CHIMERIC ANTIGEN RECEPTOR CELL LIBRARY CARRYING GENE ELEMENT COMBINATION, PREPRATION AND SCREENING METHOD, AND USE THEREOF

Abstract

A chimeric antigen receptor (CAR) cell library is established through the fusion of a cell and a vector assembly. The vector assembly carries three genetic elements corresponding to a plurality of first genetic elements encoding one or more idiotype CARs, a second genetic element carrying one or more genetic circuits, and a third genetic element encoding one or more inducible proteins, respectively. The one or more genetic circuits are pre-programmed and are each a combination of a cis-regulatory factor and a transcription factor; and the one or more inducible proteins include one or two selected from the group consisting of a drug resistance protein and a suicide protein. By designing a CAR library-genetic circuit-inducible protein coupling scheme, the cell library construction and screening for complex and unknown disease target antigens are realized, such as to solve the problems that there are complex, diverse, and variable antigens.

Claims

1. A vector assembly, comprising one or more vectors and three genetic elements inserted into the one or more vectors, wherein the three genetic elements correspond to a plurality of first genetic elements encoding one or more idiotype chimeric antigen receptors (CARs), a second genetic element carrying one or more genetic circuits, and a third genetic element encoding one or more inducible proteins, respectively; the one or more idiotype CARs each comprise an intracellular signaling domain, a transmembrane domain, and an extracellular recognition domain, and the extracellular recognition domain comprises an intact antibody, a heavy chain or light chain constituting an antibody, or an antibody fragment; when there are more than one idiotype CARs, at least three idiotype CARs are comprised; the one or more genetic circuits are pre-programmed and each comprise one or more cis-regulatory factors and/or one or more transcription factors; the activation of the one or more idiotype CARs encoded by the plurality of first genetic elements leads to an expression regulation effect on the one or more inducible proteins encoded by the third genetic element; and the one or more inducible proteins comprise one or two selected from the group consisting of a drug resistance protein and a suicide protein.

2. The vector assembly according to claim 1, wherein the antibody fragment comprises an antibody variable region, a single-chain fragment variable (scFV), a single-domain antibody, or an antigen-binding fragment (Fab); the one or more cis-regulatory factors comprise a single cis-acting factor and a fusion cis-acting factor, and the fusion cis-acting factor comprises a combination of one or more single cis-acting factors; the single cis-acting factor comprises any one or a combination of at least two selected from the group consisting of an NFAT-responsive promoter element, an NFκB-responsive promoter element, a tetracycline responsive element, an upstream activating sequence (UAS) of a galactose-metabolizing enzyme gene promoter, a PIP responsive element, a ZFHD1 responsive element, a ZF21-16 responsive element, a ZF42-10 responsive element, a ZF43-8 responsive element, a ZF54-8 responsive element, a minimal CMV promoter, a CMV promoter, an SV40 promoter, a minimal IL-2 promoter, a minimal insect heat shock protein (HSP) 70 promoter, and a minimal HIVtata promoter; the fusion cis-acting factor comprises any one or a combination of at least two selected from the group consisting of 4×NFAT, 6×NFAT, 5×NFκB, 10×NFκB, 7×TRE-P.sub.CMV-min, 5×UAS-P.sub.CMV-min, 4×PIR-P.sub.CMV-min, 8×PIR-P.sub.CMV-min, 8×PIR-P.sub.hsp70min, 4×ZFHD1RE-P.sub.CMV-min, 8×ZF21-16RE-P.sub.CMV-min, 8×ZF42-10RE-P.sub.CMV-min, 8×ZZF43-8R-.sub.CMV-min, 8×ZF54-8RE-P.sub.CMV-min, 7×TRE-P.sub.SV40, 7×TRE-Pcmv, 5×uAS-P.sub.SV40, 4×PIR-P.sub.SV40, 8×PIR-P.sub.SV40, 4×ZFHD1RE-P.sub.SV40, 8×ZF21-16RE-P.sub.SV40, 8×ZF42-10RE-P.sub.SV40, 8×ZF43-8RE-P.sub.SV40, and 8×ZF54-8RE-P.sub.SV40; the one or more transcription factors comprise any one or a combination of at least two selected from the group consisting of 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, and ZFHD1-KRAB; the expression regulation effect comprises any one or a combination of at least two selected from the group consisting of activating transcription and expression, enhancing transcription and expression, terminating transcription and expression, and inhibiting transcription and expression; the drug resistance protein comprises any one or a combination of at least two selected from the group consisting of a puromycin resistance protein, a neomycin resistance protein, a blasticidin resistance protein, and a hygromycin B resistance protein; and the suicide protein comprises any one or a combination of at least two selected from the group consisting of a herpes simplex virus (HSV) thymidine kinase (TK) protein, a cytosine deaminase (CD) protein, and an inducible caspase 9 (iCasp9) suicide system protein.

3. The vector assembly according to claim 2, wherein the one or more genetic circuits each are composed of (i) any one selected from the group consisting of a combination of Gal4-KRAB and 5×UAS-P.sub.SV40, a combination of TetR-KRAB and 7×TRE-P.sub.SV40, a combination of Gal4-VP64 and 5×UAS-P.sub.CMV-min, a combination of TetR-VP64 and 7×TRE-P.sub.CMV-min, and a combination of TetR-KRAB and 7×TRE-P.sub.cmv and (ii) any one selected from the group consisting of 4×NFAT, 6×NFAT, 5×NFκB, and 10×NFκB.

4. A CAR cell library carrying the vector assembly according to claim 1.

5. A preparation and screening method of the CAR cell library according to claim 4, comprising: inserting the plurality of first genetic elements, the second genetic element, and the third genetic element into the one or more vectors, transfecting the one or more vectors into cells to obtain the CAR cell library, and screening, wherein a method for the transfecting comprises any one or a combination of at least two selected from the group consisting of viral transfection, chemical transfection, and electroporation transfection; and the cells are mammalian immune cells or genetically-engineered immune cells.

6. The preparation and screening method according to claim 5, comprising the following steps: A. preparation of an antibody gene library establishing the antibody gene library through a healthy volunteer source, a total synthesis process, and/or a genetic engineering process; B. construction of genetic elements constructing a first genetic element comprising the antibody gene library-CAR, wherein the antibody gene library or an antibody gene sub-library thereof is constructed as the extracellular recognition domain of CAR; constructing a second genetic element comprising a first cis-regulatory factor, a transcription factor regulated by the first cis-regulatory factor, and a second cis-acting factor regulated by the transcription factor; constructing a third genetic element comprising an inducible protein gene; and inserting the three genetic elements into one or more controlled gene expression cassettes; C. introduction of the genetic elements into the cells introducing the one or more controlled gene expression cassettes into the mammalian immune cells through a lentiviral vector system to obtain the CAR cell library; D. in vitro screening of CAR cells allowing the CAR cell library to contact an antigen in vitro, and screening and enriching a target CAR cell according to the expression of an inducible protein; and E. in vivo screening of CAR cells administering an effective amount of the CAR cell library to an experimental subject, and screening and enriching a target CAR cell according to the expression of an inducible protein.

7. The preparation and screening method according to claim 6, wherein in steps D and E, after the target CAR cell is screened out and enriched, it further comprises reconstructing a secondary CAR cell library through an antibody engineering process.

8. The preparation and screening method according to claim 6, wherein in step D, the antigen comprises any one or a combination of at least two selected from the group consisting of a wild-type (WT) cell, a cell transfected with a specific antigen gene, a cell binding to a specific antigen, an antigen dissolved in a medium, an antigen coated on a petri dish, an antigen coated on a microbead, and an antigen coated on a culture scaffold; and in step E, an in vivo antigen refers to an antigen existing in a living human or animal, and comprises any one or a combination of at least two selected from the group consisting of an in vivo cell, an in vivo lesion cell, an in vivo cell transfected with a specific antigen gene, an in vivo cell infected with a specific pathogen, an in vivo cell binding to a specific antigen, and an in vivo cell transplanted into an animal model.

9. A pharmaceutical composition, comprising an active component and a pharmaceutically acceptable diluent or excipient, wherein the active component comprises any one or a combination of at least two selected from the group consisting of the vector assembly according to claim 1, the CAR cell library according to claim 4, the CAR cell obtained by the screening method according to claim 5, a CAR, and a CAR-derived antibody, and at least one medically or pharmaceutically acceptable carrier.

10. A use of the pharmaceutical composition according to claim 9 in the preparation of a drug, a reagent, or a kit for the diagnosis or treatment of a disease that requires the removal of a disease-associated mediator.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0097] FIG. 1 is a schematic diagram illustrating a structure, a construction method, and in vivo screening of the CAR cell library according to an embodiment of the present disclosure, where A shows an antibody library constructed by an existing technology; B shows a background-subtracted antibody library; C shows the construction of the CAR cell library; D shows the in vitro screening; E shows the in vivo screening; F shows the clearance of CAR cells failing to identify a target antigen; and G shows the survival of the CAR cells recognizing the target antigen.

[0098] FIG. 2 is a schematic diagram of controlled gene expression cassettes, where A is a schematic structural diagram of the controlled gene expression cassette NFAT-KRAB-iCasp9-2A-GFP; B is a schematic structural diagram of the controlled gene expression cassette CMV-scFvlab-CAR; and C is a schematic structural diagram of the controlled gene expression cassette CD19-CAR.

[0099] FIG. 3 shows the tumor volume change in each group after mouse tumor models are treated with the cell library.

[0100] FIG. 4 shows the expression of CAR in blood cells of animals in each group after treatment.

[0101] FIG. 5 shows the tumor volume change in each group after mouse tumor models are treated with the screened cells.

[0102] FIG. 6 shows the tumor tissue inhibition rate of each group after PDX models are treated with the cell library.

[0103] FIG. 7 shows the pathological score of each group with inflammatory bowel disease (IBD).

[0104] FIG. 8 is a schematic diagram of the construction of genetic elements, where A to F show the construction of the first genetic element and G to K show the construction of the second genetic element.

[0105] FIG. 9 is a schematic diagram illustrating the assembly of a vector.

[0106] FIG. 10 is a schematic diagram illustrating individualized genetic elements and vectors.

DETAILED DESCRIPTION

[0107] The following examples and experimental examples are provided to further illustrate the present disclosure, and shall be construed as a limitation to the present disclosure. The examples do not include detailed descriptions of traditional methods, such as common antibody engineering methods, methods for constructing vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, methods for introducing plasmids into host cells, and construction methods of synthetic cells, devices, and genetic circuits. Such methods are well known to those of ordinary skill in the art, and are described in many publications, including Antibody Engineering (2nd edition) edited by Dong Zhiwei and Wang Yan, Peking University Medical Press (PUMP), 2002; Sambrook, J., Fritsch, E. F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press; and Phage Display: A laboratory Manual, Cold spring Harbor Laboratory Press.

EXAMPLE 1

Totally-Synthetic Mouse-Derived CAR-T Cell Library

[0108] A construction and screening process of the library was shown in FIG. 1:

[0109] (A) Construction of a phage antibody library: A totally-synthetic mouse-derived phage scFV library was constructed through total synthesis. A method for constructing the antibody library is well known to those of ordinary skill in the art, and a method for constructing the totally-synthetic mouse-derived phage scFV library is the same as in the literature (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.). According to library capacity evaluation, the totally-synthetic mouse-derived phage scFV library had a library capacity of 1×10.sup.9. A method of the library capacity evaluation is the same as in the literature (Ridgway J B B, et al. Cancer Research, 2013, 59 (11): 2718-2723).

[0110] (B) Background removal: The totally-synthetic mouse-derived phage scFV library (1×10.sup.11 PFU) was injected into BALB/c mice through the tail vein, and 4 rounds of screening were conducted to remove phage capable of binding to mouse tissues to obtain a phage antibody sub-library. A method of the screening is the same as in the literature (Wada, Akinori, et al. Molecular Therapy-Oncolytics 12 (2019): 138-146.). The sub-library was subjected to amplification detection, and it was found that the library capacity did not change significantly. Then an antibody gene library was obtained by a PCR method.

[0111] (C) Construction of a CAR Cell Library

[0112] Mouse T lymphocytes were acquired according to the method reported in the literature [Srivastava S, et al. Cancer cell, 2019, 35 (3): 489-503. e8.]. Then a genetic circuit was constructed (FIG. 2). The genetic circuit involved two controlled gene expression cassettes: (i) Controlled gene expression cassette NFAT-KRAB-iCasp9-2A-GFP shown in FIG. 2A: The cassette included an NFAT-responsive element promoter (Uchibori R, et al. Molecular Therapy-Oncolytics, 2019, 12: 16-25.), a fusion of 6 NFAT-responsive elements and a minimal IL-2 promoter that controlled the expression of the repressive transcription factor GAL4-KRAB (Morsut L, et al. Cell, 2016, 164 (4): 780-791.), and a 5×UAS-PSV40 promoter fusion (Morsut L, et al. Cell, 2016, 164 (4): 780-791.) that controlled the iCasp9-2A-green fluorescent protein fusion gene. Construction methods of the iCasp9 gene and self-cleaving peptide 2A are the same as in the literature [Liu E, et al. Leukemia, 2018, 32 (2): 520.]. The controlled gene expression cassette NFAT-KRAB-iCasp9-2A-GFP was integrated into the mouse T lymphocytes through a lentiviral vector system. Mouse T lymphocytes successfully introduced with the cassette were sorted by FCM. ii) Controlled gene expression cassette CMV-scFvlab-CAR shown in FIG. 2B: The above mouse-derived scFV library was constructed as an extracellular recognition domain of CAR. The mammalian cell gene integration technology reported in the literature [Parthiban K, et al. mAbs. Taylor & Francis, 2019.] was used to integrate the controlled gene expression cassette CMV-scFvlab-CAR into T cells through a lentiviral vector system.

[0113] The genetic circuit was pre-programmed as follows: when the CAR bound to an antigen, the expression of the iCasp9 gene was inhibited under a change of the genetic circuit, and a corresponding cell did not undergo the induced apoptosis regulation by an iCasp9 inducer.

[0114] The obtained CAR cell library was named KRAB-iCasp9-CAR-T, which had a library capacity of 1×10.sup.6.

[0115] (D) In vitro screening: The cell library was directly incubated with a target antigen.

[0116] (E) In vivo screening: A solution of the cell library was administered to a subject at an appropriate amount and concentration.

[0117] (F) A suicide gene iCasp9 inducer was administered, the inhibition on the expression of the iCasp9 gene was detected according to the pre-programmed genetic circuit to screen out cells undergoing the induced apoptosis regulation of the iCasp9 inducer, and the cells were determined as CAR cells failing to recognize the target antigen and cleared.

[0118] (G) According to the inhibition of the expression of the iCasp9 gene in vivo, cells free from the induced apoptosis regulation of the iCasp9 inducer were screened out and enriched.

EXAMPLE 2

Treatment of Breast Cancer with the Totally-Synthetic Mouse-Derived CAR-T Cell Library

[0119] This example was implemented with the KRAB-iCasp9-CAR-T cell library obtained in Example 1.

[0120] 4T1 mouse breast cancer in situ models were constructed by a method in the literature (Paschall A V, Liu K. JoVE 2016 (114): e54040.). When an average tumor volume in mice reached 100 mm.sup.3, the mice were divided into a control group, an irrelevant CAR-T cell group, a KRAB-iCasp9-CAR-T cell library group 1, a KRAB-iCasp9-CAR-T cell library group 2, a KRAB-iCasp9-CAR-T cell library group 3, and a KRAB-iCasp9-CAR-T cell library group 4.

[0121] A CAR-positive rate of CAR-T cells was normalized to 45%. The control group was administered with PBS; the irrelevant CAR-T cell group was administered with CD19-CAR-T cells (the controlled gene expression cassette shown in FIG. 2C), and the cells were intravenously injected at a dose of 5×10.sup.6 cells once every 2 d, 3 times in total; and all KRAB-iCasp9-CAR-T cell library groups were each administered with the KRAB-iCasp9-CAR-T cell library, and the cells were diluted with the serum-free 1640 medium and then intravenously injected at a dose of 5×10.sup.6 cells once every 2 d, 3 times in total. The KRAB-iCasp9-CAR-T cell library group 1 was only subjected to cell therapy; the KRAB-iCasp9-CAR-T cell library group 2 was administered with an iCasp9 inducer from the beginning of the therapy, with a dose and route the same as in the literature [Liu E, et al. Leukemia, 2018, 32 (2): 520.]; the KRAB-iCasp9-CAR-T cell library group 3 was administered with an iCasp9 inducer from the second week of the therapy, with a dose and route the same as above; and the KRAB-iCasp9-CAR-T cell library group 4 was administered with an iCasp9 inducer from the second week of the therapy, but the iCasp9 inducer was discontinued immediately after tumor regression, and the experiment of the group was stopped accordingly.

[0122] Experimental results were shown in FIG. 3, and it can be seen that, in the control group and irrelevant CAR-T cell group, the tumor tissue volume increased rapidly; in the KRAB-iCasp9-CAR-T cell library group 1, KRAB-iCasp9-CAR-T cell library group 3, and KRAB-iCasp9-CAR-T cell library group 4, the tumor growth was significantly inhibited from the second week of the therapy, and the tumor was rapidly cleared; and in the KRAB-iCasp9-CAR-T cell library group 2 administered with the iCasp9 inducer from the beginning of the therapy, no tumor-inhibition effect was exhibited.

EXAMPLE 3

In Vivo Screening of Totally-Synthetic Mouse-Derived CAR-T Cells Targeting 4T1 Breast Cancer

[0123] Mice in all experimental groups in Example 2 were sacrificed after the experiment, blood was collected from the mice, and CAR-positive cells in the blood were detected by FCM. Proportions of CAR-positive cells in all experimental groups obtained after the experiment were shown in FIG. 4. The CAR-positive cells in each group were isolated and cultivated to complete the in vivo screening.

EXAMPLE 4

In Vitro Screening of Totally-Synthetic Mouse-Derived CAR-T Cells Targeting 4T1 Breast Cancer

[0124] This example was implemented with the KRAB-iCasp9-CAR-T cell library obtained in Example 1. The KRAB-iCasp9-CAR-T cell library (1×10.sup.7 cells, CAR positive rate: 70%) was co-cultivated with 1×10.sup.7 of 4T1 breast cancer cells for 96 h, then an iCasp9 inducer was added to a medium with an amount and manner the same as in the literature (Liu E, et al. Leukemia, 2018, 32 (2): 520.), and 5 d later, CAR-positive cells were separated by FCM to complete the in vitro screening.

EXAMPLE 5

Preparation of a CAR and Antibody Targeting 4T1 Breast Cancer Cells

[0125] The T cell genome was extracted from the CAR-T cells screened out in Examples 3 and 4 with a kit. Primers were designed to acquire the CAR gene through PCR, that is, the CAR targeting 4T1 breast cancer cells was obtained. A scFV was further obtained through PCR, the obtained scFV was constructed as mouse IgG2a through genetic engineering, and then the antibody targeting 4T1 breast cancer cells was obtained through expression and purification.

EXAMPLE 6

Identification of a Target Antigen of 4T1 Breast Cancer Cells

[0126] The antibody obtained in Example 5 was cross-linked on agarose beads, then a 4T1 breast cancer cell lysate was incubated overnight with the antibody-cross-linked beads, and the beads were rinsed, such that an antigen targeted by the antibody was enriched on the beads; and the beads were subjected to peptide mass fingerprinting (PMF) identification to obtain the target antigen.

EXAMPLE 7

Therapeutic use of Totally-Synthetic Mouse-Derived CAR-T Cells Targeting 4T1 Breast Cancer

[0127] The CAR-T cells obtained in each group in Example 3 were used once again for the treatment of breast cancer in situ in 4T1 mice. A treatment method of each group was the same as above, and an iCasp9 inducer was administered from the second week of the treatment. Results were shown in FIG. 5. A very obvious anti-tumor effect was exhibited in the KRAB-iCasp9-CAR-T cell library group 3 and KRAB-iCasp9-CAR-T cell library group 4.

EXAMPLE 8

Natural Fully Human CAR NK-92 Cell Library

[0128] A natural human-derived phage scFV library was constructed with PBMCs of 200 healthy volunteers. A method for constructing the phage scFV library is the same as in the literature (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.). According to library capacity evaluation, the natural human-derived phage scFV library had a library capacity of 1×10.sup.10.

[0129] The natural human-derived phage scFV library (1×10.sup.12PFU) was injected into NSG mice through the tail vein, and 4 rounds of screening were conducted to remove phage capable of binding to mouse tissues. A phage antibody library obtained after the screening was subjected to amplification detection, and it was found that the library capacity did not change significantly. Then an antibody gene library was obtained by a PCR method.

[0130] NK-92 cells (Schonfeld K, et al. Molecular therapy, 2015, 23 (2): 330-338.) were adopted to further construct a cell library. CARs and intracellular genetic circuits were constructed according to the same method as in Example 1 to obtain the natural fully human CAR NK-92 cell library. The obtained natural fully human CAR NK-92 cell library was named KRAB-iCasp9-CAR-NK92, which had a library capacity of 1×10.sup.6.

EXAMPLE 9

In Vivo Screening of the Natural Fully Human CAR NK-92 Cell Library

[0131] This example was implemented with the KRAB-iCasp9-CAR-NK92 library obtained in Example 8.

[0132] NSG mouse tumor-bearing models (namely PDX) were directly constructed with patient-derived tissues. According to the method in the literature (Fu W, et al. Clinical Cancer Research, 2019, 25 (9): 2835-2847.), a lung cancer PDX model L10, a breast cancer PDX model B7, and an ovarian cancer PDX model OV3 were constructed. When an average tumor volume in mice reached 400 mm.sup.3, the mice were divided into a control group, an irrelevant CAR-NK92 cell group (anti-CD19 CAR-NK92, including the controlled gene expression cassette shown in FIG. 2C), and a KRAB-iCasp9-CAR-NK92 library group. The same therapeutic dose and manner as in Example 1 were adopted. At the second week of treatment, each group was administered with an iCasp9 inducer. Mice were sacrificed at the third week of treatment, blood and tumor tissues were collected from the mice, and CAR-positive cells were detected by FCM.

[0133] In each of the lung cancer PDX model L10, the breast cancer PDX model B7, and the ovarian cancer PDX model OV3, CAR-positive cells were isolated from the KRAB-iCasp9-CAR-NK92 library group to complete the in vivo screening.

Example 10

Construction of a CAR NK-92 Cell Library Targeting a Tumor Tissue

[0134] This example was implemented with the natural fully human CAR NK-92 cells obtained from the in vivo screening in Example 9.

[0135] CARs targeting lung cancer L10, breast cancer B7, and ovarian cancer OV3 were obtained from the natural fully human CAR NK-92 cells obtained from the in vivo screening in Example 9 through a general genetic engineering technology. Then PCR was used to obtain extracellular scFV sequences of the CARs. Random mutations of the heavy chain CDR3 were designed through computer-aided design (CAD) according to the extracellular scFV sequence, and a scFV library was re-constructed through artificial synthesis, which was a genetically-engineered antibody library targeting a tumor tissue.

[0136] NK-92 cells were adopted to further prepare a cell library, and CARs and intracellular genetic circuits were constructed according to the same method as in Example 1. The extracellular recognition domain of CAR was the genetically-engineered antibody library. Finally, CAR NK-92 cell libraries respectively targeting lung cancer L10, breast cancer B7, and ovarian cancer OV3 were obtained. The obtained human CAR NK-92 cell libraries were respectively named L10-KRAB-iCasp9-CAR-NK92, B7-KRAB-iCasp9-CAR-NK92, and OV3-KRAB-iCasp9-CAR-NK92, which each had a library capacity of 1×10.sup.5.

EXAMPLE 11

Treatment of Mouse Tumor-Bearing Models with the CAR NK-92 Cell Libraries Targeting Tumor Tissues

[0137] This example was implemented with the libraries L10-KRAB-iCasp9-CAR-NK92, B7-KRAB-iCasp9-CAR-NK92, and OV3-KRAB-iCasp9-CAR-NK92 obtained in Example 10.

[0138] A lung cancer PDX model L10, a breast cancer PDX model B7, and an ovarian cancer PDX model OV3 were constructed. When an average tumor volume in mice reached 100 mm.sup.3, the mice were divided into a control group, an irrelevant CAR-NK92 cell group (anti-CD19 CAR-NK92, including the controlled gene expression cassette shown in FIG. 2C), and NK-92 cell library groups. The same therapeutic dose and manner as in Example 1 were adopted.

[0139] At the second week of treatment, each group was administered with an iCasp9 inducer. After 5 weeks of treatment, a tumor growth inhibition ratio was calculated as follows: ratio=1−average tumor volume in treatment group/average tumor volume in control group. Results were shown in FIG. 6, and it can be seen that the CAR NK-92 cell libraries targeting tumor tissues exhibited a very strong anti-tumor effect on each model.

EXAMPLE 12

Construction of an Individualized Natural Fully Human CAR-T Cell Library

[0140] This example was implemented with the natural human-derived phage scFV library constructed with PBMCs of 200 healthy volunteers in Example 8.

[0141] PBMCs of a lung cancer subject and a precancerous tissue of lung cancer obtained from surgery were taken as a control cell/tissue, and then three rounds of negative screening were conducted to remove phage capable of binding to the control cell/tissue in the phage natural human scFV library. A phage antibody library obtained after the screening was subjected to PCR amplification to obtain an antibody gene library. As tested, the library capacity did not change significantly.

[0142] The PBMCs of the above lung cancer subject were taken and then T lymphocytes were further isolated to further prepare a cell library, and CARs and genetic circuits were constructed according to the same method as in Example 1. The same genetic circuit and cell library construction methods as in Example 1 were adopted. Two controlled gene expression cassettes for the genetic circuits were shown in FIG. 2.

[0143] The obtained CAR cell library was named KRAB-iCasp9-CAR-hT, which had a library capacity of 6×10.sup.5.

EXAMPLE 13

Immunotherapy of Human Lung Cancer with the Individualized Natural Fully Human CAR-T Cell Library

[0144] The above KRAB-iCasp9-CAR-hT library was administered intravenously to the lung cancer subject in Example 12. The general administration route and dose for CAR-T cells (Fry T J, et al. Nature medicine, 2018, 24 (1): 20.) were adopted.

EXAMPLE 14

Treatment of IBD Small Animal Models with the Totally-Synthetic Mouse-Derived CAR-T Cell Library

[0145] This example was implemented with the KRAB-iCasp9-CAR-T cell library obtained in Example 1.

[0146] According to the method in the literature (Tian, Yuhua, et al. Gastroenterology 156.8 (2019): 2281-2296.), colitis was induced with trinitrobenzene sulfonic acid (TNBS) in BALB/c mice to obtain the IBD models. The model evaluation was conducted by the same method as in the literature (He C, et al. Gut, 2015.). The model mice were divided into a control group, a model group, an irrelevant CAR-T cell group, and a KRAB-iCasp9-CAR-T cell library group. The control group was not administered with TNBS, and the remaining groups were each administered with both low-dose TNBS and a therapeutic agent. The model group was administered with PBS; and the irrelevant CAR-T cell group was intravenously injected with CD19-CAR-T cells (the controlled gene expression cassette shown in FIG. 1C) at a dose of 5×10.sup.6 cells once every 2 d, 3 times in total. Four weeks later, case scoring was conducted by the same method as in the literature [Tian, Yuhua, et al. Gastroenterology 156.8 (2019): 2281-2296.]. Results were shown in FIG. 7, and it can be seen that the KRAB-iCasp9-CAR-T cell library exhibited a prominent therapeutic effect.

EXAMPLE 15

Treatment of Endometriosis Mouse Graft Models with the Natural Fully Human CAR NK-92 Cell Library

[0147] This example was implemented with the KRAB-iCasp9-CAR-NK92 library obtained in Example 8.

[0148] According to the model construction method in the literature (Masuda H, et al. N Proceedings of the National Academy of Sciences, 2007, 104 (6): 1925-1930.), an ectopic endometrium from an endometriosis subject was transplanted into NOG mice. After the mouse models were successfully constructed, the mice were divided into a control group, an irrelevant CAR-NK92 cell group (anti-CD19 CAR-NK92, including the controlled gene expression cassette shown in FIG. 2C), and a KRAB-iCasp9-CAR-NK92 library group. There were 10 mice in each group. The same therapeutic dose and manner as in Example 1 were adopted. At the second week of treatment, each group was administered with an iCasp9 inducer. Mice were sacrificed after 5 weeks of treatment to examine the transplanted endometrium. Results showed that, in the control group and irrelevant CAR-NK92 cell group, the endometrial tissue survived in each mouse; and in the KRAB-iCasp9-CAR-NK92 library treatment group, no endometrial tissue survived in mice.

EXAMPLE 16

Construction of a Natural Fully Human CAR-T Cell Library Targeting an Ectopic Endometrium

[0149] A natural human-derived phage scFV library was constructed according to the method in Example 8, and the method was briefly described as follows: a natural human-derived phage scFV library was constructed with PBMCs of 200 healthy volunteers. According to library capacity evaluation, the natural human-derived phage scFV library had a library capacity of 1×10.sup.10.

[0150] PBMCs of an endometriosis subject and an in situ endometrial tissue obtained from surgery were taken as a control cell/tissue, and then three rounds of negative screening were conducted to remove phage capable of binding to the control cell/tissue in the natural human-derived phage scFV library. A phage antibody library obtained after the screening was subjected to amplification, and then an antibody gene library was obtained through a general genetic engineering technology. As tested, the library capacity did not change significantly.

[0151] PBMCs of the above endometriosis subject were taken and then T lymphocytes were further isolated to further prepare a cell library, and CARs and genetic circuits were constructed according to the same method as in Example 1. The same genetic circuit construction as in Example 1 was adopted. Two controlled gene expression cassettes for the genetic circuits were shown in FIG. 2. The obtained natural fully human CAR-T cell library targeting an ectopic endometrium was named endo-KRAB-iCasp9-CAR-hT, which had a library capacity of 3×10.sup.5.

EXAMPLE 17

Treatment of Human Endometriosis with the Natural Fully Human CAR-T Cell Library Targeting an Ectopic Endometrium

[0152] The above endo-KRAB-iCasp9-CAR-hT library was administered intravenously to the endometriosis subject in Example 16. The general administration route and dose for CAR-T cells were adopted.

EXAMPLE 18

CAR NK-92 Cell Library including 495 Artificial Antibodies

[0153] A scFV library was first constructed through total synthesis. The antibody library was constructed from the following 495 mAbs: 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, and ZOLIMOMAB.

[0154] NK-92 cells were adopted to further construct a cell library. CARs and intracellular genetic circuits were constructed according to the same method as in Example 1 to obtain the CAR NK-92 cell library including 495 artificial antibodies. The obtained CAR NK-92 cell library was named 495-KRAB-iCasp9-CAR-NK92, which had a library capacity of 495.

EXAMPLE 19

Immunotherapy of Human Pancreatic Cancer with the CAR NK-92 Cell Library including 495 Artificial Antibodies

[0155] The above 495-KRAB-iCasp9-CAR-NK92 library was administered intravenously to a pancreatic cancer subject. The general administration dose, frequency, and route for cell therapy were adopted.

EXAMPLE 20

Example Overview of Vector Assembly Construction Methods

[0156] (1) Construction Samples of a First Genetic Element

[0157] The first genetic element is a CAR library. CAR is an artificial receptor, and the construction of the CAR library can be found in the patent document WO2015/123642. The present disclosure emphasizes the use of a randomized library of extracellular recognition domains of CARs to realize the applications of specific CARs. Examples of construction methods are as follows.

[0158] Construction as follows: A human antibody gene library is artificially constructed through DNA synthesis according to antibody coding rules of human genes. According to a CAR scheme, a human scFV gene library CD8 hinge-CD8TM-4-1BB-CD3ζ shown in FIG. 8A is constructed.

[0159] Construction as follows: A human antibody gene library is artificially constructed through DNA synthesis according to antibody coding rules of human genes. The gene library is constructed into a phage vector, and then the phage is amplified to obtain a phage antibody library. If the screening is to be conducted in an animal model, the phage antibody library is administered to the animal model, and the phage capable of binding to an antigen of the animal model is subtracted to obtain a phage antibody sub-library; and then an antibody gene library of the sub-library is obtained by a genetic engineering method. According to a CAR scheme, a scFV library IgG4-hinge-CD28TM-CD28-4-1BB-CD3ζ shown in FIG. 8B is constructed. If it is to be directly used in a subject, a control tissue of the subject (such as PBMCs and a paracancerous tissue of the subject) is collected, and the phage capable of binding to the control tissue is subtracted to obtain a phage antibody sub-library; and then an antibody gene library of the sub-library is obtained by a genetic engineering method. According to a CAR scheme, a Fab antibody library CD8-hinge-CD28TM-CD28-CD27-CD3ζ shown in FIG. 8C is constructed.

[0160] Construction as follows: An antibody gene library is constructed by a genetic engineering method with PBMCs of multiple healthy volunteers, then the gene library is constructed into a yeast vector, and the yeast is amplified to obtain a yeast antibody library. According to a CAR scheme, a human antibody gene library 2D3-CD137TM-4-1BB-CD3ζ shown in FIG. 8D is constructed.

[0161] Construction as follows: A camel antibody gene library is constructed by a genetic engineering method with PBMCs of a camel, and according to a CAR scheme, a camel antibody gene library CD8-hinge-CD8TM-4-1BB-CD3ζ shown in FIG. 8E is constructed.

[0162] Construction as follows: An antibody gene library is constructed by a genetic engineering method with PBMCs of multiple healthy volunteers, then the gene library is constructed into a yeast vector, and the yeast is amplified to obtain a yeast antibody library. The tumor cell line MDA-MB-231 is allowed to contact the antibody library, and yeast cells capable of binding to the tumor cells are screened out. A sub-library of the antibody gene library in the yeast cells is obtained by a genetic engineering method, then an antibody library is re-constructed by genetic engineering methods of CDR mutagenesis, affinity maturation, and strand exchange, and according to a CAR scheme, a genetically-engineered scFV library CD8 hinge-CD8TM-4-1BB-CD3ζ shown in FIG. 8F is constructed.

[0163] (2) Construction Samples of a Second Genetic Element

[0164] The second genetic element is a genetic circuit. Construction as follows: 6 NFAT-responsive elements and a minimal IL-2 promoter are fused (6×NFAT), as shown in FIG. 8G.

[0165] Construction as follows: 4 NFAT-responsive elements and a minimal IL-2 promoter are fused (4×NFAT), and Gal4-KRAB is included downstream of a resulting fusion; and then a 5×UAS-P.sub.SV40 fusion promoter regulated by Gal4-KRAB is constructed, as shown in FIG. 8H.

[0166] Construction as follows: 6 NFAT-responsive elements and a minimal IL-2 promoter are fused (6×NFAT), and the transcription factor TetR-KRAB is included downstream of a resulting fusion; and then a 7×TRE-P.sub.SV40 fusion promoter regulated by TetR-KRAB is constructed, as shown in FIG. 8I.

[0167] Construction as follows: 10 NFκB-binding elements and a minimal HIVtata promoter are fused (10×NFκB), and the transcription factor TetR-VP64 is included downstream of a resulting fusion; and then 7 TREs and a minimal CMV promoter are fused to obtain a fusion promoter regulated by TetR-VP64 (7×TRE-P.sub.CMV-min), as shown in FIG. 8J.

[0168] Construction as follows: 10 NFκB-binding elements, 6 NFAT-responsive elements, and a minimal IL-2 promoter are fused (10×NFκB+6×NFAT), and the transcription factor ZFHD1-VP64 is included downstream of a resulting fusion; and then a 4×ZFHD1RE-P.sub.CMV-min promoter regulated by ZFHD1-VP64 is constructed, as shown in FIG. 8K.

[0169] (3) Construction Samples of a Third Genetic Element

[0170] The third genetic element is a genetic element encoding a protein, and the protein may include a puromycin resistance protein (PuroR), a neomycin resistance protein (NeoR), a blasticidin resistance protein (Blasticidin-R), a hygromycin B resistance protein (Hygromycin B-R), an HSV-TK protein, a CD protein, or an iCasp9 suicide system protein.

[0171] (4) Examples of Vector Assembly

[0172] A vector assembly including three genetic elements can be constructed as follows: (i) A human antibody gene library is artificially constructed according to antibody coding rules of human genes. According to a CAR scheme, a human scFV gene library CD8 hinge-CD8TM-4-1BB-CD3 is constructed, that is, a first genetic element shown in FIG. 2A is constructed. (ii) 6 NFAT-responsive elements and a minimal IL-2 promoter (6×NFAT) are fused to construct a second genetic element shown in FIG. 2G. (iii) A third genetic element encoding a puromycin resistance protein (PuroR) is constructed. These elements are inserted into a vector shown in FIG. 9A as a whole, and the vector may include another regulatory element described in the prior art, such as a CMV promoter.

[0173] The vector assembly can also be constructed as follows: (i) A human antibody gene library is artificially constructed through DNA synthesis according to antibody coding rules of human genes. The gene library is constructed into a phage vector, and then the phage is amplified to obtain a phage antibody library. If the screening is to be conducted in an animal model, the phage antibody library is administered to the animal model, and the phage capable of binding to an antigen of the animal model is subtracted to obtain a phage antibody sub-library; and then an antibody gene library of the sub-library is obtained by a genetic engineering method. According to a CAR scheme, a scFV library IgG4-hinge-CD28TM-CD28-4-1BB-CD3ζ is constructed, that is, a first genetic element shown in FIG. 2B is constructed. (ii) 4 NFAT-responsive elements and a minimal IL-2 promoter (4×NFAT) are fused to obtain a fusion promoter with Gal4-KRAB downstream, then a 5×UAS-P.sub.SV40 fusion promoter regulated by Gal4-KRAB is constructed, and then a second genetic element shown in FIG. 2H is constructed with the fusion promoters. (iii) A third genetic element encoding an HSV-TK protein is constructed. These elements are inserted into two vectors shown in FIG. 9B as a whole.

[0174] The vector assembly can also be constructed as follows: (i) An antibody gene library is constructed by a genetic engineering method with PBMCs of multiple healthy volunteers, then the gene library is constructed into a yeast vector, and the yeast is amplified to obtain a yeast antibody library. The tumor cell line MDA-MB-231 is allowed to contact the antibody library, and yeast cells capable of binding to the tumor cells are screened out. A sub-library of the antibody gene library in the yeast cells is obtained by a genetic engineering method, then an antibody library is re-constructed by genetic engineering methods of CDR mutagenesis, affinity maturation, and strand exchange, and according to a CAR scheme, a genetically-engineered scFV library CD8 hinge-CD8TM-4-1BB-CD3ζ is constructed, that is, a first genetic element shown in FIG. 2F is constructed. (ii) 10 NFκB-binding elements and a minimal HIVtata promoter are fused to obtain a fusion promoter (10×NFκB) with the transcription factor TetR-VP64 downstream, then 7 TREs and a minimal CMV promoter are fused to obtain a fusion promoter regulated by TetR-VP64 (7×TRE-P.sub.CMV-min), and then a second genetic element shown in FIG. 2J is constructed with the fusion promoters. (iii) A third genetic element encoding a hygromycin B resistance protein (Hygromycin B-R) is constructed. These elements are inserted into three vectors shown in FIG. 9C as a whole.

EXAMPLE 21

Treatment of a Subject Exposed to an Unknown Pathogen

[0175] An individualized CAR cell library was first constructed. An antibody gene library was constructed by a genetic engineering method with PBMCs of healthy volunteers, and according to a CAR scheme, an individualized scFV library CD8 hinge-CD8TM-4-1BB-CD3ζ was constructed, as shown in FIG. 10A. A second genetic element including 6xNFAT with the transcription factor Gal4-KRAB downstream and 5×UAS-P.sub.SV40 regulated by Gal4-KRAB was constructed. A third genetic element iCasp9 was constructed, and a vector was constructed according to the manner in FIG. 10B. Other regulatory elements described in the prior art on the vector, such as a CMV promoter, a 2A cleaving peptide, and a green fluorescent protein, can also be seen in FIG. 10A and FIG. 10B. The above vector was transfected into T cells of a subject to obtain the CAR cell library, and the CAR cell library was cryopreserved.

[0176] In a public safety emergency or bioterrorism attack, people are exposed to unknown pathogens or toxins. The exposure is in many different modes, such as food or water intake, aerosol inhalation, or skin contact. The pathogens may include Bacillus anthracis (anthrax), influenza virus, smallpox virus, Yersinia pestis (pestis), Ebola or Marburg virus, Francisella tularensis (tularemia), Hantavirus, dengue virus, cholera toxin, botulinum toxin, ricin toxin, Salmonella, Escherichia coli (E. coli) such as E. coli 0157:H7, Shigella, and Listeria, for example. When a threatening microbe has not been identified, some patients have developed severe illness with similar symptoms, including high fever, chills, cough, severe fatigue, and diarrhea. Patients can receive a standard therapy, such as administration of an antiviral drug, an antibiotic, an antitoxin, and an immunoglobulin. The CAR cell library and the composition of the present disclosure can be intravenously administered to a patient with infection symptoms and inflammation signs (such as fever and chills) as a preventive measure or a treatment means for the patient, such as a composition with 3×10.sup.9 CAR cells as an active component. Once distributed into a body fluid (especially blood), the cell library can clear lesion cell and inflammatory mediators. By removing these disease-associated mediators, the cell library reduces the triggers of additional systemic inflammation in the patient and reduces the generation of systemic inflammatory mediators such as cytokines, thereby preventing or limiting the cell death, organ damage, multiple-organ failure (MOF), and potential death induced by cytokines or other inflammatory mediators. The cell library may be administered once or repeatedly over a period of hours to days to achieve a persistent or stable effect.

[0177] At an appropriate time, an inducer was administered to a patient, then CAR-inactivated library cells were removed, and a cell library was enriched from the patient. An extracellular recognition domain of CAR was obtained by a genetic engineering method, and accordingly, an antibody, a CAR, and an engineered cell were further prepared and used in the treatment of other patients.

EXAMPLE 22

Administration of a CAR Cell Library to an IBD Patient

[0178] A healthy human-derived CAR cell library was first constructed. An antibody gene library was constructed by a genetic engineering method with PBMCs of at least 100 healthy volunteers. Then a phage antibody library was constructed, and with PBMCs of the patient as a control tissue, the background was subtracted. Then an antibody gene library was obtained by an antibody engineering method. According to a CAR scheme, a healthy volunteer-derived scFV gene library IgG4 hinge-CD28TM-CD28-4-1BB-CD3ζ was constructed, as shown in FIG. 10C. A second genetic element including 4×NFAT with the transcription factor Gal4-KRAB downstream and 5×UAS-P.sub.SV40 regulated by Gal4-KRAB was constructed. A third genetic element HSV-TK was constructed, and a vector was constructed according to the manner in FIG. 10D. Other regulatory elements described in the prior art on the vector can also be seen in FIG. 10C and FIG. 10D. The above vector was transfected into T cells of the patient to obtain the CAR cell library, and the CAR cell library was cryopreserved.

[0179] When the IBD patient worsened to persistent severe diarrhea, the patient was administered with a standard therapy, including systemic corticosteroid and parenteral TNF blocker therapies. As a part of anti-inflammation therapy for the patient, an effective dose of the cell library was administered to the patient, such as a composition with 3×10.sup.9 CAR cells as an active component. The cell library cleared abnormal cells and neutralized inflammatory mediators generated locally (including cytokines generated in an intestinal tract), which promoted the healing of bowels and prevented the recurrence.

EXAMPLE 23

Administration of a Cell Library to a Burn Patient

[0180] A burn patient suffering from severe burns of more than 30% of his total body surface area (TBSA) and lung damage caused by prolonged smoke and chemical inhalation was selected. Although the debridement was conducted, the patient still developed severe SIRS and ARDS, requiring mechanical ventilation. The patient was administered with a standard therapy, mainly including a supportive care measure.

[0181] T cells of the patient were collected, the background was subtracted according to the method in Example 22, and then a CAR cell library was constructed. In response to systemic inflammation capable of rapidly causing MOF, the patient was administered with a composition including 3×10.sup.9 CAR cells as an active component to inhibit the systemic inflammation. The cell library can remove systemic inflammatory mediators that can result in the production of more inflammatory mediators.

EXAMPLE 24

Administration of a Cell Library to an Endometritis Patient

[0182] An endometritis patient with infertility was selected, whose conditions were not improved after the treatment with antibiotics, hormones, and traditional Chinese medicines (TCMs).

[0183] T cells of the patient were collected, the patient background was subtracted according to the method in Example 22, and then a CAR cell library was constructed. In response to endometritis, the patient was administered with a composition including 3×10.sup.9 CAR cells as an active component to inhibit the endometritis. After a therapeutic effect was achieved, the patient was administered with an inducer to remove the cell library.

EXAMPLE 25

Administration of a Cell Library to an Anti-Aging Subject

[0184] A subject who wished to remove aging cells and achieve an anti-aging effect was selected.

[0185] T cells of the subject were collected, the subject background was subtracted according to the method in Example 21, and then a CAR cell library was constructed.

[0186] In response to anti-aging, the patient was administered with a composition including 3×10.sup.7 CAR cells as an active component to achieve the anti-aging effect. Skin, biochemical indexes, and the like were detected after the treatment. After a therapeutic effect was achieved, the patient was administered with an inducer to remove the cell library. One month later, the skin elasticity of the patient was enhanced, and the liver function of the patient was significantly improved.

EXAMPLE 26

Administration of a Cell Library to an Alzheimer's Disease Patient

[0187] A high-risk Alzheimer's disease potential patient in a healthy state was selected, and an individualized CAR cell library was first constructed. According to a CAR scheme, an individualized scFV library CD8 hinge-CD8TM-4-1BB-CD3ζ was constructed, as shown in FIG. 10A. A second genetic element including 6×NFAT with the transcription factor Gal4-KRAB downstream and 5×UAS-P.sub.SV40 regulated by Gal4-KRAB was constructed. A third genetic element iCasp9 was constructed, and a vector was constructed according to the manner in FIG. 10B. Other regulatory elements described in the prior art on the vector, such as a CMV promoter, a 2A cleaving peptide, and a green fluorescent protein, can also be seen in FIG. 10A and FIG. 10B. The above vector, a Sleeping Beauty (SB) transposon vector, was transfected into T cells of a subject by an electrochemical method to obtain the CAR cell library, and the CAR cell library was cryopreserved.

[0188] After a period of time, the patient developed Alzheimer's disease, which could not be alleviated by existing supportive treatments and continued to deteriorate. An effective dose of the cell library was intradurally injected into the patient, such as a composition with 1×10.sup.5 CAR cells as an active component. The cell library cleared abnormal neurons and reduced neurological symptoms. After a therapeutic effect was achieved, the patient was administered with an inducer to remove the cell library.

EXAMPLE 27

Administration of a Cell Library to an Infertility Patient

[0189] A patient diagnosed with immune infertility that could not be cured with the existing methods was selected. T cells of the patient were collected, the patient background was subtracted according to the method in Example 21, and then a CAR cell library was constructed. In response to abnormal immune factors in the utero, the patient was administered with a composition including 3×10.sup.7 CAR cells as an active component to remove abnormal immune mediators. At an appropriate time, the patient was administered with an inducer to remove the cell library. The patient eventually became pregnant.

EXAMPLE 28

Administration of a Cell Library to a Liver Fibrosis Patient

[0190] A patient who had suffered from hepatitis B 10 years ago and was diagnosed with partial liver fibrosis was selected, and the disease failed to be cured with multiple anti-fibrosis treatments and continued to progress. T cells of the patient were collected, the patient background was subtracted according to the method in Example 22, and then a CAR cell library was constructed. In response to abnormal immune factors in the liver, the patient was administered with a composition including 3×10.sup.7 CAR cells as an active component to remove abnormal intrahepatic immune mediators and diseased cells. After a therapeutic effect was achieved, the patient was administered with an inducer to remove the cell library. The degree of fibrosis in the patient was successfully delayed.

EXAMPLE 29

Administration of a Cell Library to a Chronic Pelvic Inflammatory Disease (CPID) Patient

[0191] A CPID patient with infertility was selected, whose conditions were not improved after the treatment with antibiotics, hormones, and TCMs.

[0192] T cells of the patient were collected, the patient background was subtracted according to the method in Example 22, and then a CAR cell library was constructed. In response to CPID, the patient was administered with a composition including 3×10.sup.9 CAR cells as an active component to inhibit the CPID. After a therapeutic effect was achieved, the patient was administered with an inducer to remove the cell library.

EXAMPLE 30

Cosmetic Repair of Multiple Skin Scars

[0193] A patient with multiple skin scars was selected, and the surgery, laser, and other treatments led to an insignificant therapeutic effect for the skin scars.

[0194] T cells of the patient were collected, the patient background was subtracted according to the method in Example 22, and then a CAR cell library was constructed. In response to the multiple skin scars of the patient, the patient was administered with a composition including 1×10.sup.6 CAR cells as an active component to treat the multiple skin scars. After a therapeutic effect was achieved, the patient was administered with an inducer to remove the cell library.

EXAMPLE 31

Treatment of Lumbar Hyperosteogeny

[0195] A healthy patient was selected, and an antibody gene library of his PBMCs was preventively deposited, with a library capacity of 1×10.sup.6. A few years later, the patient developed severe lumbar hyperosteogeny, which could not be treated with a variety of existing treatments. T cells of the patient were collected, and then a CAR cell library was constructed according to the construction method in Example 21 and the gene library of the patient deposited. The patient was administered with a composition including 1×10.sup.6 CAR cells as an active component to treat the lumbar hyperosteogeny. After a therapeutic effect was achieved, the patient was administered with an inducer to remove the cell library.

EXAMPLE 32

Construction of a Common Target Library

[0196] A library was constructed according to a CAR scheme, and a scFv library of the CAR library was a small-scale library including the binding targets of 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, phosphatidylserine, cMyc, CD4, CD44v6, CD45, CD28, CD3, CD3e, CD52, CD74, CD30, CD166, CD24, EGFR/HER3 fusions, carbohydrates, Aspergillus, Dectin, Ebolavirus, fungi, GP, HERV-K, VEGF-R2, TGF-2R, IgG4, biotin, O-AcGD2, Cadherin 2, OB-cadherin, α5β1 integrin, αVβ6 integrin, Syndecan-1, Cadherin 1, Claudin 12, Claudin 7, Claudin 3, and ZO-1. A small-scale scFv library IgG4 hinge-CD28TM-CD28-4-1BB-CD3ζ was constructed. A second genetic element including 4×NFAT with the transcription factor Gal4-KRAB downstream and 5×UAS-P.sub.SV40 regulated by Gal4-KRAB was constructed. A third genetic element HSV-TK was constructed. The above vector was transfected into NK-92 cells to obtain a CAR cell library, and the CAR cell library was cryopreserved.

EXAMPLE 33

Examples of In Vitro Screening

[0197] A variety of methods can be used to construct a CAR cell library. For example, a healthy human-derived CAR cell library is first constructed; an antibody gene library is constructed by a genetic engineering method with PBMCs of at least 100 healthy volunteers, and according to a CAR scheme, a healthy volunteer-derived scFv library IgG4 hinge-CD28TM-CD28-4-1BB-CD3ζ is constructed, as shown in FIG. 10C; a second genetic element including 4×NFAT with the transcription factor Gal4-KRAB downstream and 5×UAS-P.sub.SV40 regulated by Gal4-KRAB is constructed; a third genetic element HSV-TK is constructed, and a vector is constructed according to the manner in FIG. 10D, where other regulatory elements described in the prior art on the vector can also be seen in FIG. 10C and FIG. 10D; and the above vector is transfected into Jurkat cells to obtain a CAR cell library.

[0198] A target antigen is coated on a well plate, and then 1×10.sup.9 of Jurkat cells are added to each well; and at an appropriate time, an inducer is added to remove cells that do not bind to the antigen, the remaining jurkat cells are separated, and a CAR gene is obtained by a genetic engineering method for analysis.

[0199] The unexplained parts involved in the present disclosure are the same as the prior art or implemented by the prior art. The applicants declare that the detailed methods of the present disclosure are illustrated through the above examples, but the present disclosure is not limited to the above detailed methods, that is, it does not mean that the present disclosure must be implemented relying on the above detailed methods. Those skilled in the art should understand that any improvement to the present disclosure, equivalent replacement of each raw material of the product of the present disclosure, addition of auxiliary ingredients, selection of specific methods, and the like all fall within the protection scope and disclosure scope of the present disclosure.