The application of the product in the preparation of drugs for treating tumors; (3) The application of the biomaterial in the preparation of drugs for treating tumors; (4) The application of the aforementioned tumor-treating drug in the preparation of tumor-treating drugs; (5) The application of the recombinant oncolytic influenza virus in the treatment of tumors; (6) The application of the product in the treatment of tumors; (7) The application of the biomaterial in the treatment of tumors; (8) The application of the tumor-treating drugs in the treatment of tumors.

[0026] The tumors described in this invention include, but are not limited to, colorectal cancer, and may also include lung cancer, breast cancer, liver cancer, melanoma, lymphoma, leukemia, ovarian cancer, cervical cancer, stomach cancer, kidney cancer, pancreatic cancer, prostate cancer, and glioma.

[0027] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows: Experiments have demonstrated that the gene modification and reverse genetics techniques employed in this invention can successfully rescue the recombinant oncolytic influenza virus rPR8-gpA33-CD3. This recombinant oncolytic influenza virus exhibits replication efficiency in MDCK cells and chicken embryos that is almost equivalent to that of the wild-type PR8 virus strain, while also stably expressing and secreting bispecific antibodies against gpA33×CD3. Furthermore, this virus can effectively infect various colorectal cancer cell lines, demonstrating a significant killing effect on colorectal cancer cells. Following oncolytic therapy in mice with syngeneic colorectal cancer xenografts, this recombinant virus can effectively inhibit tumor growth and metastasis, and prolong the survival of the mice. These experimental results prove that it has a good tumor-suppressive effect and can be used for tumor treatment. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1 The recombinant gene PB1-VH of the recombinant oncolytic influenza virus rPR8-gpA33-CD3 in Example 1 of this invention. gpA33 -VL CD3 and PA-VL gpA33 -VH CD3 Structural pattern diagram.

[0030] Figure 2 This is a simulation diagram of the mechanism of action of recombinant oncolytic influenza virus rPR8-gpA33-CD3 in Example 1 of the present invention.

[0031] Figure 3 The morphological structure of recombinant oncolytic influenza virus rPR8-gpA33-CD3 was identified by electron microscopy in Example 1 of this invention.

[0032] Figure 4 In Example 1 of this invention, the hemagglutination titer of recombinant oncolytic influenza virus rPR8-gpA33-CD3 was determined by a hemagglutination experiment. Here, A represents the hemagglutination titer test result of the recombinant oncolytic influenza virus after 5 consecutive passages in chicken embryos (two replicates were performed for each passage); B represents the quantitative result of the hemagglutination titer of each passage of virus.

[0033] Figure 5 The expression and secretion levels of anti-gpA33×CD3 bispecific antibody were detected by ELISA in Example 1 of this invention.

[0034] Figure 6 The purpose of this invention is to identify the replication ability of recombinant oncolytic influenza virus rPR8-gpA33-CD3 in various colorectal cancer cell lines using RT-qPCR experiments in Example 1 of this invention.

[0035] Figure 7 In Example 2 of this invention, the inhibitory activity of recombinant oncolytic influenza virus rPR8-gpA33-CD3 on the proliferation of various colorectal cancer cell lines was identified.

[0036] Figure 8 In Example 2 of this invention, the recombinant oncolytic influenza virus rPR8-gpA33-CD3 inhibits the growth of colorectal cancer in vivo. In this example, A represents a physical image of the tumor, and B represents a quantitative image of the tumor volume.

[0037] Figure 9This invention provides an example of how recombinant oncolytic influenza virus rPR8-gpA33-CD3 inhibits colorectal cancer metastasis in vivo. In this example, A represents a physical image of the tumor, and B represents a quantitative image of the number of lung metastases.

[0038] Figure 10 This is a survival curve of the xenograft mice in Example 2 of the present invention. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] The reagents required for this invention are conventional experimental reagents, purchased from commercially available channels; the experimental methods not mentioned are conventional experimental methods, and will not be described in detail here.

[0041] Example 1 Rescue and identification of recombinant oncolytic influenza virus This invention provides a recombinant oncolytic influenza virus named rPR8-gpA33-CD3, which expresses a bispecific antibody named anti-gpA33×CD3. In this bispecific antibody, the amino acid sequence of the gpA33 heavy chain variable region is the same as that in SEQ ID No. 1, encoded by nucleotides 2410-2760 of SEQ ID No. 5; the amino acid sequence of the CD3 light chain variable region is the same as that in SEQ ID No. 3, encoded by nucleotides 2806-3123 of SEQ ID No. 5; the amino acid sequence of the gpA33 light chain variable region is the same as that in SEQ ID No. 2, encoded by nucleotides 2287-2610 of SEQ ID No. 6; and the amino acid sequence of the CD3 heavy chain variable region is the same as that in SEQ ID No. 4, encoded by nucleotides 2656-3012 of SEQ ID No. 6.

[0042] EVQLVESGGGLVKPGGSLRLSCAASGFAFSTYDMSWVRQAPGKRLEWVATISSGGSYTYYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAPTTVVPFAYWGQGTLVTVSS, SEQ ID No. 1.

[0043] DIQMTQSQSSLSTSVGDRVTITCKASQNVRTVVAWYQQKPGKSPKTLIYLASNRHTGVPSRFSGSGSGTEFTLTISNVQPEDFADYFCLQHWSYPLTFGSGTKLEIKR, SEQ ID No. 2.

[0044] DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK, SEQ ID No. 3.

[0045] SEQ ID No. 4.

[0046] 1. Construct the bidirectional expression plasmid pHW2000-PB1-VH gpA33 -VL CD3 and pHW2000-PA-VL gpA33 -VH CD3 according to Figure 1 The recombinant gene fragment PB1-VH shown is gpA33 -VL CD3 and PA-VL gpA33 -VH CD3 The structure of the DNA molecule and the corresponding DNA molecules in SEQ ID No. 5 and SEQ ID No. 6 were artificially synthesized. After linearizing the DNA molecule and the bicistronic vector pHW2000, the recombinant gene fragment and the pHW2000 vector fragment were ligated using an InFusion kit to construct the target recombinant plasmid pHW2000-PB1-VH. gpA33 -VL CD3 and pHW2000-PA-VL gpA33 -VH CD3 The target recombinant plasmid obtained by homologous recombination was transformed into E. coli DH-5α competent cells for plasmid amplification, and the target recombinant plasmid was identified and screened by PCR and gene sequencing technology.

[0047] like Figure 1 As shown, the recombinant gene fragment PB1-VH gpA33 -VL CD3From the 5' end to the 3' end, the terminals are PB1, T2A, SP, and VH, respectively. gpA33 (G4S)3, VL CD3 FNRGECC, P2A, and PS, among which, PB1 represents the gene sequence encoding the PB1 protein of influenza A virus PR8 strain; T2A represents the gene sequence encoding the hydrolyzable linker T2A; SP represents the gene sequence encoding the Igκ signal peptide; VH gpA33 Gene sequence representing the heavy chain variable region encoding an antibody against the colorectal cancer cell biomarker molecule gpA33; (G4S)3 gene sequence representing the flexible linker (G4S)3; VL CD3 The gene sequence represents the light chain variable region encoding the anti-CD3 antibody; FNRGECC represents the gene sequence of the short peptide FNRGECC in the antibody hinge region; P2A represents the gene sequence encoding the hydrolyzable linker P2A; PS represents the packaging signal sequence at the 3' end of PB1 (nucleotides 1-2295, 2296-2346, 2347-2409, 2410-2760, 2761-2805, 2806-3123, 3124-3144, 3145-3210, and 3211-3374 in SEQ ID No. 5 below).

[0048] AGCGAAAGCA GGCAAACCAT TTGAATGGAT GTCAATCCGA CCTTACTTTT CTTAAAAGTGCCAGCACAAA ATGCTATAAG CAACACTTTC CCTTATACTG GAGACCCTCC TTACAGCCAT GGGACAGGAACAGGATCAC CATGGATACT GTCAACAGGA CACATCAGTA CTCAGAAAAG GGAAGATGGA CAACAAACCGAAACTGGA GCACCGCAAC TCAACCCGAT TGATGGGCCA CTGCCAGAAG ACAATGAACC AAGTGGTTATGCCCAAACAG ATTGTGTATT GGAGGCGATG GCTTTCCTTG AGGAATCCCA TCCTGGTATT TTTGAAAACTCGTGTATTGA AACGATGGAG GTTGTTCAGC AAACACGAGT AGACAAGCTG ACACAAGGCC GACAGACCTATGACTGGACT CTAAATAGAA ACCAACCTGC TGCAACAGCA TTGGCCAACA CAATAGAAGT GTTCAGATCAAATGGCCTCCA CGGCCAATGA GTCTGGAAGG CTCATAGACT TCCTTAAGGA TGTAATGGAG TCAATGAAAAAGAAAT GGGATCACA ACTCATTTTC AGAGAAAGAG ACGGGTGAGA GACAATATGA CTAAGAAAATGATAACACAG AGAACAATGG GTAAAAAGAA GCAGAGATTG AACAAAAGGA GTTATCTAAT TAGAGCATTGACCCTGAACA CAATGACCAA AGATGCTGAG AGAGGGAAGC TAAAACGGAG AGCAATTGCA ACCCCAGGGATGCAAATAAG GGGGTTTGTA TACTTTGTTG AGACACTGGC AAGGAGTATA TGTGAGAAAC TTGAACAATCAGGGTTGCCA GTTGGAGGCA ATGAGAAGAA AGCAAAGTTG GCAAATGTTG TAAGGAAGAT GATGACCAATTCTCAGGACA CCGAACTTTCTTTCACCATC ACTGGAGATA ACACCAAATG GAACGAAAAT CAGAATCCTCGGATGTTTTT GGCCATGATC ACATATATGA CCAGAAATCA GCCCGAATGG TTCAGAAATG TTCTAAGTATTGCTCCAATA ATGTTCCAA ACAAAATGGC GAGACTGGAATA GAGACTGGAATA CAAAGATGAAACTTAGAA CTCAAATACC TGCAGAAATG CTAGCAAGCA TCGATTTGAA ATATTTCAAT GATTCAACAAGAAGAT TGAAAAAATC CGACCGCTCT TAATAGGGG GACTGCATCA TTGAGCCCTG GAATGATTTGATAGCTCAGATGTAGTAGTC ATCCTGAATC TTGGACAAA GAGATACACCAAGACTACTT ACTGGTGGGA TGGTCTTCAA TCCTCTGACG ATTTTGCTCT GATTGTGAAT GCACCCAATCGAGGAT TCAAGCCGGA GTCGACAGGT TTTATCGAAC CTGTGACTAAGCTA CATAACCAATCAA GAACAGGTAC ATTTGAATTC ACAAGTTTTT TCTATCGTTA TGGGTTTGTTGCCAATTTCA GCATGGAGCT TCCCAGTTTT GGGGTGTCTG GGATCAACGA GTCAGCGGAC ATGAGTATTGGAGTTACTGT CATCAAAAAC AATATGATAAG ACAGTCAATCACT TGGCCCTTCAGTTGTTCATC AAAGATTACA GGTACACGTA CCGATGCCAT ATAGGTGACA CACAAATACA AACCCGAATTCATTTGAAA TAAAGAAACT GTGGGAGCAA ACCCGTTCCA AAGCTGGACT GCTGGTCTCC GACGGAGCCCTCAAATTCAATCATATAAT CAACCCATTCCTGAAGT CTGCCTAAA TGGGAATTGA TGGATGAGGATTACCAGGGG CGTTTATGCA ACCCACTGAA CCCATTTGTC AGCCATAAAG AAATTGAATC AATGAACAATGCAGTGATGA TGCCAGCACA TGGTCCAGCC AAAAACATGG AGTATGATGC TGTTGCAACA ACACACTCCTGGATCCCCAA AAGAAATCGA TCCATCTTGA ATACAAGTCA AAGAGGAGTA CTTGAGGATG AACAAATGTACCAAAGGTGC TGCAATTTAT TTGAAAAAATT CTTCCCCAGC AGTTCATACA GAAGACCAGT CGGGATATCCAGTATGGTGG AGGCTATGGT TTCCAGAGCC CGAATTGATG CACGGATTGA TTTCGAATCT GGAAGGATAAAAAAAGAA GTTCACTGAG ATCATGAAGA TCGTTTCCAC CATTGAAGAG CTCAGACGGCAAAAAGAGGGCCGCGGCAGC CTGCTGACCT GCGGCGACGT GGAGGAGAAC CCCGGCATGG AGACAGACAC ACTCCTGCTATGGGTACTGC TGCTCTGGGT TCCAGGTTCC ACTGGTGAC AGGTGCAGCT GGTGGAGAGC GGCGGCGGCCTGGTGAAGCC CGGCGGCAGC CTGCGCCTGA GCTGCGCCGC CAGCGGCTTC GCCTTCAGCA CCTACGACATGAGCTGGGTG CGCCAGGCCC CCGGCAAGCG CCTGGAGTGG GTGGCCACCA TCAGCAGCGG CGGCAGCTACACCTACTACC TGGACAGCGT GAAGGGCCGC TTCACCATCA GCCGCGACAA CGCCAAGAAC AGCCTGTACCTGCAGATGAA CAGCCTGCGC GCCGAGGACA CCGCCGTGTA CTACTGCGCC CCCACCACCG TGGTGCCCTTCGCCTACTGG GGCCAGGGCA CCCTGGTGACCGTGAGCAGCGGCGGCGGCG GCAGCGGCGG CGGCGGCAGCGGCGGCGGCG GCAGCGACAT CCAGCTGACC CAGAGCCCCG CCATCATGAG CGCCAGCCCC GGCGAGAAGGTGACCATGAC CTGCCGCGCC AGCAGCAGCG TGAGCTACAT GAACTGGTAC CAGCAGAAGA GCGGCACCAGCCCCAAGCGC TGGATCTACG ACACCAGCAA GGTGGCCAGC GGCGTGCCCT ACCGCTTCAG CGGCAGCGGCAGCGGCACCA GCTACAGCCT GACCATCAGC AGCATGGAGG CCGAGGACGC CGCCACCTAC TACTGCCAGCAGTGGAGCAG CAACCCCCTG ACCTTCGGCG CCGGCACCAA GCTGGAGCTG AAGTTCAACC GCGGCGAGTGCTGCGGATCT GGAGCCACGA ACTTCTCTCT GTTAAAGCAA GCAGGAGATG TTGAAGAAAACCCCGGGCCTTCCAGAGCCC GAATTGATGC ACGGATTGAT TTCGAATCTG GAAGGATAAA GAAAGAAGAGTTCACTGAGA TCATGAAGAT CTGTTCCACC ATTGAAGAGC TCAGACGGCA AAAATAGTGA ATTTAGCTTGTCCTTCATGA AAAAATGCCT TGTTTCTACT AATA, SEQ ID No. 5.

[0049] Recombinant gene fragment PA-VL gpA33 -VH CD3 From the 5' end to the 3' end, the terminals are PA, T2A, SP, and VL, respectively. gpA33 (G4S)3, VH CD3 VEPKSCC, P2A, and PS, among which, PA represents the gene sequence encoding the PA protein of influenza A virus PR8 strain; T2A represents the gene sequence encoding the hydrolyzable linker T2A; SP represents the gene sequence encoding the Igκ signal peptide; VL gpA33 The gene sequence representing the light chain variable region encoding the antibody against the colorectal cancer cell biomarker molecule gpA33; (G4S)3 represents the gene sequence of the flexible linker (G4S)3; VHCD3 The gene sequence represents the heavy chain variable region encoding the anti-CD3 antibody; VEPKSCC represents the gene sequence of the short peptide VEPKSCC in the antibody hinge region; P2A represents the gene sequence encoding the hydrolyzable linker P2A; PS represents the packaging signal sequence at the 3' end of PA (nucleotides 1-2172, 2173-2223, 2224-2286, 2287-2610, 2611-2655, 2656-3012, 3013-3033, 3034-3099, and 3100-3283 in sequence in SEQ ID No. 6 below).

[0050] AGCGAAAGCA GGTACTGATC CAAAATGGAA GATTTTGTGC GACAATGCTT CAATCCGATGATTGTCGAGC TTGCGGAAAA AACAATGAAA GAGTATGGGG AGGACCTGAA AATCGAAACA AACAAATTTGCAGCAATG CACTCACTCTG TTCAGATCAGTACATG AGCAAGGCGAGTCAATAATC GTAGAACTTG GTGATCCAAA TGCACTTTTG AAGCACAGAT TTGAAATAAT CGAGGGAGAGATCGCACAA TGGCCTGGAC AGTAGTAAAC AGTATTTGCA ACACTACAGG GGCTGAGAAA CCAAAGTTAGTTGATTCACCAGATTACT ATT AATTGGAGTA ACAAGGAGAG AAGTTCACATATACTATCTG GAAAAGGCCA ATAAAATTAA ATCTGAGAAA ACACACATCC ACATTTTCTC GTTCACTGGGGAAAATGG CCACAAAGGC GTTCACTGGGGAAGAAATGG CCACAAAGGC GATCACTCACT CTCGATGAAG AAGCAGCTGAAGA TAGGAGTAGAGTCCACACAA GCCTCTGGGA TTCCTTTCGT CAGTCCGAGA GAGGAGAGACAATTGAA GAAAGGTTTG AAATCACAGG AACAATGCGT AAGCTTGCCG ACCAAAGTCT CCCGCCGAACTTCTCCAGCC TTGAAAATTT TAGAGCCTAT GTGGATGGAT G CTGAAACCATTAA CGGACATCAA GTCCAAAGAA GTAAATGCTA GAATTGAACC TTTTTTGAAA ACAACACCAC GACCACTTAGACTTCGAAT GGGCCTCCCT GTTCTCAGCG GTCCAAATTC CTGCTGATGG ATGCCTTAAA ATTAAGCATTGAGGACCCAA GTCATGAAGGAGAGGGAATA CCGCTATATG ATGCAATCAA ATGCATGAGA ACATTCTTTGGATGGAAGGA ACCCAATGTT GTTAAACCAC ACGAAAAGGG AATAAATCCA AATTATCTC TGTCATGGAAGCAAGTACTG GCAGAACTGC AGGACATTGA GAATGAGGAAG AAAATTCAATTGA TATGAAGAAAACAAGTCAGC TAAAGTGGGC ACTTGGTGAG AACATGGCAC CAGAAAAGGT AGACTTTGAC GACTGTAAAGATGTAGGTGA TTTGAAGCAA TATGATAGTG ATGAACCAGA ATTGAGGTCG CTTGCAAGTT GGATTCAGAATGAGTTTAAC AAGGCATGACTGACTGACTGGGG ATAGAGCTCG ATGAGATTGG AGAAGATGTGGCTCCAATTG AACACATTGC AAGCATGAGA AGGAATTATT TCACATCAGA GGTGTCTCAC TGCAGAGCCACAGAATACAT AATGAAGGGA GTGTACATCA ATACTGCCTT GCTTAATGCA TCTTGTGCATGATTCATTCATTGATGAT TAAGCAAGTG TAGAACTAAG GAGGGAAGGC GAAAGACCAA CTTGTATGGTTTCATCATAA AAGGAAGATC CCACTTAAGG AATGACACCG ACGTGGTAAA CTTTGTGAGC ATGGAGTTCTCTCACTGA CCCAAGACTT GAACCACATA AATGGGAGATT CTAGACTGAGTTGAGTTGATTGATT GCCATAGGCC AGGTTTCAAG GCCCATGTTC TTGTATGTGA GAACAAATGG AACCTCAAAAATTAAAATGA AATGGGGAAT GGAGATGAGG CGTTGCCTCC TCCAGTCACT TCAACAAATT GAGAGTATGATTGAAGCTGA GTCCTCTGTC AAAGAGAAAGACATGACCAA AGAGTTCTTT GAGAACAAAT CAGAAACATGGCCCATTGGA GAGTCCCCCA AAGGAGTGGA GGAAAGTTCC ATTGGGAAGG TCTGCAGGAC TTTATTAGCAAAGTCGGT TCAACAGCTT GTATGCATCT CCACAACTAG AAGGATTTTC AGCTGAATCA AGAAAACTGCTTTTTATCGT TCAGGCTCTT AGGGACAACC TGGAACCTGG GACCTTTGAT CTTGGGGGGGGC TATATGAAGCAATTGAGGAG TGCCTGATTA ATGATCCCTG GGTTTGCTT AATGCTTCTT GGTTCAACTC CTTCCTTACACATGCATTGA GTGAGGGCCG CGGCAGCCTG CTGACCTGCG GCGACGTGGA GGAGAACCCC GGCATGGAGACAGACACACACT CCTGCTATGG GTACTGCTGGC TCTGGGTTCC AGGTTCCACT GGTGACGACA TCCAGATGACCCAGCCAG AGCAGCCTGA GCACCAGCGT GGGCGACCGC GTGACCATCA CCTGCAAGGC CAGCCAAGGTGCGCCACCAGCG TGGTGGCCTG GTACCAGCAGCA AAGCCCGGCA AGAGCCCCAA GACCCTGATC TACCTGGCCAGCAACCGCCA CACCGGCGTG CCCAGCCGCT TCAGCGGCAG CGGCAGCGGC ACCGAGTTCA CCCTGACCATCAGCAACGTG CAGCCCGAGG ACTTCGCCGA CTACTTCTGC CTGCAGCACT GGAGCTACCC CCTGACCTTCGGCAGCGGCA CCAAGCTGGA GATCAAGCGCGGCGGCGGCGCGGCGGCAGC GGCGGCGGCGGCAGCGACAT CAAGCTGCAG CAGAGCGGCG CCGAGCTGGC CCGCCCCGGC GCCAGCGTGA AGATGAGCTGCAAGACCAGC GGCTACACCT TCACCCGCTA CACCATGCACTGGGTGAAGC AGCGCCCCGG CCAGGGCCTGGAGTGGATCG GCTACATCAA CCCCAGCCGC GGCTACACCA ACTACAACCA GAAGTTCAAG GACAAGGCCACCCTGACCAC CGACAAGAGC AGCAGCACCG CCTACATGCA GCTGAGCAGC CTGACCAGCG AGGACAGCGCCGTGTACTAC TGCGCCCGCT ACTACGACGA CCACTACTGC CTGGACTACT GGGGCCAGGG CACCACCCTGACCGTGAGCA GCGTGGAGCC CAAGAGCTGC TGCGGATCTG GAGCCACGAA CTTCTCTCTG TTAAAGCAAGCAGGAGATGT TGAAGAAAAC CCCGGGCCTC TTGAACCTGG GACCTTTGAT CTTGGGGGGC TATATGAAGCAATTGAGGAG TGCCTGATTA ATGATCCCTG GGTTTTGCTT AATGCTTCTT GGTTCAACTC ATTCCTTACACATGCATTGA GTTAGTTGTG GCAGTGCTAC TATTTGCTAT CCATACTGTC CAAAAAAGTA CCTTGTTTCTACT, SEQ ID No. 6.

[0051] 2. Rescue of Recombinant Oncolytic Influenza Virus Using Polyfect Reagent reagent, the recombinant vector pHW2000-PB2 containing DNA molecules encoding PB2-RNA and the vector containing DNA molecules encoding PA-VL were recombined. gpA33 -VH CD3 -Recombinant vector for RNA-DNA molecules pHW2000-PA-VL gpA33 -VH CD3 Contains the code PB1-VH gpA33 -VL CD3 -Recombinant vector for RNA-DNA molecules pHW2000-PB1-VH gpA33 -VL CD3The recombinant vectors pHW2000-NP (encoding NP-RNA), pHW2000-NA (encoding NA-RNA), pHW2000-HA (encoding HA-RNA), pHW2000-M (encoding M-RNA), and pHW2000-NS (encoding NS-RNA) were co-transfected into 293T cells at the same ratio to obtain culture supernatant containing recombinant viruses.

[0052] 3. Amplification of recombinant oncolytic influenza virus The culture supernatant containing recombinant virus was rescued and harvested, then inoculated into chicken embryos for virus amplification. Allantoic fluid containing virus particles was harvested and concentrated and purified by sucrose density gradient centrifugation.

[0053] Figure 2 This is a simulation diagram of the mechanism of action of recombinant oncolytic influenza virus rPR8-gpA33-CD3. It can be seen that rPR8-gpA33-CD3 can stably express a bispecific anti-gpA33×CD3 antibody in colorectal cancer cells. One end of this antibody can specifically bind to the CD3 molecule on the surface of T cells, efficiently activating T cells. The other end can specifically bind to the gpA33 marker molecule on the surface of colorectal cancer cells. Through the bridging effect of the antibody, activated T cells are recruited to the surface of colorectal cancer cells, inducing T cells to target and kill colorectal cancer cells.

[0054] 4. Biological characteristics and functional identification of recombinant oncolytic influenza virus (1) The hemagglutination titer of rPR8-gpA33-CD3 was determined by hemagglutination test.

[0055] Hemagglutination assay: 1) Take a 96-well rapid reaction plate and add 50 μl of physiological saline to each well; 2) Then add 50 μl of allantoic fluid or culture supernatant containing recombinant oncolytic influenza virus particles to each well in the first column of the 96-well plate and mix thoroughly; 3) Take 50 μl of the mixture from the first column and add it to the second column, mix thoroughly, then take 50 μl from the second column and add it to the third column, mix thoroughly, and so on, to serially dilute the allantoic fluid; 4) Add 1% chicken red blood cells to the 96-well rapid reaction plate after dilution of recombinant oncolytic influenza virus, 50 μl per well, mix thoroughly, and let stand at room temperature for 30 min, then judge the results.

[0056] The results are as follows Figure 4 As shown, by Figure 4 The results showed that after culturing P1 generation recombinant oncolytic influenza virus in chicken embryos for 4 days, the hemagglutination titer of the recombinant oncolytic influenza virus HA in the allantoic fluid collected could reach 1:2. 4Subsequently, the HA hemagglutination titer in the allantoic fluid containing recombinant oncolytic influenza virus particles in generations P2, P3, P4, and P5 remained at 1:2. 5 This indicates that the recombinant oncolytic influenza virus rPR8-gpA33-CD3 was successfully rescued in this experiment, and it exhibits high replication activity and strong genetic stability when cultured in chicken embryos.

[0057] (2) Observe the morphology and structure of rPR8-gpA33-CD3 under an electron microscope.

[0058] The electron microscope was magnified 20,000 times, and the results were as follows: Figure 3 As shown in the image, the recombinant oncolytic influenza virus particles are mostly spherical, with a few being filamentous or rod-shaped. The wheel-shaped spike protein is clearly visible on the surface of the virus. The particle size is mostly between 80 and 120 nm, and its structure is clear and complete.

[0059] (3) ELISA was used to detect the expression and secretion levels of bispecific antibodies against gpA33×CD3 after chicken embryos were inoculated with rPR8-gpA33-CD3.

[0060] Chicken embryos were inoculated with wild-type influenza A virus PR8 and P2 generation recombinant oncolytic influenza virus rPR8-gpA33-CD3 (the virus solution was diluted 100 times with physiological saline, and 100 μL was inoculated into each chicken embryo). After culturing at 37°C for 3 days, the allantoic fluid of the chicken embryos was collected. The concentration of anti-gpA33×CD3 bispecific antibody in the chicken embryo allantoic fluid was detected by ELISA to determine its expression and secretion level.

[0061] The ELISA method is as follows: Centrifuge the collected chicken embryo allantoic fluid at 4℃, 1000g for 10 min; remove the CD3 antibody quantitative detection kit from 4℃ and allow it to equilibrate to room temperature; according to the instructions, first prepare 1×Washing Buffer, 1×Dilution Buffer, and HRP-conjugated antibody working solutions, and prepare a standard curve. Add the test sample and serially diluted standards to the reaction wells, 100 μL per well. Add 100 μL of Dilution Buffer to the blank control wells. After incubating at room temperature for 1.0 h, discard the liquid in the wells, add 300 μL of 1×Washing Buffer to each well, soak for 10 s, and wash the plate 3 times. Add 100 μL of diluted HRP-conjugated antibody (diluted to 0.08 μg / mL) working solution to the corresponding wells, incubate at room temperature for 1.0 h, and wash the plate 3 times. Add 100 μL of Substrate Solution to each well. After incubating at room temperature for 20 minutes, add 50 μL of Stop Solution to each well and gently shake the plate until well mixed. Measure the absorbance of each well at 450 nm and 630 nm using a microplate reader. Please read the values ​​within 10 minutes after incubation.

[0062] The results are as follows Figure 5 As shown, the average concentration of anti-gpA33×CD3 bispecific antibody in the allantoic fluid of chicken embryos infected with recombinant oncolytic influenza virus rPR8-gpA33-CD3 can reach 1250 ng / μL, indicating that rPR8-gpA33-CD3 can not only replicate effectively in chicken embryos, but also successfully express the exogenous anti-gpA33×CD3 bispecific antibody gene.

[0063] (4) RT-qPCR detection of viral replication ability of rPR8-gpA33-CD3 after inoculation with various colorectal cancer cells.

[0064] Recombinant oncolytic influenza virus rPR8-gpA33-CD3, harvested from chicken embryos and amplified from generation P3, was used to infect normal colorectal mucosal cells CCD841, as well as HT29, MC38, HCT116, CT26, SW620, and LOVO colorectal cancer cell lines (MOI=1). After culturing at 37°C for 3 days, cells were collected and RT-qPCR was performed to detect the conserved viral gene M, in order to determine the replication capacity of recombinant oncolytic influenza virus rPR8-gpA33-CD3 in various colorectal cancer cell lines.

[0065] The results are as follows Figure 6As shown, the recombinant oncolytic influenza virus rPR8-gpA33-CD3 can infect and replicate in various colorectal cancer cell lines, with the highest replication capacity observed in MC38 cells.

[0066] Example 2 In vitro and in vivo oncolytic effects of recombinant oncolytic influenza virus 1. Study on the in vitro oncolytic effect of recombinant oncolytic influenza virus (1) rPR8-gpA33-CD3 infection of colon cancer cell lines: HT29, MC38, CT26, and its effect on the proliferation of colorectal cancer cells was studied by CellTiter-Glo experiment.

[0067] The CellTiter-Glo experimental method is as follows: 1) HT29, MC38 and CT26 were plated in 96-well plates and incubated overnight at 37°C with 5% CO2. 2) The next day, once the cell density reaches 90%~95%, gently wash away the serum from the cell culture with PBS; 3) The P3 generation recombinant oncolytic influenza virus rPR8-gpA33-CD3 was diluted with virus maintenance solution so that its MOI for infecting cells was 1. 4) Add 100 μl of the diluted virus solution to each well of a 96-well plate and incubate at 37°C and 5% CO2 for 72 h. 5) Remove the buffer solution from the Cell-titerglo kit from -20℃, thaw it at room temperature, dissolve the CellTiter-Glo lyophilized powder in the buffer solution, mix well, aliquot into 1.5ml centrifuge tubes, and freeze for later use. 6) Remove the 96-well plate from the 37℃ incubator after the culture is complete, and then immediately add 25 μl of CellTiter-Glo to each well; 7) Place the 96-well plate containing CellTiter-Glo into a multi-functional microplate reader, shake for 60 seconds, then remove and incubate at 37°C for 10 minutes in the dark; 8) Use a multichannel pipette to transfer the maintenance solution, prepared in the dark, into a white, opaque, flat-bottomed 96-well plate, 80 μl per well. Be careful not to generate air bubbles during the transfer process. 9) Place the white, opaque 96-well plate in a SpectraMax M2 multi-plate reader, shake for 5 seconds, and read 20 times; 10) After exporting the experimental data, use GraphPad Prism 9.3.1 software to process and analyze the data.

[0068] The results are as follows Figure 7As shown, recombinant oncolytic influenza virus rPR8-gpA33-CD3 can inhibit the proliferation of various colorectal cancer cells. Among them, MC38 cells are the most sensitive to rPR8-gpA33-CD3, with cell viability decreasing to 20%. The results indicate that rPR8-gpA33-CD3 can significantly inhibit the proliferation and growth of colorectal cancer cells.

[0069] 2. Study on the in vivo oncolytic effect of recombinant oncolytic influenza virus (1) A subcutaneous xenograft model of the C57BL / 6 mouse colorectal cancer cell line MC38 was established. rPR8-gpA33-CD3 was injected into the tumor. The effectiveness of rPR8-gpA33-CD3 in inhibiting the growth and metastasis of colorectal cancer was evaluated by measuring the tumor volume.

[0070] MC38 cells were transplanted subcutaneously into the back of C57BL / 6 mice to establish a syngeneic colorectal cancer xenograft model. The tumor volume was allowed to grow to 100 mm. 3 To conduct oncolytic therapy, in preliminary validation experiments, we established two xenograft models: one group received oncolytic therapy using wild-type influenza virus PR8, and the other group received oncolytic therapy using recombinant oncolytic influenza virus rPR8-gpA33-CD3. Each group consisted of 3 mice. Treatment was administered via intratumoral injection, with each injection dose being 1×10⁻⁶. 7 PFU / tumor, injected every other day for a total of three times. Tumor tissue was taken for observation and size measurement 14 days later.

[0071] The results are as follows Figure 8 As shown in the results, the tumor volume in the recombinant oncolytic influenza virus rPR8-gpA33-CD3 treatment group was significantly smaller than that in the wild-type influenza virus PR8 treatment group. This indicates that the recombinant oncolytic influenza virus rPR8-gpA33-CD3 can significantly inhibit tumor growth in vivo.

[0072] (2) After infecting MC38 cells with wild-type influenza virus PR8 (MOI=1) and recombinant oncolytic influenza virus rPR8-gpA33-CD3 for 2 hours, the cells were collected and the cell concentration was adjusted to 1×10⁻⁶ cells / mL using serum-free medium. 5 / 100µL, 100µL of cell fluid was injected into the tail vein of mice to create a lung metastasis model experiment. Lung tissue was taken 14 days later to observe lung metastases.

[0073] The results are as follows Figure 9 As shown, the number of lung metastases in the recombinant oncolytic influenza virus rPR8-gpA33-CD3 group was significantly less than that in the wild-type influenza virus PR8 group.

[0074] (3) The efficacy and safety of rPR8-gpA33-CD3 were evaluated by plotting mouse survival curves.

[0075] MC38 cells were transplanted subcutaneously into the back of C57BL / 6 mice to establish a syngeneic colorectal cancer xenograft model. The tumor volume was allowed to grow to 100 mm. 3 To conduct oncolytic therapy, in preliminary validation experiments, we established two xenograft models: one group received oncolytic therapy using wild-type influenza virus PR8, and the other group received oncolytic therapy using recombinant oncolytic influenza virus rPR8-gpA33-CD3. Each group consisted of 3 mice. Treatment was administered via intratumoral injection, with each injection dose being 1×10⁻⁶. 7 PFU / mouse, injected every other day for a total of three times. By comparing the survival time of the two groups of mice, the efficacy and safety of recombinant oncolytic influenza virus rPR8-gpA33-CD3 oncolysis were preliminarily evaluated.

[0076] The results are as follows Figure 10 As shown in the figure. The results showed that the survival time of mice in the rPR8-gpA33-CD3 oncolytic therapy group was significantly prolonged.

[0077] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0078] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A recombinant oncolytic influenza virus, characterized in that, The recombinant oncolytic influenza virus is an influenza virus expressing anti-gpA33×CD3 bispecific antibodies, and its PB1 gene has been modified into the recombinant gene PB1-VH. gpA33 -VL CD3 Its PA gene was modified into the recombinant gene PA-VL. gpA33 -VH CD3 ; The recombinant gene PB1-VH gpA33 -VL CD3 The nucleotide sequence is that of the recombinant gene SEQ ID No. 5; The recombinant gene PA-VL gpA33 -VH CD3 The nucleotide sequence is that of the recombinant gene of SEQ ID No.

6.

2. The recombinant oncolytic influenza virus according to claim 1, characterized in that, The genome of the recombinant oncolytic influenza virus consists of eight single-stranded, segmented negative-strand RNAs. The negative-strand RNA of the recombinant oncolytic influenza virus is transcribed into a set of complementary positive-strand RNAs, including PB2-RNA and PB1-VH. gpA33 -VL CD3 -RNA, HA-RNA, PA-VL gpA33 -VH CD3 -RNA, NP-RNA, NA-RNA, M-RNA and NS-RNA; The PB2-RNA is the RNA encoding the PB2 gene in influenza virus strains; The PB1-VH gpA33 -VL CD3 -RNA encodes the recombinant gene PB1-VH as described in claim 1 gpA33 -VL CD3 RNA; The HA-RNA is the RNA encoding the HA gene in the influenza virus strain. The PA-VL gpA33 -VH CD3 -RNA encoding the recombinant gene PA-VL as described in claim 1 gpA33 -VH CD3 RNA; The NP-RNA is the RNA encoding the NP gene in the influenza virus strain; The M-RNA is the RNA encoding the M gene in the influenza virus strain; The NA-RNA is the RNA encoding the NA gene in the influenza virus strain; The NS-RNA is the RNA encoding the NS gene in the influenza virus strain.

3. The recombinant oncolytic influenza virus according to claim 1, characterized in that, The recombinant gene PB1-VH gpA33 -VL CD3 The sequence is defined as follows: the hydrolyzable Linker T2A gene sequence, nucleotides 2296-2346 of SEQ ID No. 5, inserted sequentially from 5' to 3' before the 3' NCR stop codon of the PB1 gene in the influenza virus strain; the Igκ signal peptide gene sequence, nucleotides 2347-2409 of SEQ ID No. 5; VH gpA33 Gene sequence, nucleotides 2410-2760 of SEQ ID No. 5; flexible linker (G4S)3 gene sequence, nucleotides 2761-2805 of SEQ ID No. 5; VL CD3 The sequence obtained was obtained by modifying the gene sequence (nucleotides 2806-3123 of SEQ ID No. 5), the antibody hinge region short peptide FNRGECC gene sequence (nucleotides 3124-3144 of SEQ ID No. 5), the hydrolyzable linker P2A gene sequence (nucleotides 3145-3210 of SEQ ID No. 5), and the PB1 gene 3'NCR end packaging signal sequence (nucleotides 3211-3374 of SEQ ID No. 5), with all other nucleotides remaining unchanged.

4. The recombinant oncolytic influenza virus according to claim 1, characterized in that, The recombinant gene PA-VL gpA33 -VH CD3 -The RNA sequence is the linker T2A gene sequence, nucleotides 2173-2223 of SEQ ID No. 6, inserted sequentially from 5' to 3' before the 3' NCR stop codon of the PA gene in the influenza virus strain; the Igκ signal peptide gene sequence, nucleotides 2224-2286 of SEQ ID No. 6; VL gpA33 Gene sequence, nucleotides 2287-2610 of SEQ ID No. 6; flexible linker (G4S)3 gene sequence, nucleotides 2611-2655 of SEQ ID No. 6; VH CD3 The sequence obtained was obtained by modifying the gene sequence (nucleotides 2656-3012 of SEQ ID No. 6), the antibody hinge region short peptide VEPKSCC gene sequence (nucleotides 3013-3033 of SEQ ID No. 6), the hydrolyzable linker P2A gene sequence (nucleotides 3034-3099 of SEQ ID No. 6), and the PA gene 3'NCR terminal packaging signal sequence (nucleotides 3100-3283 of SEQ ID No. 6), with all other nucleotides remaining unchanged.

5. The recombinant oncolytic influenza virus according to any one of claims 1 to 4, characterized in that, The influenza virus strain is either influenza A or influenza B.

6. The method for constructing recombinant oncolytic influenza virus according to any one of claims 1 to 4, characterized in that, A recombinant vector containing a DNA molecule encoding the PB2-RNA, and a vector containing a DNA molecule encoding the PA-VL... gpA33 -VH CD3 Recombinant vectors containing DNA molecules encoding HA-RNA, recombinant vectors containing DNA molecules encoding NP-RNA, recombinant vectors containing DNA molecules encoding M-RNA, recombinant vectors containing DNA molecules encoding NA-RNA, recombinant vectors containing DNA molecules encoding NS-RNA, and recombinant vectors containing DNA molecules encoding PB1-VH gpA33 -VL CD3 The recombinant vector of RNA-DNA molecules was introduced into packaging cells to obtain recombinant oncolytic influenza virus expressing bispecific antibodies against gpA33×CD3.

7. A drug for treating colorectal cancer, characterized in that, Its active ingredient is the recombinant oncolytic influenza virus as described in any one of claims 1 to 4.

8. Any of the following products, characterized in that, The product mentioned: (1) The genome of the recombinant oncolytic influenza virus according to any one of claims 1 to 4; (2) A complete set of vectors for treating tumors, namely, a vector containing the encoding gene of the anti-gpA33×CD3 bispecific antibody; (3) A complete expression cassette for treating tumors, namely, an expression cassette containing the gene encoding the anti-gpA33×CD3 bispecific antibody; (4) A complete set of genes for treating tumors, namely the encoding gene of the anti-gpA33×CD3 bispecific antibody; (5) A set of proteins for treating tumors, namely the anti-gpA33×CD3 bispecific antibody protein.

9. A biomaterial related to the recombinant oncolytic influenza virus according to any one of claims 1 to 4, characterized in that, The biomaterial is any of the following products: (1) A set of DNA molecules encoding the set of positive-sense RNA of any one of the recombinant oncolytic influenza viruses according to claims 1 to 4; the set of DNA molecules consists of a DNA molecule encoding the PB2-RNA and a DNA molecule encoding the PA-VL. gpA33 -VH CD3 -RNA DNA molecules that encode the PB1-VH gpA33 -VL CD3 It consists of a DNA molecule encoding NP-RNA, a DNA molecule encoding NA-RNA, a DNA molecule encoding HA-RNA, a DNA molecule encoding M-RNA, and a DNA molecule encoding NS-RNA; (2) A set of recombinant vectors; the set of recombinant vectors consists of a recombinant vector containing a DNA molecule encoding the PB2-RNA, and a recombinant vector containing a DNA molecule encoding the PA-VL. gpA33 -VH CD3 Recombinant vectors containing DNA molecules encoding HA-RNA, recombinant vectors containing DNA molecules encoding NP-RNA, recombinant vectors containing DNA molecules encoding M-RNA, recombinant vectors containing DNA molecules encoding NA-RNA, recombinant vectors containing DNA molecules encoding NS-RNA, and recombinant vectors containing DNA molecules encoding PB1-VH gpA33 -VL CD3 -The recombinant vector of RNA and DNA molecules; (3) Microorganisms containing any of the recombinant oncolytic influenza viruses described in claims 1 to 4; (4) Animal cells containing any one of the recombinant oncolytic influenza viruses according to claims 1 to 4; (5) Animal tissue containing any of the recombinant oncolytic influenza viruses according to claims 1 to 4; (6) Animal organs containing any of the recombinant oncolytic influenza viruses according to claims 1 to 4.

10. The following application, characterized in that, (1) The use of the recombinant oncolytic influenza virus according to any one of claims 1 to 4 in the preparation of a tumor treatment drug; (2) The use of the product of claim 8 in the preparation of a drug for treating tumors; (3) The use of the biomaterial described in claim 9 in the preparation of a drug for treating tumors.

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