CHIMERIC ANTIGEN RECEPTOR CONTAINING A TOLL-LIKE RECEPTOR INTRACELLULAR DOMAIN

Abstract

The present invention relates to a chimeric antigen receptor, a nucleic acid encoding the same and a cell expressing the same, and their use in manufacturing drugs for treating tumors. The chimeric antigen receptor of the present invention is characterized by its intracellular domain including at least Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain(s); compared to the prior art, the chimeric antigen receptors of the present invention has significant advantages in T cell expansion, cytotoxicity, T cell invasion and migration, eliminating immunosuppressive effect of regulatory T cells and promoting the formation of memory T cells, etc.

Claims

1. A chimeric antigen receptor, comprising an extracellular domain capable of binding to an antigen, a transmembrane domain and at least one intracellular domain, wherein, the at least one intracellular domain contains Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain(s).

2. The chimeric antigen receptor according to claim 1, characterized in that, the antigen is tumor associated antigen; and the extracellular domain capable of binding to the antigen is a single chain variable fragment of an antibody binding to the antigen.

3. The chimeric antigen receptor according to claim 1, characterized in that, the at least one intracellular domain further includes CD3ζ intracellular domain.

4. The chimeric antigen receptor according to claim 3, characterized in that, the intracellular domains also include CD28 intracellular domain.

5. The chimeric antigen receptor according to claim 1, characterized in that, the intracellular domains comprise two or more intracellular domains connected with each other; wherein the Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain are arranged on the C-terminal side.

6. The chimeric antigen receptor according to claim 1, characterized in that, the chimeric receptor antigen includes, in sequence from the N-terminal side, a single chain variable region of an antibody against tumor associated antigen as the extracellular domain, the transmembrane and intracellular domain of CD28 molecule, CD3ζ intracellular domain, Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain.

7. A nucleic acid encoding the chimeric antigen receptor according to claim 1.

8. A chimeric antigen receptor-expressing cell, into which the nucleic acid according to claim 7 is introduced.

9-10. (canceled)

11. The chimeric antigen receptor according to claim 3, characterized in that the Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domains are arranged on the C-terminal side of the CD3ζ intracellular domain.

12. The chimeric antigen receptor according to claim 4, characterized in that the intracellular domains include CD28 intracellular domain, CD3ζ intracellular domain and Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain(s) connected with each other in sequence from the N-terminal side.

13. The chimeric antigen receptor according to claim 4, characterized in that the intracellular domains include CD3ζ intracellular domain, Toll-like receptor 1 and/or Toll-like receptor 2 intracellular domain and CD28 intracellular domain connected with each other in sequence from the N-terminal side.

14. The chimeric antigen receptor according to claim 6, characterized in that the tumor associated antigen is CD19 or Mesothelin antigen.

15. The chimeric antigen receptor according to claim 8, wherein the cell is a T cell or a cell population containing T cells.

16. A method for treating a tumor, comprising the administration of the chimeric antigen receptor according to claim 1.

17. The method according to claim 16, wherein the tumor is hematological tumor or solid tumor.

18. A method for treating a tumor, comprising the administration of the chimeric antigen receptor-expressing cell according to claim 8.

19. The method according to claim 18, wherein the tumor is hematological tumor or solid tumor.

Description

DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 shows the in vitro killing efficacy of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells against K562-GL cells which do not express CD19.

[0034] FIG. 2 shows the in vitro killing efficacy of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells against K562-CD19-GL cells which express CD19.

[0035] FIG. 3 shows the in vitro killing efficacy of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells against NALM6-GL cells which express CD19.

[0036] FIG. 4 shows the in vitro killing efficacy of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells against REH-GL cells which express CD19.

[0037] FIG. 5 shows the level of IL-2 in supernatant after coculture of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells respectively with K562GL or K562-CD19-GL cells for 18 h.

[0038] FIG. 6 shows the transduction efficiency of CARMeso and CARMesoT1 T cells

[0039] FIG. 7 shows the in vitro killing efficacy of the CAR T cells in FIG. 6 against A549GL cells; wild-type T cells, as well as CAR T cells against CD22 molecule are used as controls.

[0040] FIG. 8 shows the transduction efficiency of GFP, CAR19, CAR19T1 and CAR19T2 T cells.

[0041] FIG. 9 shows the size of NALM6 tumors in immunodeficient mice treated with CAR T cells in FIG. 8.

[0042] FIG. 10 shows the weight of NALM6 tumors in FIG. 9.

[0043] FIG. 11 shows the weight of A549 tumors in immunodeficient mice treated with CAR T cells in FIG. 6.

EMBODIMENTS

[0044] To facilitate understanding of the present invention, the examples of the present invention are exemplified as follows. One skilled in the art should appreciate that the examples are offered to merely aid in understanding the invention and should not be regarded as particular limit to the present invention.

General Approach

[0045] In general, TLR1 and/or TLR2 signaling domain sequences are inserted into the intracellular domains of the anti-human CD19 chimeric antigen receptor for treating hematological tumor (B acute lymphoid leukemia, B-ALL) and anti-human Mesothelin chimeric antigen receptor for treating solid tumors (lung cancer) respectively to build the following four new chimeric antigen receptors: anti-CD19 ScFv-CD28-CD3ζ-TLR1/TLR2 and anti-Mesothelin ScFv-CD28-CD3ζ-TLR1/TLR2, hereinafter abbreviating the four chimeric antigen receptors as: CAR19T1, CAR19T2, CARMesoT1 and CARMesoT2 respectively. And the proliferation, anti-tumor function and the formation of memory of the CAR T cells expressing the above CAR molecules are assessed by in vivo and in vitro experiments.

Construction of CAR Plasmid

[0046] As described above, a CAR molecule comprises an extracellular region, a transmembrane domain, and an intracellular domain, and therefore, constructing steps of the CAR plasmid used by the following Examples comprises:

[0047] First, DNA encoding the respective gene required for CAR plasmid is obtained by gene synthesis, such DNA comprising: ScFv sequences of the anti-CD19 antibody, ScFv sequences of the anti-Mesothelin antibody, CD28 transmembrane and signaling sequence, TLR1 signaling sequence, TLR2 signaling sequence and CD3ζ signaling sequence;

[0048] Then, as required, the above synthesized gene sequences are connected in series with each other by steps of enzymatic digestion and connection, i.e., to obtain the novel CAR molecule of the invention. The sequence structures are as follows:

[0049] CAR19T1: anti-CD19 antibody ScFv (extracellular region), CD28 transmembrane and intracellular signaling region, CD3ζ signaling domain+TLR1 signaling domain;

[0050] CAR19T2: anti-CD19 antibody ScFv (extracellular region), CD28 transmembrane and intracellular signaling region, CD3ζ signaling domain, TLR2 signaling domain;

[0051] CAR19: anti-CD19 antibody ScFv (extracellular region), CD28 transmembrane and intracellular signaling region, CD3ζ signaling domain.

[0052] CARMesoT1: Anti-Mesothelin ScFv (extracellular region), CD28 transmembrane and intracellular signaling region, CD3ζ signaling domain, TLR1 signaling domain;

[0053] CARMesoT2: Anti-Mesothelin ScFv (extracellular region), CD28 transmembrane and intracellular signaling region, CD3ζ signaling domain, TLR2 signaling domain;

[0054] CARMeso: Anti-Mesothelin ScFv (extracellular region), CD28 transmembrane and intracellular signaling region, CD3ζ signaling domain;

[0055] Sequences of the above six kinds of CARs are inserted into the second generation of lentiviral vector pWPXLd-GFP to construct pWPXLd-CAR19T1-GFP, pWPXLd-CAR19T2-GFP, pWPXLd-CAR19-GFP, pWPXLd-CARMesoT1-GFP, pWPXLd-CARMesoT2-GFP and pWPXLd-CARMeso-GFP plasmid respectively.

SPECIFIC EXAMPLE

Example 1 Preparation of CAR19T1, CAR19T2 Plasmid

[0056] The plasmids carrying chimeric antigen receptor genes containing TLR1 and/or TLR2 intracellular domain of the present invention were prepared as follows:

[0057] (1) Plasmid pUC57-CAR19 containing CAR19 gene (SEQ ID NO.1) is obtained by gene synthesis; the CAR19 gene comprises an anti-CD19 mAb ScFv (SEQ ID NO.15), CD28 transmembrane region and intracellular region, CD3ζ intracellular region.

[0058] (2) The resultant plasmid pUC57-CAR19 was digested with endonuclease PmeI and SpeI, to obtain CAR19 gene, and then the CAR19 gene was connected into lentivirus vector pWPXLd-GFP to construct pWPXLd-CAR19-GFP.

[0059] (3) The resultant pWPXLd-CAR19-GFP plasmid was digested with endonuclease NotI and SpeI to obtain intracellular fragment 28Z of CAR19 gene.

[0060] (4) The cDNA of TLR1 and the fragment 28Z were used as templates to obtain 28ZT1 (SEQ ID NO. 9) by overlapping PCR with four primers (SEQ ID NO. 2-5); Similarly, the cDNA of TLR2 and the fragment 28Z were used as templates to obtain 28ZT2 (SEQ ID NO. 10) with four primers (SEQ ID NO. 2, 6, 7, 8).

[0061] (5) The 28zT1 and 28zT2 fragments digested with NotI and SpeI were then ligated with the ScFv of CAR19 in pUC57, generating pUC57-CAR19T1 and pUC57-CAR19T2 respectively.

[0062] (6) Finally, pWPXLd-CAR19T1-GFP or pWPXLd-CAR19T2-GFP plasmid was obtained respectively by digesting with endonuclease PmeI and SpeI, and replacing CAR19 in pWPXLD-CAR19-GFP with CAR19T1 or CAR19T2.

[0063] Intracellular signaling domains of TLR1 and TLR2 are called Toll/interleukin-1 receptor 1 (TIR1) (sequence shown in SEQ ID NO.11) and Toll/interleukin-1 receptor 2 (TIR2) (sequence shown in SEQ ID NO.12), respectively; TIR1 is the formed by 162 amino acids from the C-terminal of TLR1 (a.a. 625-786, sequence shown in SEQ ID NO.13), and TIR2 is formed by 159 amino acids from the C-terminal of TLR2 (a.a. 626-784, sequence shown in SEQ ID NO.14).

Example 2 Preparation of CARMesoT1, CARMesoT2 and CAR22 Plasmids

[0064] Mesothelin monoclonal antibody scFv domain sequence (SEQ ID NO.17) was obtained by gene synthesis, and pWPXLd-CARMesoT1-GFP or pWPXLd-CARMesoT2-GFP was obtained by digesting with endonuclease PmeI and NotI, and replacing CD19 monoclonal antibody scFv domains in pWPXLd-CAR19T1-GFP and pWPXLd-CAR19T2-GFP respectively with Mesothelin monoclonal antibody scFv domain.

[0065] In addition, CAR22, an anti-CD22 chimeric antigen receptor was used as negative control of CARMesoT1/T2. Plasmid containing CAR22 was pWPXLd-CAR22, of which the construction mainly through synthesizing anti-CD22 ScFv fragment (ie, SEQ ID NO.16), replacing anti-CD19 ScFv in CAR19 plasmid with the same by enzymatic digestion and ligation.

Example 3 Packaging of Lentiviral Vectors Expressing CARs

[0066] CAR plasmids of the present invention prepared in Example 1 and 2 and the related control plasmids were used, via lentiviral packaging, to obtain 8 kinds of recombinant lentiviruses expressing GFP (blank), CAR19T1-GFP, CAR19T2-GFP, CAR19-GFP, CARMesoT1-GFP, CARMesoT2-GFP, CARMeso-GFP, CAR22-GFP (negative control) respectively.

[0067] Specific steps were as follows: [0068] 293T cells were cultured in 150 mm dishes with the culture medium consisting of DMEM high glucose culture medium+10% FBS (fetal bovine serum)+1% penicillin/streptomycin penicillin/streptomycin; [0069] When the density of 293T cells in 150 mm dishes reached 80-90%, the culture medium was changed with DMEM high glucose medium+1% FBS+1% penicillin/streptomycinpenicillin/streptomycin; [0070] After replacing the culture medium and culturing for 2-6 hours, six kinds of pWPXLd-CARX-GFP plasmids (ie, including CAR19T1, CAR19T2, CAR19, CARMesoT1, CARMesoT2, CARMeso respectively) or blank control plasmid pWPXLd-GFP were co-transfected into 293T cells with plasmid pMD2.G and psPAX2 and the transfection reagent PEI, wherein the reagents and the doses thereof were as follows:

TABLE-US-00001 reagent dose six kinds of  9 μg pWPXLd-CARX-GFP plasmids or control plasmid pWPXLd-GFP pMD2.G  3 μg psPAX2 12 μg PEI 72 μg [0071] The lentiviral supernatant was collected and fresh culture medium (DMEM high glucose medium+1% FBS+1% penicillin/streptomycin) was added at 24, 48 and 72 hours after transfection respectively; [0072] After completing the collection of the supernatant of culture medium, the collected supernatant was centrifuged at 2500 g for 0.5 hours; [0073] The centrifuged supernatant was filtrated with 0.45 um filter, and then centrifuged at 28000 rpm for 1.5 hours with ultra high-speed centrifuge; [0074] After ultracentrifugation, the supernatant was removed gently, and 200 ul PBS was added to dissolve the precipitation under 4 degrees for 12-16 hours, and thereby to obtain six kinds of CAR lentiviruses or blank control GFP lentivirus; [0075] After the viruses were dissolved, the virus solution was subpackaged in PCR tubes, and frozen at −80 □ for use.

Example 4 Transfection of Human T Cells with CAR Lentivirus

[0076] Isolation and purification of T cells: PBMCs from healthy donors were isolated by Ficoll density gradient method, and red blood cells were depleted with red blood cell lysis buffer, followed byMACS sorting of T cells through PanT isolation Kit. [0077] The sorted T cells were resuspended with culture medium (AIM-V culture medium+5% FBS+penicillin 100 U/ml+streptomycin 0.1 mg/ml) to 2.5×10.sup.6 cells/ml for use; [0078] T cell stimulation by beads coated with anti-CD2, CD3, CD28 antibody (Origin of product: Miltenyi Biotech), ie. the beads were mixed with T cells at the ratio of 1:2, the final density of T cells was 5×10.sup.6 cells/ml/cm.sup.2; after a thorough mixing, T cells were cultured in a 37 □, 5% CO.sub.2 incubator for 48 hours. [0079] Lentiviral transfection of T cells: the beads are removed from the activated T cells and T cells were centrifuged at 300 g for 5 min, and resuspended with fresh medium, followed by addition of the lentiviruses (at MOI=10) expressing CARs or GFP, and then 8 μg/ml of polybrene and 300 IU/ml IL-2 were added. T cells were cultured in a 37 □, 5% CO.sub.2 incubator for 24 hours, and centrifuged at 300 g for 5 min and resuspended with fresh medium containing 300 IU/ml IL-2. [0080] Expansion of CAR T cells: the density of CAR T cells was maintained at 1-2×10.sup.6 cells/ml, and half of the medium was replaced once every 2-3 days. Two weeks later, CAR T cells could be amplified up to 100 times. GFP-positive cells were successfully transfected cells, and the percentages of GFP-positive cells were detected by flow cytometry. (abbreviated as CAR19T1-GFP T, CAR19T2-GFP T, CAR19-GFP T, CARMesoTLR1-GFP T, CARMesoTLR2-GFP T, CARMeso-GFP T respectively) or blank control T cells (GFP T).

Example 5 Enhanced Antitumor Efficacy In Vitro of CAR T Cells when TLR1 or TLR2 was Incorporated

[0081] GFP T (blank), CAR19T1 T, CAR19T2T and CAR19 T (control), or GFP T, CARMesoT1 T, CARMesoT2 T and CARMeso T (control) cells prepared in Example 4 are mixed with 1×10.sup.4 tumor cells respectively in different proportions and the resultant mixtures were added to 96-well U-shaped plate, with triple wells for each group, and a group containing tumor cells alone as a positive control. After centrifugation at 250 g for 5 min, cells were cultured in 37 degrees 5% CO.sub.2 incubator for 18 h.

[0082] T0 compare the effector function of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells against hematological tumor in vitro, NALM6-GL (GFP.sup.+ Luciferase), REH-GL, K562-GL and K562-CD19-GL, four kinds of leukemia or lymphoma cell line expressing luciferase, were selected as tumor cells to be tested; when validating the recognition and killing function of GFP T, CARMeso T, CARMesoT1 T and CARMesoT2 T cells on solid tumor cells in vitro, mesothelin positve A549-GL human lung adenocarcinoma cell line expressing luciferase was selected as tumor cells to be tested.

[0083] Luciferase killing assay: 18 hours after co-culture of CAR T cells with tumor cells (tumor cells cultured alone were used as control group in the experiment), 100 μl/well of luciferase substrate (1×) was added to each well of the plate, and the cells were resuspended and mixed, immediately followed by measuring RLU (relative light unit) through a multifunctional microplate reader, measuring time being set to one second. Calculation formula of killing rate was as follows: 100%×(numerical readings for control wells−numerical readings for experimental wells)/numerical readings for control wells (readings of blank control without cells could be ignored); the results were shown in FIGS. 1-4.

[0084] The results showed that, compared with CAR19 T cells, the in vitro killing capacities of CAR19T1 and CAR19T2 T cells against target tumor cells expressing CD19 were significantly higher, especially when E: T (ie, the ratio of effector T cells to target cells) was very low (see FIGS. 1-4).

[0085] After GFP T, CAR19 T, CAR19T1 T, CAR19T2 T cells were co-cultured with K562GL or K562-CD19-GL cells respectively for 18 h, the IL-2 levels in the supernatant were detected, and the results were shown in FIG. 5; FIG. 5 showed that the level of IL-2 secreted by CAR19T2 T cells was higher than that secreted by CAR19 T cells, indicating that the addition of intracellular domain of TLR2 improved the IL-2 secretion of CAR T cells.

[0086] In addition, even when the percentages of CAR T cells were very low (as shown in FIG. 6), the in vitro killing capacity of CARMesoT1 T cells against target cells expressing Mesothelin was significantly higher than CARMeso T cells (see FIG. 7).

Example 6 CAR19T1/T2 T Cells with Enhanced Anti-Tumor Efficacy In Vivo, Compared with CAR19 T Cells

[0087] For the purpose of comparing the efficacy of GFP T, CAR19 T, CAR19T1 T and CAR19T2 T cells against solid tumors, identical number (2×10.sup.5) of NALM6 cells were subcutaneously transplanted into 16 of NSI (NOD/SCIDIL2rg.sup.−/−) immunodeficient mice; 2 days and 9 days after NALM6 cell transplantation, 2×10.sup.6 T cells (four groups: GFP T, CAR19 T, CAR19T1 T, CAR19T2, four mice for each group, the proportion of positive cells as shown in FIG. 8) were intravenously injected into the NSI immunodeficient mice transplanted with NALM6 cells; on day 33, all the mice were euthanized for taking the tumors and weighed the same.

[0088] The results showed that, both CAR19T1 T and CAR19T2 T cells can significantly inhibit the growth of the subcutaneous NALM6 cells, and the tumor weight from the group of CAR19 T cells showed no difference compared with the group of GFP T cells (FIG. 9, 10).

[0089] On the other hand, although CAR19 T cells showed good efficacy in hematological cancers, they showed poor killing efficacy agianst subcutaneous solid tumors; however, after adding intracellular domain of TLR1 or TLR2, the killing capacity of CAR T cells against solid tumors was significantly improved.

Example 7 CARMesoT1 T Cells Showed Enhanced Efficacy Against A549 Tumor In Vivo

[0090] To compare CAR22, CARMesoT1 and CARMesoT2 T cells in recognizing and killing solid tumor in vivo, the identical number (1×10.sup.5) of A549 cells were subcutaneously transplanted into 12 NSI (NOD/SCID IL2rg.sup.−/−) immunodeficient mice at the flanks, 7 days and 14 days after A549 cell transplantation (the day of transplantation of tumor cells is day 0), 2×10.sup.6 T cells (three groups: CAR22 T, CARMeso T and CARMesoT1 T, four mice for each group, the proportion of positive cells as shown in FIG. 6) were intravenously injected into the NSI immunodeficient mice transplanted with A549 cells; on day 68, all the mice were euthanized for taking the tumors and weighed the same.

[0091] The results showed that, both CARMeso T and CARMesoT1 T cells can significantly inhibit the growth of the subcutaneous tumors, and compared to CARMeso T, the CARMesoT1 T cells have better effect of tumor killing in vivo (FIG. 11).

[0092] The above results comparing the effectiveness of recognizing and killing tumor of the experimental and control groups of CAR T cells indicated that both TLR1 and TLR2 signaling domains could improve the capacity of CAR T cells at killing tumors in vivo and vitro.

[0093] The applicant stated that the present invention described the product, purpose and use of the present invention by the above examples, but the present invention is not limited to the above detailed usage and use, ie. It does not mean that the present invention must rely on such detailed usage and use to implement. The one skilled in the art should be appreciated that any improvement in the present invention, the equivalent replacement of the raw materials of the product of the present invention and the addition of the auxiliary components, and the selection of specific ways, are all within the scope of protection and the scope of the disclosure of the present invention.