GLOBO SERIES ANTIGENS-BINDING CHIMERIC ANTIGEN RECEPTORS AND USES THEREOF

Abstract

The present disclosure relates to chimeric antigen receptors (CARs), which bind to Globo series antigens (e.g. Globo H, SSEA-3 or SSEA-4), including an antigen-binding fragment (Fab) or a single-chain variable fragment (scFv). Further, the present methods are also provided for administering CARs to a subject in an amount effective to inhibit cancer cells.

Claims

1. A chimeric antigen receptor (CAR), comprising: a single-chain variable fragment (scFv) or an antigen-binding fragment (Fab) that recognizes a Globo series antigen; and a first endodomain comprising CD3zeta or Fc?RI?, wherein the CAR includes an amino acid sequence with 80% to 100% sequence identity to anyone of SEQ ID Nos: 13-18.

2. The CAR of claim 1, further comprises a second endodomain including CD28, CD137, CD4, OX40, 4-1BB, CD3Z, or ICOS, wherein the scFv is fused to the second endodomain, and the second endodomain is fused to the first endodomain.

3. The CAR of claim 1, wherein the scFv comprises an amino acid sequence with 80% to 100% identity to SEQ ID No: 3 or 6.

4. (canceled)

5. The CAR of claim 1, wherein the Fab comprise: a heavy chain variable region (V.sub.H) having an amino acid sequence with 80% to 100% sequence identity to SEQ ID No: 1 or 4; and a light chain variable region (V.sub.L) having an amino acid sequence with 80% to 100% sequence identity to SEQ ID No: 2 or 5.

6. The CAR of claim 1, further comprises a second endodomain including CD28, CD137, CD4, OX40, 4-1BB, CD3Z, and ICOS, wherein the Fab is fused to the second endodomain, and the second endodomain is fused to the first endodomain.

7. The CAR of claim 2, wherein the CAR comprises: (a) a CD8 hinge region having an amino acid sequence with 90% to 100% sequence identity to SEQ ID No: 7; (b) a CD28 endodomain sequence with 90% to 100% sequence identity to SEQ ID No: 8; (c) a 4-1BB endodomain sequence with 90% to 100% sequence identity to SEQ ID No: 9; or (d) a CD3zeta domain sequence with 90% to 100% sequence identity to SEQ ID No: 10 or 11.

8. The CAR of claim 6, wherein the CAR comprises: (a) a CD8 hinge region having an amino acid sequence with 90% to 100% sequence identity to SEQ ID No: 7; (b) a CD28 endodomain sequence with 90% to 100% sequence identity to SEQ ID No: 8; (c) a 4-1BB endodomain sequence with 90% to 100% sequence identity to SEQ ID No: 9; or (d) a CD3zeta domain sequence with 90% to 100% sequence identity to SEQ ID No: 10 or 11.

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

13. The CAR of claim 1, wherein the Globo series antigen is selected from the group consisting of Globo H, stage-specific embryonic antigen 3 (SSEA-3), and stage-specific embryonic antigen 4 (SSEA-4).

14. A method for treating a subject with a tumor, comprising: (A) obtaining T cells from the subject having the tumor; (B) generating chimeric antigen receptor expression T cells (CAR-T cells) by transducing the T cells with a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR); (C) expanding the CAR-T cells; and (D) infusing the expanded CAR-T cells into the subject, whereby an immune response is raised, wherein the CAR comprises a single-chain variable fragment (scFv) or an antigen-binding fragment (Fab) that recognizes a Globo series antigen, and the CAR comprises an amino acid sequence with 80% to 100% sequence identity to anyone of SEQ ID Nos: 13-18.

15. (canceled)

16. The method of claim 14, wherein the subject is human.

17. The method of claim 14, wherein the immune response is mediated by T cells.

18. The method of claim 14, wherein the vector comprises a lentivirus, a gamma retrovirus, or an adeno-associated vims.

19. The method of claim 14, wherein the tumor expresses Globo H.

20. The method of claim 14, wherein the tumor is selected from the group consisting of breast cancer, lung cancer, esophageal cancer, rectal cancer, biliary cancer, liver cancer, buccal cancer, gastric cancer, colon cancer, nasopharyngeal cancer, kidney cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer, testicular cancer, bladder cancer, head and neck cancer, oral cancer, neuroendocrine cancer, adrenal cancer, thyroid cancer, bone cancer, skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma, and brain tumor.

21. The method of claim 14, wherein the CAR further comprises a first endodomain including CD3zeta or Fc?RI?.

22. The method of claim 14, wherein the CAR further comprises a second endodomain including CD28, CD137, CD4, OX40, 4-1BB, CD3Z, or ICOS.

23. The method of claim 14, wherein the CAR further comprises a hinge region of CD8.

24. The method of claim 14, wherein the Globo series antigen is selected from the group consisting of Globo H, stage-specific embryonic antigen 3 (SSEA-3), and stage-specific embryonic antigen 4 (S SEA-4).

Description

BRIEF DESCRIPTION OF DRAWINGS

[0034] FIG. 1. The schematic diagram of Globo-H CAR. There are total six Globo H CAR constructs [2C2-Fab CAR: SEQ ID No. 13; 2C2-scFv CAR: SEQ ID No. 14; R783-Fab CAR: SEQ ID No. 15; R783-scFv CAR: SEQ ID No. 16; 2C2-Fab CAR (CD3zeta mutant): SEQ ID No. 17; R783-scFv CAR (CD3zeta mutant: SEQ ID No. 18].

[0035] FIG. 2. In vitro cytotoxicity of Globo H CAR T cells. (A) MCF-7: breast cancer cell line-Globo H positive, (B) HCC-1428: breast cancer cell line-Globo H positive, (C) NCI-N87: gastric cancer cell line-Globo H positive, (D) SW-480: colon cancer cell line-Globo H positive, (E) SK-OV-3: ovarian cancer cell line-Globo H negative.

[0036] FIG. 3. In vitro cytotoxicity of Globo H CAR T cells with CD3zeta mutant (CD3zm). (A) HCC-1428: breast cancer cell line-Globo H positive, (B) MCF-7: breast cancer cell line-Globo H positive, (C) NCI-N87: gastric cancer cell line-Globo H positive, (D) SK-OV-3: ovarian cancer cell line-Globo H negative.

[0037] FIG. 4. In vitro persistence of Globo H CAR T cells with CD3zeta mutant (CD3zm). (A) Cytotoxicity between MCF-7 (Globo H positive) and SK-OV-3 (Globo H negative) cancer cell lines. (B) T cell number between MCF-7 (Globo H positive) and SK-OV-3 (Globo H negative) cancer cell lines.

[0038] FIG. 5. In vivo efficacy of Globo H CAR T cells in NCI-N87 gastric xenograft model. (A) Tumor bioluminescence images from Day 11 to Day 32. (B) Kinetics of tumor bioluminescence.

[0039] FIG. 6. In vivo efficacy of Globo H CAR T in different tumor models. (A) MCF-7 breast cancer orthotopic model, (B) HCC-1428 breast cancer orthotopic model, (C) SW480 colon cancer xenograft model.

[0040] FIG. 7. In vivo persistence of Globo H CAR T in NCI-N87 gastric xenograft model. (A) Kinetics of tumor bioluminescence in four tumor groups (1.sup.st tumor growth) and one tumor-free mock control from Day 0 to Day 47. The curves show data for mean?SD of three mice per group. The primary tumors treated with 2C2-Fab CAR T or 2C2-Fab (CD3zm) CAR T cells have been all eliminated at Day 25, (B) Kinetics of 2.sup.nd and 3.sup.rd tumor bioluminescence in 2C2-Fab and 2C2-Fab (CD3zm) CAR T cell therapeutic groups from Day 45 to Day 90.

DETAILED DESCRIPTION OF THE INVENTION

[0041] As used herein, the articles a and an refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, an element means one element or more than one element

[0042] An effective amount, as used herein, refers to a dose of the vaccine or pharmaceutical composition that is sufficient to reduce the symptoms and signs of cancer, such as weight loss, pain and palpable mass, which is detectable, either clinically as a palpable mass or radiologically through various imaging means. The term effective amount and therapeutically effective amount are used interchangeably.

[0043] The term subject can refer to a vertebrate having cancer or to a vertebrate deemed to be in need of cancer treatment. Subjects include all warm-blooded animals, such as mammals, such as a primate, and, more preferably, a human. Non-human primates are subjects as well. The term subject includes domesticated animals, such as cats, dogs, etc., livestock (for example, cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (for example, mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses and medical formulations are contemplated herein.

[0044] The following examples of specific aspects for carrying out the present invention are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

EXAMPLES

Example 1. Globo H CAR-T Cells Preparation

[0045] Anti-Globo H antibody 2C2 or R783 were used to perform scFv or Fab CAR constructs. Intracellular domains contain CD28, 4-1BB and CD3zeta to perform third generation CAR. CD3zeta mutant was a mutant domain with less tyrosine kinase activity to generate persistence CAR. Schematic diagram of Globo-H CAR was listed in FIG. 1. Human T cells isolate from cryopreserved human PBMCs by human Pan T Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130-096-535). Activate and expand pan T cells with Dynabeads Human T-Activator CD3/CD28 (ThemoFisher, Cat. No. 11131D) at cells/beads ratio 1:1 in RPMI-1640 medium supplemental with 10% FBS and recombinant human IL-7 (10 ng/mL, PeproTech, Cat. No. 200-07) and IL-15 (10 ng/mL, PeproTech, Cat. No. 200-15). After 2 days activation, T cells transduce with 1 MOI lentivirus for CAR expression. On 3 days later, remove Dynabeads from transduced T cells and re-fresh medium every 2-3 days with IL-7/IL-15 (10 ng/mL, respectively) under cell density at 0.5-1?10.sup.6/mL. On days 10-14 after transduction, cells were collected for in vitro and in vivo experiments. T cells were cultured in IL-7- and IL-15-depleted medium for one day before being used for in vitro assays.

Example 2. Globo H CAR-T In Vitro Cytotoxicity Assay

[0046] The cytotoxicity of Globo H CAR-T cells against Globo-H positive tumor cell line (MCF-7 and HCC-1428: breast cancer, NCI-N87: gastric carcinoma, SW480: colon cancer) and Globo-H negative tumor cell line (SK-OV-3: ovarian cancer) at serial E:T ratio (Effector CAR-T cells: Target tumor cells) in healthy donors. The luciferase stable expressed target cells pre-attached overnight in 96-well white Polystyrene plate for luminescence reading. Serial dilution of IL-7 and IL-15 depleted CAR-T cells add to target cells for 24 hours co-culture. The luminescence signal detection by Bio-Glo Luciferase Assay System (Promega, Cat. No. G7940).

[0047] In FIG. 2, it indicated 2C2-Fab, 2C2-scFv, R783-Fab and R783-scFv CAR T cells had efficacy in Globo-H positive tumor cell lines (FIG. 2A-2D) but not existed in Globo-H negative tumor cell line (FIG. 2E). It could also demonstrate the specificity of our Globo-H CAR-T construct. In FIG. 3, it also indicated 2C2-Fab, 2C2-Fab (CD3zm), R783-scFv and R783-scFv (CD3zm) CAR T cells had similar efficacy in Globo-H positive tumor cell lines (FIG. 3A-3C) but not existed in Globo-H negative tumor cell line (FIG. 3D). It could demonstrate the CD3zeta mutant (CD3zm) CAR without loss cytotoxicity.

[0048] Furthermore, another in vitro persistence assay was performed. Luciferase reporter tumor cell lines (1?10.sup.5 MCF-7 or SK-OV3) were pre-stained with CellTracker? Deep Red Dye (Thermo Fisher, Cat. No. C34565) for 15 minutes at 37? C. and then seeded in 24-well tissue culture plates for 16 hours, after which 1?10.sup.5 (E:T=1:1) CAR T cells were added to the tumor cells. After 3 days, tumor cells had been completely eradicated (round 1). All cells in the well were collected and washed with PBS, resuspended in fresh 2% FBS RPMI-1640 medium and added to a new plate seeded with 1?10.sup.5 tumor cells for 3 days (round 2). This procedure was repeated one more time, if applicable (round 3). At the end of each round, absolute cell number of residual tumor cells (APC+) and CAR T cells (CD3+) was calculated with CountBright? Absolute Counting Beads (Thermo Fisher, Cat. No. C36950) by flow cytometry.

[0049] In FIG. 4, it indicated CD3zm CAR T cells have higher cytotoxicity (FIG. 4A) and more residue CAR T cells after repetitively killing assay (FIG. 4B). It could demonstrate that 2C2-Fab CAR T with CD3zeta mutant (CD3zm) is more persistent than it with wild-type CD3zeta (CD3z).

Example 3. Globo H CAR-T In Vivo Efficacy Assay

Example 3-1: NCI-N87 Gastric Xenograft Model

[0050] Six to eight-week-old ASID mice (NOD.Cg-Prkdc.sup.scid Il2rg.sup.tm1Wjl/YckNarl) were purchased from National Laboratory Animal Center (Taipei, Taiwan) and were used in all in vivo models. ASID mice were injected with 2?10.sup.6 N87-Luc cells mixture with Matrigel (1:1, BD Bioscience) on Day 0 by subcutaneously injection on the right flank. Mice were treated intravenously with 2?10.sup.6 Globo H CAR-T cells or vector control T cells at Day 11. Flank tumor size was measured in three dimensions (mm.sup.3) with calipers. Mice were imaged on an Ami-HT optical imaging system twice per week during in vivo studies, after being intraperitoneal injected with 200 ?L of 15 mg/mL D-Luciferin (Biosynth, Cat. No. L-8220). Tumor volumes were calculated using the formula V=1/2 (length?width.sup.2).

[0051] In FIG. 5, it indicated 2C2-Fab and R783-scFv CAR T cells showed significant efficacy in NCI-N87 tumor model. FIG. 5A indicated representative tumor bioluminescence images from Day 11 to Day 32 post-tumor inoculation of CAR-T (n=3) and PBS control (n=3) groups. On Day 22, the overall nine ASID mice could observe tumor existed. However, after injection of 2C2-Fab or R783-scFv CAR-T cells for 14 days (Day 25) or 21 days (Day 32) respectively, there were only three ASID mice (vector control T cells) observe tumor existed. FIG. 5B indicated kinetics of tumor bioluminescence from three tumor groups and one tumor-free mock control. The curves show data for mean?SD of three mice per group.

Example 3-2: In Vivo Efficacy of Globo H CAR T in Different Tumor Models

[0052] MCF-7 and HCC-1428 Breast Cancer Orthotopic Models:

[0053] Six to eight-week-old ASID mice were implanted subcutaneously with Estrogen pellet (0.36 mg/pellet 17 ?-estradiol, 90-day release, Innovative Research of America). After two days, mice were orthotopically inoculated 8?10.sup.6 MCF-7 or HCC-1428 cells mixture with Matrigel (1:1, BD Bioscience) by injection at 4.sup.th mammary fat pad. Mice were treated intravenously with 2?10.sup.6 Globo H CAR-T cells or vector control T cells at Day 12. Orthotopic tumor size was measured in three dimensions (mm 3) with calipers. Tumor volumes were calculated using the formula V=1/2 (length?width.sup.2).

[0054] SW-480 Colon Cancer Xenograft Model:

[0055] Six to eight-week-old ASID mice were injected with 1?10.sup.6 SW-480 cells mixture with Matrigel (1:1, BD Bioscience) on Day 0 by subcutaneously injection on the right flank. Mice were treated intravenously with 2?10.sup.6 Globo H CAR-T cells or vector control T cells at Day 12. Flank tumor size was measured in three dimensions (mm 3) with calipers. Tumor volumes were calculated using the formula V=1/2 (length?width.sup.2).

[0056] In FIG. 6, it indicated 2C2-Fab CAR T cells showed significant efficacy in MCF-7 (FIG. 6A), HCC-1428 (FIG. 6B) breast cancer orthotopic and SW-480 (FIG. 6C) colon cancer xenograft model. Kinetics of tumor size from two tumor groups injection with control T cells or 2C2-Fab CAR T cells (n=3 per group) in these models. The curves show data for mean?SD of three mice per group.

Example 4. In Vivo Persistence of Globo H CAR T in NCI-N87 Gastric Xenograft Model

[0057] Six to eight-week-old ASID mice were injected with 2?10.sup.6 N87-Luc cells mixture with Matrigel (1:1, BD Bioscience) on day 0 by subcutaneously injection on the right flank. Mice were treated intravenously with 2?10.sup.6 Globo H CAR-T cells or vector control T cells at Day 10. For the re-challenge experiments, mice received a 2nd tumor dose 2?10.sup.6 of N87-Luc by subcutaneously injection on the left flank at Day 40. The 3.sup.rd tumor inoculated subcutaneously with 2?10.sup.6 N87-Luc cells on the right flank at Day 69. Flank tumor size was measured in three dimensions (mm 3) with calipers. Mice were imaged on an Ami-HT optical imaging system twice per week during in vivo studies, after being intraperitoneal injected with 200 ?L of 15 mg/mL D-Luciferin (BIOSYNTH, Cat. No. L-8220). Tumor volumes were calculated using the formula V=1/2 (length?width.sup.2).

[0058] In FIG. 7, it indicated 2C2-Fab and 2C2-Fab (CD3zm) CAR T cells showed significant efficacy and persistence in NCI-N87 tumor model. FIG. 7A indicated kinetics of tumor bioluminescence in four tumor groups (1.sup.st tumor growth) and one tumor-free mock control from Day 0 to Day 47. The curves show data for mean?SD of three mice per group. The primary tumors treated with 2C2-Fab CAR T or 2C2-Fab (CD3zm) CAR T cells have been all eliminated at Day 25. FIG. 7B indicated kinetics of 2nd and 3rd tumor bioluminescence in 2C2-Fab and 2C2-Fab (CD3zm) CAR T cell therapeutic groups from Day 45 to Day 90. PBS control tumor groups are tumor-free mice to inoculate with 2?10.sup.6 N87-Luc tumor cells as re-challenge control. The 2n d tumors have also been clearance both in 2C2-Fab CAR T or 2C2-Fab (CD3zm) CAR T cells treated groups at Day 60 (n=3 per group). Only 2C2-Fab (CD3zm) CAR T cells treated groups are all survival after 3.sup.rd tumor challenge (n=3).

[0059] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein.

[0060] All references cited herein are incorporated herein by reference to the full extent allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.