CHIMERIC ANTIGEN RECEPTOR COMPRISING THIRD SIGNAL RECEPTOR AND USE THEREOF

Abstract

The present invention relates a chimeric antigen receptor, which has a structure of X-Y-CD3zeta-M-N; wherein X comprises a tumor targeting antibody or a ligand or receptor capable of specifically binding to a tumor. Y is an intracellular region of a costimulatory receptor selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, and CD226; M is an intracellular region of a gamma chain family cytokine receptor, the cytokine receptor being selected from IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, and IL21Ra. N is an intracellular region of IL2Rg. The present invention further provides a CAR-T cell constructed from the recombinant expression vector of said chimeric antigen receptor, a preparation method therefor and the use thereof. The CAR-T cell of the present invention significantly improves tumor killing capacity and amplification capacity. The CAR T cell comprises a third signal receptor, has a potential effect-enhancing function, and only works on the CAR-T cell, thereby reducing the risk of causing an immune side effect.

Claims

1. A chimeric antigen receptor comprising a third signal receptor, wherein said chimeric antigen receptor comprises a structure of X-Y-CD3zeta-M-N; wherein, X comprises a tumor-targeting antibody or a ligand or receptor capable of specifically binding to a tumor; Y is an intracellular domain of a costimulatory receptor, said costimulatory receptor is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226; M is an intracellular domain of a gamma chain family cytokine receptor, said cytokine receptor is selected from IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, IL21Ra; and N is an intracellular domain of IL2Rg.

2. The chimeric antigen receptor comprising the third signal receptor according to claim 1, wherein said antibody comprises scFv.

3. The chimeric antigen receptor comprising the third signal receptor according to claim 1, wherein said X is selected from anti-CD19 antibody, anti-CD20 antibody, anti-EGFR antibody, anti-HER2 antibody, anti-EGFRVIII antibody, anti-PSMA antibody, anti-BCMA antibody, anti-CD22 antibody, anti-CD30 antibody and anti-CLDN18.2 antibody.

4. The chimeric antigen receptor comprising the third signal receptor according to claim 1, wherein said X is selected from anti-CD19 scFv, anti-CD20 scFv, anti-EGFR scFv, anti-HER2 scFv, anti-EGFRVIII scFv, anti-PSMA scFv, anti-BCMA scFv, anti-CD22 scFv, anti-CD30 scFv and anti-CLDN18.2 scFv.

5. The chimeric antigen receptor comprising the third signal receptor according to claim 4, wherein the sequence of said anti-CD20 scFv is as set forth in SEQ ID No: 1; the sequence of said anti-CD19 scFv is as set forth in SEQ ID No: 2; the sequence of said anti-CLDN18.2 scFv is as set forth in SEQ ID No: 3; and/or the sequence of said anti-EGFR scFv is as set forth in SEQ ID No: 4.

6. The chimeric antigen receptor comprising the third signal receptor according to claim 1, wherein said Y is an intracellular domain of 4-1BB.

7. The chimeric antigen receptor comprising the third signal receptor according to claim 1, wherein the sequence of said intracellular domain of IL7Ra is as set forth in SEQ ID No: 8; the sequence of said intracellular domain of IL2Rb is as set forth in SEQ ID No: 9; the sequence of said intracellular domain of IL4Ra is as set forth in SEQ ID No: 10; the sequence of said intracellular domain of IL9Ra is as set forth in SEQ ID No: 11; the sequence of said intracellular domain of IL21Ra is as set forth in SEQ ID No: 12; the sequence of said intracellular domain of IL2Rg is as set forth in SEQ ID No: 13; the sequence of said intracellular domain of 4-1BB is as set forth in SEQ ID No:6; and/or the sequence of said CD3zata is as set forth in SEQ ID No: 7.

8. The chimeric antigen receptor comprising the third signal receptor according to claim 1, wherein said X is selected from anti-CD19 scFv, anti-CD20 scFv, anti-EGFR scFv, anti-HER2 scFv, anti-EGFRVIII scFv, anti-PSMA scFv, anti-BCMA scFv, anti-CD22 scFv, anti-CD30 scFv and anti-CLDN18.2 scFv; and said Y is an intracellular domain of 4-1BB, said M is one selected from intracellular domain of IL2Rb, intracellular domain of IL4Ra, intracellular domain of IL7Ra, intracellular domain of IL9Ra, and intracellular domain of IL21Ra.

9. The chimeric antigen receptor comprising the third signal receptor according to claim 1, wherein said X is selected from anti-CD19 scFv, anti-CD20 scFv, anti-EGFR scFv, anti-HER2 scFv, anti-EGFRVIII scFv, anti-PSMA scFv, anti-BCMA scFv, anti-CD22 scFv, anti-CD30 scFv and anti-CLDN18.2 scFv; and said Y is an intracellular domain of 4-1BB, said M is an intracellular domain of IL7Ra or intracellular domain of IL21Ra.

10. The chimeric antigen receptor comprising the third signal receptor according to claim 1, wherein said chimeric antigen receptor further comprises an extracellular hinge region and a transmembrane domain, said transmembrane domain is selected from CD8a, CD28, CD137 and CD3; said extracellular hinge region is selected from CD8a or IgG.

11. The chimeric antigen receptor comprising the third signal receptor according to claim 10, wherein said extracellular hinge region and transmembrane domain are derived from the extracellular hinge region and transmembrane domain of CD8a,

12. The chimeric antigen receptor comprising the third signal receptor according to claim 11, wherein the sequence of said extracellular hinge region and transmembrane domain is as set forth in SEQ ID No: 5.

13. The chimeric antigen receptor comprising the third signal receptor according to claim 1, wherein said chimeric antigen receptor has a structure of X-H-TM-Y-CD3zeta-M-N; wherein, X comprises a tumor-targeting antibody or a ligand or receptor capable of specifically binding to a tumor; H is an extracellular hinge region, said extracellular hinge region is selected from CD8a or IgG; TM is a transmembrane domain, said transmembrane domain is selected from CD8a, CD28, CD137 and CD3; Y is an intracellular domain of a costimulatory receptor, said costimulatory receptor is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226; M is an intracellular domain of a gamma chain family cytokine receptor, said cytokine receptor is selected from IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, IL21Ra; and N is an intracellular domain of IL2Rg.

14. The chimeric antigen receptor comprising the third signal receptor according to claim 13, wherein said chimeric antigen receptor has a structure of X-H-TM-Y-CD3zeta-M-N; wherein, X is selected from anti-CD19 scFv, anti-CD20 scFv, EGFR scFv, HER2 scFv, EGFRVIII scFv, anti-PSMA scFv, anti-BCMA scFv, anti-CD22 scFv, anti-CD30 scFv and anti-CLDN18.2 scFv; H is an extracellular hinge region of CD8a; TM is a transmembrane domain of CD8a; Y is an intracellular domain of 4-1BB; M is an intracellular domain of a gamma chain family cytokine receptor, said cytokine receptor is selected from IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, IL21Ra; and N is an intracellular domain of IL2Rg.

15. The chimeric antigen receptor comprising the third signal receptor according to claim 13, wherein the sequence of said H-TM-Y-CD3zeta-M-N is as set forth in any one of SEQ ID No: 23-27.

16. The chimeric antigen receptor comprising the third signal receptor according to claim 13, wherein said X is anti-CD20 scFv.

17. The chimeric antigen receptor comprising the third signal receptor according to claim 16, wherein the sequence of said X-H-TM-Y-CD3zeta is set forth in SEQ ID NO: 15, and/or the sequence of said X-H-TM-Y-CD3zeta-M-N is set forth in SEQ ID NO: 16.

18. A expression vector, comprising the polynucleotide encoding the chimeric antigen receptor comprising the third signal receptor according to claim 1.

19. A chimeric antigen receptor-T (CAR-T) cell comprising an expression vector encoding and expressing the CAR of claim 1.

20. A method of preventing or treating a tumor, comprising administrating said CAR-T cell according to claim 19 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0171] FIG. 1 is an illustrative schematic view showing the molecular structure of chimeric antigen receptor (CAR) including a third signal receptor in embodiments of the present invention;

[0172] FIG. 2 is a schematic view showing the virus titer measured after 293 cells were infected with 20BBZIL2RbIL2Rg virus in an embodiment of the present invention.

[0173] FIG. 3 is a schematic view showing the virus titer measured after 293 cells were infected with 20BBZIL4RaIL2Rg virus in an embodiment of the present invention;

[0174] FIG. 4 is a schematic view showing the virus titer measured after 293 cells were infected with 20BBZIL7RaIL2Rg virus in an embodiment of the present invention;

[0175] FIG. 5 is a schematic view showing the virus titer measured after 293 cells were infected with 20BBZIL9RaIL2Rg virus in an embodiment of the present invention;

[0176] FIG. 6 is a schematic view showing the virus titer measured after 293 cells were infected with 20BBZIL21RaIL2Rg virus in an embodiment of the present invention;

[0177] FIG. 7 is a schematic view showing the results of phenotypic analysis of 20BBZ CAR-T cell and 20BBZIL2RbIL2Rg CAR-T cell in an embodiment of the present invention;

[0178] FIG. 8 is a schematic view showing the results of phenotypic analysis of 20BBZ CAR-T cell and 20BBZIL4RaIL2Rg CAR-T cell in an embodiment of the present invention;

[0179] FIG. 9 is a schematic view showing the results of phenotypic analysis of 20BBZ CAR-T cell and 20BBZIL7RaIL2Rg CAR-T cell in an embodiment of the present invention;

[0180] FIG. 10 is a schematic view showing the results of phenotypic analysis of 20BBZ CAR-T cell and 20BBZIL9RaIL2Rg CAR-T cell in an embodiment of the present invention;

[0181] FIG. 11 is a schematic view showing the results of phenotypic analysis of 20BBZ CAR-T cell and 20BBZIL21RaIL2Rg CAR-T cell in an embodiment of the present invention;

[0182] FIG. 12 is a schematic view showing the amplification ability of 20BBZ CAR-T cell and 20BBZIL7RaIL2Rg CAR-T cell in an embodiment of the present invention;

[0183] FIG. 13 is a schematic view showing the tumor killing ability of 20BBZ CAR-T cell, 20BBZIL21RaIL2Rg CAR-T cell, 20BBZIL9RaIL2Rg CAR-T cell and 20BBZIL7RaIL2Rg CAR-T cell in an embodiment of the present invention;

[0184] FIG. 14 is a schematic view showing the number of 20BBZ CAR-T cell and 20BBZIL7RaIL2Rg CAR-T cell in the bone marrow in an embodiment of the present invention;

[0185] FIG. 15 is a schematic view showing the in vivo survival ability of 20BBZ CAR-T cell and 20BBZIL7RaIL2Rg CAR-T cell in an embodiment of the present invention.

[0186] FIG. 16 is a schematic view showing the amplification ability of 19BBZ CAR-T cell and 19BBZIL7RaIL2Rg CAR-T cell in an embodiment of the present invention;

[0187] FIG. 17 is a schematic view showing the tumor killing ability of 19BBZ CAR-T and 19BBZIL7RaIL2Rg CAR-T cell in an embodiment of the present invention;

[0188] FIG. 18 is a schematic view showing the amplification ability of CLDN18.2BBZ CAR-T cell and CLDN18.2BBZIL21RaIL2Rg CAR-T cell in an embodiment of the present invention;

[0189] FIG. 19 is a schematic view showing the tumor killing ability of CLDN18.2BBZ CAR-T cell and CLDN18.2BBZIL21RaIL2Rg CAR-T cell in an embodiment of the present invention;

[0190] FIG. 20 is a schematic view showing the amplification ability of EGFRBBZ CAR-T cell and EGFRBBZIL21RaIL2Rg CAR-T cell in an embodiment of the present invention;

[0191] FIG. 21 is a schematic view showing the tumor killing ability of EGFRBBZ CAR-T cell and EGFRBBZIL21RaIL2Rg CAR-T cell in an embodiment of the present invention.

TERMS

[0192] To make the disclosure easier to understand, some terms are firstly defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meanings given below. Other definitions are set forth throughout the application.

[0193] As used herein, the term “about” may refer to a value or composition within an acceptable error range for a particular value or composition as determined by those skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0194] As used herein, the term “administering” refers to the physical introduction of a product of the invention into a subject using any one of various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration, such as by injection or infusion.

[0195] As used herein, the term “antibody” (Ab) may comprise, but is not limited to, an immunoglobulin that specifically binds an antigen and contains at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or an antigen binding parts thereof. Each H chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains, CH1 CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region contains a constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDR), which are interspersed within more conservative regions called framework regions (FR). Each VH and VL contains three CDRs and four FRs, which are arranged from amino terminal to carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0196] As used herein, the term “Single-chain Fv” also abbreviated as “scFv,” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0197] As used herein, the term “chimeric antigen receptor (CAR)” generally refers to an antigen receptor fused by fusing an antigen binding region of an antibody which recognizes a tumor associated antigen (TAA) or a binding fragment of other target molecules with an “immune receptor tyrosine-based activation motifs (ITAM, typically CD3ζ or FcεRIγ) of an intracellular signal domain. For example, the basic structure of CAR can include an antigen binding domain of a tumor-associated antigen (TAA) or other target molecules (typically, an scFv originated from the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an immunoreceptor tyrosine-based activation motif (ITAM) of an intracellular immune receptor.

[0198] As used herein, the term “binding domain” generally refers to a domain that (specifically) binds to a given target epitope or a given target site of a target molecule (e.g., an antigen), interacts with the given target epitope or the given target site, or recognizes the given target epitope or the given target site.

[0199] As used herein, the term “specific binding” generally refers to a measurable and reproducible interaction, such as, the binding between a target and an antibody, which can determine the presence of a target in the presence of heterogeneous populations of molecules (including biomolecules). For example, antibodies that specifically bind to targets (which can be epitopes) are antibodies that bind the target(s) with greater compatibility, affinity, easiness, and/or duration than other targets. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved in proteins of different species. In another embodiment, the specific binding includes but is not limited to exclusive binding.

[0200] As used herein, the term “transmembrane domain” generally refers to a polypeptide or protein which is encoded at a DNA level by an exon including at least an extracellular region, a transmembrane region, and an intracellular region. The transmembrane domain generally includes three different structural regions: N-terminal extracellular region, middle conserved transmembrane extension region, and C-terminal cytoplasmic region. The transmembrane domain may further include an intracellular region or a cytoplasmic region.

[0201] As used herein, the term “hinge region” generally refers to a region located between the binding domain and the transmembrane domain in the CAR structure. The hinge region usually comes from IgG family, such as IgG1 and IgG4, and some from IgD and CD8. Generally, the hinge region has a certain degree of flexibility, which affects the spatial constraints between the CAR molecule and its specific target, thereby affecting the contact between CAR T cells and tumor cells.

[0202] As used herein, the term “costimulatory” generally refers to a source of the second signal of lymphocyte activation, which is usually generated by an interaction of costimulatory molecules on the surface of immune cells (between T cells/B cells or between antigen presenting cells/T cells) involved in adaptive immunity with their receptors. For example, the complete activation of T cells depends on dual signaling and the action of cytokine. The first signal of T cell activation is derived from the specific binding of its receptors with the antigens, that is, the recognition of T cells to the antigens; and the second signal of T cell activation is derived from the costimulatory molecule, that is, the interaction of the costimulatory molecules of the antigen presenting cells with the corresponding receptors on the surfaces of T cells.

[0203] As used herein, the term “costimulatory domain” generally refers to an intracellular portion of the corresponding receptor of the costimulatory molecule, which can transduce a costimulatory signal (also known as the second signal). For example, in CAR-T cells, the costimulatory domain derived from CD137 (or receptors of other costimulatory molecules) can be activated after the binding of the extracellular binding domain in the CAR structure with the corresponding antigen, thereby transducing a costimulatory signal.

[0204] As used herein, the term “primary signal transduction domain” generally refers to an amino acid sequence within a cell that can generate signals which promote the immune effector function of CAR-containing cells such as CAR-T cells. Examples of the immune effector functions in, e.g., CAR-T cells can include cell lysis activity and auxiliary activity, including cytokine secretion. In some embodiments, the primary signal transduction domain transduces the effector functional signals and directs the cells to perform the specialization function. Although the primary signal transduction domain can be used in its entirety, it is not necessary to use the entire chain in many cases. As for the use of a truncated portion of the primary signal transduction domain, such truncated portion can be used to replace the intact chain, as long as it can transduce the effector functional signals. The term “primary signal transduction domain” is thus intended to encompass any truncated portion of an intracellular signal transduction domain that is sufficient to transduce the effector functional signals. For example, in CAR-T cells, the primary signal transduction domain derived from CD3 zeta.

[0205] As used herein, the term “tumor” generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In the present application, the tumor can be a solid tumor or a non-solid tumor. In some embodiments, a visible lump that can be detected by clinical examinations such as, X-ray radiography, CT scanning, B-ultrasound or palpation can be called solid tumor, while a tumor that cannot be seen or touched by X-ray, CT scanning, B-ultrasound and palpation, such as leukemia, can be called non-solid tumor.

[0206] As used herein, the term “pharmaceutically acceptable diluent” or “pharmaceutically acceptable excipient” generally refers to a pharmaceutically acceptable substance, composition, or vehicle involved in carrying, storing, transferring, or administering a cell preparation, e.g., liquids, semi-solid or solid fillers, diluents, osmotic agents, solvent, or encapsulating substances. The pharmaceutically acceptable diluent or excipient can include a pharmaceutically acceptable salt, wherein the term “pharmaceutically acceptable salt” includes salts of active compounds prepared by using a relatively nontoxic acid or base, e.g., sodium chloride, depending on the cell nature of the present application. The pharmaceutically acceptable carrier can further include organic acids (e.g., lactic acid), bioactive substances (e.g., polypeptides, antibodies, and the like) and antibiotics (e.g., penicillin, streptomycin), etc. The pharmaceutically acceptable carrier can further include a hydrogel, such as, a hydrogel containing polyacrylamide. The pharmaceutically acceptable diluent or excipient can include storage solution, cryopreservation solution, injection, etc., which can be used for cells. In general, the pharmaceutically acceptable diluent or excipient can maintain the activity of the cells carried by the carrier without hindering its therapeutic efficacy. The pharmaceutically acceptable diluent or excipient can also contribute to the storage, transportation, proliferation and migration of cells, and is suitable for clinical application.

[0207] As used herein, the term “subject” generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey. In some embodiments, said subject is a human.

[0208] As used herein, the term “include/including” or “comprise/comprising” generally refers to encompassing clearly specified features, but does not exclude other elements.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0209] The present invention provides a chimeric antigen receptor including a third signal receptor, and said chimeric antigen receptor have a structure of scFv(X)-(Y)CD3zeta-MN; wherein X is a tumor-targeting antibody or other protein; Y is the intracellular domain of costimulatory receptor, said costimulatory receptor is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226; M is the intracellular domain of gamma chain family cytokine receptor, said cytokine receptor is selected from IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, IL21Ra; and N is the intracellular domain of IL2Rg. The present invention also relates to a CAR-T cell constructed with a recombinant expression vector of any one of the aforesaid chimeric antigen receptor and a preparation method therefor, a formulation including the CAR-T cell, and use of the CAR-T cell.

[0210] Hereinafter the embodiments of the present invention are further described with reference to the accompanying drawings and examples. The following examples are only for more clearly illustrating the technical solutions of the present invention, but not for limiting the protective scope of the present invention.

[0211] The chimeric antigen receptors (CAR) including the third signal receptor used in the examples of the present invention are BBZIL2RbIL2Rg, BBZIL4RaIL2Rg, BBZIL7RaIL2Rg, BBZIL9RaIL2Rg, BBZIL21RaIL2Rg, respectively, and their structures are shown in FIG. 1.

EXAMPLE 1—Preparation of 20BBZIL2RbIL2Rg CAR-T Cell

[0212] The preparation of said 20BBZIL2RbIL2Rg CAR-T cell in this example includes the following steps:

[0213] 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZIL2RbIL2Rg and Production of Virus

[0214] Forming a fusion protein of scFv-antihCD20-20BBZ (SEQ ID No:1), IL2Rb intracellular domain (SEQ ID No:3) and the intracellular domain of IL2Rg (SEQ ID No:7) by overlap PCR, and adding EcoRI and BamHI restriction sites to both ends of the fusion protein to clone a pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293×. After 48 and 72 hours, collecting the supernatant, filtering it with a 0.45 μm filter, and centrifuging with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, that is, the pCDH-MSCVEF-20BBZIL2RbIL2Rg virus (briefly, 20BBZIL2RbIL2Rg virus) for use in the subsequent production of CAR-T cells. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), and infecting 293 cells with the obtained 20BBZIL2RbIL2Rg virus to determine the virus titer, as shown in FIG. 2.

[0215] 2. Preparation of 20BBZIL2RbIL2Rg CAR-T Cell and 20BBZ CAR-T Cell

[0216] Purifying human PBMC with a Stemcell T cell isolation kit, inoculating into a 96-well culture plate coated by anti-hCD3 and anti-hCD28. After 2 days, infecting the cells with 20BBZ virus and 20BBZIL2RbIL2Rg virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating with artificial antigen presenting cell or anti-hCD3/28 every 6 days. After 2 rounds of stimulation, the obtained cells are 20BBZ CAR-T cell and 20BBZIL2RbIL2Rg CAR-T cell for use in the subsequent experiments and phenotypic analysis. As shown in FIG. 7, the obtained cells are CAR-positive.

EXAMPLE 2—Preparation of 20BBZIL4RaIL2Rg CAR-T cell

[0217] The preparation of said 20BBZIL4RaIL2Rg CAR-T cell in this example includes the following steps:

[0218] 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZIL4RaIL2Rg and Production of Virus

[0219] Forming a fusion protein of scFv-antihCD20-20BBZ (SEQ ID No:1), IL4Ra intracellular domain (SEQ ID No:4) and the intracellular domain of IL2Rg (SEQ ID No:7) by overlap PCR, and adding EcoRI and BamHI restriction sites to both ends of the fusion protein to clone a pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293×. After 48 and 72 hours, collecting the supernatant, filtering it with a 0.45 μm filter, and centrifuging with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the viruses, that is, the pCDH-MSCVEF-20BBZIL4RaIL2Rg virus (briefly, 20BBZIL4RaIL2Rg virus) for use in the subsequent production of CAR-T cells. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), and infecting 293 cells with the obtained 20BBZIL4RaIL2Rg virus to determine the virus titer, as shown in FIG. 3.

[0220] 2. Preparation of 20BBZIL4RaIL2Rg CAR-T Cell and 20BBZ CAR-T Cell

[0221] Purifying human PBMC with a Stemcell T cell isolation kit, inoculating into a 96-well culture plate coated by anti-hCD3 and anti-hCD28. After 2 days, infecting the cells with 20BBZ virus and 20BBZIL4RaIL2Rg virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulated by artificial antigen presenting cell or anti-hCD3/28 every 6 days. After 2 rounds of stimulation, the obtained cells are 20BBZCAR-T cell and 20BBZIL4RaIL2RgCAR-T cell for use in the subsequent experiments and phenotypic analysis. As shown in FIG. 8, the obtained cells are CAR-positive.

EXAMPLE 3—Preparation of 20BBZIL7RaIL2Rg CAR-T cell

[0222] The preparation of said 20BBZIL7RaIL2Rg CAR-T cell in this example includes the following steps:

[0223] 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZIL7RaIL2Rg and Production of Virus

[0224] Forming a fusion protein of scFv-antihCD20-20BBZ (SEQ ID No:1), IL7Ra intracellular domain (SEQ ID No:2) and the intracellular domain of IL2Rg (SEQ ID No:7) by overlap PCR, and adding EcoRI and BamHI restriction sites to both ends of the fusion protein to clone a pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293×. After 48 and 72 hours, collecting the supernatant, filtering it with a 0.45 μm filter, and centrifuged with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the viruses, that is, the pCDH-MSCVEF-20BBZIL7RaIL2Rg virus (briefly, 20BBZIL7RaIL2Rg virus) for use in the subsequent production of CAR-T cells. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), and infecting 293 cells with 20BBZIL7RaIL2Rg virus to determine the virus titer, as shown in FIG. 4.

[0225] 2. Preparation of 20BBZIL7RaIL2Rg CAR-T Cell and 20BBZ CAR-T Cell

[0226] Purifying human PBMC with a Stemcell T cell isolation kit, inoculating into a 96-well culture plate coated by anti-hCD3 and anti-hCD28. After 2 days, infecting the cells with 20BBZ virus and 20BBZIL7RaIL2Rg virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating with artificial antigen presenting cell or anti-hCD3/28 every 6 days. After 2 rounds of stimulation, the obtained cells are 20BBZCAR-T cell and 20BBZIL7RaIL2RgCAR-T cell for use in the subsequent experiments and phenotypic analysis. As shown in FIG. 9, the obtained cells are CAR-positive.

EXAMPLE 4—Preparation of 20BBZIL9RaIL2Rg CAR-T cell

[0227] The preparation of said 20BBZIL9RaIL2Rg CAR-T cell in this example includes the following steps:

[0228] 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZIL9RaIL2Rg and Production of Virus

[0229] Forming a fusion protein of scFv-antihCD20-20BBZ (SEQ ID No:1), IL9Ra intracellular domain (SEQ ID No:5) and the intracellular domain of IL2Rg (SEQ ID No:7) by overlap PCR, and adding EcoRI and BamHI restriction sites to both ends of the fusion protein to clone a pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293×. After 48 and 72 hours, collecting the supernatant, filtering it with a 0.45 μm filter, and centrifuging with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the viruses, that is, the pCDH-MSCVEF-20BBZIL9RaIL2Rg virus (briefly, 20BBZIL9RaIL2Rg virus) for use in the subsequent production of CAR-T cells. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), and infecting 293 cells with 20BBZIL9RaIL2Rg virus to determine the virus titer, as shown in FIG. 5.

[0230] 2. Preparation of 20BBZIL9RaIL2Rg CAR-T Cell and 20BBZ CAR-T Cell

[0231] Purifying human PBMC with a Stemcell T cell isolation kit, inoculating into a 96-well culture plate coated by anti-hCD3 and anti-hCD28. After 2 days, infecting the cells with 20BBZ virus and 20BBZIL9RaIL2Rg virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating with artificial antigen presenting cell or anti-hCD3/28 every 6 days. After 2 rounds of stimulation, the obtained cells are 20BBZCAR-T cell and 20BBZIL9RaIL2RgCAR-T cell for use in the subsequent experiments and phenotypic analysis. As shown in FIG. 10, the obtained cells are CAR-positive.

EXAMPLE 5—Preparation of 20BBZIL21RaIL2Rg CAR-T cell

[0232] The preparation of the 20BBZIL21RaIL2Rg CAR-T cell in this example includes the following steps:

[0233] 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZIL21RaIL2Rg and Production of Virus

[0234] Forming a fusion protein of scFv-antihCD20-20BBZ (SEQ ID No:1), IL21Ra intracellular domain (SEQ ID No:6) and the intracellular domain of IL2Rg (SEQ ID No:7) by overlap PCR, and adding EcoRI and BamHI restriction sites to both ends of the fusion protein to clone a pCDH-MSCVEF vector. Subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol, RSV-REV) into 293×. After 48 and 72 hours, collecting the supernatant, filtering it with a 0.45 uM filter, and centrifuging with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the viruses, that is, the pCDH-MSCVEF-20BBZIL21RaIL2Rg virus (briefly, 20BBZIL21RaIL2Rg virus) for use in the subsequent production of CAR-T cells. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), and infecting 293 cells with 20BBZIL21RaIL2Rg virus to determine the virus titer, as shown in FIG. 6.

[0235] 2. Preparation of 20BBZIL21RaIL2Rg CAR-T Cell and 20BBZ CAR-T Cell

[0236] Purifying human PBMC with a Stemcell T cell isolation kit, inoculating into a 96-well culture plate coated by anti-hCD3 and anti-hCD28. After 2 days, infecting the cells with 20BBZ virus and 20BBZIL21RaIL2Rg virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating by artificial antigen presenting cell or anti-hCD3/28 every 6 days. After 2 rounds of stimulation, the obtained cells are 20BBZCAR-T cell and 20BBZIL21RaIL2RgCAR-T cell for use in the subsequent experiments and phenotypic analysis. As shown in FIG. 11, the obtained cells are CAR-positive.

EXAMPLE 6—Comparison of Expansion Abilities of 20BBZ CAR-T Cell and 20BBZIL7RaIL2Rg CAR-T Cell

[0237] Culture the 20BBZ CAR-T cell and 20BBZIL7RaIL2Rg CAR-T cell prepared in Step 2 of Example 3 continuously for 14 days, and stimulate with artificial antigen presenting cell once every 6 days. Count the cells, and the results are shown in FIG. 12. It can be seen from the figure that 20BBZIL7RaIL2Rg CAR-T cell has enhanced proliferation ability as compared with 20BBZCAR-T cell.

EXAMPLE 7—Comparison of Tumor-Killing Abilities of 20BBZ CAR-T Cell and 20BBZIL7RaIL2Rg CAR-T Cell

[0238] Inoculate the 20BBZ CAR-T cell, 20BBZIL21RaIL2Rg CAR-T cell, 20BBZIL9RaIL2Rg CAR-T cell and 20BBZIL7RaIL2Rg CAR-T cell prepared in Step 2 of Example 3 into a 96-well plate, and add the Raji tumor cells at a CAR-T:tumor cell ratio of 1:1, 1:2, 1:4. After 24 and 48 hours, compare the survival rates of tumor cells, and the results are shown in FIG. 13. It can be seen from the figure that the 20BBZIL21RaIL2Rg CAR-T cell, 20BBZIL9RaIL2Rg CAR-T cell and 20BBZIL7RaIL2Rg CAR-T cell has similar tumor killing ability as compared with 20BBZ CAR-T cell.

EXAMPLE 8—Comparison of Anti-Tumor Ability and In Vivo Survival Ability of 20BBZ CAR-T Cell and 20BBZIL7RaIL2Rg CAR-T Cell

[0239] Inoculated 10.sup.6 Nalm-6 tumor cells intravenously into B-NDG mice. Treated the mice with 10.sup.7 2 OBBZ CAR-T cells and 20BBZIL7RaIL2Rg CAR-T cells after 6 days. The mice were observed for their survival rates, and some mice were detected for the level of tumor cells and CAR-T cells in their marrow on Day 7. The results are shown in FIG. 14 and FIG. 15, respectively. It can be seen from the figures that 20BBZIL7RaIL2Rg CAR-T cell, as compared with 20BBZ CAR-T cell, substantially prolongs the survival of mice, and expanded more in vivo.

Example 9—Preparation of 19BBZIL7RaIL2Rg CAR-T Cells

[0240] The preparation of the 19BBZIL7RaIL2Rg CAR-T cells described in this Example comprises the following steps:

[0241] 1. Construction of the Lentiviral Vector pCDH-MSCVEF-19BBZIL7RaIL2Rg and Virus Production

[0242] Similar to Example 3, construct the scFv-antihCD19-BBZ (SEQ ID No: 17), IL7Ra intracellular region (SEQ ID No. 8) and IL2Rg intracellular region (SEQ ID No. 13) CAR plasmids and produce the lentivirus (referred to as 19BBZIL7RaIL2Rg virus) for subsequent CAR-T cell production. A control pCDH-MSCVEF-19BBZ virus (referred to as 19BBZ virus) was also produced.

[0243] 2. Preparation of 19BBZIL7RaIL2Rg CAR-T Cells and 19BBZ CAR-T Cells

[0244] Human PBMC were inoculated into anti-hCD3 and anti-hCD28-coated 96-well culture plates, and after 2 days, the cells were infected with 19BBZ virus and 19BBZIL7RaIL2Rg virus according to MOI=10-20, and the cell culture was continued after 1 day of fluid change, and the resulting cells were 19BBZ CAR-T cells and 19BBZIL7RaIL2RgCAR-T cells, according to every 6 days using artificial antigen-presenting cells or anti-hCD3/28 stimulation, after a total of 2 rounds of stimulation to expand cells, the cells of the expansion process species were used for proliferation and killing comparative experiments.

Example 10—Comparison of the Expansion Capacity of 19BBZ CAR-T Cells and 19BBZIL7RaIL2Rg CAR-T Cells

[0245] The 19BBZ CAR-T cells and 19BBZIL7RaIL2Rg CAR-T cells prepared in step 2 of Example 9 were cultured continuously for 12 days, and the cells were stimulated with artificial antigen presenting cells every 6 days and the cells were counted, and the results are shown in FIG. 16. From the FIG. 16, it can be seen that 19BBZIL7RaIL2Rg CAR-T cells have stronger proliferation ability relative to 19BBZCAR-T cells.

Example 11—Comparison of Tumor Killing Ability of 19BBZ CAR-T Cells and 19BBZIL7RaIL2Rg CAR-T Cells

[0246] The 19BBZ CAR-T cells and 19BBZIL7RaIL2Rg CAR-T cells prepared in step 2 of Example 9 were inoculated into 96-well plates, and Raji tumor cells were added according to the CAR-T:Raji tumor cell ratio of 1:0.5, and the survival ratio of tumor cells was compared after 24 and 48 hours, and the results are shown in FIG. 17. From the figure, it can be seen that 19BBZIL7RaIL2Rg CAR-T cells have similar tumor killing ability relative to 19BBZ CAR-T cells.

Example 12—Preparation of CLDN18.2BBZIL21RaIL2Rg CAR-T Cells

[0247] The preparation of the CLDN18.2BBZIL21RaIL2Rg CAR-T cells described in this Example comprises the following steps.

[0248] 1. Construction of Lentiviral Vector pCDH-MSCVEF-CLDN18.2BBZIL21RaIL2Rg and Virus Production

[0249] Similar to Example 3, the scFv-antihCLDN18.2-BBZ (SEQ ID No: 19), IL21Ra intracellular region (SEQ ID No. 12) and IL2Rg intracellular region (SEQ ID No. 13) CAR plasmids were constructed and lentiviruses (referred to as CLDN18.2BBZIL21RaIL2Rg viruses) were produced for subsequent CAR-T cell production. A control pCDH-MSCVEF-CLDN18.2BBZ virus (referred to as CLDN18.2BBZ virus) was also produced.

[0250] 2. Preparation of CLDN18.2BBZIL21RaIL2Rg CAR-T Cells and CLDN18.2BBZ CAR-T Cells

[0251] Human PBMC were inoculated into anti-hCD3 and anti-hCD28-coated 96-well culture plates, and after 2 days, infected with CLDN18.2BBZ virus and CLDN18.2BBZIL7RaIL2Rg virus according to MOI=10-20, and continued cell culture after 1 day of fluid change, and the resulting cells were CLDN18.2BBZ CAR-T cells and CLDN18.2BBZIL21RaIL2RgCAR-T cells, and the cells were expanded after a total of 2 rounds of stimulation using artificial antigen presenting cells or anti-hCD3/28 stimulation every 6 days, and the cells of the expansion process species were used for proliferation and killing comparison experiments.

Example 13—Comparison of the Expansion Capacity of CLDN18.2BBZ CAR-T Cells and CLDN18.2BBZIL21RaIL2Rg CAR-T Cells

[0252] The CLDN18.2BBZ CAR-T cells and CLDN18.2BBZIL21RaIL2Rg CAR-T cells prepared in step 2 of Example 12 were cultured continuously for 12 days, and the cells were stimulated with artificial antigen-presenting cells every 6 days and the results were counted as shown in FIG. 18. From the FIG. 18, it is clear that CLDN18.2BBZIL21RaIL2Rg CAR-T cells have stronger proliferation ability relative to CLDN18.2BBZ CAR-T cells.

Example 14—Comparing the Tumor Killing Ability of CLDN18.2BBZ CAR-T Cells and CLDN18.2BBZIL21RaIL2Rg CAR-T Cells

[0253] The CLDN18.2BBZ CAR-T cells and CLDN18.2BBZIL21RaIL2Rg CAR-T cells prepared in step 2 of Example 12 were inoculated into 96-well plates, and Raji tumor cells were added according to the CAR-T:Raji tumor cell ratio of 1:0.5, and the survival ratio of tumor cells was compared after 24 and 48 hours, and the results are shown in FIG. 19. From the FIG. 19, it can be seen that CLDN18.2BBZIL21RaIL2Rg CAR-T cells have a slightly elevated tumor killing ability relative to CLDN18.2BBZ CAR-T cells.

Example 15—Preparation of EGFRBBZIL21RaIL2Rg CAR-T Cells

[0254] The preparation of EGFRBBZIL21RaIL2Rg CAR-T cells described in this Example comprises the following steps.

[0255] 1. Construction of the Lentiviral Vector pCDH-MSCVEF-EGFRBBZIL21RaIL2Rg and Viral Production

[0256] Similar to Example 3, the scFv-anti-hEGFR-BBZ (SEQ ID No. 21), IL21Ra intracellular region (SEQ ID No. 12) and IL2Rg intracellular region (SEQ ID No. 13) CAR plasmids were constructed and lentiviruses (referred to as EGFRBBZIL21RaIL2Rg viruses) were produced for subsequent CAR-T cell production. A control pCDH-MSCVEF-EGFRBBZ virus (referred to as EGFRBBZ virus) was also produced.

[0257] 2. Preparation of EGFRBBZIL21RaIL2Rg CAR-T Cells and EGFRBBZ CAR-T Cells

[0258] Human PBMC were inoculated into anti-hCD3 and anti-hCD28-coated 96-well culture plates, and after 2 days, infected with EGFRBBZ virus and EGFRBBZIL21RaIL2Rg virus according to MOI=10-20, and continued cell culture by changing the liquid after 1 day, and the resulting cells were EGFRBBZ CAR-T cells and EGFRBBZIL21RaIL2RgCAR-T cells, according to every 6 days using artificial antigen-presenting cells or anti-hCD3/28 stimulation, after a total of 2 rounds of stimulation to expand cells, the cells of the expansion process species were used for proliferation and killing comparative experiments.

Example 16—Comparison of the Expansion Capacity of EGFRBBZ CAR-T Cells and EGFRBBZIL21RaIL2Rg CAR-T Cells

[0259] The EGFRBBZ CAR-T cells and EGFRBBZIL21RaIL2Rg CAR-T cells prepared in step 2 of Example 15 were cultured continuously for 12 days, and the cells were stimulated with artificial antigen presenting cells every 6 days and the results were counted as shown in FIG. 20. From the FIG. 20, it can be seen that EGFRBBZIL21RaIL2Rg CAR-T cells have stronger proliferation ability relative to EGFRBBZCAR-T cells.

Example 17—Comparison of the Tumor Killing Ability of EGFRBBZ CAR-T Cells and EGFRBBZIL21RaIL2Rg CAR-T Cells

[0260] EGFRBBZ CAR-T cells and EGFRBBZIL21RaIL2Rg CAR-T cells prepared in step 2 of Example 15 were inoculated into 96-well plates, and Raji tumor cells were added according to the CAR-T:Raji tumor cell ratio of 1:0.5, and the survival ratio of tumor cells was compared after 24 and 48 hours, and the results are shown in FIG. 21. From the FIG. 21, it can be seen that EGFRBBZIL21RaIL2Rg CAR-T cells have a slightly elevated tumor killing ability relative to EGFRBBZ CAR-T cells.

[0261] It can be seen from the aforesaid examples that the present invention constructs a novel CAR-T cells including a third signal receptor, which significantly increases the activation ability, survival ability, expansion ability of the CAR-T cells in tumors, as compared with the current CAR-T technology in clinic use, and has more superior anti-tumor therapeutic effect.

[0262] Hereinbefore the specific embodiments of the present invention are described in details. However, they are only used as examples, and the present invention is not limited to the specific embodiments as described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are encompassed in the scope of the present invention. Therefore, all the equal transformations and modifications without departing from the spirit and scope of the present invention should be covered in the scope of the present invention.