CN111675765A - Armed chimeric antigen receptor cell of targeted coronavirus SPIKE, preparation method and application - Google Patents

Armed chimeric antigen receptor cell of targeted coronavirus SPIKE, preparation method and application Download PDF

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CN111675765A
CN111675765A CN202010488988.6A CN202010488988A CN111675765A CN 111675765 A CN111675765 A CN 111675765A CN 202010488988 A CN202010488988 A CN 202010488988A CN 111675765 A CN111675765 A CN 111675765A
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CN111675765B (en
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代红久
徐慧
朱靓婧
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Nanjing Kaedi Biotech Inc
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Abstract

The invention relates to a coding amino acid sequence of armed chimeric antigen receptor of targeting SARS-CoV-2 virus SPIKE protein constructed by specific knockout of SARS-CoV-2 virus RNA polymerase, and immune response cell modified by the coding amino acid sequence, and a preparation method and application of the immune response cell. The invention constructs a recombinant vector targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor by specifically knocking out SARS-CoV-2 virus RNA polymerase, and a method for modifying immune response cells by using the recombinant vector; the novel functional immune response cell can effectively target and attack various viruses, the preparation method of the immune response cell modified by the armed chimeric antigen receptor of the target SARS-CoV-2 virus SPIKE has simple steps, and the obtained target virus SPIKE and the immune response cell modified by virus replication dependent RNA polymerase have obvious killing effect on coronavirus.

Description

Armed chimeric antigen receptor cell of targeted coronavirus SPIKE, preparation method and application
Technical Field
The invention belongs to the technical field of immunotherapy biomedicine, and relates to an amino acid coding sequence of a SPIKE protein antagonistic cytokine storm of a targeting SARS-CoV-2 virus constructed by specific knockout of SARS-CoV-2 virus RNA polymerase, a modified immune response cell, a preparation method of the amino acid coding sequence and the modified immune response cell, and application of the amino acid coding sequence and the modified immune response cell in medicine preparation.
Background
Coronaviruses are a large family of viruses known to cause the common cold and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a new strain of coronavirus that has not been previously discovered in humans. After people are infected with coronavirus, the common signs of the person are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. At present, no specific effective therapeutic drug for diseases caused by the novel coronavirus exists.
In the year 2020, 1 month and 21 days, researchers of Shanghai Pasteur institute of Chinese academy of sciences, national center of research on emergency prevention and control drug engineering technology of Chinese military medical institute and the like cooperate to disclose the genetic evolutionary relationship of the novel coronavirus, SARS coronavirus and MERS coronavirus through the model analysis of the SPIKE protein structure of 2019-nCoV (novel coronavirus pneumonia). Meanwhile, it was presumed that the novel coronavirus may have the same receptor ACE2 as SARS by the computational simulation technique, and that 2019-nCoV infects human airway epithelial cells by invasion through the mediation of the SPIKE protein and human cell surface ACE2 receptor, as with SARS-CoV (see Xu Xintian et al, Science China Life Science, (2020)). Researchers at the McLellan group analyzed the SPIKE protein structure of the novel coronavirus using cryoelectron microscopy and also analyzed the affinity of SPIKE protein to ACE2 using Surface Plasmon Resonance (SPR), and they found that the affinity of ACE2 protein to the novel coronavirus was 10 to 20 times that of SARS virus (see Daniel Wrapp et al, BioRxiv, (2020)) rather than previously being weaker than SARS virus. This also explains why the infectivity of the new coronavirus is so strong. The main infection process of the new coronavirus is as follows: SPIKE protein on the surface of the virus binds to ACE2, the receptor on the cell surface, allowing it to enter the host cell. The viral RNA is then released into the cytoplasm, and through the processes of replication, transcription, translation, etc., new viral particles are formed, released from the infected cells through exocytosis, and then new cells are infected. Therefore, ACE2 is a key molecule in this new coronavirus infection. In conclusion, the virus can be prevented from invading cells by blocking the action of the receptor ACE2 and SPIKE protein.
In addition, among the many different proteins of coronaviruses, RNA-dependent RNA polymerase, RdRp, plays a very important role in the proliferative replication phase of viruses, which on the one hand replicates genes of progeny viruses using viral RNA as a template, and on the other hand transcribes genes of proteins and enzymes required during viral propagation into mRNA, and thus it is responsible for the dual functions of replicase and transcriptase. Therefore, RdRp can be an effective drug target for the treatment of viruses.
Meanwhile, two studies on novel coronavirus are published in the recent international center medical journal "lancet": a significant number of critically infected patients develop "cytokine storms" which are a significant cause of acute respiratory distress syndrome and multiple organ failure in patients with viral infections (see Chen Nanshan et al, lace, (2020); (see Huang Chaolin et al, lace, (2020))). Many clinical experts involved in the treatment of severe patients with new coronary pneumonia have paid little attention to the phenomenon of cytokine storm, and the occurrence of the cytokine storm is closely related to the severe infection and death of virus infection. Therefore, the great impact caused by the cytokine storm should be closely paid attention to the treatment of patients infected by the novel coronavirus, which is of great significance for the treatment of severe patients and the reduction of the fatality rate. The research shows that: IL-6 plays a pivotal role in cytokine syndrome. Previous studies have confirmed that the interleukin-6 receptor (IL-6R) blocking monoclonal antibody drug tollizumab can be used for treating and improving the toxic and side effects of cytokine storm. Clinical tests prove that the toxic and side effects caused by cytokine storm can be rapidly solved after IL-6 receptor is blocked. Turkumab has been registered for a multicenter, randomized controlled clinical study (accession number: ChiCTR2000029765) of efficacy and safety in novel coronavirus pneumonia (COVID-19) at a future date.
Immunotherapy, which involves the engineering of immune cells to destroy specific cellular targets, is well known for its use in the treatment of cancer. However, over the past few years, a few research teams have been developing T cell therapies for the treatment of viral infections. In the field of antiviral immunotherapy, donated T cells are genetically modified using genetic manipulation techniques of Chimeric Antigen Receptors (CARs) against cancer targets. In essence, T cells are genetically modified to express a receptor (i.e., CAR) that recognizes a specific protein on a cancer cell or virus. Using CAR-T cell technology, one can ensure possession of T cells targeting a particular virus. In the last few years, relevant studies have been reported to target T cells to several viruses using CARs; in addition, Cell, 10.2019, an academic journal of international center, published a recent research result of the research center for treating aids in gleaston usa. This study developed a novel CAR-T named: convertible CAR-T, a new version of CAR-T, has shown great promise in combating HIV, and can greatly reduce the HIV reservoir that persists in patients under antiretroviral therapy (see Eytan Herzig et al, Cell, (2019)). Clinical researchers have shown that antiviral T cell therapy is "very exciting. This is because a very high response rate is indeed observed and it does directly affect the life of the patient ".
To sum up, the engineering immune response cell which is constructed by RNA polymerase of specific knockout SARS-CoV-2 virus and is modified by the chimeric antigen receptor of the target SARS-CoV-2 virus SPIKE armed protein has wide application prospect. The engineering cell captures the novel coronavirus through camouflage and inhibits the normal replication of the virus, and simultaneously effectively inhibits a cytokine storm; the capability of attacking human body cells is lost, so that the effects of camouflage capturing of novel coronavirus, virus removal and cytokine syndrome prevention are achieved, and finally a good clinical treatment effect is achieved.
Disclosure of Invention
In view of the above problems and/or other problems of the related art, the present invention is directed to a novel product and method for treating a novel coronavirus pneumonia by specifically knocking out SARS-CoV-2 virus RNA polymerase to construct an anti-cytokine chimeric antigen receptor targeting SARS-CoV-2 virus SPIKE protein, an amino acid coding sequence and a nucleotide coding sequence and a recombinant expression vector mainly comprising the amino acid coding sequence and the nucleotide coding sequence of the armed chimeric antigen receptor specifically knocking out SARS-CoV-2 virus RNA polymerase to construct an anti-cytokine targeting SARS-CoV-2 virus SPIKE protein, and an immunoresponsive cell modified thereby, and a method for preparing the same and a cellular immunotherapy application using the engineered immune cell.
Technical scheme
An armed chimeric antigen receptor modified immune cell targeting the SPIKE protein of SARS-CoV-2 virus, wherein said immune cell comprises a chimeric antigen receptor having the amino acid sequence:
the amino acid sequence of a guide sequence, the amino acid sequence of an extracellular identification domain targeted to bind to the SPIKE protein of SARS-CoV-2 virus, the amino acid sequence of a hinge region, the amino acid sequence of a transmembrane domain, the amino acid sequence of an intracellular signal domain and the amino acid sequence containing an anti-cytokine receptor component are sequentially connected from an amino terminal to a carboxyl terminal;
wherein the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signaling domain;
wherein the amino acid sequence of the extracellular recognition structural domain of the SPIKE protein of the targeting combined SARS-CoV-2 virus is as follows: (1) the amino acid sequence of human ACE2 protein shown by SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7 and targeted to combine with SARS-CoV-2 virus SPIKE protein; or a variant which is produced by amino acid modification and has 90-99% homology with the amino acid sequence shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No. 7.
The immune cell is characterized in that:
the transmembrane domain comprises a CD8 transmembrane domain, a CD28 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain;
the immunoreceptor tyrosine activation motif comprises an intracellular signaling domain of the CD3 zeta chain or an FcRI gamma intracellular signaling structure;
the costimulatory signaling domain comprises a CD28 intracellular signaling domain, a CD137/4-1BB intracellular signaling domain, a CD134/OX40 intracellular signaling domain, and an ICOS intracellular signaling domain;
the anti-cytokine receptor component comprises an scFv amino acid sequence of IL-6R shown in SEQ ID No. 17.
The immune cell is characterized in that:
the nucleic acid molecule of the armed chimeric antigen receptor of the targeted SARS-CoV-2 virus SPIKE protein for coding the specific knockout SARS-CoV-2 virus RNA polymerase comprises a nucleotide sequence which is connected in series from 5 'to 3' and is used for coding the guide sequence, a nucleotide sequence of human ACE2, a nucleotide sequence for coding a hinge region, a nucleotide sequence for coding the transmembrane domain, a nucleotide sequence for coding the intracellular signal domain, a nucleotide sequence for coding the anti-cytokine receptor component and a SARS-CoV-2 virus nucleotide sequence for coding the specific knockout RNA polymerase;
wherein:
the nucleotide encoding the leader sequence: as shown in SEQ ID No. 18;
the nucleotide sequence of the code human ACE2 is shown as SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23 or SEQ ID No. 24;
nucleotides encoding the hinge region include: a nucleotide sequence of human CD8 or a nucleotide sequence of human IgG 1;
encoding the transmembrane domain includes: a nucleotide sequence of the nucleotide sequence of human CD 8;
the nucleotide sequence encoding the intracellular signaling domain includes: a human 4-1BB intracellular domain, a human CD3 ζ intracellular domain, or a CD3 ζ intracellular domain; wherein, the nucleotide sequence encoding the human 4-1BB intracellular domain; the nucleotide sequence encoding the intracellular domain of human CD 28; the nucleotide sequence encoding the intracellular domain of human CD3 ζ;
the nucleotide sequence encoding the anti-cytokine receptor-containing module includes: the nucleotide sequence of Furin-2A shown as SEQ ID No.25, the nucleotide sequence of IL-2 signal peptide shown as SEQ ID No.26 or the scFv nucleotide sequence of IL-6R shown as SEQ ID No. 27.
The nucleotide sequence of the RNA polymerase for coding the specific knockout SARS-CoV-2 virus is shown as any one of SEQ ID No.32, SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.37 or SEQ ID No. 38; the specific knockout SARS-CoV-2 virus RNA polymerase is expressed by the U6 promoter as shown in SEQ ID No. 28.
A recombinant vector or expression plasmid that specifically knockdown SARS-CoV-2 viral RNA polymerase targeted SARS-CoV-2 viral SPIKE protein armed chimeric antigen receptor, comprising the nucleic acid molecule of claim 3.
The recombinant vector or the expression plasmid is characterized in that the recombinant vector or the expression plasmid contains a promoter, wherein the promoter comprises a U6 promoter shown as SEQ ID No.28 and an EF1 alpha long promoter shown as SEQ ID No.30, or the promoter is an EFS short promoter shown as SEQ ID No. 31.
A recombinant virus comprising the nucleotide sequence of the recombinant vector or expression plasmid of claim 3 or 4 and a viral particle; the virus includes lentivirus, adenovirus, adeno-associated virus or retrovirus.
The immune cell is applied to the preparation of 2019-nCoV, SARS and MERS medicaments.
Armed chimeric antigen receptor modified immune cells targeting the SPIKE protein of the SARS-CoV-2 virus, in particular
In a first aspect, the present application provides an armed chimeric antigen receptor targeting the SARS-CoV-2 virus SPIKE protein that specifically knocks out SARS-CoV-2 virus RNA polymerase, said receptor targeting the human ACE2 protein that binds to the SARS-CoV-2 virus SPIKE protein or a functional variant (analog) thereof, comprising a sequence selected from the group consisting of:
(1) an amino acid sequence shown as SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No.7, or (2) (1) a functional variant resulting from one or more amino acid modifications; wherein the functional variant modified by amino acid is polypeptide which has 90-99% homology with the amino acid sequence shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No. 7.
The inventor continuously performs amino acid sequence design and sequence arrangement combination and screening through creative work, performs random screening test and targeting function verification (for example, tests of constructing a virus vector, further infecting T cells, obtaining modified T cells, detecting in vitro killing activity of the obtained modified T cells and the like) on sequences of dozens of CAR molecules, then compares the sequences according to the results of a plurality of random combinations, performs sequence adjustment, and finally screens out the sequence with the best effect, thereby obtaining the human ACE2 amino acid sequence and functional variants thereof which are combined with SARS-CoV-2 virus SPIKE protein in a high-efficiency targeting manner.
In a second aspect, the present application provides an armed chimeric antigen receptor targeting the SARS-CoV-2 virus SPIKE protein that specifically knocks out the SARS-CoV-2 virus RNA polymerase, comprising an amino acid sequence of a leader sequence, an extracellular recognition domain amino acid sequence that targets binding the SARS-CoV-2 virus SPIKE protein, a transmembrane domain amino acid sequence, an intracellular signaling domain amino acid sequence, and an amino acid sequence of an anti-cytokine component, sequentially linked from amino terminus to carboxy terminus. The amino acid sequence targeted to bind to the extracellular recognition domain of the viral SPIKE protein comprises the human ACE2 protein or a functional variant thereof targeted to bind to the viral SPIKE protein as described in the first aspect of the application.
The extracellular recognition domain (also referred to as the extracellular domain or simply by the recognition element it contains) comprises a recognition element that specifically binds to a molecule present on the cell surface of the target cell.
In some non-limiting examples, the leader sequence is covalently linked to the 5' end of the extracellular antigen-binding domain.
In some embodiments, the armed chimeric antigen receptor constructed by specific knock-out of SARS-CoV-2 viral RNA polymerase to target the SARS-CoV-2 viral SPIKE protein comprises a hinge region.
In some embodiments, the transmembrane domain comprises a transmembrane region.
In some embodiments, the amino acid sequence of the human CD8 polypeptide of the hinge region is selected from the group consisting of the polypeptide shown in SEQ ID No.8 or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90 to 99% homology with the amino acid sequence shown in SEQ ID No. 8.
In some embodiments, the amino acid sequence of human IgG1 of the hinge region is selected from the group consisting of the polypeptide set forth in SEQ ID No.9 or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90-99% homology to the amino acid sequence set forth in SEQ ID No. 9.
In some embodiments, the amino acid sequence of human CD8 of the transmembrane region is selected from the group consisting of the polypeptide of SEQ ID No.10 or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90 to 99% homology with the amino acid sequence of SEQ ID No. 10.
In some embodiments, the human 4-1BB intracellular domain is selected from: (1) a polypeptide having an amino acid sequence as shown in SEQ ID No. 11; or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 11.
In some embodiments, the human CD28 intracellular domain is selected from the group consisting of: (1) a polypeptide having an amino acid sequence as shown in SEQ ID No. 12; or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 12.
In some embodiments, the human ICOS intracellular domain is selected from the group consisting of: (1) a polypeptide having an amino acid sequence as shown in SEQ ID No. 13; or an amino acid modified functional variant, wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 13.
In some embodiments, the CD3 ζ intracellular domain is selected from: (1) a polypeptide having an amino acid sequence as shown in SEQ ID No. 14; or a functional variant with amino acid modifications. Wherein the amino acid modified functional variant is a polypeptide having 90-99% homology with the amino acid sequence shown in SEQ ID No. 14.
In some embodiments, the amino acid sequence of the armed chimeric antigen receptor is, in order, the amino acid sequence of Furin-2A as set forth in SEQ ID No.15, the amino acid sequence of IL-2 signal peptide as set forth in SEQ ID No.16, and the scFv amino acid sequence of IL-6R as set forth in SEQ ID No. 17.
In some non-limiting embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signaling domain;
in some non-limiting embodiments, specific knock-out of SARS-CoV-2 viral RNA polymerase constructs targeting the SARS-CoV-2 viral SPIKE armed chimeric antigen receptor is recombinantly expressed or expressed from a vector.
In certain non-limiting embodiments, the specific knock-out SARS-CoV-2 virus RNA polymerase constructs of the present application target the intracellular domain of the SARS-CoV-2 virus SPIKE armed chimeric antigen receptor further comprises at least one costimulatory signaling region comprising at least one costimulatory ligand molecule that provides optimal lymphocyte activation.
In some non-limiting embodiments, specific knock-out of SARS-CoV-2 viral RNA polymerase constructs targeting the intracellular domain of the SARS-CoV-2 viral SPIKE armed chimeric antigen receptor comprises a costimulatory signaling region comprising two costimulatory molecules: CD28 and 4-1BB or CD28 and OX 40.
In some non-limiting embodiments, the specific knock-out SARS-CoV-2 viral RNA polymerase constructs armed chimeric antigen receptor intracellular domains targeted to the SPIKE protein of SARS-CoV-2 virus comprising a 4-1BB polypeptide.
In certain non-limiting embodiments, the armed chimeric antigen receptor targeted to the SPIKE protein of SARS-CoV-2 virus constructed by specific knock-out of SARS-CoV-2 virus RNA polymerase may further comprise a spacer (spacer) linking the antigen binding domain to the transmembrane domain. The spacer may be sufficiently flexible to allow the antigen binding domain to be oriented in different directions to facilitate antigen recognition. The spacer may be a hinge region from IgG1, or part of the CH2CH3 region and CD3 of an immunoglobulin.
In certain non-limiting embodiments, specific knock-out of SARS-CoV-2 viral RNA polymerase constructs targeting the intracellular domain of the armed chimeric antigen receptor of the SPIKE protein of SARS-CoV-2 virus may comprise a human CD3 ζ polypeptide that can activate or stimulate a cell (e.g., a cell of lymphoid lineage, such as a T cell).
In certain non-limiting embodiments, the specific knock-out SARS-CoV-2 viral RNA polymerase constructs an intracellular domain targeted to the SARS-CoV-2 viral SPIKE armed Chimeric Antigen Receptor (CAR) further comprises at least one costimulatory signaling region comprising at least one costimulatory molecule that provides optimal lymphocyte activation.
In some embodiments, the costimulatory signaling region of the intracellular domain of the CAR comprises two costimulatory molecules: CD28 and 4-1BB, or CD28 and OX 40.
4-1BB may act as a Tumor Necrosis Factor (TNF) ligand and has stimulatory activity. 4-1BB may act as a Tumor Necrosis Factor (TNF) ligand and has stimulatory activity.
In a non-limiting embodiment, the 4-1BB polypeptide has the amino acid sequence of the contiguous portion of SEQ ID NO. 11.
In a third aspect, the present application provides a nucleic acid molecule encoding the SARS-CoV-2 virus SPIKE armed chimeric antigen receptor targeting SARS-CoV-2 virus RNA polymerase of the specific knock-out SARS-CoV-2 virus of the second aspect, said nucleic acid molecule comprising a nucleotide sequence encoding a guide sequence, a nucleotide sequence encoding human ACE2, a nucleotide sequence encoding a transmembrane domain, a nucleotide sequence encoding an intracellular signaling domain, a nucleotide sequence of armed chimeric antigen receptor, and a nucleotide sequence of the specific knock-out SARS-CoV-2 virus RNA polymerase, connected in series in order from 5 'to 3'.
In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a hinge region. In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signaling domain;
polynucleotides encoding recognition domains that target binding to the SPIKE protein of the SARS-CoV-2 virus can be modified by codon optimization. Codon optimization can alter naturally occurring and recombinant gene sequences to achieve the highest possible level of productivity in any given expression system.
In some embodiments, the nucleotide encoding the leader sequence is set forth in SEQ ID No. 18.
In some embodiments, the nucleotide sequence encoding human ACE2 is as set forth in SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, or SEQ ID No. 24.
In some embodiments, the nucleotides encoding the hinge region comprise: a nucleotide sequence of human CD8 or a nucleotide sequence of human IgG 1;
in some embodiments, encoding the transmembrane domain comprises: a nucleotide sequence of the nucleotide sequence of human CD 8;
in some embodiments, the nucleotide sequence encoding an intracellular signaling domain comprises: a human 4-1BB intracellular domain, a human CD3 ζ intracellular domain, or a CD3 ζ intracellular domain; wherein, the nucleotide sequence encoding the human 4-1BB intracellular domain; the nucleotide sequence encoding the intracellular domain of human CD 28; the nucleotide sequence encoding the intracellular domain of human CD3 ζ;
in some embodiments, the nucleotide sequence encoding the anti-cytokine receptor component is sequentially the nucleotide sequence of Furin-2A shown as SEQ ID No.25, the nucleotide sequence of IL-2 signal peptide shown as SEQ ID No.26, and the scFv nucleotide sequence of IL-6R shown as SEQ ID No. 27.
In some embodiments, the nucleotide sequence encoding a specific knock-out SARS-CoV-2 viral RNA polymerase is designed from the full-length RNA-dependent RNAPLYLYMase (SARS-CoV-2) amino acid sequence (YP-009725307.1) as follows: the inventor finally designs 7 shRNA sequences of specificity knockout SARS-CoV-2 virus RNA polymerase according to shRNA design principle, wherein the shRNA sequences are shown as SEQ ID No.32, SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.37 or SEQ ID No. 38;
in some embodiments, the specific knockout SARS-CoV-2 viral RNA polymerase is expressed by the U6 promoter as shown in SEQ ID No.28 or the CMV promoter as shown in SEQ ID No. 29;
in a fourth aspect, the present application provides a recombinant vector or expression plasmid comprising the chimeric antigen receptor of the second aspect of the present application or the nucleic acid of the third aspect of the present application.
Genetic modification of immune-responsive cells (e.g., T cells, CTL cells, NK cells) can be achieved by transducing substantially homologous cellular compositions with recombinant DNA or RNA constructs. In one embodiment, the vector is a retroviral vector (e.g., a gammaretrovirus or lentivirus) that can introduce a DNA or RNA construct into the genome of a host cell. For example, a polynucleotide encoding human ACE2, targeting SARS-CoV-2 virus SPIKE-specific CAR, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, the retroviral long terminal repeat sequence, or from an alternative internal promoter.
Non-viral vectors or RNA may also be used. Random chromosomal integration or targeted integration can be used (e.g., using nucleases, transcription activator-like effector nucleases (TALENs), Zinc Finger Nucleases (ZFNs), and/or regularly clustered interspaced short palindromic repeats (CRISPRs), or transgene expression (e.g., using natural or chemically modified RNAs).
In some embodiments, the vector is selected from the group consisting of a gamma-retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector.
In an exemplary embodiment, the vector is a gamma-retroviral vector.
In a fifth aspect, the present application provides a recombinant virus which is a virus capable of expressing a targeted SARS-CoV-2 virus SPIKE armed chimeric antigen receptor that specifically knocks out the SARS-CoV-2 virus RNA polymerase as described in the second aspect of the invention and which is capable of infecting immunoresponsive cells.
In some embodiments, the immunoresponsive cell is a cytotoxic T lymphocyte, an NK cell, an NKT cell, a helper T cell, or the like.
In an exemplary embodiment, the immunoresponsive cell is a cytotoxic T lymphocyte.
In some embodiments, the virus is a lentivirus, adenovirus, adeno-associated virus, retrovirus, or the like.
In an exemplary embodiment, the virus is a lentivirus.
In an exemplary embodiment, the virus is a retrovirus.
In a sixth aspect, the present application provides an isolated modified immunoresponsive cell comprising a chimeric antigen receptor according to the second aspect of the present application transformed with a recombinant vector or expression plasmid according to the third aspect of the present application.
For initial genetic modification of cells to provide the specific knock-out of SARS-CoV-2 viral RNA polymerase constructs targeting SARS-CoV-2 viral SPIKE armed chimeric antigen receptor modified immunoresponsive cells, transduction is typically performed using a retroviral vector, although any other suitable viral vector or non-viral delivery system may be used. Retroviral gene transfer (transduction) has also proven effective for subsequent genetic modification of cells to provide cells comprising an antigen presenting complex comprising at least two co-stimulatory ligands. Combinations of retroviral vectors and suitable assembly lines are also suitable, wherein the capsid proteins are functional for infecting human cells.
In some embodiments, the immunoresponsive cell further comprises at least one exogenous co-stimulatory ligand.
Possible transduction methods also include direct co-culture of the cells with the producer cells. Transduction viral vectors can be used to express co-stimulatory ligands (e.g., 4-1BBL and IL-12) in immune responsive cells. Preferably, the selected vector exhibits high infection efficiency and stable integration and expression.
In some embodiments, preferably, the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, and combinations thereof, or more preferably, the co-stimulatory ligand is 4-1 BBL.
In some embodiments, the immunoresponsive cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a Cytotoxic T Lymphocyte (CTL), a regulatory T cell, a human embryonic stem cell, and a pluripotent stem cell that can differentiate into lymphoid cells, preferably a T cell and a Natural Killer (NK) cell, more preferably a T cell.
Multiple T cell subsets isolated from patients can be transduced with vectors for CAR expression.
In an exemplary embodiment, wherein the modified immunoresponsive cell is a CAR-T cell.
The specific knockout SARS-CoV-2 virus RNA polymerase can be used for constructing targeted SARS-CoV-2 virus SPIKE armed chimeric antigen receptor to prepare genetically modified central memory T cells, and then the genetically modified central memory T cells are refrigerated and stored.
In a seventh aspect, the present application provides a method of making an isolated chimeric antigen receptor modified immunoresponsive cell of the sixth aspect of the application, comprising the steps of:
firstly, the nucleic acid molecule of the third aspect is connected to an expression vector by a molecular cloning mode to obtain the specific knockout SARS-CoV-2 virus RNA polymerase to construct an expression vector targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor;
then, constructing a targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor expression vector by the obtained specificity knockout SARS-CoV-2 virus RNA polymerase to transfect 293T cells to obtain virus solution;
finally, the virus liquid is used for infecting immune response cells, and the specific knockout SARS-CoV-2 virus RNA polymerase is expressed from the infected cells to construct the immune response cells modified by the target SARS-CoV-2 virus SPIKE armed chimeric antigen receptor.
In some non-limiting embodiments, the modified immunoresponsive cells of the invention can be cells of lymphoid lineage. The cells of the lymphoid lineage are selected from B, T and Natural Killer (NK) cells, and provide functions such as antibody production, regulation of cellular immune system, detection of foreign substances in blood, detection of foreign cells in a host, and the like. Non-limiting examples of cells of lymphoid lineage include T cells, Natural Killer (NK) cells, Cytotoxic T Lymphocytes (CTLs), regulatory T cells, embryonic stem cells, and pluripotent stem cells (e.g., pluripotent stem cells that can differentiate into lymphoid cells).
In some embodiments, the immunoresponsive cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a Cytotoxic T Lymphocyte (CTL), a regulatory T cell, a human embryonic stem cell, and a pluripotent stem cell that can differentiate into lymphoid cells, preferably a T cell or a Natural Killer (NK) cell. In some exemplary embodiments, the T cells are lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system.
In some non-limiting embodiments, T cells include, but are not limited to, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells (e.g., TEM cells and TEMRA cells), regulatory T cells (also referred to as suppressor T cells), natural killer T cells, mucosa-associated constant T cells, and gamma T cells.
In some embodiments, the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, and combinations thereof. In some embodiments, the co-stimulatory ligand is 4-1 BBL.
In a preferred embodiment, the isolated modified immunoresponsive cell is a T cell.
In a preferred embodiment, the isolated modified immunoresponsive cell is a Natural Killer (NK) cell.
In some non-limiting embodiments, the isolated modified immune response cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.
In an eighth aspect, the present application provides a pharmaceutical composition comprising an effective amount of the isolated modified immunoresponsive cell of the sixth aspect of the invention and a pharmaceutically acceptable excipient.
A pharmaceutical composition comprising an isolated modified immunoresponsive cell that expresses said specific knock-out SARS-CoV-2 viral RNA polymerase to construct a SPIKE armed chimeric antigen receptor targeting SARS-CoV-2 viral antigen and a pharmaceutically acceptable carrier.
Administration of the pharmaceutical composition may be autologous or non-autologous. For example, an immune responsive cell expressing the SARS-CoV-2 virus RNA polymerase constructed armed CAR targeting the SARS-CoV-2 virus SPIKE protein and compositions comprising the same can be obtained from one subject and administered to the same subject or a different compatible subject. Peripheral blood-derived T cells of the presently disclosed subject matter or progeny thereof (e.g., derived in vivo, ex vivo, or in vitro) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When a pharmaceutical composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising the specific knock-out SARS-CoV-2 virus RNA polymerase to construct an immunoresponsive cell targeted to the SARS-CoV-2 virus SPIKE armed CAR) is administered, it is typically formulated in a unit dose injectable form (solution, suspension, emulsion).
The compositions of the present application may be formulations. Expression of the specific knock-out SARS-CoV-2 virus RNA polymerase constructs immune responsive cells targeted to the SARS-CoV-2 virus SPIKE armed Chimeric Antigen Receptor (CAR) and compositions comprising the same disclosed herein can be conveniently provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are more convenient to administer, particularly by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition can comprise a carrier, which can be a solvent or dispersion medium comprising, for example, water, physiological saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.
According to the present application, any vector, diluent or additive used must be compatible with the construction of immune responsive cells targeted to the SARS-CoV-2 virus SPIKE armed Chimeric Antigen Receptor (CAR) by the specific knockout SARS-CoV-2 virus RNA polymerase expressing the presently disclosed subject matter.
If desired, the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent. The selection of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or another liquid form, such as a time-release form or a liquid-fill form).
In a ninth aspect, the present application provides a kit for the treatment or prevention of a disease comprising an immunoresponsive cell of the sixth aspect of the invention or a nucleic acid of the third aspect of the invention.
If desired, the immunoresponsive cells are provided with instructions for administering the cells to a subject having or at risk of developing a viral infection. The instructions generally include information about the use of the composition for treating or preventing a viral infection. In other embodiments, the instructions include at least one of: a description of the therapeutic agent; dosage regimens and methods of administration for treating or preventing viral diseases; matters to be noted; contraindication; indications; non-adaptive symptoms; excess information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or affixed to the container as a label, or provided in or with the container as a separate page, booklet, card, or foldout.
In a tenth aspect, the present application provides the use of the human ACE2 protein and its variants targeted to bind to the SPIKE protein of SARS-CoV-2 virus according to the first aspect of the invention, the specific knock-out SARS-CoV-2 virus RNA polymerase according to the second aspect to construct a product targeted to the SPIKE armed chimeric antigen receptor of SARS-CoV-2 virus, the recombinant vector or expression plasmid according to the fourth aspect, the recombinant virus according to the sixth aspect, the isolated modified immunoresponsive cell according to the seventh aspect, or the kit according to the ninth aspect, in the treatment, or prevention of a disease, disorder or health disorder.
In some embodiments, the disease treated or prevented comprises a disease caused by a coronavirus such as SARS, MERS, and the like.
Principle of action
The invention adopts CAR-T cell immunization method to treat diseases caused by coronavirus such as 2019-nCoV, SARS or MERS and the like for the first time. The principle is as follows: CAR-T immune cells generally include, among other things, an extracellular recognition domain, a hinge region, a transmembrane domain, and an intracellular signaling domain. The invention designs the extracellular recognition domain of CAR-T as a functional variant of human ACE2 (i.e. the amino acid sequence of human ACE2 described in the examples), based on the suggestion that the SPIKE protein on the surface of coronaviruses binds to the cell surface receptor ACE 2. It can decoy coronavirus in vivo, SPIKE protein of coronavirus is combined with it, CAR-T cell releases cytokine and kills virus by generating immune reaction, meanwhile in order to kill residual virus particle in CAR-T cell, the invention also designs polymerase shRNA sequence interference sequence (shRNA) aiming at coronavirus, which can specifically clear polymerase of coronavirus and prevent proliferation of coronavirus. Meanwhile, after coronavirus infection, a cytokine storm can be generated, the CAR-T is also provided with the scFv of the IL-6R, and the cytokine storm is mainly IL-6, so that the CAR-T is also provided with the scFv of the IL-6R to be combined with extra IL-6 outside cells, and the harm caused by the cytokine storm is reduced.
Advantageous effects
The invention uses the chimeric antigen receptor modified T cell technology to prepare the specificity to knock off SARS-CoV-2 virus RNA polymerase to construct the engineering immune cell modified by the target SARS-CoV-2 virus SPIKE armed chimeric antigen receptor, the preparation method has simple steps, the obtained novel engineering immune cell can specifically identify the cell or virus of the virus SPIKE protein, can target and attack the virus more effectively, has high killing rate to the virus, and can be used for preparing antiviral products, in particular for preparing medicaments for treating diseases caused by coronavirus such as SARS, MERS and the like, can help to resist the cell factor storm, help the patient to get rid of severe cases, and reduce the fatality rate of virulent infectious diseases. The invention can be used for preparing antivirus products, in particular for preparing medicaments for treating diseases caused by coronavirus such as SARS, MERS and the like, and has good industrial application prospect.
Drawings
FIG. 1 shows a schematic diagram of the working mode of KD-2019 engineered cell of the present invention.
FIG. 2 is a schematic representation of the order of attachment of the portions of the knockout-targeted armed chimeric antigen receptor in example 1.
FIG. 3 shows the secondary structure diagram of the human ACE2 protein targeting SARS-CoV-2 virus SPIKE protein in the KD-2019CAR molecule of the present invention; a is a secondary structure diagram of human ACE2 protein, and B-G are secondary structure diagrams of 6 sequences which have good stability and high ligand binding force and are selected by the inventor through library screening and software analysis of biological characteristics.
FIG. 4 shows the results of flow cytometry for T cell purity in example 3.
FIG. 5 shows the flow cytometric assay results for the expression of SPIKE chimeric antigen receptor targeting SARS-CoV-2 virus in example 4, control A and KD-2019B.
FIG. 6 is a fluorescent picture of stably transfected 293T cell lines overexpressing SARS-CoV-2 virus SPIKE and SARS-CoV-2 virus RNA polymerase constructed in example 5, wherein A is bright field, B is GFP, and C is confluency.
FIG. 7 shows the results of Western blotting of FLAG protein in 293T cells (SR-293T cells) overexpressed in example 5.
FIG. 8 shows the results of 293T cell killing assay of KD-2019CAR-T cells against over-expressing viruses SARS-CoV-2SPIKE and RdRp.
FIG. 9 is the PCR results of RdRp in 293T cells after co-incubation of KD-2019CAR-T with 293T cells overexpressing the viruses SARS-CoV-2SPIKE and RdRp.
FIG. 10 schematic representation of lentiGuide-Puro vector.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The term "functional variant" is a variant of a parent structure that has been modified to have the same or similar biological function and properties, such as a structure that has the same targeted binding function as the parent. By way of non-limiting example, a "functional variant" may be obtained by making one or more conservative substitutions in the parent. A functional variant in this application is a structure that binds to the SPIKE target of SARS-CoV-2 virus, which results from a modification of the amino acid sequence of human ACE 2.
The term "analog" refers to a structurally related polypeptide that has the function of the reference polypeptide molecule. In this application, refers to a polyamino acid structure that is related to the amino acid sequence structure of the ligand SARS-CoV-2 virus SPIKE and that has the function of targeting the binding of SARS-CoV-2 virus SPIKE to human ACE 2.
The term "amino acid modification" refers to a conservative amino acid modification that does not significantly affect or alter the binding characteristics of a CAR (e.g., extracellular recognition domain) of the present disclosure comprising an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.
The term "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid within the same group.
The term "homology": refers to a high proportion of amino acids or nucleotides that are matched by comparison of a target amino sequence or target nucleotide sequence to a reference sequence. Homology in this context can be determined using standard software such as BLAST or FASTA
The term "Chimeric Antigen Receptor (CAR)"
The chimeric antigen receptor includes a leader peptide portion, an extracellular target identification domain, a transmembrane domain, and an intracellular domain.
CARs can both bind antigen and transduce functions of T cell activation that are independent of MHC restriction. Thus, CARs are "universal" immune antigen receptors that can treat a population of patients with antigen-positive tumors regardless of their HLA genotype. Adoptive immunotherapy using T lymphocytes expressing tumor-specific CARs can be a powerful therapeutic strategy for treating cancer.
The term "recognition" refers to selective binding to a target. The term "specifically binds" or "specifically binds to" or "specifically targets" as used herein refers to a polypeptide or fragment thereof that recognizes and binds to a biological molecule of interest (e.g., a polypeptide), but which does not substantially recognize other molecules in a sample, e.g., other molecules in a biological sample that naturally includes a polypeptide of the invention.
The term "specific binding" refers to the association between two molecules (e.g., a ligand and a receptor) characterized by the ability of one molecule (ligand) to bind to another specific molecule (receptor), even in the presence of many other different molecules, i.e., the ability to show preferential binding of one molecule to another in a heterogeneous mixture of molecules. Specific binding of the ligand to the receptor was also demonstrated as follows: in the presence of excess unlabeled ligand, the detectably labeled ligand has reduced binding to the receptor (i.e., a binding competition assay).
The term "co-stimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that is required for an effective response of lymphocytes to an antigen.
The term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with appropriate control elements and which can transfer gene sequences into a cell. Thus, the term includes cloning and expression vectors, as well as viral vectors and plasmid vectors.
The term "expression vector" refers to a recombinant nucleic acid sequence, i.e., a recombinant DNA molecule, which contains the desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host organism. The nucleic acid sequences necessary for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers and terminators, as well as polyadenylation signals.
The term "immunoresponsive cell" as used herein refers to a cell that plays a role in an immune response, or a progenitor cell thereof, or a progeny thereof.
The term "isolated cell" refers to an immune cell that is separated from the molecules and/or cellular components that naturally accompany the cell.
The term "modulate" as used herein refers to a change, either positively or negatively.
The term "pathogen" as used herein refers to a virus, bacterium, fungus, parasite or protozoan capable of causing a disease.
The term "treatment" refers to clinical intervention in an attempt to alter the disease process of the individual or cell being treated, and may be used prophylactically, or during the course of clinical pathology. Therapeutic effects of treatment include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, decreasing the rate of disease progression, alleviation or palliation of the disease state, and remission or improved prognosis. By preventing the progression of a disease or disorder, treatment can prevent the exacerbation of a disorder in an affected or diagnosed subject or a subject suspected of having a disorder, and treatment can prevent the onset of a disorder or symptoms of a disorder in a subject at risk of, or suspected of having, the disorder.
The term "disease" as used herein refers to any condition or disorder that disrupts or interferes with the normal function of a cell, tissue or organ. Examples of diseases include neoplasia or pathogen infection of a cell.
The term "exogenous nucleic acid molecule or polypeptide" as used herein refers to a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or in a sample obtained from a cell. The nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample.
The present invention will be further described with reference to the following examples, but the present invention is not limited to these specific embodiments. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: preparation of specific knockout SARS-CoV-2 virus RNA polymerase construction targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor expression plasmid (KD-2019 lentivirus vector)
The overall design is as follows:
1. determination of amino acid sequence of specific knockout SARS-CoV-2 virus RNA polymerase constructed targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor
First, the full-length amino acid sequence of human ACE2 (NP _068576.1) and the full-length amino acid sequence of SPIKE protein (YP _009724390.1) were searched from the Genbank database of the national library of medicine (NCBI) of the united states.
Secondly, constructing specific knockout SARS-CoV-2 virus RNA polymerase to construct targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor, namely determining the amino acid sequence of armed chimeric antigen receptor molecule:
from amino terminal to carboxyl terminal, the peptide comprises an amino acid sequence of a leader peptide (shown as SEQ ID No. 1), an amino acid sequence of human ACE2 (shown as SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No. 7; A shown as figure 3 is a secondary structure diagram of human ACE2 protein, B-G is a secondary structure diagram of 6 sequences which are screened by the inventor through library screening and analyzed by software for biological characteristics, so that the secondary structure diagram of 6 sequences with good stability and high ligand binding force is selected), an amino acid sequence of a human CD8 hinge region (shown as SEQ ID No. 8), an amino acid sequence of a human CD8 transmembrane region (shown as SEQ ID No. 10), an amino acid sequence of a human 4-1BB intracellular domain (shown as SEQ ID No. 11), an amino acid sequence of a human CD3 domain (shown as SEQ ID No. 14), an amino acid sequence of Furin-2A (shown as SEQ ID No. 15), The amino acid sequence of the IL-2 signal peptide (shown as SEQ ID No. 16) and the scFv amino acid sequence of the IL-6R (shown as SEQ ID No. 17) are connected in series in sequence.
The corresponding nucleotide sequence is:
from 5 'end to 3' end as shown in figure 2, the gene sequence comprises a nucleotide sequence (shown as SEQ ID No. 18) encoding a leader sequence, a nucleotide sequence (shown as SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23 or SEQ ID No. 24) encoding a human ACE2 sequence, a nucleotide sequence encoding a human CD8 hinge region, a nucleotide sequence encoding a human CD8 transmembrane region, a nucleotide sequence encoding a human 4-1BB intracellular domain, a nucleotide sequence encoding a CD3 zeta domain, a nucleotide sequence of Furin-2A (shown as SEQ ID No. 25), a nucleotide sequence of IL-2 signal peptide (shown as SEQ ID No. 26), a scFv nucleotide sequence of IL-6R (shown as SEQ ID No.27), a nucleotide sequence of U6 promoter (shown as SEQ ID No. 28) and a SARS-CoV-2 RNA polymerase sequence (shown as SEQ ID No. 32), SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.37 or SEQ ID No. 38) are connected in series in sequence.
2. Construction of plasmid for constructing targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor molecule by expressing specific knockout SARS-CoV-2 virus RNA polymerase
Selecting one sequence in the step 1 as a subsequent research sequence, and naming the sequence as KD-2019, wherein the specific sequence is as follows:
from amino terminal to carboxyl terminal, the gene sequence is composed of amino acid sequence of guide peptide (shown as SEQ ID No. 1), amino acid sequence of human ACE2 (shown as SEQ ID No.2), amino acid sequence of human CD8 hinge region (shown as SEQ ID No. 8), amino acid sequence of human CD8 transmembrane region (shown as SEQ ID No. 10), amino acid sequence of human 4-1BB intracellular structure domain (shown as SEQ ID No. 11), amino acid sequence of human CD3 zeta structure domain (shown as SEQ ID No. 14), amino acid sequence of Furin-2A (shown as SEQ ID No. 15), amino acid sequence of IL-2 signal peptide (shown as SEQ ID No. 16) and amino acid sequence of scFv of IL-6R (shown as SEQ ID No. 17) which are connected in series in sequence, wherein SARS-CoV-2 virus RNA polymerase sequence (shown as SEQ ID No. 33) is connected in series.
A nucleotide sequence (biosynthetic of Nanjing Optimalaceae) of a targeted SARS-CoV-2 virus SPIKE armed chimeric antigen receptor molecule is constructed by a full-gene synthesis specificity knockout SARS-CoV-2 virus RNA polymerase, and is connected to a lentiviral vector lentiGuide-Puro (Addgene, USA) in a molecular cloning mode to construct a full-length CAR sequence expression frame with a single coding frame, and an EF1 alpha promoter (shown in a sequence table SEQ ID No.30) or an EFS promoter (shown in a sequence table SEQ ID No. 31) is utilized for expression.
The specific operation steps are as follows:
the full-gene synthesis specific knockout of SARS-CoV-2 virus RNA polymerase constructs a nucleotide sequence (Nanjing one) of a targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor molecule, artificially synthesizes a CAR molecule sequence through PCR amplification, recovers the CAR molecule sequence through an Axygen gel recovery kit (Hangzhou Zealand balance), and performs homologous recombination connection with a vector lentiGuide-Puro (Addgene, USA) digested by restriction enzymes SmaI and MluI, wherein the vector schematic diagram is shown in figure 10.
The specific recombination and ligation reaction system and conditions are as follows:
recombination and connection system:
PCR product 5. mu.l recovered from the gel, SmaI and MluI enzyme digestion lentiGuide-Puro plasmid (Addgene, USA) 3. mu.l recovered from the gel; 4X 1402quick cloning Kit (Nanjing Kinuomei) 5. mu.l; 7 mu l of deionized water; the volume of the ligation reaction system is 20 μ l;
recombinant ligation conditions: placing the reaction system in a water bath at 50 ℃, reacting for 15min, and placing on ice for 1min.
10ul of the recombinant ligation product was transformed with competent Stbl3, using the following procedure.
Mu.l of the ligation product was added to 50. mu.l of competent cells (Stbl3, purchased from Invitrogen, USA) and ice-cooled for 30min at 42 ℃ for 45s for 2min, and then 500. mu.l of non-resistant LB liquid medium was added and shake-cultured at 37 ℃ and 200rpm for 40min, spread on ampicillin-resistant LB solid plates, and left overnight in a 37 ℃ incubator. After a single bacterial colony appears, selecting 5 bacterial colonies with proper size, extracting plasmids, sending the plasmids to a commercial sequencing company for sequencing, comparing a sequencing result with fitted nucleotides (namely the nucleotides for specifically knocking out SARS-CoV-2 virus RNA polymerase to construct a targeted SARS-CoV-2 virus SPIKE armed chimeric antigen receptor) to confirm that the sequence is completely correct, and proving that the plasmids (short for KD-2019 lentiviral vectors) for expressing the specifically knocked out SARS-CoV-2 virus RNA polymerase to construct the targeted SARS-CoV-2 virus SPIKE armed CAR molecules are obtained.
The specific steps of constructing the targeted SARS-CoV-2 virus SPIKE armed chimeric antigen receptor expression plasmid (KD-2019 lentiviral vector) by targeted specific knockout of SARS-CoV-2 virus RNA polymerase, refer to the instruction manual of Qiagen plasmid Midi Kit (Germany Qiagen).
Example 2: preparation of viral fluid (KD-2019 viral fluid) of lentivirus vector
The expression-specific knockdown SARS-CoV-2 virus RNA polymerase obtained in example 1 was used to construct recombinant plasmids targeting the SARS-CoV-2 virus SPIKE armed chimeric antigen receptor (KD-2019CoV lentiviral vector) and packaging vectors psPAX2 and VSVG, following a 10: 8: 5, using LipofectamineTM6000 transfection reagent (purchased from ThermoFisher company, product model 11668019) co-transfects 293T cells (the specific transfection operation process is referred to as ThermoFisher transfection instruction), the cell supernatants rich in lentiviral particles are collected after 6 hours of transfection by replacing complete culture medium (purchased from Life Technologies, product model 11995-.
Example 3: isolated culture of T cells
Taking fresh peripheral blood of a healthy donor, and centrifugally separating the fresh peripheral blood mononuclear cells by density gradient; then, paramagnetic beads (purchased from Invitrogen, USA, and the product information is shown in the specification) coupled with anti-CD 3 antibody and anti-CD 28 antibody are used
Figure RE-GDA0002599988660000211
Human T-Activator CD3/CD28) to enrich for CD3+ T cells, specifically, peripheral blood mononuclear cells are diluted to a concentration of (c10～30)×106The individual cells/ml were mixed with Magnetic beads at a ratio of 3:1, incubated at room temperature for 2-3 hours, and enriched for CD3+ T cells using a Magnetic particle collector (MPC, available from Invitrogen, USA). The enriched CD3+ T cells were finally resuspended in culture medium (purchased from Life Technologies, USA, under the product information OpTsizerTMT-CellExpansion SFM), adjusted to a cell solubility of 1 × 106One/ml, finally 5% CO at 37 ℃2The culture was carried out in an incubator for 2 days, and the results of the measurement are shown in FIG. 4.
Example 4: preparation of targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor T cell (KD-2019CAR-T) by specific knockout of SARS-CoV-2 virus RNA polymerase construction
First, the CD3+ T cells obtained in example 3 were seeded in a 24-well plate at a seeding concentration of 1 × 105Cell/ml at 37 ℃ with 5% CO2The culture is carried out in the environment for about 24 hours (the culture time depends on the specific practice, and generally, the cell confluence rate is ensured to be between 50 and 70 percent when the virus liquid is infected).
Then, the virus solution of the KD-2019 lentiviral vector collected in example 2 was taken, and added to a cell culture flask together with the virus solution according to the MOI of 1 to 10, and the flask was sealed, placed in a flat angle centrifuge, centrifuged at a low speed (500g to 1000g/min) for 1 hour, and then placed in an incubator to be cultured at 37 ℃. Expression specificity is obtained 48 hours after infection to knock out SARS-CoV-2 virus RNA polymerase, a target SARS-CoV-2 virus SPIKE armed chimeric antigen receptor T cell (KD-2019CAR-T) is constructed, a next functional experiment can be carried out, and a working schematic diagram of the engineering cell is shown in figure 1, namely the engineering cell captures a novel coronavirus through camouflage and inhibits the virus from normally replicating, so that the engineering cell loses the capability of attacking human cells; meanwhile, toxic and side effects caused by cytokine storm can be rapidly solved after IL-6 receptor is blocked; the novel coronavirus is captured by camouflage, the virus is eliminated, cytokine syndromes are prevented, and finally good clinical treatment effects are achieved.
Example 5: construction of Stable transgenic 293T cell line (SR-293T cell) overexpressing SARS-CoV-2 Virus SPIKE and SARS-CoV-2 Virus RNA polymerase
The gene is synthesized by whole gene, the overexpressed SARS-CoV-2 virus SPIKE gene and SARS-CoV-2 virus RNA polymerase are synthesized, and a nucleotide sequence with a FLAG label (Nanjing-one organism) is amplified by PCR, an artificially synthesized overexpressed molecular sequence is recovered by an Axygen gel recovery kit (Hangzhou Zhang Heng), and homologous recombination and connection are carried out with a vector pTomo-pCMV-IRES-puro-EGFP (Addgene, USA) digested by restriction enzymes XbaI and BamHI. Then 10ul of recombinant ligation products were transformed with competent Stbl3, and then positive single clones were screened and obtained, followed by extraction and purification of the overexpression plasmids. Then, a virus solution overexpressing SARS-CoV-2 virus SPIKE and SARS-CoV-2 virus RNA polymerase was obtained according to the method of example 2. Establishing a killing curve of puromycin in 293T cells to determine the optimal drug screening concentration; the obtained virus liquid is then infected into 293T cells, screening drugs are added into 6-well plates after 72 hours of infection, and the liquid is changed again and the drugs are added every 2 days. Drug screening was continued for at least 14 days until the proportion of fluorescent cells observed under a microscope was 100% (FIG. 6). Thus, a stable transgenic 293T cell line (SR-293T cell) overexpressing SARS-CoV-2 virus SPIKE and SARS-CoV-2 virus RNA polymerase was obtained.
Example 6: detection of CAR expression in KD-2019 and FLAG protein expression in SR-293T cells
1. Detection of CAR protein expression Using flow cytometry analysis
The cells were centrifuged, washed twice with PBS and resuspended in FACS fluid (PBS with 0.1% sodium azide and 0.4% BSA); adding Anti-ACE2 antibody (ab189168) to the cell suspension and incubating for 60 min at 4 ℃; after washing the cells twice, Goat Anti-Rabbit IgG H was added&L(Alexa
Figure RE-GDA0002599988660000231
488) (ab150077), 60 minutes at room temperature; bdfacscan to II was used to obtain stained cells and FlowJo was used to analyze the results. As shown in figure 5, the control group was T cells infected with empty viral fluid, and expression of the CAR molecule was barely detectable; the experimental group is T cells infected with KD-2019CAR molecule virus liquid, and the expression rate of CAR molecules is high;
2. validation of overexpression of SARS-CoV-2 Virus SPIKE and SARS-CoV-2 Virus RNA polymerase
Expression of over-expressed SARS-CoV-2 virus SPIKE and viral RNA polymerase (RdRp) was detected using western blotting.
After washing the stably transfected 293T cells overexpressing SARS-CoV-2 virus SPIKE and SARS-CoV-2 virus RNA polymerase obtained in example 5, they were lysed in RIPA lysate containing protease inhibitor for 20min on ice, and the lysate supernatant obtained after high-speed centrifugation was mixed with 4 XSDS loading buffer, separated in 4-12% Bis-Tris precast gel (NP0335BOX, Life technologies), and then the proteins were transferred to PVDF membrane by electroporation. PVDF membrane was blocked in TBST containing 5% skim milk powder for 1 hour in Anti-DDDDDDK tag (Binds to)
Figure RE-GDA0002599988660000232
tag sequence) antibody [ M2](HRP) (ab49763) overnight at 4 ℃. Membranes were washed 3 times with TBST, with 1: 10000 goat anti-mouse IgG secondary HRP-conjugated antibody (ab205718) was incubated at room temperature for 1 hour. After 5min incubation with ECL Plus luminophore, exposure was carried out in a Tanon 9500 machine. As shown in FIG. 7, the expression of FLAG protein was strong in the stably transformed 293T cells (SR-293T cells) overexpressing SARS-CoV-2 virus SPIKE and SARS-CoV-2 virus RNA polymerase obtained in example 5, relative to the 293T cell control which was not overexpressed;
because the SARS-CoV-2 virus SPIKE gene and SARS-CoV-2 virus RNA polymerase gene are connected in series and are connected with FLAG nucleotide, SR-293T cell can detect FLAG protein expression, and can prove that the SPIKE protein and RNA polymerase exist in SR-293T cell.
Example 7: killing effect of KD-2019CAR-T cell on SR-293T
The killing of SR-293T by KD-2019CAR-T was evaluated using a 7-AAD/CFSE cytotoxicity test kit (purchased from Biovision, Inc., product number K315-100).
After CSFE labelling of tumor cells, 2 × 10 per well4The cells of (a) were plated in culture plates, and the ratio of 10: 1. 5: 1 and 1: 1 ratio KD-2019CAR-T cells obtained in example 5 were added to 293T cells as described above (normal 293T cells were used as negative control), and culturedAfter 24 hours of incubation, the supernatant was centrifuged off, the cell pellet washed and stained with 7AAD, BD facs cantonii was used to obtain stained cells, FlowJo was used to analyze the results and statistically plotted (fig. 8).
Example 8: detection of the Effect of KD-2019CAR-T on mRNA levels in SR-293T
At each hole 2 × 104The over-expressing 293T cells of (a) were plated in culture plates at 10: 1 ratio KD-2019CAR-T cells obtained in example 5 were added to 293T cells as described above (normal 293T cells and KD-2019CAR-T cells were used as negative controls), and after culturing for 24 hours, the cells were collected by centrifugation; the cells were then lysed using TRIzol, followed by addition of chloroform to separate the upper aqueous phase, addition of an equal volume of isopropanol to precipitate the RNA, and washing of the RNA precipitate with 75% ethanol. Selecting high-purity RNA, detecting the absorbance (A) value of the RNA so that the ratio of A (260)/A (280) is 1.8-2.0, and performing a subsequent reverse transcription experiment after removing impurity DNA by DNase I. Add 1. mu.g RNA, 8. mu.L DEPC-H to PCR tube2O and 1. mu.L oligo-dT in a total volume of 10. mu.L, denaturing the mixture on a PCR apparatus at 70 ℃ for 5min, then rapidly cooling the PCR tube on ice, and preparing a mixture of 5 × Reaction Buffer 4. mu.L, 2.5 mmol.L-1dNTP mix 4. mu.L, RNase Inhibitor 1. mu.L, MMLVReverse Transcriptase 1. mu.L. the above Mixture (10. mu.L) was added to a PCR tube, gently whipped and mixed, and mRNA expression levels of the control and experimental groups RdRP were detected using 2 × Real Star Green Power mix at 42 ℃, 1h, 70 ℃ and 5min on a PCR instrument.
Among a plurality of different proteins of coronaviruses, RNA-dependent RNA polymerase, namely RdRp plays a very important role in the virus propagation replication period, so the coding specificity knockout SARS-CoV-2 virus RNA polymerase nucleotide (shRNA) sequence is designed, and is shown as SEQ ID No.32, SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.37 or SEQ ID No. 38; in the embodiment, the influence of KD-2019CAR-T on mRNA in SR-293T is detected to indirectly indicate the RNA polymerase knockout effect, and the experimental result (shown in FIG. 9) shows that the expression level of mRNA is remarkably reduced in the experimental group compared with the control group, and indicates that KD-2019CAR-T has the knockout effect on RdRp in SR-293T.
In conclusion, the specific knock-out SARS-CoV-2 virus RNA polymerase constructed by the invention constructs a virus vector targeting SARS-CoV-2 virus SPIKE armed chimeric antigen receptor, and the modified engineering immune cell can be applied to the treatment of coronary virus diseases, including novel coronary pneumonia, SARS, MERS and the like.
It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Sequence listing
<110> Nanjing Kaidi Biotech Co., Ltd
<120> armed chimeric antigen receptor cell of targeted coronavirus SPIKE, preparation method and application
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Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu Arg
130 135 140
Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu Arg
145 150 155 160
Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg Ala
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Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu Val
180 185 190
Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu Asp
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Ala Tyr Ala Lys Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile Ser
225 230 235 240
Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Ala Ala Trp Gly Arg
245 250 255
Phe Trp Thr Asn Leu Tyr Thr Ala Ser Ala Pro Phe Gly Gln Lys Pro
260 265 270
Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala Gln
275 280 285
Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu Pro
290 295 300
Asn Ala Ser Asn Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro Gly
305 310 315 320
Gln Ala Asn Lys Ala Val Cys His Pro Ser Val Trp Asp Leu Gly Lys
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Gly Asp Phe Arg Ile Leu Met
340
<210>8
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<212>PRT
<213> CD8 hinge region (2 Ambystoma latex x Ambystoma jeffersonia)
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<210>9
<211>20
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<213> IgG1 hinge region (2 Ambystoma latex x Ambystoma jeffersonia)
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<210>10
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35 40
<210>12
<211>107
<212>PRT
<213> CD28 Domain (2 Ambystoma latex x Ambystoma jeffersonia)
<400>12
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
1 5 10 15
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
20 25 30
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly
35 40 45
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
50 55 60
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
65 70 75 80
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
85 90 95
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
100 105
<210>13
<211>38
<212>PRT
<213> ICOS Domain (2 Ambystoma latex x Ambystoma jeffersonanium)
<400>13
Cys Trp Leu ThrLys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn
1 5 10 15
Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg
20 25 30
Leu Thr Asp Val Thr Leu
35
<210>14
<211>112
<212>PRT
<213> CD3 zeta Domain (2 Ambystoma laterale x Ambystoma jeffersonia)
<400>14
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210>15
<211>26
<212>PRT
<213>Furin-2A(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400>15
Arg Ala Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr
1 5 10 15
Cys Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210>16
<211>20
<212>PRT
<213> IL-2 Signal peptide (2 Ambystoma latex x Ambystoma jeffersonia)
<400>16
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser
20
<210>17
<211>677
<212>PRT
<213> anti-human IL-6R antibody scFv (2 Ambystoma latex x Ambystoma jeffersonia)
<400>17
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
225 230 235 240
Val Arg Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr
245 250 255
Ser Ile Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly
260 265 270
Arg Gly Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr
275 280 285
Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser
290 295 300
Lys Asn Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr
305 310 315 320
Ala Val Tyr Tyr Cys Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp
325 330 335
Tyr Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys
340 345 350
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
355 360 365
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
370 375 380
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
385 390 395 400
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
405 410 415
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
420 425 430
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
435 440 445
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
450 455 460
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
465 470 475 480
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
485 490 495
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
500 505 510
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
515 520 525
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
530 535 540
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
545 550 555 560
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
565 570 575
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
580 585 590
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
595 600 605
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
610 615 620
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
625 630 635 640
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
645 650 655
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
660 665 670
Ser Leu Ser Pro Gly
675
<210>18
<211>63
<212>DNA
<213> leader sequence (2 Ambystoma latex x Ambystoma jeffersonanium)
<400>18
atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60
ccg 63
<210>19
<211>1020
<212>DNA
<213> human ACE2 sequence 1(2 Ambystoma laterale x Ambystoma jeffersonoanum)
<400>19
cagtctacca tagaagagca agcaaaaacg tttttggata aattcaatca tgaagctgag 60
gatctgtttt atcaatcatc acttgcctcc tggaattaca atactaacat cacagaggaa 120
aacgtccaaa atatgaataa cgccggtgac aagtggagcg cgttccttaa agaacagtcc 180
acacttgcac agatgtaccc ccttcaggaa attcagaacc tcacggtgaa acttcaactg 240
caggcacttc aacagaatgg gactactgca gcaactgagg acaagtctaa aagattgaat 300
acaattctta acactatgtc tactatatac tctacgggca aagtgtgtaa tccagacaac 360
cctcaagagt gtctgctgct tgagccggga ctcaacgaga taatggcgca aacggctgag 420
tacaacgaaa ggctgtgggc ttgggagtca tggagaagtg aggtcgggaa gcagcttcgg 480
cccctgtatg aggaatacgt tgtcctcaag aacgagatgg cccgagctaa ccactacgag 540
gattatggcg actactggcg gggtgactac gaagtcaatg gtgtggacgg atacgattac 600
tcccgaggcc aagctgctga tgaagctgag cacacctttg aagaaattaa gcctctctac 660
gaacatctgc acgcatatgt gagggccaaa cttatgaacg cttacccatc ttacatatca 720
ccaatagggt gcttgccagc tcaccttttg ggcgacatgt gggggcgctt ttggacaaat 780
ctgtactctt tgaccgcccc atttggacag aagccgaaca tagatgttac ggatgctatg 840
gttgatcagg catgggatgc acagcgcatt tttaaggaag ctgaaaagtt cttcgtgtct 900
gtagggcttc cgaatatgac gcaaggattt tgggaaaact ccatgctcac cgatccgggc 960
aatgtccaaa aagctgtttg ccatcccact gcttgggatc tgggcaaagg tgattttcgc 1020
<210>20
<211>1041
<212>DNA
<213> human ACE2 sequence 2(2 Ambystoma laterale x Ambystoma jeffersonoanum)
<400>20
caatctacta tagaagagca ggctaaaact ttcctggaca agtttaacca tgaagctgcg 60
gcactgtttt atcagagctc acttgcctca tggcagtaca atacgaatgc aagcgaggag 120
caagttcaaa acatgaacaa cgctggtgac aaatggagcg cgtttgcgcg agataatacg 180
actctcgccc agatgtatcc acttcaagag atacaaaatc tgacagtgaa gttgcaactt 240
caggctcttc aacagaacgg ctctagcgtg ttgagtcctg acaaatccaa acggcttaat 300
actattctta atactatgtc tacgatttac tccacaggta aagtatgcaa ccccgataat 360
ccacaggaat gcctccttct cgagccaggt ttgaatgaag ccgctgcaaa ttccctggac 420
tataaccgac gcttgtgggc ctgggagtct tggaggtcag aggtgggaaa acagctccga 480
cctctgtacg aagaatatgc agcagcaaga aacgagatgg cacgcgctaa ccactatgag 540
gattatggtg actactggag gggagattac gaggtcaatg gcgtagatgg ctacgattac 600
tctaggggtc agctgattga ggacgttgaa cacacatttg aggagattaa gccgctctac 660
gagcaccttc atgcctacgt gcgagcaaaa ctcatgaacg catatcctag ttatatctct 720
cccatcggtt gccttccagc gcatctgttg ggtgacatgt ggggtaggtt ttggacaaat 780
ctctactctc tttctgcccc tttcgggcaa aagcctaata tagacgtgac tgatgcaatg 840
gtcgaccaag cctgggacgc ccaacgcatc tttaaagagg ctgagaaatt ttttgtaagt 900
gtcgggctgc ctaacatgac tcaaggcttt tgggagaata gtatgcttac agaccccgga 960
aatgttcaga aggccgtatg ccacccgaca gcatgggatt tgggcaaagg cgattttagg 1020
attctcatgt gtactaaagt t 1041
<210>21
<211>1026
<212>DNA
<213> human ACE2 sequence 3(2 Ambystoma laterale x Ambystoma jeffersonoanum)
<400>21
caatcaacaa tagaggaaca agcaaaaact ttcttggaca agttcaacca tgaggccgaa 60
gaccttttct atcagagcag tcttgcttct tggaactaca acactaatat aactgaagaa 120
aatgttcaga acatgaacaa tgcgggcgac aaatggtccg cttttctgaa ggaacaaagt 180
acgcttgctc agatgtatcc ccttcaagaa attcagaatc tcacagttaa gctccagttg 240
caggcccttc agcaaaacgg tagctctgtt ctctccgaag ataagtcaaa gcgacttaac 300
acaatactca acactatgtc cacgatatac tctacgggaa aggtctgtaa cccggataat 360
ccccaggaat gtttgctgct ggagcctggg ctgaatgaga tcatggcgaa ttctcttgat 420
tacaacgagc gactgtgggc ctgggagtcc tggcggagcg aggtcggcaa gcagcttagg 480
ccactgtacg aggagtatgt tgtgttgaaa aacgaaatgg cccgcgcgaa tcattatgag 540
gactacggtg actactggcg gggagattac gaagctaatg gagtcgatgg gtatgattac 600
tcacgaggac aactgataga ggacgtcgag cacacatttg acgacgccaa gcctctgtat 660
gaacacctcc acgcctatgt acgcgttcgc gctgcccaag catatcctag ctacatatcc 720
cctattggct gtctgcccgc gcacctcctg ggggacatgt gggggcggtt ctggactaac 780
ctgtatacag ctagtgcccc atttggtcaa aagcccaata tagacgtgac cgatgctatg 840
gcggaaaacg cttgggacgc ccaaagaatc tttaaagaag ctgagaaatt ttttgtttca 900
gttggtctcc caaacatgac gcagggcttc tgggaaaatt caatgctcac agatcctggt 960
aacgttcaaa aagcggtgtg ccaccctacc gcctgggacc tcggcaaagg agatttccgc 1020
atcctt 1026
<210>22
<211>1044
<212>DNA
<213> human ACE2 sequence 4(2 Ambystoma laterale x Ambystoma jeffersonoanum)
<400>22
cagtcaacta ttgaagagca ggccaaaacc ttcttggata agttcaacca cgaagcagag 60
gacctcttct accagtcttc cttggcgtct tggaactaca atacaaacat aaccgacgat 120
aatgtccaaa acgcaaataa tgcgggcgac aaatggtctg ccttccttaa ggagcagtcc 180
accctcgccc agatgtatcc ccttcaagag attcagaact tgactgttaa actgcaattg 240
caagctttgc agcaaaacgg tagctcagtc ctttcagaag ataaaagcaa gcggctgaat 300
acaatactga atacggcaac aagtgcatac tctaccggga aagtttgtaa tccagataac 360
ccccaggagt gcgctgcggc tgagccgggg ctcaacgaaa taatggcgaa ctcactcgat 420
tataacgaac ggctgtgggc gtgggagtcc tggcgcactg acgcgggaaa acaattgaga 480
cctctttacg aggaatacgt cgtactgaaa aacgaggctg taaaggtaca gcattacgaa 540
gattatggtg actactggag gggtgattac gaagttaacg gagtcgacgg ttatgactat 600
tcccgaggcc aactgatcga ggatgtagaa catacatttg aagagattaa accactttac 660
gagcatctgc acgcgtatgt acgagcaaag ctgatgaacg cataccccag ttacatcagt 720
cctattggtt gcctcccagc ccacctgctt ggcgacatgt gggggcgatt ttggacgaat 780
ctctacagct tgacggtacc ctttggacag aaaccaaaca tagatgttac tgacgcaatg 840
gttgaccaag cgtgggatgc tcaaagaatt ttcaaagagg cagaaaagtt cttcgtgtct 900
gtcggccttc cgcaggcaag taatggattc tgggaaaact ctatgcttac agacccaggc 960
aatgtccaaa aagcggtatg ccatcctact gcctgggatc tgggcaaggg agactttcgg 1020
atattgatgt gtacaaaagt cacg 1044
<210>23
<211>1035
<212>DNA
<213> human ACE2 sequence 5(2 Ambystoma laterale x Ambystoma jeffersonoanum)
<400>23
caatctacca tagaggaaca ggcgaaaact tttttggaca agttcaatca tgaggctgaa 60
gacctctttt accaatcaag tctggctagc tggaattaca acactaacat aacggaagag 120
aatgtacaga acatgaataa tgctggggat aagtggagtg cgttcctcaa agaacagtca 180
accctggctc aaatgtatcc cctccaagaa atacaaaacc tgaccgtcaa actccaactt 240
caagcattga ataataacgg tactactgct gccagcgaag acaagtccaa acgcttgaat 300
acggctgcta atacgatgtc tacgatatac tctactggca aggtttgcaa tccagataat 360
ccccaggaat gtgcgctttt ggaaccgggg cttaacgaag ccgccgcaaa cagtcttgac 420
tataatgaac gcctttgggc ctgggaatca tggcgatcag aggttggaaa acaacttaga 480
cccttgtatg aggagtacgt ggtagcgaga aacgaaatgg cgagagccaa tcactatgag 540
gattatggcg actactggcg gggggattat gcagctgccg gggtagatgg ttatgactat 600
tctaggggtc aggctgcgga tgaagtggaa catacgttcg aggagatcaa accgctgtac 660
gaacatctgc acgcttacgt tagggcgaag ctgatgaatg cctatccaag ctatattagc 720
cccataggat gcctgccggc ccatctcctt ggagacatgt ggggtcgatt ctggaccaac 780
ctctattcac ttacagttcc ctttgggcaa aagccgaaca tcgacgtaac cgacgcgatg 840
gtagaccagg cctgggacgc tcagaggata tttaaggaag ccgaaaagtt ttttgtttct 900
gttggcctgc ctaatatgac tcagggcttt tgggaaaatt ctatgctcac tgatcccgga 960
aacgtacaaa aagctgtatg ccaccctacg gcgtgggatc tcggtaaagg tgattttagg 1020
attcttatgt gtacc 1035
<210>24
<211>1029
<212>DNA
<213> human ACE2 sequence 6(2 Ambystoma laterale x Ambystoma jeffersonoanum)
<400>24
cagagtacca tagaagaaca ggctaaaaca tttctggaca agttcaacca tgaggcagag 60
gaccttttct accaatcctc tttggcatcc tggaactata acactaacat cacggaggag 120
aacgtccaaa acatgaacaa cgccggagat aagtggtctg cattcctcaa agaacagtcc 180
acattggcgc agatgtaccc ccttcaagaa atacaaaatc tcactgtgaa gctccaattg 240
caagctctcc aacaaaacgg ttcctccgtc ctttctgaag acaaaagtaa gaggctgaat 300
accattctca acaccatgtc caccatatac agcaccggaa aagtttgtaa cccggacaat 360
ccccaagaat gcctgctgct ggaacctggt ttgaacgaga tcatggccaa tagccttgac 420
tataatgaga gactgtgggc ctgggagagc tggcggagtg aggtcggcaa gcagctgcga 480
cctttgtatg aggaatatgt agtccttaag aatgagatgg ccagagctaa ccactacgag 540
gattatggag actactggag gggcgattac gaggtaaatg gcgtagacgg ctacgactac 600
tcacgaggcc agctcattga ggatgtggaa catacttttg aagaaattaa gcctctgtac 660
gaacatttgc atgcctatgc taaggctaaa ctgatgaacg cgtatccgtc atacatatct 720
ccgattggtt gtttgccagc gcacctgctg ggtgcggcct gggggcgatt ctggacgaac 780
ctctacacag cctcagctcc attcggccaa aaacccaaca ttgatgtgac ggatgcgatg 840
gtggaccaag cctgggacgc ccaacggata tttaaggagg cagagaaatt ctttgtgagc 900
gtaggtctgc ctaacgcatc caatggattc tgggaaaatt ccatgctgac agaccccgga 960
caggcaaata aggctgtttg tcatcctagc gtatgggatt tggggaaggg cgattttagg 1020
atcctcatg 1029
<210>25
<211>78
<212>DNA
<213>Furin-2A(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400>25
cgggctaaac gatctgggtc aggtgaaggc aggggcagtc tgttgacatg tggcgatgtc 60
gaagagaatc ctggacca 78
<210>26
<211>60
<212>DNA
<213> IL-2 Signal peptide (2 Ambystoma latex x Ambystoma jeffersonia)
<400>26
atgtatcgaa tgcaacttct ctcctgtatt gcgctctctc tggcacttgt gacaaactca 60
<210>27
<211>2031
<212>DNA
<213> anti-human IL-6R antibody scFv (2 Ambystoma latex x Ambystoma jeffersonia)
<400>27
gacatccaga tgacgcagag ccccagctca ctcagcgcca gtgtgggtga tcgggtcact 60
atcacttgcc gagcttcaca agacatatct agctacttga attggtacca acaaaaacct 120
gggaaagcgc ctaaactttt gacgtactat accagtcggc tgcactctgg ggtccccagc180
cggttttccg ggtctggctc agggacagat tttaccttta cgatctcctc acttcagcca 240
gaagatatag ccacctacta ttgccagcag gggaatactt tgccgtatac tttcggtcaa 300
ggcaccaaag ttgaaatcaa gcgcaccgta gcggcgccct ctgtgtttat attccctccc 360
tccgacgagc agctgaagtc cggtaccgct tcagtcgtat gtttgcttaa caacttctac 420
ccgagagagg ctaaggtaca atggaaagta gataacgcgc ttcagagtgg taactcccaa 480
gaatctgtta ctgagcagga ttctaaggac tcaacatatt ccctgagctc aacgcttaca 540
ctctccaagg ccgactacga gaagcacaaa gtatatgcct gtgaggtcac tcatcagggt 600
cttagcagcc ccgtcacaaa atcttttaac agaggcgaat gtggcggggg cgggtccggt 660
ggcggtggat ctgggggtgg aggtagcgaa gttcagctcc aagaatctgg ccccgggctg 720
gttcgaccta gtcagactct gagcttgact tgcacggtct ctggttactc aattacatct 780
gaccatgctt ggtcatgggt tcgccagcca ccaggtcgag gcctcgagtg gattggctac 840
attagctact ctggaattac tacctacaac ccgtctctta agagtcgcgt cacaatgctc 900
cgagatacga gtaagaacca gttcagcttg cgcctgtctt ctgtaaccgc agcggacacc 960
gctgtttact actgtgccag gtcactcgca cgcacgactg cgatggatta ctgggggcag 1020
ggatcccttg tcactgttag ttccgcctca acgaaaggcc cgagtgtgtt cccgcttgcg 1080
ccctctagta aaagtacgtc tgggggcacc gcggcactcg gctgtctggt aaaggactac 1140
ttcccggaac cagttacggt atcatggaat agtggggcct tgaccagcgg ggtccacact 1200
ttccccgccg tgcttcagtc tagtggcctt tacagcctct catcagtggt aaccgtaccg 1260
agcagttcac tcggcacaca gacttacata tgtaacgtta atcacaagcc atccaatacc 1320
aaagtagata agaaagttga gcctaaaagt tgcgacaaga ctcatacctg cccgccgtgt 1380
cccgcgccgg agctccttgg ggggccatca gttttcttgt ttcctcccaa gccaaaagat 1440
acccttatga tctctaggac ccccgaagta acttgtgtcg tggtggatgt gtcccatgaa 1500
gacccagaag tcaagtttaa ttggtacgtt gatggtgtgg aggttcacaa cgcgaaaacc 1560
aaaccacgcg aagagcaata caactcaact tatagagttg tgtctgttct tacagttctt 1620
caccaagatt ggctgaatgg gaaggagtat aaatgcaagg taagtaacaa agctctccca 1680
gcacctatag agaaaactat cagtaaagcc aaggggcagc cgcgggagcc acaggtttac 1740
accctgcccc cgagccgcga tgaactcacg aaaaatcagg tgtccctgac gtgtcttgtc 1800
aaggggttct acccaagcga cattgctgta gaatgggaaa gtaatgggca gcccgagaat 1860
aattataaaa cgacaccacc tgtgttggac tccgatggaa gctttttcct ttattccaaa 1920
ctcacagtcg ataagagccg atggcagcaa ggtaatgtct tttcctgtag tgtgatgcat 1980
gaagccttgc acaatcatta cactcaaaag tcattgagtc tctcaccagg a 2031
<210>28
<211>241
<212>DNA
<213> U6 promoter (2 Ambystoma laterale x Ambystoma jeffersonanum)
<400>28
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
c 241
<210>29
<211>204
<212>DNA
<213> CMV promoter (2 Ambystoma latex x Ambystoma jeffersonianum)
<400>29
agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc 60
ggagttgtta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt 120
gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt 180
gatgtactgc caaaaccgca tcac 204
<210>30
<211>1259
<212>DNA
<213> EF1 alpha promoter (2 Ambystoma laterale x Ambystoma jeffersonanum)
<400>30
tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg 60
tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 120
cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc 180
gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 240
gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 300
ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 360
gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc 420
ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc 480
gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc 540
ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg 600
ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg 660
gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt 720
cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg 780
gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa 840
ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg 900
cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac 960
aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg 1020
cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg 1080
gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc 1140
cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt 1200
tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtga 1259
<210>31
<211>256
<212>DNA
<213> EFS: EF1 alpha short promoter (2 Ambystoma laterale x Ambystomajeffersonanum)
<400>31
taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg 60
cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc ctagagaagg 120
tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt 180
gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt 240
gccgccagaa cacagg 256
<210>32
<211>58
<212>DNA
<213> RNA-dependent RNA polymerase (SARS-CoV-2 shRNA sequence 12 Ambytoma laterale x Ambytoma jeffersonanum)
<400>32
ccggaggacg aagatgacaa tttaactcga gttaaattgt catcttcgtc cttttttg 58
<210>33
<211>58
<212>DNA
<213> RNA-dependent RNA polymerase (SARS-CoV-2 shRNA sequence 22 Ambytoma laterale x Ambytoma jeffersonia)
<400>33
ccggccaaca tgaagaaaca atttactcga gtaaattgtt tcttcatgtt ggtttttg 58
<210>34
<211>58
<212>DNA
<213> RNA-dependent RNA polymerase (SARS-CoV-2 shRNA sequence 32 Ambytoma laterale x Ambytoma jeffersonanum)
<400>34
ccggacgtca acgtcttact aaatactcga gtatttagta agacgttgac gttttttg 58
<210>35
<211>58
<212>DNA
<213> RNA-dependent RNA polymerase (SARS-CoV-2 shRNA sequence 42 Ambytoma laterale x Ambytoma jeffersonanum)
<400>35
ccggaggtaa ttgtgacaca ttaaactcga gtttaatgtg tcacaattac cttttttg 58
<210>36
<211>58
<212>DNA
<213> RNA-dependent RNA polymerase (SARS-CoV-2 shRNA sequence 52 Ambytoma laterale x Ambytoma jeffersonia)
<400>36
ccggtcctgt tgtagattct tattactcga gtaataagaa tctacaacag gatttttg 58
<210>37
<211>58
<212>DNA
<213> RNA-dependent RNA polymerase (SARS-CoV-2 shRNA sequence 62 Ambytoma laterale x Ambytoma jeffersonanum)
<400>37
ccggctgcat tgtgcaaact ttaatctcga gattaaagtt tgcacaatgc agtttttg 58
<210>38
<211>58
<212>DNA
<213> RNA-dependent RNA polymerase (SARS-CoV-2 shRNA sequence 72 Ambytoma laterale x Ambytoma jeffersonanum)
<400>38
ccggttacga tggtggctgt attaactcga gttaatacag ccaccatcgt aatttttg 58

Claims (8)

1. An armed chimeric antigen receptor modified immune cell targeting SARS-CoV-2 virus SPIKE, characterized in that said immune cell comprises a chimeric antigen receptor having the amino acid sequence:
the amino acid sequence of a guide sequence, the amino acid sequence of an extracellular identification domain targeted to bind to the SPIKE protein of SARS-CoV-2 virus, the amino acid sequence of a hinge region, the amino acid sequence of a transmembrane domain, the amino acid sequence of an intracellular signal domain and the amino acid sequence containing an anti-cytokine receptor component are sequentially connected from an amino terminal to a carboxyl terminal;
wherein the amino acid sequence of the extracellular recognition structural domain of the SPIKE of the targeting combined SARS-CoV-2 virus is as follows: (1) the amino acid sequence of human ACE2 protein shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No. 7; or a variant which is produced by amino acid modification and has 90-99% homology with the amino acid sequence shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No. 7;
the transmembrane domain comprises a CD8 transmembrane domain, a CD28 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain;
the immunoreceptor tyrosine activation motif comprises an intracellular signaling domain of the CD3 zeta chain or an FcRI gamma intracellular signaling structure;
the costimulatory signaling domain comprises a CD28 intracellular signaling domain, a CD137/4-1BB intracellular signaling domain, a CD134/OX40 intracellular signaling domain, and an ICOS intracellular signaling domain;
the anti-cytokine receptor component comprises an scFv amino acid sequence of IL-6R shown in SEQ ID No. 17.
2. The immune cell of claim 1, wherein the immune cell is characterized by
A nucleic acid molecule encoding an armed chimeric antigen receptor targeting the SARS-CoV-2 virus SPIKE protein of the specific knock-out SARS-CoV-2 virus RNA polymerase of claim 1, comprising a nucleotide sequence encoding the guide sequence, a nucleotide sequence of human ACE2, a nucleotide sequence encoding the hinge region, a nucleotide sequence encoding the transmembrane domain, a nucleotide sequence encoding the intracellular signaling domain, a nucleotide sequence encoding the anti-cytokine receptor component, and a SARS-CoV-2 virus nucleotide sequence encoding a specific knock-out RNA polymerase, connected in series in order from 5 'to 3';
wherein:
the nucleotide of the coding guide sequence is shown as SEQ ID No. 18;
the nucleotide sequence for coding the human ACE2 is shown as SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23 or SEQ ID No. 24;
nucleotides encoding the hinge region include: a nucleotide sequence of human CD8 or a nucleotide sequence of human IgG 1;
encoding the transmembrane domain includes: a nucleotide sequence of human CD 8;
the nucleotide sequence encoding the intracellular signaling domain includes: a human 4-1BB intracellular domain, a human CD3 ζ intracellular domain, or a CD3 ζ intracellular domain; wherein, the nucleotide sequence encoding the human 4-1BB intracellular domain; the nucleotide sequence encoding the intracellular domain of human CD 28; the nucleotide sequence encoding the intracellular domain of human CD3 ζ;
the nucleotide sequence encoding the anti-cytokine receptor-containing module includes: the nucleotide sequence of Furin-2A shown as SEQ ID No.25, the nucleotide sequence of IL-2 signal peptide shown as SEQ ID No.26 or the scFv nucleotide sequence of IL-6R shown as SEQ ID No. 27;
the nucleotide sequence of the RNA polymerase for coding the specific knockout SARS-CoV-2 virus is shown as any one of SEQ ID No.32, SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.37 or SEQ ID No.38, or the nucleotide sequence homology of the nucleotide sequence shown as SEQ ID No.32, SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.37 or SEQ ID No.38 is 90-99%.
3. A recombinant vector that specifically knockdown SARS-CoV-2 virus RNA polymerase targeted to SARS-CoV-2 virus SPIKE armed chimeric antigen receptor, comprising the nucleic acid molecule of claim 2.
4. The recombinant vector according to claim 3, wherein the expression plasmid comprises a promoter, wherein said promoter comprises the U6 promoter as shown in SEQ ID No.28 and the EF1 α long promoter as shown in SEQ ID No.30, or said promoter is the EFS short promoter as shown in SEQ ID No. 31.
5. A recombinant virus characterized by comprising the nucleotide sequence of the recombinant vector of claim 3 or 4 and a viral particle; the virus includes lentivirus, adenovirus, adeno-associated virus or retrovirus.
6. An isolated genetically modified functionalized immune response cell obtained by infecting an immune effector cell with the recombinant virus of claim 5, which is an armed chimeric antigen receptor modified functionalized immune response cell targeted to the SARS-CoV-2 virus SPIKE protein constructed by specific knock-out of SARS-CoV-2 virus RNA polymerase; the immune effector cells include cytotoxic T lymphocytes, NK cells, NKT cells, or helper T cells.
7. A biological product comprising the amino acid of claim 1, or comprising the nucleotide of claim 2, or comprising the recombinant vector of claim 3 or 4, or comprising the recombinant virus of claim 5, or comprising the functionalized immune cell of claim 6.
8. Use of a biological product according to claim 7 in a medicament for the treatment of 2019-nCoV, SARS or MERS diseases.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112390863A (en) * 2020-09-23 2021-02-23 厚朴生物科技（苏州）有限公司 Modified new coronavirus Spike protein extracellular domain and application thereof
CN113249408A (en) * 2021-06-23 2021-08-13 深圳湾实验室 Construction and application of nucleic acid vaccine vector for targeting activation of humoral immunity and cellular immunity
WO2021201679A1 (en) * 2020-04-01 2021-10-07 Kiadis Pharma Intellectual Property B.V. Compositions and methods targeting coronaviruses
WO2022035998A1 (en) * 2020-08-11 2022-02-17 City Of Hope Compositions and uses of sars-cov-2 targeted chimeric antigen receptor modified nk cells
WO2022144040A1 (en) * 2020-12-29 2022-07-07 艾棣维欣(苏州)生物制药有限公司 Nucleotide sequence encoding novel coronavirus antigen, and use thereof
CN115040644A (en) * 2021-04-08 2022-09-13 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） New recombinant rabies virus vector vaccine for coronary pneumonia
WO2022233321A1 (en) * 2021-05-07 2022-11-10 百奥泰生物制药股份有限公司 Coronavirus antibody and use thereof
US11672829B2 (en) * 2015-09-10 2023-06-13 Sri International Genetically engineered coronavirus-specific effector cells for in situ synthesis of antiviral proteins

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109536455A (en) * 2018-12-11 2019-03-29 武汉波睿达生物科技有限公司 A kind of CAR-NK cell and its preparation method and application

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109536455A (en) * 2018-12-11 2019-03-29 武汉波睿达生物科技有限公司 A kind of CAR-NK cell and its preparation method and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SANJEETH RAJARAM等: "The Promise and Peril of Natural Killer Cell Therapies in Pulmonary Infection" *
VANESSA MONTEIL等: "Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2" *

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US11672829B2 (en) * 2015-09-10 2023-06-13 Sri International Genetically engineered coronavirus-specific effector cells for in situ synthesis of antiviral proteins
WO2021201679A1 (en) * 2020-04-01 2021-10-07 Kiadis Pharma Intellectual Property B.V. Compositions and methods targeting coronaviruses
WO2022035998A1 (en) * 2020-08-11 2022-02-17 City Of Hope Compositions and uses of sars-cov-2 targeted chimeric antigen receptor modified nk cells
CN112390863A (en) * 2020-09-23 2021-02-23 厚朴生物科技（苏州）有限公司 Modified new coronavirus Spike protein extracellular domain and application thereof
CN112390863B (en) * 2020-09-23 2022-11-01 厚朴生物科技（苏州）有限公司 Modified new coronavirus Spike protein extracellular domain and application thereof
WO2022144040A1 (en) * 2020-12-29 2022-07-07 艾棣维欣(苏州)生物制药有限公司 Nucleotide sequence encoding novel coronavirus antigen, and use thereof
CN115040644A (en) * 2021-04-08 2022-09-13 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） New recombinant rabies virus vector vaccine for coronary pneumonia
WO2022233321A1 (en) * 2021-05-07 2022-11-10 百奥泰生物制药股份有限公司 Coronavirus antibody and use thereof
CN113249408A (en) * 2021-06-23 2021-08-13 深圳湾实验室 Construction and application of nucleic acid vaccine vector for targeting activation of humoral immunity and cellular immunity
CN113249408B (en) * 2021-06-23 2021-11-02 深圳湾实验室 Construction and application of nucleic acid vaccine vector for targeting activation of humoral immunity and cellular immunity

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