WO2021093484A1 - 携带基因元件组合的嵌合抗原受体细胞文库、制备和筛选方法及用途 - Google Patents

携带基因元件组合的嵌合抗原受体细胞文库、制备和筛选方法及用途 Download PDF

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WO2021093484A1
WO2021093484A1 PCT/CN2020/119322 CN2020119322W WO2021093484A1 WO 2021093484 A1 WO2021093484 A1 WO 2021093484A1 CN 2020119322 W CN2020119322 W CN 2020119322W WO 2021093484 A1 WO2021093484 A1 WO 2021093484A1
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chimeric antigen
cells
antigen receptor
gene
library
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PCT/CN2020/119322
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French (fr)
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胡适
傅文燕
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沣潮医药科技（上海）有限公司
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Priority to US17/743,423 priority Critical patent/US20220348961A1/en

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    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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    • C12N2740/16011—Human Immunodeficiency Virus, HIV
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    • C12N2840/002—Vectors comprising a special translation-regulating system controllable or inducible

Definitions

  • the present invention relates to the fields of biomedical engineering technology and synthetic biology, in particular to a gene element combination and a chimeric antigen receptor cell library carrying the combination, and a method for preparing and constructing the gene element combination and the cell library to target in vivo antigens And/or in vitro antigen screening methods and the use of cell libraries are described in detail.
  • CAR Chimeric Antigen Receptor
  • the first generation of CAR-T cells are composed of extracellular binding region-single-chain fragment variable (scFV), transmembrane region (TM) and intracellular signal region-immunoreceptor tyrosine activation motif (immunoreceptor tyrosine-based activation motif, ITAM), in which the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD3 ⁇ .
  • This kind of CAR-T cell can stimulate the anti-tumor cytotoxic effect, but the secretion of cytokines is relatively small, and it cannot stimulate the long-lasting anti-tumor effect in the body (Zhang T.et.al, Cancer Res 2007, 67(22): 11029- 11036.).
  • the second-generation CAR-T cells that were subsequently developed added the intracellular signal region of CD28 or CD137 (also known as 4-1BB), where the parts of the chimeric antigen receptor were connected as follows: scFv-TM-CD28-ITAM or scFv -TM-CD137-ITAM.
  • the co-stimulation of B7/CD28 or 4-1BBL/CD137 in the intracellular signaling region causes the continuous proliferation of T cells, and can increase the secretion of IL-2, IFN- ⁇ and other cytokines by T cells, and at the same time increase the level of CAR-T In vivo survival cycle and anti-tumor effect [Dotti G.et.al. CD28 costimulation improvement expansion and persistence of chimeric antigen receptor modified T cells in lymphoma patients. J Clin Invest, 2011, 121(5): 1822-1826].
  • CAR-NK cells Choucair K, et al. Future Oncology, 2019, 15(26): 3053-3069.
  • CAR NK-92 cells K, et al. Molecular therapy, 2015, 23(2): 330-338.
  • CAR macrophages Zhang W, et al.. British Journal of Cancer, 2019:1-9.] and so on.
  • CAR cell technology has attractive prospects. However, which antigen-specific chimeric antigen receptors these genetically engineered cells carry is an unresolved technical problem with this technology. For example, CAR-T cells directed against a single antigen can develop drug resistance, and CAR cells cannot be directed against unknown antigens.
  • the characteristics are that the disease has a high degree of variability, individual differences, heterogeneity, and evolution. That is to say, the antigens in the lesions of patients with the same disease vary greatly, and the antigens in different parts of the lesions of a patient are also very different.
  • treatment pressure such as radiotherapy and chemotherapy, etc.
  • the evolutionary pressure of biological proliferation which makes tumor cells Enhanced gene repair capabilities
  • targeted therapies such as CAR-T therapy, antibody therapy, etc. put evolutionary pressure on specific antigens, causing tumors to reduce the expression of related antigens), and the evolution of lesion cells cannot completely eliminate the disease.
  • 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. Therefore, if treatment is the goal, the method of screening and preparing molecular identifiers for specific antigens lags far behind the disease process. In addition, due to the huge kurtosis of the human genome, the genetic background of the lesions of patients with the same type of malignant tumors varies greatly. Therapeutic strategies and preparations that target specific antigens have no significance for individualized treatment, and can only be achieved through markers and Genetic research conducts evidence-based analysis to obtain the corresponding possible effective population. The tumor antigen obtained through specific tumor tissue analysis, and the further preparation of CAR cell therapy method against the antigen also has no individualized therapeutic significance.
  • the patent document WO2015/123642 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 publication of the construction method discloses how to construct a cell library containing multiple idiotypic CARs.
  • the limitation of this technology is that the screening direction of the CAR cell library cannot follow the change of the antigen, and can only be screened for specific antigens.
  • the present invention provides a chimeric antigen receptor cell library containing new gene elements, a combination of the gene elements, a method for preparing and constructing the cell library, and a screening method for in vivo antigens and/or in vitro antigens
  • the use of the cell library is explained 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 idiotypic chimeric antigen receptors (ie chimeric antigen receptor library); (2) containing one or The second gene element of a variety of unique gene circuits; (3) the third gene element that encodes one or more unique inducible proteins.
  • three genetic elements namely: (1) multiple first genetic elements encoding one or more idiotypic chimeric antigen receptors (ie chimeric antigen receptor library); (2) containing one or The second gene element of a variety of unique gene circuits; (3) the third gene element that encodes one or more unique inducible proteins.
  • the multiple idiotype chimeric antigen receptors are at least three idiotype chimeric antigen receptors.
  • Chimeric antigen receptors include intracellular signal transduction domains, transmembrane domains, and extracellular recognition domains.
  • the extracellular recognition domains include complete antibodies, heavy or light chains or antibody fragments that make up antibodies; gene circuits are pre- Programming is a combination of regulatory homeopathic factors and transcription factors; inducible proteins include one or two of drug resistance proteins and suicide proteins.
  • the function that the gene element of the present invention can achieve is: when the chimeric antigen receptor encoded by the first gene element is activated, the gene circuit pre-programmed by the second gene element can regulate the expression of the inducible protein encoded by the third gene element .
  • the expression regulation 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 term "chimeric antigen receptor" in the first 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 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 fragments), preferably single-chain antibody (ScFv) libraries.
  • 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 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 targets targeted by the extracellular recognition domain 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, Aspergillus, Dec
  • the extracellular recognition domain library is sourced from the following monoclonal antibodies ABAGOVOMAB, ABCIXIMAB, ABELACIMAB, ABITUZUMAB, ABREZEKIMAB, ABRILUMAB, ACTOXUMAB, ADALIMUMAB, ADECATUMUMAB, ADUCANUMAB, AFASEVIKUMAB, AFELIMOMAB, ALAIMABUMA, ALFELIMOMAB, ALAIMAZUMA, ABEL, A, and B , ANETUMAB, ANIFROLUMAB, ANRUKINZUMAB, APRUTUMAB, ASCRINVACUMAB, ASELIZUMAB, ATIDORTOXUMAB, ATINUMAB, ATOLTIVIMAB, ATOROLIMUMAB, AVELUMAB, AZINTUXIZUMAB, BALSTILIMAB, bAPINEUZUMAB, bASILIXIMAB, BAVITUXIMAB, BECTUMOMAB, BEDINVETM
  • 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. Since the characteristic basis for the chimeric antigen receptor to recognize the antigen is its internal antibody structure, the chimeric antigen receptor also has a "idiotype".
  • protein domains include antigen binding domains, hinge domains, transmembrane domains, and endodomains.
  • unique means having different polypeptide (amino acid) sequences, comprising different polypeptide (amino acid) sequences, or domains composed of different polypeptide (amino acid) sequences.
  • two "unique” antigen-binding domains can bind to the same antigen (in fact, even the same epitope on the antigen); however, the antigen-binding domains are "unique” when their continuous amino acid composition differs from each other. Unique".
  • two "unique” antigen-binding domains that differ in their continuous amino acid composition can also specifically bind to different antigens and epitopes.
  • the coding domain, domain or gene includes any one or a combination of at least two of the amino acid sequence encoding the protein, the DNA sequence encoding the protein, or the RNA sequence encoding the protein.
  • the term "gene circuit" in the second genetic element is a synthetic biology concept and is preprogrammed. Broadly speaking, the gene circuit contains regulatory cis-acting factors and transcription factors. Regulatory cis-acting factors include promoters, such as T7 promoter, CMV promoter, UPS promoter, Tet promoter, etc.;
  • the complex regulatory network which can be flexibly designed according to the research purpose, is like a circuit network, so it is called “gene circuit” or “gene circuit”.
  • the gene circuit includes regulatory cis-acting factors and/or transcription factors.
  • Regulatory cis-acting factors include single homeopathic factors or fusion homeopathic factors, and transcription factors 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
  • Fusion homeopathic factors include a flexible combination of one or more single homeopathic factors, such as 4 NFAT response elements and minimal IL-2 promoter fusion (4 ⁇ NFAT), 6 NFAT response elements and minimization
  • the IL-2 promoter is fused (6 ⁇ NFAT), which includes 5 NF ⁇ B binding elements and the minimum HIVtata promoter fused (5 ⁇ NF ⁇ B), and the 10 NF ⁇ B binding elements and the minimum HIVtata promoter are fused (10 ⁇ NF ⁇ B), 7 TRE and minimal CMV promoter fused (7 ⁇ TRE-P CMV-min ), 5 UAS and minimal CMV promoter fused (5 ⁇ UAS-P CMV-min ), 4 PIR and minimal CMV promoter fusion (4 ⁇ PIR-P CMV-min ), 8 PIR and minimal CMV promoter fusion (8 ⁇ PIR-P CMV-min ), 8 PIR and insect heat shock protein 70 promoter fused (8 ⁇ PIR-P hsp70min ), 4 ZFHD1RE and minimal CMV promoter fused (4 ⁇ ZFHD1RE-
  • 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 Gal4-VP64 and 5 ⁇ UAS-P CMV-min , TetR- Any one of the combination of VP64 and 7 ⁇ TRE-P CMV-min , the combination of TetR-KRAB and 7 ⁇ TRE-P SV40 , or the combination of TetR-KRAB and 7 ⁇ TRE-P cmv and (ii) 4 ⁇ NFAT , 6 ⁇ NFAT, 5 ⁇ NF ⁇ B, and 10 ⁇ NF ⁇ B.
  • a gene circuit is "unique" it means that it includes a gene circuit composed of different design and programming schemes.
  • two “unique” 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) are "unique” when they are designed and constructed in different forms.
  • 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 "unique" genetic 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.
  • inducible protein in the third gene element means that when the protein is expressed, cells carrying the gene can die through induction methods, including drug resistance proteins and/or suicide proteins.
  • Drug resistance protein refers to a gene that encodes a protein that resists a certain drug. It is a common technology in molecular biology and genetic engineering; suicide protein is an emerging technology in recent years, and genetic engineering methods are mostly used to encode a certain prodrug hydrolase or induce Apoptosis signal to realize the death of cells carrying suicide protein under drug induction.
  • 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 at least two of the herpes simplex virus thymidine kinase protein, cytosine deaminase protein, or iCasp9 suicide system protein.
  • the inducible protein induction method includes any one or a combination of at least two of drug induction, chemical induction, physical induction, laser induction, and thermal induction.
  • a plurality of first gene elements encode a plurality of idiotypic chimeric antigen receptors
  • a plurality of second gene elements are constructed into a plurality of unique gene circuits
  • a plurality of third elements encode a plurality of Unique inducible protein.
  • a plurality of first genetic elements encode a plurality of idiotypic chimeric antigen receptors
  • a plurality of second genetic elements are constructed as a unique gene circuit
  • a plurality of third The elements encode multiple unique inducible proteins; in other embodiments, multiple first genetic elements encode multiple idiotypic chimeric antigen receptors, multiple second genetic elements are constructed into multiple unique gene circuits, and multiple This third element encodes a unique inducible protein.
  • the present invention relates to a combinatorial library of gene elements, which is a library containing a combination of different gene elements, which is randomized in terms of idiotypic chimeric antigen receptors, gene circuits, and inducible proteins.
  • the structure of the chimeric antigen receptor It can also be randomized in terms of unique antigen binding domains (ie antibody idiotypes), hinge domains and/or intramembrane domains, as in patent document WO2015/123642.
  • the library can also be randomized in terms of three genetic elements.
  • the library can be randomized in terms of idiotypic chimeric antigen receptors, gene circuits, and inducible proteins; it can also be randomized in terms of idiotypic chimeric antigen receptors. Randomization in terms of gene circuits; randomization in terms of idiotypic chimeric antigen receptors and inducible proteins; and randomization in terms of unique gene circuits and inducible proteins.
  • the second aspect of the present invention provides a method for preparing a library of chimeric antigen receptor cells.
  • the first element, the second element, and the third element are inserted into the same vector or different vectors and transfected into the cells to obtain the chimeric antigen receptor 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.
  • the antibody gene library can be established using healthy volunteers or fully synthetic methods, and genetic engineering methods can also be used to establish a functional gene library. For example, genetic engineering methods such as selecting a suitable display platform based on healthy volunteers or fully synthetic methods to establish an antibody display library, using a control tissue as a control, after multiple rounds of panning to subtract the background, obtain a phage antibody display sub-library, and then Then obtain the antibody gene library by PCR method.
  • the first gene element is constructed, the structure of which includes the single-chain antibody library-CAR, which is constructed as the extracellular recognition domain of the CAR. Construct a second gene element, including the first regulatory homeopathic factor, a transcription factor regulated by the regulatory homeopathic factor, and a second homeopathic factor regulated by the transcription factor. Construct the third element, including the inducible protein gene.
  • the above three gene elements are constructed into one or more gene control expression cassettes.
  • the lentiviral vector system is used to introduce the gene control expression cassette into mammalian immune cells to obtain a library of chimeric antigen receptor cells.
  • the third aspect of the present invention provides a library of chimeric antigen receptor cells.
  • 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 chimeric antigen receptor cells against in vitro antigens, including the following steps:
  • step (3) From the chimeric antigen receptor cells screened in step (3), use antibody engineering methods to reconstruct the secondary chimeric antigen receptor cell library; (5) repeat steps (1)-(3) to screen For target chimeric antigen receptor cells, if necessary, repeat steps (4)-(5) one or more times.
  • chimeric antigen receptor cells include monoclonal chimeric antigen receptor cells and polyclonal chimeric antigen receptor cell populations.
  • 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 of screening chimeric antigen receptor cells is to contact the library of chimeric antigen receptor cells with in vitro antigens. Only chimeric antigen receptor cells that can recognize the antigen will be based on the preprogrammed gene circuits and Inducible protein generation: such as activating the expression of the inducible protein in the cell, enhancing the expression of the inducible protein in the cell, terminating the expression of the inducible protein in the cell, and inhibiting any one or at least two of the expression of the inducible protein in the cell The combination. Select the screening method according to the expression of the inducible protein, and then screen out the target antigen receptor cells, and design flexibly according to the gene circuit and the inducible protein to achieve the screening purpose.
  • the chimeric antigen receptor response gene circuit is designed to activate and/or enhance the gene circuit that can inducible protein expression when the chimeric antigen receptor is activated.
  • the induced protein is designed as a suicide protein
  • the cells activated by the chimeric antigen receptor will undergo apoptosis in a culture environment with screening drugs, and the remaining cells will survive, so that the chimeric antigen receptor expressing cells that cannot bind to the target antigen are screened out.
  • the chimeric antigen receptor response gene circuit is designed to terminate and/or inhibit the gene circuit that can induce protein expression when the chimeric antigen receptor is activated.
  • the inducing protein is designed as a suicide protein, then the cells activated by the chimeric antigen receptor survive in a culture environment with screening drugs, and the rest of the cells undergo apoptosis, so that the chimeric antigen receptor expressing cells that can bind to the target antigen are screened out.
  • the chimeric antigen receptor response gene circuit is designed to activate and/or enhance the gene circuit that can inducible protein expression when the chimeric antigen receptor is activated.
  • the induced protein is designed as a drug-resistant protein, and then the activated cells survive in a culture environment with screening drugs, and the rest of the cells undergo apoptosis, so that chimeric antigen receptor expressing cells that can bind to the target antigen are screened out.
  • the chimeric antigen receptor response gene circuit is designed to terminate and/or inhibit the gene circuit that can induce protein expression when the chimeric antigen receptor is activated.
  • the protein is designed as a drug-resistant protein, the cells activated by the chimeric antigen receptor apoptotic in a culture environment with screening drugs, and the remaining cells survive, so that the chimeric antigen receptor expressing cells that cannot bind to the target antigen are screened out.
  • the following screening methods are further included: 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 drug for drug resistance protein includes any one of puromycin, G418, blasticidin or hygromycin B; the screening drug for suicide protein includes ganciclovir or FIAU, 5-fluorocytosine , AP1903 or AP20187 any one or a combination of at least two.
  • the fifth aspect of the present invention provides a method for screening chimeric antigen receptor cells against in vivo antigens, which includes the following steps: (1) administering an effective amount of a chimeric antigen receptor cell library to an experimental subject; 2) Implement a screening method based on the expression of the suicide protein; (3) Use general methods to recover and enrich the target chimeric antigen receptor cells from the subject.
  • the method further includes the following steps: (4) From the chimeric antigen receptor cells screened in step (3), reconstruct a secondary chimeric antigen receptor cell library using antibody engineering and/or genetic engineering methods; 5) Repeat steps (1) to (3) to screen the target chimeric antigen receptor cells, and repeat steps (4) to (5) one or more times if necessary.
  • chimeric antigen receptor cells include monoclonal chimeric antigen receptor cells and polyclonal chimeric antigen receptor cell populations.
  • 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 chimeric antigen receptor cell library with the in vivo antigen generally includes administering the chimeric antigen receptor 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 of screening chimeric antigen receptor cells is to administer the chimeric antigen receptor cell library to the target subject, so that the chimeric antigen receptor cell library is in contact with the antigen in the body, and only the chimeric antigen that can recognize the antigen Recipient cells will be based on pre-programmed gene circuits and inducible proteins in the cell: such as activating the expression of inducible proteins in the cells, enhancing the expression of inducible proteins in the cells, terminating the expression of inducible proteins in the cells, and inhibiting the cells. Any one or a combination of at least two of the inducible proteins in the expression.
  • the screening method is selected according to the expression of the inducible protein, and then the target chimeric antigen receptor cells are screened, and the design is flexibly designed according to the gene circuit and the inducible protein to achieve the screening purpose.
  • the specific screening method is the same as above.
  • the sixth aspect of the present invention provides a chimeric antigen receptor cell.
  • the chimeric antigen receptor cells are screened by the methods described in the fourth aspect and the fifth aspect.
  • the chimeric antigen receptor cells include monoclonal chimeric antigen receptor cells and polyclonal chimeric antigen receptor cells.
  • the seventh aspect of the present invention provides a chimeric antigen receptor.
  • the chimeric antigen receptor is obtained by using the chimeric antigen receptor cells described in the sixth aspect to extract related genes 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 of the chimeric antigen receptor described in the seventh aspect.
  • the ninth aspect of the present invention provides a target antigen.
  • the target antigen is a corresponding antigen obtained by the chimeric antigen receptor cell of the sixth aspect, the chimeric antigen receptor of the seventh aspect, and the antibody of the eighth aspect through general genetic engineering technology.
  • the tenth aspect of the present invention provides a pharmaceutical composition.
  • the composition comprises the chimeric antigen receptor cell library of the first aspect, the antigen receptor cell library constructed and obtained in the second aspect, the chimeric antigen receptor cell of the sixth aspect, and the chimeric antigen receptor cell of the seventh aspect.
  • An antigen receptor any one or a combination of at least two of the antibodies described in the eighth aspect, 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, and diseases related to focal cells, such as benign and malignant tumors, tissue hyperplasia, acute and chronic inflammation, etc., need to be removed.
  • 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, testis, pituitary, genitals, liver, gallbladder, eyes, ears, nose, throat, tonsils, mouth, lymph nodes and lymphatic system and other organs. In some embodiments, it also includes benign tumors and acute and chronic inflammations of these organs.
  • 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, testis, pituitary, genitals, liver, gallbladder, eyes, ears, nose, throat, tonsils, mouth, lymph nodes And the lymphatic system and other organs.
  • 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, testis, pituitary, genitalia, 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, testis, pituitary, genitalia, liver,
  • 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, The brain and its covering, spinal cord and its covering, muscle, connective tissue, adrenal gland, parathyroid, thyroid, testis, pituitary, genitals, liver, gallbladder, eyes, ears, nose, throat, tonsils, mouth, lymph nodes and lymph 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 diseases also include cosmetic repair and anti-aging of tissues.
  • 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: 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, uterine cavity and appendages, urine, and spaces , 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, uterine
  • 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 unique and creative features of the invention are: (1) The invention solves the scientific problems of unknown disease antigens, antigen changes, antigen evolution, and antigen heterogeneity.
  • the chimeric antigen receptor library of the present invention is loaded with three gene elements to realize the chimeric antigen receptor-gene circuit-inducible protein cascade reaction.
  • the gene circuit preprogrammed by the second gene element exerts expression regulation on the inducible protein encoded by the third gene element, such as activating transcription expression, enhancing transcription expression, terminating transcription expression, and inhibiting transcription expression.
  • the chimeric antigen receptor cell library of the present invention can achieve programmed changes to antigen changes in vivo, and is particularly suitable for obtaining a cell library for individualized antigens. That is, the present invention proposes a brand-new cell library that can follow changes in disease antigens to realize the preparation of individualized products. However, the CAR library in the prior art cannot respond to variable antigens.
  • the antigen receptor cell library of the present invention has the advantage that it can directly increase the pharmacodynamic index in the screening, so as to achieve the purpose of directly preparing therapeutic products.
  • the present invention also provides a preparation method and a screening method for the chimeric antigen receptor cell library.
  • the essence of the method is the construction of the vector and the method of transfecting cells.
  • the construction technology is mature and easy to master, which helps reduce the construction and screening of the library. Filter costs.
  • the chimeric antigen cell library of the present invention is based on the principle and activation characteristics of the chimeric antigen receptor library, and by designing a chimeric antigen receptor library-gene circuit-inducible protein coupling scheme, it can be targeted at complex and unknown disease targets. Spot antigen cell library and screening method. Cell libraries, construction and preparation methods, and screening methods can solve the problems of complex, diverse, variable antigens and difficulty in target identification in abnormal antigen expression diseases, and have broad application prospects.
  • Figure 1 is a schematic diagram of the structure, preparation method, and in vivo screening of the chimeric antigen receptor cell library according to an embodiment of the present invention.
  • A is the use of existing technology to construct an antibody library;
  • B is the background subtraction of the antibody library;
  • C is the construction of the chimeric antigen receptor cell library;
  • D is the schematic diagram of in vitro screening;
  • E is the schematic diagram of in vivo screening;
  • F is the unrecognized purpose The chimeric antigen receptor cells of the antigen are eliminated;
  • G is the survival of the chimeric antigen receptor cells that recognize the target antigen.
  • Figure 2 is a schematic diagram of a gene control expression cassette.
  • A is a schematic structural diagram of the NFAT-KRAB-iCasp9-2A-GFP gene control expression cassette;
  • B is a schematic structural diagram of the CMV-scFvlab-CAR gene control expression cassette;
  • C is a schematic structural diagram of the CD19-CAR gene control expression cassette.
  • Figure 3 shows the results of tumor volume changes in each group after using the cell library to treat the mouse tumor model.
  • Figure 4 shows the expression of CAR receptors in blood cells of each group of animals after treatment.
  • Figure 5 shows the changes in tumor volume of each group after the screened cells are used to treat the mouse tumor model.
  • Figure 6 shows the tumor tissue inhibition rate of each group after using the cell library to treat the PDX model.
  • Figure 7 shows the pathological scores of each group of inflammatory bowel disease.
  • Figure 8 is a schematic diagram of the construction of gene elements
  • A-F is a schematic diagram of the construction of the first gene element
  • G-K is a schematic diagram of the construction of the second gene element.
  • Fig. 9 is a schematic diagram of vector combination.
  • Figure 10 is a schematic diagram of individualized genetic elements and vectors
  • (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 Controlled Gene Expression Cassettes: 1
  • the gene control expression cassette shown in Figure 2A is named NFAT-KRAB-iCasp9-2A-GFP control expression cassette. It contains the NFAT response element promoter [Uchibori R, et al.
  • IL-2 minimal interleukin 2
  • GAL4-KRAB transcription factor
  • 5 ⁇ UAS-P SV40 promoter [Morsut L,et al.Cell,2016,164(4):780-791.]
  • iCasp9-2A-green fluorescent protein fusion gene under the control.
  • 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.].
  • Lentiviral vector system was used to integrate the NFAT-KRAB-iCasp9-2A-GFP control expression cassette into mouse T lymphocytes.
  • the successfully integrated mouse T lymphocytes were sorted by flow cytometry.
  • the gene control expression cassette shown in Figure 2B is named CMV-scFvlab-CAR.
  • the murine single-chain antibody library was constructed as the extracellular recognition domain of the CAR receptor.
  • the lentiviral vector system was used to integrate the CMV-scFvlab-CAR gene control expression cassette into T cells.
  • the pre-programming of the gene circuit is: when the CAR receptor binds to the antigen, the expression of the iCasp9 gene is inhibited under the change of the gene circuit. At this time, the cell is not regulated by the iCasp9 inducer to induce apoptosis.
  • the obtained chimeric antigen receptor cell library was named KRAB-iCasp9-CAR-T cell library, and the library capacity was 1 ⁇ 10 6 monoclonal numbers.
  • Example 2 Treatment of breast cancer with a fully synthetic mouse-derived chimeric antigen receptor T cell library
  • mice were divided into control group, irrelevant CAR-T cell group, KRAB-iCasp9-CAR-T cell library group 1, KRAB-iCasp9-CAR-T cell library group 2, KRAB -iCasp9-CAR-T cell library 3 groups; KRAB-iCasp9-CAR-T cell library 4 groups.
  • the CAR-positive rate of CAR-T cells is standardized to 45%.
  • the control group was given PBS treatment; the unrelated CAR-T cell group was given CD19-CAR-T cell therapy (the gene control expression cassette is shown in Figure 2C), with a dose of 5 ⁇ 10 6 cells intravenously injected once every 2 days, 3 injections Times; all KRAB-iCasp9-CAR-T cell library groups were treated with KRAB-iCasp9-CAR-T cell library treatment at a dose of 5 ⁇ 10 6 cells intravenously, and the cells were diluted with serum-free 1640 medium every 2 days
  • KRAB-iCasp9-CAR-T cell library group 1 was given cell therapy only;
  • KRAB-iCasp9-CAR-T cell library group 2 was given iCasp9 inducer from the start of treatment, the dosage and method were the same as those in the literature [Liu E,et al.Leukemia,2018,32(2):
  • Example 3 In vivo screening of fully synthetic murine chimeric antigen receptor T cells targeting 4T1 breast cancer
  • Figure 4 shows the ratio of CAR-positive cells obtained after the experiment in all treatment groups. The CAR-positive cells in each group were separated and cultured to complete the in vivo screening.
  • Example 4 In vitro screening of fully synthetic murine chimeric antigen receptor T cells targeting 4T1 breast cancer
  • This example was performed using the KRAB-iCasp9-CAR-T cell library described in Example 1.
  • the KRAB-iCasp9-CAR-T cell library (1 ⁇ 10 7 cells, 70% CAR positive rate) was co-cultured with 1 ⁇ 10 7 4T1 breast cancer cells, and iCasp9 inducer was added to the medium after 96 hours of culture.
  • the measurement and method are the same as those in the literature [Liu E, et al. Leukemia, 2018, 32(2): 520.]. After 5 days, the CAR-positive cells were separated by flow cytometry, and the in vitro screening was completed.
  • the T cell genome was extracted using the kit.
  • Design primers to obtain the chimeric antigen receptor gene by PCR that is, to obtain the chimeric antigen receptor targeting 4T1 breast cancer cells.
  • the single-chain antibody is further obtained by PCR, and the obtained single-chain antibody is constructed as a mouse IgG2a through genetic engineering of the antibody, and then expressed and purified to obtain an antibody targeting 4T1 breast cancer cells.
  • the antibody obtained in Example 5 was cross-linked on agarose beads, and then the 4T1 breast cancer cell lysate was incubated with the beads of the cross-linked antibody overnight. After rinsing, the corresponding antigen bound by the antibody was enriched on the beads. The beads are identified by peptide fingerprint mass spectrometry to obtain the target antigen.
  • Example 7 Therapeutic application of fully synthetic mouse chimeric antigen receptor T cells targeting 4T1 breast cancer
  • the chimeric antigen receptor T cells obtained in each group in Example 3 were again treated with 4T1 mice in situ breast cancer.
  • the treatment method of each group was the same as before, and the iCasp9 inducer was given from the second week of treatment.
  • the result is shown in Figure 5.
  • the 3 groups of KRAB-iCasp9-CAR-T cell library and the 4 groups of KRAB-iCasp9-CAR-T cell library have very obvious anti-tumor effects.
  • phage single-chain antibody library of natural human origin.
  • the method of establishing a 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.].
  • the established phage natural human single-chain antibody library has a library capacity of 1 ⁇ 10 10 .
  • 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 reacquired phage antibody library has no significant changes in the amplification and detection capacity, and then the antibody gene library is obtained by PCR.
  • NK-92 cells K, et al. Molecular therapy, 2015, 23(2):330-338.
  • CAR receptors and intracellular gene circuits according to the same method as in Example 1 to obtain natural whole-human chimera Antigen receptor NK-92 cell library.
  • the obtained natural fully human chimeric antigen receptor NK-92 cell library was named KRAB-iCasp9-CAR-NK92 library, and the library capacity was 1 ⁇ 10 6 monoclonal numbers.
  • Example 9 In vivo screening of natural fully human chimeric antigen 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
  • the literature method (Fu W, et al. Clinical Cancer Research, 2019, 25(9): 2835-2847.] established lung cancer PDX model L10, breast cancer PDX model B7, and ovarian cancer PDX model OV3.
  • group CAR-NK92 cells anti-CD19 CAR-NK92, controlling gene expression cassette comprising in FIG. 2C
  • each group was given iCasp9 inducers.
  • the mice were sacrificed at the third week of treatment, and the blood and tumor tissues of the mice were separated and the CAR-positive cells were detected by flow cytometry.
  • lung cancer PDX model L10 In lung cancer PDX model L10, breast cancer PDX model B7, and ovarian cancer PDX model OV3, CAR positive expression cells were obtained from the KRAB-iCasp9-CAR-NK92 library group. That is, the in vivo screening is completed.
  • This example was performed using the natural fully human chimeric antigen receptor NK-92 cells obtained in the in vivo screening process described in Example 9.
  • NK-92 cells were selected to further prepare a cell library, and the CAR receptor and intracellular gene circuit were constructed according to the same method as in Example 1.
  • the extracellular recognition domain of the CAR receptor was the genetically engineered antibody library.
  • the chimeric antigen receptor NK-92 cell library for lung cancer L10, breast cancer B7, and ovarian cancer OV3 was obtained.
  • the obtained human chimeric antigen receptor NK-92 cell library was named L10-KRAB-iCasp9-CAR-NK92 library, B7-KRAB-iCasp9-CAR-NK92 library, OV3-KRAB-iCasp9-CAR-NK92 library, library
  • the capacity is 1 ⁇ 10 5 single clones.
  • Example 11 Chimeric antigen receptor NK-92 cell library targeting tumor tissue to treat mouse tumor-bearing model
  • mice were divided into the control group, the unrelated CAR-NK92 cell group (anti-CD19 CAR-NK92, including the gene control expression cassette as shown in Figure 2C), the NK-92 cell library group, treatment
  • the dosage and manner are the same as in Example 1.
  • each group was given iCasp9 inducer.
  • ratio 1-average tumor volume in the treatment group/average tumor volume in the control group.
  • Example 12 Construction and preparation of individualized natural fully human chimeric antigen receptor T cell library
  • This example was carried out by using 200 healthy volunteers' peripheral monocytes as described in Example 8 to prepare a phage single-chain antibody library of natural human origin.
  • Peripheral mononuclear cells of lung cancer subjects and lung cancer adjacent tissues obtained by surgery are taken as control cells/tissues, and through three negative screenings, phage that can bind to the above-mentioned control cells/tissues in the phage natural human single-chain antibody library is removed. After the reacquired phage antibody library is amplified, the antibody gene library is obtained by PCR. At this time, there is no obvious change in the detection and expansion.
  • peripheral monocytes from the lung cancer subjects were further separated from T lymphocytes to prepare a cell library, and the CAR receptor and gene circuit were constructed according to the same method as in Example 1.
  • the method of constructing gene circuit and cell library is the same as in Example 1.
  • the two gene control expression cassettes of the gene circuit are shown in Figure 2.
  • the obtained synthetic chimeric antigen receptor cell library was named KRAB-iCasp9-CAR-hT library, and the library capacity was 6 ⁇ 10 5 monoclonal numbers.
  • Example 13 Individualized natural fully human chimeric antigen receptor T cell library for immunotherapy of human lung cancer
  • the aforementioned KRAB-iCasp9-CAR-hT library was intravenously administered to the lung cancer subject described in Example 12.
  • the administration method, dosage, and administration are implemented in accordance with the general administration method for CAR-T cells (Fry T J, et al. Nature medicine, 2018, 24(1): 20.].
  • Example 14 Fully synthetic murine chimeric antigen receptor T cell library for treatment of small animal model of inflammatory bowel disease
  • mice were used to establish trinitrobenzene sulphonic acid (TNBS) to induce colitis (ie inflammatory Enteropathy 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, irrelevant CAR-T cell group, and KRAB-iCasp9-CAR-T cell 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 CAR-T cell group was given CD19-CAR-T cell therapy (the gene control expression cassette is shown in Figure 1C). 5 ⁇ 10 6 cells were injected intravenously, once every 2 days, 3 times. The case score was performed four weeks later, and the method was the same as that in the literature [Tian, Yuhua, et al. Gastroenterology 156.8(2019):2281-2296.]. The results are shown in Figure 7. The results show that the KRAB-iCasp9-CAR-T cell library has a good therapeutic effect.
  • Example 15 Natural fully human chimeric antigen receptor NK-92 cell library for treatment of endometriosis mouse graft model
  • 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 were sacrificed to examine the transplanted endometrium. The results showed that the control group, In the irrelevant CAR-NK92 cell group, each mouse had intimal tissue surviving, and none of the mice in the KRAB-iCasp9-CAR-NK92 library treatment group survived intimal tissue.
  • Example 16 Construction and preparation of natural fully human chimeric antigen 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 8. The method is briefly described as follows: firstly, 200 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 .
  • Peripheral monocytes from subjects with endometriosis were taken to further isolate T lymphocytes to further prepare a cell library, and the CAR receptor and gene circuit were constructed according to the same method as in Example 1.
  • the gene circuit construction is the same as in Example 1.
  • the two gene control expression cassettes of the gene circuit are shown in Figure 2.
  • the obtained natural fully human chimeric antigen receptor T cell library targeting the ectopic inner membrane was named endo-KRAB-iCasp9-CAR-hT library, and the library capacity was 3 ⁇ 10 5 monoclonal numbers.
  • Example 17 Natural fully human chimeric antigen receptor T cell library targeting ectopic endometrium for the treatment of human endometriosis
  • the endo-KRAB-iCasp9-CAR-hT library described above was intravenously administered to the subjects with endometriosis described in Example 16.
  • the administration method, dosage, and administration are carried out in accordance with the general administration method of CAR-T cells.
  • Example 18 Chimeric antigen receptor NK-92 cell library containing 495 artificial antibodies
  • 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, ALAXI, A, and A, B, A, B, and B , ANRUKINZUMAB, APRUTUMAB, ASCRINVACUMAB, ASELIZUMAB, ATIDORTOXUMAB, ATINUMAB, ATOLTIVIMAB, ATOROLIMUMAB, AVELUMAB, AZINTUXIZUMAB, BALSTILIMAB, bAPINEUZUMAB, bASILIXIMAB, BAVITUXIMAB, BECTUMOMAB, BED
  • NK-92 cells were selected to further prepare a cell library, and the CAR receptor and intracellular gene circuit were constructed according to the same method as in Example 1, that is, a chimeric antigen receptor NK-92 cell library containing 495 human antibodies was obtained.
  • the obtained chimeric antigen receptor NK-92 cell library was named 495-KRAB-iCasp9-CAR-NK92 library, and the library capacity was 495 monoclonals.
  • Example 19 Chimeric antigen receptor NK-92 cell library containing 495 artificial antibodies for immunotherapy of human pancreatic cancer
  • the above 495-KRAB-iCasp9-CAR-NK92 library was intravenously administered to subjects with pancreatic cancer.
  • the dosing and frequency of administration are in accordance with the general administration method of cell therapy.
  • 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 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 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 8D;
  • 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 8E;
  • 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 by 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 8G
  • the following method can also be used to construct: 4 NFAT response elements and minimal IL-2 promoter fused to form a (4 ⁇ NFAT) promoter, and its downstream contains Gal4-KRAB; then construct a 5 ⁇ which is regulated by Gal4-KRAB UAS-PS V40 fusion promoter), as shown in Figure 8H.
  • 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 8J.
  • 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; Reconstruct 4 ⁇ ZFHD1RE-P CMV-min promoter regulated by ZFHD1-VP64, as shown in Figure 8K.
  • IL-2 minimal interleukin 2
  • the third gene element is a gene element encoding a protein, and the encoding includes puromycin resistance protein (PuroR), neomycin resistance protein (NeoR), blasticidin resistance protein (Blasticidin-R), and hygromycin B Genes for resistance protein (Hygromycin BR), herpes simplex virus thymidine kinase protein (HSV-TK), cytosine deaminase protein (CD), or iCasp9 suicide system protein (iCasp9).
  • PuroR puromycin resistance protein
  • NeoR neomycin resistance protein
  • Blasticidin-R blasticidin resistance protein
  • Hygromycin BR herpes simplex virus thymidine kinase protein
  • CD cytosine deaminase protein
  • iCasp9 suicide system protein iCasp9 suicide system protein
  • the vector combination containing the three genetic elements can be constructed using the following methods, which are briefly described as follows: (i) According to the human gene antibody coding rules, artificially synthesize a human antibody gene library. Further construct according to the chimeric antigen receptor scheme, namely: human antibody gene single chain antibody library—CD8hinge—CD8TM—4-1BB—CD3 ⁇ , construct the first gene element as shown in Figure 2A; (ii) 6 NFAT The reaction element and the minimal interleukin 2 (IL-2) promoter (6 ⁇ NFAT) were used to construct the second gene element as shown in Figure 2G; (iii) the third gene element (PuroR) encoding the puromycin resistance protein was constructed. ); On a vector as shown in Figure 9A as a whole, there can be other regulatory elements described in the prior art on the vector, such as the CMV promoter.
  • the following methods can also be used to construct: (i) The method of DNA synthesis is used to artificially synthesize a human antibody gene library according to human gene antibody coding rules.
  • 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 following methods can also be used to construct: (i) Obtain an antibody gene library from peripheral monocytes of multiple healthy volunteers by genetic engineering, construct the gene library in a yeast vector, and amplify yeast cells to obtain a 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.
  • Example 21 Treatment of subjects exposed to unknown pathogens
  • Peripheral monocytes from healthy subjects are genetically engineered to obtain antibody gene libraries and then constructed according to the chimeric antigen receptor protocol, such as: individualized single-chain antibody library—CD8 hinge—CD8TM—4-1BB—CD3 ⁇ , Such as 10A.
  • Construction element comprising a second gene 6 ⁇ NFAT, downstream of the Gal4-KRAB transcription factor, and 5 ⁇ UAS-P SV40 regulated by Gal4-KRAB.
  • the third gene element iCasp9 was constructed, and the vector was constructed in the manner shown in Figure 10B.
  • 10A and 10B also show other regulatory elements described in the prior art on the vector, such as CMV promoter, 2A cleavage peptide, green fluorescent protein, etc.
  • the above-mentioned vector is transfected into the subject's T cells to obtain a library of chimeric antigen receptor cells. Freeze storage.
  • the exposure mode is one of many different ways, such as food or water intake, aerosol inhalation, or skin contact.
  • the pathogen is one of many, such as Bacillus anthracis (anthrax), influenza virus, smallpox virus, Yersinia pestis (plague), Ebola virus or Marburg virus, Tula Francis (hare disease), Han Tan virus, dengue virus, cholera toxin, botulinum toxin, ricin, salmonella, Escherichia coli such as E.coli 0157:H7, Shigella, Listeria, etc.
  • the chimeric antigen receptor cell library and composition of the present invention are intravenously administered to the patient, for example, containing 3 ⁇ active ingredients Composition of 109 chimeric antigen receptor cell library. Once the cell library is distributed into body fluids (especially blood), the cell library removes the focus cells and inflammatory mediators.
  • the cell library By removing these disease mediators, the cell library reduces the triggers of additional systemic inflammation in patients and reduces the production of systemic inflammatory mediators (such as cytokines), thereby preventing or limiting cell death and organs induced by cytokines or other inflammatory mediators. Occurrence of injury, multiple organ failure, and potential death.
  • the cell library is administered once or repeatedly in a period of several hours to several days to have a lasting or stable effect.
  • the inducer is administered to the patient to remove the library cells in which the chimeric antigen receptor has not been activated, and then the cell library is recovered from the patient.
  • the inducer is administered to the patient to remove the library cells in which the chimeric antigen receptor has not been activated, and then the cell library is recovered from the patient.
  • genetic engineering methods to obtain the extracellular recognition domain of the chimeric antigen receptor, based on this, further prepare antibodies, chimeric antigen receptors, engineered cells, etc. for the treatment of other patients.
  • Example 22 Administration of Chimeric Antigen Receptor Cell Library to Patients with Inflammatory Bowel Disease
  • a library of chimeric antigen receptor cells from healthy people. At least 100 peripheral monocytes in healthy volunteers were genetically engineered to obtain an antibody gene library. Then establish a phage antibody library, take the patient's peripheral monocytes as a control tissue, and subtract the background. Then use the antibody engineering method to obtain the antibody gene library. Further construct according to the chimeric antigen receptor scheme, such as: healthy volunteer single chain antibody library—IgG4 hinge—CD28TM—CD28—4-1BB—CD3 ⁇ , such as 10C. Construction element comprising a second gene 4 ⁇ NFAT, downstream of the Gal4-KRAB transcription factor Gal4-KRAB, and regulated by 5 ⁇ UAS-P SV40.
  • the third genetic element HSV-TK was constructed, and the vector was constructed in the manner shown in Fig. 10D. Other regulatory elements described in the prior art on the vector are also shown in 10C and 10D.
  • the above-mentioned vector is transfected into the patient's T cells to obtain a library of chimeric antigen receptor cells. Freeze storage.
  • inflammatory bowel disease worsened to persistent severe diarrhea.
  • Standard treatments are administered to patients, which may include systemic steroids and parenteral TNF blocker treatments.
  • an effective dose of the cell library is administered to the patient, for example, a composition containing an active ingredient of 3 ⁇ 10 9 chimeric antigen receptor cell library.
  • the cell library eliminates abnormal cells and neutralizes the inflammatory mediators produced locally (including cytokines produced in the intestine). This promotes the healing of the intestines and prevents recurrence.
  • a composition containing a library of 3 ⁇ 10 9 chimeric antigen receptor cells as an active ingredient is administered to the patient to limit systemic inflammation.
  • the cell library can remove systemic inflammatory mediators that can cause more inflammatory mediators to be produced.
  • Example 24 Administration of cell library to patients with endometritis
  • a composition containing, for example, a library of 3 ⁇ 10 9 chimeric antigen receptor cells as an active ingredient is administered to the patient to limit endometrial inflammation.
  • an inducer is administered to the patient to remove the cell library.
  • Example 25 Anti-aging subject administration of cell library
  • the subject expects to remove senescent cells and achieve the effect of delaying aging.
  • a composition containing a library of 3 ⁇ 10 7 chimeric antigen receptor cells as an active ingredient is administered to the patient to achieve the effect of slowing aging.
  • Test skin and biochemical indicators after treatment After the therapeutic effect is achieved, an inducer is administered to the patient to remove the cell library.
  • an inducer is administered to the patient to remove the cell library.
  • the skin elasticity of the patient increased, and the liver function was significantly enhanced.
  • Example 26 Alzheimer's disease patient administration of cell library
  • a personalized library of chimeric antigen receptor cells is first established.
  • Construct according to the chimeric antigen receptor protocol such as: individualized single chain antibody library—CD8 hinge—CD8TM—4-1BB—CD3 ⁇ , such as 10A.
  • Construction element comprising a second gene 6 ⁇ NFAT, downstream of the Gal4-KRAB transcription factor, and 5 ⁇ UAS-P SV40 regulated by Gal4-KRAB.
  • the third gene element iCasp9 was constructed, and the vector was constructed in the manner shown in Figure 10B.
  • 10A and 10B also show other regulatory elements described in the prior art on the vector, such as CMV promoter, 2A cleavage peptide, green fluorescent protein, etc.
  • the above-mentioned vector is transfected into the subject's T cells by the electrochemical method of Sleeping Beauty vector to obtain a library of chimeric antigen receptor cells. Freeze storage.
  • an effective dose of the cell library for example, a composition containing an active ingredient of 1 ⁇ 10 5 chimeric antigen receptor cell library, is administered to the patient by intradural injection.
  • the cell library eliminates abnormal neurons and reduces neurological symptoms.
  • an inducer is administered to the patient to remove the cell library.
  • Example 27 Administration of cell library to infertile patients
  • the patient was diagnosed with immune infertility, and trying existing methods still cannot solve the problem of immune infertility.
  • a composition containing a library of 3 ⁇ 10 7 chimeric antigen receptor cells as an active ingredient is administered to patients to achieve the effect of removing abnormal immune mediators.
  • the inducer is administered to the patient to remove the cell library.
  • the patient finally became pregnant.
  • Example 28 Administration of cell library to patients with liver fibrosis
  • a patient who suffered from hepatitis B 10 years ago was diagnosed with partial fibrosis of the liver. Many anti-fibrosis treatments have failed, and the disease continues to progress.
  • Obtain the subject's T cells and construct a chimeric antigen receptor cell library after subtracting the subject's background according to the method described in Example 22.
  • a composition containing a library of 3 ⁇ 10 7 chimeric antigen receptor cells as an active ingredient is administered to the patient to remove abnormal immune mediators and diseased cells in the liver.
  • the inducer is administered to the patient to remove the cell library, and the degree of fibrosis of the patient is successfully delayed.
  • Example 29 Administration of cell library to patients with chronic pelvic inflammatory disease
  • a composition containing a library of 3 ⁇ 10 9 chimeric antigen receptor cells as an active ingredient is administered to the patient to limit pelvic inflammation.
  • an inducer is administered to the patient to remove the cell library.
  • the patient has many scars on the skin, and the effect of treatments such as surgery and laser is not significant.
  • a composition containing a library of 1 ⁇ 10 6 chimeric antigen receptor cells as an active ingredient is administered to patients to treat multiple skin scars. After the therapeutic effect is achieved, an inducer is administered to the patient to remove the cell library.
  • Example 31 Treatment of lumbar hyperosteogeny
  • the patient is healthy, and the antibody gene library of his peripheral monocytes is preserved preventively, with a storage capacity of 1 ⁇ 10 6 clones.
  • the patient developed severe lumbar bone hyperplasia, and many existing treatments and traditional Chinese medicine treatments were ineffective.
  • a composition comprising a library of 1 ⁇ 10 6 chimeric antigen receptor cells as an active ingredient is administered to the patient to treat bone hyperplasia. After the therapeutic effect is achieved, an inducer is administered to the patient to remove the cell library.
  • the library was constructed according to the chimeric antigen receptor protocol.
  • 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, 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, EG
  • Construction element comprising a second gene 4 ⁇ NFAT, downstream of the Gal4-KRAB transcription factor Gal4-KRAB, and regulated by 5 ⁇ UAS-P SV40. Construction of the third genetic element HSV-TK.
  • the above vectors are transfected into NK-92 cells to obtain a library of chimeric antigen receptor cells. Freeze storage.
  • Example 33 Examples of in vitro screening methods
  • a variety of methods can be used to establish a library of antigen receptor cells, which are briefly listed as follows: First, a library of chimeric antigen receptor cells from healthy people is established. At least 100 peripheral monocytes in healthy volunteers were genetically engineered to obtain antibody gene libraries, and constructed according to the chimeric antigen receptor protocol, such as: healthy volunteers single-chain antibody library—IgG4 hinge—CD28TM—CD28—4 -1BB-CD3 ⁇ , such as 10C. Construction element comprising a second gene 4 ⁇ NFAT, downstream of the Gal4-KRAB transcription factor Gal4-KRAB, and regulated by 5 ⁇ UAS-P SV40. The third genetic element HSV-TK was constructed, and the vector was constructed in the manner shown in Fig. 10D. Other regulatory elements described in the prior art on the vector are also shown in 10C and 10D. Transfect the above-mentioned vector into Jurkat cells to obtain a library of chimeric antigen receptor cells.
  • the inducer is applied to remove the cells that do not bind the antigen, the jurkat cells are eluted, and the chimeric antigen is obtained by genetic engineering.
  • the receptor gene is analyzed.

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Abstract

提供携带基因元件组合的嵌合抗原受体细胞文库、制备和筛选方法及用途，该嵌合抗原受体细胞文库由细胞和载体组件融合而成，载体组件携带三种基因元件，分别为编码一种或多种独特型嵌合抗原受体的多种第一基因元件；载有一种或多种基因回路的第二基因元件；编码一种或多种可诱导蛋白的第三基因元件。其中，基因回路包括是预编程性的，为调节性顺势作用因子与转录因子的组合；可诱导蛋白包括药物抗性蛋白及自杀蛋白中的一种或两种。通过设计嵌合抗原受体文库-基因回路-可诱导蛋白偶联方案，实现针对复杂、未知疾病靶点抗原的细胞文库和筛选，从而解决异常抗原表达性疾病中抗原复杂、多样、易变和靶点鉴定困难等问题，具有广阔的应用前景。

Description

携带基因元件组合的嵌合抗原受体细胞文库、制备和筛选方法及用途 技术领域
本发明涉及生物医药工程技术和合成生物学领域，具体涉及一种基因元件组合和携带该组合的嵌合抗原受体细胞文库，并对该基因元件组合、细胞文库的制备构建方法、针对体内抗原和/或体外抗原的筛选方法、细胞文库的用途进行了详细说明。
背景技术
利用基因修饰的免疫细胞治疗疾病是目前技术的前沿热点。通过嵌合抗原受体(Chimeric Antigen Receptor，CAR)的表达，向在肿瘤细胞上表达的抗原。CAR是被设计为以人白细胞抗原-非依赖性的方式识别细胞表面抗原的抗原受体。利用表达CAR的基因修饰的T细胞治疗这些类型患者的尝试已经得到一定程度的成功(Molecular Therapy,2010,18:4,666-668；Blood,2008,112:2261-2271)。随着嵌合抗原受体T细胞(Chimeric Antigen Receptor-T cell，CAR-T)技术的不断发展，目前CAR-T主要可划分为三代。第一代CAR-T细胞由胞外结合区-单链抗体(single-chain fragment variable,scFV)、跨膜区(transmembrane region,TM)和胞内信号区-免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activation motif,ITAM)组成，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-CD3ζ。该种CAR-T细胞可以激发抗肿瘤的细胞毒性效应，但是细胞因子分泌比较少，并且在体内不能激发持久的抗肿瘤效应(Zhang T.et.al,Cancer Res2007,67(22):11029-11036.)。
随后发展的第二代CAR-T细胞加入了CD28或CD137(又名4-1BB)的胞内信号区，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-CD28-ITAM或scFv-TM-CD137-ITAM。胞内信号区发生的B7/CD28或4-1BBL/CD137共刺激作用引起T细胞的持续增殖，并能够提高T细胞分泌IL-2和IFN-γ等细胞因子的水平，同时提高CAR-T在体内的存活周期和抗肿瘤效果[Dotti G.et.al.CD28costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphoma patients.J Clin Invest,2011,121(5):1822-1826]。
近些年发展的第三代CAR-T细胞，其中嵌合抗原受体各部分按如下形式连接：scFv-TM-CD28-CD137-ITAM或scFv-TM-CD28-CD134-ITAM，进一步提高了CAR-T在 体内的存活周期和其抗肿瘤效果[Carpenito C.,et al.Control of large established tumor xenografts with genetically retargeted human T cells containing CD28and CD137domains.PNAS,2009,106(9):3360-3365.]。
另外，还有利用嵌合抗原受体基因修饰其他细胞用于治疗的公开技术，如CAR-NK细胞[Choucair K,et al.Future Oncology,2019,15(26):3053-3069.]，CAR NK-92细胞[
Figure PCTCN2020119322-appb-000001
 K,et al.Molecular therapy,2015,23(2):330-338.]，CAR巨噬细胞[Zhang W,et al..British Journal of Cancer,2019:1-9.]等等。
CAR细胞技术具有诱人的前景。但是，这些基因工程细胞到底携带针对哪种抗原的嵌合抗原受体是该技术尚未解决的技术问题。如针对单一抗原的CAR-T细胞可以产生耐药、CAR细胞也不能针对未知抗原等。
但对于以恶性肿瘤为代表的人类重大疾病，其特点在于疾病具有高度的变异性、个体差异、异质性、进化性。即患有相同疾病的患者病灶内部的抗原变化极大、一个患者病灶内部不同部位的抗原也差异极大、随着治疗压力的施用(如放化疗等给与生物增殖的进化压力，使得肿瘤细胞基因修复能力增强、靶向性治疗如CAR-T疗法，抗体疗法等针对特定抗原给与进化压力，使得肿瘤调低相关抗原表达)，病灶细胞进化，无法彻底解除疾病。特异性的肿瘤抗原鉴定困难、多样性广、个体化差异极大，且恶性肿瘤组织可以随着病情发展和治疗手段压力进行生物进化，具有较大的变异度。因此，如果以治疗为目标，针对特定抗原的分子识别物筛选制备的方法大大落后于疾病进程。此外，由于人类基因组的庞大峰度，患有同类型恶性肿瘤的患者病灶的基因背景差异性极大，靶向特定的抗原的治疗策略和治疗制剂没有个体化治疗意义，只能通过标志物和遗传学研究进行循证分析以获取相应的可能有效人群。通过特定的肿瘤组织分析获得的肿瘤抗原，并进一步制备的针对该抗原的CAR细胞治疗方法也没有个体化治疗意义。
关于CAR文库的制备，专利文献WO2015/123642公开了嵌合抗原受体文库的构建制备方法，提出了制备大量CAR形成文库、以及进一步携带CAR文库的细胞文库的方法。该构建方法的公布了如何构建含有多个独特型CAR的细胞文库。但该技术的局限性在于：CAR细胞文库的筛选方向不能跟随抗原的变化而变化，只能针对特定抗原进行筛选。虽然该专利文献虽然描述了利用通用技术进行CAR文库进一步筛选方法，如使用iQue TM筛选器(Intellicyt,Albuquerque,NM)(一种高通量流式细胞仪)进行个别CAR分子的高通量测试，一方面效率低下，另一方面该系统设计的目的是快速获取可用的CAR受体，并非针对个体化治疗提出的，通过例如该文献的这些CAR文库的方法可以获取 针对特定抗原CAR受体，但无法直接靶向未知抗原，更无法作为针对异质性、变化性、进化性特点抗原疾病的治疗产品。
综上可知，本领域迫切需要建立一种克服上述缺陷的、具有个体化属性的嵌合抗原受体细胞文库和制备筛选技术方法，解决疾病抗原异质性、变化性、进化性的特点。有鉴于此，特提出本发明。
发明内容
本发明依托上述研究背景，提供了一种含有新的基因元件的嵌合抗原受体细胞文库，并对该基因元件组合、细胞文库的制备构建方法、针对体内抗原和/或体外抗原的筛选方法、细胞文库的用途进行了详细说明。
本发明的第一方面，提供了一种载体组件。其携带三种基因元件，分别为：(1)编码一种或多种独特型嵌合抗原受体的多个第一基因元件(即嵌合抗原受体文库)；(2)载有一种或多种独特的基因回路的第二基因元件；(3)编码一种或多种独特的可诱导蛋白的第三基因元件。
在本发明的某些优选实施方案中，所述多种独特型嵌合抗原受体为至少三种独特型嵌合抗原受体。嵌合抗原受体包括细胞内信号传导结构域、跨膜结构域和细胞外识别结构域，细胞外识别结构域包括完整的抗体、组成抗体的重链或轻链或抗体片段；基因回路是预编程性的，为调节性顺势作用因子与转录因子的组合；可诱导蛋白(inducible protein)包括药物抗性蛋白以及自杀蛋白中的一种或两种。
本发明中的基因元件可以实现的功能为：当第一基因元件编码的嵌合抗原受体活化时，第二基因元件预编程的基因回路发挥对第三基因元件所编码可诱导蛋白的表达调控。所述表达调控包括激活转录表达、增强转录表达、终止转录表达、抑制转录表达中的任意一种或至少两种的组合。
在本发明中，第一基因元件中的术语“嵌合抗原受体”是免疫治疗学概念，是模仿免疫细胞激活过程构建的人工受体。嵌合抗原受体包含多个免疫受体的部分，目的是设计一种无需任何帮助就能识别抗原(如BCR)，然后直接杀死被识别的细胞(如TCR)的受体。
嵌合抗原受体的结构是本领域的通用技术，包括细胞内信号传导结构域、跨膜结构域和细胞外识别结构域(细胞外识别结构域文库)。跨膜结构域还包含位于细胞外识别结构域和跨膜结构域之间的铰链区，以及位于跨膜结构域和细胞内信号传导结构域之间的 一种或多种另外的共刺激分子。
细胞内信号传导结构域包含CD3ζ；跨膜结构域包括：CD28跨膜结构域、4-1BB跨膜结构域、CD8α跨膜结构域、CD3ζ跨膜结构域中的任意一种；共刺激分子包括：CD28、CD27、OX40、4-1BB中的任意一种或至少两种的组合；细胞外识别结构域的结构包括但不限于完整的抗体、组成抗体的链(重链或轻链)、抗体的片段(抗体可变区、单链抗体、单域抗体、Fab段)所构成的文库，优选单链抗体(ScFv)文库。
本发明中嵌合抗原受体的结构以及嵌合抗原受体文库的构建方法可以有多种构建组合，并可以均构建在本发明的元件内，以实现随机化，参见专利文献WO2015/123642。
胞外识别结构域文库包括健康人来源的单链抗体文库、人工合成来源的单链抗体文库、羊驼来源的单域抗体文库或健康人来源的单链抗体文库扣除受试者外周单核细胞表达抗原后形成的亚文库。在本发明的一些具体的实施例中，胞外抗体文库还包括健康人来源的单链抗体文库扣除受试者外周单核细胞表达抗原、癌旁抗原后形成的亚文库。
相应的，嵌合抗原受体文库的来源包括但不限于来自动物、来自免疫动物制备、来自疾病人群、来自健康人群、接种过疫苗的人群、人工合成、基因工程制备中的任意一种或至少两种的组合，还包括上述来源的文库经过现有技术预处理后的亚文库，如非专利文献[尹长城,等.中国生物工程杂志,2008,28(12):82-88.]中所述的去除背景克隆后形成的亚文库。
胞外识别结构域文库可以利用抗体工程方法进一步优化，该抗体工程方法包括抗体亲和力成熟技术、抗体人源化技术、抗体动物源化技术、多功能抗体技术、多特异性抗体技术中的任意一种或至少两种的组合。其中，亲和力成熟技术包括热点定点突变、热点随机突变、CDR突变、链交换、依据三维结构的抗体突变中的任意一种或至少两种的组合。
优选的，胞外识别结构域针对的靶点包括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、LI13Rα2、CD171、ROR2、AXL、Kappa、CS1、FAP、IL-1RAP、MG7、PSMA、CD5、ROR1、CD70、HER3、Gp75、磷脂酰丝氨酸、cMyc、CD4、CD44v6、CD45、CD28、CD3、CD3e、CD52、CD74、CD30、CD166、CD24、EGFR/HER3融合物、碳水化合物、曲霉、Dectin、埃博拉、真菌、GP、HERV-K、VEGF-R2、TGF-b2R、IgG4、生物素、O-AcGD2、Cadherin 2、OB-cadherin、 α5β1integrin、αVβ6integrin、Syndecan-1、Cadherin 1、Claudin 12、Claudin 7、Claudin 3、ZO-1中的任意一种或至少两种的组合。
优选的，胞外识别结构域文库来源自如下单克隆抗体ABAGOVOMAB、ABCIXIMAB、ABELACIMAB、ABITUZUMAB、ABREZEKIMAB、ABRILUMAB、ACTOXUMAB、ADALIMUMAB、ADECATUMUMAB、ADUCANUMAB、AFASEVIKUMAB、AFELIMOMAB、ALACIZUMAB、ALEMTUZUMAB、ALIROCUMAB、AMATUXIMAB、ANATUMOMAB、ANDECALIXIMAB、ANETUMAB、ANIFROLUMAB、ANRUKINZUMAB、APRUTUMAB、ASCRINVACUMAB、ASELIZUMAB、ATIDORTOXUMAB、ATINUMAB、ATOLTIVIMAB、ATOROLIMUMAB、AVELUMAB、AZINTUXIZUMAB、BALSTILIMAB、BAPINEUZUMAB、BASILIXIMAB、BAVITUXIMAB、BECTUMOMAB、BEDINVETMAB、BEGELOMAB、BELANTAMAB、BELIMUMAB、BEMARITUZUMAB、BERLIMATOXUMAB、BERSANLIMAB、BERTILIMUMAB、BESILESOMAB、BEVACIZUMAB、BIMAGRUMAB、BIMEKIZUMAB、BIRTAMIMAB、BIVATUZUMAB、BLESELUMAB、BLINATUMOMAB、BLONTUVETMAB、BLOSOZUMAB、BOCOCIZUMAB、BRAZIKUMAB、BRIAKINUMAB、BROLUCIZUMAB、BRONTICTUZUMAB、BUDIGALIMAB、BUROSUMAB、CABIRALIZUMAB、CAMIDANLUMAB、CAMRELIZUMAB、CANAKINUMAB、CANTUZUMAB、CAPLACIZUMAB、CAPROMAB、CARLUMAB、CAROTUXIMAB、CATUMAXOMAB、CEDELIZUMAB、CEMIPLIMAB、CENDAKIMAB、CERGUTUZUMAB、CERTOLIZUMAB、CETRELIMAB、CETUXIMAB、CIBISATAMAB、CINPANEMAB、CITATUZUMAB、CIXUTUMUMAB、CLAZAKIZUMAB、CLENOLIXIMAB、CLIVATUZUMAB、COBOLIMAB、CODRITUZUMAB、COFETUZUMAB、COLTUXIMAB、CONATUMUMAB、CONCIZUMAB、COSFROVIXIMAB、CRENEZUMAB、CRIZANLIZUMAB、CROTEDUMAB、CROVALIMAB、CUSATUZUMAB、DACETUZUMAB、DACLIZUMAB、DALOTUZUMAB、DAPIROLIZUMAB、DECTREKUMAB、DEMCIZUMAB、DENINTUZUMAB、DENOSUMAB、DEPATUXIZUMAB、DETUMOMAB、DEZAMIZUMAB、DILPACIMAB、DINUTUXIMAB、DIRIDAVUMAB、DISITAMAB、DOMAGROZUMAB、DONANEMAB、DORLIMOMAB、DOSTARLIMAB、DROZITUMAB、DULIGOTUZUMAB、DUPILUMAB、 DUSIGITUMAB、DUVORTUXIZUMAB、ECROMEXIMAB、EDOBACOMAB、EDRECOLOMAB、EFALIZUMAB、EFUNGUMAB、ELDELUMAB、ELEZANUMAB、ELGEMTUMAB、ELIPOVIMAB、ELSILIMOMAB、EMACTUZUMAB、EMIBETUZUMAB、EMICIZUMAB、ENAPOTAMAB、ENAVATUZUMAB、ENFORTUMAB、ENLIMOMAB、ENOBLITUZUMAB、ENOKIZUMAB、ENOTICUMAB、ENSITUXIMAB、ENVAFOLIMAB、EPITUMOMAB、EPTINEZUMAB、ERLIZUMAB、ERTUMAXOMAB、ETIGILIMAB、ETOKIMAB、ETROLIZUMAB、EVINACUMAB、EXBIVIRUMAB、FARALIMOMAB、FARICIMAB、FARLETUZUMAB、FASINUMAB、FELVIZUMAB、FEZAKINUMAB、FICLATUZUMAB、FIGITUMUMAB、FIRIVUMAB、FLANVOTUMAB、FLETIKUMAB、FLOTETUZUMAB、FONTOLIZUMAB、FORALUMAB、FORAVIRUMAB、FRESOLIMUMAB、FROVOCIMAB、FRUNEVETMAB、FULRANUMAB、FUTUXIMAB、GALIXIMAB、GANCOTAMAB、GANITUMAB、GANTENERUMAB、GARADACIMAB、GARETOSMAB、GAVILIMOMAB、GEDIVUMAB、GEMTUZUMAB、GEVOKIZUMAB、GILVETMAB、GIMSILUMAB、GIRENTUXIMAB、GLEMBATUMUMAB、GLENZOCIMAB、GOLIMUMAB、GOSURANEMAB、IANALUMAB、IBRITUMOMAB、ICRUCUMAB、IDARUCIZUMAB、IERAMILIMAB、IFABOTUZUMAB、IGOVOMAB、ILADATUZUMAB、IMALUMAB、IMAPRELIMAB、IMCIROMAB、IMGATUZUMAB、INCLACUMAB、INDATUXIMAB、INDUSATUMAB、INEBILIZUMAB、INFLIXIMAB、INOLIMOMAB、INOTUZUMAB、INTETUMUMAB、APAMISTAMAB、DERLOTUXIMAB、IPILIMUMAB、IRATUMUMAB、ISATUXIMAB、ISCALIMAB、ISTIRATUMAB、IXEKIZUMAB、KELIXIMAB、LABETUZUMAB、LACNOTUZUMAB、LACUTAMAB、LADIRATUZUMAB、LAMPALIZUMAB、LANADELUMAB、LANDOGROZUMAB、LAPRITUXIMAB、LARCAVIXIMAB、LEBRIKIZUMAB、LEMALESOMAB、LENVERVIMAB、LENZILUMAB、LERDELIMUMAB、LERONLIMAB、LESOFAVUMAB、LETOLIZUMAB、LEVILIMAB、LEXATUMUMAB、LIBIVIRUMAB、LIFASTUZUMAB、LIGELIZUMAB、LILOTOMAB、LINTUZUMAB、LIRILUMAB、LODELCIZUMAB、LONCASTUXIMAB、LORVOTUZUMAB、LOSATUXIZUMAB、LUCATUMUMAB、LULIZUMAB、LUMILIXIMAB、LUMRETUZUMAB、LUPARTUMAB、LUTIKIZUMAB、MAFTIVIMAB、 MAGROLIMAB、MAPATUMUMAB、MARGETUXIMAB、MARSTACIMAB、MASLIMOMAB、MATUZUMAB、MAVRILIMUMAB、MEPOLIZUMAB、METELIMUMAB、MILATUZUMAB、MINRETUMOMAB、MIRIKIZUMAB、MIRVETUXIMAB、MITAZALIMAB、MITUMOMAB、MODOTUXIMAB、MOGAMULIZUMAB、MONALIZUMAB、MOROLIMUMAB、MOSUNETUZUMAB、MOTAVIZUMAB、MURLENTAMAB、NACOLOMAB、NAMILUMAB、NAPTUMOMAB、NARATUXIMAB、NARNATUMAB、NATALIZUMAB、NAVICIXIZUMAB、NAVIVUMAB、NAXITAMAB、NEBACUMAB、NEMOLIZUMAB、NERELIMOMAB、NESVACUMAB、NETAKIMAB、NIDANILIMAB、NIMACIMAB、NIMOTUZUMAB、NIRSEVIMAB、NIVOLUMAB、OBEXELIMAB、OBILTOXAXIMAB、OBINUTUZUMAB、OCARATUZUMAB、ODULIMOMAB、OFATUMUMAB、OLECLUMAB、OLENDALIZUMAB、OLINVACIMAB、OLOKIZUMAB、OMALIZUMAB、OMBURTAMAB、ONARTUZUMAB、ONTAMALIMAB、ONTUXIZUMAB、ONVATILIMAB、OPICINUMAB、OREGOVOMAB、ORILANOLIMAB、ORTICUMAB、OSOCIMAB、OTELIXIZUMAB、OTILIMAB、OTLERTUZUMAB、OXELUMAB、OZANEZUMAB、OZORALIZUMAB、PAGIBAXIMAB、PALIVIZUMAB、PAMREVLUMAB、PANITUMUMAB、PANOBACUMAB、PARSATUZUMAB、PASCOLIZUMAB、PASOTUXIZUMAB、PATECLIZUMAB、PATRITUMAB、PEMBROLIZUMAB、PEPINEMAB、PERAKIZUMAB、PERTUZUMAB、PEXELIZUMAB、PIDILIZUMAB、PINATUZUMAB、PLACULUMAB、PLAMOTAMAB、PLOZALIZUMAB、POLATUZUMAB、PONEZUMAB、PORGAVIXIMAB、POZELIMAB、PRASINEZUMAB、PREZALUMAB、PRILIXIMAB、PRITOXAXIMAB、PRITUMUMAB、PROLGOLIMAB、QUETMOLIMAB、QUILIZUMAB、RACOTUMOMAB、RADRETUMAB、RAFIVIRUMAB、RALPANCIZUMAB、RANEVETMAB、RANIBIZUMAB、RAVAGALIMAB、RAXIBACUMAB、REFANEZUMAB、REGAVIRUMAB、RELATLIMAB、RELFOVETMAB、REMTOLUMAB、RESLIZUMAB、RILOTUMUMAB、RINUCUMAB、RITUXIMAB、RIVABAZUMAB、ROBATUMUMAB、ROLINSATAMAB、ROMILKIMAB、RONTALIZUMAB、ROSMANTUZUMAB、ROVALPITUZUMAB、ROZANOLIXIZUMAB、SACITUZUMAB、SAMALIZUMAB、SAMROTAMAB、SARILUMAB、SATRALIZUMAB、SATUMOMAB、SECUKINUMAB、SELICRELUMAB、 SEMORINEMAB、SERCLUTAMAB、SERIBANTUMAB、SETOXAXIMAB、SETRUSUMAB、SIBROTUZUMAB、SIFALIMUMAB、SIMTUZUMAB、SINTILIMAB、SIRTRATUMAB、SIRUKUMAB、SOFITUZUMAB、SOLANEZUMAB、SOLITOMAB、SONTUZUMAB、SPARTALIZUMAB、SPESOLIMAB、STAMULUMAB、SULESOMAB、SUPTAVUMAB、SUTIMLIMAB、SUVIZUMAB、SUVRATOXUMAB、TABALUMAB、TABITUXIMAB、TADOCIZUMAB、TAFASITAMAB、TALACOTUZUMAB、TALIZUMAB、TAMRINTAMAB、TAMTUVETMAB、TANEZUMAB、TAPLITUMOMAB、TAREXTUMAB、TAVOLIMAB、FANOLESOMAB、NOFETUMOMAB、PINTUMOMAB、TECLISTAMAB、TEFIBAZUMAB、TELIMOMAB、TELISOTUZUMAB、TEMELIMAB、TENATUMOMAB、TENELIXIMAB、TEPLIZUMAB、TEPODITAMAB、TEPROTUMUMAB、TESIDOLUMAB、TEZEPELUMAB、TIBULIZUMAB、TIDUTAMAB、TIGATUZUMAB、TILAVONEMAB、TILDRAKIZUMAB、TIMOLUMAB、TIRAGOLUMAB、TISLELIZUMAB、TISOTUMAB、TOCILIZUMAB、TOMARALIMAB、TORALIZUMAB、TORIPALIMAB、TOSATOXUMAB、TOSITUMOMAB、TOVETUMAB、TRALOKINUMAB、TRASTUZUMAB、TREGALIZUMAB、TREMELIMUMAB、TREVOGRUMAB、TUCOTUZUMAB、TUVIRUMAB、UBLITUXIMAB、ULOCUPLUMAB、URELUMAB、URTOXAZUMAB、USTEKINUMAB、UTOMILUMAB、VADASTUXIMAB、VANDORTUZUMAB、VANTICTUMAB、VANUCIZUMAB、VAPALIXIMAB、VARISACUMAB、VARLILUMAB、VATELIZUMAB、VELTUZUMAB、VEPALIMOMAB、VESENCUMAB、VIBECOTAMAB、VISILIZUMAB、VOBARILIZUMAB、VOFATAMAB、VOLAGIDEMAB、VOLOCIXIMAB、VONLEROLIZUMAB、VOPRATELIMAB、VORSETUZUMAB、VOTUMUMAB、VUNAKIZUMAB、XENTUZUMAB、ZALIFRELIMAB、ZAMPILIMAB、ZANOLIMUMAB、ZENOCUTUZUMAB、ZIRALIMUMAB、ZOLBETUXIMAB、ZOLIMOMAB。
在本发明中，术语“独特型”是抗体工程学专业概念。抗体作为机体分子识别的最重要效应分子，具有不均一性的特点。不均一性包括同种型、同种异型和独特型。其中，独特型(idiotype)的概念是指每一个抗体形成细胞克隆所产生的抗体分子上所有的抗原特异性，是由轻链或重链可变区氨基酸序列的不同所决定的，因此与抗体结合抗原的特异性密切相关。独特型强调的是抗体结合抗原的特性区别。由于嵌合抗原受体识别抗原 的特性基础即为其内部的抗体结构，所以嵌合抗原受体也具有“独特型”。
在本发明中，蛋白质结构域包括抗原结合结构域、铰链结构域、跨膜结构域、和膜内结构域(endodomain)。术语“独特的”意味着具有不同的多肽(氨基酸)序列、包含不同的多肽(氨基酸)序列或由不同的多肽(氨基酸)序列组成的结构域。例如，两个“独特的”抗原结合结构域可以结合相同抗原(实际上，甚至所述抗原上的相同表位)；然而，抗原结合结构域在它们的连续氨基酸组成彼此不同的情况下是“独特的”。同样地，在连续的氨基酸组成方面不同的两种“独特的”抗原结合结构域也可以特异性结合不同抗原和表位。
编码域、结构域或基因包含编码蛋白质的氨基酸序列、编码蛋白的DNA序列或编码蛋白的RNA序列中的任意一种或至少两种的组合。
第二基因元件中的术语“基因回路”是合成生物学概念，是预编程性的。广义上讲，基因回路内包含由调节性的顺式作用因子和转录因子。调节性的顺式作用因子包括启动子等，如T7启动子、CMV启动子、UPS启动子、Tet启动子等；
可以根据研究目的灵活地设计的复杂调控网络，如同电路网络一般，故称为“基因回路”或“基因电路”。
在本发明中，基因回路包括调节性的顺式作用因子和/或转录因子组成。
调节性的顺式作用因子包括单个顺势作用因子或融合式顺势作用因子，转录因子包括单个转录因子或组合式转录因子。
单个顺势作用因子包括一个或多个NFAT反应启动子元件(NFAT-responsive promoter element，NFAT)、一个或多个NFκB反应启动子元件(NFκB-responsive promoter element，NFκB)、一个或多个四环素反应元件(tetracycline responsive element,TRE)、一个或多个半乳糖代谢酶系(GAL)基因启动子的UAS(upstream activating sequence，UAS)、一个或多个PIP反应元件(PIP responsive element，PIR)、一个或多个ZFHD1反应元件(ZFHD1 responsive element，ZFHD1RE)、一个或多个ZF21-16反应元件(ZF21-16 responsive element，ZF21-16RE)、一个或多个ZF42-10反应元件(responsive element，ZF42-10RE)、一个或多个ZF43-8反应元件(responsive element，ZF43-8RE)、一个或多个ZF54-8反应元件(responsive element，ZF54-8RE)、一个或多个最小化CMV启动子(minimal CMV promoter,P CMV-min)、一个或多个CMV启动子(CMV promoter，P CMV)、一个或多个SV40启动子(SV40 promoter，P SV40)、一个或多个最小化IL-2启动子(minimal IL-2 promoter，P IL-2min)、一个或多个最小化昆虫热休克蛋白70启动子 (minimal insect heat shock 70 promoter，P hsp70min)、一个或多个最小化HIVtata启动子(minimal HIVtata promoter，P HIVtatamin)中的任意一种或至少两种的组合。
融合式顺势作用因子包括一种或多种单个顺势作用因子的灵活组合，如4个NFAT反应元件和最小化IL-2启动子融合而成(4×NFAT)、6个NFAT反应元件和最小化IL-2启动子融合而成(6×NFAT)、其包括5个NFκB结合原件和最小化HIVtata启动子融合而成(5×NFκB)、10个NFκB结合原件和最小化HIVtata启动子融合而成(10×NFκB)、7个TRE和最小化CMV启动子融合而成(7×TRE-P CMV-min)、5个UAS和最小化CMV启动子融合而成(5×UAS-P CMV-min)、4个PIR和最小化CMV启动子融合而成(4×PIR-P CMV-min)、8个PIR和最小化CMV启动子融合而成(8×PIR-P CMV-min)、8个PIR和昆虫热休克蛋白70启动子融合而成(8×PIR-P hsp70min)、4个ZFHD1RE和最小化CMV启动子融合而成(4×ZFHD1RE-P CMV-min)、8个ZF21-16RE和最小化CMV启动子融合而成(8×ZF21-16RE-P CMV-min)、8个ZF42-10RE和最小化CMV启动子融合而成(8×ZF42-10RE-P CMV-min)、8个ZF43-8RE和最小化CMV启动子融合而成(8×ZZF43-8R-P CMV-min)、8个ZF54-8RE和最小化CMV启动子融合而成(8×ZF54-8RE-P CMV-min)、7个TRE和SV40启动子融合而成(7×TRE-P SV40)、7个TRE和CMV启动子融合而成(7×TRE-P cmv)、5个UAS和SV40启动子融合而成(5×UAS-P SV40)、4个PIR和SV40启动子融合而成(4×PIR-P SV40)、8个PIR和SV40启动子融合而成(8×PIR-P SV40)、4个ZFHD1RE和SV40启动子融合而成(4×ZFHD1RE-P SV40)、8个ZF21-16RE和SV40启动子融合而成(8×ZF21-16RE-P SV40)、8个ZF42-10RE和SV40启动子融合而成(8×ZF42-10RE-P SV40)、8个ZF43-8RE和SV40启动子融合而成(8×ZZF43-8RE-P SV40)、8个ZF54-8RE和SV40启动子融合而成(8×ZF54-8RE-P SV40)中的任意一种或至少两种的组合。
组合式转录因子包括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、ZFHD1-KRAB中的任意一种或至少两种的组合。优选包括TetR-VP64(tTA)、Gal4-VP64、Gal4-KRAB、TetR-KRAB中的任意一种或至少两种的组合。
在本发明的一些具体的实施例中，所述基因回路优选包括(i)Gal4-KRAB和5×UAS-P SV40的组合、Gal4-VP64和5×UAS-P CMV-min的组合、TetR-VP64和7×TRE-P CMV-min的组合、TetR-KRAB和7×TRE-P SV40的组合或TetR-KRAB和7×TRE-P cmv的组合中的任意一 种和(ii)4×NFAT、6×NFAT、5×NFκB、10×NFκB中的任意一种组合而成。
在本发明中，如提到基因回路是“独特的”意味着包含不同的设计编程方案组成的基因回路。例如，两个“独特的”基因回路可以实现完全一样的生物学效应(如控制某个下游基因表达上调，或控制某个下游基因表达下调)，然而，基因回路的多个内部顺式作用因子、多个调节基因(可以为转录因子)设计构建的形式彼此不同的情况下是“独特的”。相反，如本文中使用，相同基因回路内部顺式作用因子、多个调节基因(可以为转录因子)设计构建方案相同，仅仅在同源序列的范畴的突变并不影响顺式作用因子和调节基因的功能的变化不是“独特的”基因回路。基因回路的构建方法可以这些公开发表的文献：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.Molecular and Cellular Biology,1995,15(4):1907-1914.；杨子杰,等.生物工程学报,2018,34(12):1886–1894.；Fussenegger M,et al..Nature Biotechnology,2000,18(11):1203-1208；Pomerantz J,Sharp P,Pabo C.Science,1995,267(5194):93-96.；朱凯川,等.中国生物工程杂志,2011,31(1):81-85.Gene Transfer and Expression in Mammalian Cells.S.C.Makrides ELSEVIER 2003，不再赘述。
第三基因元件中的术语“可诱导蛋白”指该蛋白表达时，可以通过诱导方法使得携带该基因的细胞死亡，包括药物抗性蛋白和/或自杀蛋白。药物抗性蛋白指编码一种抵抗某种药物蛋白的基因，是分子生物学和基因工程中的常用技术；自杀蛋白是近年来新兴技术，多用基因工程方法编码某种前体药物水解酶或诱导凋亡信号，以实现载有自杀蛋白的细胞在药物诱导下死亡。
优选地，药物抗性蛋白包括嘌呤霉素抗性蛋白、新霉素抗性蛋白、杀稻瘟素抗性蛋白或潮霉素B抗性蛋白中的任意一种或至少两种的组合；自杀蛋白包括单纯疱疹病毒胸苷激酶蛋白、胞嘧啶脱氨酶蛋白或iCasp9自杀系统蛋白中的任意一种或至少两种的组合。
可诱导蛋白的诱导方法包括药物诱导、化学诱导、物理诱导、激光诱导、热诱导中的任意一种或至少两种的组合。
在本发明的基因元件组合中，多种第一基因元件编码多种独特型嵌合抗原受体，多种第二基因元件构建为多种独特的基因回路，并且多种第三元件编码多个独特的可诱导蛋白。在一些实施方案中，在另外一些实施方案中，多种第一基因元件编码多种独特型嵌合抗原受体，多种第二基因元件构建为一种独特的基因回路，并且多种第三元件编码 多个独特的可诱导蛋白；在其他实施方案中，多种第一基因元件编码多种独特型嵌合抗原受体，多种第二基因元件构建为多种独特的基因回路，并且多种第三元件编码一种独特的可诱导蛋白。
因此，本发明涉及基因元件组合文库，是包含不同基因元件组合的文库，其就独特型嵌合抗原受体、基因回路、可诱导蛋白而言是随机化的，其中嵌合抗原受体的结构可以就独特的抗原结合结构域(即抗体独特型)、铰链结构域和/或膜内结构域而言也是随机化的，如专利文献WO2015/123642。
当然，所述文库也可以就三种基因元件而言随机化组合，如文库就独特型嵌合抗原受体、基因回路和可诱导蛋白而言随机化；也可以就独特型嵌合抗原受体和基因回路而言随机化；也可以就独特型嵌合抗原受体和可诱导蛋白而言随机化；也可以就独特的基因回路和可诱导蛋白而言随机化。
本发明的第二方面，提供了嵌合抗原受体细胞文库的制备方法。
简言之，将第一元件、第二元件、第三元件插入同一载体或不同载体，转染至细胞内，即得到嵌合抗原受体细胞文库。
载体是本领域技术人员所熟悉的技术，如参考非专利文献中的病毒载体[Morsut L,et al.Cell,2016,164(4):780-791.]和非病毒载体[Athanasopoulos T,et al..Hematology/Oncology Clinics of North America,2017,31(5):753-770.]，还可以将基因元件插入特定位点如AAVS1位点的载体(Parthiban K,et al.mAbs.Taylor&Francis,2019.)。
转染的方法包括病毒转染、化学转染试剂转染或电击转染中的任意一种或至少两种的组合。
细胞是哺乳动物细胞，优选为免疫细胞，包括免疫细胞和/或基因工程化的免疫细胞。免疫细胞的来源包括自体免疫细胞、供体免疫细胞、健康志愿者的免疫细胞的任意一种或至少两种的组合。更优选为T淋巴细胞，特别优选NK细胞，如NK-92细胞。
具体的构建步骤如下：
A、抗体展示库制备及筛选
利用健康志愿者来源的或全合成方法建立抗体基因文库，还可以利用基因工程方法建立功能性的基因文库。举例的基因工程方法如在健康志愿者来源的或全合成方法基础上选择合适的展示平台建立抗体展示库，以对照组织作为对照，经过多轮淘选扣除背景， 获取噬菌体抗体展示亚库，然后以PCR方法再获取抗体基因文库。
B、基因元件构建
构建第一基因元件，其结构包含单链抗体库-CAR，所述单链抗体库被构建为CAR的胞外识别结构域。构建第二基因元件，包含第一种调节性顺势作用因子、以及受该调节性顺势作用因子调控的转录因子、受转录因子调控的第二种顺势作用因子。构建第三元件，包含可诱导蛋白基因。将上述三个基因元件构建到一种或多种基因控制表达盒中。
C、基因元件导入细胞
采用慢病毒载体系统将上述基因控制表达盒导入哺乳动物免疫细胞内，得到嵌合抗原受体细胞文库。
本发明的第三方面，提供一种嵌合抗原受体细胞文库。
该细胞文库携带第一方面所述的基因元件组合并通过第二方面的制备方法获取的。
本发明的第四方面，提供一种筛选针对体外抗原的嵌合抗原受体细胞的方法，包括如下步骤：
(1)将嵌合抗原受体细胞文库与抗原接触；(2)根据自杀蛋白表达的情况，实施筛选；(3)回收表达目的嵌合抗原受体细胞。
优选的，还包括以下步骤：
(4)从步骤(3)中筛选获得的嵌合抗原受体细胞中，利用抗体工程方法重新构建次级嵌合抗原受体细胞文库；(5)重复步骤(1)-(3)，筛选目标嵌合抗原受体细胞，必要时一次或多次重复步骤(4)-(5)。
其中，嵌合抗原受体细胞包括单克隆的嵌合抗原受体细胞和多克隆的嵌合抗原受体细胞群。
抗原包括野生型细胞、转染特定抗原基因的细胞、结合特定抗原的细胞、溶解在培养基中的抗原、包被在培养器皿上的抗原、包被在微珠上的抗原或包被在培养支架上的抗原中的任意一种或至少两种的组合。
本发明中，筛选嵌合抗原受体细胞的方法是将嵌合抗原受体细胞文库与体外抗原接触，只有能识别抗原的嵌合抗原受体细胞，才会根据细胞内预编程的基因回路和可诱导蛋白发生：如激活细胞中可诱导蛋白的表达、增强细胞中可诱导蛋白的表达、终止细胞中可诱导蛋白的表达、抑制细胞中可诱导蛋白的表达中的任意一种或至少两种的组合。根据可诱导蛋白的表达情况选择筛选方法，然后筛选出目标合抗原受体细胞，根据基因 回路和可诱导蛋白灵活进行设计，达到筛选目的。
在本发明的一些具体的实施例中，采取以下筛选方法：将嵌合抗原受体反应基因回路设计为当嵌合抗原受体活化时激活和/或增强可诱导蛋白表达的基因回路，将可诱导蛋白设计为自杀蛋白，则嵌合抗原受体活化的细胞在有筛选药物的培养环境中凋亡，其余的细胞存活，从而筛选出不能结合目标抗原的嵌合抗原受体表达细胞。
在本发明的一些具体的实施例中，采取以下筛选方法：将嵌合抗原受体反应基因回路设计为当嵌合抗原受体活化时终止和/或抑制可诱导蛋白表达的基因回路，将可诱导蛋白设计为自杀蛋白，则嵌合抗原受体活化的细胞在有筛选药物的培养环境中存活，其余的细胞凋亡，从而筛选出能结合目标抗原的嵌合抗原受体表达细胞。
在本发明的一些具体的实施例中，采取以下筛选方法：将嵌合抗原受体反应基因回路设计为当嵌合抗原受体活化时激活和/或增强可诱导蛋白表达的基因回路，将可诱导蛋白设计为药物抗性蛋白，则活化的细胞在有筛选药物的培养环境中存活，其余的细胞凋亡，从而筛选出能结合目标抗原的嵌合抗原受体表达细胞。
在本发明的一些具体的实施例中，采取以下筛选方法：将嵌合抗原受体反应基因回路设计为当嵌合抗原受体活化时终止和/或抑制可诱导蛋白表达的基因回路，可诱导蛋白设计为药物抗性蛋白，则嵌合抗原受体活化的细胞在有筛选药物的培养环境中凋亡，其余的细胞存活，从而筛选出不能结合目标抗原的嵌合抗原受体表达细胞。
在本发明的一些具体的实施例中，还包括以下筛选方法：药物筛选、流式细胞仪检测并分选、磁珠分选、beads分选中的任意一种或至少两种的组合。
优选地，药物抗性蛋白的筛选药物包括嘌呤霉素、G418、杀稻瘟素或潮霉素B中的任意一种；自杀蛋白的筛选药物包括更昔洛韦或FIAU、5-氟胞嘧啶、AP1903或AP20187中的任意一种或至少两种的组合。
本发明的第五方面，提供了一种筛选针对体内抗原的嵌合抗原受体细胞的方法，包括如下步骤：(1)给实验目的受试者施用有效量嵌合抗原受体细胞文库；(2)根据自杀蛋白表达的情况，实施筛选方法；(3)从受试者体内利用通用方法回收富集获得目的嵌合抗原受体细胞。
优选的，还包括以下步骤：(4)从步骤(3)中筛选获得的嵌合抗原受体细胞中，利用抗体工程和/或基因工程方法重新构建次级嵌合抗原受体细胞文库；(5)重复步骤(1)～(3)，筛选目标嵌合抗原受体细胞，必要时一次或多次重复步骤(4)-(5)。
其中，嵌合抗原受体细胞包括单克隆的嵌合抗原受体细胞和多克隆的嵌合抗原受体细胞群。
体内抗原指存在于人及动物活体内的抗原，包括在体细胞、在体病灶细胞、转染特定抗原基因的在体细胞、感染特定病原体的在体细胞、结合特定抗原的在体细胞、移植在动物模型上的在体细胞中的任意一种或至少两种的组合。
将嵌合抗原受体细胞文库与体内抗原接触的方法一般包括向体内施用所述的嵌合抗原受体细胞文库。施用的方法包括静脉输入、经胃肠道输入、肌肉注射、组织局部注射、皮下注射、腹腔注射、吸入中的任意一种或至少两种的组合。
本发明中，筛选嵌合抗原受体细胞的方法是将嵌合抗原受体细胞文库施用于目的受试者，使得嵌合抗原受体细胞文库与体内抗原接触，只有能识别抗原的嵌合抗原受体细胞，才会根据细胞内预编程的基因回路和可诱导蛋白发生：如激活细胞中可诱导蛋白的表达、增强细胞中可诱导蛋白的表达、终止细胞中可诱导蛋白的表达、抑制细胞中可诱导蛋白的表达中的任意一种或至少两种的组合。根据可诱导蛋白的表达情况选择筛选方法，然后筛选出目标嵌合抗原受体细胞，根据基因回路和可诱导蛋白灵活进行设计，达到筛选目的，具体筛选方法同上。
本发明的第六方面，提供了一种嵌合抗原受体细胞。该嵌合抗原受体细胞由第四方面以及第五方面所述的方法筛选得到，该嵌合抗原受体细胞包括单克隆的嵌合抗原受体细胞和多克隆的嵌合抗原受体细胞。
本发明的第七方面，提供一种嵌合抗原受体。该嵌合抗原受体是通过第六方面所述的嵌合抗原受体细胞利用通用基因工程技术提取相关基因获得的。
本发明的第八方面，提供了一种抗体。所述抗体是通过第七方面所述的嵌合抗原受体通用的抗体工程技术获得的。
本发明的第九方面，提供了靶点抗原。该靶点抗原是通过上述第六方面所述的嵌合抗原受体细胞、第七方面所述嵌合抗原受体、第八方面所述的抗体通过通用基因工程技术获得的对应抗原。
本发明的第十方面，提供了一种药物组合物。该组合物包含上述的第一方面所述嵌合抗原受体细胞文库、第二方面构建获得的抗原受体细胞文库、第六方面所述嵌合抗原受体细胞、第七方面所述嵌合抗原受体、第八方面所述的抗体中的任意一种或至 少两种的组合，和至少一种医学/药学上可接受的载体。此外，该药物组合物还包括医学或药学用途的稀释剂或赋形剂。
本发明的第十一方面，提供了上述药物组合物在包括预防、诊断和治疗需要去除疾病介质相关疾病药物、试剂、试剂盒用途中的任意一种或至少两种的组合。
在一些方面，所述疾病介质指病灶细胞，需要去除病灶细胞相关疾病如良恶性肿瘤、组织增生、急慢性炎症等疾病。疾病可以是例如肺、乳房、胃、胰、前列腺、膀胱、骨、卵巢及附件、子宫、皮肤、肾、窦、结肠、直肠、食道、血液、大脑和其覆盖物、脊髓和其覆盖物、肌肉、结缔组织、肾上腺、副甲状腺、甲状腺、睾丸、垂体、生殖器、肝脏、胆囊、眼、耳、鼻、喉、扁桃腺、口、淋巴结和淋巴系统及其它器官的恶性肿瘤。在某些实施方式中，还包括这些器官的良性肿瘤和急慢性炎症。
在本发明中，术语“恶性肿瘤”包括人类癌、肉瘤和黑色素瘤的所有形式，它们以低度分化、适度分化、高度分化的形式出现。
所述疾病还包括组织增生、肥大、异位或过度增长，组织包括肺、乳房、胃、胰、前列腺、膀胱、骨、卵巢及附件、子宫、皮肤、肾、窦、结肠、直肠、食道、血液、大脑和其覆盖物、脊髓和其覆盖物、肌肉、结缔组织、肾上腺、副甲状腺、甲状腺、睾丸、垂体、生殖器、肝脏、胆囊、眼、耳、鼻、喉、扁桃腺、口、淋巴结和淋巴系统及其它器官。
所述疾病还包括病毒、细菌或寄生虫改变的组织，组织包括肺、乳房、胃、胰、前列腺、膀胱、骨、卵巢及附件、子宫、皮肤、肾、窦、结肠、直肠、食道、血液、大脑和其覆盖物、脊髓和其覆盖物、肌肉、结缔组织、肾上腺、副甲状腺、甲状腺、睾丸、垂体、生殖器、肝脏、胆囊、眼、耳、鼻、喉、扁桃腺、口、淋巴结和淋巴系统及其它器官。
所述疾病包括急慢性炎症引起的组织纤维化改变，组织包括肺、乳房、胃、胰、前列腺、膀胱、骨、卵巢及附件、子宫、皮肤、肾、窦、结肠、直肠、食道、血液、大脑和其覆盖物、脊髓和其覆盖物、肌肉、结缔组织、肾上腺、副甲状腺、甲状腺、睾丸、垂体、生殖器、肝脏、胆囊、眼、耳、鼻、喉、扁桃腺、口、淋巴结和淋巴系统及其它器官。
所述疾病还包括子宫内膜异位症、扁桃体肥大、前列腺增生、银屑病、湿疹、皮肤病、痔、血管病如动脉粥样硬化或动脉硬化。或者是血管病如静脉曲张、动脉或支架的狭窄或重狭窄。
所述疾病还包括组织的整容修复、抗衰老等。如皮肤、眼、耳、鼻、喉、口、肌肉、结缔组织、毛发或乳房组织的整容修复。
所述疾病还包括神经性衰退疾病，如阿尔兹海默症、帕金森症等。
在一些方面，所述疾病介质指炎性介质。
在一些方法中，所述炎性介质包括：病毒、细菌或寄生虫等病原体、酶、细胞因子、前列腺素(prostaglandins)、类花生酸(eicosanoids)、自三烯类(Leukotrienes)、激肽类(kinins)、补体、凝血因子、毒素、内毒素、肠毒素、脂多糖、诱导细胞凋亡的物质、腐蚀性物质、胆汁盐、脂肪酸、磷脂、氧化副产物、活性氧簇、氧自由基、表面活性剂、离子、刺激性物质、细胞碎片、干扰素、以及免疫调节性抗体、生物制品(biologics)、药物中的任一种或至少两种的组合。在一些方面，所述炎性介质存在于受试者的生理性流体或载体流体中，所述生理性流体包括以下的流体:生理性流体包括以下的流体:鼻咽、口腔、食道、胃、胰腺、肝、胸膜、心包、腹膜、肠、前列腺、精液、阴道分泌物、眼泪、唾液、粘液、胆汁、血液、淋巴、血浆、血清、滑液、脑脊液、宫腔及附件、尿，以及间隙、细胞内和细胞外的流体。
所述炎性介质相关性疾病包括：全身性炎性应答综合征(SIRS)或脓毒症(例如源自病毒、细菌、真菌或寄生虫感染)、自身免疫病、外科手术、细胞毒性化疗、骨髓操作、大的组织损伤或外伤、肠系膜灌注不足、肠粘膜损伤、疟疾、胃肠道炎性疾病、肠道感染、宫腔感染、流行性感冒、急性肺炎如急性呼吸窘迫综合症或急性肺损伤、肺栓塞、胰腺炎、自身免疫和胶原血管病、输血相关疾病、烧伤、烟或吸入肺损伤、移植物抗宿主病、缺血或梗死、再灌注损伤、出血、过敏反应、药物过量、辐射损伤或化学损伤。在一些实施方案中，炎性介质由生物战的病原体、毒素或制剂导致的疾病产生，例如病毒性出血热、水母毒素等海洋毒素、登革热、埃博拉、汉坦病毒心肺综合征(汉坦病毒)、霍乱毒素、肉毒杆菌毒素、草麻毒素、Q热[博纳特氏立克次氏体(Coxiella burnetii)]、斑痊伤寒症(Rickettsia prowaszekii)或鹦鹉热[鹦鹉热衣原体(Chlamydia psittaci)]。
所述炎性介质相关性疾病还包括：接受移植，免疫不孕等需要去除目的免疫因素的疾病。
相比现有技术，本发明的技术效果如下：
本发明的独特性创造特征是：(1)本发明解决的疾病抗原未知、抗原变化、抗原进化、抗原异质性的科学问题。本发明中的嵌合抗原受体文库装载有三个基因元件，实现 了嵌合抗原受体-基因回路-可诱导蛋白的级联反应，当第一基因元件编码的嵌合抗原受体活化时，第二基因元件预编程的基因回路对第三基因元件所编码可诱导蛋白发挥表达调控，如激活转录表达、增强转录表达、终止转录表达、抑制转录表达等。由于基因回路具有预编程功能，使得本发明的嵌合抗原受体细胞文库可以在体内实现针对抗原变化的编程性变化，尤其适合获取针对个体化抗原的细胞文库。即，本发明提出了一种全新的可以跟随疾病抗原变化而变化的细胞文库，实现个体化产品的制备。而现有技术中的CAR文库无法针对变化性的抗原做出反应。(2)本发明的抗原受体细胞文库优势还在于在筛选中可以直接增加药效学指标，达到直接制备治疗性产品的用途。
此外，本发明还提供了嵌合抗原受体细胞文库的制备方法以及筛选方法，其本质为载体的构建及转染细胞的方法，其构建技术成熟，易掌握，有助于降低文库的构建和筛选成本。
因此，本发明的嵌合抗原细胞文库基于嵌合抗原受体文库原理和的活化特点，通过设计嵌合抗原受体文库-基因回路-可诱导蛋白偶联方案，从而实现针对复杂、未知疾病靶点抗原的细胞文库和筛选方法。细胞文库、构建制备方法、筛选方法可以解决异常抗原表达性疾病中抗原复杂、多样、易变和靶点鉴定困难等问题，具有广阔的应用前景。
附图说明
图1为本发明实施例所述嵌合抗原受体细胞文库的结构、制备方法、体内筛选示意图。其中，A为利用现有技术构建抗体库；B为对抗体库扣除背景；C为嵌合抗原受体细胞文库的构建；D为体外筛选示意图；E为体内筛选示意图；F为未能识别目的抗原的嵌合抗原受体细胞清除；G为识别目的抗原的嵌合抗原受体细胞存活。
图2为基因控制表达盒示意图。A为NFAT-KRAB-iCasp9-2A-GFP基因控制表达盒的结构示意图；B为CMV-scFvlab-CAR基因控制表达盒的结构示意图；C为CD19-CAR基因控制表达盒的结构示意图。
图3为利用细胞文库治疗小鼠肿瘤模型后各组肿瘤体积变化结果。
图4为治疗后各组动物血细胞CAR受体表达情况。
图5为利用筛选后的细胞治疗小鼠肿瘤模型后各组肿瘤体积变化。
图6为利用细胞文库治疗PDX模型后各组肿瘤组织抑制率。
图7为炎症性肠病各组病理评分。
图8为基因元件构建示意图，A-F为第一基因元件构建示意图，G-K为第二基因元 件构建示意图。
图9为载体组合示意图。
图10为个体化基因元件和载体示意图
具体实施方式
以下实施例、实验例对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法的详细描述，如常用的抗体工程方法、那些用于构建载体和质拉的方法，将编码蛋白的基因插入到这样的载体和质拉的方法或将质粒引入宿主细胞的方法、合成细胞、装置、基因回路的构建方法等。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括董志伟，王琰主编《抗体工程》第二版，北京医科大学出版社，2002；Sambrook,J.,Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning:A Laboratory Manual,2nd edition,Cold spring Harbor Laboratory Press；Phage Display:A laboratory Manual,Cold spring Harbor Laboratory Press。
实施例1.全合成鼠源嵌合抗原受体T细胞文库
文库的构建及筛选流程见图1：
(A)噬菌体抗体库构建：首先利用全合成方法建立全合成鼠源噬菌体单链抗体库。抗体库制备的方法是本领域普通技术的人员是众所周知，建立全合成鼠源噬菌体单链抗体库的方法同文献[Geuijen C et al..European Journal of Cancer,2005,41(1):178-187；Noronha E J,et al.Journal of Immunology,1998,161(6):2968-2976.]。经过库容量评估，所建立的全合成鼠源噬菌体单链抗体库库容量为1×10 9。库容量评估的方法同文献[Ridgway J B B,et al.Cancer Research,2013,59(11):2718-2723]。
(B)背景剔除：将全合成鼠源噬菌体单链抗体库(1×10 11PFU)从尾静脉注射BALB/c小鼠，经过4轮筛选，去除能与小鼠组织结合的所有噬菌体，方法同文献[Wada,Akinori,et al.Molecular Therapy-Oncolytics 12(2019):138-146.]，此时获取噬菌体抗体亚库。亚库扩增检测库容无明显变化。然后以PCR方法获取抗体基因文库。
(C)嵌合抗原受体细胞文库的构建
按照文献报道的方法[Srivastava S,et al..Cancer cell,2019,35(3):489-503.e8.]获取小鼠T淋巴细胞。然后构建基因回路(图2)。该基因回路包含两个基因控制表达盒(Controlled Gene Expression Cassette)：①如图2A所示的基因控制表达盒，命名为NFAT-KRAB-iCasp9-2A-GFP控制表达盒。其包含NFAT反应元件启动子[Uchibori R,et al. Molecular Therapy-Oncolytics,2019,12:16-25.]包含6个NFAT反应原件和最小化白介素2(IL-2)启动子，控制抑制性转录因子GAL4-KRAB[Morsut L,et al.Cell,2016,164(4):780-791.]的表达。5×UAS-P SV40启动子[Morsut L,et al.Cell,2016,164(4):780-791.]控制下的iCasp9-2A-绿色荧光蛋白融合基因。iCasp9基因、自切割肽2A的构建方法同文献[Liu E,et al.Leukemia,2018,32(2):520.]。利用慢病毒载体系统将NFAT-KRAB-iCasp9-2A-GFP控制表达盒整合小鼠T淋巴细胞内。利用流式细胞术分选成功整合的小鼠T淋巴细胞。②如图2B所示的基因控制表达盒，命名为CMV-scFvlab-CAR。将上述鼠源单链抗体库构建为CAR受体的胞外识别结构域。利用文献报道的哺乳动物细胞基因整合技术[Parthiban K,et al.mAbs.Taylor&Francis,2019.]，利用慢病毒载体系统将CMV-scFvlab-CAR基因控制表达盒整合入T细胞。
该基因回路的预编程是：当CAR受体结合抗原时，在基因回路变成下抑制iCasp9基因的表达，此时该细胞不受iCasp9诱导剂的诱导凋亡调控。
获得的嵌合抗原受体细胞文库命名为KRAB-iCasp9-CAR-T细胞文库，库容量为1×10 6单克隆数。
(D)体外筛选：若要进行体外筛选，直接将细胞文库与靶抗原直接孵育；
(E)若要进行体内筛选，将适量以及适当浓度的细胞文库溶液施用于受试者；
(F)给予自杀基因iCasp9诱导剂，依据预编程基因回路，检测iCasp9基因的抑制表达情况，筛选出受iCasp9诱导剂的诱导凋亡调控的细胞，并将其作为未能识别目的抗原的嵌合抗原受体细胞予以清除。
(G)根据体内iCasp9基因的抑制表达情况，筛选出不受iCasp9诱导剂的诱导凋亡调控的细胞，并进行回收。
实施例2.全合成鼠源嵌合抗原受体T细胞文库治疗乳腺癌
利用实施例1中所述KRAB-iCasp9-CAR-T细胞文库进行本实施例。
建立4T1小鼠原位乳腺癌模型：模型的建方法同文献[Paschall A V,Liu K.JoVE 2016(114):e54040.]。当小鼠肿瘤体积平均达100mm 3时将小鼠分为对照组，无关CAR-T细胞组，KRAB-iCasp9-CAR-T细胞文库1组，KRAB-iCasp9-CAR-T细胞文库2组，KRAB-iCasp9-CAR-T细胞文库3组；KRAB-iCasp9-CAR-T细胞文库4组。
CAR-T细胞的CAR阳性率标准化为45％。其中对照组给予PBS治疗；无关CAR-T细胞组给予CD19-CAR-T细胞治疗(基因控制表达盒如图2C)，剂量为5×10 6个细胞静 脉注射，每2天注射一次，注射3次；所有KRAB-iCasp9-CAR-T细胞文库组均给予KRAB-iCasp9-CAR-T细胞文库治疗，剂量为5×10 6个细胞静脉注射，使用无血清的1640培养基稀释细胞，每2天注射一次，注射3次：KRAB-iCasp9-CAR-T细胞文库1组仅进行细胞治疗；KRAB-iCasp9-CAR-T细胞文库2组从治疗开始即给予iCasp9诱导剂，剂量及方式同文献[Liu E,et al.Leukemia,2018,32(2):520.]；KRAB-iCasp9-CAR-T细胞文库3组从治疗第2周开始给予iCasp9诱导剂，量及方式同上；KRAB-iCasp9-CAR-T细胞文库4组从治疗第2周开始给予iCasp9诱导剂，但当肿瘤消退后立即停用iCasp9诱导剂，该组随即停止实验。
结果如图3所示，显示对照组、无关CAR-T细胞组肿瘤组织体积快速增长；KRAB-iCasp9-CAR-T细胞文库1组、KRAB-iCasp9-CAR-T细胞文库3组、KRAB-iCasp9-CAR-T细胞文库4组在治疗第二周开始显著抑制肿瘤生长，并迅速清除肿瘤；治疗开始即给予iCasp9诱导剂的KRAB-iCasp9-CAR-T细胞文库2组无肿瘤抑制效果。
实施例3.体内筛选靶向4T1乳腺癌的全合成鼠源嵌合抗原受体T细胞
将实施例2中的所有实验组在实验结束后处死小鼠，分离小鼠血液并用流式细胞术检测其中的CAR阳性细胞。所有处理组实验结束后获取的CAR阳性表达细胞比率如图4显示。将各组的CAR阳性细胞分离培养，即完成体内筛选。
实施例4.体外筛选靶向4T1乳腺癌的全合成鼠源嵌合抗原受体T细胞
利用实施例1中所述KRAB-iCasp9-CAR-T细胞文库进行本实施例。将KRAB-iCasp9-CAR-T细胞文库(1×10 7个细胞，CAR阳性率70％)与1×10 7个4T1乳腺癌细胞共培养，培养96小时后向培养基加入iCasp9诱导剂。计量和方式同文献[Liu E,et al.Leukemia,2018,32(2):520.]，5天后用流式细胞分离CAR阳性表达细胞，即完成体外筛选。
实施例5.靶向4T1乳腺癌细胞嵌合抗原受体、抗体的发现制备
利用实施例3和实施例4所获得的筛选完成的嵌合抗原受体T细胞，利用试剂盒提取T细胞基因组。设计引物利用PCR获取其中的嵌合抗原受体基因，即获取靶向4T1乳腺癌细胞的嵌合抗原受体。进一步利用PCR获取单链抗体，通过基因工程抗体，将获取的单链抗体构建为鼠IgG2a，然后进行表达和纯化即获得靶向4T1乳腺癌细胞的抗体。
实施例6. 4T1乳腺癌细胞靶点抗原鉴定
将实施例5中获取得抗体交联在琼脂糖beads上，然后将4T1乳腺癌细胞裂解物与交联抗体的beads孵育过夜，经过漂洗后此时抗体对应结合的抗原富集在beads上。将beads进行肽指纹质谱鉴定，即获得靶点抗原。
实施例7.靶向4T1乳腺癌的全合成鼠源嵌合抗原受体T细胞治疗应用
将实施例3中各组获取的嵌合抗原受体T细胞再次进行4T1小鼠原位乳腺癌治疗，各组治疗方式同前，从治疗第2周开始给予iCasp9诱导剂。结果如图5显示。KRAB-iCasp9-CAR-T细胞文库3组、KRAB-iCasp9-CAR-T细胞文库4组具有非常明显的抗瘤效果。
实施例8.天然全人嵌合抗原受体NK-92细胞文库
首先制备利用200名健康志愿者外周单核细胞制备天然人来源的噬菌体单链抗体库。建立噬菌体单链抗体库的方法同文献[Geuijen C et al..European Journal of Cancer,2005,41(1):178-187；Noronha E J,et al.Journal of Immunology,1998,161(6):2968-2976.]。经过库容量评估，所建立的噬菌体天然人单链抗体库库容量为1×10 10。
将噬菌体天然人单链抗体库(1×10 12PFU)从尾静脉注射NSG小鼠，经过4轮筛选，去除能与小鼠组织结合的所有噬菌体。重新获取的噬菌体抗体库扩增检测库容无明显变化，然后以PCR方法获取抗体基因文库。
选择NK-92细胞(
Figure PCTCN2020119322-appb-000002
K,et al.Mol ecular therapy,2015,23(2):330-338.]进一步制备细胞文库，根据实施例1相同的方法构建CAR受体和细胞内基因回路，即获得天然全人嵌合抗原受体NK-92细胞文库。获得的天然全人嵌合抗原受体NK-92细胞文库命名为KRAB-iCasp9-CAR-NK92文库，库容量为1×10 6单克隆数。
实施例9.体内筛选天然全人嵌合抗原受体NK-92细胞文库
利用实施例8所述KRAB-iCasp9-CAR-NK92文库进行本实施例。
利用病人来源的组织直接建立NSG小鼠荷瘤模型，即PDX。按照文献方法(Fu W,et al.Clinical Cancer Research,2019,25(9):2835-2847.]建立肺癌PDX模型L10，乳腺癌PDX模型B7，卵巢癌PDX模型OV3。当小鼠肿瘤体积平均达400mm 3时将小鼠分为对照组，无关CAR-NK92细胞组(anti-CD19 CAR-NK92，包含基因控制表达盒如图2C)，KRAB-iCasp9-CAR-NK92文库组。治疗剂量和方式同实施例1。于治疗第2周各组均给 予iCasp9诱导剂。治疗第三周处死小鼠，分离小鼠血液和肿瘤组织并用流式细胞术检测其中的CAR阳性细胞。
在肺癌PDX模型L10，乳腺癌PDX模型B7，卵巢癌PDX模型OV3中，从KRAB-iCasp9-CAR-NK92文库组获取CAR阳性表达细胞。即完成体内筛选。
实施例10.靶向肿瘤组织的嵌合抗原受体NK-92细胞文库构建
利用实施例9所述体内筛选过程中获得的天然全人嵌合抗原受体NK-92细胞进行本实施例。
首先利用通用基因工程技术从实施例9所述体内筛选过程中获得的天然全人嵌合抗原受体NK-92细胞扩增获得针对肺癌L10、乳腺癌B7、卵巢癌OV3的嵌合抗原受体。而后再利用PCR获取嵌合抗原受体的胞外单链抗体序列。根据胞外单链抗体序列计算机辅助设计重链CDR3的随机突变、并人工合成重新建立单链抗体库，即获得针对肿瘤组织的基因工程化抗体库。
选择NK-92细胞进一步制备细胞文库，根据实施例1相同的方法构建CAR受体和细胞内基因回路，CAR受体的胞外识别结构域为所述基因工程化的抗体库。即获得针对肺癌L10、乳腺癌B7、卵巢癌OV3的嵌合抗原受体NK-92细胞文库。获得的人嵌合抗原受体NK-92细胞文库分别命名为L10-KRAB-iCasp9-CAR-NK92文库、B7-KRAB-iCasp9-CAR-NK92文库，OV3-KRAB-iCasp9-CAR-NK92文库，库容量均为1×10 5单克隆数。
实施例11.靶向肿瘤组织的嵌合抗原受体NK-92细胞文库治疗小鼠荷瘤模型
利用实施例10所述的L10-KRAB-iCasp9-CAR-NK92文库、B7-KRAB-iCasp9-CAR-NK92文库，OV3-KRAB-iCasp9-CAR-NK92文库进行本实施例。
建立肺癌PDX模型L10，乳腺癌PDX模型B7，卵巢癌PDX模型OV3。当小鼠肿瘤体积平均达100mm 3时将小鼠分为对照组，无关CAR-NK92细胞组(anti-CD19 CAR-NK92，包含基因控制表达盒如图2C)，NK-92细胞文库组，治疗剂量和方式同实施例1。
于治疗第2周各组均给予iCasp9诱导剂。5周治疗后测量肿瘤生长抑制比率，计算方法为：比值＝1-治疗组肿瘤平均体积/对照组肿瘤平均体积。结果如图6所示，显示靶向肿瘤组织的嵌合抗原受体NK-92细胞文库在各个模型上均具有非常强的抗肿瘤抑制效果。
实施例12.个体化天然全人嵌合抗原受体T细胞文库构建制备
利用实施例8中所述利用200名健康志愿者外周单核细胞制备天然人来源的噬菌体单链抗体库进行本实施例。
取肺癌受试者的外周单核细胞及手术获得的肺癌癌旁组织作为对照细胞/组织，通过三次阴性筛选，去除噬菌体天然人单链抗体库中能与上述对照细胞/组织结合的噬菌体。重新获取的噬菌体抗体库扩增后以PCR方法获取抗体基因文库。此时检测扩容无明显变化。
取上述肺癌受试者的外周单核细胞进一步分离T淋巴细胞进一步制备细胞文库，根据实施例1相同的方法构建CAR受体和基因回路。基因回路和细胞文库的构建方法同实施例1。基因回路的两个基因控制表达盒如图2所示。
获得的合成嵌合抗原受体细胞文库命名为KRAB-iCasp9-CAR-hT文库，库容量为6×10 5单克隆数。
实施例13.个体化天然全人嵌合抗原受体T细胞文库用于人肺癌免疫治疗
将上述KRAB-iCasp9-CAR-hT文库静脉施用于实施例12所述的肺癌受试者。施用方法、剂量、和给药按照CAR-T细胞通用的给药方法(Fry T J,et al.Nature medicine,2018,24(1):20.]实施。
实施例14.全合成鼠源嵌合抗原受体T细胞文库治疗炎症性肠病小动物模型
利用实施例1中所述KRAB-iCasp9-CAR-T细胞文库进行本实施例。
按照文献方法[Tian,Yuhua,et al.Gastroenterology 156.8(2019):2281-2296.]，利用BALB/c小鼠建立三硝基苯磺酸(trinitrobenzene sulphonic acid，TNBS)诱导结肠炎(即炎症性肠病模型)。模型制备和评估同文献[He C,et al..Gut,2015.]。将模型鼠分为为对照组，模型组，无关CAR-T细胞组，KRAB-iCasp9-CAR-T细胞文库组。其中对照组不给予TNBS对照，其余各组在给予小剂量TNBS的同时给予治疗：给予PBS治疗，无关CAR-T细胞组给予CD19-CAR-T细胞治疗(基因控制表达盒如图1C)，剂量为5×10 6个细胞静脉注射，每2天注射一次，注射3次。四周后进行病例评分，方法同文献[Tian,Yuhua,et al.Gastroenterology 156.8(2019):2281-2296.]。结果如图7，结果显示KRAB-iCasp9-CAR-T细胞文库具有良好的治疗效果。
实施例15.天然全人嵌合抗原受体NK-92细胞文库治疗子宫内膜异位症小鼠移植物 模型
利用实施例8所述KRAB-iCasp9-CAR-NK92文库进行本实施例。
按照文献中的建模方法(Masuda H,et al.N Proceedings of the National Academy of Sciences,2007,104(6):1925-1930.]，将子宫内膜异位症受试者的异位子宫内膜移植至NOG小鼠体内。模型成功后将小鼠分为对照组，无关CAR-NK92细胞组[anti-CD19CAR-NK92，包含基因控制表达盒如图2C]，KRAB-iCasp9-CAR-NK92文库组。各组n＝10。治疗剂量和方式同实施例1。于治疗第2周各组均给予iCasp9诱导剂。5周治疗后处死小鼠检查移植的内膜。结果显示：对照组，无关CAR-NK92细胞组每只小鼠体内均有内膜组织存活，KRAB-iCasp9-CAR-NK92文库治疗组小鼠体内均无内膜组织存活。
实施例16.靶向异位内膜的天然全人嵌合抗原受体T细胞文库构建制备
如实施例8中所述方法建立天然人来源的噬菌体单链抗体库，方法简述如下：首先制备利用200名健康志愿者外周单核细胞制备天然人来源的噬菌体单链抗体库。经过库容量评估，所建立的噬菌体天然人单链抗体库库容量为1×10 10。
取子宫内膜异位症受试者的外周单核细胞及手术获得的原位子宫内膜组织作为对照细胞/组织，通过三次阴性筛选，去除噬菌体天然人单链抗体库中能与上述对照细胞/组织结合的噬菌体。重新获取的噬菌体抗体库扩增后以通用基因工程方法获取抗体基因文库。此时检测扩容无明显变化。
取上述子宫内膜异位症受试者外周单核细胞进一步分离T淋巴细胞作进一步制备细胞文库，根据实施例1相同的方法构建CAR受体和基因回路。基因回路构建同实施例1。基因回路的两个基因控制表达盒如图2所示。获得的靶向异位内膜的天然全人嵌合抗原受体T细胞文库命名为endo-KRAB-iCasp9-CAR-hT文库，库容量为3×10 5单克隆数。
实施例17.靶向异位内膜的天然全人嵌合抗原受体T细胞文库用于人子宫内膜异位症治疗
将上述endo-KRAB-iCasp9-CAR-hT文库静脉施用于实施例16所述子宫内膜异位症受试者。施用方法、剂量、和给药按照CAR-T细胞通用的给药方法实施。
实施例18.包含495个人工抗体的嵌合抗原受体NK-92细胞文库
首先利用全合成方法建立单链抗体库。抗体库来源于以下495个单克隆抗体：ABAGOVOMAB、ABCIXIMAB、ABELACIMAB、ABITUZUMAB、ABREZEKIMAB、 ABRILUMAB、ACTOXUMAB、ADALIMUMAB、ADECATUMUMAB、ADUCANUMAB、AFASEVIKUMAB、AFELIMOMAB、ALACIZUMAB、ALEMTUZUMAB、ALIROCUMAB、AMATUXIMAB、ANATUMOMAB、ANDECALIXIMAB、ANETUMAB、ANIFROLUMAB、ANRUKINZUMAB、APRUTUMAB、ASCRINVACUMAB、ASELIZUMAB、ATIDORTOXUMAB、ATINUMAB、ATOLTIVIMAB、ATOROLIMUMAB、AVELUMAB、AZINTUXIZUMAB、BALSTILIMAB、BAPINEUZUMAB、BASILIXIMAB、BAVITUXIMAB、BECTUMOMAB、BEDINVETMAB、BEGELOMAB、BELANTAMAB、BELIMUMAB、BEMARITUZUMAB、BERLIMATOXUMAB、BERSANLIMAB、BERTILIMUMAB、BESILESOMAB、BEVACIZUMAB、BIMAGRUMAB、BIMEKIZUMAB、BIRTAMIMAB、BIVATUZUMAB、BLESELUMAB、BLINATUMOMAB、BLONTUVETMAB、BLOSOZUMAB、BOCOCIZUMAB、BRAZIKUMAB、BRIAKINUMAB、BROLUCIZUMAB、BRONTICTUZUMAB、BUDIGALIMAB、BUROSUMAB、CABIRALIZUMAB、CAMIDANLUMAB、CAMRELIZUMAB、CANAKINUMAB、CANTUZUMAB、CAPLACIZUMAB、CAPROMAB、CARLUMAB、CAROTUXIMAB、CATUMAXOMAB、CEDELIZUMAB、CEMIPLIMAB、CENDAKIMAB、CERGUTUZUMAB、CERTOLIZUMAB、CETRELIMAB、CETUXIMAB、CIBISATAMAB、CINPANEMAB、CITATUZUMAB、CIXUTUMUMAB、CLAZAKIZUMAB、CLENOLIXIMAB、CLIVATUZUMAB、COBOLIMAB、CODRITUZUMAB、COFETUZUMAB、COLTUXIMAB、CONATUMUMAB、CONCIZUMAB、COSFROVIXIMAB、CRENEZUMAB、CRIZANLIZUMAB、CROTEDUMAB、CROVALIMAB、CUSATUZUMAB、DACETUZUMAB、DACLIZUMAB、DALOTUZUMAB、DAPIROLIZUMAB、DECTREKUMAB、DEMCIZUMAB、DENINTUZUMAB、DENOSUMAB、DEPATUXIZUMAB、DETUMOMAB、DEZAMIZUMAB、DILPACIMAB、DINUTUXIMAB、DIRIDAVUMAB、DISITAMAB、DOMAGROZUMAB、DONANEMAB、DORLIMOMAB、DOSTARLIMAB、DROZITUMAB、DULIGOTUZUMAB、DUPILUMAB、DUSIGITUMAB、DUVORTUXIZUMAB、ECROMEXIMAB、EDOBACOMAB、EDRECOLOMAB、EFALIZUMAB、EFUNGUMAB、ELDELUMAB、ELEZANUMAB、ELGEMTUMAB、ELIPOVIMAB、ELSILIMOMAB、EMACTUZUMAB、EMIBETUZUMAB、EMICIZUMAB、ENAPOTAMAB、ENAVATUZUMAB、 ENFORTUMAB、ENLIMOMAB、ENOBLITUZUMAB、ENOKIZUMAB、ENOTICUMAB、ENSITUXIMAB、ENVAFOLIMAB、EPITUMOMAB、EPTINEZUMAB、ERLIZUMAB、ERTUMAXOMAB、ETIGILIMAB、ETOKIMAB、ETROLIZUMAB、EVINACUMAB、EXBIVIRUMAB、FARALIMOMAB、FARICIMAB、FARLETUZUMAB、FASINUMAB、FELVIZUMAB、FEZAKINUMAB、FICLATUZUMAB、FIGITUMUMAB、FIRIVUMAB、FLANVOTUMAB、FLETIKUMAB、FLOTETUZUMAB、FONTOLIZUMAB、FORALUMAB、FORAVIRUMAB、FRESOLIMUMAB、FROVOCIMAB、FRUNEVETMAB、FULRANUMAB、FUTUXIMAB、GALIXIMAB、GANCOTAMAB、GANITUMAB、GANTENERUMAB、GARADACIMAB、GARETOSMAB、GAVILIMOMAB、GEDIVUMAB、GEMTUZUMAB、GEVOKIZUMAB、GILVETMAB、GIMSILUMAB、GIRENTUXIMAB、GLEMBATUMUMAB、GLENZOCIMAB、GOLIMUMAB、GOSURANEMAB、IANALUMAB、IBRITUMOMAB、ICRUCUMAB、IDARUCIZUMAB、IERAMILIMAB、IFABOTUZUMAB、IGOVOMAB、ILADATUZUMAB、IMALUMAB、IMAPRELIMAB、IMCIROMAB、IMGATUZUMAB、INCLACUMAB、INDATUXIMAB、INDUSATUMAB、INEBILIZUMAB、INFLIXIMAB、INOLIMOMAB、INOTUZUMAB、INTETUMUMAB、APAMISTAMAB、DERLOTUXIMAB、IPILIMUMAB、IRATUMUMAB、ISATUXIMAB、ISCALIMAB、ISTIRATUMAB、IXEKIZUMAB、KELIXIMAB、LABETUZUMAB、LACNOTUZUMAB、LACUTAMAB、LADIRATUZUMAB、LAMPALIZUMAB、LANADELUMAB、LANDOGROZUMAB、LAPRITUXIMAB、LARCAVIXIMAB、LEBRIKIZUMAB、LEMALESOMAB、LENVERVIMAB、LENZILUMAB、LERDELIMUMAB、LERONLIMAB、LESOFAVUMAB、LETOLIZUMAB、LEVILIMAB、LEXATUMUMAB、LIBIVIRUMAB、LIFASTUZUMAB、LIGELIZUMAB、LILOTOMAB、LINTUZUMAB、LIRILUMAB、LODELCIZUMAB、LONCASTUXIMAB、LORVOTUZUMAB、LOSATUXIZUMAB、LUCATUMUMAB、LULIZUMAB、LUMILIXIMAB、LUMRETUZUMAB、LUPARTUMAB、LUTIKIZUMAB、MAFTIVIMAB、MAGROLIMAB、MAPATUMUMAB、MARGETUXIMAB、MARSTACIMAB、MASLIMOMAB、MATUZUMAB、MAVRILIMUMAB、MEPOLIZUMAB、METELIMUMAB、MILATUZUMAB、MINRETUMOMAB、MIRIKIZUMAB、MIRVETUXIMAB、MITAZALIMAB、MITUMOMAB、MODOTUXIMAB、 MOGAMULIZUMAB、MONALIZUMAB、MOROLIMUMAB、MOSUNETUZUMAB、MOTAVIZUMAB、MURLENTAMAB、NACOLOMAB、NAMILUMAB、NAPTUMOMAB、NARATUXIMAB、NARNATUMAB、NATALIZUMAB、NAVICIXIZUMAB、NAVIVUMAB、NAXITAMAB、NEBACUMAB、NEMOLIZUMAB、NERELIMOMAB、NESVACUMAB、NETAKIMAB、NIDANILIMAB、NIMACIMAB、NIMOTUZUMAB、NIRSEVIMAB、NIVOLUMAB、OBEXELIMAB、OBILTOXAXIMAB、OBINUTUZUMAB、OCARATUZUMAB、ODULIMOMAB、OFATUMUMAB、OLECLUMAB、OLENDALIZUMAB、OLINVACIMAB、OLOKIZUMAB、OMALIZUMAB、OMBURTAMAB、ONARTUZUMAB、ONTAMALIMAB、ONTUXIZUMAB、ONVATILIMAB、OPICINUMAB、OREGOVOMAB、ORILANOLIMAB、ORTICUMAB、OSOCIMAB、OTELIXIZUMAB、OTILIMAB、OTLERTUZUMAB、OXELUMAB、OZANEZUMAB、OZORALIZUMAB、PAGIBAXIMAB、PALIVIZUMAB、PAMREVLUMAB、PANITUMUMAB、PANOBACUMAB、PARSATUZUMAB、PASCOLIZUMAB、PASOTUXIZUMAB、PATECLIZUMAB、PATRITUMAB、PEMBROLIZUMAB、PEPINEMAB、PERAKIZUMAB、PERTUZUMAB、PEXELIZUMAB、PIDILIZUMAB、PINATUZUMAB、PLACULUMAB、PLAMOTAMAB、PLOZALIZUMAB、POLATUZUMAB、PONEZUMAB、PORGAVIXIMAB、POZELIMAB、PRASINEZUMAB、PREZALUMAB、PRILIXIMAB、PRITOXAXIMAB、PRITUMUMAB、PROLGOLIMAB、QUETMOLIMAB、QUILIZUMAB、RACOTUMOMAB、RADRETUMAB、RAFIVIRUMAB、RALPANCIZUMAB、RANEVETMAB、RANIBIZUMAB、RAVAGALIMAB、RAXIBACUMAB、REFANEZUMAB、REGAVIRUMAB、RELATLIMAB、RELFOVETMAB、REMTOLUMAB、RESLIZUMAB、RILOTUMUMAB、RINUCUMAB、RITUXIMAB、RIVABAZUMAB、ROBATUMUMAB、ROLINSATAMAB、ROMILKIMAB、RONTALIZUMAB、ROSMANTUZUMAB、ROVALPITUZUMAB、ROZANOLIXIZUMAB、SACITUZUMAB、SAMALIZUMAB、SAMROTAMAB、SARILUMAB、SATRALIZUMAB、SATUMOMAB、SECUKINUMAB、SELICRELUMAB、SEMORINEMAB、SERCLUTAMAB、SERIBANTUMAB、SETOXAXIMAB、SETRUSUMAB、SIBROTUZUMAB、SIFALIMUMAB、SIMTUZUMAB、SINTILIMAB、SIRTRATUMAB、SIRUKUMAB、SOFITUZUMAB、SOLANEZUMAB、SOLITOMAB、SONTUZUMAB、SPARTALIZUMAB、SPESOLIMAB、STAMULUMAB、SULESOMAB、 SUPTAVUMAB、SUTIMLIMAB、SUVIZUMAB、SUVRATOXUMAB、TABALUMAB、TABITUXIMAB、TADOCIZUMAB、TAFASITAMAB、TALACOTUZUMAB、TALIZUMAB、TAMRINTAMAB、TAMTUVETMAB、TANEZUMAB、TAPLITUMOMAB、TAREXTUMAB、TAVOLIMAB、FANOLESOMAB、NOFETUMOMAB、PINTUMOMAB、TECLISTAMAB、TEFIBAZUMAB、TELIMOMAB、TELISOTUZUMAB、TEMELIMAB、TENATUMOMAB、TENELIXIMAB、TEPLIZUMAB、TEPODITAMAB、TEPROTUMUMAB、TESIDOLUMAB、TEZEPELUMAB、TIBULIZUMAB、TIDUTAMAB、TIGATUZUMAB、TILAVONEMAB、TILDRAKIZUMAB、TIMOLUMAB、TIRAGOLUMAB、TISLELIZUMAB、TISOTUMAB、TOCILIZUMAB、TOMARALIMAB、TORALIZUMAB、TORIPALIMAB、TOSATOXUMAB、TOSITUMOMAB、TOVETUMAB、TRALOKINUMAB、TRASTUZUMAB、TREGALIZUMAB、TREMELIMUMAB、TREVOGRUMAB、TUCOTUZUMAB、TUVIRUMAB、UBLITUXIMAB、ULOCUPLUMAB、URELUMAB、URTOXAZUMAB、USTEKINUMAB、UTOMILUMAB、VADASTUXIMAB、VANDORTUZUMAB、VANTICTUMAB、VANUCIZUMAB、VAPALIXIMAB、VARISACUMAB、VARLILUMAB、VATELIZUMAB、VELTUZUMAB、VEPALIMOMAB、VESENCUMAB、VIBECOTAMAB、VISILIZUMAB、VOBARILIZUMAB、VOFATAMAB、VOLAGIDEMAB、VOLOCIXIMAB、VONLEROLIZUMAB、VOPRATELIMAB、VORSETUZUMAB、VOTUMUMAB、VUNAKIZUMAB、XENTUZUMAB、ZALIFRELIMAB、ZAMPILIMAB、ZANOLIMUMAB、ZENOCUTUZUMAB、ZIRALIMUMAB、ZOLBETUXIMAB、ZOLIMOMAB。
选择NK-92细胞进一步制备细胞文库，根据实施例1相同的方法构建CAR受体和细胞内基因回路，即获得包含495个人工抗体的嵌合抗原受体NK-92细胞文库。获得的嵌合抗原受体NK-92细胞文库命名为495-KRAB-iCasp9-CAR-NK92文库，库容量为495单克隆数。
实施例19.包含495个人工抗体的嵌合抗原受体NK-92细胞文库用于人胰腺癌免疫治疗
将上述495-KRAB-iCasp9-CAR-NK92文库静脉施用于胰腺癌受试者。计量和给药频率按照细胞治疗通用的给药方法。
实施例20.载体组合构建方法举例概述
(1)第一基因元件的构建举例
第一基因元件即为嵌合抗原受体文库。嵌合抗原受体是一种人工受体，其文库的构建可以参见专利文献WO2015/123642。本发明强调利用嵌合抗原受体胞外识别结构域的随机化文库实现特定嵌合抗原受体的应用，构建方法举例如下。
可以采用如下的方法构建，简述如下：利用DNA合成的方法，根据人基因抗体编码规则，人工合成人类抗体基因文库。进一步按照嵌合抗原受体方案进行构建，即：人类抗体基因单链抗体文库—CD8 hinge—CD8TM—4-1BB—CD3ζ，如图8A；
也可以采用如下的方法构建，简述如下：利用DNA合成的方法，根据人基因抗体编码规则，人工合成人类抗体基因文库。将基因文库构建在噬菌体载体中，扩增噬菌体即获得噬菌体抗体库。如要在动物模型上的应用筛选方法，则将噬菌体抗体库施用于动物，扣除能够结合动物模型抗原的噬菌体，此时得到噬菌体抗体亚库，利用基因工程方法，获取亚库的抗体基因文库。进一步按照嵌合抗原受体方案进行构建，即：单链抗体文库—IgG4-hinge—CD28TM—CD28—4-1BB—CD3ζ如图8B；如要直接用于受试者，则获取受试者的对照组织，如受试者的外周单核细胞、癌旁组织等，扣除能够结合对照组织的噬菌体，此时得到噬菌体抗体亚库，利用基因工程方法，获取亚库的抗体基因文库。进一步按照嵌合抗原受体方案进行构建，即：Fab抗体文库—CD8-hinge—CD28TM—CD28—CD27—CD3ζ如图8C；
也可以采用如下的方法构建，简述如下：从多名健康志愿者外周单核细胞利用基因工程方法获取抗体基因文库，将基因文库构建在酵母载体中，扩增酵母细胞即获得酵母抗体库。进一步按照嵌合抗原受体方案进行构建，即：人类抗体基因文库—2D3—CD137TM—4-1BB—CD3ζ，如图8D；
也可以采用如下的方法构建，简述如下：利用骆驼外周血单核细胞利用基因工程方法获取骆驼抗体基因文库，进一步按照嵌合抗原受体方案进行构建，即：骆驼抗体基因文库—CD8-hinge—CD8TM—4-1BB—CD3ζ，如图8E；
也可以采用如下的方法构建，简述如下：从多名健康志愿者外周单核细胞利用基因工程方法获取抗体基因文库，将基因文库构建在酵母载体中，扩增酵母细胞即获得酵母抗体库。将肿瘤细胞系MDA-MB-231接触抗体库，淘选能够结合细胞的酵母。利用基 因工程方法获取酵母中的抗体基因文库亚库，再利用CDR突变、亲和力成熟、链交换的基因工程方法重新建立抗体库，进一步按照嵌合抗原受体方案进行构建，即：基因工程单链抗体文库—CD8 hinge—CD8TM—4-1BB—CD3ζ，如图8F；
(2)第二基因元件的构建举例
第二元件为基因回路。可以采用如下的方法构建，简述如下：6个NFAT反应原件和最小化白介素2(IL-2)启动子(6×NFAT)，如图8G
也可以采用如下的方法构建：4个NFAT反应元件和最小化IL-2启动子融合而成(4×NFAT)的启动子，其下游包含Gal4-KRAB；再构建受到Gal4-KRAB调控的5×UAS-PS V40融合启动子)，如图8H。
也可以采用如下的方法构建：6个NFAT反应原件和最小化白介素2(IL-2)启动子(6×NFAT)，其下游的TetR-KRAB转录因子；再构建受到TetR-KRAB调控的7×TRE-P SV40融合启动子，如图8I。
也可以采用如下的方法构建：10个NFκB结合原件和最小化HIVtata启动子融合而成的启动子(10×NFκB)，其下游的转录因子TetR-VP64；再构建受到TetR-VP64调控的7个TRE和最小化CMV启动子融合而成(7×TRE-P CMV-min)启动子，如图8J。
也可以采用如下的方法构建：10个NFκB结合原件、6个NFAT反应原件和最小化白介素2(IL-2)启动子融合(10×NFκB+6×NFAT)而成的启动子，其下游的转录因子ZFHD1-VP64；再构建受到ZFHD1-VP64调控的4×ZFHD1RE-P CMV-min启动子，如图8K。
(3)第三基因元件的构建举例
第三基因元件为编码蛋白的基因元件，编码包括嘌呤霉素抗性蛋白(PuroR)、新霉素抗性蛋白(NeoR)、杀稻瘟素抗性蛋白(Blasticidin-R)、潮霉素B抗性蛋白(Hygromycin B-R)、单纯疱疹病毒胸苷激酶蛋白(HSV-TK)、胞嘧啶脱氨酶蛋白(CD)或iCasp9自杀系统蛋白(iCasp9)的基因。
(4)载体组合举例
包含三种基因元件的载体组合可以采用如下的方法构建，简述如下：(i)据人基因抗体编码规则，人工合成人类抗体基因文库。进一步按照嵌合抗原受体方案进行构建，即：人类抗体基因单链抗体文库—CD8 hinge—CD8TM—4-1BB—CD3ζ，构建如图2A所示的第一基因元件；(ii)6个NFAT反应原件和最小化白介素2(IL-2)启动子(6×NFAT)，构建如图2G所示的第二基因元件；(iii)构建编码嘌呤霉素抗性蛋白的第三基因元件(PuroR)；整体如图9A所示的一个载体上，载体上可以有其他现有技术描述的调控元 件，如CMV启动子。
也可以采用如下的方法构建：(i)利用DNA合成的方法，根据人基因抗体编码规则，人工合成人类抗体基因文库。将基因文库构建在噬菌体载体中，扩增噬菌体即获得噬菌体抗体库。如要在动物模型上的应用筛选方法，则将噬菌体抗体库施用于动物，扣除能够结合动物模型抗原的噬菌体，此时得到噬菌体抗体亚库，利用基因工程方法，获取亚库的抗体基因文库。进一步按照嵌合抗原受体方案进行构建，即：单链抗体文库—IgG4-hinge—CD28TM—CD28—4-1BB—CD3ζ，构建如图2B所示的第一基因元件；(ii)4个NFAT反应元件和最小化IL-2启动子融合而成(4×NFAT)的启动子，其下游包含Gal4-KRAB；再构建受到Gal4-KRAB调控的5×UAS-PS V40融合启动子)，构建如图2H所示第二基因元件。(iii)构建编码单纯疱疹病毒胸苷激酶蛋白的第三基因元件(HSV-TK)；整体如图9B所示的两个载体上
也可以采用如下的方法构建：(i)从多名健康志愿者外周单核细胞利用基因工程方法获取抗体基因文库，将基因文库构建在酵母载体中，扩增酵母细胞即获得酵母抗体库。将肿瘤细胞系MDA-MB-231接触抗体库，淘选能够结合细胞的酵母。利用基因工程方法获取酵母中的抗体基因文库亚库，再利用CDR突变、亲和力成熟、链交换的基因工程方法重新建立抗体库，进一步按照嵌合抗原受体方案进行构建，即：基因工程单链抗体文库—CD8 hinge—CD8TM—4-1BB—CD3ζ，构建如图2F所示的第一基因元件；(ii)10个NFκB结合原件和最小化HIVtata启动子融合而成的启动子(10×NFκB)，其下游的转录因子TetR-VP64；再构建受到TetR-VP64调控的7个TRE和最小化CMV启动子融合而成(7×TRE-P CMV-min)启动子，构建如图2J所示的第二基因元件。(iii)构建编码潮霉素B抗性蛋白的第三基因元件(Hygromycin B-R)；整体如图9C所示的三个载体上
实施例21.向暴露于未知病原体的受试者治疗
首先建立个体化的嵌合抗原受体细胞文库。将处于健康状态的受试者外周单核细胞利用基因工程方法获取抗体基因文库进一步按照嵌合抗原受体方案进行构建，如：个体化单链抗体文库—CD8 hinge—CD8TM—4-1BB—CD3ζ，如10A。构建第二基因元件包括6×NFAT，下游的Gal4-KRAB转录因子，和受到Gal4-KRAB调控的5×UAS-P SV40。构建第三基因元件iCasp9，按照图10B的方式构建载体，10A和10B中还显示了载体上的其他现有技术描述的调控元件，如CMV启动子，2A切割肽，绿色荧光蛋白等。将上 述载体转染入受试者的T细胞中，即获得嵌合抗原受体细胞文库。冻存。
在突发公共安全或生物恐怖主义袭击中，人们暴露于未知的病原体或毒素。暴露模式为很多不同方式中的一种，例如食物或水摄取、气雾剂吸入或皮肤接触。病原体为众多中的一种，例如炭疽杆菌(炭疽热)、流感病毒、天花病毒、鼠疫耶尔森菌(鼠疫)、埃博拉病毒或马尔堡病毒、土拉弗朗西斯菌(野兔病)、汉坦病毒、登革病毒、霍乱毒素、肉毒杆菌毒素、蓖麻毒素、沙门氏菌、大肠杆菌如E.coli 0157:H7、志贺氏杆菌、李斯特菌等。当威胁性的微生物尚未确定时，一些病人已经迅速开始患有相似症状的严重疾病，包括高烧、寒颤、咳嗽、严重疲劳和腹泻。患者可接受标准治疗，例如抗病毒药、抗生素、抗毒素、免疫球蛋白。然后，作为发生感染症状和炎症体征(发烧、寒颤等)的患者的预防措施或治疗手段，向患者静脉施用本发明所述嵌合抗原受体细胞文库和组合物，例如包含活性成分为3×10 9个嵌合抗原受体细胞文库的组合物。一旦细胞文库分布到体液(尤其是血液)中，细胞文库清除病灶细胞、清除炎症介质。通过去除这些疾病介质，细胞文库减少患者体内额外全身性炎症的引发物，减少全身性炎性介质(如细胞因子)的产生，从而防止或限制细胞因子或其他炎性介质诱导的细胞死亡、器官损伤、多器官衰竭和潜在死亡的发生。在数小时至数天的时间中施用一次或重复施用细胞文库，以具有持久或稳定的效果。
在合适的时机，向患者施用诱导剂，去除嵌合抗原受体未激活的文库细胞，然后从患者体内回收细胞文库。利用基因工程方法获取嵌合抗原受体胞外识别域，以此为依据，进一步制备抗体、嵌合抗原受体、工程细胞等用于其他患者的治疗。
实施例22.向具有炎性肠病的患者施用嵌合抗原受体细胞文库
首先建立健康人来源的嵌合抗原受体细胞文库。将至少100位处于健康志愿者的外周单核细胞利用基因工程方法获取抗体基因文库。然后建立噬菌体抗体库，取患者的外周单核细胞作为对照组织，扣除背景。然后再用抗体工程方法获取抗体基因文库。进一步按照嵌合抗原受体方案进行构建，如：健康志愿者单链抗体文库—IgG4 hinge—CD28TM—CD28—4-1BB—CD3ζ，如10C。构建第二基因元件包括4×NFAT，下游的Gal4-KRAB转录因子，和受到Gal4-KRAB调控的5×UAS-P SV40。构建第三基因元件HSV-TK，按照图10D的方式构建载体，10C和10D中还显示了载体上的其他现有技术描述的调控元件。将上述载体转染入患者的T细胞中，即获得嵌合抗原受体细胞文库。冻存。
具有炎性肠病的患者恶化为持续的严重腹泻。对患者施用标准治疗，其可包括全身 性类固醇和胃肠外TNF阻断剂治疗。作为患者抗炎症疗法的一部分，向患者施用有效剂量的所述细胞文库，例如包含活性成分为3×10 9个嵌合抗原受体细胞文库的组合物。细胞文库清除异常细胞、中和局部产生的炎性介质(包括肠道产生的细胞因子)。这促进了肠的愈合并且免于复发。
实施例23.向烧伤患者施用细胞文库
烧伤患者受到超过他总体表面积的30％的严重烧伤，以及长时间的烟和化学物质的吸入肺损伤。尽管有烧伤的清创术，但是患者还是发生了严重的全身性炎性应答综合征，并有急性呼吸窘迫综合症的发作，需要机械通气。对患者施用标准治疗，主要包括支持性护理措施。
获取患者的T细胞，按照实施例22所述的方法扣除背景后构建嵌合抗原受体细胞文库。为了应对迅速导致多器官衰竭的全身性炎症，向患者施用例如包含活性成分为3×10 9个嵌合抗原受体细胞文库的组合物以限制全身性炎症。细胞文库能够去除可导致产生更多炎性介质的全身性炎性介质。
实施例24.向子宫内膜炎患者施用细胞文库
子宫内膜炎患者不孕，利用抗生素、激素、中药治疗未见好转。
获取患者的T细胞，按照实施例22所述的方法扣除患者背景后构建嵌合抗原受体细胞文库。为了应对子宫内膜炎，向患者施用例如包含活性成分为3×10 9个嵌合抗原受体细胞文库的组合物以限制内膜炎症。达到治疗效果后向患者施用诱导剂去除细胞文库。
实施例25.抗衰老受试者施用细胞文库
受试者期望去除衰老细胞，达到延缓衰老效果。
获取受试者的T细胞，按照实施例21所述的方法扣除受试者背景后构建嵌合抗原受体细胞文库。
了应对子宫内膜炎，向患者施用例如包含活性成分为3×10 7个嵌合抗原受体细胞文库的组合物以达到减缓衰老效果。治疗后检测皮肤、生化指标等。达到治疗效果后向患者施用诱导剂去除细胞文库。一个月后患者皮肤弹性增强，肝功能明显增强。
实施例26.阿尔兹海默病患者施用细胞文库
高风险的阿尔兹海默病潜在患者处于健康状态，首先建立个体化的嵌合抗原受体细胞文库。按照嵌合抗原受体方案进行构建，如：个体化单链抗体文库—CD8 hinge—CD8TM—4-1BB—CD3ζ，如10A。构建第二基因元件包括6×NFAT，下游的Gal4-KRAB转录因子，和受到Gal4-KRAB调控的5×UAS-P SV40。构建第三基因元件iCasp9，按照图10B的方式构建载体，10A和10B中还显示了载体上的其他现有技术描述的调控元件，如CMV启动子，2A切割肽，绿色荧光蛋白等。将上述载体以睡美人载体电化学发方法转染入受试者的T细胞中，即获得嵌合抗原受体细胞文库。冻存。
一段时间后，患者发生阿尔兹海默病，按照现有支持治疗进行治疗无法缓解，不断恶化。向患者施用有效剂量的所述细胞文库，例如包含活性成分为1×10 5个嵌合抗原受体细胞文库的组合物，通过硬膜内注射。细胞文库清除异常神经元、减少神经症状。达到治疗效果后向患者施用诱导剂去除细胞文库。
实施例27.不孕患者施用细胞文库
患者被诊断为免疫性不孕，尝试现有方法依然无法解决免疫不孕问题。获取受试者的T细胞，按照实施例21所述的方法扣除受试者背景后构建嵌合抗原受体细胞文库。为了应对子宫内的异常免疫因素，向患者施用例如包含活性成分为3×10 7个嵌合抗原受体细胞文库的组合物以达到去除异常免疫介质的效果。在合适的时机后向患者施用诱导剂去除细胞文库。患者最终怀孕成功。
实施例28.肝纤维化患者施用细胞文库
曾经10年前患乙型肝炎的患者诊断为肝脏部分纤维化，多种抗纤维化治疗无效，疾病继续进展。获取受试者的T细胞，按照实施例22所述的方法扣除受试者背景后构建嵌合抗原受体细胞文库。为了应对肝内的的异常免疫因素，向患者施用例如包含活性成分为3×10 7个嵌合抗原受体细胞文库的组合物以去除异常的肝内免疫介质和病态细胞。达到治疗效果后向患者施用诱导剂去除细胞文库，成功延缓患者纤维化程度。
实施例29.慢性盆腔炎患者施用细胞文库
慢性盆腔炎患者不孕，利用抗生素、激素、中药治疗盆腔炎症未见好转。
获取患者的T细胞，按照实施例22所述的方法扣除患者背景后构建嵌合抗原受体细胞文库。为了应对盆腔炎，向患者施用例如包含活性成分为3×10 9个嵌合抗原受体细 胞文库的组合物以限制盆腔炎症。达到治疗效果后向患者施用诱导剂去除细胞文库。
实施例30.多处皮肤瘢痕整容修复
患者多处皮肤瘢痕，经手术、激光等治疗效果不显著。
获取患者的T细胞，按照实施例22所述的方法扣除患者背景后构建嵌合抗原受体细胞文库。为了应对患者多处皮肤瘢痕，向患者施用例如包含活性成分为1×10 6个嵌合抗原受体细胞文库的组合物以治疗多发性皮肤瘢痕。达到治疗效果后向患者施用诱导剂去除细胞文库。
实施例31.腰椎骨质增生治疗
患者健康，预防性保藏其外周单核细胞的抗体基因文库，库容达1×10 6克隆数。数年后患者发生严重腰椎骨质增生，多种现有治疗、中药治疗无效。获取获取患者的T细胞，，按照实施例21所述的构建方法和患者自身保藏的基因文库制备嵌合抗原受体细胞文库。向患者施用例如包含活性成分为1×10 6个嵌合抗原受体细胞文库的组合物以治疗骨质增生。达到治疗效果后向患者施用诱导剂去除细胞文库。
实施例32.常用靶点文库构建
按照嵌合抗原受体方案进行构建文库，CAR文库的scFv文库为包含结合靶点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、LI13Rα2、CD171、ROR2、AXL、Kappa、CS1、FAP、IL-1RAP、MG7、PSMA、CD5、ROR1、CD70、HER3、Gp75、磷脂酰丝氨酸、cMyc、CD4、CD44v6、CD45、CD28、CD3、CD3e、CD52、CD74、CD30、CD166、CD24、EGFR/HER3融合物、碳水化合物、曲霉、Dectin、埃博拉、真菌、GP、HERV-K、VEGF-R2、TGF-b2R、IgG4、生物素、O-AcGD2、Cadherin 2、OB-cadherin、α5β1integrin、αVβ6integrin、Syndecan-1、Cadherin 1、Claudin 12、Claudin 7、Claudin 3、ZO-1的小规模文库，建立如：小规模单链抗体文库—IgG4 hinge—CD28TM—CD28—4-1BB—CD3ζ。构建第二基因元件包括4×NFAT，下游的Gal4-KRAB转录因子，和受到Gal4-KRAB调控的5×UAS-P SV40。构建第三基因元件HSV-TK。将上述载体转染入NK-92细胞中，即获得嵌合抗原受体细胞文库。冻存。
实施例33.体外筛选方法举例
可以采用多种方法建立抗原受体细胞文库，简单列举如下：首先建立健康人来源的嵌合抗原受体细胞文库。将至少100位处于健康志愿者的外周单核细胞利用基因工程方法获取抗体基因文库，按照嵌合抗原受体方案进行构建，如：健康志愿者单链抗体文库—IgG4 hinge—CD28TM—CD28—4-1BB—CD3ζ，如10C。构建第二基因元件包括4×NFAT，下游的Gal4-KRAB转录因子，和受到Gal4-KRAB调控的5×UAS-P SV40。构建第三基因元件HSV-TK，按照图10D的方式构建载体，10C和10D中还显示了载体上的其他现有技术描述的调控元件。将上述载体转染入Jurkat细胞中，即获得嵌合抗原受体细胞文库。
在将目的抗原分别包被在孔板中，然后每孔孵育jurkat细胞文库1×10 9，合适的时间后施用诱导剂去除没有结合抗原的细胞，洗脱jurkat细胞，基因工程方法获取嵌合抗原受体基因进行分析。
本发明中涉及的未说明部分与现有技术相同或采用现有技术加以实现。申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种载体组件，其特征在于，包括一种或多种载体，以及插入在所述载体上的三种基因元件，该三种基因元件分别为编码一种或多种独特型嵌合抗原受体的多种第一基因元件；载有一种或多种基因回路的第二基因元件；编码一种或多种可诱导蛋白的第三基因元件，
    其中，所述嵌合抗原受体包括细胞内信号传导结构域、跨膜结构域和细胞外识别结构域，所述细胞外识别结构域包括完整的抗体、组成抗体的重链、轻链或抗体片段；所述多种独特型嵌合抗原受体为至少三种独特型嵌合抗原受体。
    所述基因回路是预编程性的，包括一种或多种调节性顺势作用因子和/或一种或多种转录因子，在所述第一基因元件编码的嵌合抗原受体活化时，对所述第三基因元件所编码的可诱导蛋白产生表达调控效应；
    所述可诱导蛋白包括药物抗性蛋白及自杀蛋白中的一种或两种。
  2. 根据权利要求1所述的载体组件，其特征在于：
    其中，所述抗体片段包括抗体可变区、单链抗体、单域抗体或Fab段；
    所述调节性顺势作用因子包括单个顺势作用因子和融合式顺势作用因子，融合式顺势作用因子包括一种或多种单个顺势作用因子的组合；
    所述单个顺势作用因子包括NFAT反应启动子元件、NFκB反应启动子元件、四环素反应元件、半乳糖代谢酶系基因启动子的UAS、PIP反应元件、ZFHD1反应元件、ZF21-16反应元件、ZF42-10反应元件、ZF43-8反应元件、ZF54-8反应元件、最小化CMV启动子、CMV启动子、SV40启动子、最小化IL-2启动子、最小化昆虫热休克蛋白70启动子、最小化HIVtata启动子中的任意一种或至少两种的组合；
    所述融合式顺势作用因子包括4×NFAT、6×NFAT、5×NFκB、10×NFκB、7×TRE-P CMV-min、5×UAS-P CMV-min、4×PIR-P CMV-min、8×PIR-P CMV-min、8×PIR-P hsp70min、4×ZFHD1RE-P CMV-min、8×ZF21-16RE-P CMV-min、8×ZF42-10RE-P CMV-min、8×ZZF43-8R-P CMV-min、8×ZF54-8RE-P CMV-min、7×TRE-P SV40、7×TRE-Pcmv、5×UAS-P SV40、4×PIR-P SV40、8×PIR-P SV40、4×ZFHD1RE-P SV40、8×ZF21-16RE-P SV40、8×ZF42-10RE-P SV40、8×ZF43-8RE-P SV40、8×ZF54-8RE-P SV40中的任意一种或至少两种的组合；
    所述转录因子包括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、 ZFHD1-KRAB中的任意一种或至少两种的组合；
    所述表达调控效应包括激活转录表达、增强转录表达、终止转录表达、抑制转录表达中的任意一种或至少两种的组合；
    所述药物抗性蛋白包括嘌呤霉素抗性蛋白、新霉素抗性蛋白、杀稻瘟素抗性蛋白或潮霉素B抗性蛋白中的任意一种或至少两种的组合；
    所述自杀蛋白包括单纯疱疹病毒胸苷激酶蛋白、胞嘧啶脱氨酶蛋白或iCasp9自杀系统蛋白中的任意一种或至少两种的组合。
  3. 根据权利要求2所述的载体组件，其特征在于：
    其中，所述基因回路由(i)Gal4-KRAB和5×UAS-PS V40的组合、TetR-KRAB和7×TRE-P SV40的组合、Gal4-VP64和5×UAS-P CMV-min的组合、TetR-VP64和7×TRE-P CMV-min的组合、以及TetR-KRAB和7×TRE-P cmv的组合中的任意一种与(ii)4×NFAT、6×NFAT、5×NFκB、10×NFκB中的任意一种组合而成。
  4. 携带权利要求1～3任一项所述载体组件的嵌合抗原受体细胞文库。
  5. 权利要求4所述的嵌合抗原受体细胞文库的制备和筛选方法，其特征在于：
    将第一基因元件、第二基因元件、第三基因元件插入同一载体或不同载体，而后转染至细胞内，即得到嵌合抗原受体细胞文库，然后进行筛选。
    其中，转染方法包括病毒转染、化学试剂转染或电击转染中的任意一种或至少两种的组合；所述细胞是哺乳动物的免疫细胞或基因工程化的免疫细胞。
  6. 根据权利要求5所述的嵌合抗原受体细胞文库的制备和筛选方法，其特征在于，包括如下步骤：
    A、抗体库制备
    利用健康志愿者来源、全合成方法和/或基因工程方法建立抗体基因文库；
    B、基因元件构建
    构建第一基因元件，其结构包含抗体库-CAR，所述抗体库或抗体亚库被构建为CAR的胞外识别结构域；构建第二基因元件，包含第一种调节性顺势作用因子、受该调节性顺势作用因子调控的转录因子、受转录因子调控的第二种顺势作用因子；构建第三元件，包含可诱导蛋白基因，而后将上述三种基因元件构建到一种或多种基因控制表达盒中；
    C、基因元件导入细胞
    采用慢病毒载体系统将上述基因控制表达盒导入哺乳动物免疫细胞内，得到嵌合抗原受体细胞文库；
    D、嵌合抗原受体细胞体外筛选
    将嵌合抗原受体细胞文库体外与抗原接触，根据可诱导蛋白的表达情况，筛选目标嵌合抗原受体表达细胞，并对其进行回收，
    E、嵌合抗原受体细胞体内筛选
    给实验目的受试者施用有效量嵌合抗原受体细胞文库，根据可诱导蛋白的表达情况，筛选目标嵌合抗原受体表达细胞，并对其进行回收。
  7. 根据权利要求6所述的嵌合抗原受体细胞文库的制备方法，其特征在于：
    其中，在步骤D和步骤E中，还包括筛选获得的嵌合抗原受体细胞后，利用抗体工程方法重新构建次级嵌合抗原受体细胞文库的步骤。
  8. 根据权利要求6所述的嵌合抗原受体细胞文库的制备方法，其特征在于：
    其中，步骤D中，抗原包括野生型细胞、转染特定抗原基因的细胞、结合特定抗原的细胞、溶解在培养基中的抗原、包被在培养器皿上的抗原、包被在微珠上的抗原或包被在培养支架上的抗原中的任意一种或至少两种的组合；
    步骤E中，体内抗原指存在于人及动物活体内的抗原，包括在体细胞、在体病灶细胞、转染特定抗原基因的在体细胞、感染特定病原体的在体细胞、结合特定抗原的在体细胞、移植在动物模型上的在体细胞中的任意一种或至少两种的组合。
  9. 一种药物组合物，其特征在于，包括活性组分以及药学上可接受的稀释剂或赋形剂，该活性组分包括权利要求1所述的载体组件、权利要求4-6任一项所述的嵌合抗原受体细胞文库、权利要求5-8任一项所述筛选方法获得的嵌合抗原受体细胞、嵌合抗原受体、嵌合抗原受体来源的抗体中的任意一种或至少两种的组合，和至少一种医学或药学上可接受的载体。
  10. 权利要求9所述的药物组合物在制备诊断或治疗需要去除疾病介质相关疾病药物、试剂、试剂盒中的用途。
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