CHIMERIC ANTIGEN RECEPTOR SPECIFICALLY BINDING TO CD138, IMMUNE CELL EXPRESSING SAME, AND ANTICANCER USE THEREOF

Abstract

Provided is a chimeric antigen receptor (CAR) specifically binding to CD138, an immune cell expressing same, and a pharmaceutical composition for the treatment or prevention of cancer including same as an active ingredient. It was confirmed that the CD138 chimeric antigen receptor (CAR)-expressing immune cell of the presently claimed subject matter efficiently exhibits strong cytotoxic ability against CD138-expressing (positive) cancer cells. Accordingly, it is expected that the CD138 chimeric antigen receptor (CAR)-expressing immune cell of the presently claimed subject matter can be utilized for the treatment of CD138-expressing (benign) cancer diseases.

Claims

1. A chimeric antigen receptor comprising: (i) an antigen-binding domain; (ii) a hinge region; (iii) a transmembrane domain; (iv) an intracellular co-stimulatory domain; and (v) an intracellular main stimulatory signal domain, wherein the antigen-binding domain specifically binds to CD138.

2. The chimeric antigen receptor of claim 1, wherein the antigen-binding domain is an antibody or an antibody fragment, wherein the antigen-binding domain comprises a light chain variable region and a heavy chain variable region.

3. (canceled)

4. The chimeric antigen receptor of claim 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 5, and the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 6.

5. The chimeric antigen receptor of claim 2, further comprising a linker polypeptide positioned between the light chain variable region and the heavy chain variable region.

6. (canceled)

7. The chimeric antigen receptor of claim 1, wherein the antigen-binding domain is linked to the transmembrane domain by a hinge region, wherein the hinge region comprises a hinge region of IgG1, IgG4, or CD8.

8. The chimeric antigen receptor of claim 7, wherein: the IgG1 hinge region comprises the amino acid sequence of SEQ ID NO: 9; the IgG4 hinge region comprises the amino acid sequence of SEQ ID NO: 10; and the CD8 hinge region comprises the amino acid sequence of SEQ ID NO: 11.

9. The chimeric antigen receptor of claim 1, wherein the transmembrane domain comprises a transmembrane domain of CD8 or CD28.

10. The chimeric antigen receptor of claim 9, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 12, and the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 13.

11. The chimeric antigen receptor of claim 1, wherein the intracellular co-stimulatory domain comprises an intracellular co-stimulatory domain of CD28, DAP10, or CD137 (4-1BB).

12. The chimeric antigen receptor of claim 11, wherein; the CD28 intracellular co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 14; the DAP10 intracellular co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 15; and the CD137 (4-1BB) intracellular co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 16.

13. The chimeric antigen receptor of claim 1, wherein the intracellular stimulatory signal domain comprises a CD3 zeta (ζ) intracellular domain.

14. The chimeric antigen receptor of claim 13, wherein the CD3 zeta (ζ) intracellular domain comprises the amino acid sequence of SEQ ID NO: 17.

15. The chimeric antigen receptor of claim 1, further comprising a signal peptide, wherein the signal peptide comprises a signal peptide of CD16, human IgG, CD8, or GM-CSF.

16. (canceled)

17. The chimeric antigen receptor according to claim 15, wherein: the CD16 signal peptide comprises the amino acid sequence of SEQ ID NO: 1; the human IgG signal peptide comprises the amino acid sequence of SEQ ID NO: 2; the CD8 signal peptide comprises the amino acid sequence of SEQ ID NO: 3; and the GM-CSF signal peptide comprises the amino acid sequence of SEQ ID NO: 4.

18. A polynucleotide encoding the chimeric antigen receptor described in claim 1.

19. A recombinant vector comprising the polynucleotide described in claim 18.

20. A cell comprising the recombinant vector described in claim 19, wherein the cell is an NK cell, a T cell, a cytotoxic T cell, or a regulatory T cell.

21. (canceled)

22. A method for treating or preventing cancer, comprising administering a pharmaceutical composition comprising an effective amount of the cells according to claim 20 as an active ingredient to a subject in need thereof.

23. The method of claim 22, wherein the cancer is a cancer expressing CD138.

24. The method of claim 22, wherein the cancer is a cancer selected from the group consisting of multiple myeloma, ovarian cancer, kidney cancer, gallbladder cancer, breast cancer, prostate cancer, lung cancer, colon cancer, Hodgkin and non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), solid tissue sarcoma, ovarian adenocarcinoma, bladder transitional cell carcinoma, renal clear cell carcinoma, squamous cell lung cancer, and uterine cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0106] FIG. 1 shows a graph illustrating the results of flow cytometric analysis of NK cells, in which CD138 chimeric antigen receptors (CARs) were expressed.

[0107] FIG. 2 shows a graph illustrating the cytotoxic ability of CD138 chimeric antigen receptor (CAR)-expressing NK cells against CD138-overexpressing K562 cancer cells.

[0108] FIG. 3 shows graphs illustrating the changes in the secretion of granzyme B and interferon gamma (IFN-γ) in a reaction between CD138 chimeric antigen receptor (CAR)-expressing NK cells and target cancer cells(K562) expressing CD138 (T138) or not expressing CD138 (Tneo).

[0109] FIG. 4 shows graphs illustrating the cytotoxic effect of CD138 chimeric antigen receptor (CAR)-expressing NK cells against CD138-expressing (positive) multiple myeloma (MM) cell lines RPMI8226, IM9, and MM.1R.

MODE FOR CARRYING OUT THE INVENTION

[0110] The specific embodiments described herein represent preferred embodiments or examples of the present invention, and the scope of the present invention is not limited thereto. It will be apparent to those skilled in the art that variations and other uses of the invention do not depart from the scope of the invention described in the claims of this specification.

EXAMPLES

[0111] Experimental Method

[0112] 1. Cell Line Culture

[0113] Chronic myelogenous leukemia cancer cells K562 and three multiple myeloma (MM) cell lines of RPMI 8226, IM9, and MM1.R were cultured in an environment of 37° C. and 5% CO.sub.2 using RPMI 1640 containing 10% FBS. The cells were used as target cells for the experiment to confirm the cytotoxic ability of NK cells. NK cells were cultured in α-MEM medium containing 12.5% FBS, 12.5% horse serum, and 0.1 mM 2-mercaptoethanol after adding 100 U/mL of recombinant IL-2, in an environment of 37° C. and 5% CO.sub.2.

[0114] 2. Design of CD138 Chimeric Antigen Receptor (CAR)

[0115] The chimeric antigen receptor (CAR) of the present invention consisted of third generation chimeric antigen receptors (CARS).

[0116] The chimeric antigen receptor (CAR) of the present invention is a third generation chimeric antigen receptor comprising the following: (i) a signal peptide; (ii) a CD138 antigen recognition and binding domain; (iii) a hinge region; (iv) a transmembrane domain; and as an intracellular stimulatory signal domain (v) a CD3 zeta (ζ) stimulatory signal domain and (vi) two co-stimulatory domains.

[0117] The amino acid sequence of each of the domains or peptides is shown in Table 1 below. The polypeptide sequence of the CD138 chimeric antigen receptor (CAR) comprising the above construction was converted into a nucleic acid sequence by codon optimization so as to be suitable for protein expression in animal cells, and the nucleic acid sequence was synthesized and cloned for use.

TABLE-US-00001 TABLE 1 SEQ ID NO Sequence Information Description 1 MWQLLLPTALLLLVSA CD16 signal peptide 2 MDWTWRILFLVAAATGAHS human IgG signal peptide 3 MALPVTALLLPLALLLHAARP CD8 signal peptide 4 MWLQSLLLLGTVACSIS GM-CSF signal peptide 5 DIQMTQSTSSLSASLGDRVTISCSASQGINNYLNWYQQKPD variable GTVELLIYYTSTLQSGVPSRFSGSGSGTDYSLTISNLEPED light chain IGTYYCQQYSKLPRTFGGGTKLEIK of scFv (VL) 6 QVQLQQSGSELMMPGASVKISCKATGYTFSNYWIEWVKQR variable PGHGLEWIGEILPGTGRTIYNEKFKGKATFTADISSNTVQ heavy chain MQLSSLTSEDSAVYYCARRDYYGNFYYAMDYWGQGTSVTV of scFv (VH) SS 7 GSTSGSGKPSGEGSTKG linker peptide 1 8 GGGGS linker peptide 2 9 EPKSCDKTHTCPPCP IgG1 alpha hinge region 10 ESKYGPPCPSCP IgG4 alpha hinge region 11 ALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQP CD8 alpha LSLRPEASRPAAGGAVHTRGLD hinge region 12 AWVSACDTEDTVGHLGPWRDKDPALWCQLCLSSQHQAIER CD8 alpha FYDKMQNAESGRGQVMSSLAELEDDFKEGYLETVAAYYEE transmembrane domain 13 KPFWVLVWGGVLACYSLLVTVAFIIFWV CD28 transmembrane domain 14 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 co-stimulatory domain 15 LCARPRRSPAQEDGKVYINMPGRG DAP10 co-stimulatory domain 16 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD137(4-1BB) co- stimulatory domain 17 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD CD3 zeta PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK stimulatory GHDGLYQGLSTATKDTYDALHMQALPPR signal domain

[0118] Specifically, each domain of the chimeric antigen receptor (CAR) in the examples of the present invention was designed as follows. That is, the chimeric antigen receptor (CAR) was prepared to include a CD16 signal peptide; a single chain variant fragment (scFv) targeting CD138 as an antigen recognition and binding domain; an extracellular domain comprising a CD8 hinge region; a transmembrane domain of CD28; an intracellular domain of CD28 and an intracellular domain of CD137 (4-1BB) as a co-stimulatory domain; and an intracellular domain of CD3 zeta (ζ) as a main stimulatory signal domain.

[0119] 3. Gene Transduction

[0120] The method of introducing a cloned gene into cells was performed according to the manufacturer's manual using Lonza's Nucleofector 2B (one of the electroporation methods) and the Cell Line Nucleofector® Kit for each cell. After transformation, the cells were stabilized for 48 hours in α-MEM medium containing 12.5% FBS, 12.5% horse serum, and 0.1 mM 2-mercaptoethanol, and the transformed cells were selected by treating an antibiotic at an appropriate concentration for 2 weeks or more according to the antibiotic resistance gene of the vector used.

[0121] 4. NK Cell-Mediated Cytotoxicity Assay (CFSE-7AAD Assay)

[0122] Carboxyfluorescein succinimidyl ester (CFSE) was added to target cells (T) to a final concentration of 0.5 ∞M per 1×10.sup.6 cells, and stained for 30 minutes in an environment of 5% CO.sub.2 and 37° C., washed three times with PBS, 4×10.sup.4 cells each were dispensed into a 96-well round bottom plate. NK cells (effector cells; E) were dispensed into wells in accordance with the E:T ratio of 1:1 and various ratios thereof, and then reacted for 4 hours in an environment of 5% CO.sub.2 and 37° C. After washing three times with PBS, the cells were suspended in 100 μL of 1% BSA/PBS, and 5 μL of 7-AAD was added to each well, reacted at 4° C. for 30 minutes with the light blocked, and washed again twice with PBS. After the NK cells were suspended in 1% BSA/PBS, the data were compared and analyzed using a flow cytometry.

[0123] 5. Enzyme-Linked Immunosorbent Assay of Granzyme B and IFN-γ

[0124] After washing the NK cells once with PBS, the NK cells were added into a 96-well round bottom plate in which target cells had been seeded (4×10.sup.4 cells/well) with various effector target cell ratio, and then reacted in an environment of 5% CO.sub.2 and 37° C. The level of granzyme B secreted from NK cells was measured using a human granzyme B ELISA kit, and the level of IFN-γ was measured using a human IFN-γ ELISA kit. 100 μL of the capture antibody diluted in a coating buffer was added into each well of a 96-well plate for ELISA, and the plate was sealed, coated at 4° C. overnight, washed three times with a wash buffer, and all the remaining buffer was removed. 200 μL of an assay diluent was added to each well, blocked by reacting at room temperature for one hour, washed three times with a washing buffer, and all remaining buffer was removed. Prepared 100 μL granzyme B, interferon-gamma (IFN-γ) standards or each of the samples (cell-free supernatants from each well reacted) were added thereto, and the plate was sealed, reacted at room temperature for two hours, washed five times with a wash buffer, and all the remaining buffer was removed. After adding 100 μL of a working detector (Detection Antibody+SAv-HRP reagent) to each well, the plate was sealed, reacted at room temperature for one hour, washed five times with a wash buffer, and all remaining buffer was removed. 100 μL of a substrate solution was added to each well, and after reacting at room temperature for 30 minutes with the light blocked, 100 μL of the reaction stop solution was added to each well, and the absorbance was measured at 450 nm within 20 minutes.

[0125] Experiment Results

[0126] 1. Preparation and Confirming Expression of CD138 Chimeric Antigen Receptor-Expressing NK Cells (CD138CAR-NK)

[0127] In this experiment, for more stable and efficient gene expression of NK cells, NK cells expressing CD138 chimeric antigen receptor (CAR) were produced by introducing the CD138 chimeric antigen receptor (CAR) gene by electroporation using the Lonza's Nucleofector. For the purpose of selecting only the NK cells into which the CD138 chimeric antigen receptor (CAR) gene was introduced, puromycin was used at a concentration of 1 μg/mL. As a result of confirming the expression of the CD138 chimeric antigen receptor (CAR) through flow cytometry, it was confirmed that the expression of the chimeric antigen receptor (CAR) in the finally selected NK cells was 80% or higher compared to the control NK cells (see FIG. 1).

[0128] 2. Cytotoxicity of CD138 Chimeric Antigen Receptor (CAR) Expressing NK Cells (CD138CAR-NK)

[0129] In order to measure the cytotoxic ability of CD138 chimeric antigen receptor (CAR)-expressing NK cells to target cancer cells, the same was confirmed through CFSE-7AAD analysis.

[0130] The cytotoxicity ability of CD138 chimeric antigen receptor (CAR)-expressing NK cells on K562 cells (Tneo) which do not express CD138, or K562 cells (T138) in which the CD138 antigen was artificially overexpressed, was evaluated by the E:T ratio of 0:1, 1:1, and 5:1. As a result it was confirmed that the cytotoxic effect of the CD138 chimeric antigen receptor (CAR)-expressing NK cells on K562 cells (Tneo) not expressing CD138 was about 0.3%, 5%, and 24%, whereas the cytotoxic effect on K562 cells (T138) artificially overexpressing the CD138 antigen was 0.8%, 82.6%, and 82.3%, thus showing stronger cytotoxicity. That is, it was confirmed that the CD138 chimeric antigen receptor (CAR)-expressing NK cells exhibit a cytotoxic effect specific to CD138 antigens (see FIG. 2).

[0131] Next, the proteins such as perforin and granzyme that play an important role in destroying target cells are present in the granules of NK cells, and interferon-gamma (IFN-γ) is also an important protein whose cytotoxicity can be evaluated. As a result of measuring these proteins using ELISA, it was confirmed that granzyme B and interferon-gamma (IFN-γ) in an amount of about 5 times or more were produced specifically only in the NK cells expressing CD138 chimeric antigen receptor (CAR) that reacted with K562 (T138) expressing CD138 (see FIG. 3).

[0132] 3. Anticancer Efficacy of CD138-CAR NK Cells (CD138CAR-NK) on Multiple Myeloma

[0133] The cytotoxic effect of CD138-CAR NK cells was confirmed in RPMI8226, IM9, and MM.1R, among multiple myeloma (MM) cell lines, which are CD138-expressing (positive) cells. As a control, the cytotoxic effect on CD138 non-expressing (negative) K562 cells was compared. As a result, the NK cells expressing CD138 chimeric antigen receptor (CAR) showed a high cytotoxicity of 50% or higher against all of the three types of multiple myeloma cells, and this is a result showing that the NK cells can more effectively remove CD138-expressing (positive) cancer cells by allowing the CD138 chimeric antigen receptor designed in the present invention (CAR) to effectively recognize and bind to CD138 (i.e., a target antigen) (see FIG. 4).

[0134] As described above, specific parts of the present invention have been described in detail, and it is apparent that these specific descriptions are merely preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Accordingly, it should be noted that the substantial scope of the present invention is defined by the appended claims and equivalents thereof.