NOVEL CO-STIMULATORY DOMAIN AND USE THEREOF

Abstract

A chimeric antigen receptor, containing a ligand binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain, the co-stimulatory domain containing CD94 and/or LTβ intracellular regions. The present invention further relates to engineered immune cells containing such a chimeric antigen receptor, and uses thereof in the treatment of diseases, such as cancer, autoimmune diseases, and infections.

Claims

1. A chimeric antigen receptor, comprising a ligand binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain, wherein the co-stimulatory domain comprises a CD94 intracellular region and/or an LTβ intracellular region.

2. The chimeric antigen receptor according to claim 1, wherein the CD94 intracellular region has at least 90%, 95%, 97% or 99%, or 100% sequence identity to an amino acid sequence represented by SEQ ID NO: 25, and the LTβ intracellular region has at least 90%, 95%, 97% or 99%, or 100% sequence identity to an amino acid sequence represented by SEQ ID NO: 27.

3. The chimeric antigen receptor according to claim 1, wherein the co-stimulatory domain further comprises a signaling domain of a protein selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof.

4. The chimeric antigen receptor according to claim 1, wherein the co-stimulatory domain further comprises a signaling domain of CD27, CD28, CD134, CD137 or CD278 or a combination thereof.

5. The chimeric antigen receptor according to claim 1, wherein the ligand binding domain is an antibody or an antigen binding portion thereof.

6. The chimeric antigen receptor according to claim 5, wherein the antigen binding portion is selected from the group consisting of Fab, Fab′, F(ab′)2, Fv fragment, scFv antibody fragment, linear antibody, sdAb or nanobody.

7. The chimeric antigen receptor according to claim 1, wherein the ligand binding domain binds to a target selected from the group consisting of: TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 1Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, ERBB2 (Her2/neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor J, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos associated antigen 1, p53, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal tract carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGF R, APRIL, NKG2D and any combination thereof.

8. The chimeric antigen receptor according to claim 1, wherein the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

9. The chimeric antigen receptor according to claim 1, wherein the intracellular signaling domain is a signaling domain of a protein selected from the group consisting of: FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b, and CD66d.

10-11. (canceled)

12. An engineered immune cell, comprising the chimeric antigen receptor according claim 1.

13. The engineered immune cell according to claim 12, wherein the immune cell is selected from the group consisting of a T cell, a macrophage, a dendritic cell, a monocyte, a NK cell or a NKT cell.

14. The engineered immune cell according to claim 13, wherein the T cell is a CD4+/CD8+ double positive T cell, a CD4+ helper T cell, a CD8+ T cell, a tumor infiltrating cell, a memory T cell, a naive T cell, a γδ-T cell or an αβ-T cell.

15. The engineered immune cell of claim 12, wherein the immune cell is derived from an adult stem cell, an embryonic stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell.

16. A pharmaceutical composition, comprising the engineered immune cell according to claim 12, and one or more pharmaceutically acceptable excipients.

17. (canceled)

18. The pharmaceutical composition according to claim 16, wherein the composition is for treating cancers, infections or autoimmune diseases.

19. The chimeric antigen receptor according to claim 2, wherein the co-stimulatory domain further comprises a signaling domain of a protein selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP70, and combinations thereof.

20. The chimeric antigen receptor according to claim 2, wherein the ligand binding domain is an antibody or an antigen binding portion thereof.

21. The chimeric antigen receptor according to claim 2, wherein the ligand binding domain binds to a target selected from the group consisting of: TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 1Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-0, SSEA-4, CD20, AFP, Folate receptor α, ERBB2 (Her2/neu), MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos associated antigen 1, p53, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal tract carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LTLRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGF β, APRIL, NKG2D and any combination thereof.

22. The chimeric antigen receptor according to claim 2, wherein the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of: TCR α chain, TCR β chain, TCR γ chain, TCR δ chain, CD3 ζ subunit, CD3 ε subunit, CD3 γ subunit, CD3 δ subunit, CD45, CD4, CD5, CD8 α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

23. The chimeric antigen receptor according to claim 2, wherein the intracellular signaling domain is a signaling domain of a protein selected from the group consisting of: FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD22, CD79a, CD79b, and CD66d.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0095] FIG. 1 shows structural schematic view of the chimeric antigen receptor of the present disclosure.

[0096] FIG. 2 shows scFv expression levels in z-CAR, 94z-CAR and LTBz-CAR T cells.

[0097] FIG. 3 shows the expansion levels of CAR-T cells expressing z-CAR, 94z-CAR and LTBz-CAR.

[0098] FIG. 4 shows scFv expression levels in CAR-T cells expressing bbz-CAR, bbz94-CAR and bbzLTB-CAR.

[0099] FIG. 5 shows the killing effect of CAR-T cells expressing bbz-CAR, bbz94-CAR and bbzLTB-CAR on target cells at concentrations of various effector-to-target ratios.

[0100] FIG. 6 shows the cytokine release levels of CAR-T cells expressing bbz-CAR, bbz94-CAR and bbzLTB-CAR.

[0101] FIG. 7 shows the in vivo expansion levels of CAR-T cells expressing bbz-CAR, bbz94-CAR and bbzLTB-CAR.

[0102] FIG. 8 shows the survival rate of tumor-bearing mice in each group treated with CAR-T cells.

SPECIFIC EMBODIMENTS

[0103] The T cells used in all the examples of the present disclosure are primary human CD4+CD8+ T cells isolated from healthy donors by leukapheresis using Ficoll-Paque™ PREMIUM (GE Healthcare, Cat. No. 17-5442-02).

Example 1. Preparation of CAR T Cells

[0104] The following coding sequences were synthesized and cloned into pGEM-T Easy vector (Promega, Cat. No. A1360) to prepare CAR constructs: CD8a signal peptide (SEQ ID NO: 18), anti-CD19 scFv (SEQ ID NO: 2), CD8a hinge region (SEQ ID NO: 20), CD8a transmembrane region (SEQ ID NO: 4), costimulatory domain, CD3ζ intracellular signaling domain (SEQ ID NO: 12), wherein the costimulatory domain is absent (z-CAR), CD94 intracellular region (SEQ ID NO: 26, 94z-CAR) or LTβ intracellular region (SEQ ID NO: 28, LTBz-CAR), and the correct insertion of the target sequence was confirmed by sequencing. The structure of CAR is shown in FIG. 1.

[0105] After the above plasmids were diluted by adding 3 ml Opti-MEM (Gibco, Cat. No. 31985-070) in a sterile tube, the packaging vector psPAX2 (Addgene, Cat. No. 12260) and the envelope vector pMD2.G (Addgene, Cat. No. 12259) were added according to the ratio of plasmid: viral packaging vector: viral envelope vector=4:2:1. Then, 120 ul X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001) was added, mixed immediately, and incubated at room temperature for 15 min. Then, the plasmid/vector/transfection reagent mixture was added dropwise to the culture flask of 293T cells. Viruses were collected at 24 hours and 48 hours, pooled, and ultracentrifuged (25000g, 4° C., 2.5 hours) to obtain concentrated lentiviruses.

[0106] T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco, Cat. No. 40203D) and cultured at 37° C. and 5% CO2 for 1 day. Then, the concentrated lentivirus was added, and after continuous culture for 3 days, the traditional z-CAR T cells targeting CD19 and the 94z-CAR T and LTBz-CAR T cells of the present disclosure were obtained. Unmodified wild-type T cells were used as negative controls (NT).

[0107] After culturing at 37° C. and 5% CO2 for 11 days, Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab′).sub.2 Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 115-065-072) were used as the primary antibody, and APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) was used as the secondary antibody, and the expression level of scFv on CAR-T cells was detected by flow cytometry. The results are shown in FIG. 2.

[0108] It can be seen that the scFv in the CAR T cells prepared by the present disclosure can be effectively expressed.

Example 2: In Vitro Expansion Level of CAR-T Cells

[0109] In order to determine the expansion ability of CAR-T cells in vitro, Nalm6 target cells carrying the green fluorescent protein gene were first plated in a 24-well plate at 1×10.sup.6/well, and then, CAR-T cells expressing 94z-CAR, LTBz-CAR or z-CAR were plated into the 24-well plate at an effector-to-target ratio (i.e., the ratio of effector T cells to target cells) of 1:1 for co-culture (DO), and on D4, D8, D12, D16, and D20, the target cells were further added at an effector-to-target ratio of 1:1. The number of expanded CAR T cells was counted regularly, and the results are shown in FIG. 3.

[0110] It can be seen that the expansion level of z-CAR T cells without co-stimulatory domain gradually decreased after D12, while 94z-CAR and LTBz-CAR T cells could continue to expand, with an in vitro expansion level after D12 significantly higher than that of the z-CAR T group. This indicates that the CD94 intracellular region and the LTβ intracellular region can serve as co-stimulatory domains to efficiently provide stimulating signals to T cells, thereby increasing the expansion level of CAR-T cells.

Example 3: Killing Effect of CAR T Cells on Target Cells and Release of Cytokines

[0111] 3.1 Preparation of CAR-T Cells

[0112] The following coding sequences were synthesized and cloned into pGEM-T Easy vector (Promega, Cat. No. A1360) to prepare CAR constructs: CD8α signal peptide (SEQ ID NO: 18), anti-CD19 scFv (SEQ ID NO: 30), CD8α hinge region (SEQ ID NO: 20), CD8a transmembrane region (SEQ ID NO: 4), 4-1BB co-stimulatory domain (SEQ ID NO: 10), CD3ζ intracellular signaling domain (SEQ ID NO: 12), and the correct insertion of the target sequence was confirmed by sequencing to obtain bbz-CAR.

[0113] An additional costimulatory domain CD94 intracellular region (SEQ ID NO: 26) or LTβ intracellular region (SEQ ID NO: 28) was further added to bbz-CAR to obtain bbz94-CAR and bbzLTB-CAR, respectively. The structure of CAR is shown in FIG. 1.

[0114] According to the method described in Example 1, flow cytometry was used to detect the expression level of scFv in the above CAR-T cells, and the results are shown in FIG. 4.

[0115] It can be seen that the scFv in the bbz-CAR, bbz94-CAR and bbzLTB-CAR T cells prepared by the present disclosure can all be effectively expressed.

[0116] 3.2 The Killing Effect of CAR-T Cells on Target Cells

[0117] When T cells kill target cells, the number of target cells decreases. After co-culturing T cells with target cells expressing luciferase, the number of target cells decreases and the secretion of luciferase decreases accordingly. Luciferase can catalyze the conversion of luciferin to oxidized luciferin, and during this oxidation process, bioluminescence will be generated, and the intensity of this luminescence will depend on the level of luciferase expressed by the target cells. Therefore, the detected fluorescence intensity can reflect the ability of T cells to kill target cells.

[0118] In order to detect the killing ability of CAR-T cells on target cells, Nalm6 target cells carrying the fluorescein gene were first plated into a 96-well plate at 1×10.sup.4/well, and then cells expressing bbz94-CAR, bbzLTB-CAR or bbz-CAR and NT cells were plated into the 96-well plate at an effector-to-target ratio (i.e. the ratio of effector T cells to target cells) of 10:1, 5:1 or 2.5:1, respectively for co-culture. After 16-18 hours, a microplate reader was used to measure the fluorescence value. The killing efficiency was calculated according to the calculation formula: (average fluorescence value of target cells−average fluorescence value of samples)/average fluorescence value of target cells×100%, and the results are shown in FIG. 5.

[0119] It can be seen that compared with NT, the specific killing effect of the CAR T cells of the present disclosure on target cells is equivalent to that of traditional bbz-CAR T cells at various concentrations of effector-target ratios.

[0120] 3.3 Cytokine Release of CAR-T Cells

[0121] When T cells kill target cells, the number of target cells decreases, and at the same time, cytokines IL-2 and IFN-γ are released. According to the following steps, enzyme-linked immunosorbent assay (ELISA) was used to measure the release levels of cytokines IL-2 and IFN-γ when target cells were killed by CAR T cells.

[0122] (1) Collection of Supernatant of the Cell Co-Culture

[0123] Target cells Nalm6 were plated in a 96-well plate at a concentration of 1×10.sup.5/well, and then bbz94-CAR, bbzLTB-CAR, bbz-CAR T cells and NT cells were co-cultured with target cells at a ratio of 1:1, and the supernatants of the cell co-cultures were collected after 18-24 hours.

[0124] (2) ELISA Detection of the Secretion of IL-2 and IFN-γ in the Supernatant

[0125] Purified anti-human IL2 Antibody (Biolegend, Cat. No. 500302) or Purified anti-human IFN-γ Antibody (Biolegend, Cat. No. 506502) were used as capture antibody to coat the 96-well plate by incubating overnight at 4° C. After removing the antibody solution, 250 μL PBST (1×PBS containing 0.1% Tween) containing 2% BSA (sigma, Cat. No. V900933-1 kg) was added and incubated at 37° C. for 2 hours. Plates were then washed 3 times with 250 μL PBST (1×PBS containing 0.1% Tween). 50 μL of cell co-culture supernatant or standards per well were added and incubated at 37° C. for 1 h, then the plate was washed 3 times with 250 μL of PBST (1×PBS with 0.1% Tween). Then 50 μL Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, Cat. No. ab25017) as detection antibody was added to each well and after incubating at 37° C. for 1 hour, the plate was washed 3 times with 250 μL PBST (1×PBS containing 0.1% Tween). Then HRP Streptavidin (Biolegend, Cat. No. 405210) was added, and incubated at 37° C. for 30 minutes. The supernatant was discarded, and 250 μL PBST (1×PBS containing 0.1% Tween) was added for washing 5 times. 50 μL of TMB substrate solution was added to each well. Reactions were allowed to occur at room temperature in the dark for 30 minutes, and then 50 μL of 1 mol/L H.sub.2SO.sub.4 was added to each well to stop the reaction. Within 30 minutes after the reaction was stopped, the absorbance at 450 nm was detected using a microplate reader, and the content of cytokines was calculated according to the standard curve (drawn according to the reading value and concentration of the standard), and the results are shown in FIG. 6.

[0126] It can be seen that the release of IL-2 and IFN-γ was not detected in the NT group, indicating that the killing of target cells by bbz-CAR T cells and the CAR T cells of the present disclosure is specific. In addition, compared with traditional bbz-CAR T cells, the release level of IL-2 in bbz94-CAR T cells did not change significantly, but the release level of IFN-γ was significantly higher; while the release levels of IL-2 and IFN-γ of bbzLTB-CAR T cells were higher than those of bbz-CAR T group. Therefore, in general, the cytokine release levels of bbz94-CAR T cells and bbzLTB-CAR T cells of the present disclosure are higher than those of traditional bbz-CAR T cells.

Example 3 Verification of the Tumor Suppressive Effect of CAR-T Cells

[0127] The suppressive effect of CAR-T cells on tumors was verified in a mouse model.

[0128] Twenty 8-week-old healthy female NCG mice were divided into four groups: NT group (negative control), bbz-CAR T group, bbz94-CAR T group, and bbzLTB-CAR T group. On day 0 (DO), 1×10.sup.6 Nalm6 cells were injected intravenously into the tail of each mouse. Seven days later (D7), each mouse was injected intravenously with PBS solution or 2×10.sup.6 NT cells, bbz-CAR T cells, bbz94-CAR T cells or bbzLTB-CAR T cells at the tail according to the grouping.

[0129] On D21, submandibular vein blood was taken from mice in the bbz-CAR T, bbz94-CAR T and bbzLTB-CAR T groups, and the expression levels of hCD8 and hCD4 were analyzed by Trucount FACS to detect the expansion of CAR T cells in the mice. The results are shown in FIG. 7.

[0130] It can be seen that T cell expansion was detected in all CAR T group mice tested, and the expansion of CD4+ T cells and CD8+ T cells in bbz94-CAR T group and bbzLTB-CAR T group was significantly higher than that in bbzCAR T group.

[0131] In addition, the inventors also counted the survival percentage of mice in each group by the end of the experiment (i.e., day 53 after inoculation of tumor cells Nalm6) (FIG. 8). All the mice in the NT group died on day 21, only one mouse (20%) treated with bbz-CAR T cells survived, and 3 mice treated with bbz94-CAR T cells survived (accounted for 60%), and 4 mice survived in the bbzLTB-CAR T group with a survival rate as high as 80%. This shows that the addition of CD94 and LTβ co-stimulatory domains can significantly improve the inhibitory effect of CAR-T cells on tumors and improve the survival rate of tumor-bearing mice.

[0132] In summary, compared with traditional CAR T cells, the CAR-T cells containing CD94 intracellular region and LTβ intracellular region in the present disclosure can greatly promote the expansion of T cells due to the introduction of a new co-stimulatory domain structure, so as to improve the sustained killing effect on tumor cells and improve the tumor suppression effect.