Chimeric antigen receptors targeting tumor antigens

Abstract

Nucleic acid constructs encoding a chimeric antigen receptor (CAR) and a truncated human epidermal growth factor receptor (huEGFRt) are described. The encoded CAR includes a tumor antigen specific monoclonal antibody, such as a glypican-3 (GPC3)-specific, GPC2-specific or mesothelin-specific monoclonal antibody, fused to a CD8 [alpha] hinge region, a CD8 [alpha] transmembrane region, a 4-1BB co-stimulatory domain, and a CD3 [zeta] signaling domain. Also described are isolated host cells, such as isolated T cells that co-express the disclosed CAR and huEGFRt. The T cells transduced with the disclosed CAR constructs can be used in cancer immunotherapy.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of Chinese patent application CN201880073043.9, filed on November 7, 2018. This application claims the benefit of U.S. Provisional Application No. 62 / 584,421, filed on November 10, 2017, the entirety of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to chimeric antigen receptors specific for tumor antigens and their use in cancer immunotherapy.

[0004] Acknowledgement of Government Support

[0005] This invention was made with government support under Grant No. Z01 BC010891 awarded by the National Institutes of Health, National Cancer Institute. The U.S. Government has certain rights in this invention. Background Art

[0006] Chimeric antigen receptor (CAR) is composed of an antibody fragment specific to tumor antigens fused with a transmembrane domain and a T cell signaling moiety. When the receptor is expressed on the surface of T cells, it mediates the binding to the target and activates T cells, ultimately inducing target cell lysis. CAR is becoming one of the most promising methods for treating hematological malignancies (Kochenderfer et al., Blood 119: 2709-2720, 2012; Kochenderfer and Rosenberg, Nat Rev Clin Oncol 10: 267-276, 2013; Porter et al., New Engl J Med 365: 725-733, 2011; Maude et al., New Engl J Med 371: 1507-1517, 2014; Grupp et al., New Engl J Med 368: 1509-1518, 2013). Two CD19-targeted CARs have been approved in the United States: axicabtagene ciloleucel (Yescarta TM ) and tisagenlecleucel (Kymriah TM), which are used to treat B-cell non-Hodgkin's lymphoma and B-cell acute lymphoblastic leukemia, respectively. Clinical trials are currently underway to test various CAR T cell therapies for the treatment of solid tumors (Yu et al., J Hematol Oncol 10(1):78, 2017). Summary of the Invention

[0007] Disclosed herein are nucleic acid constructs encoding chimeric antigen receptors (CAR) and truncated human epidermal growth factor receptors (huEGFRt). Encoded CAR includes a tumor antigen-specific monoclonal antibody fragment fused to an extracellular hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signaling domain. HuEGFRt includes two EGFR extracellular domains (domain III and domain IV) and an EGFR transmembrane domain, but lacks two membrane distal extracellular domains and all intracellular domains. Also disclosed are cells, such as T lymphocytes, that co-express the disclosed CAR and huEGFRt separations. T cells transduced with CAR constructs can be used for cancer immunotherapy.

[0008] Provided herein are nucleic acid molecules encoding CAR and huEGFRt. In some embodiments, the nucleic acid molecule includes a nucleic acid encoding a first signal sequence in a 5' to 3' direction; a nucleic acid encoding an antigen-specific antibody or its antigen-binding fragment; a nucleic acid encoding an extracellular hinge region; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an intracellular costimulatory domain; a nucleic acid encoding an intracellular signaling domain; a nucleic acid encoding a self-cleavage 2A peptide; a nucleic acid encoding a second signal sequence; and a nucleic acid encoding huEGFRt. In some instances, the first and / or second signal sequence is a granulocyte-macrophage colony-stimulating factor receptor signal sequence (GMCSFRss), the extracellular hinge region is a CD8α hinge region, the transmembrane domain is a CD8α transmembrane domain, the intracellular costimulatory domain is a 4-1BB costimulatory domain, and the intracellular signaling domain is a CD3ζ signaling domain. In some instances, the antibody or antigen-binding fragment specifically binds to a tumor antigen, such as glypican-3 (GPC3), GPC2, or mesothelin. Also provided are vectors, such as viral vectors, comprising the nucleic acid molecules disclosed herein. In a specific, non-limiting example, the viral vector is a lentiviral vector. Further provided are isolated host cells comprising the nucleic acid molecules disclosed herein.

[0009] Also provided are isolated host cells that co-express CAR and huEGFRt. In some embodiments, CAR includes an antigen-specific antibody or its antigen-binding fragment, an extracellular hinge region, a transmembrane domain, an intracellular costimulatory domain, and an intracellular signaling domain; and / or huEGFRt includes domain III, domain IV, and a transmembrane domain from human EGFR, but lacks epidermal growth factor (EGF)-binding domain and cytoplasmic domain. In some instances, the extracellular hinge region includes a CD8α hinge region, the transmembrane domain includes a CD8α transmembrane domain, the intracellular costimulatory domain includes a 4-1BB costimulatory domain, and the intracellular signaling domain includes a CD3ζ signaling domain. In some instances, the antibody or antigen-binding fragment specifically binds to a tumor antigen, such as GPC3, GPC2, or mesothelin.

[0010] Further provided are compositions comprising the isolated host cells disclosed herein and a pharmaceutically acceptable carrier.In some embodiments, the isolated host cells are T lymphocytes.

[0011] Further provided are methods of treating a GPC3-positive cancer, a GPC2-positive cancer, or a mesothelin-positive cancer in a subject by administering to the subject an isolated host cell disclosed herein. In some embodiments, the isolated host cell is a T lymphocyte, such as an autologous T lymphocyte.

[0012] The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Schematic diagram of a lentiviral construct for producing a tumor-targeted chimeric antigen receptor (CAR). The lentiviral construct includes a CAR coding region and a region encoding a truncated human epidermal growth factor receptor (huEGFRt), each preceded by a granulocyte-macrophage colony-stimulating factor receptor signal sequence (GMCSFRss). The two regions are separated by a self-cleavage T2A sequence so that after the construct is expressed, the CAR is cut off from the huEGFRt. The expression of the construct is driven by the human elongation factor 1α (EF1α) promoter. The CAR includes an antigen binding region, a CD8α hinge region, a CD8α transmembrane (TM) domain, a 4-1BB costimulatory region, and a CD3ζ signaling domain. The huEGFRt includes two extracellular domains (domain III and domain IV) and a TM domain.

[0014] Figures 2A-2C The vector map of the following constructs: pMH288 encoding CAR.HN3 ( Figure 2A), pMH289 encoding CAR.hYP7 ( Figure 2B ) and pMH290 encoding CAR.LH7 ( Figure 2C ).

[0015] Figures 3A-3C Figure 1 is a flow cytometric graph showing the transduction efficiency of GPC3-targeted CAR T cells. Transduction efficiency was determined using the anti-huEGFRt antibody cetuximab. Figure 3A ) and CAR.hYP7( Figure 3B ) lentiviral vectors transduced 65% and 45.4% of T cells, respectively. Figure 3C ) Control human serum IgG.

[0016] Figures 4A-4G Figure 2 shows the cytotoxicity of GPC3-targeted CAR T cells against human cell lines. Effector:target ratios of 1:2, 1.5:1, 5:1, and 16:1 were used to determine the cytotoxicity of GPC3-targeted CAR T cells against human cell lines. + G1 cells ( Figure 4A ), GPC3 + Hep3B cells ( Figure 4B ), GPC3 + HepG2 cells ( Figure 4C ), GPC3 + Huh7 cells ( Figure 4D ), GPC3 - A431 cells ( Figure 4E ), GPC3 - T3M4 cells ( Figure 4F ) and GPC3 - IMR32 cells ( Figure 4G CAR.hYP7 was tested on GPC3-positive cell lines, but not on GPC3-negative cell lines.

[0017] Figure 5 is a graph showing that CAR.hYP7 T cells induce interferon (IFN)-γ secretion from target GPC-positive Hep3B, Huh7, and G1 tumor cells.

[0018] Figure 6Bioluminescent images showing Hep3B tumor suppression in mice treated with GPC3-targeted T cells. Mice were injected intravenously with 4 million Hep3B cells on day 0. On day 10, mice were injected with either a blank injection or PBS, 10 million CAR.HN3 T cells (HN3-10 M), 10 million CAR.hYP7 T cells (hYP7-10 M), 20 million CAR.hYP7 T cells (hYP7-20 M), or 40 million CAR.hYP7 T cells (hYP7-40 M). Tumor size was measured by bioluminescent imaging.

[0019] Figures 7A-7C This figure shows that CAR.hYP7 T cells have durable anti-tumor activity against Hep3B xenograft tumors in mice. Figure 7A ) Tumor volume of Hep3B tumor-bearing mice treated with PBS, vehicle treatment, 10 million CAR.HN3 T cells, 10 million CAR.hYP7 T cells, 20 million CAR.hYP7 T cells, or 40 million CAR.hYP7 T cells up to 3 weeks after treatment. ( Figure 7B ) Tumor volume in Hep3B tumor-bearing mice treated with PBS, 10 million CAR.HN3 T cells, 10 million CAR.hYP7 T cells, or 40 million CAR.hYP7 T cells up to 7 weeks after treatment. ( Figure 7C ) Survival curves of mice bearing Hep3B tumors. Ten days after Hep3B inoculation, mice were injected with PBS, 10 million CAR.hYP7 T cells, or 40 million CAR.hYP7 T cells, and survival was assessed for 70 days. Treatment with 40 million CAR.hYP7 T cells resulted in 100% survival.

[0020] Figures 8A-8D is to show that the mock-treated ( Figure 8A ) or treated with 10 million CAR.hYP7 T cells ( Figure 8B ) or 40 million CAR.hYP7 T cell therapy ( Figure 8C ) of tumor volumes in NSG mice bearing HepG2 xenografts. Figures 10A-10C Each row represents a single animal. Figure 8D Mean tumor volumes for all three treatment groups are shown.

[0021] Figure 9 Figure 3. GPC3 mRNA levels in normal human tissues measured by quantitative real-time PCR. Relative GPC3 levels in different normal tissues were compared with GPC3 expression in placenta.

[0022] Figures 10A-10E Showing the generation and expression of GPC3 CAR T cells. ( Figure 10A ) Schematic structures of HN3 and hYP7 antibodies binding to the N-lobe and C-lobe of mature GPC3, respectively. ( Figure 10B ) Schematic diagram of a bicistronic lentiviral construct expressing a GPC3-targeting CAR and huEGFRt using a T2A ribosomal skipping sequence. ( Figure 10C ) CAR expression on healthy donor-derived T cells transduced with lentiviral particles was analyzed by flow cytometry by detecting EGFR expression. ( Figure 10D ) CD3 expression of blank T cells and CAR(hYP7) T cells from healthy donors and CAR(hYP7) T cells from HCC patients + 、CD4 + and CD8 + T cell population analysis. ( Figure 10E ) Proliferation of CAR(hYP7) T cells in 8 different healthy donors and 4 different HCC patients was assessed by trypan blue exclusion assay.

[0023] Figures 11A-11G Graph showing that GPC3-targeted CAR T cells kill GPC3-positive HCC cells in vitro. ( Figure 11A ) As measured by luciferase activity, GPC3-positive target cells lyse (G1), but GPC3-negative target cells do not lyse (A431 and T3M4). Blank or GPC3-targeted CAR T cells were co-cultured with luciferase-expressing target cells at the specified effector (E): target (T) ratio for 24 hours, and specific lysis was measured using a fluorescence-based cell lysis assay. ( Figures 11B-11C ) were co-cultured with Hep3B cells for 24 hours, and the Figure 11B ) or HCC patients ( Figure 11C )'s cytolytic activity of CAR(HN3)T cells and CAR(hYP7)T cells. ( Figure 11D ) After stimulation with anti-CD3 / CD28 beads, CAR(hYP7) T cells proliferated strongly for 35 days. ( Figure 11E ) Cytolytic activity of CAR(hYP7) T cells on days 14 and 28 after activation after 24 hours of co-culture with Hep3B cells. Figure 11F ) Cytolytic activity of GPC3-specific CAR T cells from healthy donors after 24 hours of co-culture with HepG2 and Huh-7 cells. ( Figure 11G) GPC3-targeted CAR T cell-mediated killing of HepG2 cells measured using IncuCyte zoom. HepG2 cells were incubated with CAR T cells at an E:T ratio of 2:1 for up to 140 hours.

[0024] Figure 12 The cytokine / chemokine profiles and polyfunctionality of T cells redirected with GPC3-CAR are shown. Hep3B and HepG2 cells were co-cultured with GPC3-targeted CAR T cells at various E:T ratios for 24 hours, and the indicated cytokine / chemokine levels in the supernatants were measured using Luminex. Bars from left to right: blank, hYP7, and HN3. Mean and SD are shown. *p < 0.05; **p < 0.01; ***p < 0.001.

[0025] Figures 13A-13F Targeting GPC3 induced apoptosis in HCC cells by inhibiting Wnt / β-catenin signaling. Figure 13A ) After 6 hours of treatment, CAR(hYP7) T cells inhibited the expression of β-catenin and increased the expression of apoptosis markers (cleaved PARP and cleaved caspase-9) in Hep3B cells. ( Figure 13B )CAR(hYP7) T cells inhibited β-catenin expression in Hep3B cells in a time-dependent manner. ( Figure 13C ) GPC3 protein expression in Hep3B cells after CRISPR / Cas9-mediated GPC3 knockout. ( Figure 13D ) Antitumor activity of sgRNA5-2 targeting exon 5 of GPC3. 5×10 6 Hep3B cells. When the average tumor volume reaches 150mm 3 At the same time, mice were treated with intratumoral injections of sgRNA5-2 plasmid or empty vector every other day for 6 injections. Figure 13E ) Knockout of GPC3 reduces β-catenin expression in mouse tumors. ( Figure 13F ) Serum AFP levels before and after treatment with sgRNA5-2 plasmid or empty vector control. Mean and SD are shown. *p < 0.05; **p < 0.01.

[0026] Figures 14A-14F CAR(hYP7) T cells were shown to eradicate tumors in a Hep3B peritoneal dissemination xenograft mouse model. Figure 14A Schematic diagram of the experiment. On day 12 after tumor cell inoculation, Hep3B tumor-bearing NSG mice were peritoneally injected with blank T cells and 5×10 6CAR(HN3)T cells, 5×10 6 CAR(hYP7)T cells, 10×10 6 CAR(hYP7)T cells or 20×10 6 CAR(hYP7) T cells were used for treatment. Tumor burden was monitored by bioluminescence imaging. Figure 14B )CAR(hYP7) T cells regressed established Hep3B xenografts at a high dose (20 million cells) and inhibited tumor growth at a low dose (5 million or 10 million cells). Figure 14C ) Figure 14B Tumor bioluminescence in mice treated with β-catenin was measured as mean photon counts. Figure 14D ) Kaplan-Meier survival curves of tumor-bearing mice treated with 5 million or 20 million CAR(hYP7) T cells. ( Figure 14E ) Two or six weeks after CAR T treatment, Figure 14B AFP levels in serum collected from the indicated groups. Sera from three different mice in each group were collected for ELISA analysis. Figure 14F ) CAR T cell persistence in xenograft tumor tissues after 3 weeks of treatment, as measured by droplet digital PCR (ddPCR). Values ​​represent mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

[0027] Figures 15A-15D CAR(hYP7) T cells were shown to eliminate tumor cells in a HepG2 peritoneal dissemination xenograft mouse model. Figure 15A ) Experimental diagram. On day 21, NSG mice bearing Hep3B tumors were peritoneally injected with either blank T cells or 20×10 6 CAR(hYP7)T cells for treatment. Figure 15B )CAR(hYP7) T cells demonstrated potent anti-tumor activity and mediated eradication of HepG2 xenograft tumors. ( Figure 15C ) Figure 15B Tumor bioluminescence in mice treated with β-catenin was measured as mean photon counts. Figure 15D ) CAR T cell persistence in xenograft tumor tissues and mouse spleens after 5 weeks of treatment, as measured by ddPCR.

[0028] Figures 16A-16D HCC eradication by CAR(hYP7) T cells in a Hep3B orthotopic xenograft mouse model was demonstrated. Figure 16A ) Experimental schematic. On day 21, NSG mice bearing Hep3B orthotopic tumors were injected intraperitoneally or intravenously with 20×10 6CAR(hYP7) T cells. Mice were imaged weekly. Figure 16B ) Mice treated with CAR(hYP7) T cells via the tail vein demonstrated tumor eradication, while intraperitoneal treatment resulted in tumor growth inhibition. ( Figure 16C ) Figure 16B Tumor bioluminescence in mice treated with β-catenin was measured as mean photon counts. Figure 16D ) CAR T cell persistence in tumor tissue and mouse spleens after 5 weeks of treatment, as measured by ddPCR. Values ​​represent mean ± SD. **p < 0.01.

[0029] Figure 17 is a series of flow cytometric and Scatchard plots showing binding of GPC3-targeted (HN3 and hYP7) Jurkat CAR T cells to GPC3-human Fc (hFc) fusion protein.

[0030] Figure 18 Figure 2 is a series of graphs showing differential cytokine and chemokine secretion measured by Luminex after 24 hours of incubation of GPC3-targeted CAR T cells with Hep3B and HepG2 tumor cells. Bars from left to right: blank, hYP7, and HN3. *P < 0.05, **P < 0.01, ***P < 0.001.

[0031] Figures 19A-19B Shown are the body weights of Hep3B and HepG2 tumor xenograft mice after treatment with GPC3-targeted CAR T cells. Figure 19A ) Body weight of Hep3B tumor model mice after intraperitoneal injection of PBS, blank T-cells, CAR(hYP7)T cells, or CAR(HN3)T cells. ( Figure 19B ) Body weight of HepG2 tumor model mice after intraperitoneal injection of 20 million blank T cells or CAR(hYP7) T cells.

[0032] Sequence Listing

[0033] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are presented using the standard nucleotide base abbreviations and amino acid three-letter codes defined in 37 CFR 1.822. Each nucleic acid sequence is shown for only one strand, but it is understood that the complementary strand is included by any reference to the strand shown. The sequence listing is submitted as an ASCII text file, generated on October 29, 2018, 59.3 KB, and is incorporated herein by reference. In the accompanying sequence listing:

[0034] SEQ ID NO: 1 is the nucleotide sequence encoding GMCSFRss.

[0035] SEQ ID NO: 2 is the amino acid sequence of GMCSFRss.

[0036] SEQ ID NO: 3 is the nucleotide sequence encoding the CD8α hinge.

[0037] SEQ ID NO: 4 is the amino acid sequence of the CD8α hinge.

[0038] SEQ ID NO: 5 is the nucleotide sequence encoding the CD8α transmembrane domain.

[0039] SEQ ID NO: 6 is the amino acid sequence of the CD8α transmembrane domain.

[0040] SEQ ID NO: 7 is a nucleotide sequence encoding 4-1BB.

[0041] SEQ ID NO: 8 is the amino acid sequence of 4-1BB.

[0042] SEQ ID NO: 9 is the nucleotide sequence encoding CD3ζ.

[0043] SEQ ID NO: 10 is the amino acid sequence of CD3ζ.

[0044] SEQ ID NO: 11 is a nucleotide sequence encoding the self-cleaving T2A peptide.

[0045] SEQ ID NO: 12 is the amino acid sequence of the self-cleaving T2A peptide.

[0046] SEQ ID NO: 13 is the nucleotide sequence encoding huEGFRt.

[0047] SEQ ID NO: 14 is the amino acid sequence of huEGFRt.

[0048] SEQ ID NO: 15 is the nucleotide sequence encoding CAR.hYP7, having the following characteristics: Nucleotides 1-66 = GMCSFRss coding sequence

[0049] Nucleotides 67-72 = NdeI restriction site

[0050] Nucleotides 73-807 = humanized YP7 scFv coding sequence

[0051] Nucleotides 808-813 = SpeI restriction site

[0052] Nucleotides 814-948 = CD8α hinge region coding sequence

[0053] Nucleotides 949-1011 = CD8α transmembrane domain coding sequence Nucleotides 1012-1137 = 4-1BB costimulatory domain coding sequence Nucleotides 1138-1473 = CD3ζ signaling domain coding sequence Nucleotides 1474-1527 = T2A coding sequence

[0054] Nucleotides 1528-1593 = GMCSFRss coding sequence

[0055] Nucleotides 1594-2598 = huEGFRt coding sequence.

[0056] SEQ ID NO: 16 is the amino acid sequence of CAR.hYP7, having the following features: Residues 1-22 = GMCSFRss

[0057] Residues 23-24 = HM (encoded by NdeI restriction site)

[0058] Residues 25-269 = humanized YP7 scFv

[0059] Residues 270-271 = TS (encoded by SpeI restriction site)

[0060] Residues 272-316 = CD8α hinge region

[0061] Residues 317-337 = CD8α transmembrane domain

[0062] Residues 338-379 = 4-1BB costimulatory domain

[0063] Residues 380-491 = CD3ζ signaling domain

[0064] Residues 492-509 = self-cleaving T2A peptide

[0065] Residues 510-531 = GMCSFRss

[0066] Residues 532-866 = huEGFRt coding sequence.

[0067] SEQ ID NO: 17 is the nucleotide sequence encoding CAR.HN3, having the following characteristics: Nucleotides 1-66 = GMCSFRss coding sequence

[0068] Nucleotides 67-72 = NdeI restriction site

[0069] Nucleotides 73-420 = HN3 coding sequence

[0070] Nucleotides 421-426 = SpeI restriction site

[0071] Nucleotides 427-561 = CD8α hinge region coding sequence

[0072] Nucleotides 562-624 = CD8α transmembrane domain encoding sequence Nucleotides 625-750 = 4-1BB costimulatory domain encoding sequence Nucleotides 751-1086 = CD3ζ signaling domain encoding sequence

[0073] Nucleotides 1087-1140 = T2A coding sequence

[0074] Nucleotides 1141-1206 = GMCSFRss coding sequence

[0075] Nucleotides 1207-2211 = huEGFRt coding sequence.

[0076] SEQ ID NO: 18 is the amino acid sequence of CAR.HN3, having the following features: Residues 1-22 = GMCSFRss

[0077] Residues 23-24 = HM (encoded by NdeI restriction site)

[0078] Residues 25-140 = HN3 single domain antibody

[0079] Residues 141-142 = TS (encoded by SpeI restriction site)

[0080] Residues 143-187 = CD8α hinge region

[0081] Residues 188-208 = CD8α transmembrane domain

[0082] Residues 209-250 = 4-1BB costimulatory domain

[0083] Residues 251-362 = CD3ζ signaling domain

[0084] Residues 363-380 = self-cleaving T2A peptide

[0085] Residues 381-402 = GMCSFRss

[0086] Residues 403-737 = huEGFRt coding sequence.

[0087] SEQ ID NO: 19 is the nucleotide sequence encoding CAR.LH7, having the following characteristics: Nucleotides 1-66 = GMCSFRss coding sequence

[0088] Nucleotides 67-72 = NdeI restriction site

[0089] Nucleotides 73-432 = LH7 coding sequence

[0090] Nucleotides 433-438 = SpeI restriction site

[0091] Nucleotides 439-573 = CD8α hinge region coding sequence

[0092] Nucleotides 574-636 = CD8α transmembrane domain coding sequence Nucleotides 637-762 = 4-1BB costimulatory domain coding sequence Nucleotides 763-1098 = CD3ζ signaling domain coding sequence Nucleotides 1099-1152 = T2A coding sequence

[0093] Nucleotides 1153-1218 = GMCSFRss coding sequence

[0094] Nucleotides 1219-2223 = huEGFRt coding sequence.

[0095] SEQ ID NO: 20 is the amino acid sequence of CAR.LH7, having the following features: Residues 1-22 = GMCSFRss

[0096] Residues 23-24 = HM (encoded by NdeI restriction site)

[0097] Residues 25-144 = LH7 single domain antibody

[0098] Residues 145-146 = TS (encoded by SpeI restriction site)

[0099] Residues 147-191 = CD8α hinge region

[0100] Residues 192-212 = CD8α transmembrane domain

[0101] Residues 213-254 = 4-1BB costimulatory domain

[0102] Residues 255-366 = CD3ζ signaling domain

[0103] Residues 367-384 = self-cleaving T2A peptide

[0104] Residues 385-406 = GMCSFRss

[0105] Residues 407-741 = huEGFRt coding sequence.

[0106] SEQ ID NO: 21 is the nucleotide sequence of the YP7 VH domain.

[0107] SEQ ID NO: 22 is the amino acid sequence of the YP7 VH domain.

[0108] SEQ ID NO: 23 is the nucleotide sequence of the YP7 VL domain.

[0109] SEQ ID NO: 24 is the amino acid sequence of the YP7 VL domain.

[0110] SEQ ID NO: 25 is the nucleotide sequence of the hYP7 VH domain.

[0111] SEQ ID NO: 26 is the amino acid sequence of the hYP7 VH domain.

[0112] SEQ ID NO: 27 is the nucleotide sequence of the hYP7 VL domain.

[0113] SEQ ID NO: 28 is the amino acid sequence of the hYP7 VL domain.

[0114] SEQ ID NO: 29 is the nucleotide sequence of the HN3 single domain antibody.

[0115] SEQ ID NO: 30 is the amino acid sequence of the HN3 single domain antibody.

[0116] SEQ ID NO: 31 is the nucleotide sequence of the LH7 single domain antibody.

[0117] SEQ ID NO: 32 is the amino acid sequence of the LH7 single domain antibody.

[0118] SEQ ID NO: 33 is the nucleotide sequence of the LH4 single domain antibody.

[0119] SEQ ID NO: 34 is the amino acid sequence of the LH4 single domain antibody.

[0120] SEQ ID NO: 35 is the nucleotide sequence of the LH6 single domain antibody.

[0121] SEQ ID NO: 36 is the amino acid sequence of the LH6 single domain antibody.

[0122] SEQ ID NO: 37 is the nucleotide sequence of the YP218 VH domain.

[0123] SEQ ID NO: 38 is the amino acid sequence of the YP218 VH domain.

[0124] SEQ ID NO: 39 is the nucleotide sequence of the YP218 VL domain.

[0125] SEQ ID NO:40 is the amino acid sequence of the YP218 VL domain.

[0126] SEQ ID NO: 41 is the nucleotide sequence of the SD1 single domain antibody.

[0127] SEQ ID NO: 42 is the amino acid sequence of the SD1 single domain antibody.

[0128] SEQ ID NOs: 43-51 are sgRNA sequences. DETAILED DESCRIPTION

[0129] I. Abbreviations

[0130] ADCC antibody-dependent cell-mediated cytotoxicity

[0131] CAR chimeric antigen receptor

[0132] CDR complementarity determining region

[0133] CTL cytotoxic T lymphocyte

[0134] ddPCR (droplet digital PCR)

[0135] DMEM Dulbecco's modified Eagle medium

[0136] EF1α elongation factor 1α

[0137] EGF epidermal growth factor

[0138] EGFR epidermal growth factor receptor

[0139] ELISA enzyme-linked immunosorbent assay

[0140] FACS fluorescence activated cell sorting

[0141] FBS fetal bovine serum

[0142] GPC2 glypican-2

[0143] GPC3 glypican-3

[0144] GMCSFRss granulocyte-macrophage colony stimulating factor receptor signalsequence

[0145] HCC hepatocellular carcinoma

[0146] HLA human leukocyte antigen

[0147] huEGFRt human truncated epidermal growth factor receptor

[0148] IFN interferon

[0149] Ig immunoglobulin

[0150] IL interleukin

[0151] ip intraperitoneal

[0152] ITAM immunoreceptor tyrosine-based activation motif

[0153] PBMC peripheral blood mononuclear cell

[0154] PBS Phosphate-buffered saline

[0155] scFv single-chain variable fragment

[0156] TM transmembrane

[0157] VH or V H variable heavy chain

[0158] VL or V L variable light chain

[0159] YST yolk sac tumor

[0160] II. Terminology and Methodology

[0161] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in the following documents: Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0162] To facilitate review of the various embodiments of the present disclosure, the following explanations of specific terms are provided:

[0163] 4-1BB: A co-stimulatory molecule expressed by T cell receptor (TCR)-activated lymphocytes and other cells, including natural killer cells. Ligation of 4-1BB induces a signaling cascade that leads to cytokine production, expression of anti-apoptotic molecules, and enhanced immune responses.

[0164] Acute lymphoblastic leukemia (ALL): An acute form of leukemia characterized by an overproduction of lymphoblasts. ALL is most common in children, with incidence peaking in children aged 2 to 5 years.

[0165] Antibody: A polypeptide ligand containing at least one variable region that recognizes and binds (e.g., specifically recognizes and binds) an epitope of an antigen. Mammalian immunoglobulin molecules are composed of heavy (H) chains and light (L) chains, each of which has a variable region, referred to as the heavy chain variable (VH ) region and light chain variable (V L ) area. H Area and V L The two regions are collectively responsible for binding to the antigen recognized by the antibody. There are five major heavy chain classes (or isotypes) of mammalian immunoglobulins, which determine the functional activity of the antibody molecules IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is the predominant antibody produced by birds and reptiles and is somewhat similar in function to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0166] The variable region of an antibody consists of a "framework" region and hypervariable regions, called "complementarity determining regions" or "CDRs." The CDRs are primarily responsible for binding to the epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; the "Kabat" numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., (JMB 273, 927-948, 1997; the "Chothia" numbering scheme), and the ImMunoGeneTics (IMGT) database (see Lefranc, Nucleic Acids Res 29:207-9, 2001; the "IMGT" numbering scheme). The Kabat and IMGT databases are maintained online.

[0167] "Single-domain antibody" refers to an antibody with a single domain (variable domain) that is capable of specifically binding to an antigen or an epitope of an antigen in the absence of other antibody domains. Single-domain antibodies include, for example, V H Domain antibodies, V NAR Antibodies, camelid V HH antibody and V L Domain antibodies. V NAR Antibodies are produced by cartilaginous fish such as nurse sharks, wobbegong sharks, spiny dogfish, and bamboo sharks. H H antibodies are produced by several species, including camel, llama, alpaca, dromedary, and guanaco, which naturally produce heavy chain antibodies lacking light chains.

[0168] A "monoclonal antibody" is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence for a single antibody has been transfected. The antibodies are produced by methods known to those skilled in the art. Monoclonal antibodies include humanized monoclonal antibodies.

[0169] "Chimeric antibodies" have framework residues from one species, such as human, and the CDRs (which typically confer antigen binding) from another species.

[0170] "Humanized" antibodies are antibodies that include a human framework region and one or more CDRs from a non-human (e.g., mouse, rabbit, rat, shark, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is referred to as the "donor," and the human immunoglobulin that provides the framework is referred to as the "acceptor." In one embodiment, all CDRs in the humanized immunoglobulin are from a donor immunoglobulin. Constant regions do not need to exist, but if present, they must be substantially identical to human immunoglobulin constant regions, i.e., have at least about 85-90%, such as about 95% or higher identity. Therefore, all parts of the humanized immunoglobulin, possibly with the exception of the CDRs, are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. Humanized antibodies bind to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies may have other conservative amino acid substitutions that have little or no effect on antigen binding or other immunoglobulin functions.

[0171] Binding affinity: The affinity of an antibody for an antigen. In one embodiment, affinity is calculated by the modified Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by antigen / antibody dissociation rate. In another embodiment, high binding affinity is measured by competitive radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. In another embodiment, antibody affinity is measured by flow cytometry. An antibody that "specifically binds" to an antigen (e.g., GPC3) is one that binds to that antigen with high affinity and does not significantly bind to other unrelated antigens.

[0172] Breast cancer: A type of cancer that develops in breast tissue, typically the ducts (tubes that carry milk to the nipple) and lobules (glands that produce milk). Triple-negative breast cancer refers to a type of breast cancer in which the cancer cells do not express estrogen receptors, progesterone receptors, or significant levels of the HER2 / neu protein. Triple-negative breast cancer is also called ER-negative, PR-negative, and HER2 / neu-negative breast cancer.

[0173] Chemotherapeutic agent: Any chemical agent that has therapeutic activity in treating diseases characterized by abnormal cell growth. These diseases include tumors, neoplasms, and cancers, as well as diseases characterized by hyperplastic growth, such as psoriasis. In one embodiment, the chemotherapeutic agent is a radioactive compound. One skilled in the art can readily determine the chemotherapeutic agent to use (see, for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2). nd ed., 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, DS, Knobf, MF, Durivage, HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. An example is the administration of CAR T cells in combination with radioactive or chemical compounds.

[0174] Chimeric antigen receptor (CAR): a chimeric molecule comprising an antigen binding portion (e.g., a single domain antibody or scFv) and a signaling domain (e.g., a signaling domain from a T cell receptor (e.g., CD3ζ)). Typically, CAR consists of an antigen binding portion, a transmembrane domain, and an intracellular domain. The intracellular domain typically includes a signaling chain with an immunoreceptor tyrosine activation motif (ITAM), such as CD3ζ or FcεRIγ. In some cases, the intracellular domain also includes the intracellular portion of at least one other costimulatory domain, such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or DAP10.

[0175] Cholangiocarcinoma: A type of cancer that develops in the cells lining the bile ducts of the liver.

[0176] Complementarity Determining Region (CDR): A region of highly variable amino acid sequence that determines the binding affinity and specificity of an antibody. The light and heavy chains of mammalian immunoglobulins each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively.

[0177] Conservative variants: "Conservative" amino acid substitutions are those that do not substantially affect or reduce the affinity of a protein, such as an antibody, for GPC3. For example, a monoclonal antibody that specifically binds to GPC3 may include up to about 1, up to about 2, up to about 5, up to about 10, or up to about 15 conservative substitutions and specifically bind to a GPC3 polypeptide. The term "conservative variant" also encompasses the use of a substituted amino acid in place of the unsubstituted parent amino acid, as long as the variant retains activity. Non-conservative substitutions are those that reduce the activity of the protein.

[0178] Providing conservative amino acid substitution tables for functionally similar amino acids is well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered conservative substitutions for each other:

[0179] 1) Alanine (A), serine (S), threonine (T);

[0180] 2) Aspartic acid (D), glutamic acid (E);

[0181] 3) Asparagine (N), glutamine (Q);

[0182] 4) Arginine (R), Lysine (K);

[0183] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0184] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0185] In some embodiments herein, an amino acid sequence is provided that includes no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitution relative to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:32.

[0186] Cytotoxic agent: Any drug or compound that kills cells.

[0187] Cytotoxicity: The toxicity of a molecule to the cells to which it is intended to be targeted, as opposed to cells in the rest of the organism.

[0188] Degenerate variants: Polynucleotides encoding polypeptides with sequences that are degenerate due to the genetic code. There are 20 naturally occurring amino acids, most of which are specified by more than one codon. Therefore, as long as the amino acid sequence of the polypeptide remains unchanged, all degenerate nucleotide sequences are included.

[0189] Desmoplastic small round cell tumor (DRCT): A soft tissue sarcoma that primarily occurs in childhood, particularly in boys. DRCT is an aggressive and rare cancer that primarily presents as an abdominal mass but can also be found in the lymph nodes, abdominal lining, diaphragm, spleen, liver, chest wall, skull, spinal cord, intestine, bladder, brain, lungs, testicles, ovaries, and pelvis.

[0190] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic, meaning they elicit a specific immune response. Antibodies specifically bind to specific epitopes on polypeptides.

[0191] Ewing's sarcoma: A rare type of cancerous tumor found in bone or soft tissue. Ewing's sarcoma is a small, blue, round cell tumor.

[0192] Framework region: The amino acid sequence between the CDRs. The framework region includes both the light chain variable and heavy chain variable framework regions. The framework region serves to maintain the CDRs in the proper orientation for antigen binding.

[0193] Fusion protein: A protein that contains at least portions of two different (heterologous) proteins.

[0194] Glypican-2 (GPC2): A member of the six-member glypican family of heparan sulfate (HS) proteoglycans, which are attached to the cell surface via a GPI anchor (Filmus et al., Genome Biol 9:224, 2008). GPC2 is uniquely expressed in the nervous system (Stipp et al., J Cell Biol 124:149-160, 1994), where it participates in cell adhesion and is thought to regulate axonal growth and guidance. In addition, GPC2 mRNA is highly expressed in neuroblastoma and other pediatric cancers (Orentas et al., Front Oncol 2:194, 2012). GPC2 is also known as cerebroglycan and glypican 2. The GPC2 genome, mRNA, and protein sequences are publicly available (see, e.g., NCBI Gene ID 221914).

[0195] GPC2-positive cancer: A cancer that overexpresses GPC2. Examples of GPC2-positive cancers include, but are not limited to, neuroblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

[0196] Glypican-3 (GPC3): A member of the glypican family of heparan sulfate (HS) proteoglycans, which are attached to the cell surface via a glycosylphosphatidylinositol anchor (Filmus and Selleck, J Clin Invest 108:497-501, 2001). The GPC3 gene encodes a core protein of approximately 70 kD that can be cleaved by furin to produce a 40 kD N-terminal fragment and a 30 kD C-terminal fragment. Two HS chains are attached to the C-terminal portion of GPC3. GPC3 and other glypican family proteins play a role in cell division and cell growth regulation. GPC3 is highly expressed in HCC and some other human cancers, including melanoma, lung squamous cell carcinoma, and ovarian clear cell carcinoma (Ho and Kim, Eur J Cancer 47(3):333-338, 2011), but is not expressed in normal tissues. GPC3 is also known as SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1, and GTR2-2.

[0197] Human GPC3 has four known isoforms (isoforms 1-4). The nucleic acid and amino acid sequences of the four isoforms of GPC3 are known, including GenBank accession numbers: NM_001164617 and NP_001158089 (isoform 1); NM_004484 and NP_004475 (isoform 2); NM_001164618 and NP_001158090 (isoform 3); and NM_001164619 and NP_001158091 (isoform 4).

[0198] GPC3-positive cancer: A cancer that overexpresses GPC3. Examples of GPC3-positive cancers include, but are not limited to, HCC, melanoma, ovarian clear cell carcinoma, yolk sac tumor (YST), neuroblastoma, hepatoblastoma, Wilms tumor, lung squamous cell carcinoma, testicular non-seminomatous germ cell tumor, liposarcoma, cervical intraepithelial neoplasia, adrenal adenoma, schwannoma, and embryonal tumor (Ho and Kim, Eur J Cancer 47(3):333-338, 2011; Baumhoer et al., Am J Clin Pathol 129(6):899-906, 2008; Saikali and Sinnett, Int J Cancer 89(5):418-422, 2000).

[0199] HAMA (human anti-murine antibody) response: An immune response in a human subject to the variable and constant regions of a murine antibody that has been administered to that patient. Repeated administration of an antibody may result in an increased rate of clearance of the antibody from the patient's serum and may also cause an allergic reaction in the patient.

[0200] Hepatocellular carcinoma (HCC): A primary malignant tumor of the liver that usually occurs in patients with inflamed livers caused by viral hepatitis, hepatotoxins, or cirrhosis (often caused by alcoholism). HCC is also known as malignant liver cancer.

[0201] Heterologous: Derived from a single genetic resource or species.

[0202] Immune response: A response by cells of the immune system (e.g., B cells, T cells, or monocytes) to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, e.g., a CD4 + response or CD8 + In another embodiment, the response is a B cell response and results in the production of specific antibodies.

[0203] Isolated: An "isolated" biological component (e.g., a nucleic acid, protein (including antibodies), or organelle) has been substantially separated or purified away from other biological components (i.e., other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles) in the environment (e.g., a cell) in which the component naturally occurs. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0204] Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule (such as an antibody or protein) to facilitate the detection of that molecule. Non-limiting specific examples of labels include fluorescent tags, enzyme linkages, and radioisotopes. In one example, a "labeled antibody" refers to the introduction of another molecule into an antibody. For example, a label is a detectable marker, such as an amino acid that is incorporated with a radiolabel or is linked to a biotin moiety polypeptide that can be detected by labeled avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (for example 35 S. 11 C. 13 N. 15 O. 18 F. 19 F. 99m Tc, 131 I. 3 H. 14 C. 15 N. 90 Y. 99 Tc, 111 In and 125 I), fluorescent labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide fluorescent agents), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotin groups, predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, the labels are attached via spacer arms of various lengths to reduce potential steric hindrance.

[0205] Linker: In some cases, a linker is a peptide within an antibody binding fragment (e.g., an Fv fragment) that is used to indirectly bind the variable heavy chain to the variable light chain. A "linker" may also refer to a peptide used to connect a targeting moiety, such as an antibody, to an effector molecule, such as a cytotoxin or a detectable marker.

[0206] The terms "conjugating," "joining," "bonding," or "linking" refer to making two polypeptides into one continuous polypeptide molecule, or covalently attaching a radionuclide or other molecule to a polypeptide, such as an scFv. In certain contexts, the terms include reference to linking a ligand, such as an antibody moiety, to an effector molecule. Linking can be performed chemically or recombinantly. "Chemical" refers to a reaction between the antibody moiety and the effector molecule such that a covalent bond forms between the two molecules to form one molecule.

[0207] Lung cancer: A cancer that forms in lung tissue, typically in the cells lining the airways. The two main types are small cell lung cancer and non-small cell lung cancer (NSCLC). These types are diagnosed based on how the cells look under a microscope.

[0208] Mammal: This term includes human and non-human mammals. Similarly, the term "subject" includes human and veterinary subjects.

[0209] Melanoma: A cancer that originates in melanocytes (cells that produce the pigment melanin). Melanocytes are found primarily in the skin but are also present in the intestine and eyes. Melanomas of the skin include superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, and lentigo maligna (melanomas). Any of these types may produce melanin or may be amelanotic. Also, any subtype may show desmoplasia (a dense fibrous reaction with a neurotropic effect), a hallmark of aggressive behavior and a tendency to recur locally. Other melanomas include clear cell sarcoma, mucosal melanoma, and uveal melanoma.

[0210] Mesothelin: A 40 kDa cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein. Human mesothelin is synthesized as a 70 kDa precursor, which is then proteolytically processed. The 30 kDa amino terminus of mesothelin is secreted and is known as megakaryocyte potentiating factor (Yamaguchi et al., J. Biol. Chem. 269:805-808, 1994). As mature mesothelin, the 40 kDa carboxyl terminus remains membrane-bound (Chang et al., Natl. Acad. Sci. USA 93:136-140, 1996). Exemplary nucleic acid and amino acid sequences of mesothelin are described in PCT Publication No. WO 97 / 25,068; U.S. Pat. No. 6,083,502; Chang and Pastan, Int. J. Cancer 57:90, 1994; Chang and Pastan, Proc. Natl. Acad. Sci USA 93:136, 1996; Brinkmann et al., Int. J. Cancer 71:638, 1997; and Chowdhury et al., Mol. Immunol. 34:9, 1997. Mesothelin also refers to mesothelin proteins or polypeptides retained within cells as well as extracellular mesothelin proteins that are secreted and / or isolated.

[0211] Mesothelin-positive cancer: A cancer that overexpresses mesothelin. Examples of mesothelin-positive cancers include, but are not limited to, mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, triple-negative breast cancer, and ovarian cancer.

[0212] Mesothelioma: A type of tumor that originates from the cells lining the pleura and peritoneum. It grows into thick sheets covering the internal organs and is composed of spindle-shaped cells or fibrous tissue that may surround glandular spaces lined with cuboidal cells. Mesothelioma usually originates in the lining of the lungs, heart, or abdomen. In some cases, mesothelioma is caused by exposure to asbestos.

[0213] Neoplasia, malignancy, cancer, or tumor: A neoplasm is an abnormal growth of tissue or cells due to excessive cell division. Neoplastic growth can give rise to a tumor. The amount of tumor in an individual is the "tumor burden," which can be measured by the number, volume, or weight of tumors. Tumors that do not metastasize are called "benign." Tumors that invade surrounding tissues and / or can metastasize are called "malignant."

[0214] Neuroblastoma: A solid tumor that arises from embryonic neural crest cells. Neuroblastomas usually develop in and around the adrenal glands but can also occur anywhere sympathetic nerve tissue is found, such as in the abdomen, chest, neck, or near the spine. Neuroblastomas usually develop in children under the age of 5.

[0215] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first and second nucleic acid sequences are in a functional relationship. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, in the case of joining two protein coding regions, in the same reading frame.

[0216] Osteosarcoma: A type of cancerous tumor found in the bones. Osteosarcoma is an aggressive cancer that originates from primitive transformed cells of mesenchymal origin. This type of cancer is most common in children and young adults.

[0217] Ovarian cancer: Cancer that develops in the tissues of the ovary (one of the pair of female reproductive glands where eggs, or ova, are formed). Most ovarian cancers are either epithelial ovarian cancer (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in the egg cells).

[0218] Ovarian clear cell carcinoma: A distinct histopathological subtype of epithelial ovarian cancer that accounts for less than 5% of all ovarian malignancies. When viewed under a microscope, the cells of this type of tumor appear clear inside.

[0219] Pancreatic cancer: A disease in which malignant (cancer) cells are found in the tissues of the pancreas. Also called exocrine cancer.

[0220] Pediatric cancer: Cancer that develops in children between the ages of 0 and 14. Major types of pediatric cancer include, for example, neuroblastoma, acute lymphoblastic leukemia (ALL), embryonal rhabdomyosarcoma (ERMS), alveolar rhabdomyosarcoma (ARMS), Ewing's sarcoma, desmoplastic small round cell tumor (DRCT), osteosarcoma, brain and other CNS tumors, Wilm's tumor, non-Hodgkin lymphoma, and retinoblastoma.

[0221] Pharmaceutical Agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or cell.

[0222] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers used are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein.

[0223] In some embodiments, the present invention provides the pharmaceutical composition of the present invention.Usually, the character of carrier will depend on the specific mode of administration adopted.For example, parenteral preparation generally includes injectable fluid, and it includes pharmaceutically and physiologically acceptable fluid, for example water, normal saline, balanced salt solution, glucose aqueous solution, glycerol etc. are as vehicle.For solid composition (for example powder, pill, tablet or capsule form), conventional non-toxic solid carrier can include, for example pharmaceutical grade mannitol, lactose, starch or magnesium stearate.Except biological neutral carrier, pharmaceutical composition to be used can contain a small amount of nontoxic auxiliary substance, for example wetting agent or emulsifying agent, preservative and pH buffer etc., for example sodium acetate or sorbitan monolaurate.

[0224] Preventing, treating, or ameliorating a disease: "Preventing" a disease means inhibiting the overall development of the disease. "Treatment" refers to therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has already developed, such as reducing tumor burden or decreasing the number and size of metastases. "Amelioration" refers to a decrease in the number or severity of signs or symptoms of a disease, such as cancer.

[0225] Prostate cancer: Cancer that develops in the tissues of the prostate (a gland in the male reproductive system located below the bladder and in front of the rectum). Prostate cancer usually occurs in older men.

[0226] Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than in its natural environment within a cell. In one embodiment, the preparation is purified so that the protein or peptide constitutes at least 50% of the total peptide or protein content of the preparation. Substantially purified refers to purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95%, or 98% pure. Thus, in a specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.

[0227] Recombinant: A recombinant nucleic acid is a nucleic acid having a non-naturally occurring sequence or a sequence that is the result of the artificial combination of two separate fragments of sequence. This artificial combination is usually achieved by chemical synthesis or by artificial manipulation of separate nucleic acid fragments, such as through genetic engineering techniques.

[0228] Rhabdomyosarcoma (RMS): A soft tissue malignancy originating from skeletal muscle. The most common primary sites of rhabdomyosarcoma are the head and neck (e.g., parameningeal, orbital, pharyngeal, etc.), genitourinary tract, and extremities. Other less common primary sites include the trunk, chest wall, abdomen (including the retroperitoneum and biliary tract), and perineal / anal region. There are at least two types of RMS. The most common forms are alveolar RMS (ARMS) and embryonal histological RMS (ERMS). Approximately 20% of children with rhabdomyosarcoma have the ARMS subtype. This subtype is found with increased frequency in adolescents and in patients whose primary sites involve the extremities, trunk, and perineal / perianal region. ARMS is associated with a chromosomal translocation encoding a fusion gene involving FKHR and PAX family members on chromosome 13. The embryonal subtype is the most common subtype in children, accounting for approximately 60-70% of childhood rhabdomyosarcoma. Tumors with embryonal histology typically arise in the head and neck or genitourinary tract, although they may occur at any primary site. ERMS are characterized by younger age at diagnosis, loss of heterozygosity, and altered genomic imprinting.

[0229] Sample (or biological sample): A biological sample obtained from a subject and containing genomic DNA, RNA (including mRNA), protein, or a combination thereof. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsies, fine needle aspirates, surgical specimens, and autopsy material. In one example, the sample comprises a tumor biopsy, such as a tumor tissue biopsy.

[0230] Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed as the similarity between the sequences (or sequence identity). Sequence identity is often measured as a percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of polypeptides or nucleic acid molecules will have a relatively high degree of sequence identity when aligned using standard methods.

[0231] Methods for alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994 presents detailed considerations of sequence alignment methods and homology calculations.

[0232] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the internet, and is used in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to use this program to determine sequence identity is available on the NCBI website on the internet.

[0233] V of an antibody that specifically binds to a GPC3 polypeptide L or V HHomologues and variants are generally characterized in that, according to the full-length comparison calculation of the antibody amino acid sequence using NCBIBlast2.0, gapped Blastp is set to default parameters, there is at least about 75%, for example, at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In order to be more than about 30 amino acid whose amino acid sequences are compared, the default BLOSUM62 matrix ((gap existence cost is 11 for gaps, and 1 for every residue gap cost) is used to use the Blast2 sequence function) that is set to default parameters. When comparing short peptides (less than about 30 amino acids), the comparison should be carried out using the Blast2 sequence function, using the PAM30 matrix (open gap 9, extension gap 1 penalty) that is set to default parameters. When evaluated by this method, proteins with even greater similarity to the reference sequence will show increasing percentages of identity, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When comparing sequence identity over less than the entire sequence, homologs and variants will typically have at least 80% sequence identity over a short window of 10-20 amino acids, and may have at least 85%, or at least 90%, or 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity over these short windows are available on the NCBI website on the internet. Those skilled in the art will recognize that these sequence identity ranges are provided as a guide only; it is entirely possible to obtain strongly significant homologs that fall outside the provided ranges.

[0234] Squamous cell carcinoma: A type of cancer that begins in the squamous cells, thin, flat cells that line the skin, eyes, the surface of various internal organs, and the hollow organs and ducts of certain glands. Squamous cell carcinoma is also called epidermoid carcinoma. One type of squamous cell carcinoma is squamous cell carcinoma of the lung. Squamous cell carcinoma is the most common type of skin cancer.

[0235] Stomach cancer: Cancer that develops in the tissues lining the stomach. Also called gastric cancer.

[0236] Subject: Living multicellular vertebrate organisms, including humans and the category of veterinary subjects, including human and non-human mammals.

[0237] Synthetic: Produced artificially in a laboratory, such as a synthetic nucleic acid or protein (eg, an antibody) that can be chemically synthesized in a laboratory.

[0238] Therapeutically effective amount: The amount of a particular substance sufficient to achieve the desired effect in the subject being treated. For example, this may be the amount required to inhibit or prevent tumor growth. In one embodiment, a therapeutically effective amount is the amount necessary to eliminate, reduce the size of a tumor, or prevent tumor metastasis. When administered to a subject, a dose will generally be used that achieves a target tissue concentration (e.g., in a tumor) that has been shown to achieve the desired in vitro effect.

[0239] Vector: A nucleic acid molecule introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that allow it to replicate in the host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.

[0240] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present disclosure belongs. Unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include the plural. "Comprising A or B" means comprising A or B, or A and B. It should also be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for descriptive purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, particularly suitable methods and materials are described below. In the event of conflict, the present specification (including explanation of terms) shall prevail. In addition, these materials, methods and examples are illustrative only and are not intended to be limiting.

[0241] III. Overview of Several Implementations

[0242] Disclosed herein are nucleic acid molecules encoding chimeric antigen receptor (CAR) and truncated human epidermal growth factor receptor (huEGFRt).The coded CAR includes a tumor antigen-specific monoclonal antibody fused with an extracellular hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signal transduction domain.The huEGFRt includes two EGFR extracellular domains (domain III and domain IV) and a transmembrane domain, but lacks two membrane distal extracellular domains (domain I and domain II) and all intracellular domains (near membrane domain, tyrosine kinase domain, and C- end tail). Also provided are cells separated by co-expression of the disclosed CAR and huEGFRt, such as T lymphocytes.T cells transduced with the CAR construct can be used for cancer immunotherapy.

[0243] Provided herein are nucleic acid molecules encoding CAR and huEGFRt. In some embodiments, the nucleic acid molecule comprises, in a 5' to 3' direction, a nucleic acid encoding a first signal sequence; a nucleic acid encoding an antigen-specific antibody or antigen-binding fragment thereof; a nucleic acid encoding an extracellular hinge region; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an intracellular costimulatory domain; a nucleic acid encoding an intracellular signaling domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second signal sequence; and a nucleic acid encoding huEGFRt.

[0244] The first and second signal sequences can be any suitable signal sequences known in the art. The first and second signal sequences can be the same signal sequence or they can be different signal sequences. In some embodiments, the first and / or second signal sequence is a granulocyte-macrophage colony stimulating factor receptor signal sequence (GMCSFRss).

[0245] In some embodiments, the extracellular hinge region comprises a CD8α hinge region, a CD28 hinge region, or a sequence from another immunoglobulin molecule, such as IgG1, IgG4, or IgD (e.g., a CH2 and / or CH3 domain from an immunoglobulin molecule). The hinge region is sometimes also referred to in the art as a "spacer region."

[0246] In some embodiments, the transmembrane domain comprises a CD8α, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154 transmembrane domain. The transmembrane domain can also be the transmembrane region of the α, β or ζ chain of the T cell receptor.

[0247] In some embodiments, the intracellular costimulatory domain includes 4-1BB (CD137, TNFRSF9), CD28, ICOS, OX40 (CD134), CD27, CD30, CD40, PD-1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 or DAP10 costimulatory domain. In some instances, the intracellular costimulatory domain includes 4-1BB and CD28.

[0248] In some embodiments, the intracellular signaling domain is a domain having an immunoreceptor tyrosine-based activation motif (ITAM), such as a CD3ζ or FcεRIγ signaling domain.

[0249] In a specific embodiment, the first and second signal sequences comprise GMCSFRss, the extracellular hinge region comprises a CD8α hinge region, the transmembrane domain comprises a CD8α transmembrane domain, the intracellular costimulatory domain comprises a 4-1BB costimulatory domain, and the intracellular signaling domain comprises a CD3ζ signaling domain.

[0250] In some examples, the nucleic acid encoding the CD8 alpha hinge comprises a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In a non-limiting example, the nucleic acid encoding the CD8 alpha hinge comprises the sequence of SEQ ID NO: 3.

[0251] In some examples, the nucleic acid encoding the CD8α transmembrane domain comprises a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5. In one non-limiting example, the nucleic acid encoding the CD8α transmembrane domain comprises the sequence of SEQ ID NO: 5.

[0252] In some examples, the nucleic acid molecule encoding the 4-1BB costimulatory domain comprises a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7. In one non-limiting example, the nucleic acid molecule encoding the 4-1BB costimulatory domain comprises the sequence of SEQ ID NO: 7.

[0253] In some examples, the nucleic acid encoding the CD3 zeta signaling domain comprises a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9. In one non-limiting example, the nucleic acid encoding the CD3 zeta signaling domain comprises the sequence of SEQ ID NO: 9.

[0254] In some examples, the nucleic acid encoding the first GMCSFRss and the nucleic acid encoding the second GMCSFRss each comprise a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In a non-limiting example, the nucleic acid encoding the first GMCSFRss and the nucleic acid encoding the second GMCSFRss each comprise the sequence of SEQ ID NO: 1.

[0255] In some examples, the self-cleaving 2A peptide is a T2A peptide from adenovirus 2A. In other examples, the self-cleaving 2A peptide is a F2A peptide from foot and mouth disease virus 2A, an E2A peptide from equine rhinitis virus 2A, or a P2A peptide from porcine teschovirus-1. In specific examples, the nucleic acid encoding the T2A peptide comprises a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11. In one non-limiting example, the nucleic acid encoding the T2A peptide comprises the sequence of SEQ ID NO: 11.

[0256] In some examples, the nucleic acid encoding huEGFRt comprises a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13. In one non-limiting example, the nucleic acid encoding huEGFRt comprises the sequence of SEQ ID NO: 13.

[0257] In some embodiments, the nucleic acid molecule further comprises a human elongation factor 1α (EF1α) promoter sequence 5′ of the nucleic acid encoding the first GMCSFRss. However, those skilled in the art can select any suitable promoter sequence.

[0258] In some embodiments, the antigen-binding fragment is a single-chain variable fragment (scFv) or a single-domain antibody.

[0259] In some embodiments, the antibody or antigen-binding fragment specifically binds to a tumor antigen. In particular examples, the tumor antigen is GPC3, GPC2, or mesothelin.

[0260] In some examples, wherein the tumor antigen is GPC3, the nucleic acid encoding the antibody binding fragment includes the heavy chain variable (VH) domain complementarity determining region 1 (CDR1), CDR2, and CDR3 nucleic acid sequences of SEQ ID NO: 25 (hYP7 VH domain nucleotide sequence) and the light chain variable (VL) domain CDR1, CDR2, and CDR3 nucleic acid sequences of SEQ ID NO: 27 (hYP7 VL domain nucleotide sequence). CDR sequences can be determined using any known numbering scheme, such as IMGT, Kabat, or Chothia. In specific examples, the VH domain CDR1, CDR2, and CDR3 nucleic acid sequences each include nucleotides 91-105, 148-204, and 301-318 of SEQ ID NO: 25; and / or the VL domain CDR1, CDR2, and CDR3 nucleic acid sequences each include nucleotides 70-120, 166-186, and 283-309 of SEQ ID NO: 27. In other specific examples, the VH domain CDR1, CDR2, and CDR3 nucleic acid sequences each include nucleotides 76-99, 151-180, and 295-318 of SEQ ID NO: 25; and / or the VL domain CDR1, CDR2, and CDR3 nucleic acid sequences each include nucleotides 79-114, 166-174, and 283-309 of SEQ ID NO: 27. In a non-limiting example, the nucleic acid encoding the antibody binding fragment includes the sequence of nucleotides 73-807 of SEQ ID NO: 15.

[0261] In other examples, wherein the tumor antigen is GPC3, the nucleic acid encoding the antibody binding fragment includes the CDR1, CDR2 and CDR3 nucleic acid sequences of SEQ ID NO: 29 (HN3 single domain antibody nucleotide sequence). Any known numbering scheme, such as IMGT, Kabat or Chothia, can be used to determine the CDR sequence. In a specific example, the CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 91-105, 148-195 and 286-315 of SEQ ID NO: 29. In other specific examples, the CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 76-99, 151-171 and 286-315 of SEQ ID NO: 29. In a non-limiting example, the nucleic acid encoding the antibody binding fragment includes the sequence of nucleotides 73-420 of SEQ ID NO: 17.

[0262] In other examples, where the tumor antigen is GPC3, the nucleic acid encoding the antibody binding fragment includes the CDR nucleic acid sequences of the GPC3-specific monoclonal antibodies disclosed in WO 2013 / 181543 or WO 2012 / 145469, which are incorporated herein by reference in their entireties.

[0263] In some instances, wherein the tumor antigen is GPC2, the nucleic acid encoding the antibody binding fragment includes the CDR1, CDR2 and CDR3 nucleic acid sequences of SEQ ID NO: 31 (LH7 single domain antibody nucleotide sequence). Any known numbering scheme such as IMGT, Kabat or Chothia can be used to determine the CDR sequence. In a specific example, the CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 91-105, 148-195 and 286-327 of SEQ ID NO: 31. In other specific examples, the CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 76-99, 151-171 and 286-327 of SEQ ID NO: 31. In a non-limiting example, the nucleic acid encoding the antibody binding fragment includes the sequence of nucleotides 73-432 of SEQ ID NO: 19.

[0264] In other examples, wherein the tumor antigen is GPC2, the nucleic acid encoding the antibody binding fragment includes the CDR1, CDR2 and CDR3 nucleic acid sequences of SEQ ID NO: 33 (LH4 single domain antibody nucleotide sequence). Any known numbering scheme, such as IMGT, Kabat or Chothia, can be used to determine the CDR sequence. In a specific example, the CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 91-105, 148-195 and 286-327 of SEQ ID NO: 33. In other specific examples, the CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 76-99, 151-171 and 286-327 of SEQ ID NO: 33.

[0265] In other examples, wherein the tumor antigen is GPC2, the nucleic acid encoding the antibody binding fragment includes the CDR1, CDR2 and CDR3 nucleic acid sequences of SEQ ID NO: 35 (LH6 single domain antibody nucleotide sequence). Any known numbering scheme such as IMGT, Kabat or Chothia can be used to determine the CDR sequence. In a specific example, the CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 91-105, 148-198 and 289-330 of SEQ ID NO: 35. In other specific examples, the CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 76-99, 151-174 and 289-330 of SEQ ID NO: 35.

[0266] In other examples, wherein the tumor antigen is GPC2, the nucleic acid encoding the antibody binding fragment includes the CDR nucleic acid sequence of the GPC2-specific monoclonal antibody disclosed in Li et al., Proc Natl Acad Sci USA 114(32):E6623-E6631, 2017.

[0267] In some examples, wherein the tumor antigen is mesothelin, the nucleic acid encoding the antibody binding fragment includes the VH domain CDR1, CDR2, and CDR3 nucleic acid sequences of SEQ ID NO: 37 (YP218 VH domain nucleotide sequence) and the VL domain CDR1, CDR2, and CDR3 nucleic acid sequences of SEQ ID NO: 39 (YP218 VL domain nucleotide sequence). CDR sequences can be determined using any well-known numbering scheme, such as IMGT, Kabat, or Chothia. In specific examples, the VH domain CDR1, CDR2, and CDR3 nucleic acid sequences each include nucleotides 91-108, 101-204, and 298-36 of SEQ ID NO: 37; and / or the VL domain CDR1, CDR2, and CDR3 nucleic acid sequences each include nucleotides 70-102, 148-168, and 265-303 of SEQ ID NO: 39. In other specific examples, the VH domain CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 79-102, 154-177 and 292-336 of SEQ ID NO:37; and / or the VL domain CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 79-96, 148-156 and 265-303 of SEQ ID NO:39.

[0268] In other examples, wherein the tumor antigen is mesothelin, the nucleic acid encoding the antibody binding fragment includes the CDR1, CDR2 and CDR3 nucleic acid sequences of SEQ ID NO: 41 (SD1 single domain antibody nucleotide sequence). Any well-known numbering scheme such as IMGT, Kabat or Chothia can be used to determine the CDR sequence. In a specific example, the CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 91-105, 151-198 and 295-306 of SEQ ID NO: 41. In other specific examples, the CDR1, CDR2 and CDR3 nucleic acid sequences each include nucleotides 78-105, 151-174 and 289-309 of SEQ ID NO: 41. Further provided herein are vectors comprising the CAR-encoding nucleic acid molecules disclosed herein. In some embodiments, the vector is a viral vector, for example, but not limited to a lentiviral vector.

[0269] In other examples, wherein the tumor antigen is mesothelin, the nucleic acid encoding the antibody binding fragment includes the CDR nucleic acid sequence of a mesothelin-specific monoclonal antibody disclosed in WO 2014 / 031476, WO 2014 / 052064, U.S. Patent No. 8,460,660, U.S. Patent No. 6,809,184, or U.S. Patent No. 7,081,518, each of which is herein incorporated by reference in its entirety.

[0270] Also provided are isolated host cells comprising a CAR-encoding nucleic acid molecule disclosed herein. In some embodiments, the isolated host cell is a T lymphocyte.

[0271] The present disclosure also provides an isolated host cell that co-expresses a chimeric antigen receptor (CAR) and a truncated human epidermal growth factor receptor (huEGFRt). In some embodiments, the CAR includes an antigen-specific antibody or its antigen-binding fragment, an extracellular hinge region, a transmembrane domain, an intracellular costimulatory domain, and an intracellular signaling domain; and / or the huEGFRt includes domain III, domain IV, and a transmembrane domain from human EGFR, but lacks an epidermal growth factor (EGF)-binding domain and a cytoplasmic domain.

[0272] In some embodiments, the extracellular hinge region comprises a CD8α hinge region, a CD28 hinge region, or a sequence from another immunoglobulin molecule, such as IgG1, IgG4, or IgD (e.g., from the CH2 and / or CH3 domains of an immunoglobulin molecule).

[0273] In some embodiments, the transmembrane domain comprises a CD8α, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154 transmembrane domain. The transmembrane domain can also be the transmembrane region of the α, β or ζ chain of the T cell receptor.

[0274] In some embodiments, the intracellular co-stimulatory domain includes 4-1BB (CD137, TNFRSF9), CD28, ICOS, OX40 (CD134), CD27, CD30, CD40, PD-1, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 or DAP10 co-stimulatory domain.

[0275] In some embodiments, the intracellular signaling domain is an ITAM-bearing domain, such as a CD3ζ or FcεRIγ signaling domain.

[0276] In a specific embodiment, the extracellular hinge region comprises a CD8α hinge region, the transmembrane domain comprises a CD8α transmembrane domain, the intracellular costimulatory domain comprises a 4-1BB costimulatory domain, and the intracellular signaling domain comprises a CD3ζ signaling domain.

[0277] In some examples, the amino acid sequence of the CD8α hinge region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In specific examples, the amino acid sequence of the CD8α hinge region includes SEQ ID NO: 4.

[0278] In some examples, the amino acid sequence of the CD8α transmembrane domain is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In specific examples, the amino acid sequence of the CD8α transmembrane domain includes SEQ ID NO: 6.

[0279] In some examples, the amino acid sequence of the 4-1BB costimulatory domain is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8. In specific examples, the amino acid sequence of the 4-1BB costimulatory domain includes SEQ ID NO: 8.

[0280] In some examples, the amino acid sequence of the CD3 zeta signaling domain is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In specific examples, the amino acid sequence of the CD3 zeta signaling domain includes SEQ ID NO: 10.

[0281] In some examples, the amino acid sequence of huEGFRt is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14. In specific examples, the amino acid sequence of huEGFRt includes SEQ ID NO: 14.

[0282] In some embodiments, the antigen-binding fragment is a scFv or a single domain antibody.

[0283] In some embodiments, the antibody or antigen-binding fragment specifically binds to a tumor antigen. In some instances, the tumor antigen is GPC3, GPC2, or mesothelin.

[0284] In some examples, where the tumor antigen is GPC3, the amino acid sequence of the antigen-binding fragment comprises the VH domain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 26 (hYP7 VH domain) and the VL domain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 28 (hYP7 VL domain). CDR sequences can be determined using any well-known numbering scheme, such as IMGT, Kabat, or Chothia. In specific examples, the VH domain CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 31-35, 50-68, and 101-106 of SEQ ID NO: 26 and / or the VL domain CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 24-40, 56-62, and 95-103 of SEQ ID NO: 28. In other specific examples, the VH domain CDR1, CDR2, and CDR3 amino acid sequences each include residues 26-33, 51-60, and 99-106 of SEQ ID NO: 26 and / or the VL domain CDR1, CDR2, and CDR3 amino acid sequences each include residues 27-38, 56-58, and 95-103 of SEQ ID NO: 28. In a non-limiting example, the amino acid sequence of the antibody-binding fragment includes residues 25-269 of SEQ ID NO: 16.

[0285] In other examples, wherein the tumor antigen is GPC3, the amino acid sequence of the antigen-binding fragment comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 30 (HN3 single domain antibody sequence). CDR sequences can be determined using any well-known numbering scheme, such as IMGT, Kabat, or Chothia. In specific examples, the CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 31-35, 50-65, and 96-105 of SEQ ID NO: 30. In other specific examples, the CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 26-33, 51-57, and 96-105 of SEQ ID NO: 30. In one non-limiting example, the amino acid sequence of the antibody-binding fragment comprises residues 25-140 of SEQ ID NO: 18.

[0286] In other examples, wherein the tumor antigen is GPC3, the amino acid sequence of the antibody binding fragment includes the CDR sequences of the GPC3-specific monoclonal antibodies disclosed in WO 2013 / 181543 or WO 2012 / 145469, which are incorporated herein by reference in their entireties.

[0287] In some instances, wherein the tumor antigen is GPC2, the amino acid sequence of the antigen-binding fragment comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 32 (LH7 single domain antibody sequence). CDR sequences can be determined using any well-known numbering scheme, such as IMGT, Kabat, or Chothia. In a specific example, the CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 26-33, 51-57, and 96-109 of SEQ ID NO: 32. In other specific examples, the CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 31-35, 50-65, and 96-109 of SEQ ID NO: 32. In a non-limiting example, the amino acid sequence of the antibody-binding fragment comprises residues 25-144 of SEQ ID NO: 20.

[0288] In other examples, wherein the tumor antigen is GPC2, the amino acid sequence of the antigen-binding fragment comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 34 (LH4 single domain antibody sequence). CDR sequences can be determined using any well-known numbering scheme, such as IMGT, Kabat, or Chothia. In specific examples, the CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 31-35, 50-65, and 96-109 of SEQ ID NO: 34. In other specific examples, the CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 26-33, 51-57, and 96-109 of SEQ ID NO: 34.

[0289] In other examples, wherein the tumor antigen is GPC2, the amino acid sequence of the antigen-binding fragment comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 36 (LH6 single domain antibody sequence). CDR sequences can be determined using any well-known numbering scheme, such as IMGT, Kabat, or Chothia. In specific examples, the CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 31-35, 50-66, and 97-110 of SEQ ID NO: 36. In other specific examples, the CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 26-33, 51-58, and 97-110 of SEQ ID NO: 36.

[0290] In other examples, wherein the tumor antigen is GPC2, the amino acid sequence of the antibody binding fragment includes the CDR sequence of the GPC2-specific monoclonal antibody disclosed in Li et al., Proc Natl Acad Sci USA 114(32):E6623-E6631, 2017.

[0291] In some examples, wherein the tumor antigen is mesothelin, the amino acid sequence of the antigen-binding fragment comprises the VH domain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 38 (YP218 VH domain) and the VL domain CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 40 (YP218 VL domain). CDR sequences can be determined using any well-known numbering scheme, such as IMGT, Kabat, or Chothia. In specific examples, the VH domain CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 31-36, 51-68, and 100-112 of SEQ ID NO: 38 and / or the VL domain CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 24-34, 50-56, and 89-101 of SEQ ID NO: 40. In other specific examples, the VH domain CDR1, CDR2 and CDR3 amino acid sequences each include residues 27-34, 52-59 and 98-112 of SEQ ID NO:38 and / or the VL domain CDR1, CDR2 and CDR3 amino acid sequences each include residues 27-32, 50-52 and 89-101 of SEQ ID NO:40.

[0292] In other examples, wherein the tumor antigen is mesothelin, the amino acid sequence of the antigen-binding fragment comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 42 (SD1 single domain antibody sequence). CDR sequences can be determined using any well-known numbering scheme, such as IMGT, Kabat, or Chothia. In specific examples, the CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 31-35, 51-66, and 99-102 of SEQ ID NO: 42. In other specific examples, the CDR1, CDR2, and CDR3 amino acid sequences each comprise residues 26-35, 51-58, and 97-103 of SEQ ID NO: 42.

[0293] In other examples, wherein the tumor antigen is mesothelin, the amino acid sequence of the antibody binding fragment includes the CDR sequences of the mesothelin-specific monoclonal antibodies disclosed in WO 2014 / 031476, WO 2014 / 052064, U.S. Patent No. 8,460,660, U.S. Patent No. 6,809,184, or U.S. Patent No. 7,081,518, each of which is herein incorporated by reference in its entirety.

[0294] In some embodiments, the isolated host cell is a T lymphocyte. In some instances, the T lymphocyte is an autologous T lymphocyte. In other instances, the T lymphocyte is an allogeneic T lymphocyte.

[0295] Also provided herein are compositions comprising the isolated (CAR-expressing) host cells disclosed herein and a pharmaceutically acceptable carrier.

[0296] Further provided herein are methods for treating GPC3-positive cancers in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an isolated host cell comprising a nucleic acid molecule encoding a GPC3-targeting CAR disclosed herein, or administering a therapeutically effective amount of an isolated host cell co-expressing a GPC3-targeting CAR and huEGFRt, as disclosed herein. In some instances, the GPC3-positive cancer is hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, yolk sac tumor, neuroblastoma, hepatoblastoma, or Wilms tumor.

[0297] Also provided are methods for treating GPC2-positive cancers in subjects. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an isolated host cell comprising a nucleic acid molecule encoding a GPC2-targeting CAR disclosed herein, or using a therapeutically effective amount of an isolated host cell co-expressing a GPC2-targeting CAR and huEGFRt, as disclosed herein. In some instances, the GPC2-positive cancer is neuroblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

[0298] Further provided are methods for treating a mesothelin-positive cancer in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an isolated host cell comprising a nucleic acid molecule encoding a mesothelin-targeting CAR disclosed herein, or administering a therapeutically effective amount of an isolated host cell co-expressing a mesothelin-targeting CAR and huEGFRt, as disclosed herein. In some instances, the mesothelin-positive cancer is mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, triple-negative breast cancer, or ovarian cancer.

[0299] In some embodiments of the methods of treatment, the isolated host cell is a T lymphocyte. In some instances, the T lymphocyte is an autologous T lymphocyte. In other instances, the T lymphocyte is an allogeneic T lymphocyte.

[0300] In one embodiment herein, a nucleic acid molecule encoding a CAR is provided, which comprises, in a 5' to 3' direction, a nucleic acid encoding a first GMCSFRss; a nucleic acid encoding an antigen-specific antibody or an antigen-binding fragment thereof; a nucleic acid encoding a CD8α hinge region; a nucleic acid encoding a CD8α transmembrane domain; a nucleic acid encoding a 4-1BB costimulatory domain; a nucleic acid encoding a CD3ζ signaling domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second GMCSFRss; and a nucleic acid encoding huEGFRt. In some examples, the nucleic acid encoding the antibody binding fragment comprises a sequence of nucleotides 73-807 of SEQ ID NO: 15, nucleotides 73-420 of SEQ ID NO: 17, or nucleotides 73-432 of SEQ ID NO: 19. In specific examples, the nucleic acid molecule is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19. In specific non-limiting examples, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19.

[0301] In one embodiment herein, there is provided an isolated host cell that co-expresses CAR and huEGFRt, wherein the CAR includes an antigen-specific antibody or antigen-binding fragment thereof, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain; and the huEGFRt includes domain III, domain IV, and a transmembrane domain from human EGFR, but lacks an EGF-binding domain and a cytoplasmic domain. In some instances, the amino acid sequence of the antigen-binding fragment includes residues 25-269 of SEQ ID NO: 16, residues 25-140 of SEQ ID NO: 18, or residues 25-144 of SEQ ID NO: 20. In specific examples, the amino acid sequence of CAR is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 25-491 of SEQ ID NO: 16, residues 25-362 of SEQ ID NO: 18, or residues 25-366 of SEQ ID NO: 20, and the amino acid sequence of huEGFRt is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14. In other specific examples, the amino acid sequence of the CAR comprises no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitution relative to residues 25-491 of SEQ ID NO: 16, residues 25-362 of SEQ ID NO: 18, or residues 25-366 of SEQ ID NO: 20, and the amino acid sequence of the huEGFRt comprises no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitution relative to SEQ ID NO: 14. In specific, non-limiting examples, the amino acid sequence of the CAR comprises residues 25-491 of SEQ ID NO: 16, residues 25-362 of SEQ ID NO: 18, or residues 25-366 of SEQ ID NO: 20, and the amino acid sequence of the huEGFRt comprises SEQ ID NO: 14.

[0302] IV. Antibodies specific for tumor antigens

[0303] CAR disclosed herein can be targeted to tumor cells expressing or overexpressing specific antigens by selecting appropriate tumor antigen-specific monoclonal antibodies or their antigen-binding fragments. In some embodiments, the antigen-binding portion of CAR is an antigen-binding fragment of a monoclonal antibody. In a specific example, the antigen-binding fragment is a scFv or a single domain (VH domain) antibody. Although CAR disclosed herein can be used with any antigen-specific antibody (or its antigen-binding fragment), exemplary antibodies include GPC3-specific, GPC2-specific and mesothelin-specific monoclonal antibodies.

[0304] A. GPC3-specific antibodies

[0305] The CAR construct disclosed herein can be modified to include any GPC3-specific monoclonal antibody or its antigen-binding fragment. Several GPC3-specific monoclonal antibodies are known in the art, including, but not limited to YP6, YP7, YP8, YP9 and YP9.1 disclosed in PCT Publication No. WO2013 / 181543, and HN3 disclosed in WO 2012 / 145469, which are incorporated herein by reference in their entirety. In some embodiments herein, the CAR includes an antigen-binding fragment comprising the CDR sequence of the GPC3-specific monoclonal antibody YP7 (disclosed in WO 2013 / 181543) or its humanized form. The nucleotide and amino acid sequences of YP7 and humanized YP7 (hYP7) are provided below. Tables 1A-1D show the CDR1, CDR2 and CDR3 of YP7 and hYP7. In other embodiments, the CAR comprises a single-domain monoclonal antibody comprising the CDR sequences of the GPC3-specific antibody HN3 (disclosed in WO 2012 / 145469). The nucleotide and amino acid sequences of HN3 are provided below. Tables 2A-2B show the positions of CDR1, CDR2, and CDR3 in HN3.

[0306] YP7 VH nucleotide sequence (SEQ ID NO: 21)

[0307] GAGGTGCAGCTTGTTGAGACTGGTGGAGGAATGGTGCAGCCTGAAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGAATGCCATGAATTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAATAAAACTAATAATTATGCAACATATTATGCCGATTCAGTGAAAGCCAGGTTTACCATCTCCAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAACAACTTGAAAATTGAGGACACAGCCATGTACTATTGTGTGGCTGGTAACTCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0308] YP7 VH amino acid sequence (SEQ ID NO:22)

[0309] EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTNNYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYCVAGNSFAYWGQGTLVTVSA

[0310] YP7 VL nucleotide sequence (SEQ ID NO:23)

[0311] GACATTGTGATGTCACAGTCTCCATCCTCCCTAGTTGTGTCAATTGGAGAGAAGGTTACTATGACCTGCAAGTCCAGTCAGAGCCTTTTATATAGCAGCAATCAAAAGAACTACTTGGCCTGGTACCAACAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCAGTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAACTATCCGCTCACGTTCGGTGCTGGGACCAAGTTGGAGCTGAAA

[0312] YP7 VL amino acid sequence (SEQ ID NO:24)

[0313] DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWAS SRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLELK

[0314] hYP7 VH nucleotide sequence (SEQ ID NO:25)

[0315] GAGGTGCAGCTTGTTGAGTCTGGTGGAGGATTGGTGCAGCCTGGAGGGTCATTGAGACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGAATGCCATGAATTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGGCCGCATAAGAAATAAAACTAATAATTATGCAACATATTATGCCGATTCAGTGAAAGCCAGGTTTACCATCTCCAGAGATGATTCAAAGAACTCACTCTATCTGCAAATGAACAGCTTGAAAACCGAGGACACAGCCGTGTACTATTGTGTGGCTGGTAACTCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0316] hYP7 VH amino acid sequence (SEQ ID NO:26)

[0317] EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTLVTVSA

[0318] hYP7 VL nucleotide sequence (SEQ ID NO:27)

[0319] GACATTTGTGATGACCCAGTCTCCAGACTCCCTAGCTGTGTCACTGGGAGAGAGGGCCACTATCAACTGCAAGTCCAGTCAGAGCCTTTTATATAGCAGCAATCAAAAGAACTACTTGGCCTGGTACCAACAGAAACCAGGGCAGCCTCCTAAACTGCTGATTTACTGGG CATCCAGTAGGGAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGTCAGCAATATTATAACTATCCGCTCACGTTCGGTCAGGGGACCAAGTTGGAGATCAAA

[0320] hYP7 VL amino acid sequence (SEQ ID NO: 28)

[0321] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWAS SRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIK

[0322] Table 1A. Position of CDRs in YP7 / hYP7 VH sequences (according to Kabat)

[0323]

[0324] Table 1B. Positions of CDRs in YP7 / hYP7 VH sequences (according to IMGT)

[0325]

[0326] Table 1C. Position of CDRs in YP7 / hYP7 VL sequences (according to Kabat)

[0327]

[0328] Table 1D. Positions of CDRs in YP7 / hYP7 VL sequences (according to IMGT)

[0329]

[0330] HN3 DNA sequence (SEQ ID NO:29)

[0331] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTTATTTCGATTTCGATTCTTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTAGAGTGGATTGGGAGTATCTATCATAGTGGGAGCACCTACT ACAACCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAGAGTAAATATGGACCGATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAAGT

[0332] HN3 protein sequence (SEQ ID NO: 30)

[0333] QVQLVQSGGGLVQPGGSLRLSCAASYFDFDSYEMSWVRQAPGKGLEWIGSIYHSGSTYYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTATYYCARVNMDRFDYWGQGTLVTVSSS

[0334] Table 2A. Positions of CDRs in HN3 sequences (according to Kabat)

[0335] CDR DNA sequence (SEQ ID NO:29) Protein sequence (SEQ ID NO: 30) CDR1 Nucleotides 91-105 Amino acids 31-35 CDR2 Nucleotides 148-195 Amino acids 50-65 CDR3 Nucleotides 286-315 Amino acids 96-105

[0336] Table 2B. Positions of CDRs in HN3 sequences (according to IMGT)

[0337] CDR DNA sequence (SEQ ID NO:29) Protein sequence (SEQ ID NO: 30) CDR1 Nucleotides 76-99 Amino acids 26-33 CDR2 Nucleotides 151-171 Amino acids 51-57 CDR3 Nucleotides 286-315 Amino acids 96-105

[0338] B. GPC2-specific antibodies

[0339] The CAR construct disclosed herein can also be transformed to include any GPC2-specific monoclonal antibody or its antigen binding fragment. In some embodiments herein, the CAR includes an antigen binding fragment, which includes the CDR sequences of GPC2-specific single domain monoclonal antibodies LH7, LH4, LH6, LH1, LH2 or LH3 (disclosed in Li et al., Proc Natl Acad Sci USA 114 (32): E6623-E6631, 2017). The nucleotide and amino acid sequences of LH7, LH4 and LH6 are provided below. Tables 3A-5B show the positions of CDR1, CDR2 and CDR3 of H7, LH4 and LH6.

[0340] LH7 DNA (SEQ ID NO: 31)

[0341] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGATTTCTATTTCTATGATTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTGGAGTGGATTGGGACTGTCTCCTATAGTGGGAGCACCTACTACAAC CCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTAAGAGCCGAGGACACAGCCATGTATTACTGTGCGAGAGGTTACAGCTATGATGACTCCCGATATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0342] LH7 protein (SEQ ID NO: 32)

[0343] QVQLVQSGGGLVQPGGSLRLSCAASDFYFYDYEMSWVRQAPGKGLEWIGTVSYSGSTYYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAMYYCARGYSYDDSRYFDYWGQGTLVTVSS

[0344] Table 3A. Position of CDRs in LH7 sequence (according to Kabat)

[0345] CDR DNA sequence (SEQ ID NO:31) Protein sequence (SEQ ID NO: 32) CDR1 91-105 31-35 CDR2 148-195 50-65 CDR3 286-327 96-109

[0346] Table 3B. Positions of CDRs in the LH7 sequence (according to IMGT)

[0347] CDR DNA sequence (SEQ ID NO:31) Protein sequence (SEQ ID NO: 32) CDR1 76-99 26-33 CDR2 151-171 51-57 CDR3 286-327 96-109

[0348] LH4 DNA (SEQ ID NO: 33)

[0349] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTTCTTTCTATTTCGATGATTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGCCCTGGAGTGGATTGGGCGTATCTATACCAGTGGGAGCACCAACTACAAC CCCTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAGGGGATATTGTAGTGGTGGTAGCTGCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0350] LH4 protein (SEQ ID NO: 34)

[0351] QVQLVQSGGGLVQPGGSLRLSCAASSFYFDDYEMSWVRQAPGKALEWIGRIYTSGSTNYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTATYYCARGYCSGGSCYFDYWGQGTLVTVSS

[0352] Table 4A. Position of CDRs in LH4 sequence (according to Kabat)

[0353] CDR DNA sequence (SEQ ID NO:33) Protein sequence (SEQ ID NO: 34) CDR1 91-105 31-35 CDR2 148-195 50-65 CDR3 286-327 96-109

[0354] Table 4B. Positions of CDRs in the LH4 sequence (according to IMGT)

[0355] CDR DNA sequence (SEQ ID NO:33) Protein sequence (SEQ ID NO: 34) CDR1 76-99 26-33 CDR2 151-171 51-57 CDR3 286-327 96-109

[0356] LH6 DNA (SEQ ID NO: 35)

[0357] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGATTTCTATTTCGATGATTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAGTGGTAGTGGTGGTGGCACATACTACG CAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCACATATTACTGTGCGAGAGGTTACAGTTATGACGACTCCCGATATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0358] LH6 protein (SEQ ID NO: 36)

[0359] QVQLVQSGGGLVQPGGSLRLSCAASDFYFDDYEMSWVRQAPGKGLEWVSTISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNTLRAEDTATYYCARGYSYDDSRYFDYWGQGTLVTVSS

[0360] Table 5A. Position of CDRs in LH6 sequence (according to Kabat)

[0361] CDR DNA sequence (SEQ ID NO:35) Protein sequence (SEQ ID NO: 36) CDR1 91-105 31-35 CDR2 148-198 50-66 CDR3 289-330 97-110

[0362] Table 5B. Positions of CDRs in the LH6 sequence (according to IMGT)

[0363] CDR DNA sequence (SEQ ID NO:35) Protein sequence (SEQ ID NO: 36) CDR1 76-99 26-33 CDR2 151-174 51-58 CDR3 289-330 97-110

[0364] C. Mesothelin-specific antibodies

[0365] The CAR constructs disclosed herein can also be modified to include any mesothelin-specific monoclonal antibody or its antigen-binding fragment. Several mesothelin-specific monoclonal antibodies are known in the art, including, but not limited to, YP218, YP223, YP3, YP158, and YP187 disclosed in PCT Publication No. WO 2014 / 031476, SD1 disclosed in PCT Publication No. WO 2014 / 052064, HN1 disclosed in U.S. Patent No. 8,460,660, SS disclosed in U.S. Patent No. 6,809,184, and SS1 disclosed in U.S. Patent No. 7,081,518, each of which is incorporated herein by reference in its entirety. The nucleotide and amino acid sequences of YP218 and SD1 are provided below. Tables 6A-7B show the positions of the CDRs of YP218 and SD1.

[0366] YP218 VH nucleotide sequence (SEQ ID NO: 37)

[0367] CAGCAGCAGCTGGAGGAGTCCGGGGGAGGCCTGGTCAAGCCTGAGGGATCCCTGACACTCACCTGCAAAGCCTCTGGATTCGACCTCGGTTTCTACTTTTACGCCTGTTGGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGATCGCATGCATTTATACTGCTGGTAGTGGTAGCACGTACT ACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAGCCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGGCAGCCGCGGACACGGCCACCTATTTCTGTGCGAGATCTACTGCTAATACTAGAAGTACTTATTATCTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0368] YP218 VH amino acid sequence (SEQ ID NO: 38)

[0369] QQQLEESGGGLVKPEGSLTLTCKASGFDLGFYFYACWVRQAPGKGLEWIACIYTAGSGSTYYASWAKGRFTISKASSTTVTLQMTSLAAADTATYFCARSTANTRSTYYLNLWGPGTLVTVSS

[0370] YP218 VL nucleotide sequence (SEQ ID NO: 39)

[0371] GACGTCGTGATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGGATTAGTAGTTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTTTTGGTGCATCCACTCTGGCA TCTGGGGTCCCCTCGCGGTTCAAAGGCAGTGGATCTGGGACAGAATACACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAGAGTTATGCTTATTTTGATAGTAATAATTGGCATGCTTTCGGCGGAGGGACCGAGGTGGTGGTC

[0372] YP218 VL amino acid sequence (SEQ ID NO: 40)

[0373] DVVMTQTPASVSEPVGGTVTIKCQASQRISSYLSWYQQKPGQRPKLLIFGASTLASGV PSRFKGSGSGTEYTLTISDLECADAATYYCQSYAYFDSNNWHAFGGGTEVVV

[0374] Table 6A. Position of CDRs in the YP218 VH sequence (according to Kabat)

[0375] CDR DNA sequence (SEQ ID NO:37) Protein sequence (SEQ ID NO: 38) CDR1 Nucleotides 91-108 Amino acids 31-36 CDR2 Nucleotides 101-204 Amino acids 51-68 CDR3 Nucleotides 298-336 Amino acids 100-112

[0376] Table 6B. Positions of CDRs in the YP218 VH sequence (according to IMGT)

[0377] CDR DNA sequence (SEQ ID NO:37) Protein sequence (SEQ ID NO: 38) CDR1 Nucleotides 79-102 Amino acids 27-34 CDR2 Nucleotides 154-177 Amino acids 52-59 CDR3 Nucleotides 292-336 Amino acids 98-112

[0378] Table 6C. Positions of CDRs in the YP218 VL sequence (according to Kabat)

[0379] CDR DNA sequence (SEQ ID NO:39) Protein sequence (SEQ ID NO:40) CDR1 Nucleotides 70-102 Amino acids 24-34 CDR2 Nucleotides 148-168 Amino acids 50-56 CDR3 Nucleotides 265-303 Amino acids 89-101

[0380] Table 6D. Positions of CDRs in the YP218 VL sequence (according to IMGT)

[0381] CDR DNA sequence (SEQ ID NO:39) Protein sequence (SEQ ID NO:40) CDR1 Nucleotides 79-96 Amino acids 27-32 CDR2 Nucleotides 148-156 Amino acids 50-52 CDR3 Nucleotides 265-303 Amino acids 89-101

[0382] SD1 nucleotide sequence (SEQ ID NO:41):

[0383] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGATTTCGATTTCGCTGCTTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGACAAGGCCTTGAGTGGGTGGCAATTATATCACATGATGGAATCG ATAAATACTACACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCACGTATTACTGTTTAAGGCTTGGTGCTGTAGGCCAGGGAACCCTGGTCACCGTCTCCTCAAGT

[0384] SD1 amino acid sequence (SEQ ID NO:42):

[0385] QVQLVQSGGGLVQPGGSLRLSCAASDFDFAAYEMSWVRQAPGQGLEWVAIISHDGID KYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTATYYCLRLGAVGQGTLVTVSSS

[0386] Table 7A. Position of CDRs in SD1 sequence (according to Kabat)

[0387] CDR DNA sequence (SEQ ID NO:41) Protein sequence (SEQ ID NO:42) CDR1 91-105 31-35 CDR2 151-198 51-66 CDR3 295-306 99-102

[0388] Table 7B. Positions of CDRs in SD1 sequences (according to IMGT)

[0389] CDR DNA sequence (SEQ ID NO:41) Protein sequence (SEQ ID NO:42) CDR1 78-105 26-35 CDR2 151-174 51-58 CDR3 289-309 97-103

[0390] V. Chimeric Antigen Receptor (CAR)

[0391] Disclosed herein are CARs (also referred to as chimeric T cell receptors, artificial T cell receptors, or chimeric immunoreceptors) and T cells engineered to express CARs. Typically, CARs include a binding moiety, an extracellular hinge / spacer element, a transmembrane region, and an intracellular domain that performs a signaling function (Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010; Dai et al., J Natl Cancer Inst 108(7):djv439, 2016). In many cases, the binding moiety is an antigen-binding fragment of a monoclonal antibody, such as a scFv or single-domain antibody. The spacer / hinge region typically includes sequences from IgG subclasses, such as IgG1, IgG4, IgD, and CD8 domains. The transmembrane domain can be derived from a variety of different T cell proteins, such as CD3ζ, CD4, CD8, or CD28.

[0392] Although the entire intracellular T cell signaling domain can be used in CAR, it is not necessary to use the entire chain in many cases. In terms of using a truncated portion of the intracellular T cell signaling domain, as long as it transduces the relevant T cell effector function signal, the truncated portion can be used instead of the complete chain. Examples of intracellular T cell signaling domains for CAR include cytoplasmic sequences of T cell receptors (TCRs) and costimulatory molecules that jointly initiate signal transduction after antigen receptor binding, as well as derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities. Several different intracellular domains have been used to produce CAR. For example, the intracellular domain can be composed of a signaling chain such as CD3ζ or FcεRIγ with ITAM. In some cases, the intracellular domain also includes the intracellular portion of at least one additional costimulatory domain. A costimulatory domain refers to a part of a CAR comprising an intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules required for lymphocytes to effectively respond to antigens other than antigen receptors or their ligands. Co-stimulatory molecules include, for example, CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27, and / or DAP10.

[0393] CAR can also include a signal peptide sequence, for example, the N- end of the antigen binding domain. The signal peptide sequence can be any suitable signal peptide sequence, such as the signal sequence from granulocyte-macrophage colony stimulating factor receptor (GMCSFR), immunoglobulin light chain κ or IL-2. Although the signal peptide sequence can promote the expression of CAR on the cell surface, the presence of the signal peptide sequence in the expressed CAR is not necessary for CAR to work. When CAR is expressed on the cell surface, the signal peptide sequence can be cut off from CAR. Therefore, in some embodiments, CAR lacks a signal peptide sequence.

[0394] The CAR disclosed herein is expressed from a construct (e.g., from a lentiviral vector) that also expresses a truncated form of human EGFR (huEGFRt; as discussed in detail in Section VI below). The CAR and huEGFRt are separated by a self-cleaving peptide sequence (e.g., T2A) such that upon expression in transduced cells, the CAR is cleaved from the huEGFRt (see Figure 1 ).

[0395] In some embodiments disclosed herein, the CAR construct encodes the following amino acid sequence in the N-terminal to C-terminal direction:

[0396] GMCSFRss:MLLLVTSLLLCELPHPAFLLIP(SEQ ID NO:2)

[0397] NdeI:HM

[0398] Antigen binding: scFv or single domain antibody sequences

[0399] SpeI:TS

[0400] CD8α hinge: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 4)

[0401] CD8αTM:IYIWAPLAGTCGVLLLSLVIT(SEQ ID NO:6)

[0402] 4-1BB:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:8)

[0403] CD3ζ:

[0404] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:10)

[0405] T2A:EGRGSLLTCGDVEENPGP(SEQ ID NO:12)

[0406] GMCSFRss:MLLLVTSLLLCELPHPAFLLIP(SEQ ID NO:2)

[0407] huEGFRt:

[0408] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKT

[0409] VKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGD

[0410] VIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPR

[0411] DCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQC

[0412] AHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM(SEQ ID NO:14)

[0413] The CAR-expressing T cells disclosed herein can be used to target specific cell types, such as tumor cells, for example, GPC3-positive, GPC2-positive, or mesothelin-positive tumor cells. Compared to standard CTL-based immunotherapy, the use of CAR-expressing T cells is more universal because CAR-expressing CTLs are HLA-unrestricted and can therefore be used in any patient with a tumor that expresses the target antigen.

[0414] Therefore, CAR is provided herein, which includes tumor-specific antibodies (or their binding fragments), such as GPC3-specific antibodies, GPC2-specific antibodies or mesothelin-specific antibodies. Also provided are isolated nucleic acid molecules and vectors encoding CAR, and host cells expressing CAR, such as T lymphocytes. T cells expressing CARs including GPC3-specific, GPC2-specific or mesothelin-specific monoclonal antibodies can be used to treat cancers that express GPC3, GPC2 and mesothelin, respectively.

[0415] VI. Truncated human EGFR (huEGFRt)

[0416] The human epidermal growth factor receptor (EGFR) is composed of four extracellular domains, one transmembrane domain, and three intracellular domains. The EGFR domains are arranged in the following order from N-terminus to C-terminus: Domain I – Domain II – Domain III – Domain IV – transmembrane (TM) domain – juxtamembrane domain – tyrosine kinase domain – C-terminal tail. Domains I and III are leucine-rich domains involved in ligand binding. Domains II and IV are cysteine-rich domains and do not contact EGFR ligands. Domain II mediates the formation of homo- or heterodimers with similar domains from other EGFR family members, and Domain IV can form disulfide bonds with Domain II. The EGFR TM domain undergoes a single pass through the cell membrane and may play a role in protein dimerization. The intracellular domain includes the juxtamembrane domain, tyrosine kinase domain and C-terminal tail that mediate EGFR signaling (Wee and Wang, Cancers 9(52), doi:10.3390 / cancers9050052; Ferguson, Annu Rev Biophys 37:353-373, 2008; Wang et al., Blood 118(5):1255-1263, 2011).

[0417] A truncated form of human EGFR, also referred to herein as "huEGFRt," includes only Domain III, Domain IV, and the TM domain. Thus, huEGFRt lacks Domain I, Domain II, and all three intracellular domains. huEGFRt cannot bind to EGF and lacks signaling activity. However, the molecule retains the ability to bind to certain EGFR-specific monoclonal antibodies, such as FDA-approved cetuximab (PCT Publication No. WO 2011 / 056894, which is incorporated herein by reference).

[0418] Transduction of T cells with constructs disclosed herein encoding huEGFRt and tumor antigen-specific CARs (e.g., lentiviral vectors) allows for the use of labeled EGFR monoclonal antibody cetuximab (ERBITUXTM ) select transduced T cells. For example, cetuximab can be labeled with biotin, and transduced T cells can be selected using commercially available anti-biotin magnetic beads (e.g., from Miltenyi Biotec). The co-expression of huEGFRt also allows in vivo tracking of adoptively transferred CAR-expressing T cells. In addition, the combination of cetuximab and huEGFRt-expressing T cells induces the cytotoxicity of ADCC effector cells, thereby providing a mechanism for eliminating transduced T cells in vivo (Wang et al., Blood 118 (5): 1255-1263, 2011), such as at the end of treatment.

[0419] In some embodiments herein, the nucleic acid molecule encoding huEGFRt is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13. In some instances, the nucleic acid molecule encoding huEGFRt comprises or consists of the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the amino acid sequence of huEGFRt is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14. In some instances, the amino acid sequence of huEGFRt comprises or consists of SEQ ID NO: 14. In other embodiments, the amino acid sequence of huEGFRt comprises no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitution relative to SEQ ID NO: 14. In some instances, the amino acid substitutions are conservative substitutions.

[0420] VII. CAR-Expressing Cell Compositions

[0421] Compositions are provided that include CAR-expressing cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. The CAR-expressing cells can be T cells, such as CD3 + T cells, such as CD4 + and / or CD8 + T cells, and / or NK cells. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, dextran, or mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The cells may be autologous to the recipient. However, the cells may also be heterologous (allogeneic).

[0422] Regarding cells, various aqueous carriers, such as buffered saline solutions, can be used to introduce cells. These solutions are sterile and generally free of unwanted substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances required for approximate physiological conditions, such as pH regulators and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentrations in these preparations can vary over a wide range and will be selected primarily based on the specific mode of administration selected and the needs of the subject, according to fluid volume, viscosity, body weight, etc.

[0423] The exact amount of the composition to be administered can be determined by the physician taking into account individual differences in age, weight, tumor size, degree of metastasis, and condition of the patient (subject). In general, the pharmaceutical composition comprising CAR-expressing T cells (and / or NK cells) described herein can be administered at the following doses: 10 4 to 10 9 cells / kg body weight, e.g. 10 5 to 10 6 cells / kg body weight, including all integer values ​​within these ranges. An exemplary dose is 10 6 cells / kg to about 10 8 cells / kg, for example, about 5 x 10 6 cells / kg to approximately 7.5 x 10 7 cells / kg, e.g., about 2.5 x 10 7 cells / kg or approximately 5.0 x 10 7 cells / kg.

[0424] The composition can be administered once or repeatedly at these dosages, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. Compositions can be administered by using well-known infusion techniques in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Compositions can be administered every day, every week, every half month (bimonthly) or every month. In some non-limiting examples, compositions are formulated for intravenous administration and repeatedly administered. The quantity and frequency of administration are determined by factors such as the type and severity of the subject's condition, subject's disease, although appropriate dosage can be determined by clinical trials.

[0425] In some embodiments, CAR- encoding nucleic acid molecules are introduced into cells such as T cells or NK cells, and the subject receives initial cell administration, and one or more subsequent cell administrations, wherein the subsequent one or more administrations are less than 15 days after the previous administration, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days are administered. In one embodiment, more than one CAR- expressing cell administration is given to the subject (for example, people) weekly, for example, 2, 3 or 4 times of CAR- expressing cell administration of the present disclosure are given weekly. In one embodiment, the subject receives more than one CAR- expressing T cell administration (for example, 2, 3 or 4 times of administration per week) (also referred to as cycle) weekly, then does not carry out CAR expression cell administration in one week, then gives one or more additional CAR- expressing cell administrations (for example, more than one CAR T cell administration per week) to the subject. In another embodiment, the subject (such as human subject) receives more than one CAR- expressing cell cycle, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4 or 3 days. In one embodiment, CAR-expressing cells are administered every other day, 3 times a week. In another embodiment, CAR-expressing cells are administered for at least two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks or longer. The dosage of the above-mentioned treatment administered to the patient will vary with the exact nature of the condition being treated and the treatment recipient. The dosage for human administration can be scaled according to practices recognized in the art.

[0426] In some embodiments, CAR-expressing T cells are able to replicate in vivo, resulting in long-term persistence, thereby enabling sustained tumor control. In various aspects, after T cells are administered to a subject, the T cells or descendants of these cells administered to the subject continue in the subject for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months or several years. In other embodiments, after CAR-expressing T cells are administered to a subject, the presence of the cell and its descendants continues to be less than 6 months, 5 months, 4 months, 3 months, 2 months or 1 month, for example, 3 weeks, 2 weeks, 1 week.

[0427] The administration of the subject composition can be carried out in any convenient manner, including by injection, ingestion, infusion, implantation or transplantation. The disclosed composition can be administered to the patient via intravenous injection, intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullaryly, intramuscularly or intraperitoneally. In some embodiments, the composition is administered to the patient via intradermal or subcutaneous injection. In other embodiments, the composition of the present invention is administered via intravenous injection. The composition can also be injected directly into the tumor or lymph node.

[0428] In some embodiments, the subject can undergo leukolysis (leukapheresis), wherein leukocytes are collected, enriched or exhausted in vitro to select and / or separate cells of interest, such as T cells and / or NK cells. These cell isolates can be amplified and processed by methods known in the art so that one or more CAR constructs can be introduced to produce autologous cells expressing CAR. In some embodiments herein, CAR-expressing cells are produced using a lentiviral vector expressing CAR and a truncated form of human EGFR (huEGFRt). The co-expression of huEGFRt allows the use of antibodies (such as cetuximab, referring to PCT Publication No. WO 2011 / 056894, which are incorporated herein by reference) identifying huEGFRt to select and purify CAR-expressing T cells, as described in Section VI above.

[0429] In some embodiments, the monocytes are depleted by lysing the red blood cells, for example, by PERCOLL TM T cells are separated from peripheral blood lymphocytes by gradient centrifugation or counterflow centrifugal elutriation. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, by conjugating beads conjugated with anti-CD3 / anti-CD28 (e.g., 3×28) M-450CD3 / CD28 T cells are incubated for a time sufficient to perform positive selection of the desired T cells, and T cells can be isolated, see U.S. Published Application No. US20140271635 A1. In a non-limiting example, the time is about 30 minutes. In other non-limiting examples, the time ranges from 30 minutes to 36 hours or longer, and integer values ​​therebetween. In another non-limiting example, the time is at least 1, 2, 3, 4, 5, 6 hours, 10 to 24 hours, 24 hours or longer. In any case where there are very few T cells compared to other cell types (e.g., separation from immunocompromised individuals), longer incubation times can be used to isolate T cells. In addition, using longer incubation times can improve the efficiency of capturing CD8+ T cells. Therefore, by simply shortening or extending the time that T cells are allowed to bind to CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as further described herein), a subpopulation of T cells can be preferentially selected or not selected at the start of culture or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subsets of T cells can be preferentially selected or deselected at the beginning of the culture or at other desired time points. Multiple rounds of selection can also be used.

[0430] Enrichment of T cell populations by negative selection can be accomplished using a combination of antibodies against surface markers specific to the negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, using a mixture of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells by negative selection, a monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. T cell populations expressing one or more cytokines can be selected. Methods for screening for cell expression are disclosed in PCT Publication No. WO 2013 / 126712.

[0431] To isolate a desired cell population by positive or negative selection, the cell concentration and surface (e.g., particles such as beads) can be varied to ensure maximum contact between cells and beads. In some embodiments, 1×10 9 In other embodiments, a concentration of greater than 1×10 cells / ml is used. 8 In other embodiments, 10, 15, 20, 25, 30, 35, 40, 45, 50, 65, 70, 75, 80, 85, 90, 95, or 100×10 6cells / ml. Without being bound by theory, using a high concentration can result in increased cell yield, cell activation, and cell expansion. Lower concentrations of cells can also be used. Without being bound by theory, the mixture of T cells and surface (e.g., particles such as beads) is significantly diluted to minimize interactions between the particles and cells. This allows cells expressing large amounts of the desired antigen to be selected for binding to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured at dilute concentrations than CD8+ T cells. In some embodiments, the cell concentration used is 5×10 6 In other embodiments, the concentration used can be about 1×10 5 / ml to 1×10 6 / ml, and any integer value in between.

[0432] VIII. Treatment Methods

[0433] Provided herein are methods for treating cancer in a subject by administering to the subject a therapeutically effective amount of a tumor-targeted CAR T cell disclosed herein. Also provided herein are methods for inhibiting tumor growth or metastasis in a subject by administering to the subject a therapeutically effective amount of a tumor-targeted CAR T cell disclosed herein.

[0434] Specifically provided are methods for treating a GPC3-positive cancer in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an isolated host cell comprising a nucleic acid molecule encoding a GPC3-targeting CAR and huEGFRt, or administering a therapeutically effective amount of an isolated host cell co-expressing a GPC3-targeting CAR and huEGFRt. In some embodiments, the GPC3-positive cancer is HCC, melanoma, ovarian clear cell carcinoma, YST, neuroblastoma, hepatoblastoma, or Wilms tumor.

[0435] Also provided are methods for treating a GPC2-positive cancer in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an isolated host cell comprising a nucleic acid molecule encoding a GPC2-targeting CAR and huEGFRt, or administering a therapeutically effective amount of an isolated host cell co-expressing a GPC2-targeting CAR and huEGFRt. In some embodiments, the GPC2-positive cancer is neuroblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

[0436] Further provided are methods for treating a mesothelin-positive cancer in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an isolated host cell comprising a nucleic acid molecule encoding a mesothelin-targeting CAR and huEGFRt, or administering a therapeutically effective amount of an isolated host cell co-expressing a mesothelin-targeting CAR and huEGFRt. In some embodiments, the mesothelin-positive cancer is mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, triple-negative breast cancer, or ovarian cancer.

[0437] In some embodiments of the methods disclosed herein, the isolated host cell is a T lymphocyte. In some instances, the T lymphocyte is an autologous T lymphocyte.

[0438] The therapeutically effective amount of CAR-expressing T cells will depend on the severity of the disease, the type of disease, and the overall health of the patient. A therapeutically effective amount of CAR-expressing T cells and compositions thereof is one that provides subjective relief of symptoms or objectively determinable improvement (e.g., reduction in tumor volume or metastasis) as indicated by a clinician or other qualified observer.

[0439] The administration of CAR-expressing T cells and compositions disclosed herein can also be accompanied by the administration of other anticancer drugs or therapeutic treatments (such as surgical resection of tumors).Any suitable anticancer drug can be administered in combination with the compositions disclosed herein.Exemplary anticancer drugs include, but are not limited to, chemotherapeutics, such as mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, antisurvival agents, biological response modifiers, antihormones (such as antiandrogens) and anti-angiogenic agents.Other anticancer treatments include radiotherapy and other antibodies that specifically target cancer cells.

[0440] Non-limiting examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, semustine, streptozotocin, or dacarbazine).

[0441] Non-limiting examples of antimetabolites include folate analogs (eg, methotrexate), pyrimidine analogs (eg, 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.

[0442] Non-limiting examples of natural products include vinca alkaloids (e.g., vinblastine, vincristine, or vindesine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., actinomycin d, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (L-asparaginase).

[0443] Non-limiting examples of miscellaneous agents include platinum coordination complexes (e.g., cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrenocortical suppressants (e.g., mitotane and aminoglutethimide).

[0444] Non-limiting examples of hormones and antagonists include adrenocortical steroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate and magestrol acetate), estrogens (such as diethylstilbestrol and ethinylestradiol), antiestrogens (such as tamoxifen) and androgens (such as testosterone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include doxorubicin, melphalan (Alkeran), cytarabine (Ara-C), BiCNU, busulfan, CCNU, carboplatinum, cisplatin, cyclophosphamide (Cytoxan), daunomycin, DTIC, 5-FU, fludarabine, hydroxyurea (Hydrea), idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes, such as Newer drugs include gemcitabine (Gemzar), Herceptin, irinotecan (Camptosar, CPT-11), cladribine (Leustatin), Navelbine, rituximab (STI-571), Taxotere, topotecan (Hycamtin), Xeloda (capecitabine), Zevelin, and calcitriol.

[0445] Non-limiting examples of immunomodulators that can be used include AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony-stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0446] Another common treatment for certain types of cancer is surgery, such as surgical removal of the cancer or a portion thereof. Another example of treatment is radiation therapy, such as administering radioactive material or energy (e.g., external beam therapy) to the tumor site prior to surgical removal to help eliminate or shrink the tumor.

[0447] The following examples are provided to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the disclosure to the specific features or embodiments described.

[0448] Example

[0449] Example 1: CAR-expressing lentiviral constructs

[0450] This example describes the generation of three lentiviral vectors encoding a tumor-targeting chimeric antigen receptor (CAR) and a truncated human EGFR (huEGFRt).

[0451] The pWPT backbone lentiviral vector (Addgene) was used to generate three CAR constructs targeting GPC3 or GPC2. The vector also encodes huEGFRt that can be recognized by the FDA-approved anti-EGFR antibody cetuximab, enabling CART cell labeling and removal. Figure 1A schematic diagram of a lentiviral construct for producing a tumor-targeted CAR is provided. The lentiviral construct includes a CAR coding region and a region encoding huEGFRt, each preceded by a granulocyte-macrophage colony-stimulating factor receptor signal sequence (GMCSFRss). The two regions are separated by a self-cleavage T2A sequence so that after the construct is expressed, CAR is cut off from huEGFRt. The expression of the construct is driven by the human elongation factor 1α (EF1α) promoter. The CAR includes an antigen binding region, a CD8α hinge region, a CD8α transmembrane (TM) domain, a 4-1BB costimulatory region, and a CD3ζ signaling domain. The huEGFRt includes two extracellular domains (domain III and domain IV) and a TM domain.

[0452] There are two targeting GPC3 in the CAR construct. Lentiviral vector pMH228 encodes the GPC3-specific single domain monoclonal antibody HN3 (disclosed in WO 2012 / 145469, which is incorporated herein by reference). The vector map of pMH228 is shown in Figure 2A The nucleotide and amino acid sequences of CAR.HN3 are shown herein as SEQ ID NOs: 17 and 18. The second GPC3-specific CAR is expressed from the lentiviral vector pMH289, which encodes a humanized scFv of the mouse GPC3-specific antibody YP7. Mouse antibody YP7 is disclosed in WO 2013 / 18154, which is incorporated herein by reference. The vector map of pMH289 is shown in Figure 2B ; The nucleotide and amino acid sequences of CAR.hYP7 are shown herein as SEQ ID NOs: 15 and 16. The third CAR construct targets GPC2 and is encoded by the lentiviral vector pMH290, which encodes the GPC2-specific single-domain monoclonal antibody LH7 (disclosed in Li et al., Proc Natl Acad Sci USA 114(32):E6623-E6631, 2017). The vector map of pMH290 is shown in Figure 2C The nucleotide and amino acid sequences of CAR.LH7 are set forth herein as SEQ ID NOs: 19 and 20. The nucleotide and amino acid sequences of HN3, humanized YP7 (hYP7), and LH7 are provided in Section IV above and are set forth as SEQ ID NOs: 25-32.

[0453] Example 2: Materials and Methods

[0454] This example describes lentiviral production and titration methods, T cell activation and transduction methods, and functional assays relevant to the studies described in Example 3.

[0455] Lentivirus production, concentration, and titration

[0456] 293T cells were seeded into 10 cm culture dishes (7.0 × 10 6 Cells / dish) were plated to an optimal monolayer density of 90% confluency at the time of transfection. Approximately 30-60 minutes before transfection, the cell supernatant was removed and replaced with 5 ml of complete medium (Dulbecco's modified Eagle's medium; DMEM) supplemented with serum and antibiotics.

[0457] For each dish, a total of 16 μg of DNA (8 μg of lentiviral vector plasmid, 2 μg of envelope plasmid MD2G and 6 μg of packaging plasmid PAX28) was diluted into 500 μl of serum-free DMEM medium and vortexed gently. TM (SignaGen Laboratories; DNA: CalFectin TM =1:3) was added to 500 μl serum-free DMEM and mixed gently. TM The reagent is immediately mixed with the diluted DNA solution and vortexed to form CalFectin TM -DNA complex. The mixture was incubated at room temperature for 10 minutes. Next, CalFectin TM The α-DNA complex was added dropwise to the culture medium of each dish and homogenized by gently swirling the plate.

[0458] Viruses were collected from the cell supernatant 48-72 hours after transfection. The supernatant was centrifuged at 500 x g for 5 minutes and then filtered through a 0.45 μm filter. To concentrate the lentivirus, the clarified supernatant was transferred to a sterile container and 3 volumes of clarified supernatant were combined with 1 volume of Lenti-X Concentrator (Clontech). The mixture was incubated at 4 ° C for 30 minutes to overnight. The sample was then centrifuged at 1,500 x g for 45 minutes at 4 ° C to form an off-white precipitate. The supernatant was removed and the precipitate was resuspended in 1 / 10 to 1 / 100 of the original volume using complete DMEM.

[0459] Lentivirus titration

[0460] 293T cells were cultured at a rate of 1 to 5 x 10 5The density of cells / well was seeded in a 12-well plate with 1 ml of growth medium (DMEM supplemented with 10% FBS) per well. One well was used to count cells, and the other well was used as a non-transduction control (NI). Other wells were transduced in duplicate with 500 μL, 100 μL, 50 μL, 20 μL or 10 μL of crude (unconcentrated) supernatant. The volume of each well was increased to 500 μL with growth medium. For concentrated virus samples, cells were transduced with 1 μL, 0.1 μL, 0.01 μL, 0.001 μL or 0.0001 μL of vector in 500 μL of fresh growth medium.

[0461] After three days, the cells were washed with 1 ml PBS and detached with 200 μl trypsin / EDTA per well and incubated at 37°C for 1 minute. Growth medium (800 μl) was added to each well to resuspend the cells. This step inactivates trypsin and EDTA. The cells were then transferred to a 5 ml FACS tube, centrifuged at 500 x g and 4°C for 5 minutes, and the supernatant was removed. The cells were stained with 1 μg / ml cetuximab in FACS buffer (5% BSA, 0.01% sodium azide in PBS) on ice for 1 hour. The cells were washed with PBS 1X, centrifuged at 500 x g and 4°C for 5 minutes, and the pellet was resuspended in a secondary antibody with an appropriate fluorescent dye. The secondary antibody was also diluted in FACS buffer.

[0462] The cells were stained on ice for 1 hour, then washed once in PBS and centrifuged at 500 x g and 4°C for 5 minutes. The pellet was resuspended in 500 μl of 1% formaldehyde in PBS and incubated at room temperature for 5 minutes. This step fixed the cells and inactivated the vector particles.

[0463] The fixed cells were washed in 1X PBS and centrifuged at 500 x g and 4°C for 5 minutes. The pellet was resuspended in 1 ml of PBS. The cells were analyzed for CAR expression using flow cytometry.

[0464] T cell activation and transduction protocols

[0465] DYNABEADS TM Human T-activator CD3 / CD28 (DYNABEADS TM Resuspend the vial with Human T-Activator CD3 / CD28 (Life Technologies) and transfer the required volume to a test tube. Add 1 ml of PBS or growth medium and mix by vortexing for 5 seconds. Centrifuge the vial and discard the supernatant. Washed DYNABEADS TMResuspend in culture medium (RPMI1640 + 10% FBS) with the same volume as the initial volume of beads removed from the vial.

[0466] Frozen peripheral blood mononuclear cells (PBMC) were thawed and resuspended in growth medium (RPMI1640 + 10% FBS). Cells were counted using trypan blue reagent. Cells (1×10 6 ) were seeded in 1 ml of culture medium in a 24-well plate. DYNABEADS were added at a ratio of 2:1 between beads and cells. TM Human T-activator CD3 / CD28 and 50 U / ml IL-2 were added.

[0467] After 24 hours of incubation, PBMCs were inoculated with lentivirus (MOI of 5) at 1000 g for 60 minutes in the presence of 10 μg / mL protamine sulfate. The cells were then resuspended in viral supernatant and incubated overnight with 50 U / mL IL-2.

[0468] The next day, the cells were centrifuged and resuspended in fresh RPMI1640+10% FBS medium, and 100 IU / mL IL-2 was added.

[0469] Over the next 10 days, examine the culture daily, noting cell size and shape. Cell shrinkage and decreased proliferation rates are commonly observed in exhausted cell cultures. After thorough resuspension, count the cells every other day. When the cell density exceeds 2.0 × 10 6 cells / ml or when the medium turns yellow, split the culture to a density of 0.5-1 × 10 cells / ml in medium containing 100 U / ml IL-2. 6 When cell growth kinetics and volume indicate that the cells have been quiescent from activation, they can be used for functional assays or cryopreserved.

[0470] Functional assays

[0471] Luciferase-expressing target cells (2×10 3) were seeded in 50 μl of culture medium in each well of a 96-well plate. CAR T cells (effector cells) were prepared at different effector (E) / target (T) ratios. 50 μl of CAR T cells were added to each well and incubated at 37 ° C overnight. Each effector: target (E: T) ratio was performed in triplicate. The next day, supernatants were collected to measure cytokine levels by ELISA and stored at -20 ° C. Stable Glo luciferase reagent (Promega) was added to each well to lyse tumor cells, and the plates were incubated in the dark at room temperature for at least 5 minutes. Luminescence was read on Victor (PerkinElmer). The results were analyzed as the percentage of killing based on luciferase activity in the wells of individual tumor cells: [% kill = 100-((RLU from wells with effector and target cells) / (RLU from wells with target cells) × 100)].

[0472] Example 3: GPC3-targeted CAR induces cytotoxicity in GPC3-expressing cell lines and reduces tumor volume of GPC3-positive tumors in animal models

[0473] This example describes the in vitro and in vivo cytotoxicity of T cells expressing CAR.HN3 and CAR.hYP7.

[0474] The T cell transduction efficiency of the lentiviral vectors pMH288 (expressing CAR.HN3) and pMH289 (expressing CAR.hYP7) was assessed by flow cytometry using the anti-huEGFRt antibody cetuximab. Figure 3A ) and CAR.hYP7( Figure 3B ) lentiviral vectors transduced 65% and 45.4% of T cells, respectively. Human serum IgG was used as a control ( Figure 3C ).

[0475] The cytotoxicity of CAR.hYP7 T cells against several human cell lines, including GPC3 + G1 cells, Hep3B cells, HepG2 cells, Huh7 cells, and GPC3 - A431 cells, T3M4 cells, and IMR32 cells. For each cell line, effector:target ratios of 1:2, 1.5:1, 5:1, and 16:1 were used. CAR.hYP7 T cells were cytotoxic to all GPC3-positive cell lines ( Figures 4A-4D ), but had no cytotoxicity against GPC3-negative cell lines ( Figures 4E-4G ).

[0476] Another study was performed to determine whether treatment with CAR.hYP7 T cells induces IFN-γ production in GPC3-positive cells in culture. Hep3B, Huh7, and G1 cells were mock-treated or treated with CAR.hYP7 T cells. Figure 5 As shown, CAR.hYP7 T cells induced IFN-γ secretion from all three target GPC-positive tumor cells.

[0477] A study was conducted to test the ability of GPC3-targeted CAR T cells to inhibit the growth of GPC3-positive tumors in mice. Mice were injected intraperitoneally with 4 million Hep3B cells on day 0. On day 10, mice were injected with a blank injection or PBS, 10 million CAR.HN3 T cells (HN3-10 M), 10 million CAR.hYP7 T cells (hYP7-10 M), 20 million CAR.hYP7 T cells (hYP7-20 M), or 40 million CAR.hYP7 T cells (hYP7-40 M). Tumor size was measured by bioluminescence imaging. Treatment with CAR.hYP7 T cells (at all doses tested) resulted in a significant reduction in tumor volume ( 100 μg / mL) compared to PBS-treated and blank-treated animals. Figure 6 ).

[0478] The durability of the anti-tumor effect of CAR.hYP7 T cells on Hep3B xenograft tumors in mice was also evaluated. Mice were injected intraperitoneally with 4 million Hep3B cells on day 0. On day 10, mice were injected with a blank injection or PBS, 10 million CAR.HN3 T cells (HN3-10 M), 10 million CAR.hYP7 T cells (hYP7-10 M), 20 million CAR.hYP7 T cells (hYP7-20 M), or 40 million CAR.hYP7 T cells (hYP7-40 M). First, tumor volume was measured for up to 3 weeks after treatment. Tumors increased steadily over time in PBS-treated, blank-treated, and CAR.HN3-treated mice. In contrast, at doses of 10 and 20 million CAR.hYP7 T cells, tumor volume remained almost unchanged over three weeks, while at a dose of 40 million CAR.hYP7 T cells, tumor volume decreased significantly ( Figure 7AThe second study evaluated anti-tumor activity in mice bearing Hep3B tumors over the course of 7 weeks. The study evaluated mice treated with PBS, 10 million CAR.HN3 T cells, 10 million CAR.hYP7 T cells, or 40 million CAR.hYP7 T cells. The results showed that the dose of 40 million CAR.hYP7 T cells resulted in a reduction in tumor volume and maintained the reduced tumor volume over the 7-week study ( Figure 7B The survival of mice bearing Hep3B tumors was also assessed for up to 70 days after Hep3B cell inoculation. The study tracked mice injected with PBS, 10 million CAR.hYP7 T cells, or 40 million CAR.hYP7 T cells. Treatment with 10 million and 40 million CAR.hYP7 T cells resulted in 50% and 100% survival, respectively. None of the mice treated with PBS survived ( Figure 7C ).

[0479] Next, CAR.hYP7 T cells were tested in another GPC3-positive tumor model. HepG2 xenografted NSG mice were treated with either a placebo or with 10 million CAR.hYP7 T cells or 40 million CAR.hYP7 T cells. Figures 8A-8D As shown, treatment with either dose of CAR.hYP7 T cells resulted in a reduction in tumor volume over the 20-day study period.

[0480] Example 4: Materials and Methods for GPC3-Targeted CAR Studies

[0481] This example provides the experimental procedures for the studies described in Example 5.

[0482] Cell culture

[0483] The human HCC cell line Hep3B was obtained from the National Cancer Institute (NCI), Bethesda, Maryland. HepG2 (hepatoblastoma), A431 (epidermal carcinoma), and HEK-293T cell lines were purchased from the American Type Culture Collection (ATCC). G1 is a transfected A431 cell line stably expressing human GPC3. Hep3B and HepG2 were transduced with lentivirus expressing firefly luciferase obtained from NCI Frederick (Day et al., Pigment Cell Melanoma Res 22:283-295, 2009). The luciferase-expressing Huh-7 cell line (an HCC cell line) was obtained from Baylor College of Medicine. The above cell lines were cultured in DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO2 at 37°C. T3M4 (human pancreatic cancer cell line) cells were obtained from NCI and engineered to express luciferase. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors using Ficoll (GE Healthcare) according to the manufacturer's instructions. PBMCs from HCC patients were obtained from NCI. Jurkat cells were also purchased from ATCC. These cells were grown in RPMI-1640 medium supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO2 at 37°C. All cell lines were authenticated by morphology and growth rate and were mycoplasma-free.

[0484] Generation of GPC3-targeted CAR

[0485] The GPC3-specific scFv and single-domain antibody fragment HN3 from hYP7 were subcloned in frame into the lentiviral expression vector pWPT (Addgene) based on the EF-1α promoter. The construct contains the expression cassette encoding the CD8α hinge and transmembrane regions, the 4-1BB costimulatory domain, the intracellular CD3ζ, the self-cleaving T2A sequence, and the huEGFRt. Figure 10B The final product was confirmed by sequence analysis.

[0486] Lentivirus production, T-cell transduction, and expansion

[0487] Using Calfectin (SignaGen), recombinant GPC3-CAR lentiviral vectors were produced by co-transfection into HEK-293T cells with packaging plasmid psPAX2 and envelope plasmid pMD2.G obtained from Addgene. After 72 hours of transfection, lentiviral particles were collected from the supernatant and concentrated 100 times with Lenti-X concentrator (Clontech) according to the manufacturer's instructions. PBMCs were purchased from the Oklahoma Blood Institute (Oklahoma Blood Institute) and stimulated PBMCs for 24 hours using anti-CD3 / anti-CD28 antibody-coated beads (Invitrogen) at a 2: 1 bead / cell ratio in the presence of IL-2 according to the manufacturer's instructions. CAR T cells were produced as previously described (Li et al., Proc Natl Acad Sci USA 114: E6623-E6631, 2017). In order to track the number of T cells over time, surviving cells were counted using trypan blue.

[0488] Flow cytometry

[0489] The transduction efficiency of GPC3 CAR on T cells was detected by anti-EGFR human monoclonal antibody cetuximab (Erbitux) and goat-anti-human IgG-phycoerythrin (PE) or allophycocyanin (APC)-conjugated antibodies (Jackson ImmunoResearch). CAR expression on T cells was measured using GPC3-hFc fusion protein and goat-anti-human IgG-PE-conjugated antibodies. PE-conjugated anti-CD3, anti-CD4 and anti-CD8 antibodies were obtained from eBioscience. Data acquisition was performed using FACSCanto II (BD Biosciences) and analyzed using FlowJo software (Tree Star).

[0490] Cytotoxicity assay

[0491] The cytotoxicity of T cells transduced with GPC3-specific CAR was determined by a luciferase-based assay as described previously (Li et al., Proc Natl Acad Sci USA 114: E6623-E6631, 2017). In short, CAR T cells and luciferase-expressing GPC3-positive (G1, Hep3B, HepG2, Huh-7) and GPC3-negative (A431, T3M4) tumor cells were incubated for 24 hours at different effector / target (E: T) ratios. Luciferase activity was measured using a luciferase assay system (Promega) on Victor (PerkinElmer). The cytotoxicity of CAR-expressing T cells was also tested using IncuCyte-FLR-Platform (Essen BioScience). In short, T cells were added to GFP-expressing HepG2 tumor cells at an E: T ratio of 2: 1. Images were taken every 2 minutes for 140 hours. The number of viable cells was quantified based on GFP expression, and cell killing activity was analyzed using the IncuCyte Zoom Hepatocyte Imaging System.

[0492] Cytokine assay

[0493] Cytokine levels in supernatants collected after 24 h of coculture of T cells and tumor cells were analyzed using a human cytokine 22-plex panel (granzyme B, GM-CSF, IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-21, CCL-3, CCL-4, CCL-19, CCL-20, CX3CL1, CXCL-11, and CXCL-8) on a Luminex system (Thermo Fisher Scientific).

[0494] Assessment of T cell polyfunctionality by single-cell cytokine profiling

[0495] The cryopreserved CAR T cell products were thawed and cultured in complete RPMI 1640 medium containing IL-2 (10 ng / ml). After overnight recovery, live CAR T cells were enriched using Ficoll. CD4 T cells were isolated using anti-CD4 or anti-CD8 microbeads (Miltenyi Biotec). + / CD8 +T cell subsets were stimulated with Hep3B or G1 cells at a 1:1 ratio for 20 hours. Next, single cell functional profiles were determined using previously described methods (Ma et al., Cancer Discov 3:418-429, 2013; Rossi et al., Blood 132:804-814, 2018; Xue et al., J Immunother Cancer 5:85, 2017). The functional profiles were classified into effector group (granzyme B, IFN-γ, CCL-3, perforin, TNF-α, TNF-β), stimulatory group (GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21), regulatory group (IL-4, IL-10, IL-13, IL-22, TGF-β1, sCD137, sCD40L), chemoattractant group (CCL-11, IP-10, CCL-4, RANTES) and inflammatory group (IL-1β, IL-6, IL-17A, IL-17F, MCP-1, MCP-4). Multifunctional CAR product T cells were defined as cells that co-secrete at least two proteins from a pre-specified panel per cell, based on the amount of each protein produced. In addition, the PSI of each sample was calculated using a pre-specified formula (Ma et al., Cancer Discov 3:418-429, 2013), defined as the percentage of multipotent cells multiplied by the mean fluorescence intensity (MFI) of the protein secreted by these cells.

[0496] Immunohistochemistry

[0497] Human HCC tissue and normal tissue microarrays were purchased from USBiomax and immunostained with anti-GPC3 antibody YP7. All tissue samples were sent to Histoserv Inc. (Germantown, MD) for staining.

[0498] Human normal tissue cDNA array

[0499] The human normal tissue array was purchased from Origene. This panel contains 48 samples, covering all major normal human tissues from various locations. GPC3 primers and RT2 SYBR Green qPCR Mastermix were purchased from Qiagen. Real-time quantification was performed on an Applied Biosystems 7900HT Real-Time PCR System. Results were analyzed using the 2-ΔΔCt method.

[0500] Western blotting

[0501] Cells were lysed with ice-cold lysis buffer (Cell Signaling Technology) and clarified by centrifugation at 10,000 g for 10 minutes at 4°C. Protein concentration was measured using a bicinchoninic acid assay (Pierce) according to the manufacturer's instructions. 20 μg of cell lysate was loaded onto a 4-20% SDS-PAGE gel for electrophoresis. The anti-GPC3 antibody YP7 is described in PCT Publication No. WO 2013 / 181543 and Phung et al. (MAbs 4(5):592-599, 2012). Anti-active-β-catenin antibody was obtained from Millipore. All other antibodies were obtained from Cell Signaling Technology.

[0502] CRISPR / Cas9-mediated GPC3 editing

[0503] The following table lists the sgRNAs targeting different exons of GPC3. The lentiCRISPRv2 expression vector is a product of Addgene (plasmid #52961). In short, the vector was digested with BsmBI and gel purified using a gel extraction kit (Qiagen). Following the previously described protocol (Sanjana et al., Nat Methods 11:783-784, 2014; Shalemet al., Science 343:84-87, 2014), a pair of oligonucleotides for each targeting site was annealed and ligated into a linearized lentiCRISPRv2 vector to generate a plasmid expressing gRNA. The sgRNA targeting GPC2 was used as a control.

[0504] Hep3B cells were transfected with gRNA-expressing plasmids using LIPOFECTAMINE 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. The cells were then incubated at 37°C for 72 hours after transfection. The effects of different gRNA-expressing plasmids on cell proliferation were determined using a crystal violet assay.

[0505] sgRNA SEQ ID NO: sequence GPC3 exon sgRNA 1-1 43 GCAGTCTCTGGAAGAAGGAG 1 sgRNA 1-2 44 TGGTGACAGGTGGCGTCCGG 1 sgRNA 2 45 CGGTTTTCCAAGGTGAGTTC 2 sgRNA 3-1 46 GGTCACGTCTTGCTCCTCGG 3 sgRNA 3-2 47 GACATCAATGAGTGCCTCCG 3 sgRNA 4 48 GATAATAAGCAGATCTATAT 4 sgRNA 5-1 49 CGTTTTCCGCCACAGGGCTA 5 sgRNA 5-2 50 AGGGTGTCGTTTTCCGCCAC 5 Control sgRNA 51 GAGGCAGAGCAGGTAGTCAG GPC2

[0506] AFP assay

[0507] Serum AFP levels were determined using an enzyme-linked immunosorbent assay (GenWay Biotech) according to the manufacturer's instructions.

[0508] Droplet digital PCR

[0509] Genomic DNA was isolated from T cells using the FlexiGene DNA Kit (QIAGEN). Droplet digital PCR was performed on a QX200 Droplet Digital PCR System (Bio-Rad) according to the manufacturer's instructions.

[0510] Animal studies

[0511] Five-week-old female NOD / SCID (NSG) mice (NCI Frederick) were bred and treated according to approved protocols. For the established intraperitoneal (ip) models Hep3B and HepG2, 3 million luciferase-expressing Hep3B or 2 million luciferase-expressing HepG2 tumor cells were injected intraperitoneally (ip) into mice. To deplete the host lymphocyte compartment, all mice were injected intraperitoneally with 200 mg / kg cyclophosphamide 24 hours before CAR T cell infusion. For the Hep3B model, mice were randomly divided into six groups and injected once intraperitoneally with different CAR T cells, as follows: (a) saline without T cells (PBS); (b) 5 million untransduced T cells (blank); (c) 5 million CAR(HN3) T cells; (d) 5 million CAR(hYP7) T cells; (e) 10 million CAR(hYP7) T cells; or (f) 20 million CAR(hYP7) T cells. For the HepG2 model, mice were randomly divided into two groups, including blank and CAR(hYP7). For the established orthotopic Hep3B model, mice were inoculated with 500,000 luciferase-expressing Hep3B cells in the liver. Three weeks after tumor formation, mice were infused intraperitoneally or intravenously with CAR(hYP7) T cells. To monitor tumor growth and survival in mice bearing HCC xenografts, all mice were injected intraperitoneally with 3 mg of D-luciferin (PerkinElmer) and imaged weekly using a Xenogen IVIS Lumina (PerkinElmer) 10 minutes later. The bioluminescent signal flux of each mouse was analyzed using Living Image software and expressed as photons / s / cm² / steradian. 2 When the bioluminescence signal reaches 5×10 10 When , the mice were euthanized.

[0512] To measure the effect of GPC3 knockout on HCC tumor cell growth, 2 million Hep3B cells were subcutaneously injected into nude mice. 3After the tumor size was determined, treatment was initiated by intratumoral injection of a plasmid expressing sgRNA5-2 or an empty vector every other day for a total of 5 injections. Tumor dimensions were measured twice a week using a caliper. Tumor volume in cubic millimeters was calculated using the following formula: (a) × (b 2 )×0.5, where “a” is the tumor length and “b” is the tumor width in millimeters.

[0513] Toxicology analysis

[0514] Three NSG mice were selected from each group for toxicology studies. Complete blood counts (CBCs), comprehensive serum chemistry profiles (VetScan, Abaxis Veterinary Diagnostics, Union City, CA), and internal organ weights were performed on the samples. These analyses were performed by the Pathology / Histotechnology Laboratory in NCI-Frederick, MD.

[0515] statistics

[0516] All experiments were repeated at least three times to determine the reproducibility of the results. Data were analyzed using Prism (GraphPad Software) and presented as mean ± SEM. Results were analyzed using an unpaired Student's t-test (2-tailed). P < 0.05 was considered statistically significant. All statistical analyses were performed using Prism software.

[0517] Example 5: Glypican 3-targeted chimeric antigen receptor T cells for the treatment of hepatocellular carcinoma

[0518] This example describes the discovery that T cells expressing huEGFRt and a binding fragment of the anti-GPC3 antibody hYP7 exhibited significant anticancer activity and robust T cell activation and expansion in a mouse model of hepatocellular carcinoma.

[0519] GPC3 expression in HCC and normal tissues

[0520] To analyze GPC3 expression in tumors and normal tissues, 46 paired tumor tissues and adjacent non-tumor tissues (cirrhotic or hepatitis) from HCC patients were examined by immunohistochemistry using the YP7 antibody. GPC3 protein was highly expressed in 50% (23 / 46) of primary HCCs, compared with only 2% (1 / 46) of matched tumor-adjacent tissues. Strong GPC3 staining was found in 24 (2%) of an additional 40 HCC tissues but not in any normal liver tissue. A concern with CAR T-cell therapy is the potential for on-target, off-tumor toxicities due to antigen expression on normal tissues. Here, GPC3 expression was analyzed in 30 human normal tissues. Notably, GPC3 protein was absent from all essential normal tissues, including brain, heart, lung, stomach, small intestine, colon, kidney, pancreas, spleen, nerves, and skin. Among all normal tissues, low levels of GPC3 protein expression were detected only in the testis. GPC3 mRNA levels were also measured in human normal tissue arrays by quantitative real-time PCR. Consistent with the protein profile, GPC3 mRNA expression was not found in most normal tissues except placenta ( Figure 9 ), which is consistent with previous reports of GPC3 expression in human placenta (Khan et al., Histol Histopathol 16:71-78, 2001). GPC3 cell surface expression was then examined on a panel of cancer cell lines by flow cytometry. YP7 showed strong binding to HCC cell lines (Hep3B, HepG2, and Huh-7) and the GPC3-overexpressing A431 cell line (G1). In contrast, YP7 showed no binding to A431 and T3M4 cells, indicating that the GPC3 expression detected by the YP7 antibody is highly tumor-specific.

[0521] Generation of GPC3-specific CAR T cells

[0522] Human single-domain antibody HN3 recognizes the N-lobule of GPC3 ( Figure 10A (Feng et al., Proc Natl Acad Sci USA 110:E1083-1091, 2013). The YP7 antibody (hYP7) targeting the C-lobule of GPC3 was also humanized to reduce the risk of immunogenicity (Zhang and Ho, Sci Reports 6:33878, 2016). The variable regions of the HN3 or hYP7 antibodies were cloned in frame into a lentiviral vector containing an expression cassette with the 4-1BB and CD3ζ intracellular domains ( Figure 10B) (See also Example 1). To facilitate cell tracking and ablation, a truncated human epidermal growth factor receptor (huEGFRt) was incorporated into the construct and separated from the CAR by a T2A ribosomal skipping sequence. huEGFRt lacks the domains necessary for ligand binding and tyrosine kinase activity, but retains the binding epitope of the anti-EGFR monoclonal antibody cetuximab (Wang et al., Blood 118: 1255-1263, 2011). Figure 2B As shown, the CAR plasmids were transduced into primary T cells from healthy donors or HCC patients, expanded in vitro for 10-12 days, and then tested in HCC cells and animal models. The expression of GPC3-targeted CAR was determined by flow cytometry. Both CARs were efficiently expressed on the surface of human Jurkat T cells as detected by recombinant GPC3-human Fc (hFc) fusion protein. In addition, the expression of CAR on human primary T cells was demonstrated by cell surface expression of huEGFRt. Figure 10C As shown, the transduction efficiencies of CAR(HN3) and CAR(hYP7) were 76% and 58%, respectively. After 11 days of in vitro expansion, more than 99% of healthy donor-derived CAR(hYP7) T cells became CD3 positive, including a similar frequency of CD4 + (43%) and CD8 + (56%) T cell subsets ( Figure 10D ). It is worth noting that in HCC patient-derived CAR(hYP7)T cells, CD4 + The proportion of T cells (14.9%) was significantly lower than that of CD8 + Surprisingly, 27.9% of CD3 T cells were detected in HCC patient-derived CAR(hYP7) T cells. + CD4 - CD8 - T cells, called "double negative" (DN) T cells. Figure 10E As shown in Figure 3, CAR(hYP7) T cells from eight different healthy donors showed 15- to 60-fold expansion 11 days after initial sensitization with anti-CD3 / CD28 beads. In contrast, CAR(hYP7) T cells from four HCC patients expanded only 5- to 25-fold on day 11 after activation. In summary, CAR T cells based on HN3 and hYP7 antibodies have significant differences in expression levels, binding affinity to GPC3, transduction efficiency, and CD4 + / CD8 + Similar values ​​were found for the proportion of T cells. Primary T cells derived from HCC patients were able to express CAR and expand in culture.

[0523] In vitro antitumor activity of GPC3-targeted CAR T cells

[0524] To determine whether T cells targeting GPC3 can specifically recognize and kill GPC3-positive tumor cells, a cytolytic assay was established using tumor cells expressing luciferase. Figure 11A As shown, A431(G1) cells overexpressing GPC3 were effectively lysed by CAR(HN3) and CAR(hYP7) T cells in a dose-dependent manner. In contrast, both CAR T cells showed minimal cytolytic activity against GPC3-negative cells, including A431 and T3M4, indicating the specificity of CAR T cells. The cytolytic capacity of GPC3-targeted CAR T cells from healthy donors and HCC patients was also compared. At different E:T ratios on Hep3B cells, CAR(hYP7) T cells appeared to have higher lytic activity than CAR(HN3) T cells ( Figure 11B and Figure 11C At an E:T ratio of 5, the lytic activity of HCC patient-derived CAR (hYP7) T cells against Hep3B cells ranged from 30% to 70%, with an average of 50% ( Figure 11C ), which is lower than the average activity of healthy donor-derived CAR(hYP7) T cells (90%) ( Figure 11B ). In contrast, the lowest degree of cytolysis was observed in Hep3B cells treated with naive T cells from any source. Interestingly, CAR(hYP7) T cells were able to undergo long-term expansion by initial activation with anti-CD3 / CD28 beads ( Figure 11D CAR(hYP7) T cells induced similar levels of cell death in Hep3B cells on day 14 or day 28 ( Figure 11E In addition to Hep3B cells, GPC3 CAR T cells were also tested in other HCC cell lines including HepG2 and Huh-7. Figure 11F As shown, HepG2 and Huh-7 cells were lysed by CAR(HN3) and CAR(hYP7) T cells to a lesser extent than Hep3B cells expressing high levels of GPC3. To determine whether GPC3-targeted CAR T cells would lead to increased tumor cell lysis during long-term co-culture, CAR(HN3) or CAR(hYP7) CAR T cells were incubated with HepG2 cells at an E:T ratio of 2:1 for more than 140 hours. Initially, neither CAR T cell killed HepG2 cells compared to blank T cells. At 40 hours and beyond, CAR(hYP7) T cells were more effective than CAR(HN3) T cells in clearing HepG2 cells. Figure 11G). Overall, CAR(hYP7) T cells showed better cytolytic capacity than CAR(HN3) T cells when co-cultured with GPC3-positive tumor cells.

[0525] In vitro multiplex cytokine and chemokine profiling of GPC3-targeted CAR T cells

[0526] Having established that GPC3-targeted CAR T cells can recognize GPC3-positive tumor cells in an antigen-specific manner, studies were performed to determine the effect of 4-1BB on the cytokine and chemokine profiles of CAR(HN3) or CAR(hYP7) T cells after exposure to Hep3B or HepG2 cells. Figure 12 As shown in Figure 3, in the presence of GPC3-positive tumor cells, GPC3-specific CAR T cells produced a significant amount of all cytokines compared to mock-transduced T cells. When co-cultured with Hep3B or HepG2 cells for 24 hours, both CAR T cells secreted significantly higher levels of granzyme B (2500-13000 pg / mL) ( Figure 12 ). It was also determined that 4-1BB co-stimulation induced high levels of Th1 cytokines (GM-CSF, IFN-γ, TNF-α, and IL-12) and Th2 cytokines (IL-5 and IL-13), which is consistent with a Th1 / Th2 phenotype. However, GPC3-specific CAR T cells only expressed low levels of IL-21 (<10 pg / mL), which is produced by Th17 cells ( Figure 18 In addition to cytokines, CAR(HN3) and CAR(hYP7) T cells secrete very high levels of CCL-3 and CCL-4 chemokines (400-2225 pg / mL), which may promote lymphocyte infiltration into tumors ( Figure 12 The complete spectrum of cytokines and chemokines analyzed in this study is shown in Figure 2 . Figure 18 Overall, CAR(hYP7) T cells produced more cytokines and chemokines than CAR(HN3) T cells, which is consistent with the difference in antitumor activity between the two CARs.

[0527] Single-cell-based polyfunctional analysis of GPC3-targeted CAR T cells

[0528] Recent studies have shown that T cells that can co-produce multiple cytokines / chemokines at the single cell level, called "multifunctional" T cells, are key effector cells that contribute to the development of effective and lasting cellular immunity against cancer (Ahmadzadeh et al., Blood 114:1537-1544, 2009; Baitsch et al., J Clin Invest 121:2350-2360, 2011). To determine the multifunctionality of our CAR T cell products, a high-content single-cell multiplex cytokine analysis was applied (Lu et al., Proc Natl Acad Sci USA 112:E607-615, 2015; Ma et al., Cancer Discov 3:418-429, 2013), which enables the identification of multifunctional T cell subsets that produce two or more cytokines after stimulation with GPC3 antigen in vitro. The 32-plex panel includes key immune elements of T cells. Hep3B cell-stimulated CAR T cells showed an increased percentage of multifunctional cells compared to blank T cells. When stimulated with Hep3B cells, CAR(hYP7) T cells had higher multifunctionality than CAR(HN3) T cells. Similarly, when stimulated with G1 cells, compared to A431 cells, the percentage of CD4 + and CD8 + Enhanced polyfunctionality was observed in both CAR T cells. It was also noted that CD8 + T cells than CD4 + T cells are more multifunctional. In addition, the previously described multifunctional strength index (PSI) is used to quantify the collective impact of multifunctional T cells (Ma et al., Cancer Discov 3: 418-429, 2013). The PSI of a sample is defined as the percentage of multifunctional cells multiplied by the average signal intensity of the cytokines secreted by these cells. The PSI is broken down by cytokine function - effector, stimulatory, regulatory and inflammatory to highlight the contribution of each group to the overall multifunctionality of the sample. Although effector cytokines contribute to CD4 + CAR(hYP7) T cells and most CD8 + CAR(hYP7) T cells have all the pluripotency but exhibit the same pluripotency in CD8 T cells stimulated by Hep3B cells. + A small proportion of the regulatory cytokine sCD137 was observed in CAR(hYP7) T cells. +When CAR(hYP7) T cells were co-cultured with G1 cells, they produced more effector and chemotactic molecules (such as CCL-3 and CCL-4), which was consistent with the cytokine release measured using Luminex assays. To distinguish all polyfunctional subpopulations in the sample, polyfunctional heat map visualization was used. Compared with CAR(HN3) T cells after stimulation with Hep3B or G1 cells, CAR(hYP7) T cells had a higher frequency of the most expressed functional groups. + CAR(hYP7) T cells expressed a 4-plex group containing granzyme B, INF-γ, perforin, and sCD137. G1 stimulated CD8 + CAR (hYP7) T cells are more multifunctional and secrete a 7-plex group containing granzyme B, INF-γ, CCL-3, CCL-4, perforin, TNF-α and sCD137. In summary, CAR (hYP7) stimulates multifunctional T cells (especially CD8 + Cytotoxic T cells) more robust activation and expansion.

[0529] Effects of Wnt signaling on GPC3-targeted CAR T-cell therapy

[0530] Previous studies have shown that GPC3 interacts with Wnt ligands and promotes HCC cell proliferation by promoting Wnt / Frizzled binding (Capurro et al., Cancer Res 65:6245-6254, 2005; Gao et al., Hepatology 60:576-587, 2014; Gao et al., Nat Comm 6:6536, 2015). To determine whether GPC3-targeted CAR T cells can affect Wnt signaling, the levels of active and total β-catenin were measured. Figure 13A As shown in Figure 3, CAR (hYP7) T cells significantly reduced the expression of active-β-catenin and total β-catenin compared with blank T cells after 6 hours of co-culture with Hep3B cells at an E:T ratio of 10:1. The reduction of active-β-catenin expression even began 2 hours after CAR (hYP7) T cell treatment in Hep3B cells ( Figure 13B ). In addition, CAR(hYP7) T cells induced apoptosis of Hep3B cells, as evidenced by elevated expression of cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase 9. However, after 6 hours of incubation, CAR(HN3) T cells neither inhibited β-catenin expression nor induced apoptosis in Hep3B cells.

[0531] To further investigate whether targeting GPC3 downregulates HCC tumor growth by inhibiting Wnt signaling, CRISPR / Cas9 technology was used to edit the GPC3 gene in Hep3B cells. Transfection of cells with constructs encoding small guiding RNAs (sgRNAs) targeting different exons of GPC3 resulted in a substantial reduction in GPC3 protein, especially exon 5-targeting sgRNA (5-2), which reduced GPC3 expression by more than 95% ( Figure 13C ). This 5-2sgRNA treatment downregulated the expression of active β-catenin and total β-catenin ( Figure 13C ), and resulted in significant cell death. Therefore, both CAR(hYP7) T cells and CRISPR / Cas9-mediated gene editing of GPC3 inhibited Wnt / β-catenin signaling in HCC cells. Following in vitro experiments, it was tested whether targeting GPC3 with the CRISPR / Cas9 platform would hinder HCC tumor growth in mice. Hep3B cells were subcutaneously inoculated into nude mice. Four weeks after tumor inoculation, mice were intravenously injected with an empty plasmid or a plasmid encoding 5-2sgRNA. Figure 13D As shown in Figure 3, tumor growth was significantly inhibited in mice treated with the 5-2sgRNA plasmid compared to mice treated with the empty plasmid. Importantly, 5-2sgRNA treatment also resulted in a decrease in both active-β-catenin and total β-catenin levels in the tumors ( Figure 13E ). Alpha-fetoprotein (AFP) has been the most widely used biomarker for HCC over the past few decades (IuS, Vopr Med Khim 10:90-91,1964). A serum concentration of 20 ng / mL is a commonly used cutoff value for distinguishing HCC patients from healthy adults (Trevisani et al., J Hepatol 34:570-575,2001). HCC patients with high AFP concentrations (≥400 ng / mL) tend to have larger tumor size, massive or diffuse type, and lower median survival rate (Fujioka et al., Hepatology 34:1128-1134,2001; Tangkijvanich et al., J Clin Gastroenterol 31:302-308,2000). Serum AFP was measured before and after CRISPR / Cas9-mediated GPC3 editing. As Figure 13F As shown, AFP serum levels in mice injected with 5-2sgRNA were significantly lower than those in mice injected with empty plasmid, indicating a positive correlation between AFP levels and tumor size. Overall, these data suggest that targeting GPC3 may inhibit HCC tumor growth by suppressing Wnt / β-catenin signaling.

[0532] CAR(hYP7) T cells induce HCC tumor regression in a xenograft mouse model

[0533] To evaluate the in vivo antitumor activity of GPC3-specific CAR T cells, NSG mice were intraperitoneally injected with luciferase-expressing Hep3B cells (Hep3B-luc). Twelve days later, a single intraperitoneal infusion of blank or CAR T cells ( Figure 14A Two weeks after treatment, the groups treated with different doses of CAR (hYP7) T cells all showed reduced tumor burden compared with the blank T cell treatment group ( Figure 14B and Figure 14C Although CAR(HN3)T cells showed moderate cytolytic activity against Hep3B cells in vitro, no significant tumor growth inhibition was observed in mice treated with CAR(HN3)T cells. Notably, by day 70, 100% of NSG mice receiving 20 million CAR(hYP7)T cells survived without relapse, while the survival rate of the group treated with 5 million CAR(hYP7)T cells was only 50% ( Figure 14D Although GPC3-targeted CAR T cells initially caused weight loss, the mice gradually gained weight ( Figure 19A In addition, two weeks after treatment, the serum AFP levels of mice treated with 5 million (average: 400 ng / mL) or 10 million (average: 300 ng / mL) CAR(hYP7) T cells were significantly lower than those of mice treated with blank T cells (average: 20,000 ng / mL) ( Figure 14E Notably, AFP levels in mice treated with 20 million CAR(hYP7) T cells ranged from 25 to 78 ng / mL, close to the cutoff value for adults (20 ng / mL).

[0534] The robust in vivo expansion and persistence of genetically modified T cells are also considered key predictors of durable clinical remission in cancer patients. To understand the persistence of infused CAR T cells, droplet digital PCR (ddPCR) using CAR-specific amplicons was used to assess the percentage of CAR T cells. Figure 14FAs shown, after 3 weeks of treatment, 22.1% of CAR expression was found in the 5 million CAR (hYP7) group, while only 1.3% of CAR was detected in the 5 million CAR (HN3) group. In addition, 26.5% of CAR integration was detected in the 10 million CAR (hYP7) group, indicating an inverse correlation between tumor burden and T cell persistence over time. In contrast, only 2.2% of CAR expression was detected in mice treated with 10 million CD19 CAR T cells, indicating that tumor antigen recognition drives the survival of infused T cells in vivo.

[0535] In the Hep3B peritoneal dissemination mouse model, mice developed tumor lesions in the liver and other tissues and organs in the abdominal cavity. Interestingly, in mice treated with 5 million CAR(hYP7) T cells, Hep3B tumors grew locally and were confined to adipose tissue away from the mouse liver, suggesting that CAR(hYP7) T cells can prevent tumor seeding and growth in the liver and spread to other organs such as the kidneys, lungs, and heart.

[0536] The efficacy of CAR(hYP7)T cells was also evaluated in a HepG2 peritoneal dissemination xenograft mouse model. NSG mice were injected intraperitoneally with luciferase-expressing HepG2 cells (HepG2-luc), followed by injection of 20 million CAR(hYP7)T cells ( Figure 15A ).like Figure 15B and Figure 15C As shown, CAR( Figure 15A )T cells reduced tumor burden to background levels, and on day 21, tumor flux was much lower than that of mice treated with blank T cells, further demonstrating that CAR(hYP7)T cells have superior anti-tumor efficacy. The body weight of mice treated with CAR(hYP7)T cells temporarily decreased, but the body weight returned to baseline levels and remained stable thereafter ( Figure 19B ), which is consistent with transient cytokine release syndrome. After 5 weeks of CAR ( Figure 19B ) T cell therapy, ddPCR detected 35.6% and 19.5% CAR expression in genomic DNA from tumors and mouse spleens, respectively ( Figure 15D In contrast, CD19 CAR T cells showed no signs of gene integration in either tissue. Furthermore, human HepG2 cells migrated to the mouse liver, and CAR(hYP7) T cells limited the spread of tumor cells, similar to what was observed in a Hep3B peritoneal xenograft mouse model.

[0537] The anti-tumor activity of CAR(hYP7) T cells was further examined in an orthotopic HCC mouse model because it is more clinically relevant. 500,000 Hep3B-luc cells were injected into the livers of NSG mice, and tumor engraftment was confirmed by bioluminescence imaging ( Figure 16A On day 21, CAR (hYP7) T cells were infused intraperitoneally or intravenously into mice. Although both routes of administration of CAR (hYP7) T cells resulted in a reduction in tumor size and a significant inhibition of tumor growth compared with the control group, intravenous injection of CAR T cells (hYP7 IV) resulted in stronger tumor regression in mice than intraperitoneal injection of CAR T cells (hYP7 IP) ( Figures 16B-16C At the end of the study, three of the four mice in the hYP7 IV group had no liver tumors, while all the mice in the blank T cell group had large tumors. Figure 16D As shown, 5 weeks after CAR T cell infusion, 30.3% of CAR was detected in the spleens of mice from the hYP7IV group, and only 8.6% of CAR was present in the spleens of mice from the hYP7IP group, which is consistent with the anti-tumor efficacy of each administration route in mice. In addition, luminescence imaging showed that Hep3B cells grew in the livers of mice, and the infusion of CAR(hYP7)T cells could significantly inhibit tumor growth in mice or completely eliminate tumor cells. Finally, toxicology studies were performed to evaluate the side effects of CAR(hYP7)T cell treatment. Mice in the hYP7 intravenous injection group showed an increase in white blood cells and neutrophils, which may be related to the rapid immune response in vivo (Table 8). In addition, alanine aminotransferase (ALT) activity was elevated in one mouse that received CAR(hYP7)T cells via the tail vein. However, no obvious evidence of liver damage was found after autopsy of the mice. Except for the lungs, the weights of all organs in the treated mice were similar to those in the control group. No significant differences were detected in any other measured parameters. Together, these results demonstrate that CAR(hYP7) T cells can induce complete regression of HCC tumors in mice.

[0538] Table 8. Toxicity of CAR(hYP7) T cells in Hep3B orthotopic xenograft mice

[0539]

[0540]

[0541] Given that the principles of the disclosed invention can be applied to many possible embodiments, it should be recognized that the illustrated embodiments are merely examples of the invention and should not be construed as limiting the scope of the invention. Rather, the scope of the invention is defined by the appended claims. We therefore claim protection for all inventions that fall within the scope and spirit of these claims.

Claims

1. A nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising, in 5' to 3' direction: a nucleic acid encoding the first granulocyte-macrophage colony-stimulating factor receptor signal sequence (GMCSFRss); a nucleic acid encoding an antigen-specific antibody or an antigen-binding fragment thereof; a nucleic acid encoding an extracellular hinge region; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an intracellular costimulatory domain; a nucleic acid encoding an intracellular signaling domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second GMCSFRss; and a nucleic acid encoding a truncated human epidermal growth factor receptor (huEGFRt), The antibody or antigen-binding fragment specifically binds to a tumor antigen, and the tumor antigen is glypican-2 (GPC2) or mesothelin.

2. The nucleic acid molecule of claim 1, wherein: The extracellular hinge region includes a CD8α hinge region or a CD28 hinge region; The transmembrane domain includes a CD8α transmembrane domain or a CD28 transmembrane domain; The intracellular costimulatory domain includes a 4-1BB, CD28, ICOS, OX40, CD27 or DAP10 costimulatory domain; and / or The intracellular signaling domain includes a CD3ζ or FcεRIγ signaling domain.

3. The nucleic acid molecule of claim 1 or 2, wherein the extracellular hinge region comprises a CD8α hinge region, the transmembrane domain comprises a CD8α transmembrane domain, the intracellular costimulatory domain comprises a 4-1BB costimulatory domain, and the intracellular signaling domain comprises a CD3ζ signaling domain.

4. The nucleic acid molecule of claim 3, wherein: The nucleic acid encoding the CD8α hinge comprises the sequence of SEQ ID NO: 3; The nucleic acid encoding the CD8α transmembrane domain includes the sequence of SEQ ID NO: 5; The nucleic acid encoding the 4-1BB costimulatory domain comprises the sequence of SEQ ID NO: 7; and / or The nucleic acid encoding the CD3 zeta signaling domain includes the sequence of SEQ ID NO:

9.

5. The nucleic acid molecule of any one of claims 1 to 4, wherein: The nucleic acid encoding the first GMCSFRss and the nucleic acid encoding the second GMCSFRss each comprise the sequence of SEQ ID NO: 1; The self-cleaving 2A peptide is a T2A peptide and the nucleic acid encoding the self-cleaving 2A peptide comprises the sequence of SEQ ID NO: 11; and / or The nucleic acid encoding the huEGFRt includes the sequence of SEQ ID NO:

13.

6. The nucleic acid molecule of any one of claims 1 to 5, further comprising a human elongation factor 1α (EF1α) promoter sequence 5' to the nucleic acid encoding the first GMCSFRss.

7. The nucleic acid molecule of any one of claims 1 to 6, wherein the antigen-binding fragment is a single-chain variable fragment (scFv) or a single domain antibody.

8. The nucleic acid molecule of any one of claims 1-7, wherein the tumor antigen is GPC2, and the nucleic acid encoding the antibody binding fragment comprises the CDR1, CDR2, and CDR3 nucleic acid sequences of SEQ ID NO:

31.

9. The nucleic acid molecule of claim 8, wherein the nucleic acid encoding the antibody binding fragment comprises the sequence of nucleotides 73-432 of SEQ ID NO:

19.

10. The nucleic acid molecule of any one of claims 1 to 7, wherein the tumor antigen is mesothelin.

11. A vector comprising the nucleic acid molecule of any one of claims 1 to 10.

12. The vector of claim 11, wherein the vector is a viral vector.

13. The vector of claim 12, wherein the viral vector is a lentiviral vector.

14. An isolated host cell comprising the nucleic acid molecule of any one of claims 1 to 10 or the vector of any one of claims 11 to 13.

15. An isolated host cell co-expressing a chimeric antigen receptor (CAR) and a truncated human epidermal growth factor receptor (huEGFRt), wherein: The CAR comprises an antigen-specific antibody or an antigen-binding fragment thereof, an extracellular hinge region, a transmembrane domain, an intracellular costimulatory domain, and an intracellular signaling domain; and The huEGFRt includes domain III, domain IV, and transmembrane domain from human EGFR, but lacks the epidermal growth factor (EGF)-binding domain and cytoplasmic domain. The antibody or antigen-binding fragment specifically binds to a tumor antigen, and the tumor antigen is glypican-2 (GPC2) or mesothelin.

16. The isolated host cell of claim 15, wherein: The extracellular hinge region includes a CD8α hinge region or a CD28 hinge region; The transmembrane domain includes a CD8α transmembrane domain or a CD28 transmembrane domain; The intracellular costimulatory domain includes a 4-1BB, CD28, ICOS, OX40, CD27 or DAP10 costimulatory domain; and / or The intracellular signaling domain includes a CD3ζ or FcεRIγ signaling domain.

17. The isolated host cell of claim 15 or 16, wherein the extracellular hinge region comprises a CD8α hinge region, the transmembrane domain comprises a CD8α transmembrane domain, the intracellular costimulatory domain comprises a 4-1BB costimulatory domain, and the intracellular signaling domain comprises a CD3ζ signaling domain.

18. The isolated host cell of claim 17, wherein: The CD8α hinge region includes the amino acid sequence of SEQ ID NO: 4; The CD8α transmembrane domain includes the amino acid sequence of SEQ ID NO: 6; The 4-1BB costimulatory domain comprises the amino acid sequence of SEQ ID NO: 8; The CD3ζ signaling domain comprises the amino acid sequence of SEQ ID NO: 10; and / or The huEGFRt comprises the amino acid sequence of SEQ ID NO:

14.

19. The isolated host cell of any one of claims 15-18, wherein the antigen binding fragment is a single chain variable fragment (scFv) or a single domain antibody.

20. The isolated host cell of any one of claims 15-19, wherein the tumor antigen is GPC2, and the amino acid sequence of the antigen-binding fragment comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:

32.

21. The isolated host cell of claim 20, wherein the amino acid sequence of the antibody binding fragment comprises residues 25-144 of SEQ ID NO:

20.

22. The isolated host cell of any one of claims 15-19, wherein the tumor antigen is mesothelin.

23. The isolated host cell of any one of claims 14-22, wherein the cell is a T lymphocyte.

24. The isolated host cell of claim 23, wherein the T lymphocytes are autologous T lymphocytes or allogeneic T lymphocytes.

25. A composition comprising the isolated host cell of any one of claims 14 to 24 and a pharmaceutically acceptable carrier.

26. Use of a therapeutically effective amount of an isolated host cell comprising the nucleic acid molecule of claim 8 or 9, or a therapeutically effective amount of an isolated host cell of claim 20 or 21, in the preparation of a medicament for treating a GPC2-positive cancer in a subject.

27. The use of claim 26, wherein the GPC2-positive cancer is neuroblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

28. Use of a therapeutically effective amount of an isolated host cell comprising the nucleic acid molecule of claim 10, or a therapeutically effective amount of an isolated host cell of claim 22, in the preparation of a medicament for treating a mesothelin-positive cancer in a subject.

29. The use of claim 28, wherein the mesothelin-positive cancer is mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, bile duct cancer, triple-negative breast cancer or ovarian cancer.

30. The use according to any one of claims 26 to 29, wherein the isolated host cell is a T lymphocyte.

31. The use of claim 30, wherein the T lymphocytes are autologous T lymphocytes or allogeneic T lymphocytes.

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