CHIMERIC ANTIGEN RECEPTOR COMPRISING NOVEL CO-STIMULATORY DOMAIN AND USE THEREOF

Abstract

Provided is a chimeric antigen receptor, comprising a ligand-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. The co-stimulatory domain comprises an intracellular region of an NK-activated receptor or a ligand thereof. Also provided are an engineered immune cell comprising the chimeric antigen receptor and a use thereof in treatment of diseases, such as cancers, 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 an intracellular region of an NK activating receptor or a ligand thereof, wherein the NK activating receptor is selected from the group consisting of 2B4, DNAM-1 and LFA-1, and the ligand of the NK activating receptor is selected from the group consisting of CD48, CD112, CD155, ICAM1, ICAM2 and ICAM3.

2. The chimeric antigen receptor of claim 1, wherein the costimulatory domain comprises an intracellular region of a protein selected from the group consisting of CD155, ICAM3, and 2B4.

3. The chimeric antigen receptor of claim 2, wherein the CD155 intracellular region has at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 29 The intracellular region of ICAM3 has at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:27; the intracellular region of 2B4 is shown in SEQ ID NO: 31 The amino acid sequences of have at least 90%, 95%, 97% or 99% or 100% sequence identity.

4. The chimeric antigen receptor of claim 1, wherein the costimulatory domain further comprises a signaling domain selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD27, CD28, CD30, CD40, CD83, CD134, CD137, CD270, CD272, CD276, CD278, CD357, DAP10, DAP12, LAT, NKG2C, SLP76, PD 1, LIGHT, TRIM, CD94, LTB, ZAP70, and combinations thereof.

5. The chimeric antigen receptor of claim 1, wherein the costimulatory domain further comprises a signaling domain of CD27, CD28, CD134, CD137, or CD278, or a combination thereof.

6. The chimeric antigen receptor of claim 1, wherein the ligand binding domain is selected from the group consisting of immunoglobulin molecules, Fab, Fab, F(ab)2, Fv fragments, scFv, linear Antibodies, heavy chain antibodies, sdAbs or Nanobodies.

7. The chimeric antigen receptor of claim 1, wherein the ligand binding domain binds to one or more targets selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD46, CD70, 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-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-, SSEA-4, CD20, AFP, Folate receptor alpha, 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 beta, 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, Pod Protein, 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 body, 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 carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAI R1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGF, APRIL, NKG2D, NKG2D ligands, and/or pathogen-specific antigens, biotinylated molecules , molecules expressed by HIV, HCV, HBV and/or other pathogens; and/or neo-epitopes or neo-antigens.

8. The chimeric antigen receptor of claim 1, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR chain, TCRf chain, TCR chain, TCR chain, CD3 subunit, CD3 subunit, CD3 subunit, CD3 subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

9. The chimeric antigen receptor of claim 1, wherein the intracellular signaling domain is selected from the signaling domains of the following proteins: FcR, FcR, CD3, CD3, CD3, CD3, CD22, CD79a, CD79b and CD66d.

10-11. (canceled)

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

13. The engineered immune cell of claim 12, wherein expression of the corresponding endogenous NK activating receptor or ligand as a costimulatory domain in the engineered immune cell is inhibited or silenced.

14. (canceled)

15. The engineered immune cell of claim 12, wherein the immune cell further comprises suppressed or silenced expression of at least one gene selected from the group consisting of TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4.

16. The engineered immune cell of claim 12, wherein the immune cell is selected from T cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells.

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

18. The engineered immune cell of claim 12, wherein the immune cell further expresses a chimeric antigen receptor or a recombinant T cell receptor.

19. The engineered immune cell of claim 18, wherein the immune cell further expresses a chimeric antigen receptor comprising a ligand binding domain, a transmembrane domain, a costimulatory domain, and an intracellular A signaling domain, wherein the costimulatory domain is selected from the group consisting of the signaling domains of CD27, CD28, CD134, CD137, or CD278, or a combination thereof.

20. A pharmaceutical composition comprising the engineered immune cell of claim 12, and a plurality of pharmaceutically acceptable excipients.

21. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0092] FIG. 1 shows the expression levels of scFv in bbzi3-CAR T cells and bbz155-CAR T cells.

[0093] FIG. 2 shows the killing effect of bbzi3-CAR T cells and bbz155-CAR T cells on target cells.

[0094] FIG. 3 shows the cytokine release levels of bbzi3-CAR T cells and bbz155-CAR T cells.

[0095] FIG. 4 shows the expression levels of scFv of CAR-T cells in which the corresponding endogenous ligands of the NK activating receptors were knocked out.

[0096] FIG. 5 shows the killing effect of CAR-T cells in which the corresponding endogenous ligands of the NK activating receptors are knocked out on target cells.

[0097] FIG. 6 shows the cytokine release levels of CAR-T cells in which the corresponding endogenous ligands of the NK activating receptors were knocked out.

[0098] FIG. 7 shows the expression levels of scFv of bbz2B4-CAR T cells and CAR-T cells in which CD48 was knocked out.

[0099] FIG. 8 shows the killing effect of bbz2B4-CAR T cells and CAR-T cells in which CD48 is knocked out on target cells.

[0100] FIG. 9 shows the changes of the proliferation level of i3KO-bbz-CAR T cells and bbz-CAR T cells in mice over time.

[0101] FIG. 10 shows the change in tumor burden over time in mice treated with i3KO-bbz-CAR T cells and bbz-CAR T cells.

DETAILED DESCRIPTION OF EMBODIMENTS

[0102] 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

[0103] Sequences encoding the following proteins were synthesized and cloned into pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): CD8 signal peptide (SEQ ID NO: 15), anti-CD19 scFv (SEQ ID NO: 1), CD8 hinge region (SEQ ID NO: 19), CD8 transmembrane region (SEQ ID NO: 3), 4-1BB intracellular region (SEQ ID NO: 9), CD3 intracellular region (SEQ ID NO: 11) and the intracellular region of the NK activating ligand as an additional co-stimulatory domain (CD155 intracellular region (SEQ ID NO: 29) or ICAM3 intracellular region (SEQ ID NO: 27)), and the correct insertion of the target sequence was confirmed by sequencing.

[0104] Three ml Opti-MEM (Gibco, Cat. No. 31985-070) was added to a sterile tube to dilute the above plasmid, and a packaging vector psPAX2 (Addgene, Cat. No. 12260) and an envelope vector pMD2.G (Addgene, Cat. No. 12259) were added at a 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, incubated at room temperature for 15 min, and then the plasmid/vector/transfection reagent mixture was added dropwise to the culture flask containing 293T cells. Viruses were collected at 24 hours and 48 hours, pooled, and ultracentrifuged (25000 g, 4 C., 2.5 hours) to obtain concentrated lentiviruses.

[0105] T cells were activated with DyneBeads CD3/CD28 CTSTM (Gibco, Cat. No. 40203D) and cultured at 37 C. and 5% CO.sub.2 for 1 day. Then, the concentrated lentivirus was added, and after 3 days of continuous culture, T cells expressing bbzi3-CAR and bbz155-CAR were obtained. bbz-CAR T cells without additional co-stimulatory domains (i.e., CD8 signal peptide-anti-CD19 scFv-CD8 hinge region-CD8 transmembrane region-4-1BB intracellular region-CD3 intracellular region) and unmodified wild-type T cells (NT) were used as controls.

[0106] After culturing at 37 C. and 5% CO.sub.2 for 11 days, the expression of scFv on CAR-T cells was detected by flow cytometry level by using Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab).sub.2 Fragment Specific (min X Hu, Boy, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 115-065-072) as the primary antibody and APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) as the secondary antibody, and the results are shown in FIG. 1.

[0107] It can be seen that the scFv in the CAR T cells prepared by the present disclosure is effectively expressed.

EXAMPLE 2: THE KILLING EFFECT OF CAR T CELLS ON TARGET CELLS AND THE RELEASE OF CYTOKINE

2.1 Detection of the Ability to Kill Target Cells

[0108] Firstly, the Nalm6 target cells carrying the fluorescein gene were plated in a 96-well plate at 110.sup.4 cells/well, and then NT cells, bbz-CAR T cells, bbzi3-CAR T cells and bbz155-CAR T cells were plated into the 96-well plate at a 2:1 effector-to-target ratio (i.e. the ratio of effector T cells to target cells) for co-culture, and the fluorescence value was measured with a microplate reader after 16-18 hours. According to the calculation formula: (average fluorescence value of target cellsaverage fluorescence value of samples)/average fluorescence value of target cells100%, the killing efficiency was calculated, and the results are shown in FIG. 2.

[0109] It can be seen that, compared with bbz-CAR T cells with traditional structure, the further inclusion of the intracellular region of NK activating ligand ICAM3 or CD155 as the co-stimulatory domain significantly enhances the killing ability of CAR T cells.

2.2 Detection of Cytokine Release Levels

(1) Collection of Cell Co-Culture Supernatant

[0110] Target cells Nalm6 or non-target cells Jurkat were plated in a 96-well plate at a concentration of 110.sup.5 cells/well, and then NT cells, bbz-CAR T cells, bbzi3-CAR T cells and bbz155-CAR T cells of the present disclosure were co-cultured with target cells or non-target cells at a ratio of 1:1, and the cell co-culture supernatant was collected after 18-24 hours.

(2) ELISA Detection of Secretion of IL-2 and IFN- in the Supernatant

[0111] A 96-well plate was coated with capture antibody Purified anti-human IL2 Antibody (Biolegend, Cat. No. 500302) or Purified anti-human IFN- Antibody (Biolegend, Cat. No. 506502) and incubated overnight at 4 C. Then the antibody solution was removed, and 250 L of PBST (1PBS containing 0.1% Tween) solution 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 L PBST (1PBS containing 0.1% Tween). 50 L of cell co-culture supernatant or standards per well was added and incubated at 37 C. for 1 h, then the plate was washed 3 times with 250 L of PBST (1PBS with 0.1% Tween). Then 50 L detection antibody, Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, Cat. No. ab25017) was added to each well, and incubated at 37 C. for 1 hour, then the plate was washed 3 times with 250 L PBST (1PBS containing 0.1% Tween). Then HRP Streptavidin (Biolegend, Cat. No. 405210) was added and incubate at 37 C. for 30 minutes. The supernatant was discard, and 250 L PBST (1PBS containing 0.1% Tween) was added for washing 5 times. 50 L of TMB substrate solution was added to each well. Reactions were allowed to occur at room temperature in the dark for 30 minutes, after which 50 L of 1 mol/L H.sub.2SO.sub.4 was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, a microplate reader was used to detect the absorbance at 450 nm, 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. 3.

[0112] It can be seen that compared with bbz-CAR T cells with traditional structure, the further inclusion of the intracellular region of NK activating ligand ICAM3 or CD155 as the co-stimulatory domain significantly increases the cytokine release level of CAR T cells.

EXAMPLE 3. CART CELLS IN WHICH NK ACTIVATING LIGANDS HAVE BEEN KNOCKED OUT

[0113] The corresponding NK activating ligands in bbzi3-CAR and bbz155-CAR T cells were knocked out by CRISP/Cas9 system to obtain ICAM3 knockout i3KO-bbzi3-CAR T cells and CD155 knockout 155KO-bbz155-CAR T cells. ICAM3 or CD155 in bbz-CAR T cells was also knocked out in the same way to obtain ICAM3 knockout i3KO-bbz-CAR T cells and CD155 knockout 155KO-bbz-CAR T cells.

[0114] After the CAR T cells were cultured at 37 C. and 5% CO.sub.2 for 11 days, the gene editing efficiency of CD155 and ICAM3 was detected by flow cytometry by using APC-anti human CD155 (biolegend, Cat. No. 337618) and PE-anti human ICAM3 (biolegend, Cat. No. 330005) antibodies, and the results are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Efficiency of gene editing Expression level Cell name Knockout gene NT after knockout 155KO-bbz-CAR T CD155 87.6% 23.6% 155KO-bbz155-CAR T CD155 87.6% 25.8% i3KO-bbz-CAR T ICAM3 93.5% .sup.18% i3KO-bbzi3-CAR T ICAM3 93.5% 19.8%

[0115] It can be seen that various NK activating ligands were efficiently knocked out.

[0116] After culturing at 37 C. and 5% CO.sub.2 for 11 days, the expression of scFv on CAR T cells was detected by flow cytometry level by using Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab).sub.2 Fragment Specific (min X Hu, Boy, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 115-065-072) as the primary antibody and APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) as the secondary antibody, and the results are shown in FIG. 4.

[0117] It can be seen that the scFv in the CAR T cells prepared by the present disclosure can be effectively expressed.

[0118] The killing ability of the above CAR T cells on Nalm6 target cells was detected according to the method described in Example 2.1, and the results are shown in FIG. 5.

[0119] It can be seen that after the corresponding NK activating ligand is knocked out, the further inclusion of the intracellular region of NK activating ligand can significantly enhance the killing ability of CAR T cells.

[0120] According to the method described in Example 2.2, the cytokine release level after the above CAR T cells were co-cultured with Nalm6 target cells was detected, and the results are shown in FIG. 6.

[0121] It can be seen that after the corresponding NK activating ligand is knocked out, CAR T cells that further comprise the intracellular region of the NK activating ligand have a stronger ability to secrete IFN-. In addition, compared with the i3KO-bbz-CAR T group, the secretion level of IL-2 in the cells of the i3KO-bbzi3-CAR T group was also significantly enhanced. The above results indicate that in the context of knocking out NK activating ligands, the further inclusion of the intracellular region of the corresponding NK activating ligands can enhance the cytokine secretion ability of CAR-T cells.

EXAMPLE 4 CAR T CELLS EXPRESSING THE INTRACELLULAR SEGMENT OF NK ACTIVATING RECEPTORS

[0122] bbz2B4-CAR T cell was prepared according to the method in Example 1, which is different from bbz-CAR T cell only in that it further comprises the intracellular region of NK activating receptor 2B4 (SEQ ID NO: 31) as an additional co-stimulatory domain in the CAR structure. At the same time, the CD48 gene (i.e., the ligand of 2B4) in bbz-CAR T cells and bbz2B4-CAR T cells was knocked out by the CRISP/Cas9 system to obtain 48KO-bbz-CAR T cells and 48KO-bbz2B4-CAR T cells in which CD48 was knocked out.

[0123] After the CAR T cells were cultured at 37 C. and 5% CO.sub.2 for 11 days, PE-anti human CD48 (biolegend, product number 336708) was used to detect the gene editing efficiency against CD48 to confirm that CD48 was effectively knocked out. The results are shown in the following table 2.

TABLE-US-00002 TABLE 2 Efficiency of gene editing Expression level Cell name Knockout gene NT after knockout 48KO-bbz-CAR T CD48 97.3% 17.4% 48KO-bbz2B4-CAR T CD48 97.3% 16.1%

[0124] After culturing at 37 C. and 5% CO.sub.2 for 11 days, the expression of scFv on CAR T cells was detected by flow cytometry level by using Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab).sub.2 Fragment Specific (min X Hu, Boy, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 115-065-072) as the primary antibody and APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) as the secondary antibody, and the results are shown in FIG. 7.

[0125] It can be seen that the scFv in the CAR T cells prepared by the present disclosure is effectively expressed.

[0126] The killing effect of the above-mentioned CAR T cells on Nalm6 cells was detected according to the method of 2.1 in Example 2 (FIG. 8). The results showed that, whether compared with CAR T cells with traditional structure (i.e., bbz-CAR) or compared with CAR-T cells in which the NK activating ligand CD48 was knocked out (i.e., 48KO-bbz-CAR), the further inclusion of the intracellular region of NK activating ligand receptor 2B4 in the CAR structure as an additional co-stimulatory domain significantly enhances the killing ability of CAR T cells.

[0127] In addition, the applicant also unexpectedly found that compared with traditional CAR-T cells, further knocking out the NK activating ligand CD48 also significantly improves the killing ability of CAR-T cells on target cells (see FIG. 8, bbz-CAR vs 48KO-bbz-CAR).

EXAMPLE 5 IN VIVO ANTI-TUMOR EFFECT OF CAR-T CELLS WITH KNOCKED OUT OF NK ACTIVATING LIGAND

[0128] In order to further verify the effect of knocking out NK activating ligands on the function of CAR-T cells in vivo, the inventors conducted the following experiments.

[0129] Fifteen 8-week-old healthy female NCG mice were divided into three groups: NT group (negative control), bbz-CAR T group (positive control), and i3KO-bbz-CAR T group. On day 0 (D0), 510.sup.5 Raji cells were injected into the tail vein of each mouse. Three days later (D3), 210.sup.6 NT cells, bbz-CAR T cells or i3KO-bbz-CAR T cells were injected into the tail vein of each mouse according to the group. The change of Tumor burden and CAR T cell proliferation in vivo was regularly assessed by flow cytometry.

[0130] FIG. 9 shows the expansion level of CAR T cells in mice over time. It can be seen that from D21 onwards, the proliferation level of i3KO-bbz-CAR T cells is much higher than that of traditional bbz-CAR T cells. Moreover, bbz-CAR T cells could not be detected in mice at D28, while i3KO-bbz-CAR T cells continued expanding in mice until D48. This indicates that knocking out the NK activating ligand ICAM3 increases the expansion level of CAR T cells in vivo and prolongs the survival time of CAR T cells in vivo.

[0131] FIG. 10 shows the change of tumor burden in mice over time. It can be seen that compared with the NT group, both bbz-CAR T cells and i3KO-bbz-CAR T cells effectively inhibits the growth of tumors. But starting from D39, the tumors in the bbz-CAR T group mice began to recur, while the tumors in the i3KO-bbz-CAR T group mice continued being suppressed.

[0132] The above data show that knocking out only NK activating ligands (such as ICAM3) prolongs the persistence of CAR T cells in vivo, thereby improving the sustained killing effect on tumor cells, improving the tumor suppression effect in vivo, and increasing the survival of mice.

[0133] It should be noted that the above-mentioned are merely for preferred examples of the present disclosure and not used to limit the present disclosure. For one skilled in the art, various modifications and changes may be made to the present disclosure. Those skilled in the art should understand that any amendments, equivalent replacements, improvements, and so on, made within the spirit and principle of the present disclosure, should be covered within the scope of protection of the present disclosure.