Chimeric antigen receptor comprising third signal receptor and use thereof

Abstract

The present invention provides a chimeric antigen receptor having a structure of scFv(X)-(Y)CD 3zeta-MN.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, 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.

Claims

1. A chimeric antigen receptor (CAR), wherein said chimeric antigen receptor has the structure of scFv(X)-(Y)CD3zeta-MN, wherein said scFv(X)-(Y)CD3zeta is scFv-antihCD20-20BBZ with a sequence as set forth in SEQ ID No.1; wherein M is an intracellular domain of IL7Ra, wherein the sequence of said intracellular domain of IL7Ra is as set forth in SEQ ID No.2; and N is an intracellular domain of IL2Rg, and the sequence of said intracellular domain of IL2Rg is as set forth in SEQ ID No.7.

2. A chimeric antigen receptor-T cell expressing the CAR of claim 1.

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

4. A composition comprising the chimeric antigen receptor T cells of claim 2.

5. A method of treating a CD20 expressing tumor in vivo comprising administering an effective amount of the composition of claim 4.

6. A method of making a chimeric antigen receptor-T cell (CAR-T cell) expressing the chimeric antigen receptor of claim 1 comprising: constructing a lentiviral vector comprising a nucleic acid encoding the chimeric antigen receptor of claim 1; isolating human peripheral blood mononuclear cells and purifying T cells therefrom; inoculating the purified T cells to a culture plate under suitable stimulation conditions; culturing the culture plate for a predetermined period of time; after the predetermined period of time infecting the T cells with the lentiviral vector encoding the chimeric antigen receptor and subjecting to infected cells to cell expansion under suitable stimulator conditions to obtain the CAR-T cell expressing the chimeric antigen receptor of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) 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;

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

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

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

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

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

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

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

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

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

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

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

(13) FIG. 13 is a schematic view showing the tumor killing ability of BBZ CAR-T cell and BBZIL7RaIL2Rg CAR-T cell in an embodiment of the present invention;

(14) FIG. 14 is a schematic view showing the anti-tumor ability of BBZ CAR-T cell and BBZIL7RaIL2Rg CAR-T cell in an embodiment of the present invention;

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

DETAILED DESCRIPTION OF THE EMBODIMENTS

(16) 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.

(17) 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.

(18) 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

(19) The preparation of said 20BBZIL2RbIL2Rg CAR-T cell in this example includes the following steps:

(20) 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZIL2RbIL2Rg and Production of Virus

(21) 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 293X. 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 25000RPM 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.

(22) 2. Preparation of 20BBZIL2RbIL2Rg CAR-T Cell and 20BBZ CAR-T Cell

(23) 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

(24) The preparation of said 20BBZIL4RaIL2Rg CAR-T cell in this example includes the following steps:

(25) 1. Construction of lentiviral vector pCDH-MSCVEF-20BBZIL4RaIL2Rg and production of virus

(26) 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 293X. 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 25000RPM 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.

(27) 2. Preparation of 20BBZIL4RaIL2Rg CAR-T Cell and 20BBZ CAR-T Cell

(28) 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

(29) The preparation of said 20BBZIL7RaIL2Rg CAR-T cell in this example includes the following steps:

(30) 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZIL7RaIL2Rg and Production of Virus

(31) 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 293X. After 48 and 72 hours, collecting the supernatant, filtering it with a 0.45 uM filter, and centrifuged with Beckman ultracentrifuge and SW28 head at 25000RPM 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.

(32) 2. Preparation of 20BBZIL7RaIL2Rg CAR-T Cell and 20BBZ CAR-T Cell

(33) 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

(34) The preparation of said 20BBZIL9RaIL2Rg CAR-T cell in this example includes the following steps:

(35) 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZIL9RaIL2Rg and Production of Virus

(36) 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 293X. 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 25000RPM 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.

(37) 2. Preparation of 20BBZIL9RaIL2Rg CAR-T Cell and 20BBZ CAR-T Cell

(38) 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

(39) The preparation of the 20BBZIL21RaIL2Rg CAR-T cell in this example includes the following steps:

(40) 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZIL21RaIL2Rg and Production of Virus

(41) 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 293X. 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 25000RPM 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.

(42) 2. Preparation of 20BBZIL21RaIL2Rg CAR-T Cell and 20BBZ CAR-T Cell

(43) 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

(44) 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

(45) Inoculate the 20BBZ 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 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

(46) 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.

(47) 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.

(48) 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.