Method for preparing chimeric antigen receptor libraries

Abstract

The invention discloses a method for preparing a chimeric antigen receptor library. In combination with an original construction mode of the CAR library, a segment of consensus sequence is arranged in front of and behind each structural domain part of the CAR structure, and random combination is performed in a Golden Gate mode to form the CAR molecular library with different consensus sequences in front of and behind each structural domain part. The different consensus sequences are subjected to random cleavage through a CRISPR technology, and more library combinations are generated.

Description

Technical Field

[0001] This application generally relates to the field of biotechnology, and particularly to a method for preparing a chimeric antigen receptor library and its uses. Background Art

[0002] CAR-T therapy involves removing a patient's T cells in vitro and introducing specific chimeric antigen receptors (CARs) through genetic engineering techniques. These receptors can recognize specific antigens on the surface of tumor cells. After the modified T cells are amplified in vitro, they are reinfused into the patient's body to recognize and kill cancer cells.

[0003] The CAR structure generally consists of three parts: an extracellular region (a single-chain antibody scFv responsible for recognizing and binding to the target antigen), a transmembrane region (TM, which anchors the CAR molecule to the cell membrane of T cells), and an intracellular signaling domain (ICD, one or two stimulatory signaling domains that provide activation signals). When the antigen is recognized and bound, a stimulatory signal is transmitted to the intracellular signaling domain, and the T cell is activated and exerts effector functions.

[0004] How to construct a rich and diverse CAR library for modifying T cells is an important problem that urgently needs to be solved in this field. Summary of the Invention

[0005] Based on this, the present application provides a method for preparing a chimeric antigen receptor library, including:

[0006] 1) Construct different consensus sequences between each different module of the CAR molecule to form a CAR construct, which includes an extracellular recognition domain, a transmembrane domain, one or more intracellular signaling domains, and one or more additional functional domains;

[0007] 2) Design different CRISPR systems targeting different consensus sequences between different modules of the CAR molecule, and use the CRISPR systems to randomly cleave the CAR construct;

[0008] 3) By adjusting the CRISPR system, obtain a CAR molecule library containing different CAR constructs.

[0009] In another aspect, the present application also provides the use of the method for preparing a chimeric antigen receptor library described herein in constructing a CAR molecule library.

[0010] In another aspect, the present application also provides a CAR molecule library obtained by the method for preparing a chimeric antigen receptor library described herein.

[0011] In another aspect, the present application also provides a nucleic acid sequence library encoding the CAR molecule library described herein.

[0012] On the other hand, the present application also provides a plasmid library for constructing a CAR molecular library as described herein.

[0013] On the other hand, the present application also provides a CAR cell library modified with the CAR molecular library described herein.

[0014] On the other hand, the present application also provides the use of the CAR molecular library described herein, the nucleic acid sequence library described herein, the plasmid library described herein, or the CAR cell library described herein in the preparation of a drug or kit for treating tumors, infectious diseases, or autoimmune diseases.

[0015] The present application combines the original construction method of the CAR library, sets a consensus sequence before and after each domain part of the CAR structure, and randomly combines them by the Golden Gate method to form a CAR molecular library in which the consensus sequences before and after each domain part are unique (the consensus sequences of different domains are different). Random shearing is performed on the different consensus sequences by CRISPR technology to generate more library combinations.

[0016] Other features and advantages of the present application will be described in the subsequent specification, and in part, will become apparent from the specification, or will be understood by implementing the present application. Other advantages of the present application can be achieved and obtained through the solutions described in the specification and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings are used to provide an understanding of the technical solutions of the present application, and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solutions of the present application, and do not constitute a limitation on the technical solutions of the present application.

[0018] Figure 1 It is a schematic diagram of the design and library preparation of the CAR structure in Example 1 of the present application.

[0019] Figure 2 It is a schematic diagram of the construction of the CAR library containing diverse suicide sequences in Example 2 of the present application.

[0020] Figure 3 It is a schematic diagram of the construction of the CAR library with optimized transmembrane domain in Example 3 of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application belongs. When a quantity, concentration, or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood that it specifically discloses any range formed by combining any pair of upper range limits or preferred numerical values with any lower range limit or preferred numerical value, regardless of whether the range is specifically disclosed. Unless otherwise specified, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0022] The terms "about" or "approximately", when used in combination with a numerical variable, generally refer to the numerical value of the variable and all numerical values of the variable within the experimental error (e.g., within the 95% confidence interval of the mean) or within ±10% of the specified numerical value, or within a wider range.

[0023] The expressions "comprising" or similar expressions synonymous therewith, such as "including", "containing", and "having", are open-ended and do not exclude additional unlisted elements, steps, or components. The expression "consisting of" excludes any unstated element, step, or component. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps, or components, plus optionally existing elements, steps, or components that do not substantially affect the basic and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

[0024] The expression "at least one" or "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0025] As used herein, when referring to "CAR molecules", "different modules of CAR molecules", or "CAR constructs", it refers to genes or mRNAs encoding chimeric antigen receptors (CARs) or their different domains.

[0026] During the process of CAR-T cell therapy, several of the most common problems are:

[0027] 1. Toxic and side effects: cytokine storm and neurotoxicity;

[0028] 2. Off-target effects: gene-modified T cells cause damage to normal tissues and organs that do not express the target molecule;

[0029] 3. Persistence: The retention time of CAR-T cells in the body is too short.

[0030] For 1 and 2, in addition to optimizing the CAR structure during design, increasing the accuracy of off-target detection, and reducing the generation of side effects by blocking excessive inflammatory responses with antibodies, when necessary, the method of reducing CAR should be adopted, such as designing a "suicide switch" to rapidly degrade CAR cells and reduce the immune response. However, at the same time, all CARs will be killed and the treatment process will stop.

[0031] For 3, the method of optimizing CAR is generally adopted, such as blocking the signals related to CAR-T cell exhaustion (which can be achieved by abolishing the TOX and NR4A transcription factors or blocking the antibody-mediated co-inhibitory receptor such as PD-1), etc.

[0032] In order to improve the existing CAR library to meet the requirements of the field for CAR-T therapy, in this application, different consensus sequences are inserted into each domain part of the CAR library, and CRISPR cleavage elements targeting different consensus sequences are designed. The existing library is randomly re-edited by random cleavage to generate a library of a higher magnitude.

[0033] This application provides a method for preparing a chimeric antigen receptor library, including:

[0034] 1) Different consensus sequences are respectively constructed between each different module of the CAR molecule to form a CAR construct, and the CAR construct includes an extracellular recognition domain, a transmembrane domain, one or more intracellular signaling domains, and one or more additional functional domains;

[0035] 2) Different CRISPR systems targeting different consensus sequences between different modules of the CAR molecule are designed, and the CRISPR systems are used to randomly cleave the CAR construct;

[0036] 3) By adjusting the CRISPR system, a CAR molecule library containing different CAR constructs is obtained.

[0037] "CRISPR system" is a highly precise gene editing tool that, through the cooperative action of Cas proteins and guide RNA (gRNA), locates and cleaves specific DNA sequences. Its core components include Cas nucleases for cleaving double-stranded or single-stranded DNA, and gRNA that guides the Cas nuclease to target and cleave the target DNA. The CRISPR / Cas nuclease or CRISPR / Cas nuclease system may include a non-coding RNA molecule (guide RNA) whose sequence specifically binds to DNA, and a Cas protein (such as Cas9) with nuclease function (such as two nuclease domains). One or more elements of the CRISPR system may be derived from type I, type II, or type III CRISPR systems. For example, they may be derived from a specific organism containing an endogenous CRISPR system, such as Streptococcus pyogenes. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, Cpf1 (Cas12a), their homologs, or their modified versions.

[0038] The CRISPR nuclease system can direct the cleavage of one or both strands at the position of the target sequence (such as within the target sequence and / or within the complement of the target sequence). In some embodiments, Cas9 nickase can be used in combination with guide sequences (such as two guide sequences that respectively target the sense strand and the antisense strand of the DNA target).

[0039] In some embodiments, the common sequence is constructed between each different module of the CAR molecule by the Golden Gate method, and the common sequence contains a sequence that can be cleaved by the CRISPR system. In some embodiments, the common sequence can also be constructed between each different module by other methods known in the art (such as molecular cloning, Gibson Assembly, etc.). The common sequence can be designed according to methods known in the art as long as it contains a sequence that can be cleaved by the CRISPR system. After inserting different common sequences between different modules of the CAR molecule, different CAR molecule modules can be cleaved by using different CRISPR systems. Moreover, by adjusting the types and concentrations of different CRISPR systems, a CAR molecule library containing CAR constructs with different CAR molecule modules cleaved off can be obtained.

[0040] In some embodiments, the extracellular recognition domain is an antibody or an antigen-binding fragment thereof; optionally, the extracellular recognition domain is an scFv.

[0041] As used herein, "scFv" or "single-chain variable fragment" includes a molecule in which the variable heavy chain (VH) and variable light chain (VL) of an antibody are linked by a flexible oligopeptide. Thus, an scFv is a fusion between at least one variable heavy chain and at least one variable light chain. The flexible oligopeptide that typically links the variable heavy and light chains can be 5 amino acids in length, particularly 8, 9, 10, or 11 amino acids to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length, such as 10 - 20, 12 - 18, or 14 - 17, or 14 - 16 amino acids in length. The flexible oligopeptide can contain glycine, serine, and / or threonine residues, and particularly can contain at least 50%, 60%, 70%, or 80% glycine residues. The flexible linker can generally link the C-terminus of the variable heavy chain to the N-terminus of the variable light chain, or the C-terminus of the variable light chain to the N-terminus of the variable heavy chain. Engineered antibodies, such as scFv antibodies, can be prepared using techniques and methods known in the art. An scFv can be bivalent / trivalent or tetravalent (other than monovalent), and an scFv can contain more than one variable heavy chain and more than one variable light chain, such as two, three, or four variable heavy chains or variable light chains. More than one variable heavy and / or variable light chain can be the same or can be from different antibodies. An scFv can be a diabody, triabody, or tetrabody. In this regard, the flexible linker used can be shorter than that used in the monovalent scFv above.

[0042] In some embodiments, a "transmembrane domain" or "transmembrane region" can be based on or derived from the transmembrane domain of any transmembrane protein. The transmembrane region can be from, for example, CD28, the alpha chain of the T cell receptor, the beta chain of the T cell receptor, the zeta chain of the T cell receptor, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, DAP10, DAP12, killer immunoglobulin-like receptor (KIR) such as KIR2DL2, LILRB1, LILRB2 or any combination thereof. In some embodiments, the transmembrane domain is the CD28 transmembrane domain. In some embodiments, the transmembrane domain is the CD8 transmembrane domain.

[0043] In other embodiments, the transmembrane domain can be synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine. Thus, the transmembrane domain is capable of spanning or being present within the cell membrane of a cell. As discussed above, the transmembrane domain can be derived from a protein that contains extracellular and / or intracellular portions, and thus in addition to the portion within or spanning the cell membrane, the transmembrane domain as used herein can be attached to extracellular and / or intracellular residues derived from the original protein. For example, the transmembrane domain can be attached to a hinge or spacer region derived from the originating protein, e.g., a transmembrane domain derived from CD8 alpha can be attached to a spacer region or hinge domain derived from CD8 alpha. Any suitable method known in the art can be used to assess the presence of the transmembrane domain within the cell membrane, including fluorescence labeling and fluorescence microscopy.

[0044] An "intracellular signaling domain", "intracellular signaling region" or "intracellular domain" refers to a portion of the CAR protein that is involved in transducing the information of the effective binding of the CAR to the target antigen into the interior of the cell (host cell, e.g., an immune effector cell) to initiate a cellular function (e.g., effector cell function), e.g., activation of cytokine production, increased cytotoxic activity (including release of cytotoxic factors into the target cell bound by the CAR), or other cellular responses caused by antigen binding to the extracellular CAR domain.

[0045] The term "effector function" refers to the specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or help or activity including cytokine secretion. Thus, an "effector cell" is a cell having such effector functions. Accordingly, the term "intracellular signaling domain" refers to a portion of a protein that transduces signals for effector function and directs a cell to perform a specialized function. While the entire intracellular signaling domain of a naturally occurring protein can be used in the present invention, in many cases, it is not necessary to use the entire domain. In terms of using variants, such as truncated portions of a naturally occurring intracellular signaling domain, such variants (e.g., truncated portions) can be used in place of the entire domain as long as it transduces signals for effector function, e.g., has at least 50, 60, 70, 80, 90 or 95% of the ability of the full-length domain to transduce effector function. Compared to the full-length intracellular signaling domain, a variant (e.g., truncated) intracellular signaling domain can further have an increased ability to transduce signals for effector function, e.g., at least 105, 110, 120, 130 or 140% of the ability to transduce effector function. The ability to transduce effector function can be measured by measuring the effector function of a cell after interaction with a target, e.g., by measuring cytokine release, cell proliferation, etc. Thus, the term intracellular signaling domain means any truncated portion of an intracellular signaling domain that is sufficient to transduce signals for effector function.

[0046] A variant intracellular signaling domain can have at least 70%, 80%, 90% or 95% sequence identity with a naturally occurring intracellular signaling domain. It should be understood that if a truncated domain is used, the % sequence identity can be less than 70% compared to the full-length sequence. The intracellular signaling domain is also referred to as the "intracellular signal domain" and generally derives from a portion of the human CD3ζ or FcRy chain.

[0047] In some embodiments, the intracellular domain can be the intracellular domain from, for example, CD3ζ, CD27, CD28, 4-1BB, DAP12, NKG2D, OX-40 (CD134), DAP10, CD40L, 2B4, DNAM, CS1, CD48, NKp30, NKp44, NKp46 or NKp80 or any combination thereof. In some embodiments, the intracellular domain is the CD3ζ intracellular domain. In some embodiments, the intracellular domain is the CD28 intracellular domain. In some embodiments, two or more intracellular domains include the CD3ζ intracellular domain and an additional intracellular domain selected from the CD28, DAP10, DAP12, 4-1BB, NKG2D and 2B4 intracellular domains. In certain cases, two or more intracellular domains include the CD3ζ intracellular domain and the CD28 intracellular domain.

[0048] In some embodiments, the CAR constructs described herein further comprise additional functional domains. In some embodiments, one or more functional domains are used, such as therapeutic proteins and / or proteins that enhance cell activity, proliferation, and / or persistence. In some embodiments, the additional functional domains are suicide genes, cytokines, and / or human or viral proteins that enhance proliferation, expansion, and / or metabolic fitness. In certain embodiments, the additional functional domain is a cytokine, such as IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, or IL-7. In one specific embodiment, the cytokine is IL-15.

[0049] In some embodiments, the term "suicide gene" as used herein is defined as a gene that converts a gene product into a compound that kills its host cell upon administration of a prodrug or other agent. In some embodiments, the suicide gene encodes a gene product that is targeted by an agent (such as an antibody) that targets the suicide gene product when needed.

[0050] Examples of suicide gene / prodrug combinations that can be used are herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytarabine. Escherichia coli purine nucleoside phosphorylase can be used, which is a suicide gene that converts the prodrug 6-methylpurine deoxynucleoside into the toxic purine 6-methylpurine. Other examples of suicide genes used in prodrug therapy are the Escherichia coli cytosine deaminase gene and the HSV thymidine kinase gene.

[0051] Exemplary suicide genes also include CD20, CD52, EGFRv3, or inducible caspase 9 (iCasp9). Additional suicide genes known in the art that can be used in the present disclosure include purine nucleoside phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), xanthine-guanine ribosyltransferase (XGRTP), glycosidase, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0052] In some embodiments, the CAR construct comprises multiple functional domains, for example, multiple cytokines and multiple suicide genes, which can form a library of CAR molecules containing CAR constructs with different cytokines and suicide genes upon cleavage by the CRISPR system.

[0053] In some embodiments, after being cleaved by the CRISPR system, the CAR construct may comprise one or more different cytokines and / or one or more different suicide genes. Cells transformed with a library of CAR molecules comprising different CAR constructs can be killed with different prodrugs as needed, one or more of them.

[0054] In some embodiments, the transmembrane domain further comprises a hairpin structure. The length of the transmembrane domain determines the rigidity of the CAR molecule. By designing a hairpin structure, the originally longer transmembrane domain can be shortened. If its flexibility needs to be increased subsequently, a nucleic acid sequence that can open the hairpin structure (a specific nucleic acid sequence can be complementary paired with the loop region of the hairpin structure, thereby triggering the opening of the hairpin structure) is added to make it longer, so as to increase the extracellular sequence length, increase the flexibility of the scFv, and increase its recognition performance.

[0055] On the other hand, the present application also provides the use of the method for preparing a library of chimeric antigen receptors described herein in constructing a library of CAR molecules.

[0056] On the other hand, the present application also provides a library of CAR molecules obtained by the method for preparing a library of chimeric antigen receptors described herein.

[0057] On the other hand, the present application also provides a library of nucleic acid sequences encoding the library of CAR molecules described herein.

[0058] On the other hand, the present application also provides a library of plasmids for constructing a library of CAR molecules as described herein.

[0059] On the other hand, the present application also provides a library of CAR cells modified with the library of CAR molecules described herein.

[0060] On the other hand, the present application also provides the use of the library of CAR molecules described herein, the library of nucleic acid sequences described herein, the library of plasmids described herein, or the library of CAR cells described herein in the preparation of drugs or kits for treating tumors, infectious diseases or autoimmune diseases.

[0061] The present application describes multiple embodiments, but the description is exemplary rather than restrictive, and it will be apparent to those of ordinary skill in the art that there can be more embodiments and implementation schemes within the scope of the embodiments described in the present application. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically restricted, any feature or element of any embodiment can be combined with any other feature or element in any other embodiment, or can replace any other feature or element in any other embodiment.

[0062] This application includes and contemplates combinations of features and elements known to those of ordinary skill in the art. The embodiments, features, and elements disclosed in this application can also be combined with any conventional features or elements to form unique inventive solutions. Any feature or element of any embodiment can also be combined with features or elements from other inventive solutions to form another unique inventive solution. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented alone or in any suitable combination. Therefore, the embodiments are not subject to other limitations except those made in accordance with the appended claims and their equivalents. In addition, various modifications and changes can be made within the scope of the appended claims.

[0063] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, to the extent that the method or process does not depend on the specific order of the steps described herein, the method or process should not be limited to the specific order of steps described. As will be understood by those of ordinary skill in the art, other step sequences are possible. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation on the claims. In addition, the claims directed to the method and / or process should not be limited to performing their steps in the order written, and those skilled in the art can easily understand that these orders can be varied and still remain within the spirit and scope of the embodiments of this application.

[0064] For the experimental methods without specific conditions noted in the following examples, they are generally determined according to national standards. The experimental materials not indicated the source in the following examples are all commercially available raw materials. The equipment used in each step of the following examples are all conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer. Unless otherwise defined or stated, all professional and scientific terms used in this application have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the methods of this application.

[0065] Examples

[0066] Example 1. Design and Library Preparation of CAR Structure

[0067] Example 1 of this application provides a CAR structure (TM + ICD1 + ICD2 + cytokine), and each domain is constructed by the Golden Gate connection method, so that there are different common sequences between each domain.

[0068] See Figure 1 , and the structure of the CAR is described as follows:

[0069] From left to right are the overlapping regions TM-F and TM library of 30 - 45 nt, the overlapping region TM-ICD1 and ICD1 library of 30 - 45 nt, the overlapping region ICD1-ICD2 (region A) of 30 - 45 nt, the ICD2 library, the overlapping region ICD2-cytokine (region B) of 30 - 45 nt, the cytokine library, and the overlapping region cytokine-R of 30 - 45 nt.

[0070] CRISPR cleavage libraries were designed respectively to identify the characteristic sequence CRI1 of region A and the characteristic sequence CRI2 of region B.

[0071] When CRISPR cleavage elements targeting CRI1 and CRI2 are introduced simultaneously and the reaction time is controlled (unsaturated), three major types of sequences can be probabilistically obtained: the uncut group (fourth-generation CAR, TM + ICD1 + ICD2 + cytokine), the CAR after region B is cut off (third-generation CAR, TM + ICD1 + ICD2), and the CAR after region A is cut off (second-generation CAR, TM + ICD1). This significantly increases the variety of the library at one time.

[0072] Example 2. Construction of a CAR library containing diverse suicide sequences

[0073] Different suicide sequences were designed on the CAR (for example, Figure 2 contains 3 suicide sequences, namely recognition sequences 1 - 3). Using a principle similar to that in Example 1, the recognition sequences are equivalent to the domains in Example 1, combined by the golden gate method, and randomly cleaved by crisper targeting the common sequence to obtain different categories of CAR sequences containing different suicide sequences. n different types of CAR sequences can be designed, and for different CAR sequence categories, prodrugs targeting different suicide sequences can be used (for example, CRI1 - CRI3 in Figure 2 ). CRI1 can only kill the first category, CRI2 can target the first and second categories... CIRn can target categories 1 to n.

[0074] For example, see Figure 2, the recognition sequence 1 corresponds to the suicide sequence of the iCasp9 system, the recognition sequence 2 corresponds to the suicide sequence of the CD20 system, and the recognition sequence 3 corresponds to the suicide sequence of the RQR8 (Rituximab-CD34 Quasi-Receptor 8) system. Different from Example 1, CRI1-CRI3 represent 3 different prodrugs for suicide genes. CRI1 is rituximab, CRI2 is rituximab, and CRI3 is the drug AP1903. Using a principle similar to that in Example 1, the recognition sequences in Example 2 are equivalent to the domains in Example 1, combined by the golden gate method, and randomly sheared by crisper for the consensus sequence. We can obtain three major categories of CAR molecules, namely those with three suicide sequences, two suicide sequences, and one suicide sequence. In the first stage, for mild side effects, "add CRI1 with recognition sequence 3", specifically add rituximab that recognizes the suicide sequence of the RQR8 system, which can kill cells containing the first type of CAR (i.e., cells with CARs having three suicide sequences). In the second stage, for moderate side effects, "add CRI2 with recognition sequence 2", specifically add rituximab that recognizes the suicide sequence of the CD20 system, which can further kill cells containing the first type and the second type of CAR (i.e., cells with CARs having three suicide sequences and cells with CARs having two suicide sequences). In the third stage, for strong side effects, "add CRI3 with recognition sequence 1", specifically add the drug AP1903 that recognizes the suicide sequence of the iCasp9 system, which can kill all types of cells (i.e., cells with CARs having three suicide sequences, cells with CARs having two suicide sequences, and cells with CARs having one suicide sequence).

[0075] Example 3. Construction of a CAR library with optimized transmembrane domains

[0076] The length of the transmembrane domain determines the rigidity of the CAR molecule. By designing a hairpin structure, the originally longer transmembrane domain is shortened. Subsequently, if its flexibility needs to be increased, a nucleic acid sequence that can open the hairpin structure is added (a specific nucleic acid sequence can complementarily pair with the loop region of the hairpin structure, thereby triggering the opening of the hairpin structure), making it longer to increase the extracellular sequence length, increase the flexibility of the scFv, and increase its recognition performance (see Figure 3 ).

[0077] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present application.

Claims

1. A method for preparing a chimeric antigen receptor library, characterized in that, Comprising: 1) Constructing different consensus sequences between each different module of the CAR molecule to form a CAR construct, the CAR construct comprising an extracellular recognition domain, a transmembrane domain, one or more intracellular signaling domains, and one or more additional functional domains; 2) Designing different CRISPR systems for different consensus sequences between different modules of the CAR molecule and randomly cleaving the CAR construct using the CRISPR systems; 3) Obtaining a CAR molecule library comprising different CAR constructs by modulating the CRISPR systems.

2. The method according to claim 1, wherein The consensus sequences are constructed between each different module of the CAR molecule by the Golden Gate method, and the consensus sequences contain sequences that can be cleaved by the CRISPR systems.

3. The method according to claim 1 or 2, characterized in that, The extracellular recognition domain is an antibody or an antigen-binding fragment thereof; optionally, the extracellular recognition domain is an scFv.

4. The method according to any one of claims 1 to 3, characterized in that The transmembrane domain is a transmembrane domain from CD28, the α chain of the T cell receptor, the β chain of the T cell receptor, the ζ chain of the T cell receptor, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, DAP10, DAP12, or any inhibitory or activating KIR.

5. The method according to any one of claims 1 to 4, characterized in that, The intracellular signaling domain is an intracellular domain from CD3ζ, CD27, CD28, 4-1BB, DAP12, NKG2D, OX-40 (CD134), DAP10, CD40L, 2B4, DNAM, CS1, CD48, NKp30, NKp44, NKp46, or NKp80.

6. The method according to any one of claims 1-5, characterized in that, The additional functional domain is a suicide gene, a cytokine, or a human or viral protein that enhances proliferation, expansion, and / or metabolic fitness.

7. The method according to claim 6, wherein The cytokine is IL-15, IL-2, IL-12, IL-18, IL-21, IL-23, or IL-7.

8. The method according to claim 6, characterized in that, The suicide gene is CD20, CD52, EGFRv3, or inducible caspase 9.

9. The method according to any one of claims 1-8, characterized in that, The transmembrane domain further comprises a hairpin structure.

10. Use of the method according to any one of claims 1-9 in constructing a CAR molecule library.

11. A CAR molecule library constructed by the method according to any one of claims 1-9.

12. A nucleic acid sequence library encoding the CAR molecule library according to claim 11.

13. A plasmid library for constructing the CAR molecule library according to claim 11.

14. A library of CAR cells modified with the CAR molecule library according to claim 11.

15. Use of the CAR molecule library according to claim 11, the nucleic acid sequence library according to claim 12, the plasmid library according to claim 13 or the CAR cell library according to claim 14 in the preparation of a medicament or a kit for treating tumors, infectious diseases or autoimmune diseases.

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