CN117025555A - Oncolytic adenovirus expressing dual-specificity T cell adapter, construction method and application - Google Patents
Oncolytic adenovirus expressing dual-specificity T cell adapter, construction method and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to an oncolytic adenovirus for expressing a bispecific T cell adapter, a construction method and application thereof. The oncolytic adenovirus comprises oncolytic adenovirus E1A and E1B gene expression cassettes and a B7H3 xCD 3BiTE exogenous gene expression cassette. Adenovirus E1A is controlled by the human telomerase reverse transcriptase promoter; IRES is linked between the E1A and E1B genes, and IRES initiates the expression of the E1B gene. After the exogenous gene expression cassette is inserted into the adenovirus E1A and E1B gene expression cassettes, the exogenous gene is a fusion gene of B7H3scFv and CD3scFv, and the fusion gene is controlled by a constitutive strong promoter, such as CMV, mCMV, CAG promoter and the like. For simple gene therapy or oncolytic virus therapy, the oncolytic adenovirus constructed by the recombinant method obviously enhances the inhibition capability on malignant tumors.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an oncolytic adenovirus for expressing a bispecific T cell adapter, a construction method and application thereof.
Background
Oncolytic Viruses (OVs) are a class of natural or genetically modified viruses capable of specifically killing tumor cells, which are able to replicate preferentially in tumor cells, ultimately leading to tumor cell lysis, and activating anti-tumor immune responses. The onset of antitumor action of this virus in the body is generally thought to be by two mechanisms: firstly, oncolytic viruses are specifically replicated in tumor cells to cause the tumor cells to be directly lysed, and new viruses are released to infect and destroy other tumor cells after the tumor cells are lysed; secondly, oncolytic viruses release a large amount of tumor antigens after cracking tumor cells, and immune cells are activated through antigen presentation to generate anti-tumor response. Oncolytic virus tumor treatment technology is the leading direction of tumor immunotherapy and shows good safety and subversion curative effect in clinical trials. At present, representative products of oncolytic viruses are marketed in batches, and have great potential and application prospect. Common oncolytic viruses include herpes simplex virus, adenovirus, vesicular stomatitis virus, poxvirus, measles virus, and the like. The most common oncolytic virus type currently being developed for clinical trials is oncolytic adenovirus. As of 2022, at least four oncolytic viral therapies have been marketed worldwide by half a year, namely, rigvir (ECHO-7 virus), an Kerui (recombinant human adenovirus type 5), T-Vec (herpes simplex virus), delytact (herpes simplex virus), respectively. Oncolytic viruses exert an anti-tumor effect by specifically killing tumor cells and delivering tumor antigens. However, in addition to a large number of tumor cells, the tumor tissue contains a large number of interstitial cells, including tumor vascular endothelial cells, fibroblasts, suppressive immune cells and the like, and these cells form a tumor microenvironment to regulate the proliferation of the tumor cells. Therefore, oncolytic viral monotherapy has limited efficacy.
Initial studies of B7-H3, also known as CD276, showed that B7-H3 is capable of inducing expression on human DC cells and has the functions of stimulating proliferation of T cells, enhancing induction of cytotoxic T lymphocytes and secretion of gamma interferon. However, subsequent studies have reported that human B7-H3 is a T cell co-inhibitor molecule capable of significantly inhibiting secretion of IFN-gamma, IL-2, IL-10 and IL-13 during T cell activation. B7-H3 knockout or blocking antibody antagonism can promote T cell proliferation and inhibit tumor growth. B7-H3 is highly expressed in lung cancer, melanoma, pancreatic cancer, intestinal cancer, prostate cancer, ovarian cancer, renal cancer, glioma, head and neck tumor and other solid tumors and blood system tumors. As a pan-tumor antigen and a potential immunosuppressive regulatory molecule, in recent years, development of targeted therapeutic research against B7-H3 is one of the hot spots in the field of anti-tumor research.
Currently, B7-H3 targeted biological drugs entering the clinic mainly comprise Fc engineering antibodies, antibody coupling drugs, antibody coupling nuclides, CAR-T and other types. Among these, the radionuclide-labeled B7-H3 mab 8H9 (Omburtmaab) of Y-mAbs Therapeutics is rapidly developed and is used for treating neuroblastoma of central nervous system/pia mater metastasis of children, and due to the excellent clinical treatment effect, the European drug administration has been filed with the market application and is communicated with the FDA to supplement the market application data. Meanwhile, B7-H3 targeted ADC drugs also obtain encouraging curative effects, such as B7-H3 ADC drug DS-7300 developed by Daiichi Sankyo corporation of Japan, coupled toxin is DNA topoisomerase I inhibitor Dxd, clinical I/II data show that the drug has good safety on metastatic castration resistant prostate cancer and obtains definite encouraging curative effects; macrogeneics corporation also reported early clinical data at the ASCO and ESMO university of 2021, with their B7-H3 ADC drug MGC018, MGC018 conjugated toxin was a DNA synthesis inhibitor duocarmycin, and as a result, MGC018 was shown to have a controllable safety profile, and a reduction in metastases was observed for metastatic melanoma. The macrogeneics company also develops an Fc engineering antibody Enoblituzumab targeting B7-H3, and a clinical phase II test is also currently being carried out to evaluate the treatment effect of single drugs and combined PD-1 antibodies on various solid tumors.
Nisonoff in 1960 first proposed the concept of bispecific antibody (BsAb), and along with the rapid progress of related fields such as genetic engineering antibodies, immunology, etc., the construction concept, technical platform and product development of BsAb are also developing and innovating continuously. The bispecific antibody is an artificial antibody containing two specific antigen binding sites, can erect a bridge stress between target cells and functional molecules (cells), excite immune response with guidance, is one of genetic engineering antibodies, becomes a hotspot in the field of antibody engineering, and has wide application prospect in the immunotherapy of tumors. Bispecific T cell adapter (Bispecific T cell engagers, biTE) is a bispecific antibody, which is formed by fusion of scFv segments of two kinds of antibodies, and can be respectively combined with CD3 molecules or other activating factors on the surface of T cells and tumor cell surface antigen molecules, so that the T cells are induced to kill the tumor cells, and the process is not limited by MHC. At present, this therapy works well in hematological malignancies, one of which, biTE, blinatumomab, CD3 x CD19, has been approved by the FDA for treatment of B cell malignancies. However, biTE generally has a short half-life in serum and may have severe off-target toxicity. In addition, the use of BiTE is limited due to the physical barrier present in solid tumors and the immunosuppressive microenvironment.
Oncolytic viruses can be used as a platform for genetic engineering to express exogenous therapeutic genes. The oncolytic virus (OVs-BiTE) capable of specifically expressing the BiTE is developed, the self-replication and cell killing capacity of the OVs are utilized, the BiTE can be continuously expressed and secreted, and the OVs can specifically target tumor cells through specific genetic modification and transformation, so that more BiTE can permeate to tumor tissue parts, the off-target toxicity of the BiTE is reduced, the anti-tumor therapeutic capacity of the OVs is improved, and the limitations of oncolytic viruses and the BiTE can be effectively solved. There is no report of oncolytic adenoviruses expressing B7H3 xcd 3 BiTE.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. The invention aims to provide oncolytic adenoviruses expressing dual-specificity T cell adapters, which comprise oncolytic adenovirus E1A and E1B gene expression cassettes and a B7H3 xCD 3 BiTE exogenous gene expression cassette.
Further, the oncolytic adenovirus E1A is controlled by the human telomerase promoter hTERTP.
Still further, E1A is linked by an internal ribosome entry site IRES and initiates E1B expression, forming an oncolytic adenovirus of the hTERTP-E1A-IRES-E1B structure.
Further, the oncolytic adenovirus E1A and E1B gene expression cassettes comprise the following operably linked elements: human telomerase promoter hTERTp, E1A gene, IRES sequence, E1B gene, terminator.
Further, the nucleotide sequence of the human telomerase promoter hTERTP is shown as SEQ ID NO. 1.
The nucleotide sequence of the oncolytic adenovirus E1A gene is shown as SEQ ID NO. 2.
The amino acid sequence of the oncolytic adenovirus E1A gene is shown as SEQ ID NO. 3.
The IRES nucleotide sequence is shown as SEQ ID NO. 4.
The nucleotide sequence of the oncolytic adenovirus E1B gene is shown as SEQ ID NO. 5.
The amino acid sequences of E1B19K and E1B55K coded by the oncolytic adenovirus E1B gene are shown as SEQ ID NO. 6 and SEQ ID NO. 7.
The terminator of the oncolytic adenovirus E1A and E1B gene expression cassette is SV40 PolyA, and the nucleotide sequence is shown in SEQ ID NO. 8.
Further, the B7H3 xCD 3 BiTE exogenous gene comprises fusion genes of B7H3 scFv and CD3 scFv.
Wherein the B7H3 scFv comprises a B7H3 scFv light chain variable region coding sequence and a B7H3 scFv heavy chain variable region coding sequence.
Wherein the CD3 scFv comprises a CD3 scFv heavy chain variable region coding sequence and a CD3 scFv light chain variable region coding sequence.
Still further, the B7H3 xcd 3 BiTE exogenous gene expression cassette comprises the following operably linked elements: B7H3 scFv light chain variable region encoding sequence, B7H3 scFv heavy chain variable region encoding sequence, CD3 scFv light chain variable region encoding sequence.
Preferably, the B7H3 xcd 3 BiTE exogenous gene expression cassette comprises the following operably linked elements: a promoter, a signal peptide coding sequence, a B7H3 scFv light chain variable region coding sequence, a connecting sequence 1, a B7H3 scFv heavy chain variable region coding sequence, a connecting sequence 2, a CD3 scFv heavy chain variable region coding sequence, a connecting sequence 3, a CD3 scFv light chain variable region coding sequence and a terminator.
Further, the B7H3 xCD 3 BiTE exogenous gene expression cassette also comprises a Kozak sequence, and the promoter comprises a eukaryotic promoter. Preferably, the eukaryotic promoter is CMV, mCMV, CAG.
Further, the signal peptide is Ig kappa signal peptide, and the nucleotide sequence of the signal peptide is shown as SEQ ID NO. 9.
Further, the signal peptide is Ig kappa signal peptide, and the amino acid sequence of the signal peptide is shown as SEQ ID NO. 10;
further, the nucleotide sequence of the variable region of the B7H3 scFv light chain is shown as SEQ ID NO. 11 or SEQ ID NO. 12.
Further, the amino acid sequence of the variable region of the B7H3 scFv light chain is shown as SEQ ID NO. 13 or SEQ ID NO. 14.
Further, the nucleotide sequence of the heavy chain variable region of the B7H3 scFv is shown as SEQ ID NO. 15 or SEQ ID NO. 16;
further, the amino acid sequence of the heavy chain variable region of the B7H3 scFv is shown as SEQ ID NO. 17 or SEQ ID NO. 18.
Further, the connecting peptide between the light chain variable region and the heavy chain variable region of the B7H3 scFv is (G4S) 3, and the nucleotide sequence of the connecting peptide is shown as SEQ ID NO. 19.
Further, the amino acid sequence of the connecting peptide (G4S) 3 is shown as SEQ ID NO. 20.
Further, the nucleotide sequence of the CD3 scFv is shown as SEQ ID NO. 21.
Further, the amino acid sequence of the CD3 scFv is shown as SEQ ID NO. 22.
Further, the nucleotide sequence of the connecting peptide between the B7H3 scFv and the CD3 scFv is shown as SEQ ID NO. 23.
Further, the amino acid sequence of the connecting peptide between the B7H3 scFv and the CD3 scFv is shown as SEQ ID NO. 24.
The invention also provides a recombinant oncolytic adenovirus vector which is operably inserted into or contains the B7H3 xCD 3 BiTE exogenous gene expression cassette.
The invention also provides a construction method of the oncolytic adenovirus for expressing the dual-specificity T cell adapter, which comprises the following steps: cloning the synthesized E1A-E1B gene expression cassette into a shuttle vector to construct a plasmid containing E1A-E1B gene expression, cloning the synthesized B7H3 xCD 3 BiTE gene into the plasmid containing E1A-E1B gene expression, and packaging the recombinant adenovirus to obtain the recombinant oncolytic adenovirus expressing the B7H3 xCD 3 BiTE.
Preferably, the present invention also provides a construction method of the oncolytic adenovirus expressing the bispecific T cell adapter, comprising the steps of: E1A-E1B gene expression cassettes are synthesized, and E1A-E1B gene expression cassettes are cloned into shuttle vectors by using a Gibson cloning method to construct plasmids containing E1A-E1B gene expression, then B7H3 xCD 3 BiTE genes are cloned into pDC316 or pDC516 shuttle plasmids, pDC316 or pDC516 shuttle plasmids carrying B7H3 xCD 3 BiTE genes and skeleton plasmids of an AdMax adenovirus packaging system are co-transfected into HEK293 cells to carry out packaging of recombinant adenovirus, and thus the recombinant oncolytic adenovirus expressing B7H3 xCD 3 BiTE is obtained.
More preferably, the backbone plasmid comprises pBHGlox_E1,3Cre or pBHGfrt_E1,3Flp.
The invention also provides application of the oncolytic adenovirus or recombinant oncolytic adenovirus vector expressing the dual-specificity T cell adapter in preparing medicines for treating and/or preventing and/or assisting in treating cancers or tumors.
In the application, the medicine is a preparation prepared by taking oncolytic adenovirus or recombinant oncolytic adenovirus vector as an anticancer active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
Wherein the preparation comprises but is not limited to injection preparation and freeze-dried preparation.
Preferably, the formulation is an injectable formulation.
The cancer or tumor is a tumor with high expression of B7H3, and comprises at least one of solid tumor, childhood tumor or blood system tumor.
The solid tumor comprises at least one of melanoma, pancreatic cancer, intestinal cancer, prostate cancer, ovarian cancer, renal cancer, glioma or head and neck tumor.
The childhood susceptible neoplasm comprises at least one of neuroblastoma, medulloblastoma or osteosarcoma.
The beneficial effects are that: the invention discloses an oncolytic adenovirus expressing a dual-specificity T cell adapter, which is modified on the basis of a 5-type adenovirus genome and comprises oncolytic adenovirus E1A and E1B gene expression cassettes and a B7H3 xCD 3BiTE exogenous gene expression cassette. Oncolytic adenovirus E1A is controlled by a human telomerase promoter (hTERTP), and E1A is linked by an Internal Ribosome Entry Site (IRES) and initiates E1B expression, and the oncolytic virus is inserted into an expression cassette comprising a bispecific T cell adapter BiTE against B7H3, and is capable of continuously expressing a B7H3 xCD 3BiTE bifunctional antibody, blocking the tumor cell surface B7H3 molecule, and stimulating T lymphocyte activation to target killing B7H3 positive tumor cells. In vitro and animal experiments show that the bispecific T cell adapter is combined with the oncolytic virus, and compared with the simple gene therapy or oncolytic virus therapy, the oncolytic virus obviously enhances the inhibition capability on malignant tumors.
Drawings
FIG. 1 is a graph showing the results of the in vitro oncolytic capacity of oAd-B7H 3-BiTE; wherein, (a) oAd-B7H3-BiTE quantitatively detects the survival rate of tumor cells 72 hours after infection of HCT116 tumor cells with moi=0, 1,5, 10, 20; (B) oAd-B7H3-BiTE the survival rate of tumor cells was quantitatively determined by CCK8 after 72 hours of infection of SW480 tumor cells with MOI=0, 1,5, 10, 20;
FIG. 2 is a graph showing the blocking ability of B7H3-CD3 BiTE expressed after oAd-B7H3-BiTE infects tumor cells to B7H3 on the surface of tumor cells; wherein oAd-B7H3-BiTE infects (A) DLD-1, (B) RKO, (C) HCT116, (D) SW620 and (E) SW480 tumor cells with MOI=10, and after 48 hours, detecting the expression condition of B7H3 on the surface of the tumor cells by flow cytometry, and evaluating the blocking capacity of B7H3-CD3 BiTE expressed after oAd-B7H3-BiTE infects the tumor cells on the surface of the tumor cells;
FIG. 3 is a graph showing the results of the activation ability of B7H3-CD3 BiTE expressed after oAd-B7H3-BiTE infection of tumor cells to human T lymphocytes; (A) CD4 + CD25 + T cell ratio, (B) CD4 + CD69 + T cell ratio, (C) CD8 + CD25 + T cell ratio, (D) CD8 + CD69 + T cell ratio;
FIG. 4 is a graph showing the results of targeted killing effect of B7H3-CD3 BiTE expressed by oAd-B7H3-BiTE on tumor cells; (a) an LDH-cytotoxicity assay to detect cytotoxic killing effect of T cells on HCT116, (B) an LDH-cytotoxicity assay to detect cytotoxic killing effect of T cells on SW480, and (C) an LDH-cytotoxicity assay to detect cytotoxic killing effect of T cells on DLD-1.
FIG. 5 is a graph showing the proliferation potency of B7H3-CD3 BiTE expressed after oAd-B7H3-BiTE infects tumor cells to human T lymphocytes; (A) Fluorescence intensity of cd3+ T cell CFSE, (B) cd3+ T cell number;
FIG. 6 is a graph showing the results of the ability of B7H3-CD3 BiTE expressed by oAd-B7H3-BiTE to secrete cytokines into human T lymphocytes; (a) IFN- γ secretion status, b.il-2 secretion status;
FIG. 7 is a graph showing the effect of oAd-B7H3-BiTE on tumor-bearing mice; (a) dosing regimen, (B) tumor volume growth curve, (C) tumor-bearing mice tumor photograph, (D) tumor weight statistic;
FIG. 8 shows T cell infiltration in tumor tissue; (A) CD4 in tumor tissue after T cell injection alone + ，CD8 + T cell ratio; (B) Intratumoral administration of oAd-ON and tail vein injection of CD4 in tumor tissue after T + ，CD8 + T cell ratio; (C) The proportion of CD4+, CD8+ T cells in tumor tissue after intratumoral administration of oAd-FHA-BiTE and tail vein injection of T; (D) Intratumoral administration of oAd-B7H3-BiTE and tail vein injection of CD4 in tumor tissue after T + ，CD8 + T cell ratio; (E) Histogram shows oAd-B7H3-BiTE induces CD4 + T cell infiltration; (F) Histogram shows oAd-B7H3-BiTE induces CD8 + T cell infiltration;
fig. 9 shows infiltration of Tregs in tumor tissue; (A) The polychromatic flow chart reflects the proportion of Tregs cells in the tumor tissue, and the histogram (B) represents the proportion of Tregs cells in the tumor tissue.
Detailed Description
The inventor is dedicated to the development and optimization of oncolytic adenovirus, and the oncolytic adenovirus of recombinant exogenous therapeutic gene is obtained through intensive research and creative work. Specifically, the adenovirus E1A is controlled by a human telomerase reverse transcriptase promoter; IRES is linked between E1A and E1B genes, and IRES starts the expression of E1B gene. After the exogenous gene expression cassette is inserted into the adenovirus E1A and E1B gene expression cassettes, the exogenous gene is a fusion gene of B7H3 scFv and CD3 scFv, and the fusion gene is controlled by a constitutive strong promoter (such as CMV, mCMV, CAG promoter and the like).
The inventor finds that the oncolytic adenovirus has the capability of selectively proliferating and replicating in tumor cells by effectively controlling proliferation essential genes of the oncolytic adenovirus, namely adenovirus E1A and E1B through a tumor specific promoter, such as a human telomerase reverse transcriptase promoter, and specifically lyses the tumor cells; the oncolytic adenovirus can efficiently express exogenous genes B7H3 xCD 3 BiTE in tumor cells, can be respectively combined with CD3 molecules on the surfaces of the T cells and activate the T cells, and can be combined with B7H3 antigen molecules on the surfaces of the tumor cells to block the inhibition of the B7H3 molecules on the surfaces of the tumor cells to the T cells, so that the activated T cells are induced to target and kill the tumor cells, the anti-tumor immunity of an organism is stimulated, the anti-tumor efficacy is enhanced, and the anti-tumor effect is synergistically exerted. The recombinant oncolytic adenovirus provided by the invention can treat various malignant tumors.
In one embodiment of the invention, the B7H3 scFv single chain antibody is obtained by phage display technology. Hybridoma cells are obtained through immune animal experiments, cDNA is extracted from the hybridoma cells, heavy chain variable region VH and light chain variable region VL of the whole set of antibody are obtained through reverse transcription PCR (RT-PCR), and single chain antibody variable region gene fragments (scFv) are obtained through splicing and amplifying the VH fragments and the VL fragments through overlap PCR (SOE-PCR). Cloning scFv genes into a proper phage vector, electrically transforming competent escherichia coli, and performing secondary phage supercotaining to obtain a supernatant which is a single-chain antibody library. The specific antigen is used as solid phase, and the enriched single-chain antibody capable of being specifically combined with the antigen can be obtained through 3-5 rounds of 'adsorption-elution-amplification' screening. In certain embodiments, the nucleotide sequence of the light chain variable region (VL) of the B7H3 scFv is set forth in SEQ ID NO:11 and SEQ ID NO:12, the amino acid sequences are shown in SEQ ID NO:13 and SEQ ID NO: 14; the nucleotide sequence of the heavy chain variable region (VH) of the B7H3 scFv is set forth in SEQ ID NO:15 and SEQ ID NO:16, the amino acid sequence is shown as SEQ ID NO:17 and SEQ ID NO: shown at 18. B7H3 scFv antibodies include heavy chain variable domain sequences, light chain variable domain sequences, or CDR sequences, which may have at least 70% homology, at least 80% homology, at least 90% homology, at least 95% homology with the amino acid or nucleic acid sequences disclosed herein.
In one embodiment of the invention, there is provided an oncolytic adenovirus expressing B7H3 xcd 3 BiTE (oAd-B7H 3-BiTE), the oncolytic adenovirus E1A being controlled by a human telomerase promoter (hTERTp), and E1A being linked by an Internal Ribosome Entry Site (IRES) and initiating E1B expression, resulting in an oncolytic adenovirus of the hTERTp-E1A-IRES-E1B structure; the B7H3 XCD 3 BiTE exogenous gene expression cassette is inserted after the hTERTP-E1A-IRES-E1B expression cassette.
The oncolytic adenovirus carrying the B7H3 xCD 3 BiTE gene can be specifically and rapidly copied and proliferated in tumor cells to lyse the tumor cells; meanwhile, the oncolytic adenovirus can continuously express the B7H3 xCD 3 BiTE bifunctional antibody, can seal B7H3 molecules on the surface of tumor cells and stimulate T lymphocyte activation; oncolytic adenoviruses expressing B7H3 xcd 3 BiTE can target killing B7H3 positive tumor cells.
The invention relates to a recombinant oncolytic adenovirus vector, which structurally comprises the following characteristics:
the oncolytic adenovirus E1A and E1B gene expression cassette comprises the following operably linked elements: human telomerase promoter (hTERTp), E1A gene, IRES sequence, E1B gene, terminator.
The nucleotide sequence of the human telomerase promoter (hTERTP) is shown as SEQ ID NO. 1;
The nucleotide sequence of the oncolytic adenovirus E1A gene is shown as SEQ ID NO. 2;
the amino acid sequence of the oncolytic adenovirus E1A gene is shown as SEQ ID NO. 3;
the IRES nucleotide sequence is shown as SEQ ID NO. 4;
the nucleotide sequence of the oncolytic adenovirus E1B gene is shown as SEQ ID NO. 5;
the amino acid sequences of E1B19K and E1B55K coded by the oncolytic adenovirus E1B gene are shown as SEQ ID NO. 6 and SEQ ID NO. 7;
the nucleotide sequences of the oncolytic adenovirus E1A and E1B gene expression cassettes SV40 PolyA are shown in SEQ ID NO. 8.
In another aspect, the invention relates to a recombinant oncolytic adenoviral vector operably inserted or containing the following exogenous genes:
a B7H3 xcd 3 BiTE exogenous gene expression cassette comprising the following operably linked elements: a promoter, a signal peptide coding sequence, a B7H3 scFv light chain variable region coding sequence, a connecting sequence 1, a B7H3 scFv heavy chain variable region coding sequence, a connecting sequence 2, a CD3 scFv heavy chain variable region coding sequence, a connecting sequence 3, a CD3 scFv light chain variable region coding sequence and a terminator.
The B7H3 xCD 3 BiTE exogenous gene expression cassette also comprises a Kozak sequence, and the promoter comprises eukaryotic promoters such as CMV, mCMV, CAG and the like.
The signal peptide is Igkappa signal peptide, and the nucleotide sequence of the signal peptide is shown as SEQ ID NO. 9;
the signal peptide is Igkappa signal peptide, and the amino acid sequence of the signal peptide is shown as SEQ ID NO. 10;
the nucleotide sequence of the variable region of the B7H3 scFv light chain is shown as SEQ ID NO. 11 and SEQ ID NO. 12;
the amino acid sequence of the variable region of the B7H3 scFv light chain is shown as SEQ ID NO. 13 and SEQ ID NO. 14;
the nucleotide sequence of the variable region of the heavy chain of the B7H3 scFv is shown as SEQ ID NO. 15 and SEQ ID NO. 16;
the amino acid sequence of the heavy chain variable region of the B7H3 scFv is shown as SEQ ID NO. 17 and SEQ ID NO. 18;
the connecting peptide between the light chain variable region and the heavy chain variable region of the B7H3 scFv is (G4S) 3, and the nucleotide sequence of the connecting peptide is shown as SEQ ID NO. 19;
the connecting peptide between the light chain variable region and the heavy chain variable region of the B7H3 scFv is (G4S) 3, and the amino acid sequence of the connecting peptide is shown as SEQ ID NO. 20;
the nucleotide sequence of the CD3 scFv is shown as SEQ ID NO. 21;
the amino acid sequence of the CD3 scFv is shown as SEQ ID NO. 22;
the nucleotide sequence of the connecting peptide between the B7H3 scFv and the CD3 scFv is shown as SEQ ID NO. 23;
the amino acid sequence of the connecting peptide between the B7H3 scFv and the CD3 scFv is shown as SEQ ID NO. 24;
The invention also provides a construction method of the oncolytic adenovirus for expressing the dual-specificity T cell adapter, which comprises the following steps: E1A-E1B gene expression cassettes are synthesized, E1A-E1B gene expression cassettes are cloned into shuttle vectors by using a Gibson cloning method, plasmids containing E1A-E1B gene expression are constructed, then B7H23XCD3 BiTE genes are cloned into pDC316 or pDC516 shuttle plasmids, pDC316 or pDC516 shuttle plasmids carrying B7H23XCD3 BiTE genes and skeleton plasmids pBHGlox_E1,3Cre or pBHGfrt_E1,3Flp of an AdMax adenovirus packaging system are used for co-transfecting HEK293 cells to carry out packaging of recombinant adenovirus, and thus the recombinant oncolytic adenovirus for expressing B7H23XCD3 BiTE is obtained.
Another aspect of the invention relates to the use of a recombinant oncolytic adenoviral vector or a recombinant oncolytic adenovirus according to the invention for the preparation of a medicament for the treatment and/or prevention and/or co-treatment of cancer or tumors.
The cancer or tumor is B7H3 high expression tumor, including lung cancer, melanoma, pancreatic cancer, intestinal cancer, prostatic cancer, ovarian cancer, renal cancer, glioma, head and neck tumor and other solid tumors, neuroblastoma, medulloblastoma, osteosarcoma and other tumors which are easy to occur in children, and even blood system tumor.
The nucleotide or amino acid sequences related to the present invention are as follows
SEQ ID NO. 1 human telomerase promoter (hTERTP) nucleotide sequence
TGGCCCCTCCCTCGGGTTACCCCACAGCCTAGGCCGATTCGACCTCTCTCCGCTGGGGCCCTCGCTGGCGTCCCTGCACCCTGGGAGCGCGAGCGGCGCGCGGGCGGGGAAGCGCGGCCCAGACCCCCGGGTCCGCCCGGAGCAGCTGCGCTGTCGGGGCCAGGCCGGGCTCCCAGTGGATTCGCGGGCACAGACGCCCAGGACCGCGCTTCCCACGTGGCGGAGGGACTGGGGACCCGGGCACCCGTCCTGCCCCTTCACCTTCCAGCTCCGCCTCCTCCGCGCGGACCCCGCCCCGTCCCGACCCCTCCCGGGTCCCCGGCCCAGCCCCCTCCGGGCCCTCCCAGCCCCTCCCCTTCCTTTCCGCGGCCCCGCCCTCTCCTCGCGGCGCGAGTTTCAGGCAGCGCTGCGTCCTGCTGCGCACGTGGGAAGCCCTGGCCCCGGCCACCCCCGCG
Nucleotide sequence of oncolytic adenovirus E1A gene of SEQ ID NO 2
ATGAGACATATTATCTGCCACGGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAAGAGGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGACGTGACGGCCCCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTCCCGACTCTGTAATGTTGGCGGTGCAGGAAGGGATTGACTTACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCTCACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTTGGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCTTACCTGCCACGAGGCTGGCTTTCCACCCAGTGACGACGAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGGAGCACCCCGGGCACGGTTGCAGGTCTTGTCATTATCACCGGAGGAATACGGGGGACCCAGATATTATGTGTTCGCTTTGCTATATGAGGACCTGTGGCATGTTTGTCTACAGTAAGTGAAAATTATGGGCAGTGGGTGATAGAGTGGTGGGTTTGGTGTGGTAATTTTTTTTTTAATTTTTACAGTTTTGTGGTTTAAAGAATTTTGTATTGTGATTTTTTTAAAAGGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAGCCAGAACCGGAGCCTGCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGCCCGACATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGAGTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGCCTGGGCAACCTTTGGACTTGAGCTGTAAACGCCCCAGGCCATAA
SEQ ID NO. 3 amino acid sequence of oncolytic adenovirus E1A gene
MRHIICHGGVITEEMAASLLDQLIEEVLADNLPPPSHFEPPTLHELYDLDVTAPEDPNEEAVSQIFPDSVMLAVQEGIDLLTFPPAPGSPEPPHLSRQPEQPEQRALGPVSMPNLVPEVIDLTCHEAGFPPSDDEDEEGEEFVLDYVEHPGHGCRSCHYHRRNTGDPDIMCSLCYMRTCGMFVYSPVSEPEPEPEPEPEPARPTRRPKMAPAILRRPTSPVSRECNSSTDSCDSGPSNTPPEIHPVVPLCPIKPVAVRVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP
The E1A gene is transcribed into RNA and the intron sequences are excised and translated into protein.
SEQ ID NO. 4IRES nucleotide sequence
GCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACC
Nucleotide sequence of 5 oncolytic adenovirus E1B gene of SEQ ID NO
ATGGAGGCTTGGGAGTGTTTGGAAGATTTTTCTGCTGTGCGTAACTTGCTGGAACAGAGCTCTAACAGTACCTCTTGGTTTTGGAGGTTTCTGTGGGGCTCATCCCAGGCAAAGTTAGTCTGCAGAATTAAGGAGGATTACAAGTGGGAATTTGAAGAGCTTTTGAAATCCTGTGGTGAGCTGTTTGATTCTTTGAATCTGGGTCACCAGGCGCTTTTCCAAGAGAAGGTCATCAAGACTTTGGATTTTTCCACACCGGGGCGCGCTGCGGCTGCTGTTGCTTTTTTGAGTTTTATAAAGGATAAATGGAGCGAAGAAACCCATCTGAGCGGGGGGTACCTGCTGGATTTTCTGGCCATGCATCTGTGGAGAGCGGTTGTGAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATAATACCGACGGAGGAGCAGCAGCAGCAGCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGCCCATGGAACCCGAGAGCCGGCCTGGACCCTCGGGAATGAATGTTGTACAGGTGGCTGAACTGTATCCAGAACTGAGACGCATTTTGACAATTACAGAGGATGGGCAGGGGCTAAAGGGGGTAAAGAGGGAGCGGGGGGCTTGTGAGGCTACAGAGGAGGCTAGGAATCTAGCTTTTAGCTTAATGACCAGACACCGTCCTGAGTGTATTACTTTTCAACAGATCAAGGATAATTGCGCTAATGAGCTTGATCTGCTGGCGCAGAAGTATTCCATAGAGCAGCTGACCACTTACTGGCTGCAGCCAGGGGATGATTTTGAGGAGGCTATTAGGGTATATGCAAAGGTGGCACTTAGGCCAGATTGCAAGTACAAGATCAGCAAACTTGTAAATATCAGGAATTGTTGCTACATTTCTGGGAACGGGGCCGAGGTGGAGATAGATACGGAGGATAGGGTGGCCTTTAGATGTAGCATGATAAATATGTGGCCGGGGGTGCTTGGCATGGACGGGGTGGTTATTATGAATGTAAGGTTTACTGGCCCCAATTTTAGCGGTACGGTTTTCCTGGCCAATACCAACCTTATCCTACACGGTGTAAGCTTCTATGGGTTTAACAATACCTGTGTGGAAGCCTGGACCGATGTAAGGGTTCGGGGCTGTGCCTTTTACTGCTGCTGGAAGGGGGTGGTGTGTCGCCCCAAAAGCAGGGCTTCAATTAAGAAATGCCTCTTTGAAAGGTGTACCTTGGGTATCCTGTCTGAGGGTAACTCCAGGGTGCGCCACAATGTGGCCTCCGACTGTGGTTGCTTCATGCTAGTGAAAAGCGTGGCTGTGATTAAGCATAACATGGTATGTGGCAACTGCGAGGACAGGGCCTCTCAGATGCTGACCTGCTCGGACGGCAACTGTCACCTGCTGAAGACCATTCACGTAGCCAGCCACTCTCGCAAGGCCTGGCCAGTGTTTGAGCATAACATACTGACCCGCTGTTCCTTGCATTTGGGTAACAGGAGGGGGGTGTTCCTACCTTACCAATGCAATTTGAGTCACACTAAGATATTGCTTGAGCCCGAGAGCATGTCCAAGGTGAACCTGAACGGGGTGTTTGACATGACCATGAAGATCTGGAAGGTGCTGAGGTACGATGAGACCCGCACCAGGTGCAGACCCTGCGAGTGTGGCGGTAAACATATTAGGAACCAGCCTGTGATGCTGGATGTGACCGAGGAGCTGAGGCCCGATCACTTGGTGCTGGCCTGCACCCGCGCTGAGTTTGGCTCTAGCGATGAAGATACAGATTGA
SEQ ID NO. 6 oncolytic adenovirus E1B gene encoded E1B19K amino acid sequence
MEAWECLEDFSAVRNLLEQSSNSTSWFWRFLWGSSQAKLVCRIKEDYKWEFEELLKSCGELFDSLNLGHQALFQEKVIKTLDFSTPGRAAAAVAFLSFIKDKWSEETHLSGGYLLDFLAMHLWRAVVRHKNRLLLLSSVRPAIIPTEEQQQQQEEARRRRQEQSPWNPRAGLDPRE
SEQ ID NO. 7 oncolytic adenovirus E1B gene encoded E1B55K amino acid sequence
MERRNPSERGVPAGFSGHASVESGCETQESPATVVFRPPGDNTDGGAAAAAGGSQAAAAGAEPMEPESRPGPSGMNVVQVAELYPELRRILTITEDGQGLKGVKRERGACEATEEARNLAFSLMTRHRPECITFQQIKDNCANELDLLAQKYSIEQLTTYWLQPGDDFEEAIRVYAKVALRPDCKYKISKLVNIRNCCYISGNGAEVEIDTEDRVAFRCSMINMWPGVLGMDGVVIMNVRFTGPNFSGTVFLANTNLILHGVSFYGFNNTCVEAWTDVRVRGCAFYCCWKGVVCRPKSRASIKKCLFERCTLGILSEGNSRVRHNVASDCGCFMLVKSVAVIKHNMVCGNCEDRASQMLTCSDGNCHLLKTIHVASHSRKAWPVFEHNILTRCSLHLGNRRGVFLPYQCNLSHTKILLEPESMSKVNLNGVFDMTMKIWKVLRYDETRTRCRPCECGGKHIRNQPVMLDVTEELRPDHLVLACTRAEFGSSDEDTD
Transcription of the E1B gene into RNA also cleaves intron sequences and then translates into protein.
SEQ ID NO. 8SV40 PolyA nucleotide sequence
CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA
Nucleotide sequence of SEQ ID NO 9Ig kappa Signal peptide
ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC
Amino acid sequence of SEQ ID NO 10Ig kappa Signal peptide
METDTLLLWVLLLWVPGSTGD
SEQ ID NO. 11B7H3 scFv light chain variable region nucleotide sequence (1)
GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCATTCACGTTCGGCTCGGGTACCAAGGTGGAGATCAAG
SEQ ID NO. 12B7H3 scFv light chain variable region nucleotide sequence (2)
CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCCTCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGTTCAAGTGTAAGTCTCATGCACTGGTACCAGCAGAAGTCGGACACCTCCCCCAAAAGATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTCGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAGCAGTGGAGTGATAACCCGCTCACGTTCGGTGCTGGTACCAAGGTGGAGATCAAG
SEQ ID NO. 13B7H3 scFv light chain variable region amino acid sequence (1)
DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPFTFGSGTKVEIK
SEQ ID NO. 14B7H3 scFv light chain variable region amino acid sequence (2)
QIVLTQSPAIMSASPGEKVTMTCSASSSVSLMHWYQQKSDTSPKRWIYDTSKLASGVPARFSGSRSGTSYSLTISSMEAEDAATYYCQQWSDNPLTFGAGTKVEIK
15B7H3 scFv heavy chain variable region nucleotide sequence (1)
GAGGTGCAGCTGCAGGAGTCTGGAGCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGACTTCTGGCTACACCTTCACCAACTACTGGATTCAGTGGATAAAACAGAGGCCTGGACAGGGCCTTGGATGGATTGGAGAGATATTTCCTGTAACTGGCACTACTTACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTATAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCTGTCTATTTCTGTGCAAGAACGGGGACAGCTCGGGCTCAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCGAGT
SEQ ID NO. 16B7H3 scFv heavy chain variable region nucleotide sequence (2)
GAGGTGCAGCTGCAGGAGTCTGGACCTGAGAAGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACCATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAACAGTGGTGGTACTACCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAACTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTACAAGAGTGGGGGGACCATGGCCCACGACGAGGGGTATGGACTACTGGGGTGAAGGAACCTCAGTCACCGTCTCGAGT
SEQ ID NO:17B7H3 scFv heavy chain variable region amino acid sequence (1)
EVQLQESGAELVKPGASVKLSCKTSGYTFTNYWIQWIKQRPGQGLGWIGEIFPVTGTTYYNEKFKGKATLTIDTSSSTAYMQLSSLTSEDSAVYFCARTGTARAQFAYWGQGTLVTVSS
SEQ ID NO. 18B7H3 scFv heavy chain variable region amino acid sequence (2)
EVQLQESGPEKVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKSLEWIGGINPNSGGTTYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCTRVGGPWPTTRGMDYWGEGTSVTVSS
SEQ ID NO. 19B7H3 scFv connecting peptide (G4S) 3 nucleotide sequence between light chain variable region and heavy chain variable region
GGCGGCGGCGGCTCCGGAGGAGGAGGAAGCGGAGGAGGCGGCAGC
The connecting peptide (G4S) 3 amino acid sequence between the 20B7H3 scFv light chain variable region and the heavy chain variable region of SEQ ID NO
GGGGSGGGGSGGGGS
Nucleotide sequence of SEQ ID NO:21CD3 scFv
The nucleotide sequence of GACATCAAGCTGCAGCAGTCAGGGGCTGAACTGGCCAGGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGACCTCTGGCTACACCTTCACCAGATACACCATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGATACATTAACCCTTCTAGAGGCTATACTAACTACAATCAAAAGTTCAAGGACAAGGCCACATTGACTACCGACAAGTCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCCAGATATTACGACGACCACTATTGCCTGGACTACTGGGGCCAAGGCACCACGCTGACCGTCAGCAGCGTGGAGGGCGGTTCAGGCGGAAGCGGCGGGAGCGGTGGCAGCGGAGGCGTGGACGACATCCAGCTGACCCAGAGCCCAGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGTAGGGCCAGCTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGAGCGGTACCAGCCCAAAGAGATGGATCTACGACACATCCAAGGTGGCTTCTGGTGTGCCATACAGATTCAGCGGTAGCGGTAGCGGTACCAGCTACAGCCTCACCATCAGCAGCATGGAGGCTGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGCGCTGGGACCAAGCTGGAACTGAAACD scFv is CD3 scFv heavy chain variable region coding sequence, connecting sequence 1 and CD3 scFv light chain variable region coding sequence, wherein, the connecting sequence 1 is connecting peptide (G2S) 4.
Amino acid sequence of SEQ ID NO:22CD3 scFv
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK
Nucleotide sequence of the connecting peptide between the SEQ ID NO: 237H 3 scFv and the CD3 scFv
GAATTCAGCAGCGGCGGAGGCGGAAGC
Amino acid sequence of the connecting peptide between the 24B7H3 scFv and the CD3 scFv
EFSSGGGGS
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 construction of recombinant oncolytic adenovirus expressing B7H3 XCD 3 BiTE (oAd-B7H 3-BiTE)
(1) The constructed plasmids were named pDC316-hTERTP-E1AE1B or pDC516-hTERTP-E1AE1B according to the combination of hTERTP (shown in SEQ ID NO: 1), E1A gene (shown in SEQ ID NO: 2), IRES sequence (shown in SEQ ID NO: 4), E1B gene (shown in SEQ ID NO: 5) and SV40 PolyA terminator (shown in SEQ ID NO: 8) as the E1A-E1B gene expression cassettes, i.e., SEQ ID NO:1+SEQ ID NO:2+SEQ ID NO:4+SEQ ID NO:5+SEQ ID NO:8, and cloned into shuttle vectors such as pDC316 or pDC516 by Gibson cloning.
(2) The sequence of the B7H3 xCD 3 BiTE gene (SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:19 and SEQ ID NO:21, SEQ ID NO: 23) was synthesized based on the total gene sequence of the B7H3 xCD 3 BiTE gene, i.e., the combination of SEQ ID NO:9+SEQ ID NO:12+SEQ ID NO:19+SEQ ID NO:16+SEQ ID NO:23+SEQ ID NO:21, and the B7H3 xCD 3 BiTE gene was cloned into pDC316-hTERTP-E1AE1B or pDC516-hTERTP-E1AE1B vector by the molecular cloning method, and the successfully constructed plasmid was named pDC316-E1AE1B-B7H3-BiTE or pDC516-E1AE1B-B7H3-BiTE. Meanwhile, a fusion gene of pertussis Filamentous Hemagglutinin (FHA) scFv and CD3 scFv is synthesized, and is taken as a control BiTE to be named as FHA-BiTE, wherein the nucleotide sequence of the FHA-BiTE is as follows: SEQ ID NO. 25:
CAAGTGCAGCTGCAGCAGCCCGGCAGCGAGCTGGTGAGACCCGGCGCTAGCGTGAAGCTGAGCTGCAAGGCTAGCGGCTACAAGTTCACAAGCTACTGGATGCACTGGGTGAAGCAGAGACCCGGCCAAGGCCTGGAGTGGATCGGCAACATCTTCCCCGGCAGCGGGAGCACCAATTACGACGAGAAGTTCAACAGCAAGGCCACCCTCACCGTGGACACAAGCAGCAACACCGCCTACATGCAGCTGAGCAGCCTGACAAGCGAGGACAGCGCCGTGTACTACTGCACAAGATGGCTGAGCGGCGCCTACTTCGACTATTGGGGCCAAGGCACAACCCTGACAGTGAGCAGCGGCGGCGGGGGCTCCGGCGGGGGCGGCAGCGGCGGGGGCGGCAGCCAAATTGTGCTGACACAGAGCCCCGCCCTGATGAGCGCTAGCCCCGGCGAGAAGGTGACCATGACCTGCAGCGCTAGCAGCAGCGTGAGCTTCATGTACTGGTATCAGCAGAAGCCTAGAAGCAGCCCCAAGCCCTGGATCTACCTGACAAGCAACCTGCCTAGCGGCGTGCCCGCTAGATTCAGCGGCAGCGGCAGCGGCACATCCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGTCAGCAGTGGAGCAGCCACCCCCCCACCTTCGGGTCCGGCACCAAGCTGGAGATCAAGGGCGGAGGCGGAAGCGACATCAAGCTGCAGCAGTCAGGGGCTGAACTGGCCAGGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGACCTCTGGCTACACCTTCACCAGATACACCATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGATACATTAACCCTTCTAGAGGCTATACTAACTACAATCAAAAGTTCAAGGACAAGGCCACATTGACTACCGACAAGTCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCCAGATATTACGACGACCACTATTGCCTGGACTACTGGGGCCAAGGCACCACGCTGACCGTCAGCAGCGTGGAGGGCGGTTCAGGCGGAAGCGGCGGGAGCGGTGGCAGCGGAGGCGTGGACGACATCCAGCTGACCCAGAGCCCAGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGTAGGGCCAGCTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGAGCGGTACCAGCCCAAAGAGATGGATCTACGACACATCCAAGGTGGCTTCTGGTGTGCCATACAGATTCAGCGGTAGCGGTAGCGGTACCAGCTACAGCCTCACCATCAGCAGCATGGAGGCTGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGCGCTGGGACCAAGCTGGAACTGAAA. The specific experimental steps are as follows:
1) Firstly, sal I restriction enzyme is utilized to cut plasmids pUC-B7H3-BiTE or pUC-FHA-BiTE with B7H3 xCD 3 BiTE or FHA xCD 3 BiTE genes, agarose gel electrophoresis is followed by gel cutting to recover B7H3-BiTE or FHA-BiTE gene fragments.
2) pDC316-hTERTP-E1AE1B or pDC516-hTERTP-E1AE1B plasmid is digested with Sal I restriction enzyme, agarose gel electrophoresis is followed by gel cutting to recover pDC316-hTERTP-E1AE1B or pDC516-hTERTP-E1AE1B vector fragment.
3) 50ng of the digested pDC316-hTERTP-E1AE1B or pDC516-hTERTP-E1AE1B vector fragment and 3 times of the molar amount of B7H3-BiTE or FHA-BiTE insert were taken, the total volume was adjusted to 10. Mu.L by double evaporation, 10. Mu.L of 2 Xligation buffer solution and 1. Mu. L T4 DNA ligase were added, and DH 5. Alpha. Competent cells were transformed after 10 minutes reaction at room temperature.
4) The next day clones were picked and verified by colony PCR, and clones positive for PCR results were shaken and plasmids were extracted for restriction verification, and restriction correct plasmid sequencing was further verified. The plasmids with correct sequencing were designated pDC316-E1AE1B-B7H3-BiTE or pDC516-E1AE1B-B7H3-BiTE, and the control BiTE shuttle plasmids were designated pDC316-E1AE1B-FHA-BiTE or pDC516-E1AE1B-FHA-BiTE.
(3) Packaging of recombinant adenoviruses
1) And co-transfecting HEK293 cells with the constructed shuttle plasmids pDC316-E1AE1B-B7H3-BiTE or pDC516-E1AE1B-B7H3-BiTE carrying the B7H3-BiTE genes respectively by contrast with the BiTE shuttle plasmids pDC316-E1AE1B-FHA-BiTE or pDC516-E1AE1B-FHA-BiTE and skeleton plasmids pBHGlox_E1,3Cre or pBHGfrt_E1 and 3Flp of an AdMax adenovirus system to package recombinant adenovirus. The process is as follows:
1) Will be 0.8-1×10 6 The HEK293 cells were inoculated into six-well plates, high-sugar DMEM+10% FBS medium, and placed at 37℃with 5% CO 2 The cells were cultured overnight in a cell incubator.
2) The following day HEK293 cells were co-transfected with 4. Mu.g of backbone plasmid (pBHGlox_E1, 3Cre or pBHGfrt_E1,3 Flp) and 2. Mu.g of shuttle plasmid using lipofectamine3000, with high sugar DMEM+2% FBS. The cells were observed daily for signs of cell toxicity and when they were full, they were introduced at 25cm 2 In a cell culture flask, 75cm of cells are introduced after the cells grow fully 2 In the cell culture flask, the cell is subjected to toxin collection until obvious plaques appear on the cell, and most of the cells are diseased and fall off from the bottom.
3) The virus-free cell culture was collected, centrifuged at 1200rpm for 3 minutes, the virus-containing supernatant was aspirated, the cell pellet was resuspended in 1/10 of the culture volume of the virus-containing supernatant, and the suspension was repeatedly freeze-thawed three times in a-80℃refrigerator and 37℃water bath. And (3) centrifuging at 3000rpm for 20 minutes, collecting the supernatant containing the viruses, and combining the supernatant with the supernatant containing the viruses, thus obtaining the virus seed of the adenovirus vaccine.
4) Taking 50 mu L of vaccine candidate strain virus seed liquid, adding 2 mu L of proteinase K, digesting for 30min at 50 ℃ to release virus genome, taking the virus genome as a template for PCR amplification of target genes, and sequencing and identifying after the recovery of PCR product electrophoresis gel. Identifying the correct target virus named oAd-B7H3-BiTE and comparing the target virus named oAd-FHA-BiTE; oAd-FHA-BiTE is an oncolytic adenovirus of control BiTE that expresses pertussis Filamentous Hemagglutinin (FHA) scFv and CD3 scFv, only CD3 scFv being active, without B7H3 targeting.
Example 2, oAd-B7H3-BiTE in vitro oncolytic Capacity
The tumor-dissolving capacity of oAd-B7H3-BiTE to tumor cells in vitro is detected by using a CCK8 method, and the specific experimental steps are as follows: 1.5X10 4 Inoculating HCT116 or SW480 tumor cells into a 96-well plate, wherein each well comprises 100 mu L of each well and 3 compound wells, and infecting the tumor cells with oAd-B7H3-BiTE, oAd-FHA-BiTE and oAd according to MOI=0, 1,5, 10 and 20 respectively after cell inoculation and culture for 24 hours; after 72h of virus infection of tumor cells, 10% CCK8 was prepared with complete medium, the original culture well liquid was discarded, 100 μl was added per well, incubated at 37 ℃ for 30 min, absorbance was detected at 450nm, and cell viability was calculated according to the following formula:
cell viability (%) = [ (As-Ab)/(Ac-Ab) ]x100, as = experimental well absorbance (absorbance of wells containing cells, medium, CCK-8 and test compound), ab = blank well absorbance (absorbance of wells containing medium and CCK-8), ac = control well absorbance (absorbance of wells containing cells, medium and CCK-8). The results show that: along with the increase of MOI values, the survival rate of tumor cells after being infected by oAd-B7H3-BiTE, oAd-FHA-BiTE and oAd viruses is obviously reduced; the oncolytic capacity of the HCT116 tumor cells of the oAd-B7H3-BiTE, oAd-FHA-BiTE and the control virus oAd pair is not obviously different; oAd-FHA-BiTE has no obvious difference with the control virus oAd on the oncolytic capacity of SW480 tumor cells, while oAd-B7H3-BiTE has slightly reduced oncolytic capacity on SW480 tumor cells, and the specific result is shown in figure 1.
EXAMPLE 3 oAd-B7H3-BiTE blocking ability of B7H3 on tumor cell surface by expressed B7H3-CD3BiTE after infection of tumor cells
HCT116, SW480, RKO, SW620, DLD-1 cells were used at 1X 10 5 The cells/wells were seeded in 24-well plates at 37℃with 5% CO 2 Culturing overnight in incubator; oAd-B7H3-BiTE infected 5 cells with MOI=10, leaving one well untreated as a negative control; at 37 ℃,5% CO 2 Culturing in incubator for 48 hr and collecting the cells; after washing the cells twice with 1 XPBS solution, 100. Mu.LCell pellet was resuspended in 1 XPBS solution, and after incubation for 30min in the dark with APC-B7H3 flow antibody, the cells were washed twice with 1 XPBS solution and assayed for tumor cell B7H3 expression on-press. The experimental results show that: after oAd-B7H3-BiTE infection of various tumor cells, the expression rate of B7H3 protein on the surface of HCT116 tumor cells is 24.13%, the expression rate of B7H3 protein on the surface of SW620 tumor cells is 31.18%, the expression rate of B7H3 protein on the surface of DLD-1 tumor cells is 28.49%, the expression rate of B7H3 protein on the surface of SW480 tumor cells is 41.77%, and the expression rate of B7H3 protein on the surface of RKO tumor cells is 57.18%. It was found that B7H3-CD3BiTE expressed after oAd-B7H3-BiTE infection of B7H3 positive tumor cells can bind to B7H3 tumor cells, thereby blocking binding of APC-B7H3 streaming antibody to B7H3 indicated by tumor cells, indicating that B7H3-CD3BiTE expressed after oAd-B7H3-BiTE infection of tumor cells can block B7H3 on the surface of tumor cells, and the specific results are shown in FIG. 2.
EXAMPLE 4 activation of T lymphocytes by B7H3-CD3 BiTE expressed after infection of tumor cells by oAd-B7H3-BiTE
Viruses oAd-B7H3-BiTE, oAd-FHA-BiTE and oAd were infected with MOI 10 for 293A for 48H, and supernatants were collected and stored at-20deg.C for further use. Will be 1.5X10 4 The HCT116 tumor cells were then plated in 96-well plates, followed by infection of the tumor cells with 100. Mu.L of viral supernatant followed by addition of 7.5X10 4 T cells (E: t=5:1), CD3/CD28 antibody treated group served as positive control group. Cells were collected after day 4, and expression of CD3, CD4, CD8, CD25 and CD69 was examined by flow cytometry to assess T activation. The research results show that: after CD3/CD28 antibody treatment, CD4 + CD25 + T cells, CD4 + CD69 + T cells, CD8 + CD25 + T cells and CD8 + CD69 + The proportion of T cells is increased, and the T cells are activated to a certain extent. CD4 in T cells stimulated with supernatant of oAd, oAd-FHA-BiTE and oAd-B7H3-BiTE viruses compared to untreated group + CD25 + T cells, CD4 + CD69 + T cells, CD8 + CD25 + T cells and CD8 + CD69 + The proportion of T cells was elevated and compared to oAd, oAd-FHA-BiTE and oAd-B7H3-BiTE and supernatant stimulated CD4 in T cells + CD25 + T cells, CD4 + CD69 + T cells, CD8 + CD25 + T cells and CD8 + CD69 + The proportion of T cells was significantly increased, but there was no significant difference in activation of T cells between the two groups of oAd-FHA-BiTE and oAd-B7H3-BiTE, suggesting that both oAd-FHA-BiTE and oAd-B7H3-BiTE expressed BiTE were able to stimulate T cell activation, and the specific results are shown in FIG. 3. Wherein, FIG. 3 is a graph showing the evaluation of BiTE-mediated T cell activation in different viral supernatants by multicolor flow analysis of T cell surface CD4, CD8, CD25 and CD69 expression levels when CD3+ T effector cells (E) were co-cultured with HCT116 target cells (T) at E:T=5:1 and BiTE-containing oncolytic virus supernatants for 24h, 48h and 96 h.
EXAMPLE 5, oAd-B7H 3-CD3 BiTE expressed B7H3-CD3 BiTE against tumor cell-targeted killing Effect
2×10 4 Inoculating tumor cells into 96-well plate, arranging 3 multiple wells each, culturing for 24 hr, adding 1×10 5 Individual T lymphocytes, lymphocytes as effector cells (E), effector cells (E): target cells (T) =5:1, then oAd-B7H3-BiTE, oAd-FHA-BiTE, oAd virus supernatant 10 μl each, untreated group served as control. After further incubation at 37℃for 24 hours, 500g of the 96-well plate was centrifuged for 5 minutes, the culture supernatant was aspirated, and the LDH content in the cell culture supernatant was measured using an LDH-cytotoxicity detection kit (Promega) to evaluate the targeted killing effect of oAd-B7H3-BiTE on tumor cells. The experimental results show that: the cytotoxicity of T cells was most pronounced in the supernatant containing oAd-B7H3-BiTE virus, oAd-FHA-BiTE mediated cytotoxicity was much smaller than in the oAd-B7H3-BiTE group, while the killing of T cells induced by the supernatant containing oAd virus was similar to the killing of tumor cells by T cells alone, as shown in FIG. 4, wherein CD3+ T effector cells (E) were co-cultured with tumor target cells (T) at E:T=5:1 and with BiTE oncolytic virus-containing supernatant for 24H, and the LDH content in the cell culture supernatant was measured using an LDH-cytotoxicity assay kit to evaluate the targeted killing effect of oAd-B7H3-BiTE on tumor cells.
Example 6, oAd Effect of B7H3-CD3 BiTE expressed by B7H3-BiTE on proliferation of human T lymphocytes
Tumor cells HCT116 at 1X 10 5 Individual cells/well were seeded into 24-well plates at 37 ℃,5% CO 2 Applying the mixture in a box overnight; t cells with good status were taken at 1: CFSE was added at a ratio of 1000-2000 (V/V) and incubated at 37℃in the absence of light. After incubation for 30min, the labeling was stopped. Infecting oAd-B7H3-BiTE, oAd-FHA-BiTE, oAd-ON with tumor cells, followed by adding 7.5X10 4 T cells (E: t=5:1), CD3/CD28 antibody treated group served as positive control group. Cells were collected on day 4 and fluorescence intensity of cd3+ T cell CFSE was detected by flow cytometry. The research results show that: compared with the control virus oAd-ON, the fluorescence intensity of CFSE of the T cells of the oAd-B7H3-BiTE and oAd-FHA-BiTE treatment group is obviously reduced, the number of CD3+ T cells is obviously increased, but the conditions of the T cells stimulated by oAd-B7H3-BiTE and oAd-FHA-BiTE have no obvious difference. The specific results are shown in FIG. 5, wherein, after CD3+ T effector cells (E) and tumor target cells (T) were co-cultured for 96 hours according to E:T=5:1 and the supernatant containing BiTE oncolytic virus, the effect of oAd-B7H3-BiTE on proliferation of human T lymphocytes was evaluated by flow-detection of fluorescence intensity of CFSE.
Example 7, oAd-influence of B7H3-CD3 BiTE expressed by B7H3-BiTE on the ability of T cells to secrete cytokines
Tumor cells HCT116 at 1X 10 5 Individual cells/well were seeded into 24-well plates at 37 ℃ with 5% CO 2 Applying the mixture in a box overnight; mu.L of virus supernatant was used to infect tumor cells and 5X 10 was added 5 T cells (E: t=5:1), CD3/CD28 antibody treated group served as positive control, and T cells alone served as negative control. Each group was provided with 3 duplicate wells. Supernatants were collected 3 days later and assayed for ifnγ, IL2 production according to ELisa instructions. The research results show that: compared with the control viruses oAd-ON and oAd-FHA-BiTE, oAd-B7H3-BiTE stimulates T cells to secrete IFN-gamma, but under the stimulation of three viruses, IL-2 secretion conditions have no statistical difference, but have significant difference from untreated groups. The specific results are shown in fig. 6, in which, after co-culturing cd3+ T effector cells (E) with tumor target cells (T) at E: t=5:1 and BiTE oncolytic virus-containing supernatant for 72 hours, the ability of human T lymphocytes to produce ifnγ, IL2 was evaluated by ELisa assay.
EXAMPLE 8 therapeutic Effect of B7H3-BiTE expressed by oAd-B7H3-BiTE on HCT116 subcutaneously transplanted mouse model
An NSG mouse was used to construct a HCT116 subcutaneous tumor model. Subcutaneous inoculation of tumor cells HCT116 on the right dorsal part of mice, each 3X 10 6 Tumor cells, after 14 days, tumor volume was about 120mm 3 Treatment was started at that time. Intratumoral injection of virus or PBS with a viral load of 3.5X10 8 pfu/dose, injection volume was 100 μl/dose, administered once every 3 days for a total of 3 doses. And after 48h of each virus or PBS injection, the activated T cells were injected into the tail vein, the cell number was 1×10 7 The injection volume was 200 μl/dose. Mice were sacrificed 5 days after the third T cell injection. Fresh tumor tissue was excised and weighed for reconstitution. The research results show that: compared with PBS group, oAd-B7H3-BiTE and oAd-FHA-BiTE single virus administration has a certain inhibition effect on tumor, and the tumor inhibition effect of tail vein injection T cells is stronger than that of single virus group. As shown in FIG. 7, the tumor volume of the single virus administration of oAd-B7H3-BiTE and oAd-FHA-BiTE is obviously reduced compared with that of the PBS group, when T cells are injected into tail vein after the administration of oncolytic adenovirus oAd-B7H3-BiTE and oAd-FHA-BiTE, the tumor growth can be effectively inhibited compared with the single virus administration, and the tumor inhibition effect of oAd-B7H3-BiTE+T is stronger than that of the oAd-FHA-BiTE+T group. After mice were sacrificed, the tumor weights in the oAd-B7H3-BiTE+T group were significantly different from those in the T cell group. The therapeutic effect of B7H3-BiTE expressed by oAd-B7H3-BiTE on tumor-bearing mice was evaluated by constructing a HCT116 subcutaneous tumor model.
Example 9, oAd Effect of B7H3-BiTE expressed by B7H3-BiTE on T lymphocyte infiltration
The mice were sacrificed after intratumoral administration of oAd-B7H3-BiTE and tail vein injection of T cells, about 0.2g of fresh tumor tissue was scraped off, sheared, and transferred to a 15ml centrifuge tube. 5ml of digestive juice was added to each tube, and the mixture was shaken in a shaker at 37℃until no tissue mass was visible in the digestive juice. The digestate was filtered with a 70. Mu.M filter screen, centrifuged at 1500rpm for 3min and the supernatant discarded. The cell pellet was washed with PBS, centrifuged, and the supernatant was discarded, and 1ml of PBS containing dead living cell dye was added to resuspend the cells, and incubated at room temperature for 15min in the dark. After washing with PBS, the supernatant was centrifuged off, and the cells were resuspended in PBS and dispensed into flow tubes. 1 mu.l of Fc block was added to each tube and incubated at room temperature for 10min in the dark. The antibodies CD3, CD4, CD8 were added and incubated at 4℃for 30min in the absence of light. Each tube was resuspended in PBS, centrifuged and the supernatant discarded and washed again. After being resuspended with PBS, the samples were checked on-board. The research results show that: oAd-B7H3-BiTE+T and oAd-FHA-BiTE+T can significantly improve the infiltration of CD4+ T cells at the tumor site, but does not improve the infiltration of CD8+ T cells. The oAd-B7H3-BiTE+T group was significantly different from the oAd-ON+T group. The proportion of cd8+t was low and there was no significant difference in infiltration of cd8+ T cells in each group. As shown in FIG. 8, the specific results show that the CD4+ T cell ratio of the oAd-B7H3-BiTE+T group is 58.81 + -12.15%, the CD4+ T cell ratio of the oAd-FHA-BiTE+T group is 45.99+ -7.89%, the CD4+ T cell ratio of the oAd-ON+T group is 25.11+ -11.72%, and the CD4+ T cell ratio of the T group is 24.11+ -11.71%, which indicates that oAd-B7H3-BiTE+T and oAd-FHA-BiTE+T can significantly improve the infiltration of CD4+ T cells at the tumor site. And oAd-B7H3-BiTE+T group is significantly different from oAd-ON+T group. Compared with the infiltration condition of CD4+T, the proportion of CD8+T is lower, the proportion of CD8+T in the oAd-B7H3-BiTE+T group is 2.24+/-0.89%, but the infiltration of CD8+T cells in each group is not obviously different. Tumor infiltrating T lymphocytes were examined by flow cytometry.
EXAMPLE 10 influence of B7H3-BiTE expressed by oAd-B7H3-BiTE on the infiltration of Tregs in tumor tissue
Intratumoral administration of oAd-B7H3-BiTE and tail vein injection of T cells followed by sacrifice of mice, tumor tissue removal was sheared and transferred to a centrifuge tube. Each tube was filled with 5ml of digest and shaken on a shaker at 37 ℃. The digestate was filtered with a 70. Mu.M filter screen, centrifuged at 1500rpm for 3min and the supernatant discarded. The cell pellet was washed with PBS, centrifuged, and the supernatant was discarded, and 1ml of PBS containing dead living cell dye was added to resuspend the cells, and incubated at room temperature for 15min in the dark. After washing with PBS, the supernatant was centrifuged off, and the cells were resuspended in PBS and dispensed into flow tubes. Mu.l of Fc block was added to each tube and incubated at room temperature for 10min in the dark. After membrane rupture for 1h, the flow antibodies CD4, CD25 and FOXP3 are added and incubated for 30min at 4 ℃ in the absence of light. Each tube was resuspended in PBS, centrifuged and the supernatant discarded and washed again. After being resuspended with PBS, the samples were checked on-board. Research results show that oAd-B7H3-BiTE+T and oAd-FHA-BiTE+T can obviously reduce infiltration of Tregs cells at tumor positions. Specific results are shown in FIG. 9, wherein the ratio of the Tregs cells in the oAd-B7H3-BiTE+T group is 9.51%, the ratio of the Tregs cells in the oAd-FHA-BiTE+T group is 22.22%, and the ratio of the Tregs cells in the oAd-ON+T group is 32.15%, which indicates that the ratio of the Tregs cells in the tumor sites can be significantly reduced by oAd-B7H3-BiTE+T and oAd-FHA-BiTE+T, and the ratios of the Tregs cells in the oAd-B7H3-BiTE+T and oAd-FHA-BiTE+T groups have no significant difference.
Claims (11)
1. An oncolytic adenovirus expressing a bispecific T cell adaptor, characterized in that: comprises oncolytic adenovirus E1A and E1B gene expression cassettes and B7H3 xCD 3 BiTE exogenous gene expression cassettes.
2. The oncolytic adenovirus expressing a bispecific T cell adapter according to claim 1, wherein: oncolytic adenovirus E1A is controlled by the human telomerase promoter hTERTP; preferably, E1A is linked by an internal ribosome entry site IRES and initiates E1B expression, forming an oncolytic adenovirus of the hTERTP-E1A-IRES-E1B structure.
3. The oncolytic adenovirus expressing a bispecific T cell adapter according to claim 2, wherein: the oncolytic adenovirus E1A and E1B gene expression cassettes comprise the following operably linked elements: human telomerase promoter hTERTp, E1A gene, IRES sequence, E1B gene, terminator.
4. An oncolytic adenovirus expressing a bispecific T cell adapter according to claim 3 wherein: at least one of the following is satisfied:
the nucleotide sequence of the human telomerase promoter hTERTP is shown as SEQ ID NO. 1;
the nucleotide sequence of the oncolytic adenovirus E1A gene is shown as SEQ ID NO. 2;
the amino acid sequence of the oncolytic adenovirus E1A gene is shown as SEQ ID NO. 3;
The IRES nucleotide sequence is shown as SEQ ID NO. 4;
the nucleotide sequence of the oncolytic adenovirus E1B gene is shown as SEQ ID NO. 5;
the amino acid sequences of E1B19K and E1B55K coded by the oncolytic adenovirus E1B gene are respectively shown as SEQ ID NO. 6 and SEQ ID NO. 7;
the terminator of the oncolytic adenovirus E1A and E1B gene expression cassette is SV40 PolyA, and the nucleotide sequence is shown in SEQ ID NO. 8.
5. The oncolytic adenovirus expressing a bispecific T cell adapter according to claim 1, wherein: B7H3 xCD 3 BiTE exogenous genes include fusion genes of B7H3 scFv and CD3 scFv;
preferably, the B7H3 scFv comprises a B7H3 scFv light chain variable region coding sequence and a B7H3 scFv heavy chain variable region coding sequence;
preferably, the CD3 scFv comprises a CD3 scFv heavy chain variable region coding sequence and a CD3 scFv light chain variable region coding sequence.
6. The oncolytic adenovirus expressing a bispecific T cell adapter according to claim 1, wherein: the B7H3 xCD 3 BiTE exogenous gene expression cassette comprises the following operably linked elements: B7H3 scFv light chain variable region encoding sequence, B7H3 scFv heavy chain variable region encoding sequence, CD3 scFv light chain variable region encoding sequence;
Preferably, the B7H3 xcd 3 BiTE exogenous gene expression cassette comprises the following operably linked elements: a promoter, a signal peptide coding sequence, a B7H3 scFv light chain variable region coding sequence, a connecting sequence 1, a B7H3 scFv heavy chain variable region coding sequence, a connecting sequence 2, a CD3 scFv heavy chain variable region coding sequence, a connecting sequence 3, a CD3 scFv light chain variable region coding sequence and a terminator.
7. The oncolytic adenovirus expressing a bispecific T cell adapter according to claim 6, wherein: the B7H3 xCD 3 BiTE exogenous gene expression cassette also comprises a Kozak sequence, and the promoter comprises a eukaryotic promoter; preferably, the eukaryotic promoter is CMV, mCMV, CAG.
8. The oncolytic adenovirus expressing a bispecific T cell adapter according to claim 6, wherein: at least one of the following is satisfied:
the signal peptide is Igkappa signal peptide, and the nucleotide sequence of the signal peptide is shown as SEQ ID NO. 9;
the amino acid sequence of the Ig kappa signal peptide is shown as SEQ ID NO. 10;
the nucleotide sequence of the variable region of the B7H3 scFv light chain is shown as SEQ ID NO. 11 or SEQ ID NO. 12;
the amino acid sequence of the variable region of the light chain of the B7H3 scFv is shown as SEQ ID NO. 13 or SEQ ID NO. 14;
The nucleotide sequence of the heavy chain variable region of the B7H3 scFv is shown as SEQ ID NO. 15 or SEQ ID NO. 16;
the amino acid sequence of the heavy chain variable region of the B7H3 scFv is shown as SEQ ID NO. 17 or SEQ ID NO. 18;
the connecting peptide between the light chain variable region and the heavy chain variable region of the B7H3 scFv is (G4S) 3, and the nucleotide sequence of the connecting peptide is shown as SEQ ID NO. 19;
the amino acid sequence of the connecting peptide (G4S) 3 is shown as SEQ ID NO. 20;
the nucleotide sequence of the CD3 scFv is shown as SEQ ID NO. 21;
the amino acid sequence of the CD3 scFv is shown as SEQ ID NO. 22;
the nucleotide sequence of the connecting peptide between the B7H3 scFv and the CD3 scFv is shown as SEQ ID NO. 23;
the amino acid sequence of the connecting peptide between B7H3 scFv and CD3 scFv is shown as SEQ ID NO. 24.
9. A recombinant oncolytic adenoviral vector characterized in that: operably inserted into or containing the B7H3 xcd 3 BiTE exogenous gene expression cassette of any one of claims 5-8.
10. A method of constructing an oncolytic adenovirus expressing a bispecific T cell adapter according to any one of claims 1-8, wherein: the method comprises the following steps: cloning a synthesized E1A-E1B gene expression cassette into a shuttle vector to construct a plasmid containing E1A-E1B gene expression, cloning a synthesized B7H3 xCD 3 BiTE gene into the plasmid containing E1A-E1B gene expression, and packaging recombinant adenovirus to obtain recombinant oncolytic adenovirus expressing B7H3 xCD 3 BiTE;
Preferably, the present invention also provides a construction method of the oncolytic adenovirus expressing the bispecific T cell adapter, comprising the steps of: synthesizing E1A-E1B gene expression cassettes, cloning the E1A-E1B gene expression cassettes into a shuttle vector by using a Gibson cloning method, constructing plasmids containing E1A-E1B gene expression, cloning B7H3 xCD 3 BiTE genes into pDC316 or pDC516 shuttle plasmids, co-transfecting HEK293 cells with the shuttle plasmids carrying B7H3 xCD 3 BiTE genes and skeleton plasmids of an AdMax adenovirus packaging system, and packaging recombinant adenovirus, thus obtaining recombinant oncolytic adenovirus expressing B7H3 xCD 3 BiTE;
more preferably, the backbone plasmid comprises pBHGlox_E1,3Cre or pBHGfrt_E1,3Flp.
11. Use of an oncolytic adenovirus expressing a bispecific T cell adapter according to any one of claims 1-8 or a recombinant oncolytic adenovirus vector according to claim 9 for the preparation of a medicament for the treatment and/or prevention and/or co-treatment of cancer or tumors.
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