CHIMERIC ANTIGEN RECEPTOR TARGETING CLL1 AND USE THEREOF

Abstract

Provided is a chimeric antigen receptor targeting CLL1 and an application thereof. The chimeric antigen receptor targeting CLL1 comprises an antigen binding domain, a hinge region, a transmembrane domain and a signal transduction domain; the antigen binding domain is an anti-CLL1 antibody. The present application uses an anti-CLL1 antibody as the antigen binding domain to construct a chimeric antigen receptor molecule, the chimeric antigen receptor targeting CLL1 has specific targeting effect on CLL1 positive tumor cells, and immune cells expressing chimeric antigen receptor targeting CLL1 have a significant killing effect in vitro and in vivo, and secrete a large amount of cytokine IFN-γ after co-cultured with CLL1 positive tumor cells, which has a specific clearance effect on CLL1 positive tumor cells.

Claims

1. A chimeric antigen receptor targeting CLL1, comprising an antigen binding domain, a hinge region, a transmembrane domain and a signal transduction domain; wherein the antigen binding domain is an anti-CLL1 antibody.

2. The chimeric antigen receptor targeting CLL1 according to claim 1, wherein, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2; or the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4; or the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6; or the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 7 and the amino acid sequence shown in one of SEQ ID NO: 8 to 10; or the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 11 and SEQ ID NO: 12.

3. The chimeric antigen receptor targeting CLL1 according to claim 1, wherein the hinge region comprises a CD8α hinge region.

4. The chimeric antigen receptor targeting CLL1 according to claim 1, wherein the transmembrane domain comprises a CD8α transmembrane region and/or a CD28 transmembrane region.

5. The chimeric antigen receptor targeting CLL1 according to claim 1, wherein the signal transduction domain comprises CD3ζ; preferably, the signal transduction domain further includes any one or a combination of at least two of 4-1BB, CD28 intracellular region, DAP10 or OX40.

6. The chimeric antigen receptor targeting CLL1 according to claim 1, wherein the chimeric antigen receptor targeting CLL1 further comprises a signal peptide; preferably, the signal peptide comprises a CD8α signal peptide and/or an IgGκ light chain signal peptide.

7. The chimeric antigen receptor targeting CLL1 according to claim 1, wherein, the chimeric antigen receptor targeting CLL1 comprises a signal peptide, an anti-CLL1 antibody, a CD8α hinge region, a CD8α transmembrane region, 4-1BB and CD3.

8. The chimeric antigen receptor targeting CLL1 according to claim 1, wherein: the chimeric antigen receptor targeting CLL1 comprises the amino acid sequence shown in SEQ ID NO: 13; or the chimeric antigen receptor targeting CLL1 comprises the amino acid sequence shown in SEQ ID NO: 14; or the chimeric antigen receptor targeting CLL1 comprises the amino acid sequence shown in SEQ ID NO: 15; or the chimeric antigen receptor targeting CLL1 comprises the amino acid sequence shown in SEQ ID NO: 16; or the chimeric antigen receptor targeting CLL1 comprises the amino acid sequence shown in SEQ ID NO:17.

9. A nucleic acid molecule, comprising a coding gene of the chimeric antigen receptor targeting CLL1 according to claim 1.

10. The nucleic acid molecule according to claim 9, wherein: the nucleic acid molecule includes the nucleic acid sequence shown in SEQ ID NO: 18; or the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 19; or the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 20; or the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 21; or the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 22.

11. An expression vector, comprising the nucleic acid molecule of claim 9; preferably, the expression vector is a viral vector or a non-viral vector containing the nucleic acid molecule; preferably, the viral vector comprises any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector; preferably, the non-viral vector comprises any one of a Piggybac transposon system, a Sleeping Beauty transposon system or a nanocarrier.

12. recombinant lentivirus, wherein the recombinant lentivirus is prepared from a mammalian cell transfected with the expression vector of claim 11 and a helper plasmid.

13. A chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the chimeric antigen receptor targeting CLL1 according to claim 1; preferably, the immune cell comprises any one of a T cell, an NK cell or a macrophage; preferably, the T cell comprises an αβT cell and/or a γδT cell.

14. A pharmaceutical composition, comprising the chimeric antigen receptor immune cell of claim 13; optionally, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

15. Use of the chimeric antigen receptor targeting CLL1 of claim 1 in the preparation of a medicine for treating a malignant tumor; preferably, the malignant tumor comprises acute myeloid leukemia.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0051] FIG. 1 shows the expression of CLL1 in different tumor cells;

[0052] FIG. 2 shows a schematic diagram of the structure of a chimeric antigen receptor targeting CLL1;

[0053] FIG. 3A shows a map of the lentiviral expression vector containing the H27H4 CAR gene, and FIG. 3B shows the position of the H27H4 CAR in the lentiviral expression vector;

[0054] FIG. 4 shows the positive rate of CAR expression in different CAR-T cells;

[0055] FIG. 5 shows the killing rate of CAR-T targeting CLL1 on Raji-CLL1 cells;

[0056] FIG. 6 shows the IFN-γ secretion of CAR-T targeting CLL1 to kill target cells.

DETAILED DESCRIPTION

[0057] In order to further illustrate the technical means adopted by the present application and effects thereof, the application will be further described below in conjunction with examples and drawings. It can be understood that the specific embodiments described here are only used to explain the application, but not to limit the application.

[0058] If the specific technology or conditions are not indicated in the examples, the embodiments shall be carried out according to the technology or conditions described in the literature in the field or according to the product specification. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through formal channels.

Example 1 Source of Anti-CLL1 Antibodies

[0059] In the present example, anti-CLL1 antibodies 19C1, 23D7, 27H4, humanized 27H4 (H27H4) and 1075.7 (U.S. Pat. No. 8,536,310B2) were selected as antigen binding domains for the construction of CAR molecules, wherein, 19C1 included the variable regions shown in SEQ ID NO: 1-2, 23D7 included the variable regions shown in SEQ ID NOs: 3-4, 27H4 included the variable regions shown in SEQ ID NOs: 5-6, H27H4 included the variable regions shown in SEQ ID NOs: 7-8, and 1075.7 included the variable regions shown in SEQ ID NOs: 11-12.

Example 2 Expression of CLL1 by Tumor Cells

[0060] In the present example, FITC anti-human CD371 (CLL1) antibody (biolegend) was incubated with target cells Jurkat and KG-la, separately. Then the expression of CLL1 by the target cells was detected by flow cytometry.

[0061] The results are shown in FIG. 1. THP-1, U937 and HL-60 were CLL1 positive cells, and Jurkat and KG-la were CLL1 negative cells.

Example 3 Design of Chimeric Antigen Receptor

[0062] In the present example, a chimeric antigen receptor targeting CLL1 was designed. The schematic diagram of the structure is shown in FIG. 2, including the CD8α signal peptide, single-chain antibody that specifically binds to CLL1 antigen (Anti-CLL1 scFv), CD8α hinge region (Hinge) and transmembrane region (Transmembrane), 4-1BB costimulatory domain and CD3ζ signaling domain. The specific CAR molecules are as follows.

[0063] (1) 19C1-CAR: CD8α signal peptide, Anti-CLL1 scFv (19C1), CD8α Hinge+TM, 4-1BB and CD3ζ;

[0064] (2) 23D7-CAR: CD8α signal peptide, Anti-CLL1 scFv (23D7), CD8α Hinge+TM, 4-1BB and CD3ζ;

[0065] (3) 27H4-CAR: CD8α signal peptide, Anti-CLL1 scFv (27H4), CD8α Hinge+TM, 4-1BB and CD3ζ;

[0066] (4) H27H4-CAR: IgGκ light chain signal peptide, Anti-CLL1 scFv (H27H4), CD8α Hinge+TM, 4-1BB and CD3ζ;

[0067] (5) 1075.7-CAR: CD8α signal peptide, Anti-CLL1 scFv (1075.7), CD8α Hinge+TM, 4-1BB and CD3ζ;

[0068] wherein, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 23, and the nucleic acid sequence is shown in SEQ ID NO: 24; the amino acid sequence of the IgGκ light chain signal peptide is shown in SEQ ID NO: 25, and the nucleic acid sequence is shown in SEQ ID NO: 26; the amino acid sequence of CD8α Hinge is shown in SEQ ID NO: 27, and the nucleic acid sequence is shown in SEQ ID NO: 28; the amino acid sequence of CD8α TM is shown in SEQ ID NO: 29, and the nucleic acid sequence is shown in SEQ ID NO: 30; the amino acid sequence of 4-1BB is shown in SEQ ID NO: 31, and the nucleic acid sequence is shown in SEQ ID NO: 32; the amino acid sequence of CD3 is shown in SEQ ID NO: 33, and the nucleic acid sequence is shown in SEQ ID NO: 34;

TABLE-US-00016 SEQ ID NO: 23: MALPVTALLLPLALLLHAARP; SEQ ID NO: 24: atggcactgccagtgacagccctgctgctgccactggccctgctgctgca cgcagcacgccct; SEQ ID NO: 25: MDMRVPAQLLGLLLLWLRGARC; SEQ ID NO: 26: atggatatgagggttcctgcacaactcctgggactcctcctgctctggct gagaggcgcaagatgt; SEQ ID NO: 27: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD; SEQ ID NO: 28: accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtc gcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcg cagtgcacacgagggggctggacttcgcctgtgat; SEQ ID NO: 29: IYIWAPLAGTCGVLLLSLVITLYC; SEQ ID NO: 30: atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtc actggttatcaccctttactgc; SEQ ID NO: 31: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL; SEQ ID NO: 32: aagagaggcaggaagaagctgctgtacatcttcaagcagcccttcatgcg ccccgtgcagacaacccaggaggaggacggctgc agctgtcggttcccagaggaggaggagggaggatgtgagctg; SEQ ID NO: 33: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR; SEQ ID NO: 34: agggtgaagttttctcggagcgccgatgcaccagcatatcagcagggaca gaatcagctgtacaacgagctgaatctgggcaggcgcgaggagtacgacg tgctggataagcggagaggcagagatcccgagatgggaggcaagccaagg aggaagaaccctcaggagggcctgtataatgagctgcagaaggacaagat ggccgaggcctactctgagatcggcatgaagggagagcggagaaggggca agggacacgatggcctgtatcagggcctgagcacagccaccaaggacacc tacgatgcactgcacatgcaggccctgccacctagg.

Example 4 Construction of an Expression Vector of the Chimeric Antigen Receptor Targeting CLL1

[0069] (1) According to the CAR molecule designed in Example 3, the CAR encoding gene was codon-optimized to promote its high-efficiency expression in human cells, and the whole gene of the CAR encoding gene was synthesized (Guangzhou Ige BIOTECHNOLOGY Co., Ltd.);

[0070] (2) The full-length CAR gene and the empty vector pCDH-EF1-MCS were digested with EcoRI and BamHI. After digestion in a 37° C. water bath for 30 min, DNA electrophoresis was performed on a 1.5% agarose gel, and agarose gel purification kit (Tiangen BIOTECH Co., Ltd.) was used to purify and recover the digested products;

[0071] (3) The ligation system shown in Table 1 was prepared, and the CAR gene fragment and linearized pCDH-EF1-MCS were ligated at 22° C. for 1 h, and the ligation product was directly transformed into Stb13 E. coli competent cells. 200 μL of the transformed product was coated on an ampicillin-resistant LB plate which was inverted and incubated overnight in an incubator at 37° C. The next morning, 3 single clones were randomly selected for colony PCR identification, and positive clones were identified by sequencing;

TABLE-US-00017 TABLE 1 Component Addition Amount Linearized pCDH-EF1-MCS vector 50 ng CAR gene 150 ng  T4 DNA ligation buffer  2 μL T4 DNA ligase (NEB)  1 μL ddH.sub.2O To 20 μL

[0072] Illustratively, the constructed lentiviral expression vector pBG-27H4 containing the 27H4 CAR gene is shown in FIG. 3A and FIG. 3B.

Example 5 Lentivirus Packaging

[0073] In the present example, a four-plasmid system was used to perform lentiviral packaging on the lentiviral expression vector constructed in Example 4. The specific steps are as follows:

[0074] (1) the four-plasmid system consisting of a lentiviral expression vector, a helper plasmid gag/pol, a Rev, and a VSV-G was mixed with a PEI transfection reagent, then added to a certain volume of serum-free DMEM, mixed and placed for 15 min;

[0075] (2) the above mixture was added to a T75 cell culture flask with 293T cells, mixed gently, and cultured in a 37° C., 5% CO.sub.2 cell incubator for 6 hours;

[0076] (3) after 6 h, the culture medium was replaced with fresh medium and the culture was continued with the addition of 10 mM sodium butyrate solution; the lentivirus culture supernatant was collected after 72 h for purification assay.

Example 6 Acquisition and Expansion of T Cells

[0077] 30 mL of whole blood was collected from each volunteer, and the peripheral blood was diluted with saline in proportion of 1:1; Ficoll's lymphocyte isolate was added to a centrifuge tube, and the diluted peripheral blood was slowly added and centrifuged at 1500 rpm for 30 min, and the PBMC layer was gently aspirated and transferred into another centrifuge tube;

[0078] PBMCs were washed several times with saline and transferred to X-VIVO medium (containing 50 ng/mL OKT3, 300 IU/mL IL-2) for culture. PBMCs were isolated and activated with X-VIVO (containing 50 ng/mL OKT3, 300 IU/mL IL-2), and the medium was changed to X-VIVO containing 300 IU/mL for expanded culture after 2 days. Then cells were counted every two days and the medium was replaced by fresh X-VIVO containing 300 IU/mL to maintain the cell concentration at (0.5-1)×10.sup.6 cells/mL. The observation lasted for ten days.

Example 7 Preparation of CAR-T Cells

[0079] In the present example, RetroNectin was used to improve the infection efficiency of lentivirus on T cells, and the steps were as follows:

[0080] 30 μg RetroNectin was coated on a 6-well plate and maintained in a 37° C. cell incubator for 2 h; RetroNectin was pipetted, the coated 6-well plate was blocked with Hank's solution containing 2.5% BSA and placed in a cell incubator at 37° C. for 0.5 h; the blocking solution was pipetted, the 6-well plate was washed with Hank's solution containing 2% Hepes, added with X-VIVO medium, added with appropriate amount of lentivirus solution, centrifuged at 2000 g for 2 hours, and the supernatant was discarded; 1×10.sup.6 T cells (CD3 positive>90%) were added, centrifuged at 1000 g for 10 min, and cultured in a cell incubator at 37° C., 5% CO.sub.2, and a certain humidity. Repeat the above steps the next day.

[0081] The amount of lentivirus added is shown in Table 2.

TABLE-US-00018 TABLE 2 Virus Number Infection of Lentivirus CAR Titer Starting Usage Lentivirus (TU/mL) Cells (pcs) MOI Amount (μL)  19C1-CAR 2.41E+07 4.00E+06 5 830.91  23D7-CAR 4.70E+07 4.00E+06 5 425.99  27H4-CAR 1.76E+08 4.00E+06 5 113.51 H27H4-CAR 3.91E+08 4.00E+06 5 51.22  1075.7-CAR 9.88E+07 4.00E+06 5 202.43

[0082] Flow cytometry was used to detect the expression or CAR molecules on the surface of T cells, and the CLL1-Fc fusion protein (Acrobiosystems) was used to detect the expression of CAR. The secondary antibody was FITC-Labeled anti-human Fc, and T cell (T mock) not transfected with CAR was used as a negative control.

[0083] The results are shown in FIG. 4 and Table 3. The positive rate of CAR expression in 19C1-CAR-T cells was 13.28%, the positive rate of CAR expression in 23D7-CAR-T cells was 37.49%, the positive rate of CAR expression in 27H4-CAR-T cells was 39.44%, the positive rate of CAR expression in H27H4-CAR-T cells was 33.59%, and the positive rate of CAR expression in 1075.7-CAR-T cells was 5.09%.

TABLE-US-00019 TABLE 3 T cell type T mock 19C1 23D7 27H4 H27H4 1075.7 CAR Positive Rate 1.23% 13.28% 37.49% 39.44% 33.59% 5.09%

Example 8 the Killing Function of CAR-T Cells

[0084] In the present example, the xCELLigence Real Time Cell Analysis (RTCA) was used to automatically detect the cell killing effect in the whole process. The xCELLigence® Real Time Cell Analyzer (RTCA) was based on microelectronic impedance technology. There were a large number of micro-gold electrodes integrated at the bottom of the E-plate. When the adherent cells adhered to the micro-gold electrodes, the number, diameter and adhesion ability of the cells will affect the current conduction between the micro-gold electrodes, thereby causing the impedance value to change. This change was extremely delicate and sensitive. Under the toxic negative effect, the cell directly or indirectly affected the impedance value. Therefore, xCELLigence can monitor the cytotoxic effects caused by molecular targets.

[0085] Steps are as follows:

[0086] (1) the anti-CD40 was diluted with a certain volume of 1× Tether Buffer, 50 μL of diluted anti-CD40 was added to each well of an E-Plate View 96-well plate as a coating solution, incubated at room temperature in the dark for 3 hours, and each group was set with three replicate wells;

[0087] (2) the coating solution was discarded, the wells were gently washed twice with 200 μL PBS, 50 μL of 1640 medium containing 2% FBS was added to each well, and the E-Plate View 96-well plate was placed in the xCELLigence instrument (the instrument was placed in the incubator 1 h in advance) and equilibrated at 37° C. for 1 h to measure the background values;

[0088] (3) Raji cells stably overexpressing CLL1 (Raji-CLL1) were used as a target cell, the target cell suspension was prepared and cell density was measured, 50,000 cells/50 μL were added to each well, the final volume of each well was 100 μL, incubated at room temperature for 30 min. The E-Plate View 96-well plate was put back into the instrument, and the software was operated for data collection, and electrical impedance was detected every 5 min for 2 h;

[0089] (4) effector cell suspensions (i.e. CAR-T cells) and negative control cell suspensions (i.e. untransfected T cells) with different effector-to-target ratios were prepared separately. 50 μL of diluted effector cell suspension or negative control cell suspension was added to each well, 50 μL of culture medium was added to the blank control group and 50 μL of 1× Cytolysis Solution was added to the positive control group. Then all groups were incubated for 30 min at room temperature to uniformly distribute the effector cells on the fixed target cells;

[0090] (5) the E-Plate View 96-well plate was put back into the instrument and the software was operated for data collection, and the electrical impedance was detected every 5 min for 16 h. The data were saved and analyzed after the experiment.

[0091] The results are shown in FIG. 5. After 19C1-CAR-T, 23D7-CAR-T, 27H4-CAR-T, H27H4-CAR-T or 1075.7-CAR-T were incubated with Raji-CLL1 for 16 h under different effector-to-target ratios (1:1, 2:1, 4:1), all of them could effectively kill Raji-CLL1 cells. The larger the effector-to-target ratio, the stronger the killing ability. Wherein, H27H4-CAR-T had the strongest killing ability, 27H4-CAR-T had similar killing ability to H27H4-CAR-T, 23D7-CAR-T was inferior, 19C1-CAR-T and 1075.7-CAR-T had relatively weak killing ability, and the untransfected CAR T mock could not kill Raji-CLL1, indicating that the killing activity of anti-CLL1 CAR-T was dependent on the CAR element.

Example 9 Secretion of IFN-γ by Co-Cultured CAR-T Cells and Tumor Cells

[0092] In the present example, the Human IFN-γ ELISA kit (Neobioscience) was used to detect the concentration of IFN-γ cytokine released by CAR-T cells, and the secretion of IFN-γ was analyzed after CAR-T and target cells were co-cultured. Specifically, CLL1 positive cells Raji-CLL1, HL60, U937 were used as positive target cells, and CLL1 negative cells Raji were used as negative target cells.

[0093] CAR-T cells and different target cells were incubated for 16 h according to an effector-to-target ratio of 1:1, and the supernatant was taken to detect the secretion of IFN-γ in the culture supernatant by enzyme-linked immunosorbent assay (ELISA). The principle of the assay was based on a double antibody sandwich ELISA in which anti-human IFN-γ antibody was coated on an enzyme standard plate, human IFN-γ in the sample or standard was bound to the coating antibody during the experiment, and the free components were washed away; biotinylated anti-human IFN-γ antibody and horseradish peroxidase labeled avidin were added sequentially, the anti-human IFN-γ antibody bound to human IFN-γ that bound to the coating antibody, and biotin specifically bound to avidin to form an immune complex, and the free components were washed away; the chromogenic substrate (TMB) was added, which appeared blue under the catalysis of horseradish peroxidase, and turned yellow after adding the termination solution, and the OD value was measured at 450 nm with Microplate Reader, and there was a positive correlation between IFN-γ concentration and OD450, and the concentration of IFN-γ in the sample was calculated by plotting the standard curve.

[0094] The results are shown in FIG. 6. 19C1-CAR-T, 23D7-CAR-T, 27H4-CAR-T, H27H4-CAR-T or 1075.7-CAR-T released large amounts of IFN-γ after co-culture with Raji-CLL1, HL60 or U937, while no large amount of IFN-γ was released after co-culture with Raji cells, indicating that the killing effect of CAR-T cells targeting CLL1 is specific.

[0095] In summary, the CAR-T cells targeting CLL1 of the present application have a significant killing effect on CLL1 positive tumor cells at different effector-to-target ratios, secrete a large amount of cytokine IFN-γ after co-cultured with tumor cells, and have promising applications in the field of CLL1 positive tumor therapy.

[0096] The applicant has stated that although the detailed method of the present application is described through the examples described above, the present application is not limited to the detailed method described above, which means that implementation of the present application does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients to the product of the present application, and selections of specific manners, etc., all fall within the protection scope and the disclosed scope of the present application.