WO2021093482A1 - Ensemble vecteur comprenant une combinaison d'éléments géniques, banque de cellules receveuses, préparation et procédé de criblage et utilisation associés - Google Patents

Ensemble vecteur comprenant une combinaison d'éléments géniques, banque de cellules receveuses, préparation et procédé de criblage et utilisation associés Download PDF

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WO2021093482A1
WO2021093482A1 PCT/CN2020/119248 CN2020119248W WO2021093482A1 WO 2021093482 A1 WO2021093482 A1 WO 2021093482A1 CN 2020119248 W CN2020119248 W CN 2020119248W WO 2021093482 A1 WO2021093482 A1 WO 2021093482A1
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gene
library
cells
synnotch
receptor
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傅文燕
胡适
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沣潮医药科技（上海）有限公司
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001166—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/82—Colon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Definitions

  • the present invention relates to the fields of biomedical engineering technology and synthetic biology, in particular to a gene element combination and a synNotch receptor and an artificial receptor cell library carrying the combination, and a method for preparing and constructing the gene element combination and cell library.
  • the methods for screening in vivo antigens and/or in vitro antigens, and the use of cell libraries are described in detail.
  • malignant tumors are characterized by a high degree of variability, individual differences, heterogeneity, and evolution.
  • the identification of specific tumor antigens is difficult, diverse, and individualized.
  • the malignant tumor tissue can undergo biological evolution with the development of the disease and the pressure of treatment methods, and has a large degree of variability.
  • Phage display technology and other library technologies can use molecular biology and genetic engineering methods to construct peptide libraries in vitro, and then screen them.
  • in vitro screening or in vivo screening methods are generally used.
  • In vitro screening is the most commonly used, such as coupling a specific antigen to a solid support (magnetic beads or enzyme-linked plate), adding a phage peptide library or other antibody library for elution, enrichment screening. But in fact, once in vitro, no matter what kind of antigens (such as cells, tissues, etc.) are used, they will change and lose because they are separated from the environment in the body. There are also some public reports on in vivo screening methods.
  • Patent document CN 109576292 A discloses an antibody library using synthetic Notch receptor (synNotch receptor) cells as a carrier and a screening method for solid-phase antigens in vitro.
  • synthetic Notch receptor seNotch receptor
  • the synNotch receptor library binds to known or unknown antigens and activates in the cell, allowing the cell to produce artificial markers such as fluorescent proteins or molecular tags and suicide proteins.
  • the expression of the substance isolates cells carrying activated synNotch receptors. Through this process, synNotch receptors that bind to certain known or unknown antigens can be obtained, and synNotch receptors can be further used to obtain antibodies.
  • whether the antibody obtained through such a screening method can be used for disease treatment is unknown.
  • antibody library technologies such as phage display can also obtain antibodies with known or unknown antigen binding ability through panning, but it is impossible to determine whether these antibodies with binding ability are used for disease treatment. Therefore, compared with traditional systems such as phage display, this system has no improvement in function, and it cannot improve the effectiveness of the existing technology for the preparation of functional molecules such as antibodies.
  • Patent document WO2015/123642 is a background technology of the present invention.
  • This patent discloses a method for constructing and preparing a chimeric antigen receptor library, and proposes a method for preparing a large number of CAR formation libraries and further carrying a CAR library cell library.
  • the patent document describes the use of general technology for further screening of CAR libraries, such as the use of iQue TM filters (Intellicyt, Albuquerque, NM) (a high-throughput flow cytometer) for high-throughput testing of individual CAR molecules
  • iQue TM filters Intelligent Cell Library
  • Albuquerque, NM a high-throughput flow cytometer
  • the present invention provides a synNotch receptor and chimeric antigen receptor combined cell library containing new gene elements, and a method for preparing and constructing the gene element combination and the cell library, targeting in vivo antigens and/ Or in vitro antigen screening methods and the use of cell libraries are described in detail.
  • a carrier assembly In the first aspect of the present invention, a carrier assembly is provided. It carries three genetic elements, namely: (1) multiple first genetic elements encoding one or more idiotype synNotch receptors (ie synNotch receptor library); (2) containing one or more idiotypes The second gene element of the gene circuit; (3) the third gene element encoding one or more idiotypic chimeric antigen receptors.
  • the third gene element when the first gene element encodes an idiotype synNotch receptor, the third gene element must encode at least three idiotype chimeric antigen receptors, and when the third gene element encodes an idiotype chimeric antigen receptor , The first gene element must encode three idiotypic synNotch receptors to achieve screening.
  • the gene circuit is pre-programmed. It is a combination of regulatory homeopathic factors and transcription factors.
  • synNotch receptor encoded by the first gene element When activated, it controls the expression of the chimeric antigen receptor encoded by the third gene element. .
  • the synNotch receptor includes the core regulatory domain of the intercellular signaling receptor Notch, a synthetic extracellular recognition domain, and a synthetic intracellular transcription domain.
  • the chimeric antigen receptor includes an intracellular signaling domain, Transmembrane domain and extracellular recognition domain.
  • the extracellular recognition domains of both receptors include complete antibodies, heavy chains, light chains, or antibody fragments that make up the antibody.
  • the "idiotype" synNotch receptor and chimeric antigen receptor are both in terms of their extracellular recognition domains.
  • the invention is not limited to the extracellular recognition domain.
  • the gene element combination in the present invention can achieve the following functions: when the synNotch receptor encoded by the first gene element is activated, the second gene element can be pre-programmed through the gene circuit to achieve simultaneous controlled expression of the third gene element.
  • the controlled expression includes any one or a combination of at least two of activating transcriptional expression, enhancing transcriptional expression, terminating transcriptional expression, and inhibiting transcriptional expression.
  • the combination of genetic elements in the present invention further includes: (4) a fourth genetic element encoding one or more idiotypic selectable marker proteins.
  • the function that the gene element combination can achieve is: when the synNotch receptor encoded by the first gene element is activated, the second gene element can be pre-programmed through the gene circuit to achieve simultaneous control of the third gene element and the fourth gene element sexual expression.
  • the selection marker protein in the fourth gene element includes any one or a combination of at least two of drug resistance protein, suicide protein, fluorescent protein gene, or molecular tag protein.
  • synNotch receptor is a synthetic biology concept.
  • the synNotch receptor contains the core regulatory domain of the natural intercellular signal transduction receptor Notch, as well as a synthetic extracellular recognition domain and a synthetic intracellular transcription domain.
  • the synthetic extracellular recognition domain can be constructed as a single-chain antibody. When the single-chain antibody recognizes and binds to an antigen, the synNotch system undergoes inductive transmembrane cleavage, thereby releasing the intracellular transcription domain into the nucleus and binding upstream cis Activator to activate the expression of the regulated target gene.
  • Notch Due to the conservation of the Notch gene, Notch of multiple species can be used for construction.
  • the patent CN 109180805 A describes Notch receptors derived from Notch genes such as humans, mice, zebrafish, and fruit flies.
  • Patent CN 109576292 A discloses a method for generating a synNotch receptor library for randomization of extracellular recognition domains.
  • idiotype means having different polypeptides.
  • amino acid sequences domains comprising different polypeptide (amino acid) sequences or composed of different polypeptide (amino acid) sequences.
  • two "idiotypic" antigen-binding domains can bind to the same antigen (in fact, even the same epitope on the antigen); however, the antigen-binding domains are "in the case where their contiguous amino acid composition differs from each other). Idiotype”.
  • two "idiotype” antigen binding domains that differ in their continuous amino acid composition can also specifically bind to different antigens and epitopes.
  • the extracellular recognition domain library its structure includes, but is not limited to, complete antibodies, the chains (heavy or light chains) that make up the antibody, and antibody fragments (antibody variable regions, single chain antibodies, single domain antibodies, Fab segments)
  • the constituted library is preferably a single chain antibody (ScFv) library.
  • the source of the library includes, but is not limited to, any one or a combination of at least two of animal, prepared from immunized animals, from diseased population, from healthy population, vaccinated population, artificial synthesis, genetic engineering preparation; also includes the above
  • the source library is a sub-library that has been preprocessed by the prior art, such as the one described in the non-patent literature [Yin Changcheng, et al. Chinese Journal of Bioengineering, 2008,28(12):82-88.] after removing background clones. Sub library.
  • the extracellular recognition domain library in the present invention includes a single-chain antibody library derived from healthy people, a single-chain antibody library derived from artificial synthesis, a single-domain antibody library derived from alpaca, and a single-chain antibody library derived from healthy people.
  • the extracellular recognition domain library can be further optimized using antibody engineering methods, including antibody affinity maturation technology, antibody humanization technology, antibody animalization technology, multifunctional antibody technology, and multispecific antibody technology. Any one or a combination of at least two of them.
  • the affinity maturation technology includes any one or a combination of at least two of hotspot-directed mutations, hotspot random mutations, CDR mutations, chain exchanges, and antibody mutations based on three-dimensional structures.
  • the source of the extracellular recognition domain library includes monoclonal antibodies ABAGOVOMAB, ABCIXIMAB, ABELACIMAB, ABITUZUMAB, ABREZEKIMAB, ABRILUMAB, ACTOXUMAB, ADALIMUMAB, ADECATUMUMAB, ADUCANUMAB, AFASEVIKUMAB, ACIELIMUMB, aLEMTUZUMAB, ALIROCUMAB, AMATUXIMAB, ANATUMOMAB, ANDECALIXIMAB, ANETUMAB, ANIFROLUMAB, ANRUKINZUMAB, APRUTUMAB, ASCRINVACUMAB, ASELIZUMAB, ATIDORTOXUMAB, ATINUMAB, ATOLTIVIMAB, ATOROLIMUMAB, AVELUMAB, AZINTUXIZUMAB, BALSTILIMAB, bAPINEUZUMAB, bASILIXIMAB, BAVITUXIMAB, BECTUM
  • idiotype is a professional concept of antibody engineering. As the most important effector molecule recognized by the body's molecules, antibodies have the characteristics of heterogeneity. Heterogeneity includes isotype, allotype and idiotype. Among them, the concept of idiotype refers to the specificity of all antigens on the antibody molecule produced by each antibody-forming cell clone, which is determined by the difference in the amino acid sequence of the variable region of the light chain or the heavy chain. The specificity of binding antigen is closely related. The idiotype emphasizes the difference in the characteristics of the antibody binding to the antigen. Because synNotch receptors and chimeric antigen receptors recognize antigens based on their internal antibody structure, these artificial receptors also have "idiotypes.”
  • Notch core regulatory domain library its sources include but are not limited to human Notch genes (UniProtKB: P46531; Q04721; Q04721; Q99466), mouse Notch genes (UniProtKB: Q01705; P31695; O35516; Q61982); rat Notch genes (UniProtKB: Q07008; Q9QW30; Q9R172), zebrafish Notch gene (UniProtKB: P46530), Drosophila Notch gene (UniProtKB: P07207).
  • human Notch genes UniProtKB: P46531; Q04721; Q04721; Q99466
  • mouse Notch genes UniProtKB: Q01705; P31695; O35516; Q61982
  • rat Notch genes UniProtKB: Q07008; Q9QW30; Q9R172
  • zebrafish Notch gene UniProtKB: P46530
  • the synthetic intracellular transcription domain library its sources include but are not limited to TetR-VP64 (tTA), Gal4-VP64, PIP-VP64, ZF21-16-VP64, ZF-42-10-VP64, ZF43-8- VP64, ZF54-8-VP64, ZFHD1-VP64, Gal4-KRAB, TetR-KRAB, PIP-KRAB, ZF21-16-KRAB, ZF-42-10-KRAB, ZF43-8-KRAB, ZF54-8-KRAB, Any one or a combination of at least two of ZFHD1-KRAB.
  • the term "gene circuit” is a synthetic biology concept. Broadly speaking, the gene circuit contains regulatory cis-acting factors and regulated genes. Regulatory cis-acting factors include promoters, such as T7 promoter, CMV promoter, UPS promoter, Tet promoter and so on. The regulated genes are mostly genes encoding proteins.
  • the complex regulatory network of multiple cis-acting factors and multiple regulatory genes (in this case, the regulatory genes can be further designed as trans-acting factors, such as transcription factors, etc.) can be flexibly designed according to the research purpose, which is like a circuit network, so it is called It is “gene circuit” or “gene circuit”.
  • a gene circuit is "idiotype"
  • idiotype it means a gene circuit composed of different design and programming schemes.
  • two “idiotypic” gene circuits can achieve exactly the same biological effects (such as controlling the up-regulation of a certain downstream gene expression, or controlling the down-regulation of a certain downstream gene expression), however, multiple internal cis-acting factors of the gene circuit ,
  • multiple regulatory genes which can be transcription factors
  • they are "idiotypes”.
  • the cis-acting factors and multiple regulatory genes (which can be transcription factors) within the same gene loop have the same design and construction schemes, and mutations in the category of homologous sequences do not affect cis-acting factors and regulatory genes.
  • the change in function is not a "idiotypic" gene circuit.
  • the method for constructing gene circuits can be found in these publicly published documents: Kulemzin S V, et al. BMC Medical Genomics, 2019, 12 (S2).; Uchibori R, et al. Molecular Therapy—Oncolytics, 2019, 12: 16-25; Morsut L,et al.Cell,2016,164(4):780-791.; Deuschle U, Meyer W K, Thiesen H J.
  • Regulatory cis-acting factors include single homeopathic factors or fusion homeopathic factors, and regulated genes include single transcription factors or combined transcription factors.
  • a single homeopathic factor includes one or more NFAT-responsive promoter elements (NFAT), one or more NF ⁇ B-responsive promoter elements (NF ⁇ B), and one or more tetracycline response elements (tetracycline responsive element, TRE), UAS (upstream activating sequence, UAS) of one or more galactose metabolizing enzyme system (GAL) gene promoters, one or more PIP response elements (PIP responsive element, PIR), one or Multiple ZFHD1 responsive elements (ZFHD1 responsive element, ZFHD1RE), one or more ZF21-16 responsive elements (ZF21-16 responsive element, ZF21-16RE), one or more ZF42-10 responsive elements (ZF42-10RE) ), one or more ZF43-8 response elements (ZF43-8RE), one or more ZF54-8 response elements (ZF54-8RE), one or more minimal CMV promoters (minimal CMV promoter, P CMV-min ), one or more CMV promoters (CMV promoter, P CMV ), one or
  • Combined transcription factors include TetR-VP64 (tTA), Gal4-VP64, PIP-VP64, ZF21-16-VP64, ZF-42-10-VP64, ZF43-8-VP64, ZF54-8-VP64, ZFHD1-VP64, Any one or at least two of Gal4-KRAB, TetR-KRAB, PIP-KRAB, ZF21-16-KRAB, ZF-42-10-KRAB, ZF43-8-KRAB, ZF54-8-KRAB, ZFHD1-KRAB The combination. Preferably, it includes any one or a combination of at least two of TetR-VP64 (tTA), Gal4-VP64, Gal4-KRAB, and TetR-KRAB.
  • the gene circuit preferably includes (i) a combination of Gal4-KRAB and 5 ⁇ UAS-P SV40, a combination of TetR-KRAB and 7 ⁇ TRE-P SV40 , or a combination of TetR-KRAB and Any one of the combination of 7 ⁇ TRE-P cmv and (ii) any one of 4 ⁇ NFAT, 6 ⁇ NFAT, 5 ⁇ NF ⁇ B, and 10 ⁇ NF ⁇ B are combined.
  • chimeric antigen receptor in the third gene element is an immunotherapy concept, which is an artificial receptor constructed by mimicking the activation process of immune cells.
  • the chimeric antigen receptor contains multiple immune receptor parts, and the purpose is to design a receptor that can recognize the antigen (such as BCR) without any help, and then directly kill the recognized cells (such as TCR).
  • the targets targeted by the chimeric antigen receptor of the third gene element of the present invention include CD19, BCMA, Mesothelin, GD2, EGFR, HER2, CD22, CD123, Glypican 3, CD30, MUC1, CD33, CD20, CD38, EpCAM, CD56, CD138, CD7, CD133, CEA, CD34, CD117, Claudin18.2, PSCA, cMET, Lewis Y, EphA2, NKG2D ligands, ErbB, NY-ESO-1, CLL-1, CD10, LI13R ⁇ 2, CD171, ROR2, AXL, Kappa, CS1, FAP, IL-1RAP, MG7, PSMA, CD5, ROR1, CD70, HER3, Gp75, phosphatidylserine, cMyc, CD4, CD44v6, CD45, CD28, CD3, CD3e, CD52, CD74, CD30, CD166, CD24, EGFR/HER3 fusion, carbohydrate,
  • the structure of chimeric antigen receptors is a common technique in the art, including intracellular signal transduction domains, transmembrane domains, and extracellular recognition domains (extracellular recognition domain library).
  • the transmembrane domain also includes a hinge region located between the extracellular recognition domain and the transmembrane domain, and one or more additional costimulatory molecules located between the transmembrane domain and the intracellular signaling domain.
  • the intracellular signal transduction domain includes CD3 ⁇ ; the transmembrane domain includes: any one of CD28 transmembrane domain, 4-1BB transmembrane domain, CD8 ⁇ transmembrane domain, and CD3 ⁇ transmembrane domain; costimulatory molecules include : Any one or a combination of at least two of CD28, CD27, OX40, 4-1BB; the structure of the extracellular recognition domain includes, but is not limited to, complete antibodies, chains (heavy or light chains) that make up the antibody, and antibodies A library composed of fragments (antibody variable regions, single-chain antibodies, single-domain antibodies, Fab segments), preferably a single-chain antibody (ScFv) library, the source of which is the same as the source of the extracellular recognition domain library in the synNotch receptor .
  • ScFv single-chain antibody
  • the extracellular recognition domain library includes a single-chain antibody library derived from healthy people, a single-chain antibody library derived from artificial synthesis, a single-domain antibody library derived from alpaca, or a single-chain antibody library derived from healthy people to subtract the peripheral monocytes of the subject
  • the sub-library formed after the expression of the antigen is included in some specific embodiments of the present invention.
  • the extracellular antibody library also includes a sub-library formed by deducting the antigens and para-cancer antigens expressed by the peripheral monocytes of the subject from the single-chain antibody library derived from healthy people.
  • the source of the chimeric antigen receptor library includes but is not limited to any one or at least one of animal, prepared from immunized animals, from diseased population, from healthy population, vaccinated population, artificial synthesis, genetic engineering preparation
  • the combination of the two also includes the sub-library after the library of the above source is pre-processed by the prior art, such as the non-patent literature [Yin Changcheng, et al. Chinese Journal of Bioengineering, 2008,28(12):82-88.] The sub-library formed after removing the background cloning.
  • the extracellular recognition domain library can be further optimized using antibody engineering methods, which include any of antibody affinity maturation technology, antibody humanization technology, antibody animalization technology, multifunctional antibody technology, and multispecific antibody technology. Or a combination of at least two.
  • the affinity maturation technology includes any one or a combination of at least two of hotspot-directed mutations, hotspot random mutations, CDR mutations, chain exchanges, and antibody mutations based on three-dimensional structures.
  • the structure of the chimeric antigen receptor and the method for constructing the chimeric antigen receptor library in the present invention can have a variety of construction combinations, and all of them can be constructed in the elements of the present invention to achieve randomization, see patent document WO2015/123642.
  • the selection marker protein of the fourth gene element species includes any one or a combination of at least two of drug resistance protein, suicide protein, fluorescent protein or molecular tag protein.
  • the drug resistance protein includes any one or a combination of at least two of puromycin resistance protein, neomycin resistance protein, blasticidin resistance protein or hygromycin B resistance protein; suicide;
  • the protein includes any one or a combination of herpes simplex virus thymidine kinase gene, cytosine deaminase gene or iCasp9 suicide system gene; fluorescent protein includes any one of EGFP, YFP, mCherry, DsRed or BFP Or a combination of at least two;
  • the molecular tag protein includes any one or a combination of at least two of His-tag, Flag-tag, HA-tag, Myc-tag, Sun-tag or Strep-tag.
  • a plurality of first gene elements encode a plurality of unique synNotch receptors
  • a plurality of second gene elements construct a plurality of unique gene circuits
  • a plurality of third elements encode a plurality of unique synNotch receptors. It combines antigen receptors
  • a plurality of fourth elements encode a plurality of selectable marker proteins.
  • a plurality of first gene elements encode a unique synNotch receptor
  • a plurality of second gene elements construct a plurality of unique gene circuits
  • a plurality of third elements encode a plurality of unique chimeric antigen receptors
  • multiple fourth elements encode multiple unique selectable marker proteins.
  • a plurality of first gene elements encode a plurality of unique synNotch receptors
  • a plurality of second gene elements construct a unique gene circuit
  • a plurality of third elements encode a plurality of synNotch receptors.
  • Unique chimeric antigen receptor, and multiple fourth elements encode multiple unique selectable marker proteins.
  • a plurality of first gene elements encode a plurality of unique synNotch receptors
  • a plurality of second gene elements construct a plurality of unique gene circuits
  • a plurality of third elements encode a unique chimeric antigen receptor
  • multiple fourth elements encode multiple unique selectable marker proteins.
  • a plurality of first gene elements encode a plurality of unique synNotch receptors
  • a plurality of second gene elements construct a plurality of unique gene circuits
  • a plurality of third elements encode a plurality of unique chimeric antigen receptors.
  • Body, and multiple fourth elements encode a unique selectable marker protein.
  • the gene element combination of the present invention includes gene elements encoding different synNotch receptors (according to the prior art, the synNotch receptor has a unique extracellular recognition domain, Notch core regulatory domain and/or synthetic cell
  • the internal transcription domain can also be randomized, such as patent documents CN109180805A, CN109576292A and non-patent documents Morsut, et al. 2016, etc.), elements carrying different gene circuits, encoding different chimeric antigen receptors, and / Or a collection of elements encoding different selectable marker proteins, the element collection encoding multiple unique synNotch receptors, carrying multiple unique gene circuits, encoding multiple unique chimeric antigen receptors, and/or encoding multiple A unique selection marker protein.
  • the collection is randomized with respect to the genetic elements.
  • the present invention relates to a combinatorial library of gene elements
  • the combinatorial library of gene elements is a library containing a combination of different gene elements
  • the library is a unique synNotch receptor (according to the prior art, the synNotch receptor is unique
  • the extracellular recognition domain, Notch core regulatory domain and/or synthetic intracellular transcription domain can also be randomized, such as patent documents CN109180805A, CN109576292A and non-patent documents Morsut, et al. 2016, etc.)
  • Gene circuit, chimeric antigen receptor, and selection marker protein are randomized.
  • the library is randomized with respect to any one or a combination of at least two of unique synNotch receptors, gene circuits, chimeric antigen receptors, and selectable marker proteins.
  • the second aspect of the present invention provides a method for preparing an artificial cell library carrying the above-mentioned combination of genetic elements.
  • the first gene element, the second gene element, the third gene element, and the fourth gene element are inserted into the same vector or different vectors and transfected into cells to obtain a cell library, which is the artificial cell library.
  • Vectors are techniques familiar to those skilled in the art, such as viral vectors in non-patent literature [Morsut L, et al. Cell, 2016, 164(4): 780-791.] and non-viral vectors [Athanasopoulos T, et al.. Hematology/Oncology Clinics of North America, 2017, 31(5): 753-770.], it is also possible to insert genetic elements into a specific site such as the vector of the AAVS1 site (Parthiban K, et al. mAbs. Taylor & Francis, 2019.).
  • the method of transfection includes any one or a combination of at least two of virus transfection, chemical transfection reagent transfection or electric shock transfection.
  • the cells are mammalian cells, preferably immune cells, including immune cells and/or genetically engineered immune cells.
  • the source of immune cells includes any one or a combination of at least two of autologous immune cells, donor immune cells, and immune cells from healthy volunteers. T lymphocytes are more preferred, and NK cells such as NK-92 cells are particularly preferred.
  • Use healthy volunteer sources, total synthesis methods, and genetic engineering methods to build antibody gene libraries are as follows: use healthy volunteer sources, total synthesis methods, etc. to establish an antibody gene library, select phage, yeast or mammalian cells to further establish an antibody display library, and select control tissues. After multiple rounds of panning, the background is subtracted to obtain antibodies. Display the sub-library, and then obtain a background-subtracted antibody gene library by PCR;
  • a first gene element whose structure includes the antibody library-synNotch, construct a second gene element including one or more regulatory homeopathic factors, and/or one or more transcription factors; construct a third gene element, which The structure includes the antibody library-CAR, the antibody library or antibody sublibrary is constructed as synNotch receptor, the extracellular recognition domain of CAR receptor; the fourth gene element is constructed, including the selection marker protein gene, and then the above four genes
  • the elements are constructed into one or more gene control expression cassettes;
  • A phage single-chain antibody library preparation and screening
  • the phage single-chain antibody library was established by using healthy volunteers or fully synthetic methods. Normal tissue was used as a control. After multiple rounds of panning, the background was subtracted to obtain the phage antibody sub-library, and then the antibody gene library was obtained by PCR.
  • a first gene element whose structure includes a single-chain antibody library-synNotch, which is constructed as an extracellular recognition domain of synNotch; construct a second gene element, including the first regulatory homeopathic factor, and The transcription factor regulated by the regulatory homeopathic factor, the second homeopathic factor regulated by the transcription factor; the third gene element is constructed, the structure of which includes the single-chain antibody library-CAR, and the single-chain antibody library is constructed as CAR The extracellular recognition domain of the; construct the fourth gene element, including the selection marker protein gene, and then construct the above four gene elements into one or more gene control expression cassettes.
  • the lentiviral vector system is used to introduce the above-mentioned gene control expression cassette into mammalian immune cells to obtain an artificial receptor cell library.
  • the third aspect of the present invention provides an artificial cell library
  • the cell library carries the combination of gene elements described in the first aspect and is obtained by the preparation method of the second aspect.
  • the fourth aspect of the present invention provides a method for screening artificial cells against in vitro antigens, including the following steps:
  • step (3) From the artificial receptor cells screened in step (3), use antibody engineering methods to reconstruct the secondary artificial receptor cell library; (5) Repeat steps (1)-(3) to screen the target artificial receptor Cells, if necessary, repeat steps (4)-(5) one or more times.
  • artificial receptor cells include monoclonal artificial receptor cells and polyclonal artificial receptor cell populations.
  • the "monoclonal” and “polyclonal” also include the synNotch receptor encoded by the first gene element, the gene circuit constructed by the second gene element, the chimeric antigen receptor encoded by the third element, and the fourth element encoding screening In terms of marker protein, it includes the synNotch receptor encoded by the monoclonal first gene element, the gene circuit constructed by the second gene element, the chimeric antigen receptor encoded by the third element and the polyclonal first gene element.
  • synNotch receptor the gene circuit constructed by the second gene element, the chimeric antigen receptor encoded by the third element, and the synNotch receptor encoded by the first gene element, the gene circuit constructed by the second gene element, and the chimeric antigen receptor encoded by the third element A collection of combined antigen receptors.
  • Antigens include wild-type cells, cells transfected with specific antigen genes, cells that bind to specific antigens, antigens dissolved in culture medium, antigens coated on culture vessels, antigens coated on beads or coated in culture Any one or a combination of at least two of the antigens on the scaffold.
  • the method for screening artificial cells is to contact the artificial cell library with in vitro antigens. Only artificial cells that can recognize the antigen can regulate the chimeric antigen receptor and/or select the marker protein according to the preprogrammed gene circuit in the cell.
  • the expression of includes any one or a combination of at least two of activating expression, enhancing expression, terminating expression, and inhibiting expression.
  • a suitable screening method is selected, and then the target artificial cells are screened out. It can be designed flexibly according to gene circuits, chimeric antigen receptors, and/or screening marker proteins to achieve screening purposes.
  • the screening methods for screening marker proteins include any one or a combination of at least two of drug screening, flow cytometry detection and sorting, magnetic bead sorting, and beads sorting;
  • the screening drugs for drug resistance proteins include puromycin, Any one of G418, blasticidin or hygromycin B;
  • the screening drug for suicide protein includes any one or a combination of ganciclovir or FIAU, 5-fluorocytosine, AP1903 or AP20187 .
  • the fifth aspect of the present invention provides a method for screening artificial receptor cells against in vivo antigens, which includes the following steps: (1) administering an effective amount of artificial receptor cell library to the experimental target subject to make it compatible with the in vivo antigen Contact; (2) Implement a screening method based on the expression of the suicide protein; (3) Use general methods to recover and enrich the target artificial recipient cells from the subject.
  • step (3) From the artificial receptor cells screened in step (3), reconstruct a secondary artificial receptor cell library using antibody engineering and/or genetic engineering methods; (5) repeat the steps (1) ⁇ (3), screen the target artificial recipient cells, and repeat steps (4)-(5) one or more times if necessary.
  • the artificial cells include monoclonal synthetic cells and polyclonal synthetic cell populations.
  • the "monoclonal” and “polyclonal” also include the synNotch receptor encoded by the first gene element, the gene circuit constructed by the second gene element, the chimeric antigen receptor encoded by the third element, and the fourth element encoding screening In terms of marker protein, it includes the synNotch receptor encoded by the monoclonal first gene element, the gene circuit constructed by the second gene element, the chimeric antigen receptor encoded by the third element and the polyclonal first gene element.
  • synNotch receptor, the gene circuit constructed by the second gene element, the chimeric antigen receptor encoded by the third element, and the synNotch receptor encoded by the first gene element, the gene circuit constructed by the second gene element, and the third element encodes A collection of chimeric antigen receptors.
  • In vivo antigens refer to antigens that exist in humans and animals, including in somatic cells, in vivo lesion cells, in vivo cells transfected with specific antigen genes, in vivo cells infected with specific pathogens, in vivo cells in combination with specific antigens, transplantation Any one or a combination of at least two in somatic cells on an animal model.
  • the method of contacting the artificial cell library with in vivo antigens generally includes administering the artificial cell library into the body.
  • the method of administration includes any one or a combination of at least two of intravenous infusion, gastrointestinal infusion, intramuscular injection, local tissue injection, subcutaneous injection, intraperitoneal injection, and inhalation.
  • the method for screening synthetic somatic cells is to administer the synthetic somatic cell library to the target subject, so that the synthetic somatic cell library is in contact with the antigen in the body, and only artificial cells that can recognize the antigen will be based on the preprogrammed genes in the cell.
  • the loop regulating the expression of the chimeric antigen receptor and/or the selection marker protein includes any one or a combination of at least two of activating expression, enhancing expression, terminating expression, and inhibiting expression.
  • a suitable screening method is selected, and then the target artificial cells are screened out. It can be designed flexibly according to gene circuits, chimeric antigen receptors, and/or screening marker proteins to achieve screening purposes.
  • the screening methods for screening marker proteins include any one or a combination of at least two of drug screening, flow cytometry detection and sorting, magnetic bead sorting, and beads sorting;
  • the screening drugs for drug resistance proteins include puromycin, Any one of G418, blasticidin or hygromycin B;
  • the screening drug for suicide protein includes any one or a combination of ganciclovir or FIAU, 5-fluorocytosine, AP1903 or AP20187 .
  • the sixth aspect of the present invention provides an artificial recipient cell.
  • the artificial receptor cells are screened by the methods described in the fourth aspect and the fifth aspect, and the artificial receptor cells include monoclonal artificial receptor cells and polyclonal artificial receptor cells.
  • the seventh aspect of the present invention provides a synthetic receptor.
  • the artificially synthesized receptor is obtained by extracting relevant genes from the artificial receptor cells described in the sixth aspect using universal genetic engineering technology.
  • the eighth aspect of the present invention provides an antibody.
  • the antibody is obtained through the general antibody engineering technology for synthetic receptors described in the seventh aspect.
  • the ninth aspect of the present invention provides a target antigen.
  • the target antigen is a corresponding antigen obtained through the artificial recipient cell described in the sixth aspect, the antibody described in the seventh aspect, and the eighth aspect described above through general genetic engineering technology.
  • the tenth aspect of the present invention provides a pharmaceutical composition.
  • the composition comprises the artificial receptor cell library of the first aspect, the antigen receptor cell library constructed and obtained in the second aspect, the artificial receptor cell of the sixth aspect, the artificial receptor cell of the seventh aspect, and the artificial receptor cell library of the seventh aspect. Any one or a combination of at least two of the antibodies described in the eight aspects, and at least one medically/pharmaceutically acceptable carrier.
  • the pharmaceutical composition also includes a diluent or excipient for medical or pharmaceutical use.
  • the eleventh aspect of the present invention provides any one or a combination of at least two of the above-mentioned pharmaceutical composition including drugs, reagents, and kits for preventing, diagnosing and treating diseases related to diseases requiring removal of disease mediators.
  • the disease mediator refers to focal cells. It is necessary to remove focal cell related diseases such as benign and malignant tumors, tissue proliferation, acute and chronic inflammation and other diseases.
  • the disease can be, for example, lung, breast, stomach, pancreas, prostate, bladder, bone, ovary and appendages, uterus, skin, kidney, sinus, colon, rectum, esophagus, blood, brain and its covering, spinal cord and its covering, Malignant tumors of muscle, connective tissue, adrenal gland, parathyroid, thyroid, uterus, testis, pituitary, genitals, liver, gallbladder, eyes, ears, nose, throat, tonsils, mouth, lymph nodes and lymphatic system and other organs. In some embodiments, benign tumors of these organs are also included.
  • malignant tumor includes all forms of human cancer, sarcoma, and melanoma, which appear in the form of poorly differentiated, moderately differentiated, and highly differentiated.
  • the disease also includes tissue hyperplasia, hypertrophy, ectopic or excessive growth, tissues including lung, breast, stomach, pancreas, prostate, bladder, bone, ovary and appendages, uterus, skin, kidney, sinus, colon, rectum, esophagus, Blood, brain and its covering, spinal cord and its covering, muscle, connective tissue, adrenal gland, parathyroid, thyroid, uterus, testis, pituitary, genitals, liver, gallbladder, eyes, ears, nose, throat, tonsils, mouth , Lymph nodes and lymphatic system and other organs.
  • tissues including lung, breast, stomach, pancreas, prostate, bladder, bone, ovary and appendages, uterus, skin, kidney, sinus, colon, rectum, esophagus, Blood, brain and its covering, spinal cord and its covering, muscle, connective tissue, adrenal gland, parathyroid, thyroid, uterus, testis, pituitary, genitals, liver, gallbladder
  • the disease also includes tissues altered by viruses, bacteria or parasites, including lungs, breasts, stomach, pancreas, prostate, bladder, bones, ovaries and appendages, uterus, skin, kidneys, sinuses, colon, rectum, esophagus, blood , Brain and its covering, spinal cord and its covering, muscle, connective tissue, adrenal gland, parathyroid, thyroid, uterus, testis, pituitary, genitals, liver, gallbladder, eyes, ears, nose, throat, tonsils, mouth, Lymph nodes and lymphatic system and other organs.
  • tissues altered by viruses, bacteria or parasites including lungs, breasts, stomach, pancreas, prostate, bladder, bones, ovaries and appendages, uterus, skin, kidneys, sinuses, colon, rectum, esophagus, blood , Brain and its covering, spinal cord and its covering, muscle, connective tissue, adrenal gland, parathyroid, thyroid, uterus, testis, pituitary
  • the diseases include tissue fibrotic changes caused by acute and chronic inflammation, tissues including lung, breast, stomach, pancreas, prostate, bladder, bone, ovary and appendages, uterus, skin, kidney, sinus, colon, rectum, esophagus, blood, Brain and its covering, spinal cord and its covering, muscle, connective tissue, adrenal gland, parathyroid, thyroid, uterus, testis, pituitary, genitals, liver, gallbladder, eyes, ears, nose, throat, tonsils, mouth, lymph nodes And the lymphatic system and other organs.
  • the diseases also include endometriosis, tonsillar hypertrophy, prostate hyperplasia, psoriasis, eczema, skin diseases, hemorrhoids, vascular diseases such as atherosclerosis or arteriosclerosis. Or vascular disease such as varicose veins, stenosis or severe stenosis of arteries or stents.
  • the disease also includes cosmetic repair of tissues, such as skin, eye, ear, nose, throat, mouth, muscle, connective tissue, hair or breast tissue.
  • the diseases also include neurological degeneration diseases, such as Alzheimer's disease, Parkinson's disease and the like.
  • the disease mediator refers to an inflammatory mediator.
  • the inflammatory mediators include pathogens such as viruses, bacteria or parasites, enzymes, cytokines, prostaglandins, eicosanoids, Leukotrienes, kinins, etc. (kinins), complements, coagulation factors, toxins, endotoxins, enterotoxins, lipopolysaccharides, substances that induce apoptosis, corrosive substances, bile salts, fatty acids, phospholipids, oxidation by-products, reactive oxygen species, oxygen free radicals, Any one or a combination of at least two of surfactants, ions, irritating substances, cell debris, interferons, and immunomodulatory antibodies, biologics, and drugs.
  • pathogens such as viruses, bacteria or parasites, enzymes, cytokines, prostaglandins, eicosanoids, Leukotrienes, kinins, etc. (kinins), complements, coagulation factors, toxins, endotoxins, enterotoxins, lipopoly
  • the inflammatory mediator is present in the physiological fluid or carrier fluid of the subject, and the physiological fluid includes the following fluids: nasopharyngeal, oral cavity, esophagus, stomach, pancreas, liver, pleura, pericardium, Peritoneum, intestine, prostate, semen, vaginal secretions, tears, saliva, mucus, bile, blood, lymph, plasma, serum, synovial fluid, cerebrospinal fluid, urine, as well as interstitial, intracellular and extracellular fluids.
  • the physiological fluid includes the following fluids: nasopharyngeal, oral cavity, esophagus, stomach, pancreas, liver, pleura, pericardium, Peritoneum, intestine, prostate, semen, vaginal secretions, tears, saliva, mucus, bile, blood, lymph, plasma, serum, synovial fluid, cerebrospinal fluid, urine, as well as interstitial, intracellular and extracellular fluids.
  • the inflammatory mediator-related diseases include: systemic inflammatory response syndrome (SIRS) or sepsis (for example, derived from viral, bacterial, fungal or parasitic infection), autoimmune diseases, surgery, cytotoxic chemotherapy, Bone marrow manipulation, large tissue injury or trauma, mesenteric hypoperfusion, intestinal mucosal injury, malaria, gastrointestinal inflammatory disease, intestinal infection, uterine infection, influenza, acute pneumonia such as acute respiratory distress syndrome or acute lung Injury, pulmonary embolism, pancreatitis, autoimmune and collagen vascular diseases, blood transfusion-related diseases, burns, smoke or inhalation lung injury, graft versus host disease, ischemia or infarction, reperfusion injury, hemorrhage, allergic reactions, drug overdose, Radiation damage or chemical damage.
  • SIRS systemic inflammatory response syndrome
  • sepsis for example, derived from viral, bacterial, fungal or parasitic infection
  • autoimmune diseases surgery, cytotoxic chemotherapy, Bone marrow manipulation, large tissue injury or trauma, mes
  • inflammatory mediators are produced by diseases caused by pathogens, toxins or agents of biological warfare, such as viral hemorrhagic fever, marine toxins such as jellyfish toxins, dengue fever, Ebola, Hantavirus cardiopulmonary syndrome (hantan Virus), cholera toxin, botulinum toxin, flax toxin, Q fever [Coxiella burnetii], typhoid fever (Rickettsia prowaszekii), or psittacosis (Chlamydia psittaci) psittaci)].
  • diseases caused by pathogens, toxins or agents of biological warfare such as viral hemorrhagic fever, marine toxins such as jellyfish toxins, dengue fever, Ebola, Hantavirus cardiopulmonary syndrome (hantan Virus), cholera toxin, botulinum toxin, flax toxin, Q fever [Coxiella burnetii], typhoid fever (Ricketts
  • the inflammatory mediator-related diseases also include: transplantation, immune infertility and other diseases that require removal of target immune factors.
  • the artificial cell library of the present invention is loaded with four gene elements.
  • the synNotch receptor encoded by the first gene element is activated, the second gene element can be pre-programmed through the gene circuit to achieve simultaneous controlled expression of the third gene element.
  • the gene loop was successfully used to realize the information coupling between the synNotch library and the chimeric antigen receptor library.
  • the artificial cell library of the present invention combines the SynNotch receptor cell library with high-precision binding to cells or tissues to achieve antibody screening, even
  • the screening of unknown antigens and the benefit of gene circuit programming can achieve the advantage of following antigen changes, and the chimeric antigen receptor library can prepare a large number of activating immune cell CARs for abnormal cell killing. It is especially suitable for the preparation of personalized medicines, reagents, and reagents.
  • the kit has very broad application prospects.
  • Figure 1 is a schematic diagram illustrating the structure, preparation method, and in vivo screening of the artificial cell library of the present invention, where A is the construction of an antibody library using existing technology; B is the background subtraction of the antibody library; C is the construction of the artificial cell library; D For administration to subjects, in vivo screening is performed; E is the removal of artificial cells that fail to recognize the target antigen; F is the survival of artificial cells that recognize the target antigen.
  • Figure 2 is a schematic diagram of the gene control expression cassette:
  • A is UAS- ⁇ EpCAM.CAR-2A-TerR.KRAB-iCasp9-2A-GFP control expression cassette, the expression of CAR-2A-TerR-KRAB fusion protein controlled by 5 ⁇ UAS-PCMV-min promoter, and 7 ⁇ TRE -ICasp9-2A-green fluorescent protein fusion gene under the control of PCMV promoter.
  • B is the CMV-scFvlab-synNotch control expression cassette, which contains the synNotch receptor controlled by the CMV promoter, the antibody library is constructed in the synNotch extracellular recognition domain, and the intracellular transcription domain of the synNotch receptor is Gal4-VP64.
  • C is the anti-CD19 synNotch receptor.
  • Figure 3 shows the tumor tissue inhibition rate of each group.
  • Figure 4 shows the tumor tissue inhibition rate of each group.
  • Figure 5 shows the UAS- ⁇ EpCAM.CAR-IL-12-2A-TerR.KRAB-iCasp9-2A-GFP control expression cassette. It contains the expression of ⁇ EpCAM.CAR-IL12FLAG-2A-TerR-KRAB fusion protein controlled by the 5 ⁇ UAS-PCMV-min promoter, and the iCasp9-2A-green fluorescent protein fusion gene controlled by the 7 ⁇ TRE-PCMV promoter.
  • Figure 6 shows the expression levels of IL-2FLAG at different times in each group.
  • Figure 7 shows the number of metastases in the tissues detected by pathology in each group.
  • FIG. 8 is a schematic diagram of a gene control expression cassette.
  • A is the UAS-CARlab-iCasp9-2A-GFP control expression cassette. It contains CAR library genes controlled by the 5 ⁇ UAS-Psv40 promoter and an inhibitory transcription factor controlled by the reverse NFAT response element promoter.
  • B is the CMV-scFvlab-synNotch-VP64 control expression cassette. It contains the synNotch receptor controlled by the CMV promoter, and the chain antibody library is constructed in the synNotch extracellular recognition region.
  • the intracellular transcription domain of the iCasp9-2A-green fluorescent protein fusion gene synNotch receptor under the control of the reverse 7 ⁇ TRE-PCMV promoter is Gal4-VP64.
  • C is the anti-CD19 synNotch receptor. The intracellular transcription domain of synNotch receptor is Gal4-KRAB.
  • Figure 9 shows the inhibition rate of tumor tissues in each group.
  • Figure 10 is the UAS-ctxCAR-2A-TerR.KRAB-iCasp9-2A-GFP control expression cassette. It contains the expression of the ctxCAR-inhibitory transcription factor-TerR-KRAB fusion protein, the scFv region of the chimeric antigen receptor is constructed from the monoclonal antibody cetuximab, and the iCasp9-2A-green fluorescent protein fusion under the control of the 7 ⁇ TRE-PCMV promoter gene.
  • Figure 11 shows the pathological scores of each group of inflammatory bowel disease.
  • Figure 12 is a schematic diagram of the construction of genetic elements
  • A-F is a schematic diagram of the construction of the first genetic element
  • G-K is a schematic diagram of the construction of the second genetic element.
  • Figure 13 is a schematic diagram of the construction of the third gene element
  • Fig. 14 is a schematic diagram of vector combination.
  • (A) Construction of phage antibody library First, a fully synthetic murine phage single-chain antibody library is established using a total synthesis method. The method of antibody library preparation is well known to those of ordinary skill in the art. The method of establishing a fully synthetic murine phage single-chain antibody library is the same as in the literature [Geuijen C et al.. European Journal of Cancer, 2005, 41(1): 178- 187; Noronha EJ, et al. Journal of Immunology, 1998, 161(6): 2968-2976.]. After the library capacity evaluation, the library capacity of the fully synthetic murine phage single-chain antibody library is 1 ⁇ 10 9 . The method of library capacity evaluation is the same as that in the literature [Ridgway J B B, et al. Cancer Research, 2013, 59(11): 2718-2723].
  • the gene circuit contains two gene control expression cassettes: 1
  • the gene control expression cassette as shown in Figure 2A named UAS- ⁇ EpCAM.CAR-2A-TerR.KRAB-iCasp9-2A-GFP control expression cassette. It contains the ⁇ EpCAM.CAR-2A-TerR-KRAB fusion protein controlled by the 5 ⁇ UAS-P CMV-min promoter (Deuschle U, Meyer WK, Thiesen H J..
  • iCasp9-2A under the control of the 7 ⁇ TRE-P CMV promoter [Deuschle U, Meyer WK, Thiesen H J. Molecular and Cellular Biology, 1995, 15(4):1907-1914.] -Green fluorescent protein fusion gene.
  • the construction methods of iCasp9 gene and self-cleaving peptide 2A are the same as those in the literature [Liu E, et al. Leukemia, 2018, 32(2):520.].
  • Anti-EpCAM scFv reference [Shivani Srivastava, et al.
  • the pre-programming of the gene circuit is: when the synNotch receptor binds to the antigen, the gene circuit is programmed to express ⁇ EpCAM.CAR while inhibiting the expression of the iCasp9 gene. At this time, the cell is not regulated by the iCasp9 inducer to induce apoptosis. ⁇ EpCAM.CAR recognizes antigens and exerts anti-tumor effects.
  • the obtained synthetic cell library was named KRAB-iCasp9- ⁇ EpCAM.CAR-synNotch-T library, and the library capacity was 1 ⁇ 10 6 monoclonal numbers.
  • mice were divided into control group, unrelated synNotch-T cell group, and KRAB-iCasp9- ⁇ EpCAM.CAR-synNotch-T library group.
  • the receptor positive rate of synNotch-T cells was standardized to 40%.
  • the control group was given PBS treatment, and the unrelated synNotch-T cell group was given CD19-synNotch-T cell treatment (the gene control expression cassette is shown in Figure 2C).
  • the dose of 5 ⁇ 10 6 cells was injected intravenously, once every 2 days, and 3 injections .
  • KRAB-iCasp9- ⁇ EpCAM.CAR-synNotch-T library group the dose is 5 ⁇ 10 6 cells intravenously, and the cell therapy uses serum-free 1640 medium to dilute the cells.
  • each group was given iCasp9 inducer.
  • the tumor growth inhibition ratio was measured after 5 weeks of treatment.
  • the result is shown in Figure 3.
  • the results show that the KRAB-iCasp9- ⁇ EpCAM.CAR-synNotch-T library group has a strong tumor suppressor effect.
  • Example 3 In vivo screening of a fully synthetic murine synNotch receptor cell library targeting 4T1 breast cancer
  • Example 2 All the experimental groups in Example 2 were sacrificed after the experiment, the mouse blood and tumor tissue were separated, and the CAR positive cells in the iCasp9- ⁇ EpCAM.CAR-synNotch-T library group were isolated and cultured by flow cytometry. That is, the in vivo screening is completed.
  • the T cell genome was extracted with a kit.
  • the obtained single-chain antibody is constructed into mouse IgG2a, and then expressed and purified. That is, antibodies targeting 4T1 breast cancer cells were obtained.
  • Example 4 The antibody obtained in Example 4 was cross-linked on agarose beads.
  • the 4T1 breast cancer cell lysate was incubated with cross-linked antibody beads overnight. After rinsing, the pooled antigen corresponding to the antibody is now enriched on the beads.
  • the beads are subjected to peptide fingerprint identification to obtain the target antigen.
  • the synNotch receptor T cells obtained in each group in Example 3 were again subjected to 4T1 mouse in situ breast cancer treatment, and the treatment method of each group was the same as in Example 2.
  • mice When the tumor volume reached 100mm 3 on average, the mice were divided into control group, unrelated synNotch-T cell group, iCasp9- ⁇ EpCAM.CAR-synNotch-T library group.
  • the receptor positive rate of synNotch-T cells was standardized to 40%.
  • the control group was given PBS treatment, and the unrelated synNotch-T cell group was given CD19-synNotch-T cell treatment (the gene control expression cassette is shown in Figure 2C).
  • the dose of 5 ⁇ 10 6 cells was injected intravenously, once every 2 days, and 3 injections .
  • Example 1 The gene control expression cassette shown in FIG. 2A in Example 1 was re-engineered, as shown in FIG. 5. Then construct a synthetic cell library according to the method as in Example 1, briefly as follows:
  • a fully synthetic method was used to establish a fully synthetic mouse phage single-chain antibody library.
  • the method of establishing a fully synthetic murine phage single-chain antibody library is the same as in the literature.
  • the library capacity of the fully synthetic murine phage single-chain antibody library is 1 ⁇ 10 9 .
  • the fully synthetic mouse-derived phage single-chain antibody library (1 ⁇ 10 11 PFU) was injected into BALB/c mice from the tail vein, and after 4 rounds of screening, all phages that could bind to mouse tissues were removed, and the newly acquired phage antibody library was expanded. There is no significant change in the increased detection storage capacity.
  • the antibody gene library was obtained by PCR method. Obtain mouse T lymphocytes. Select mouse T lymphocytes to further prepare cell library.
  • the gene circuit was constructed according to the synNotch receptor structure design scheme ( Figure 2A, 2B).
  • the gene circuit contains two gene control expression cassettes: 1
  • the gene control expression cassette shown in Figure 5 named UAS- ⁇ EpCAM.CAR-IL-12-2A-TerR.KRAB-iCasp9-2A-GFP control expression cassette. It contains the ⁇ EpCAM.CAR-IL12FLAG-2A-TerR-KRAB fusion protein controlled by the 5 ⁇ UAS-P CMV-min promoter (Deuschle U, Meyer WK, Thiesen H J. Molecular and Cellular Biology, 1995, 15(4): 1907-1914.], and the iCasp9-2A-green fluorescent protein fusion gene under the control of the 7 ⁇ TRE-P CMV promoter.
  • UAS- ⁇ EpCAM.CAR-IL-12-2A-TerR.KRAB -iCasp9-2A-GFP control expression cassette integrates mouse T lymphocytes.
  • the successfully integrated mouse T lymphocytes are sorted by flow cytometry.
  • the gene control expression cassette shown in Figure 2B is named CMV-scFvlab- synNotch.
  • the murine phage single-chain antibody library was constructed in the extracellular recognition region of synNotch.
  • the CMV-scFvlab-synNotch gene control expression cassette was inserted into the AVVS1 site of T cells.
  • the pre-programming of the gene circuit is: when the synNotch receptor binds
  • the gene circuit is programmed to express anti-EpCAM CAR and secrete IL-12FLAG, while inhibiting the expression of iCasp9 gene.
  • the cell is not regulated by the apoptosis induced by iCasp9 inducer.
  • IL-12 itself can activate macrophages, Enhance the anti-tumor effect of the cell library.
  • IL-2 fused with FLAG tag can be detected from the blood.
  • the obtained cell library was named iCasp9- ⁇ EpCAM.CAR-IL12-synNotch-T library.
  • a cell library for 4T1 tumors was obtained and named 4T1.KRAB-iCasp9- ⁇ EpCAM.CAR-IL12-synNotch-T library.
  • the 4T1 orthotopic tumor model was established according to the method of Example 1.
  • the orthotopic tumor was surgically removed 50 days later, and then the mice were divided into control group, unrelated synNotch-T cell group, 4T1.KRAB-iCasp9- ⁇ EpCAM.CAR-IL12- SynNotch-T library 1 group, 4T1.KRAB-iCasp9- ⁇ EpCAM.CAR-IL12-synNotch-T library 2 groups.
  • the dose is 5 ⁇ 10 6 cells intravenously, once every 2 days, 3 times.
  • the level of IL-2-FLAG in the blood of mice was measured weekly (Figure 6).
  • phage single-chain antibody library 300 healthy volunteers' peripheral monocytes were used to prepare a phage single-chain antibody library of natural human origin.
  • the method for preparing the antibody library is well known to those of ordinary skill in the art, and the method for establishing a phage single-chain antibody library is the same as in the literature [Ridgway BB, et al. Cancer Research, 2013, 59(11): 2718-2723).
  • the established phage natural human single-chain antibody library has a library capacity of 1 ⁇ 10 10 .
  • the method of library capacity evaluation is the same as that in the literature [Ridgway BB, et al. Cancer Research, 2013, 59(11): 2718-2723).
  • the phage natural human single-chain antibody library (1 ⁇ 10 12 PFU) was injected into NSG mice from the tail vein, and after 4 rounds of screening, all phages that could bind to mouse tissues were removed.
  • the method was the same as that in the literature [Wada, Akinori, et al. Molecular Therapy-Oncolytics 12(2019):138-146.].
  • the reacquired phage antibody library had no significant changes in the volume of the amplification test. Then the antibody gene library was obtained by PCR method.
  • NK-92 cells K, et al. Molecular therapy, 2015, 23(2):330-338.] further prepare a cell library.
  • the gene circuit was constructed according to the synNotch receptor structure design scheme ( Figure 8A, 8B).
  • the gene circuit contains two gene control expression cassettes: 1
  • the gene control expression cassette as shown in Figure 8A named UAS-CARlab-iCasp9-2A-GFP control expression cassette. It contains CAR library genes under the control of the 5 ⁇ UAS-P sv40 promoter.
  • the scFv library of the CAR library contains binding targets CD19, BCMA, Mesothelin, GD2, EGFR, HER2, CD22, CD123, Glypican 3, CD30, MUC1, CD33, CD20, CD38, EpCAM, CD56, CD138, CD7, CD133, CEA, CD34, CD117, Claudin18.2, PSCA, cMET, Lewis Y, EphA2, NKG2D ligands, ErbB, NY-ESO-1, CLL- 1.
  • This promoter contains 6 NFAT response elements and minimizes interleukin 2 (IL-2) promoter.
  • UAS-CARlab-iCasp9-2A-GFP control expression cassette was integrated into NK-92 cells using lentivirus system. The successfully integrated NK-92 cells were sorted by flow cytometry. 2
  • the gene control expression cassette shown in Figure 8B was named CMV-scFvlab-synNotch-VP64.
  • the above-mentioned natural human single-chain antibody library was constructed in the extracellular recognition region of synNotch. It also contains the iCasp9-2A-green fluorescent protein fusion gene under the control of the reversed 7 ⁇ TRE-P CMV promoter.
  • the CMV-scFvlab-synNotch-VP64 gene control expression cassette was inserted into the AVVS1 site of NK-92 cells.
  • the pre-programming of the gene circuit is: when the synNotch receptor binds to the antigen, the gene circuit activates the CAR receptor library. When the CAR receptor can also be activated at the same time, it continues to inhibit the expression of the downstream iCasp9 gene, and the cell is not affected by iCasp9. Apoptosis-inducing regulation of inducer.
  • the obtained fully human synNotch receptor NK-92 cell library was named CARlab-iCasp9-synNotch-NK92 library, and the library capacity was 1 ⁇ 10 6 monoclonal numbers.
  • Example 9 In vivo screening of natural whole human synNotch receptor NK-92 cell library
  • mice Use patient-derived tissues to directly establish NSG mouse tumor-bearing model, namely PDX.
  • PDX NSG mouse tumor-bearing model
  • literature methods [Fu W, et al. Clinical Cancer Research, 2019, 25(9): 2835-2847.], lung cancer PDX model L10, breast cancer PDX model B7, and ovarian cancer PDX model OV3 were established.
  • the tumor volume of the mice reached an average of 400mm 3
  • the mice were divided into the control group, the unrelated synNotch-NK92 cell group (anti-CD19 synNotch-NK92, containing the gene control expression cassette as shown in Figure 8C), CARlab-iCasp9-synNotch-NK92 library group.
  • the treatment dosage and method are the same as in Example 1.
  • each group was given iCasp9 inducer.
  • the mice were sacrificed, and the blood and tumor tissues of the mice were separated and the CAR-positive cells were detected by flow cytometry.
  • CAR-positive expression cells were isolated from the CARlab-iCasp9-synNotch-NK92 library group of lung cancer PDX model L10, breast cancer PDX model B7, and ovarian cancer PDX model OV3. That is, the in vivo screening is completed.
  • the chimeric antigen receptor NK-92 cell library for lung cancer L10, breast cancer B7, and ovarian cancer OV3 was obtained.
  • the human chimeric antigen receptor NK-92 cell library obtained at this time was named L10-CARlab-iCasp9-synNotch-NK92 library, B7-CARlab-iCasp9-synNotch-NK92 library, OV3-CARlab-iCasp9-synNotch-NK92 library, respectively .
  • the storage capacity is 6 ⁇ 10 4 single clones.
  • Example 11 Chimeric antigen receptor NK-92 cell library targeting tumor tissue to treat mouse tumor-bearing model
  • mice Use patient-derived tissues to directly establish NSG mouse tumor-bearing model, namely PDX.
  • literature method Fe W, et al. Clinical Cancer Research, 2019, 25(9): 2835-2847.
  • the tumor volume of mice is average
  • the mice are divided into control group, irrelevant CAR-NK92 cell group (anti-CD19CAR-NK92, including gene control expression cassette as shown in Figure 8C), body NK-92 cell library group.
  • the treatment dose and method are the same as in the example 1.
  • each group was given iCasp9 inducers.
  • ratio 1-average tumor volume of the treatment group/average tumor volume of the control group.
  • Figure 9 The results show that the chimeric antigen receptor NK-92 cell library targeting tumor tissue has a very strong anti-tumor inhibitory effect.
  • Example 12 Construction and preparation of individualized natural whole human synNotch receptor T cell library
  • Example 8 Using 300 healthy volunteers' peripheral monocytes as described in Example 8 to prepare a natural human-derived phage single-chain antibody library for this example. After the library capacity evaluation, the established phage natural human single-chain antibody library has a library capacity of 1 ⁇ 10 10 .
  • the gene circuit contains two gene control expression cassettes: 1
  • the gene control expression cassette shown in Figure 10 named UAS-ctxCAR-2A-TerR.KRAB-iCasp9-2A-GFP control expression cassette. It contains the expression of ctxCAR-inhibitory transcription factor-TerR-KRAB fusion protein controlled by the 5 ⁇ UAS-P CMV-min promoter, and the scFv region of the chimeric antigen receptor is constructed from the monoclonal antibody cetuximab (reference Fu W, et al . Nat Commun. 2019 Sep 25; 10(1): 4355.), and 7 ⁇ TRE-P CMV promoter (Deuschle U, Meyer WK, Thiesen H J.
  • iCasp9-2A-green fluorescent protein fusion gene under control UAS-ctxCAR-2A-TerR.KRAB-iCasp9-2A-GFP control expression cassette was integrated into the subject’s T lymphocytes using the lentiviral system. The successfully integrated T lymphocytes were sorted by flow cytometry. 2The gene control expression cassette as shown in Figure 2B was named CMV-scFvlab-synNotch. The above-mentioned natural human-derived single-chain antibody library was constructed in the extracellular recognition region of synNotch. The lentiviral system was used to integrate CMV-scFvlab-synNotch into the subject’s T lymphocytes, and flow cytometry was used to sort the successfully integrated T lymphocytes.
  • the pre-programming of the gene circuit is: when the synNotch receptor binds to the antigen, the gene circuit is programmed to express the chimeric antigen receptor that can bind to EGFR while inhibiting the expression of iCasp9 gene. At this time, the cell is not affected by the iCasp9 inducer. Death regulation. Chimeric antigen receptors can mediate T cells to kill tumor cells and exert anti-tumor effects.
  • the obtained synNotch receptor cell library was named ctxCAR-iCasp9-synNotch-hT library, and the library capacity was 6 ⁇ 10 5 monoclonal numbers.
  • the aforementioned ctxCAR-iCasp9-synNotch-hT library was intravenously administered to the lung cancer subject described in Example 12.
  • the method of administration, dosage, etc. are in accordance with the general administration method of cell therapy[Fry T J, et al. Nature medicine, 2018, 24(1): 20.]
  • Example 14 Fully synthetic murine synNotch receptor cell library for treatment of small animal model of inflammatory bowel disease
  • mice were used to establish trinitrobenzene sulphonic acid (TNBS)-induced colitis (ie, inflammatory bowel disease model).
  • TNBS trinitrobenzene sulphonic acid
  • Model preparation and evaluation are the same as those in the literature [He C, et al.. Gut, 2015.].
  • the model mice were divided into control group, model group, unrelated synNotch-T cell group, and KRAB-iCasp9- ⁇ EpCAM.CAR-synNotch-T library group.
  • the control group was not given TNBS control, and the other groups were given small doses of TNBS at the same time treatment: PBS was given, and the unrelated synNotch-T cell group was given CD19-synNotch-T cell therapy (the gene control expression cassette is shown in Figure 2C). 5 ⁇ 10 6 cells were injected intravenously, once every 2 days, 3 times. The case score was performed after four weeks, and the method was the same as that in the literature. The results are shown in Figure 11. The results show that the KRAB-iCasp9- ⁇ EpCAM.CAR-synNotch-T cell library has a good therapeutic effect.
  • Example 15 In vivo screening of a natural fully human synNotch receptor NK-92 cell library targeting ectopic inner membrane
  • the treatment dose and method are the same as in Example 1. In the second week of treatment, each group was given iCasp9 inducer.
  • mice After 5 weeks of treatment, the mice were sacrificed and the intima of each group was obtained for examination. The blood and blood of each group were separated. The ectopic endometrial tissue was separated and obtained by flow cytometry to obtain CAR-positive expression cells in the CARlab-iCasp9-synNotch-NK92 library treatment group, and the in vivo screening was completed.
  • Example 16 Construction and preparation of a natural fully human synNotch receptor T cell library targeting ectopic inner membrane
  • the phage single-chain antibody library of natural human origin was established by the method described in Example 7. The method is briefly described as follows: firstly, 300 healthy volunteers' peripheral monocytes are used to prepare a phage single-chain antibody library of natural human origin. After the library capacity evaluation, the established phage natural human single-chain antibody library has a library capacity of 1 ⁇ 10 10 .
  • Example 17 Natural whole human synNotch receptor T cell library targeting ectopic endometrium for the treatment of human endometriosis
  • the endo-CARlab-iCasp9-synNotch-hT library described above was intravenously administered to subjects with endometriosis.
  • the method of administration, dosage, etc. are in accordance with the general administration method of cell therapy[Fry T J, et al. Nature medicine, 2018, 24(1): 20.]
  • the antibody library is derived from the following 495 monoclonal antibodies: ABAGOVOMAB, ABCIXIMAB, ABELACIMAB, ABITUZUMAB, ABREZEKIMAB, ABRILUMAB, ACTOXUMAB, ADALIMUMAB, ADECATUMUMAB, ADUCANUMAB, AFASEVIKUMAB, AFELIMOMAB, ALACIZUMA, AB, MATALEMTUIMAB, ALACIZUMA, AB, ALAB, , ANRUKINZUMAB, APRUTUMAB, ASCRINVACUMAB, ASELIZUMAB, ATIDORTOXUMAB, ATINUMAB, ATOLTIVIMAB, ATOROLIMUMAB, AVELUMAB, AZINTUXIZUMAB, BALSTILIMAB, bAPINEUZUMAB, bASIL
  • NK-92 cells K,et al.Molecular therapy,2015,23(2):330-338.
  • a cell library construct synNotch receptor and intracellular gene circuit according to the same method in Example 1, that is, obtain a cell library containing 495 artificial antibodies Artificial NK-92 cell library.
  • the obtained NK-92 cell library was named KRAB-iCasp9- ⁇ EpCAM.CAR-495synNotch-NK92 library, and the library capacity was 495 monoclonals in terms of synNotch receptor.
  • KRAB-iCasp9- ⁇ EpCAM.CAR-495synNotch-NK92 library was intravenously administered to subjects with pancreatic cancer.
  • the administration method and dosage are in accordance with the general administration method of cell therapy [Fry T J, et al. Nature medicine, 2018, 24(1): 20.].
  • the first gene element is the synNotch receptor library.
  • the synNotch receptor is an artificial receptor.
  • the present invention emphasizes the use of a randomized library of synNotch extracellular recognition domains to realize the application of a specific synNotch library, and the construction method is exemplified as follows.
  • the following methods can also be used to construct, briefly as follows: the use of DNA synthesis methods, according to human gene antibody coding rules, artificially synthesize a human antibody gene library.
  • the gene library is constructed in a phage vector, and the phage is amplified to obtain a phage antibody library. If the screening method is to be applied to an animal model, the phage antibody library is applied to the animal, and the phages that can bind to the animal model antigen are subtracted. At this time, the phage antibody sub-library is obtained, and the antibody gene library of the sub-library is obtained by genetic engineering methods.
  • synNotch receptor protocol namely: single chain antibody library-human synNotch core domain-Gal4-KRAB as shown in Figure 12B; if it is to be used directly in the subject, the control tissue of the subject is obtained, such as the test subject Peripheral monocytes, para-cancerous tissues, etc., are subtracted from the phage that can bind to the control tissue, and the phage antibody sub-library is obtained at this time, and the antibody gene library of the sub-library is obtained using genetic engineering methods.
  • the synNotch receptor scheme namely: Fab antibody library-zebrafish synNotch core-PIP-VP64 as shown in Figure 12C;
  • the antibody gene library is obtained from the peripheral monocytes of healthy volunteers by genetic engineering, the gene library is constructed in a yeast vector, and the yeast cells are amplified to obtain the yeast antibody library. Further construct according to the synNotch receptor protocol, namely: human antibody gene library-rat synNotch core-ZFHD1-VP64, as shown in Figure 12D;
  • camel peripheral blood mononuclear cells to obtain camel antibody gene library by genetic engineering method, and further construct according to the synNotch receptor protocol, namely: camel antibody gene library-mouse synNotch core structure Domain—Gal4-VP64, as shown in Figure 12E;
  • the antibody gene library is obtained from the peripheral monocytes of healthy volunteers by genetic engineering
  • the gene library is constructed in a yeast vector
  • the yeast cells are amplified to obtain the yeast antibody library.
  • the tumor cell line MDA-MB-231 was contacted with the antibody library, and yeasts that could bind to the cells were panned.
  • the second element is the gene circuit. It can be constructed using the following method, which is briefly described as follows: 6 NFAT reaction elements and minimal interleukin 2 (IL-2) promoter (6 ⁇ NFAT), as shown in Figure 12G
  • the following method can also be used to construct: construct 7 TRE and minimize CMV promoter fusion (7 ⁇ TRE-P CMV-min ), then construct 4 NFAT response elements and minimize IL-2 promoter fusion (4 ⁇ NFAT) promoter, containing Gal4-KRAB downstream; and then construct a 5 ⁇ UAS-PS V40 fusion promoter regulated by Gal4-KRAB), as shown in Figure 12H.
  • the following method can also be used to construct: a promoter (10 ⁇ NF ⁇ B) formed by fusion of 10 NF ⁇ B binding originals and a minimal HIVtata promoter, and the downstream transcription factor TetR-VP64; and then construct 7 regulated by TetR-VP64 TRE and minimal CMV promoter are fused to form (7 ⁇ TRE-P CMV-min ) promoter, as shown in Figure 12J.
  • NF ⁇ B binding elements 10 NF ⁇ B binding elements, 6 NFAT response elements and a minimal interleukin 2 (IL-2) promoter fusion (10 ⁇ NF ⁇ B+6 ⁇ NFAT) promoter, and its downstream Transcription factor ZFHD1-VP64;
  • IL-2 minimal interleukin 2
  • the first genetic element is the chimeric antigen receptor library.
  • the chimeric antigen receptor is an artificial receptor, and the library construction can be found in patent document WO2015/123642.
  • the present invention emphasizes the use of a randomized library of chimeric antigen receptor extracellular recognition domains to realize the application of specific chimeric antigen receptors. Examples of construction methods are as follows.
  • the method of DNA synthesis is used to artificially synthesize a human antibody gene library according to the rules of human gene antibody coding. Further construct according to the chimeric antigen receptor scheme, namely: human antibody gene single chain antibody library—CD8hinge—CD8TM—4-1BB—CD3 ⁇ , as shown in Figure 13A;
  • the following methods can also be used to construct, briefly as follows: the use of DNA synthesis methods, according to human gene antibody coding rules, artificially synthesize a human antibody gene library.
  • the gene library is constructed in a phage vector, and the phage is amplified to obtain a phage antibody library. If the screening method is to be applied to an animal model, the phage antibody library is applied to the animal, and the phages that can bind to the animal model antigen are subtracted. At this time, the phage antibody sub-library is obtained, and the antibody gene library of the sub-library is obtained by genetic engineering methods.
  • the antibody gene library is obtained from the peripheral monocytes of multiple healthy volunteers by genetic engineering, the gene library is constructed in a yeast vector, and the yeast cells are amplified to obtain the yeast antibody library. Further construct according to the chimeric antigen receptor scheme, namely: human antibody gene library-2D3-CD137TM-4-1BB-CD3 ⁇ , as shown in Figure 13D;
  • camel peripheral blood mononuclear cells to obtain camel antibody gene library by genetic engineering method, and further construct according to the chimeric antigen receptor protocol, namely: camel antibody gene library-CD8-hinge —CD8TM—4-1BB—CD3 ⁇ , as shown in Figure 13E;
  • the antibody gene library is obtained from the peripheral monocytes of healthy volunteers by genetic engineering
  • the gene library is constructed in a yeast vector
  • the yeast cells are amplified to obtain the yeast antibody library.
  • the tumor cell line MDA-MB-231 was contacted with the antibody library, and yeasts that could bind to the cells were panned.
  • the third gene element is the gene element encoding the protein.
  • the encoding includes green fluorescent protein GFP, blue fluorescent protein BFP, Myc tag, FLAG tag, puromycin resistance protein (PuroR), neomycin resistance protein (NeoR), Blasticidin resistance protein (Blasticidin-R), hygromycin B resistance protein (Hygromycin BR), herpes simplex virus thymidine kinase protein (HSV-TK), cytosine deaminase protein (CD) or iCasp9 suicide System protein (iCasp9) gene.
  • the vector combination containing the three genetic elements can be constructed using the following methods, which are briefly described as follows: (i) The method of DNA synthesis, according to the human gene antibody coding rules, artificially synthesize a human antibody gene library. Further construct according to the synNotch receptor protocol, namely: human antibody gene single-chain antibody library-mouse synNotch core domain-TetR-VP64; (ii) use the following method to construct: construct 7 TRE and minimize CMV promoter fusion (7 ⁇ TRE-P CMV-min ), and then construct 4 NFAT response elements and minimize IL-2 promoter fusion to form a (4 ⁇ NFAT) promoter, which contains Gal4-KRAB downstream; Gal4-KRAB regulated 5 ⁇ UAS-P SV40 fusion promoter.
  • camel peripheral blood mononuclear cells to obtain camel antibody gene library by genetic engineering method, and further construct it according to the synNotch receptor protocol, namely: camel antibody gene library—mouse synNotch core domain—Gal4-VP64, (ii) Construct 5 UAS and minimal CMV promoter fusion (5 ⁇ UAS-P CMV-min ), then construct 6 NFAT reaction elements and minimal interleukin 2 (IL-2) promoter (6 ⁇ NFAT), which The downstream TetR-KRAB transcription factor; the 7 ⁇ TRE-P SV40 fusion promoter regulated by TetR-KRAB was constructed, (iii) the antibody gene library was obtained from the peripheral monocytes of multiple healthy volunteers using genetic engineering methods, and the genes The library is constructed in a yeast vector, and yeast cells are amplified to obtain a yeast antibody library.
  • camel antibody gene library mimouse synNotch core domain—Gal4-VP64
  • the tumor cell line MDA-MB-231 was contacted with the antibody library, and yeasts that could bind to the cells were panned.

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Abstract

L'invention concerne un ensemble vecteur comprenant une combinaison d'éléments géniques, une banque de cellules receveuses, une préparation et un procédé de criblage et une utilisation associés. La banque de cellules receveuses comprend une cellule fusionnée avec l'ensemble vecteur. L'ensemble vecteur comprend au moins trois éléments géniques, à savoir, de multiples types d'un premier élément de gène codant pour un ou plusieurs récepteurs de synNotch idiotypiques, un second élément de gène comprenant un ou plusieurs circuits de gène, et un troisième élément de gène codant pour un ou plusieurs récepteurs d'antigène chimériques idiotypiques, lorsque le premier élément de gène code un récepteur de synNotch idiotypique, le troisième élément de gène doit coder au moins trois récepteurs d'antigène chimériques idiotypiques, et lorsque le troisième élément de gène code un récepteur d'antigène chimère idiotypique, le premier élément de gène doit coder trois récepteurs de synNotch idiotypiques. Le circuit de gène est préprogrammé et comprend une combinaison d'un facteur cis-actif régulateur et d'un facteur de transcription, et peut obtenir une expression contrôlée d'un récepteur d'antigène chimèrique codé par le troisième élément de gène lors de l'activation du récepteur de synNotch codé par le premier élément de gène.
PCT/CN2020/119248 2019-11-13 2020-09-30 Ensemble vecteur comprenant une combinaison d'éléments géniques, banque de cellules receveuses, préparation et procédé de criblage et utilisation associés WO2021093482A1 (fr)

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