Chimeric antigen receptor comprising co-stimulatory receptor and application thereof

Abstract

Provided by the present invention is a chimeric antigen receptor comprising a co-stimulatory receptor, the chimeric antigen receptor having a structure of scFv(X)-(Y)CD3zeta-2A-(Z); X comprises a tumortargeting antibody or a ligand or receptor capable of specifically binding to a tumor; Y is an intracellular region of the co-stimulatory receptor, and Z is a co-stimulatory receptor that is selected from among ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIMI, SLAM, CD2, CD226. Further provided by the present invention are CAR-T cells that are constructed by means of a recombinant expression vector of the described chimeric antigen receptor, a preparation method therefor and an application thereof. The CAR-T cells described in the present invention significantly improve the tumor-killing abilities and amplification abilities thereof.

Claims

1. A fusion protein, which comprises, from N-terminus to C-terminus, (a) a chimeric antigen receptor (CAR) that specifically binds to a CD20, (b) a 2A peptide and (c) a OX40 protein wherein; said fusion protein has a structure of scFv(X)-(H)-(TM)-(Y)CD3zeta-2A-(Z); the amino acid sequence of said scFv(X)-(H)-(TM)-(Y)CD3zeta comprises SEQ ID No.1; the amino acid sequence of said 2A peptide comprises SEQ ID No.7, SEQ ID No.8, SEQ ID No.9 or SEQ ID No.10; (Z) is OX40 and the amino acid sequence of said OX40 protein comprises SEQ ID No.2.

2. A CAR-T cell comprising; an expression vector, wherein the expression vector comprises a nucleic acid encoding said fusion protein of claim 1.

3. A method of preparing said CAR-T cell of claim 2, comprising the following steps: step 1: incorporating a nucleic acid encoding the 2A peptide between a nucleic acid encoding the CAR and a nucleic acid encoding the OX40 protein to form a nucleic acid encoding the fusion protein, adding a lentiviral vector to both ends of the nucleic acid encoding the fusion protein, and co-transfecting with a lentiviral packaging plasmid to obtain a virus comprising the nucleic acid encoding the fusion protein; and step 2: culturing purified human PBMC, and infecting said PBMC with the virus comprising the nucleic acid encoding the fusion protein obtained in step 1, subjecting the infected PBMC to cell expansion under suitable conditions to prepare the CAR-T cell.

4. The method of preparing said CAR-T cell according to claim 3, comprising: incorporating the nucleic acid encoding 2A peptide between the nucleic acid encoding the CAR and the nucleic acid encoding the OX40 protein by overlap PCR to form the nucleic acid encoding the fusion protein, and adding restriction sites to both ends of the nucleic acid encoding the fusion protein to clone a lentiviral vector; subjecting the clones sequenced correctly to a large scale endotoxin-free extraction, and co-transfecting with the lentiviral packaging plasmid; after a predetermined time period, collecting a supernatant, filtering, and centrifuging to concentrate the virus to obtain a virus comprising the nucleic acid encoding the fusion protein.

5. The method of preparing said CAR-T cell according to claim 3, wherein the step 2 comprises: isolating human PBMC for purification, inoculating into a culture plate under suitable stimulation conditions, culturing them for a predetermined period of time, infecting said PBMC with the virus comprising the nucleic acid encoding the fusion protein produced in step_1, and subjecting the infected PBMC to cell expansion under suitable stimulation conditions, after 2 rounds of expansion under stimulation, obtaining the CAR-T cell.

6. A method of treating a B cell lymphoma expressing CD20 in a human subject, comprising administrating to the subject a CAR-T cell comprising an expression vector comprising a nucleotide sequence encoding a fusion protein comprising, from N-terminus to C-terminus, a chimeric antigen receptor (CAR) that specifically binds to human CD20 which comprises SEQ ID NO: 1, a 2A peptide which comprises SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, and a costimulatory protein which comprises SEQ ID NO: 2.

7. An expression vector, comprising a nucleotide sequence encoding the fusion protein 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 the 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 BBZ-2A-OX40 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 BBZ-2A-HVEM 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 BBZ-2A-ICOS 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 BBZ-2A-CD27 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 BBZ-2A-4-1BB 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 BBZ-2A-OX40 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 BBZ-2A-HVEM 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 BBZ-2A-ICOS 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 BBZ-2A-CD27 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 BBZ-2A-4-1BB CAR-T cell in an embodiment of the present invention;

(12) FIG. 12 is a schematic view showing the expansion ability of BBZ CAR-T cell and BBZ-2A-OX40 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 BBZ-2A-OX40 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 BBZ-2A-OX40 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 BBZ-2A-OX40 CAR-T cell in an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

(16) The present invention provides a chimeric antigen receptor including a co-stimulatory receptor having a structure of scFv(X)-(Y)CD3zeta-2A-(Z); wherein X is a tumor-targeting antibody or other protein; Y is an intracellular domain of a co-stimulatory receptor, and said co-stimulatory receptor is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226; Z is a co-stimulatory receptor, and said co-stimulatory receptor is selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226. The present invention also relates to a CAR-T cell constructed by 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 a 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 receptor (CAR) molecules including a co-stimulatory receptor used in the following examples of the present invention are BBZ-2A-OX40, BBZ-2A-HVEM, BBZ-2A-ICOS, BBZ-2A-CD27, BBZ-2A-4-1BB, respectively, and their structures are shown in FIG. 1.

Example 1—Preparation of 20BBZ-2A-OX40 CAR-T Cell

(19) The preparation of the 20BBZ-2A-OX40 CAR-T cell in this example includes the following steps:

(20) 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZ-2A-OX40 and Production of Virus

(21) incorporating 2A (SEQ ID No. 7) sequence between scFv-antihCD20-20BBZ (SEQ ID No.1) and OX40 (SEQ ID No.2) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone the 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 by a 0.45 uM filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-OX40 virus (briefly, 20BBZ-2A-OX40 virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), and infecting 293 cells with the obtained virus to measure the virus titer, as shown in FIG. 2.

(22) 2. Preparation of 20BBZ-2A-OX40 CAR-T Cell and 20BBZ CAR-T Cell

(23) purifying human PBMC by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28. After 2 days, infecting the cells with 20BBZ virus and 20BBZ-2A-OX40 virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them 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 20BBZ-2A-OX40 CAR-T cell for subsequent experiments and phenotypic analysis. The results are shown in FIG. 7. It can be seen that the obtained cells are CAR-POSITIVE.

Example 2—Preparation of 20BBZ-2A-HVEM CAR-T Cell

(24) The preparation of the 20BBZ-2A-HVEM CAR-T cell in in this example includes the following steps:

(25) 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZ-2A-HVEM and Production of Virus

(26) incorporating 2A (SEQ ID No. 8) sequence between scFv-antihCD20-20BBZ (SEQ ID No.1) and HVEM (SEQ ID No.3) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone 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 by a 0.45 uM filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-HVEM virus (briefly, 20BBZ-2A-HVEM virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus). Infecting 293 cells with the obtained virus to measure the virus titer, as shown in FIG. 3.

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

(28) purifying human PBMC by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28. After 2 days, infecting the cells were infecte with 20BBZ virus and 20BBZ-2A-HVEM virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them 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 20BBZ-2A-HVEM CAR-T cell for subsequent experiments and phenotypic analysis. The results are shown in FIG. 8. It can be seen from the figure that the obtained cells are CAR-POSITIVE.

Example 3—Preparation of 20BBZ-2A-ICOS CAR-T Cell

(29) The preparation of the 20BBZ-2A-ICOS CAR-T cell in this example includes the following steps:

(30) 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZ-2A-ICOS and Production of Virus

(31) incorporating 2A (SEQ ID No. 9) sequence between scFv-antihCD20-20BBZ (SEQ ID No.1) and ICOS (SEQ ID No.4) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone 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 by a 0.45 uM filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-ICOS virus (briefly, 20BBZ-2A-ICOS virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), and infecting 293 cells with the obtained virus to measure the virus titer, as shown in FIG. 4.

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

(33) purifying human PBMCs by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28. After 2 days, infecting the cells with 20BBZ virus and 20BBZ-2A-ICOS virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them 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 20BBZ-2A-ICOS CAR-T cell for subsequent experiments and phenotypic analysis. The results are shown in FIG. 9. It can be seen from the figure that the obtained cells are CAR-POSITIVE.

Example 4—Preparation of 20BBZ-2A-CD27 CAR-T Cell

(34) The preparation of 20BBZ-2A-CD27 CAR-T cell in this example includes the following steps:

(35) 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZ-2A-CD27 and Production of Virus

(36) incorporating 2A (SEQ ID No. 10) sequence between scFv-antihCD20-20BBZ (SEQ ID No.1) and CD27 (SEQ ID No.5) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone 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 by a 0.45 uM filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-CD27 virus (briefly, 20BBZ-2A-CD27 virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), and infecting 293 cells with the obtained virus to measure the virus titer, as shown in FIG. 5.

(37) 2. Preparation of 20BBZ-2A-CD27 CAR-T Cell and 20BBZ CAR-T Cell

(38) purifying human PBMC by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28. After 2 days, infecting the cells with 20BBZ virus and 20BBZ-2A-CD27 virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them 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 20BBZ-2A-CD27 CAR-T cell for subsequent experiments and phenotypic analysis. The results are shown in FIG. 10. It can be seen from the figure that the obtained cells are CAR-POSITIVE.

Example 5—Preparation of 20BBZ-2A-4-1BB CAR-T Cell

(39) The preparation of the 20BBZ-2A-4-1BB CAR-T cell in this example includes the following steps:

(40) 1. Construction of Lentiviral Vector pCDH-MSCVEF-20BBZ-2A-4-1BB and Production of Virus

(41) incorporating 2A (SEQ ID No. 7) sequence between scFv-antihCD20-20BBZ (SEQ ID No.1) and 4-1BB (SEQ ID No.6) by overlap PCR, and adding EcoRI and SalI restriction sites to both ends to clone 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 by a 0.45 uM filter, and performing centrifugation with Beckman ultracentrifuge and SW28 head at 25000 RPM for 2 hours to concentrate the virus, which is pCDH-MSCVEF-20BBZ-2A-4-1BB virus (briefly, 20BBZ-2A-4-1BB virus) for the subsequent production of CAR-T cell. Meanwhile, producing the control pCDH-MSCVEF-20BBZ virus (briefly, 20BBZ virus), infecting 293 cells with the obtained virus to measure the virus titer, as shown in FIG. 6.

(42) 2. Preparation of 20BBZ-2A-4-1BB CAR-T Cell and 20BBZ CAR-T Cell

(43) purifying human PBMC by a Stemcell T cell isolation kit, and inoculating into a 96-well culture plate coated with anti-hCD3 and anti-hCD28. After 2 days, infecting the cells with 20BBZ virus and 20BBZ-2A-4-1BB virus at MOI=10-20. After 1 day, continuing to culture the cells with the medium changed, and stimulating them 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 20BBZ-2A-4-1BB CAR-T cell for subsequent experiments and phenotypic analysis. The results are shown in FIG. 11. It can be seen from the figure that the obtained cells are CAR-POSITIVE.

Example 6—Comparison of Expansion Abilities of 20BBZ CAR-T Cell and 20BBZ-2A-OX40 CAR-T Cell

(44) 20BBZ CAR-T cell and 20BBZ-2A-OX40 CAR-T cell prepared in Step 2 of Example 1 were continuously cultured for 14 days, and stimulated with artificial antigen presenting cell once every 6 days. The cells were counted, and the results are shown in FIG. 12. It can be seen from the figure that 20BBZ-2A-OX40 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 20BBZ-2A-OX40 CAR-T Cell

(45) 20BBZ CAR-T cell and 20BBZ-2A-OX40 CAR-T cell obtained in Step 2 of Example 1, 20BBZ-2A-ICOS CAR-T cell obtained in Step 2 of Example 3, and 20BBZ-2A-CD27 CAR-T cell obtained in Step 2 of Example 4 were inoculated into a 96-well plate, and Raji tumor cells were added at a CAR-T:tumor cell ratio of 1:1, 1:2, 1:4. After 24 and 48 hours, the survival rates of tumor cells were compared, and the results are shown in FIG. 13. It can be seen from the figure that 20BBZ-2A-OX40/ICOS/CD27 CAR-T cell has similar tumor killing ability as compared with 20BBZ CAR-T cell, and some CAR-T including the co-stimulatory receptor has a stronger tumor killing ability.

Example 8—Comparison of Anti-Tumor Ability and In Vivo Survival Ability of 20BBZ CAR-T Cell and 20BBZ-2A-OX40 CAR-T Cell

(46) 10.sup.6 Nalm-6 tumor cells were intravenously inoculated into B-NDG mice, which were treated with 10.sup.7 20BBZ CAR-T cells and 20BBZ-2A-OX40 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 figure that 20BBZ-2A-OX40 CAR-T cell, as compared with 20BBZ CAR-T cell, significantly 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 cell including a co-stimulatory 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 a 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.