Chimeric Antigen Receptors Targeting Tumor Antigens

Abstract

Nucleic acid constructs encoding chimeric antigen receptors (CARs) and truncated human epidermal growth factor receptors (huEGFRt) are described. The encoded CARs include tumor antigen-specific monoclonal antibodies fused to CD8α hinge regions, CD8α transmembrane regions, 4-1BB costimulatory domains, and CD3ζ signaling domains, such as glypican-3 (GPC3)-specific, GPC2-specific, or mesothelin-specific monoclonal antibodies. Separated host cells are also described, such as separated T cells co-expressing the disclosed CARs and huEGFRt. T cells transduced with the disclosed CAR constructs can be used for cancer immunotherapy.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 584,421, filed on November 10, 2017, which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to chimeric antigen receptors specific for tumor antigens and their use for 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 antibody fragments specific to tumor antigens fused with transmembrane domains and T cell signaling moieties. When the receptor is expressed on the surface of T cells, it mediates 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 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). The encoded CAR includes a tumor antigen-specific monoclonal antibody fragment fused to an extracellular hinge region, a transmembrane region, an intracellular co-stimulatory 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 separated from the disclosed CAR and huEGFRt, such as T lymphocytes. 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 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 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 an antigen-binding fragment thereof, an extracellular hinge region, a transmembrane domain, an intracellular co-stimulatory 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 co-stimulatory domain includes a 4-1BB co-stimulatory 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 cell is a T lymphocyte.

[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 It is a 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, 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.

[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 2 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 is a graph showing 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 detect 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 blank or PBS, 10 million CAR.HN3 T cells (HN3-10 M), 10 million CAR.hYP7 T cells (hYP7-10 M), 20 million CAR.hYP7T 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 is a graph showing that CAR.hYP7 T cells have durable anti-tumor activity against Hep3B xenograft tumors in mice. Fig. 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. Mice were injected with PBS, 10 million CAR.hYP7 T cells, or 40 million CAR.hYP7 T cells 10 days after Hep3B inoculation, 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 Fig. 8A ) or treated with 10 million CAR.hYP7 T cells ( Figure 8B ) or 40 million CAR.hYP7 T cells ( Figure 8C ) of tumor volume in NSG mice with HepG2 xenografts. Figures 10A-10C Each row represents a single animal. Fig.8D Mean tumor volumes for all three treatment groups are shown.

[0021] Fig. 9 Shown are the levels of GPC3 mRNA in human normal tissues measured by quantitative real-time PCR. The relative GPC3 levels in different normal tissues were compared with the expression of GPC3 in the placenta.

[0022] Figures 10A-10E Showing the generation and expression of GPC3 CAR T cells. ( Fig. 10A ) Schematic structure of HN3 and hYP7 antibodies binding to the N-lobe and C-lobe of mature GPC3, respectively. Fig. 10B ) Schematic diagram of a bicistronic lentiviral construct expressing a GPC3-targeting CAR and huEGFRt using a T2A ribosomal skipping sequence. Fig. 10C ) CAR expression on healthy donor-derived T cells transduced with lentiviral particles was analyzed by flow cytometry by detecting EGFR expression. ( Fig. 10D ) CD3 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. Fig. 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 is a graph showing that GPC3-targeted CAR T cells kill GPC3-positive HCC cells in vitro. ( Fig.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) ratios for 24 hours, and specific lysis was measured using a fluorescence-based cytolysis assay. ( Figures 11B-11C ) were co-cultured with Hep3B cells for 24 hours. Fig. 11B ) or HCC patients ( Fig. 11C )'s cytolytic activity of CAR(HN3)T cells and CAR(hYP7)T cells. Fig.11D )After stimulation with anti-CD3 / CD28 beads, CAR(hYP7) T cells proliferated strongly for 35 days. ( Fig.11E ) Cytolytic activity of CAR(hYP7) T cells on days 14 and 28 after activation after co-culture with Hep3B cells for 24 hours. ( Fig.11F ) Cytolytic activity of GPC3-specific CAR T cells from healthy donors after co-culture with HepG2 and Huh-7 cells for 24 hours. ( Fig.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] Fig.12 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 h, and the indicated cytokine / chemokine levels in the supernatant 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. Fig.13A ) After 6 hours of treatment, CAR(hYP7) T cells inhibited the expression of β-catenin and increased the expression of apoptotic markers (cleaved PARP and cleaved caspase-9) in Hep3B cells. ( Fig. 13B )CAR(hYP7) T cells inhibited the expression of β-catenin in Hep3B cells in a time-dependent manner. Fig. 13C ) GPC3 protein expression in Hep3B cells after CRISPR / Cas9-mediated GPC3 knockout. ( Fig.13D ) Antitumor activity of sgRNA5-2 targeting exon 5 of GPC3. Athymic nu / nu mice were subcutaneously inoculated with 5×10 6 Hep3B cells. When the average tumor volume reaches 150mm 3 At 4 dpi, mice were treated with intratumoral injections of sgRNA5-2 plasmid or empty vector every other day for 6 injections. Fig.13E ) Knockout of GPC3 reduced β-catenin expression in mouse tumors. Fig.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. Fig.14A ) Experimental schematic diagram. On day 12 after tumor cell inoculation, blank T cells, 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. Fig. 14B )CAR(hYP7) T cells regressed established Hep3B xenografts at high doses (20 million cells) and inhibited tumor growth at low doses (5 million or 10 million cells). Fig. 14C ) Fig. 14B Tumor bioluminescence in mice treated with , as measured by mean photon counts. Fig.14D ) Kaplan-Meier survival curves of tumor-bearing mice after treatment with 5 million or 20 million CAR(hYP7) T cells. ( Fig.14E ) Two or six weeks after CAR T therapy, Fig. 14B AFP levels in sera collected from the indicated groups. Sera from three different mice in each group were collected for ELISA analysis. Fig.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. Fig.15A ) Experimental schematic. On day 21, NSG mice bearing Hep3B tumors were injected intraperitoneally with blank T cells or 20×10 6 CAR(hYP7)T cells for treatment. Fig. 15B )CAR(hYP7) T cells demonstrated potent antitumor activity and mediated eradication of HepG2 xenograft tumors. Fig. 15C ) Fig. 15B Tumor bioluminescence in mice treated with , as measured by mean photon counts. Fig.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 shown. Fig.16A ) Experimental schematic diagram. 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. Fig. 16B ) Mice treated with CAR(hYP7) T cells via tail vein demonstrated tumor eradication, while intraperitoneal treatment resulted in tumor growth inhibition. ( Fig. 16C ) Fig. 16B Tumor bioluminescence in mice treated with , as measured by mean photon counts. Fig.16D ) CAR T cell persistence in tumor tissue and mouse spleen after 5 weeks of treatment as measured by ddPCR. Values ​​represent mean ± SD. **p < 0.01.

[0029] Fig.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] Fig.18 is a series of graphs showing differential cytokine and chemokine secretion measured by Luminex after 24 h 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 mice bearing Hep3B and HepG2 tumor xenografts after treatment with GPC3-targeted CAR T cells. Fig.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. ( Fig.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 attached sequence listing are shown using the standard nucleotide base letter abbreviations and amino acid three-letter codes defined in 37 CFR 1.822. Each nucleic acid sequence shows only one strand, but it should be 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, 59.3KB, which is incorporated herein by reference. In the attached 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 CD8 alpha hinge.

[0038] SEQ ID NO: 5 is a 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 a nucleotide sequence encoding CAR.hYP7, having the following characteristics:

[0049] Nucleotides 1-66 = GMCSFRss coding sequence

[0050] Nucleotides 67-72 = NdeI restriction site

[0051] Nucleotides 73-807 = humanized YP7 scFv coding sequence

[0052] Nucleotides 808-813 = SpeI restriction site

[0053] Nucleotides 814-948 = CD8α hinge region coding sequence

[0054] Nucleotides 949-1011 = CD8α transmembrane domain coding sequence

[0055] Nucleotides 1012-1137 = 4-1BB co-stimulatory domain coding sequence

[0056] Nucleotides 1138-1473 = CD3 zeta signaling domain coding sequence

[0057] Nucleotides 1474-1527 = T2A coding sequence

[0058] Nucleotides 1528-1593 = GMCSFRss coding sequence

[0059] Nucleotides 1594-2598 = huEGFRt coding sequence.

[0060] SEQ ID NO: 16 is the amino acid sequence of CAR.hYP7, having the following characteristics:

[0061] Residues 1-22 = GMCSFRss

[0062] Residues 23-24 = HM (encoded by NdeI restriction site)

[0063] Residues 25-269 = humanized YP7 scFv

[0064] Residues 270-271 = TS (encoded by SpeI restriction site)

[0065] Residues 272-316 = CD8α hinge region

[0066] Residues 317-337 = CD8α transmembrane domain

[0067] Residues 338-379 = 4-1BB co-stimulatory domain

[0068] Residues 380-491 = CD3ζ signaling domain

[0069] Residues 492-509 = self-cleaving T2A peptide

[0070] Residues 510-531 = GMCSFRss

[0071] Residues 532-866 = huEGFRt coding sequence.

[0072] SEQ ID NO: 17 is a nucleotide sequence encoding CAR.HN3, having the following characteristics:

[0073] Nucleotides 1-66 = GMCSFRss coding sequence

[0074] Nucleotides 67-72 = NdeI restriction site

[0075] Nucleotides 73-420 = HN3 coding sequence

[0076] Nucleotides 421-426 = SpeI restriction site

[0077] Nucleotides 427-561 = CD8α hinge region coding sequence

[0078] Nucleotides 562-624 = CD8α transmembrane domain coding sequence

[0079] Nucleotides 625-750 = 4-1BB co-stimulatory domain coding sequence

[0080] Nucleotides 751-1086 = CD3 zeta signaling domain coding sequence

[0081] Nucleotides 1087-1140 = T2A coding sequence

[0082] Nucleotides 1141-1206 = GMCSFRss coding sequence

[0083] Nucleotides 1207-2211 = huEGFRt coding sequence.

[0084] SEQ ID NO: 18 is the amino acid sequence of CAR.HN3, having the following characteristics:

[0085] Residues 1-22 = GMCSFRss

[0086] Residues 23-24 = HM (encoded by NdeI restriction site)

[0087] Residues 25-140 = HN3 single domain antibody

[0088] Residues 141-142 = TS (encoded by SpeI restriction site)

[0089] Residues 143-187 = CD8α hinge region

[0090] Residues 188-208 = CD8α transmembrane domain

[0091] Residues 209-250 = 4-1BB co-stimulatory domain

[0092] Residues 251-362 = CD3ζ signaling domain

[0093] Residues 363-380 = self-cleaving T2A peptide

[0094] Residues 381-402 = GMCSFRss

[0095] Residues 403-737 = huEGFRt coding sequence.

[0096] SEQ ID NO: 19 is a nucleotide sequence encoding CAR.LH7, having the following characteristics:

[0097] Nucleotides 1-66 = GMCSFRss coding sequence

[0098] Nucleotides 67-72 = NdeI restriction site

[0099] Nucleotides 73-432 = LH7 coding sequence

[0100] Nucleotides 433-438 = SpeI restriction site

[0101] Nucleotides 439-573 = CD8α hinge region coding sequence

[0102] Nucleotides 574-636 = CD8α transmembrane domain coding sequence

[0103] Nucleotides 637-762 = 4-1BB co-stimulatory domain coding sequence

[0104] Nucleotides 763-1098 = CD3 zeta signaling domain coding sequence

[0105] Nucleotides 1099-1152 = T2A coding sequence

[0106] Nucleotides 1153-1218 = GMCSFRss coding sequence

[0107] Nucleotides 1219-2223 = huEGFRt coding sequence.

[0108] SEQ ID NO:20 is the amino acid sequence of CAR.LH7, having the following characteristics:

[0109] Residues 1-22 = GMCSFRss

[0110] Residues 23-24 = HM (encoded by NdeI restriction site)

[0111] Residues 25-144 = LH7 single domain antibody

[0112] Residues 145-146 = TS (encoded by SpeI restriction site)

[0113] Residues 147-191 = CD8α hinge region

[0114] Residues 192-212 = CD8α transmembrane domain

[0115] Residues 213-254 = 4-1BB co-stimulatory domain

[0116] Residues 255-366 = CD3ζ signaling domain

[0117] Residues 367-384 = self-cleaving T2A peptide

[0118] Residues 385-406 = GMCSFRss

[0119] Residues 407-741 = huEGFRt coding sequence.

[0120] SEQ ID NO:21 is the nucleotide sequence of the YP7 VH domain.

[0121] SEQ ID NO:22 is the amino acid sequence of the YP7 VH domain.

[0122] SEQ ID NO:23 is the nucleotide sequence of the YP7 VL domain.

[0123] SEQ ID NO:24 is the amino acid sequence of the YP7 VL domain.

[0124] SEQ ID NO:25 is the nucleotide sequence of the hYP7 VH domain.

[0125] SEQ ID NO:26 is the amino acid sequence of the hYP7 VH domain.

[0126] SEQ ID NO:27 is the nucleotide sequence of the hYP7 VL domain.

[0127] SEQ ID NO:28 is the amino acid sequence of the hYP7 VL domain.

[0128] SEQ ID NO: 29 is the nucleotide sequence of the HN3 single domain antibody.

[0129] SEQ ID NO: 30 is the amino acid sequence of the HN3 single domain antibody.

[0130] SEQ ID NO: 31 is the nucleotide sequence of the LH7 single domain antibody.

[0131] SEQ ID NO: 32 is the amino acid sequence of the LH7 single domain antibody.

[0132] SEQ ID NO: 33 is the nucleotide sequence of the LH4 single domain antibody.

[0133] SEQ ID NO: 34 is the amino acid sequence of the LH4 single domain antibody.

[0134] SEQ ID NO: 35 is the nucleotide sequence of the LH6 single domain antibody.

[0135] SEQ ID NO: 36 is the amino acid sequence of the LH6 single domain antibody.

[0136] SEQ ID NO:37 is the nucleotide sequence of the YP218 VH domain.

[0137] SEQ ID NO:38 is the amino acid sequence of the YP218 VH domain.

[0138] SEQ ID NO:39 is the nucleotide sequence of the YP218 VL domain.

[0139] SEQ ID NO:40 is the amino acid sequence of the YP218 VL domain.

[0140] SEQ ID NO:41 is the nucleotide sequence of the SD1 single domain antibody.

[0141] SEQ ID NO:42 is the amino acid sequence of the SD1 single domain antibody.

[0142] SEQ ID NO:43-51 are sgRNA sequences. DETAILED DESCRIPTION OF THE INVENTION

[0144] I. Abbreviations

[0145] ADCC antibody-dependent cell-mediated cytotoxicity

[0146] CAR chimeric antigen receptor

[0147] CDR complementarity determining region

[0148] CTL Cytotoxic T lymphocyte

[0149] ddPCR droplet digital PCR

[0150] DMEM Dulbecco's modified Eagle medium

[0151] EF1α elongation factor 1α

[0152] EGF epidermal growth factor

[0153] EGFR epidermal growth factor receptor

[0154] ELISA enzyme-linked immunosorbent assay

[0155] FACS fluorescence activated cells sorting

[0156] FBS fetal bovine serum

[0157] GPC2 glypican-2

[0158] GPC3 glypican-3

[0159] GMCSFRss granulocyte-macrophage colony stimulating factor receptor signal sequence

[0160] HCC hepatocellular carcinoma

[0161] HLA human leukocyte antigen

[0162] huEGFRt human truncated epidermal growth factor receptor

[0163] IFN interferon

[0164] Ig immunoglobulin

[0165] IL interleukin

[0166] ip intraperitoneal

[0167] ITAM immunoreceptor tyrosine-based activation motif

[0168] PBMC peripheral blood mononuclear cell

[0169] PBS Phosphate-buffered saline

[0170] scFv single-chain variable fragment

[0171] TM transmembrane

[0172] VH or V H Variable heavy chain

[0173] VL or V L Variable light

[0174] YST Yolk sac tumor

[0175] II. Terminology and Methodology

[0176] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of commonly used 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).

[0177] To facilitate review of the various embodiments of the present disclosure, the following explanations of specific terms are provided:

[0178] 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.

[0179] Acute lymphoblastic leukemia (ALL): An acute form of leukemia characterized by an overproduction of lymphoblasts. ALL is most common in children, peaking in children aged 2-5 years.

[0180] Antibody: A polypeptide ligand containing at least one variable region that recognizes and binds (e.g., specifically recognizes and specifically 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 (V H ) 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 main 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.

[0181] The variable region of an antibody contains a "framework" region and a hypervariable region, called a "complementarity determining region" or "CDR." The CDR is primarily responsible for binding to the epitope of an antigen. The framework region of an antibody serves to position and align the CDR 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 in 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.

[0182] "Single-domain antibody" refers to an antibody with a single domain (variable domain) that is able to specifically bind to an antigen or an epitope of an antigen in the absence of other antibody domains. H Domain antibodies, V NAR Antibodies, camelid V H H 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 produce heavy chain antibodies that naturally lack light chains.

[0183] 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.

[0184] "Chimeric antibodies" have framework residues from one species, such as human, and the CDRs (which typically confer antigen binding) from another species.

[0185] "Humanized" antibodies are CDRs that include human framework regions and one or more immunoglobulins from non-human (e.g., mouse, rabbit, rat, shark, or synthetic) antibodies. The non-human immunoglobulin that provides the CDRs is called the "donor," and the human immunoglobulin that provides the framework is called the "acceptor." In one embodiment, all CDRs in the humanized immunoglobulin are from the donor immunoglobulin. The constant region need not be present, but if present, they must be substantially identical to the human immunoglobulin constant region, i.e., have at least about 85-90%, such as about 95% or higher identity. Therefore, all parts of the humanized immunoglobulin, possibly except 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 effect on antigen binding or other immunoglobulin functions.

[0186] 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" an antigen (e.g., GPC3) is an antibody that binds to the antigen with high affinity and does not significantly bind to other unrelated antigens.

[0187] Breast cancer: A type of cancer that forms in breast tissue, which is usually the ducts (tubes that carry milk to the nipple) and lobules (glands that make 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, HER2 / neu-negative breast cancer.

[0188] 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, e.g., 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 used in combination with radioactive or chemical compounds.

[0189] 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 an intracellular portion of at least one other costimulatory domain, such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or DAP10.

[0190] Cholangiocarcinoma: A type of cancer that develops in the cells lining the bile ducts in the liver.

[0191] 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, referred to as L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively.

[0192] 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 includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, as long as the variant retains activity. Non-conservative substitutions are those that reduce the activity of a protein.

[0193] Conservative amino acid substitution tables that provide functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered conservative substitutions of each other:

[0194] 1) Alanine (A), serine (S), threonine (T);

[0195] 2) Aspartic acid (D), glutamic acid (E);

[0196] 3) Asparagine (N), glutamine (Q);

[0197] 4) Arginine (R), Lysine (K);

[0198] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0199] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0200] 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.

[0201] Cytotoxic agent: Any drug or compound that kills cells.

[0202] Cytotoxicity: The toxicity of a molecule to the cells it is intended to be targeted against the cells of the rest of the organism.

[0203] Degenerate variant: A polynucleotide encoding a polypeptide containing a sequence that is degenerate due to the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, as long as the amino acid sequence of the polypeptide is unchanged, all degenerate nucleotide sequences are included.

[0204] Desmoplastic small round cell tumor (DRCT): A soft tissue sarcoma that occurs primarily in childhood, especially in boys. DRCT is an aggressive and rare type of 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, intestines, bladder, brain, lungs, testicles, ovaries, and pelvis.

[0205] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on a molecule that are antigenic, i.e., that elicit a specific immune response. Antibodies specifically bind to specific epitopes on a polypeptide.

[0206] Ewing's sarcoma: A rare type of malignant tumor found in bone or soft tissue. Ewing's sarcoma is a small, blue, round cell tumor.

[0207] Framework region: The amino acid sequence between the CDRs. The framework region includes the light chain variable and heavy chain variable framework regions. The framework region is used to keep the CDRs in the appropriate orientation for antigen binding.

[0208] Fusion protein: A protein that contains at least portions of two different (heterologous) proteins.

[0209] Glypican-2 (GPC2): A member of the six-member glypican family of heparan sulfate (HS) proteoglycans that 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), is involved 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 proteoglycan and glypican proteoglycan 2. The GPC2 genome, mRNA, and protein sequences are publicly available (see, e.g., NCBI Gene ID 221914).

[0210] 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.

[0211] Glypican-3 (GPC3): A member of the glypican family of heparan sulfate (HS) proteoglycans, which are attached to the cell surface via glycosylphosphatidylinositol anchors (Filmus and Selleck, J Clin Invest 108: 497-501, 2001). The GPC3 gene encodes a core protein of approximately 70 kD, which 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.

[0212] There are four known isoforms of human GPC3 (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).

[0213] 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).

[0214] HAMA (human anti-murine antibody) response: An immune response of a human subject to the variable and constant regions of a murine antibody that has been administered to the 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.

[0215] Hepatocellular carcinoma (HCC): A primary malignant tumor of the liver that usually occurs in patients with an inflamed liver caused by viral hepatitis, hepatotoxins, or cirrhosis (often caused by alcoholism). HCC is also called malignant liver cancer.

[0216] Heterologous: Derived from a single genetic source or species.

[0217] Immune response: The response of a cell of the immune system (e.g., a B cell, T cell, or monocyte) 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, such as a CD4 + Response or CD8 +In another embodiment, the response is a B cell response and results in the production of specific antibodies.

[0218] Isolated: An "isolated" biological component (e.g., a nucleic acid, protein (including antibodies), or organelle) has been substantially separated or purified 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.

[0219] Label: A detectable compound or composition that is directly or indirectly conjugated to another molecule (such as an antibody or protein) to facilitate the detection of the 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, the label is a detectable marker, such as an amino acid incorporated with a radiolabel or linked to a polypeptide with a biotin moiety that can be detected by a labeled avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods for 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 markers (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide fluorescent agents), enzyme markers (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 of 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.

[0220] 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. "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.

[0221] 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 a scFv. In certain contexts, the term includes reference to linking a ligand, such as an antibody portion, to an effector molecule. The linking can be performed chemically or recombinantly. "Chemical" refers to the reaction between the antibody portion and the effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

[0222] Lung cancer: Cancer that forms in lung tissue, usually 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.

[0223] Mammal: This term includes human and non-human mammals. Similarly, the term "subject" includes human and veterinary subjects.

[0224] Melanoma: A cancer that originates in melanocytes (cells that produce the pigment melanin). Melanocytes are found primarily in the skin, but are also found in the intestines and eyes. Melanomas in 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 amelanin-free. 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.

[0225] Mesothelin: A 40 kDa cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein. Human mesothelin protein is synthesized as a 70 kD precursor and then proteolytically processed. The 30 kD amino terminus of mesothelin is secreted and is called megakaryocyte potentiating factor (Yamaguchi et al., J. Biol. Chem. 269: 805 808, 1994). As mature mesothelin, the 40 kD 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.

[0226] 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.

[0227] 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 tissue of the lungs, heart, or abdomen. In some cases, mesothelioma is caused by exposure to asbestos.

[0228] 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 the tumors. Tumors that do not metastasize are called "benign." Tumors that invade surrounding tissues and / or can metastasize are called "malignant."

[0229] 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.

[0230] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, in the case of two protein coding regions to be linked, in the same reading frame.

[0231] 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 prevalent in children and young adults.

[0232] 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 cancers (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in the egg cells).

[0233] 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 tumor cells of this type appear clear inside.

[0234] Pancreatic cancer: A disease in which malignant (cancer) cells are found in the tissues of the pancreas. Also called exocrine cancer.

[0235] Pediatric cancer: Cancer that occurs 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.

[0236] Pharmaceutical Agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or cell.

[0237] 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.

[0238] Usually, the character of carrier will depend on the specific mode of administration adopted.For example, parenteral preparations generally include injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solution, aqueous glucose solution, glycerol, etc. as vehicles.For solid compositions (such as powder, pill, tablet or capsule form), conventional non-toxic solid carriers can include, such as pharmaceutical grade mannitol, lactose, starch or magnesium stearate.Except for biological neutral carriers, pharmaceutical compositions to be administered can contain a small amount of nontoxic auxiliary substances, such as wetting agents or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0239] Preventing, treating or ameliorating a disease: "Preventing" a disease means inhibiting the overall development of a disease. "Treatment" refers to therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop, such as reducing tumor burden or reducing 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.

[0240] Prostate cancer: Cancer that develops in the tissue 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.

[0241] Purified: The term "purified" does not require absolute purity, but rather, it is intended to be 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 accounts for at least 50% of the total peptide or protein content of the preparation. Substantially purified refers to purification from other proteins or cellular components. Substantially purified proteins are 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.

[0242] Recombinant: A recombinant nucleic acid is a nucleic acid having a non-naturally occurring sequence or a nucleic acid having a sequence that is made by the artificial combination of two separate fragments of a sequence. This artificial combination is usually achieved by chemical synthesis or by artificial manipulation of separate nucleic acid fragments, such as by genetic engineering techniques.

[0243] Rhabdomyosarcoma (RMS): A soft tissue malignancy of skeletal muscle origin. 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 bile duct), and perineal / anal region. There are at least two types of RMS. The most common forms are alveolar RMS (ARMS) and embryonic histological RMS (ERMS). Approximately 20% of children with rhabdomyosarcoma have the ARMS subtype. This subtype is found to have an increased frequency in adolescents and in patients whose primary sites involve the extremities, trunk, and perineal / perianal regions. ARMS is associated with a chromosomal translocation encoding a fusion gene involving FKHR and PAX family members on chromosome 13. The embryonic 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.

[0244] 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 biopsy, fine needle aspirate, surgical specimens, and autopsy material. In one example, the sample comprises a tumor biopsy, such as a tumor tissue biopsy.

[0245] Sequence identity: Similarity between amino acid or nucleic acid sequences is expressed as 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. Homologues or variants of polypeptides or nucleic acid molecules will have a relatively high degree of sequence identity when aligned using standard methods.

[0246] Methods of sequence alignment 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.

[0247] 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.

[0248] 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 NCBI Blast2.0, 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 compare the amino acid sequence greater than about 30 amino acids, the default BLOSUM62 matrix ((gap existence cost is 11 for gaps, and 1 for each residue gap) is used as default parameters) to use the Blast2 sequence function. When comparing short peptides (less than about 30 amino acids), the comparison should be performed using the Blast2 sequence function, using the PAM30 matrix (open gap 9, extension gap 1 penalty) set as default parameters. When evaluated by this method, proteins with even higher similarity to the reference sequence will show increasing percentages of identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity. When comparing sequence identities of less than the entire sequence, homologues and variants will generally have at least 80% sequence identity within 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 only as a guide; it is entirely possible to obtain strongly significant homologues that fall outside the provided ranges.

[0249] Squamous cell carcinoma: A type of cancer that begins in the squamous cells, thin, flat cells that form the lining of 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.

[0250] Stomach cancer: Cancer that forms in the tissues lining the stomach. Also called gastric cancer.

[0251] Subject: Living multicellular vertebrate organisms, including humans and the category of veterinary subjects, including human and non-human mammals.

[0252] Synthetic: Produced by artificial means in a laboratory, such as synthetic nucleic acids or proteins (such as antibodies) that can be chemically synthesized in a laboratory.

[0253] Therapeutically effective amount: The amount of a particular substance sufficient to obtain a desired effect in a treated subject. 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 tumor size, or prevent tumor metastasis. When administered to a subject, a dose that achieves a target tissue concentration (e.g., in a tumor) that has been shown to achieve the desired in vitro effect will generally be used.

[0254] Vector: A nucleic acid molecule introduced into a host cell, thereby producing a transformed host cell. A vector may include a nucleic acid sequence that allows 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.

[0255] 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 including A or B, or A and B. It should also be understood that all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weights or molecular mass values ​​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 a conflict, the present specification (including explanations of terms) shall prevail. In addition, these materials, methods and embodiments are illustrative only and are not intended to be limited.

[0256] III. Overview of Several Implementations

[0257] Disclosed herein are nucleic acid molecules encoding chimeric antigen receptors (CAR) and truncated human epidermal growth factor receptors (huEGFRt). The encoded CAR includes a tumor antigen-specific monoclonal antibody fused to an extracellular hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signaling 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 domains, tyrosine kinase domains, and C-terminal tails). Also provided are cells separated from the disclosed CAR and huEGFRt, such as T lymphocytes. T cells transduced with the CAR construct can be used for cancer immunotherapy.

[0258] Provided herein are nucleic acid molecules encoding CAR and huEGFRt. In some embodiments, the nucleic acid molecule includes, in a 5' to 3' direction, a nucleic acid encoding a first signal sequence; 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 signal sequence; and a nucleic acid encoding huEGFRt.

[0259] 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).

[0260] In some embodiments, the extracellular hinge region includes 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 as a "spacer region" in the art.

[0261] In some embodiments, the transmembrane domain includes 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.

[0262] 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.

[0263] In some embodiments, the intracellular signaling domain is a domain having an immunoreceptor tyrosine-based activation motif (ITAM), such as a CD3 zeta or FcεRIγ signaling domain.

[0264] 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 co-stimulatory domain comprises a 4-1BB co-stimulatory domain, and the intracellular signaling domain comprises a CD3ζ signaling domain.

[0265] 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.

[0266] 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 a non-limiting example, the nucleic acid encoding the CD8α transmembrane domain comprises the sequence of SEQ ID NO: 5.

[0267] In some examples, the nucleic acid molecule encoding the 4-1BB co-stimulatory 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 a non-limiting example, the nucleic acid molecule encoding the 4-1BB co-stimulatory domain comprises the sequence of SEQ ID NO: 7.

[0268] In some examples, the nucleic acid encoding the CD3 zeta signaling domain includes 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 a non-limiting example, the nucleic acid encoding the CD3 zeta signaling domain includes the sequence of SEQ ID NO: 9.

[0269] In some examples, the nucleic acid encoding the first GMCSFRss and the nucleic acid encoding the second GMCSFRss each include 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 include the sequence of SEQ ID NO: 1.

[0270] In some examples, the self-cleaving 2A peptide is a T2A peptide. In other examples, the self-cleaving 2A peptide is a F2A peptide, an E2A peptide, or a P2A peptide. In a specific example, the nucleic acid encoding the self-cleaving T2A peptide includes 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 a non-limiting example, the nucleic acid encoding the self-cleaving T2A peptide includes the sequence of SEQ ID NO: 11.

[0271] In some examples, the nucleic acid encoding huEGFRt includes 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 a non-limiting example, the nucleic acid encoding huEGFRt includes the sequence of SEQ ID NO: 13.

[0272] 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.

[0273] In some embodiments, the antigen binding fragment is a single chain variable fragment (scFv) or a single domain antibody.

[0274] In some embodiments, the antibody or antigen-binding fragment specifically binds to a tumor antigen. In a specific example, the tumor antigen is GPC3, GPC2 or mesothelin.

[0275] 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). Any well-known numbering scheme, such as IMGT, Kabat or Chothia, can be used to determine the CDR sequence. In a specific example, 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.

[0276] 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 a sequence of nucleotides 73-420 of SEQ ID NO:17.

[0277] In other examples, wherein the tumor antigen is GPC3, the nucleic acid encoding the antibody binding fragment includes the CDR nucleic acid sequence of a GPC3-specific monoclonal antibody disclosed in WO 2013 / 181543 or WO 2012 / 145469, which are incorporated herein by reference in their entirety.

[0278] 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 a sequence of nucleotides 73-432 of SEQ ID NO:19.

[0279] 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.

[0280] In other examples, wherein the tumor antigen is GPC2, the nucleic acid encoding the antibody binding fragment includes 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.

[0281] 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.

[0282] 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). Any well-known numbering scheme, such as IMGT, Kabat or Chothia, can be used to determine the CDR sequence. In a specific example, 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.

[0283] In other examples, wherein the tumor antigen is mesothelin, the nucleic acid encoding the antibody binding fragment includes 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 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.

[0284] 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. Pat. No. 8,460,660, U.S. Pat. No. 6,809,184, or U.S. Pat. No. 7,081,518, each of which is incorporated herein by reference in its entirety.

[0285] Also provided is an isolated host cell comprising a CAR-encoding nucleic acid molecule disclosed herein. In some embodiments, the isolated host cell is a T lymphocyte.

[0286] The present disclosure also provides an isolated host cell, which 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 an antigen-binding fragment thereof, 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.

[0287] 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 (eg, from the CH2 and / or CH3 domains of an immunoglobulin molecule).

[0288] In some embodiments, the transmembrane domain includes 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.

[0289] 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.

[0290] In some embodiments, the intracellular signaling domain is an ITAM-bearing domain, such as a CD3 zeta or FcεRIγ signaling domain.

[0291] In a specific embodiment, the extracellular hinge region comprises a CD8α hinge region, the transmembrane domain comprises a CD8α transmembrane domain, the intracellular co-stimulatory domain comprises a 4-1BB co-stimulatory domain, and the intracellular signaling domain comprises a CD3ζ signaling domain.

[0292] 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 a specific example, the amino acid sequence of the CD8α hinge region includes SEQ ID NO: 4.

[0293] 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.

[0294] 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 a specific example, the amino acid sequence of the 4-1BB costimulatory domain includes SEQ ID NO: 8.

[0295] 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.

[0296] 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.

[0297] In some embodiments, the antigen binding fragment is a scFv or a single domain antibody.

[0298] 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.

[0299] In some examples, where the tumor antigen is GPC3, the amino acid sequence of the antigen-binding fragment includes 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). Any well-known numbering scheme, such as IMGT, Kabat or Chothia, can be used to determine the CDR sequence. In a specific example, the VH domain CDR1, CDR2 and CDR3 amino acid sequences each include 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 include 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.

[0300] In other examples, wherein the tumor antigen is GPC3, the amino acid sequence of the antigen binding fragment includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:30 (HN3 single domain antibody 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 amino acid sequences each include 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 include residues 26-33, 51-57 and 96-105 of SEQ ID NO:30. In a non-limiting example, the amino acid sequence of the antibody binding fragment includes residues 25-140 of SEQ ID NO:18.

[0301] In other examples, wherein the tumor antigen is GPC3, the amino acid sequence of the antibody binding fragment includes the CDR sequence of a GPC3-specific monoclonal antibody disclosed in WO 2013 / 181543 or WO 2012 / 145469, which are incorporated herein by reference in their entirety.

[0302] In some instances, wherein the tumor antigen is GPC2, the amino acid sequence of the antigen binding fragment includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:32 (LH7 single domain antibody 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 amino acid sequences each include 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 include 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 includes residues 25-144 of SEQ ID NO:20.

[0303] In other examples, wherein the tumor antigen is GPC2, the amino acid sequence of the antigen-binding fragment includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:34 (LH4 single domain antibody 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 amino acid sequences each include 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 include residues 26-33, 51-57 and 96-109 of SEQ ID NO:34.

[0304] In other examples, wherein the tumor antigen is GPC2, the amino acid sequence of the antigen-binding fragment includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:36 (LH6 single domain antibody 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 amino acid sequences each include 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 include residues 26-33, 51-58 and 97-110 of SEQ ID NO:36.

[0305] 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.

[0306] In some examples, wherein the tumor antigen is mesothelin, the amino acid sequence of the antigen-binding fragment includes 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). Any well-known numbering scheme, such as IMGT, Kabat or Chothia, can be used to determine the CDR sequence. In a specific example, the VH domain CDR1, CDR2 and CDR3 amino acid sequences each include 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 include 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.

[0307] In other examples, wherein the tumor antigen is mesothelin, the amino acid sequence of the antigen-binding fragment includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:42 (SD1 single domain antibody 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 amino acid sequences each include 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 include residues 26-35, 51-58 and 97-103 of SEQ ID NO:42.

[0308] In other examples, wherein the tumor antigen is mesothelin, the amino acid sequence of the antibody binding fragment includes the CDR sequence of a mesothelin-specific monoclonal antibody disclosed in WO 2014 / 031476, WO 2014 / 052064, U.S. Pat. No. 8,460,660, U.S. Pat. No. 6,809,184, or U.S. Pat. No. 7,081,518, each of which is incorporated herein by reference in its entirety.

[0309] 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.

[0310] Also provided herein are compositions comprising the isolated (CAR-expressing) host cells disclosed herein and a pharmaceutically acceptable carrier.

[0311] Further provided herein is a method 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-targeted CAR disclosed herein, or administering a therapeutically effective amount of an isolated host cell co-expressing a GPC3-targeted 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.

[0312] Also provided is a method for treating a GPC2-positive cancer in a subject. In some embodiments, the method includes administering to the subject a therapeutically effective amount of an isolated host cell comprising a nucleic acid molecule encoding a GPC2-targeted CAR disclosed herein, or using a therapeutically effective amount of an isolated host cell co-expressing a GPC2-targeted CAR and huEGFRt, as disclosed herein. In some instances, the GPC2-positive cancer is a neuroblastoma, acute lymphoblastic leukemia, embryonic rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

[0313] Further provided is a method 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.

[0314] In some embodiments of the method 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.

[0315] In one embodiment herein, a nucleic acid molecule encoding CAR is provided, which includes a nucleic acid encoding a first GMCSFRss in the 5' to 3' direction; 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 co-stimulatory 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 includes 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 a specific example, 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.

[0316] In one embodiment herein, there is provided an isolated host cell co-expressing CAR and huEGFRt, wherein the CAR includes an antigen-specific antibody or an antigen-binding fragment thereof, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB co-stimulatory 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 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 substitutions 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 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 substitutions relative to SEQ ID NO: 14. In specific, non-limiting examples, the amino acid sequence of 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 huEGFRt comprises SEQ ID NO: 14.

[0317] IV. Antibodies specific for tumor antigens

[0318] CAR disclosed herein can be targeted to express or overexpress tumor cells of 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.

[0319] A. GPC3-specific antibodies

[0320] The CAR construct disclosed herein can be transformed 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, which includes 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 CDR1, CDR2 and CDR3 of YP7 and hYP7. In other embodiments, the CAR comprises a single domain monoclonal antibody comprising a CDR sequence 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.

[0321] YP7 VH nucleotide sequence (SEQ ID NO:21)

[0322] GAGGTGCAGCTTGTTGAGACTGGTGGAGGAATGGTGCAGCCTGAAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGAATGCCATGAATTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAATAAAACTAATAATTATGCAACATATTATGCCGATTCAGTGAAAGCCAGGTTTACCATCTCCAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAACAACTTGAAAATTGAGGACACAGCCATGTACTATTGTGTGGCTGGTAACTCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0323] YP7 VH amino acid sequence (SEQ ID NO:22)

[0324] EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTNNYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYCVAGNSFAYWGQGTLVTVSA

[0325] YP7 VL nucleotide sequence (SEQ ID NO:23)

[0326] GACATTGTGATGTCACAGTCTCCATCCTCCCTAGTTGTGTCAATTGGAGAGAAGGTTACTATGACCTGCAAGTCCAGTCAGAGCCTTTTATATAGCAGCAATCAAAAGAACTACTTGGCCTGGTACCAACAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCAGTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAACTATCCGCTCACGTTCGGTGCTGGGACCAAGTTGGAGCTGAAA

[0327] YP7 VL amino acid sequence (SEQ ID NO:24)

[0328] DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASSRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLELK

[0329] hYP7 VH nucleotide sequence (SEQ ID NO:25)

[0330] GAGGTGCAGCTTGTTGAGTCTGGTGGAGGATTGGTGCAGCCTGGAGGGTCATTGAGACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGAATGCCATGAATTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGGCCGCATAAGAAATAAAACTAATAATTATGCAACATATTATGCCGATTCAGTGAAAGCCAGGTTTACCATCTCCAGAGATGATTCAAAGAACTCACTCTATCTGCAAATGAACAGCTTGAAAACCGAGGACACAGCCGTGTACTATTGTGTGGCTGGTAACTCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0331] hYP7 VH amino acid sequence (SEQ ID NO:26)

[0332] EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTLVTVSA

[0333] hYP7 VL nucleotide sequence (SEQ ID NO:27)

[0334] GACATTTGTGATGACCCAGTCTCCAGACTCCCTAGCTGTGTCACTGGGAGAGAGGGCCACTATCAACTGCAAGTCCAGTCAGAGCCTTTTATATAGCAGCAATCAAAAGAACTACTTGGCCTGGTACCAACAGAAACCAGGGCAGCCTCCTAAACTGCTGATTTACTGGG CATCCAGTAGGGAATCTGGGGTCCCTGATCGCTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAGGCTGAAGACGTGGCAGTTTATTACTGTCAGCAATATTATAACTATCCGCTCACGTTCGGTCAGGGGACCAAGTTGGAGATCAAA

[0335] hYP7 VL amino acid sequence (SEQ ID NO: 28)

[0336] DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIK

[0337] Table 1A. Position of CDRs in YP7 / hYP7 VH sequences (according to Kabat)

[0338]

[0339] Table 1B. Position of CDRs in YP7 / hYP7 VH sequences (according to IMGT)

[0340]

[0341] Table 1C. Position of CDRs in YP7 / hYP7 VL sequences (according to Kabat)

[0342]

[0343] Table 1D. Position of CDRs in YP7 / hYP7 VL sequences (according to IMGT)

[0344]

[0345] HN3 DNA sequence (SEQ ID NO:29)

[0346] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTTATTTCGATTTCGATTCTTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGCCTAGAGTGGATTGGGAGTATCTATCATAGTGGGAGCACCTACT ACAACCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAGAGTAAATATGGACCGATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAAGT

[0347] HN3 protein sequence (SEQ ID NO:30)

[0348] QVQLVQSGGGLVQPGGSLRLSCAASYFDFDSYEMSWVRQAPGKGLEWIGSIYHSGSTYYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTATYYCARVNMDRFDYWGQGTLVTVSSS

[0349] Table 2A. Position of CDRs in HN3 sequence (according to Kabat)

[0350] 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

[0351] Table 2B. Positions of CDRs in HN3 sequences (according to IMGT)

[0352] 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

[0353] B. GPC2-specific antibodies

[0354] 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.

[0355] LH7 DNA (SEQ ID NO:31)

[0356] CAGGTGCAGTCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGATTTCTATTTCTATGATTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTGGAGTGGATTGGGACTGTCTCCTATAGTGGGAGCACCTACTACAAC CCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTAAGAGCCGAGGACACAGCCATGTATTACTGTGCGAGAGGTTACAGCTATGATGACTCCCGATATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0357] LH7 protein (SEQ ID NO:32)

[0358] QVQLVQSGGGLVQPGGSLRLSCAASDFYFYDYEMSWVRQAPGKGLEWIGTVSYSGSTYYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAMYYCARGYSYDDSRYFDYWGQGTLVTVSS

[0359] Table 3A. Position of CDRs in LH7 sequence (according to Kabat)

[0360] 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

[0361] Table 3B. Position of CDRs in LH7 sequence (according to IMGT)

[0362] 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

[0363] LH4 DNA (SEQ ID NO:33)

[0364] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTTCTTTCTATTTCGATGATTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGCCCTGGAGTGGATTGGGCGTATCTATACCAGTGGGAGCACCAACTACAAC CCCTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCACGTATTACTGTGCGAGGGGATATTGTAGTGGTGGTAGCTGCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0365] LH4 protein (SEQ ID NO:34)

[0366] QVQLVQSGGGLVQPGGSLRLSCAASSFYFDDYEMSWVRQAPGKALEWIGRIYTSGSTNYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTATYYCARGYCSGGSCYFDYWGQGTLVTVSS

[0367] Table 4A. Position of CDRs in LH4 sequence (according to Kabat)

[0368] 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

[0369] Table 4B. Position of CDRs in LH4 sequence (according to IMGT)

[0370] 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

[0371] LH6 DNA (SEQ ID NO:35)

[0372] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGATTTCTATTTCGATGATTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAGTGGTAGTGGTGGTGGCACATACTACG CAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCACATATTACTGTGCGAGAGGTTACAGTTATGACGACTCCCGATATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

[0373] LH6 protein (SEQ ID NO:36)

[0374] QVQLVQSGGGLVQPGGSLRLSCAASDFYFDDYEMSWVRQAPGKGLEWVSTISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNTLRAEDTATYYCARGYSYDDSRYFDYWGQGTLVTVSS

[0375] Table 5A. Position of CDRs in LH6 sequence (according to Kabat)

[0376] 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

[0377] Table 5B. Position of CDRs in LH6 sequence (according to IMGT)

[0378] 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

[0379] C. Mesothelin-specific antibodies

[0380] The CAR construct disclosed herein can also be transformed 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. WO2014 / 031476, SD1 disclosed in PCT Publication No. WO2014 / 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.

[0381] YP218 VH nucleotide sequence (SEQ ID NO:37)

[0382] CAGCAGCAGCTGGAGGAGTCCGGGGGAGGCCTGGTCAAGCCTGAGGGATCCCTGACACTCACCTGCAAAGCCTCTGGATTCGACCTCGGTTTCTACTTTTACGCCTGTTGGGGTCCGCCAGGCTCCAGGGAAGGGCCTGGAGTGGATCGCATGCATTTATACTGCTGGTAGTGGTAGCACGTACT ACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAGCCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGGCAGCCGCGGACACGGCCACCTATTTCTGTGCGAGATCTACTGCTAATACTAGAAGTACTTATTATCTTAACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0383] YP218 VH amino acid sequence (SEQ ID NO:38)

[0384] QQQLEESGGGLVKPEGSLTLTCKASGFDLGFYFYACWVRQAPGKGLEWIACIYTAGSGSTYYASWAKGRFTISKASSTTVTLQMTSLAAADTATYFCARSTANTRSTYYLNLWGPGTLVTVSS

[0385] YP218 VL nucleotide sequence (SEQ ID NO:39)

[0386] GACGTCGTGATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGGATTAGTAGTTACTTATCCTGGTATCAGCAGAAACCAGGGCAGCGTCCCAAGCTCCTGATCTTTTGGTGCATCCACTCTGGCA TCTGGGGTCCCCTCGCGGTTCAAAGGCAGTGGATCTGGGACAGAATACACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAGAGTTATGCTTATTTTGATAGTAATAATTGGCATGCTTTCGGCGGAGGGACCGAGGTGGTGGTC

[0387] YP218 VL amino acid sequence (SEQ ID NO:40)

[0388] DVVMTQTPASVSEPVGGTVTIKCQASQRISSYLSWYQQKPGQRPKLLIFGASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQSYAYFDSNNWHAFGGGTEVVV

[0389] Table 6A. Position of CDRs in the YP218 VH sequence (according to Kabat)

[0390] 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

[0391] Table 6B. Position of CDRs in the YP218 VH sequence (according to IMGT)

[0392] 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

[0393] Table 6C. Position of CDRs in the YP218 VL sequence (according to Kabat)

[0394] 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

[0395] Table 6D. Position of CDRs in the YP218 VL sequence (according to IMGT)

[0396] 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

[0397] SD1 nucleotide sequence (SEQ ID NO:41):

[0398] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGATTTCGATTTCGCTGCTTATGAAATGAGCTGGGTCCGCCAGGCTCCAGGACAAGGCCTTGAGTGGGTGGCAATTATATCACATGATGGAATCG ATAAATACTACACAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCACGTATTACTGTTTAAGGCTTGGTGCTGTAGGCCAGGGAACCCTGGTCACCGTCTCCTCAAGT

[0399] SD1 amino acid sequence (SEQ ID NO:42):

[0400] QVQLVQSGGGLVQPGGSLRLSCAASDFDFAAYEMSWVRQAPGQGLEWVAIISHDGIDKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTATYYCLRLGAVGQGTLVTVSSS

[0401] Table 7A. Position of CDRs in SD1 sequence (according to Kabat)

[0402] 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

[0403] Table 7B. Position of CDRs in SD1 sequence (according to IMGT)

[0404] 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

[0405] V. Chimeric Antigen Receptor (CAR)

[0406] Disclosed herein are CARs (also referred to as chimeric T cell receptors, artificial T cell receptors, or chimeric immune receptors) and T cells modified 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 scFv or a 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.

[0407] 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 an 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 co-stimulatory molecules that co-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 co-stimulatory domain. The co-stimulatory domain refers to a part of a CAR comprising an intracellular domain of a co-stimulatory molecule. Co-stimulatory 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.

[0408] CAR can also include a signal peptide sequence, for example, the N-terminus of the antigen binding domain. The signal peptide sequence can be any suitable signal peptide sequence, such as a signal sequence from a granulocyte-macrophage colony stimulating factor receptor (GMCSFR), an 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 from CAR. Therefore, in some embodiments, CAR lacks a signal peptide sequence.

[0409] 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 after expression in the transduced cells, the CAR is cleaved from the huEGFRt (see Figure 1 ).

[0410] In some embodiments disclosed herein, the CAR construct encodes the following amino acid sequence in the N-terminal to C-terminal direction:

[0411] GMCSFRss:MLLLVTSLLLCELPHPAFLLIP(SEQ ID NO:2)

[0412] NdeI:HM

[0413] Antigen binding: scFv or single domain antibody sequences

[0414] SpeI:TS

[0415] CD8α hinge: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 4)

[0416] CD8αTM:IYIWAPLAGTCGVLLLSLVIT(SEQ ID NO:6)

[0417] 4-1BB:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:8)

[0418] CD3ζ:

[0419] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:10)

[0420] T2A:EGRGSLLTCGDVEENPGP(SEQ ID NO:12)

[0421] GMCSFRss:MLLLVTSLLLCELPHPAFLLIP(SEQ ID NO:2)

[0422] huEGFRt:

[0423] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKAT GQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM(SEQ ID NO:14)

[0424] The CAR-expressing T cells disclosed herein can be used to target specific cell types, such as tumor cells, such as GPC3-positive, GPC2-positive or mesothelin-positive tumor cells. Compared with 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.

[0425] Therefore, CAR is provided herein, which includes a tumor-specific antibody (or its binding fragment), such as a GPC3-specific antibody, a GPC2-specific antibody or a mesothelin-specific antibody. 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 expressing GPC3, GPC2 and mesothelin, respectively.

[0426] VI. Truncated human EGFR (huEGFRt)

[0427] Human epidermal growth factor receptor consists of four extracellular domains, one transmembrane domain and three intracellular domains. The EGFR domains are found in the following order from N-terminus to C-terminus: domain I-domain II-domain III-domain IV-transmembrane (TM) domain-near membrane domain-tyrosine kinase domain-C-terminal tail. Domain I and domain III are leucine-rich domains involved in ligand binding. Domain II and domain IV are cysteine-rich domains and do not contact EGFR ligands. Domain II mediates the formation of homo- or hetero-dimers with similar domains from other EGFR family members, and domain IV can form a disulfide bond with domain II. The EGFR TM domain passes through the cell membrane once 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).

[0428] 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 specific EGFR-specific monoclonal antibodies, such as FDA-approved cetuximab (PCT Publication No. WO 2011 / 056894, which is incorporated herein by reference).

[0429] Transduction of T cells with constructs disclosed herein (e.g., lentiviral vectors) encoding huEGFRt and tumor antigen-specific CARs 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.

[0430] 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 examples, 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 examples, 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 examples, the amino acid substitutions are conservative substitutions.

[0431] VII. CAR-Expressing Cell Compositions

[0432] 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 (such as aluminum hydroxide); and preservatives. The cells may be autologous to the recipient. However, the cells may also be heterologous (allogeneic).

[0433] With respect to cells, various aqueous carriers, such as buffered saline solutions, etc., 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 according to the specific mode of administration selected and the needs of the subject, according to fluid volume, viscosity, body weight, etc.

[0434] 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 Up to 10 9 cells / kg body weight, e.g. 10 5 Up to 10 6 cells / kg body weight, including all integer values ​​within these ranges. An exemplary dosage is 10 6 Cells / kg to about 10 8 cells / kg, e.g. about 5 x 10 6 Cells / kg to about 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.

[0435] The composition can be administered once or multiple times at these dosages, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. The composition 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). The composition can be administered daily, weekly, bimonthly or monthly. In some non-limiting examples, the composition is formulated for intravenous administration and multiple administrations. The quantity and frequency of administration are determined by factors such as the type and severity of the subject's condition, the subject's disease, although appropriate dosage can be determined by clinical trials.

[0436] 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. In one embodiment, more than one CAR- expressing cell administration is given to the subject (e.g., 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 (e.g., 2, 3 or 4 administrations per week) (also referred to as cycle), and then CAR expression cell administration is not performed for one week, and then one or more additional CAR- expressing cell administrations (e.g., more than one CAR T cell administration per week) are given to the subject. In another embodiment, the subject (e.g., 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.

[0437] In some embodiments, CAR-expressing T cells can replicate in vivo, resulting in long-term persistence, so that tumors can be continuously controlled. 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.

[0438] 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 via an artery, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, 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 a tumor or lymph node.

[0439] In some embodiments, the subject may undergo leukapheresis, wherein leukocytes are collected, enriched or depleted 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, a lentiviral vector expressing CAR and a truncated form of human EGFR (huEGFRt) is used to produce CAR-expressing cells. The co-expression of huEGFRt allows the use of antibodies (e.g., cetuximab, see PCT Publication No. WO 2011 / 056894, which is incorporated herein by reference) identifying huEGFRt to select and purify CAR-expressing T cells, as described in Section VI above.

[0440] In some embodiments, the erythrocytes are lysed and the monocytes are depleted, for example by PERCOLL TM Gradient centrifugation or counterflow centrifugal elutriation is used to separate T cells from peripheral blood lymphocytes. Specific subsets 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 such as anti-CD3 / anti-CD28 (e.g., 3×28)- M-450CD3 / CD28T is incubated for a time sufficient to perform positive selection of the desired T cells, and T cells can be separated, see US Publication No. US20140271635A1. 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 T cells are rare compared to other cell types (e.g., separated from immunocompromised individuals), longer incubation times can be used to separate T cells. In addition, the use of longer incubation times can improve the efficiency of capturing CD8+T cells. Therefore, by simply shortening or extending the time allowing T cells to bind CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as further described herein), subpopulations of T cells can be preferentially selected or not selected at the start of culture or at other time points in 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.

[0441] Enrichment of T cell populations by negative selection can be accomplished using a combination of antibodies against surface markers specific to negatively selected cells. One method is to sort and / or select cells by negative magnetic immunoadhesion or flow cytometry, using a mixture of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, in order to enrich CD4+ cells by negative selection, a mixture of monoclonal antibodies 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.

[0442] 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. 9 In other embodiments, a concentration of greater than 1×10 8 In other embodiments, 10, 15, 20, 25, 30, 35, 40, 45, 50, 65, 70, 75, 80, 85, 90, 95 or 100×10 cells / ml are used. 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 particles and cells. This allows cells that express 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 than CD8+ T cells at dilute concentrations. In some embodiments, the cell concentration used is 5×10 6 In other embodiments, the concentration used may be about 1×10 5 / ml to 1×10 6 / ml, and any integer values ​​in between.

[0443] VIII. Treatment Methods

[0444] 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.

[0445] Specifically provided is a method 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.

[0446] Also provided is a method 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 a neuroblastoma, acute lymphoblastic leukemia, embryonic rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

[0447] Further provided is a method 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.

[0448] 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.

[0449] 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 an objectively determinable improvement (e.g., a reduction in tumor volume or metastasis) as indicated by a clinician or other qualified observer.

[0450] The administration of CAR-expressing T cells and compositions disclosed herein may also be accompanied by the administration of other anticancer drugs or therapeutic treatments (e.g., surgical resection of tumors). Any suitable anticancer drug may 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 (e.g., antiandrogens), and antiangiogenic agents. Other anticancer treatments include radiotherapy and other antibodies that specifically target cancer cells.

[0451] Non-limiting examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozotocin, or dacarbazine).

[0452] 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.

[0453] 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).

[0454] 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).

[0455] 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 (Idarubicin), ifosfamide, methotrexate, mithramycin (Mithramycin), mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes, such as docetaxel), vinblastine (Velban), vincristine, VP-16, and some newer drugs include gemcitabine (Gemzar), Herceptin, irinotecan (Camptosar, CPT-11), cladribine (Leustatin), Navelbine, rituximab STI-571, taxotere, topotecan (Hycamtin), Xeloda (capecitabine), Zevelin, and calcitriol.

[0456] 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).

[0457] 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 eradicate or shrink the tumor.

[0458] 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. Example

[0459] Example 1: CAR-expressing lentiviral constructs

[0460] This example describes the generation of three lentiviral vectors encoding a tumor-targeting chimeric antigen receptor (CAR) and a truncated human EGFR (huEGFRt).

[0461] pWPT backbone lentiviral vector (Addgene) was used to produce 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 of which is 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 a 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.

[0462] 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 pMH288 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. The 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 a lentiviral vector pMH290 encoding a 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 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 shown as SEQ ID NOs: 25-32.

[0463] Example 2: Materials and Methods

[0464] 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.

[0465] Lentivirus production, concentration, and titration

[0466] 293T cells were seeded into 10 cm culture dishes (7.0 × 10 6 Cells / dish) were placed in a monolayer to achieve an optimal 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.

[0467] 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 to mix. In addition, 48 μl of CalFectin 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 -DNA complexes were added dropwise to the culture medium of each dish and homogenized by gently swirling the plate.

[0468] 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 at 1 / 10 to 1 / 100 of the original volume using complete DMEM.

[0469] Lentivirus titration

[0470] 293T cells were cultured at 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) in each well. One well was used to count cells, and the other well was used as a non-transduced 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.

[0471] After three days, the cells were washed with 1 ml PBS and detached by adding 200 μl trypsin / EDTA to each 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 for 1 hour on ice in FACS buffer (5% BSA, 0.01% sodium azide in PBS). The cells were washed with PBS 1X, centrifuged at 500 x g and 4°C for 5 minutes, and the precipitate was resuspended in a secondary antibody with an appropriate fluorescent dye. The secondary antibody was also diluted in FACS buffer.

[0472] 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.

[0473] Wash the fixed cells in PBS 1X and centrifuge at 500 x g and 4 °C for 5 minutes. Resuspend the pellet in 1 ml PBS. Analyze the cells for CAR expression using flow cytometry.

[0474] T cell activation and transduction protocol

[0475] DYNABEADS TM Human T-activator CD3 / CD28 (DYNABEADS TM Human T-Activator CD3 / CD28, Life Technologies) in a vial 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.

[0476] 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 inoculated in 1 ml of culture medium in a 24-well plate. DYNABEADS was added at a ratio of 2:1 beads to cells. TM Human T-activator CD3 / CD28, and 50U / ml IL-2 were added.

[0477] 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. Cells were then resuspended in viral supernatant and incubated overnight with 50 U / ml IL-2.

[0478] The next day, the cells were centrifuged and resuspended in fresh RPMI1640+10% FBS medium and 100 IU / mL IL-2 was added.

[0479] During the next 10 days, check the cultures 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 6 When cell growth kinetics and volume indicate that cells have been quiescent from activation, they can be used for functional assays or cryopreserved.

[0480] Functional assays

[0481] Target cells expressing luciferase (2×10 3) were inoculated 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 overnight at 37 ° C. 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 wells of individual tumor cells: [% Killing = 100-((RLU from wells with effector and target cells) / (RLU from wells with target cells) × 100)].

[0482] Example 3: GPC3-targeted CAR induces cytotoxicity of GPC3-expressing cell lines and reduces tumor volume of GPC3-positive tumors in animal models

[0483] This example describes the in vitro and in vivo cytotoxicity of T cells expressing CAR.HN3 and CAR.hYP7.

[0484] The T cell transduction efficiency of 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 ).

[0485] The cytotoxicity of CAR.hYP7 T cells was tested 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 ( Figure 4E-4G ).

[0486] Another study was performed to determine whether treatment with CAR.hYP7 T cells induces IFN-γ production by 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.

[0487] 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 given a blank injection or injected with 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 ( 1.04 × 10-cm2) compared to PBS-treated and blank-treated animals. Figure 6 ).

[0488] The durability of the antitumor effect of CAR.hYP7 T cells on mouse Hep3B xenograft tumors was also evaluated. Mice were injected intraperitoneally with 4 million Hep3B cells on day 0. On day 10, mice were injected with blank injections 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 was significantly reduced ( Fig. 7A). The 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 evaluated 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 ).

[0489] Next, CAR.hYP7 T cells were tested in another GPC3-positive tumor model. HepG2 xenografted NSG mice were treated with either sham treatment 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.

[0490] Example 4: Materials and Methods for GPC3-Targeted CAR Studies

[0491] This example provides the experimental procedures for the studies described in Example 5.

[0492] Cell culture

[0493] The human HCC cell line Hep3B was obtained from the National Cancer Institute (NCI), Bethesda, Maryland, USA. 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 that stably expresses 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 Huh-7 cell line (a HCC cell line) expressing luciferase 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 modified to express luciferase. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by 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 at 37°C in a humidified environment containing 5% CO2. All cell lines were verified by morphology and growth rate and were mycoplasma-free.

[0494] Generation of GPC3-targeted CAR

[0495] The GPC3-specific scFv from hYP7 and the single-domain antibody fragment HN3 were subcloned in frame into the EF-1α promoter-based lentiviral expression vector pWPT (Addgene). The construct contains an expression cassette encoding the CD8α hinge and transmembrane regions, the 4-1BB co-stimulatory domain, the intracellular CD3ζ, the self-cleaving T2A sequence, and the huEGFRt, as shown in Figure 2. Fig. 10B The final product was confirmed by sequence analysis.

[0496] Lentivirus production, T-cell transduction and expansion

[0497] Calfectin (SignaGen) was used to generate recombinant GPC3-CAR lentiviral vectors by cotransfection 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 Oklahoma Blood Institute (Oklahoma Blood Institute), and according to the manufacturer's instructions, PBMCs were stimulated 24 hours in the presence of IL-2 using anti-CD3 / anti-CD28 antibody-coated beads (Invitrogen) at a 2:1 bead / cell ratio. CAR T cells were generated as described previously (Li et al., Proc Natl Acad Sci USA 114: E6623-E6631, 2017). In order to track the number of T cells over time, trypan blue was used to count surviving cells.

[0498] Flow cytometry

[0499] 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 Immuno Research). 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).

[0500] Cytotoxicity assay

[0501] 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, CART cells and GPC3-positive (G1, Hep3B, HepG2, Huh-7) and GPC3-negative (A431, T3M4) tumor cells expressing luciferase were incubated for 24 hours at different effector / target (E: T) ratios. Luciferase activity was measured on Victor (PerkinElmer) using a luciferase assay system (Promega). The cytotoxicity of CAR-expressing T cells was also tested using IncuCyte-FLR-Platform (Essen BioScience). In short, T cells were added to HepG2 tumor cells expressing GFP at an E: T ratio of 2: 1. Images were taken every 2 minutes for up to 140 hours. The number of viable cells was quantified based on GFP expression. Cell killing activity was analyzed using the IncuCyte Zoom Hepatocyte Imaging System.

[0502] Cytokine assay

[0503] Cytokine levels in supernatants collected after 24 h of coculture of T cells and tumor cells were analyzed using a human cytokine 22-plexpanel (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, CXCL-8) on a Luminex system (Thermo Fisher Scientific).

[0504] Assessment of T cell polyfunctionality by single-cell cytokine profiling

[0505] 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 ratio of 1:1 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 groups (granzyme B, IFN-γ, CCL-3, perforin, TNF-α, TNF-β), stimulatory groups (GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21), regulatory groups (IL-4, IL-10, IL-13, IL-22, TGF-β1, sCD137, sCD40L), chemoattractant groups (CCL-11, IP-10, CCL-4, RANTES) and inflammatory groups (IL-1β, IL-6, IL-17A, IL-17F, MCP-1, MCP-4). Multifunctional CAR product T cells are defined as cells that co-secrete at least 2 proteins from a pre-specified panel per cell, combined with 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 multifunctional cells multiplied by the mean fluorescence intensity (MFI) of the proteins secreted by these cells.

[0506] Immunohistochemistry

[0507] Human HCC tissue and normal tissue microarrays were purchased from US Biomax and immunostained with anti-GPC3 antibody YP7. All tissue samples were sent to Histoserv Inc. (Germantown, MD) for staining.

[0508] Human Normal Tissue cDNA Array

[0509] Human normal tissue array was purchased from Origene. The panel contains 48 samples covering all major human normal tissues at different 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. The results were analyzed using the 2-ΔΔCt method.

[0510] Western blotting

[0511] 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 the 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. 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.

[0512] CRISPR / Cas9-mediated editing of GPC3

[0513] 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.

[0514] 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.

[0515] 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

[0516] AFP assay

[0517] Serum AFP levels were determined using an enzyme-linked immunosorbent assay (GenWay Biotech) according to the manufacturer's instructions.

[0518] Droplet Digital PCR

[0519] Genomic DNA was isolated from T cells using the FlexiGene DNA Kit (QIAGEN). Droplet digital PCR was performed on the QX200 Droplet Digital PCR System (Bio-Rad) according to the manufacturer's instructions.

[0520] Animal studies

[0521] Five-week-old female NOD / SCID (NSG) mice (NCI Frederick) were bred and handled 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 with different CAR T cells only once by intraperitoneal injection, 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. After 3 weeks of tumor formation, mice were infused with CAR(hYP7) T cells intraperitoneally or intravenously. 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 / cm 2 / sr). When the bioluminescence signal reaches 5×10 10 When , the mice were euthanized.

[0522] To measure the effect of GPC3 knockout on HCC tumor cell growth, 2 million Hep3B cells were injected subcutaneously into nude mice. 3After the size of the tumor was determined, treatment was started by intratumoral injection of plasmid expressing sgRNA5-2 or empty vector every other day for a total of 5 injections. Tumor size was measured twice a week with a caliper. Tumor volume in cubic millimeters was calculated by the following formula: (a) × (b 2 )×0.5, where “a” is the tumor length and “b” is the tumor width in millimeters.

[0523] Toxicology Analysis

[0524] Three NSG mice were selected from each group for toxicology studies. Complete blood count (CBC), comprehensive serum chemistry analysis (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.

[0525] statistics

[0526] All experiments were repeated at least three times to determine the repeatability 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.

[0527] Example 5: Glypican 3-targeted chimeric antigen receptor T cells for the treatment of hepatocellular carcinoma

[0528] 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 potent T cell activation and expansion capabilities in a mouse model of hepatocellular carcinoma.

[0529] GPC3 expression in HCC and normal tissues

[0530] To analyze GPC3 expression in tumors and normal tissues, 46 pairs of tumor tissues and adjacent non-tumor tissues (cirrhosis 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) in matched tumor-adjacent tissues. Strong GPC3 staining was found in 24 of the other 40 HCC tissues (2% (1 / 46), but not in any normal liver tissue. One problem with CAR T cell therapy is that on-target, off-tumor toxicities may occur due to the expression of antigens on normal tissues. Here, GPC3 expression in 30 human normal tissues was analyzed. Notably, GPC3 protein was absent in 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 a human normal tissue array by quantitative real-time PCR. Consistent with the protein profile, GPC3 mRNA expression was not found in most normal tissues except placenta ( Fig. 9 ), which is consistent with previous reports of GPC3 expression in human placenta (Khan et al., Histol Histopathol 16:71-78, 2001). The cell surface expression of GPC3 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 A431 cell line (G1) that overexpresses GPC3. In contrast, YP7 did not show binding to A431 and T3M4 cells, indicating that the GPC3 expression detected by the YP7 antibody is highly tumor specific.

[0531] Generation of GPC3-specific CAR T cells

[0532] The human single-domain antibody HN3 recognizes the N-leaflet of GPC3 ( Fig. 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 HN3 or hYP7 antibodies were cloned in frame into a lentiviral vector containing an expression cassette with 4-1BB and CD3ζ intracellular domains ( Fig. 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 huEGFRt expression. As shown Fig. 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 ( Fig. 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. Fig. 10E As shown, CAR(hYP7)T cells from 8 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 4 HCC patients only expanded 5- to 25-fold at day 11 after activation. In summary, CAR T cells based on HN3 and hYP7 antibodies have good 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.

[0533] In vitro antitumor activity of GPC3-targeted CAR T cells

[0534] 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. Fig.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 ( Fig. 11B and Fig. 11C ). When the E:T ratio was 5, the lytic activity of HCC patient-derived CAR(hYP7)T cells on Hep3B cells ranged from 30% to 70%, with an average of 50% ( Fig. 11C ), which is lower than the average activity (90%) of healthy donor-derived CAR(hYP7) T cells ( Fig. 11B ). In contrast, minimal cytolysis was observed in Hep3B cells treated with blank 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 ( Fig.11D CAR(hYP7) T cells induced similar levels of cell death in Hep3B cells at day 14 or day 28 ( Fig.11E In addition to Hep3B cells, GPC3 CAR T cells were also tested in other HCC cell lines including HepG2 and Huh-7. Fig.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. Fig.11G) was more effective. Overall, CAR(hYP7)T cells showed better cytolytic ability than CAR(HN3)T cells when co-cultured with GPC3-positive tumor cells.

[0535] In vitro multiplex cytokine and chemokine profiling of GPC3-targeted CAR T cells

[0536] 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. Fig.12 As shown in A, 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) ( Fig.12 A). 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 ( Fig.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 ( Fig.12 A) The complete spectrum of cytokines and chemokines analyzed in this study is shown in Fig.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.

[0537] Single-cell-based polyfunctional analysis of GPC3-targeted CAR T cells

[0538] Recent studies have shown that T cells that can co-produce multiple cytokines / chemokines at the single cell level, called "polyfunctional" T cells, are key effector cells that contribute to the development of effective and durable 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 versatility of our CAR T cell products, 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 polyfunctional T cell subsets that produce 2 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 polyfunctional cells compared to blank T cells. When stimulated with Hep3B cells, CAR(hYP7) T cells had higher polyfunctionality than CAR(HN3) T cells. Similarly, when stimulated with G1 cells, there was an increase in the percentage of CD4 + and CD8 + Enhanced polyfunctionality was observed in both CAR T cells. It was also noted that CD8 + T cells vs 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 versatility but in CD8 + 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 (e.g., CCL-3 and CCL-4), which was consistent with cytokine release measured using the Luminex assay. To distinguish all polyfunctional subsets in the samples, polyfunctional heatmap visualization was used. CAR(hYP7) T cells had a higher frequency of the most expressed functional groups compared to CAR(HN3) T cells after stimulation with Hep3B or G1 cells. + CAR(hYP7) T cells expressed a 4-plex panel containing granzyme B, INF-γ, perforin, and sCD137. G1 stimulated CD8 + CAR(hYP7) T cells were more polyfunctional and secreted a 7-plex containing granzyme B, INF-γ, CCL-3, CCL-4, perforin, TNF-α, and sCD137. In summary, CAR(hYP7) stimulated polyfunctional T cells (especially CD8 + Cytotoxic T cells) more robust activation and expansion.

[0539] Effects of Wnt signaling on GPC3-targeted CAR T-cell therapy

[0540] 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. Fig.13A As shown, after 6 hours of co-culture with Hep3B cells at an E:T ratio of 10:1, CAR(hYP7)T cells significantly reduced the expression of active-β-catenin and total β-catenin compared with blank T cells. The reduction of active-β-catenin expression even began at 2 hours of CAR(hYP7)T cell treatment in Hep3B cells ( Fig. 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 h of incubation, CAR(HN3)T cells neither inhibited β-catenin expression nor induced apoptosis of Hep3B cells.

[0541] 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 RNA (sgRNA) 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% ( Fig. 13C ). This 5-2sgRNA treatment downregulated the expression of active β-catenin and total β-catenin ( Fig. 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 empty plasmid or plasmid encoding 5-2sgRNA. Fig.13D As shown, 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 active-β-catenin and total β-catenin levels in tumors ( Fig.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 Fig.13F As shown, the AFP serum levels of mice injected with 5-2sgRNA were significantly lower than those of mice injected with empty plasmid, indicating that AFP levels were positively correlated with tumor size. Overall, this data suggests that targeting GPC3 may inhibit HCC tumor growth by inhibiting Wnt / β-catenin signaling.

[0542] CAR(hYP7) T cells induce HCC tumor regression in xenograft mouse models

[0543] To evaluate the in vivo antitumor activity of GPC3-specific CAR T cells, NSG mice were injected intraperitoneally with luciferase-expressing Hep3B cells (Hep3B-luc). Twelve days later, a single intraperitoneal infusion of blank or CAR T cells ( Fig.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 ( Fig. 14B and Fig. 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 recurrence, while the survival rate of the group treated with 5 million CAR(hYP7)T cells was only 50% ( Fig.14D Although GPC3-targeted CAR T cells initially caused weight loss, the mice gradually gained weight ( Fig.19A In addition, after two weeks of 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) ( Fig.14E Notably, the AFP levels of mice treated with 20 million CAR(hYP7) T cells were in the range of 25-78 ng / mL, close to the cutoff value for adults (20 ng / mL).

[0544] 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. Fig.14FAs shown, 22.1% CAR expression was found in the 5 million CAR(hYP7) group after 3 weeks of treatment, while only 1.3% CAR was detected in the 5 million CAR(HN3) group. In addition, 26.5% CAR integration was detected in the 10 million CAR(hYP7) group, indicating a negative correlation between tumor burden and T cell persistence over time. In contrast, only 2.2% 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.

[0545] In the Hep3B peritoneal dissemination mouse model, mice were found to develop 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.

[0546] 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 ( Fig.15A ).like Fig. 15B and Fig. 15C As shown, CAR( Fig.15A )T cells reduced tumor burden to background levels, and the tumor flux on day 21 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 ( Fig.19B ), which is consistent with transient cytokine release syndrome. After 5 weeks of CAR ( Fig.19B ) T cell therapy, ddPCR detected 35.6% and 19.5% CAR expression in genomic DNA from tumors and mouse spleens, respectively ( Fig.15D ). In contrast, CD19 CAR T cells showed no signs of genetic integration in either tissue. In addition, 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 the Hep3B peritoneal xenograft mouse model.

[0547] The antitumor activity of CAR(hYP7) T cells was further examined in an orthotopic HCC mouse model because it is more clinically relevant. Half a million Hep3B-luc cells were injected into the livers of NSG mice, and tumor engraftment was confirmed by bioluminescence imaging ( Fig.16A ). On day 21, CAR(hYP7)T cells were infused intraperitoneally or intravenously into mice. Although both administration routes 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 mice in the blank T cell group had large tumors. Fig.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 hYP7 IP group, which is consistent with the anti-tumor efficacy of each administration route in mice. In addition, luminescent imaging showed that Hep3B cells grew in the liver of mice, and the infusion of CAR (hYP7) T cells significantly inhibited tumor growth in mice or completely eliminated tumor cells. Finally, toxicology studies were performed to evaluate the side effects of CAR (hYP7) T cell therapy. Mice in the hYP7 intravenous injection group showed an increase in leukocytes and neutrophils, which may be related to the rapid immune response in vivo (Table 8). In addition, a mouse that received CAR (hYP7) T cells through the tail vein had elevated alanine aminotransferase (ALT) activity. However, no obvious evidence of liver damage was found after the mice were autopsied. 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.

[0548] Table 8. Toxicity of CAR(hYP7) T cells in Hep3B orthotopic xenograft mice

[0549]

[0550]

[0551] In view of the fact that the principles of the disclosed invention can be applied to many possible embodiments, it should be recognized that the embodiments described are merely examples of the present invention and should not be construed as limiting the scope of the present invention. On the contrary, the scope of the present invention is defined by the appended claims. Therefore, we claim protection for all inventions that fall within the scope and spirit of these claims. Sequence Listing <110> The United States Government (represented by the Secretary of Health and Human Services) <120> Chimeric Antigen Receptors Targeting Tumor Antigens <130> 4239-99836-02 <150> US 62 / 584,421 <151> 2017-11-10 <160> 51 <170> PatentIn version 3.5 <210> 1 <211> 66 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (GMCSFRss) <400> 1 atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60 atccca 66 <210> 2 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide (GMCSFRss) <400> 2 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro 20 <210> 3 <211> 135 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (CD8alpha hinge) <400> 3 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgac 135 <210> 4 <211> 45 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide (CD8alpha hinge) <400> 4 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 5 <211> 63 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (CD8alpha TM) <400> 5 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 acc 63 <210> 6 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide (CD8alpha TM) <400> 6 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <210> 7 <211> 126 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (4-1BB) <400> 7 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 8 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide (4-1BB) <400> 8 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 9 <211> 336 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (CD3zeta signaling domain) <400> 9 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttggggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 10 <211> 112 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide (CD3zeta signaling domain) <400> 10 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 11 <211> 54 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (T2A) <400> 11 gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54 <210> 12 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide (T2A) <400> 12 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <210> 13 <211> 1005 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (huEGFRt) <400> 13 cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60 acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120 gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat 180 attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac 240 aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300 ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 360 aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420 acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac 480 agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540 ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 600 gaatgcgtgg acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct 660 gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga 720 cggggaccag acaactgtat ccagtgtgcc cactacattg acggccccca ctgcgtcaag 780 acctgcccgg caggagtcat gggagaaaac aacaccctgg tctggaagta cgcagacgcc 840 ggccatgtgt gccacctgtg ccatccaaac tgcacctacg gatgcactgg gccaggtctt 900 gaaggctgtc caacgaatgg gcctaagatc ccgtccatcg ccactgggat ggtgggggcc 960 ctcctcttgc tgctggtggt ggccctgggg atcggcctct tcatg 1005 <210> 14 <211> 335 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide (huEGFRt) <400> 14 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile 20 25 30 Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe 35 40 45 Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr 50 55 60 Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn 65 70 75 80 Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg 85 90 95 Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile 100 105 110 Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val 115 120 125 Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp 130 135 140 Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn 145 150 155 160 Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu 165 170 175 Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser 180 185 190 Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu 195 200 205 Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln 210 215 220 Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly 225 230 235 240 Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro 245 250 255 His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr 260 265 270 Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His 275 280 285 Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro 290 295 300 Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala 305 310 315 320 Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 325 330 335 <210> 15 <211> 2601 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct (pMH289) <400> 15 atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60 atcccacata tggaggtgca gcttgttgag tctggtggag gattggtgca gcctggaggg 120 tcattgagac tctcatgtgc agcctctgga ttcaccttca ataagaatgc catgaattgg 180 gtccgccagg ctccaggaaa gggtttggaa tgggttggcc gcataagaaa taaaactaat 240 aattatgcaa catattatgc cgattcagtg aaagccaggt ttaccatctc cagagatgat 300 tcaaagaact cactctatct gcaaatgaac agcttgaaaa ccgaggacac agccgtgtac 360 tattgtgtgg ctggtaactc gtttgcttac tggggccaag ggactctggt cactgtctct 420 gcaggcggag gcggatcagg tggtggcgga tctggaggtg gcggaagcga cattgtgatg 480 acccagtctc cagactccct agctgtgtca ctgggagaga gggccactat caactgcaag 540 tccagtcaga gccttttata tagcagcaat caaaagaact acttggcctg gtaccaacag 600 aaaccagggc agcctcctaa actgctgatt tactgggcat ccagtaggga atctggggtc 660 cctgatcgct tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg 720 caggctgaag acgtggcagt ttattactgt cagcaatatt ataactatcc gctcacgttc 780 ggtcaggga ccaagttgga gatcaaaact agtaccacga cgccagcgcc gcgaccacca 840 acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900 gcggcgggg gcgcagtgca cacgagggg ctggacttcg cctgtgacat ctacatctgg 960 gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac caaacggggc 1020 agaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1080 gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1140 gtgaagttca gcaggagcgc agacgcccc gcgtaccagc agggccagaa ccagctctat 1200 aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1260 gaccctgaga tgggggaaa gccgagaagg aagaaccctc aggaagcct gtacaatgaa 1320 1380 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1440 gacgcccttc acatgcaggc cctgccccct cgcgagggca gaggaagtct tctaacatgc 1500 ggtgacgtgg aggagaatcc cggccctatg cttctcctgg tgacaagcct tctgctctgt 1560 gagttaccac acccagcatt cctcctgatc ccacgcaaag tgtgtaacgg aataggtatt 1620 ggtgaattta aagactcact ctccataaat gctacgaata ttaaacactt caaaaactgc 1680 acctccatca gtggcgatct ccacatcctg ccggtggcat ttaggggtga ctccttcaca 1740 catactcctc ctctggatcc acaggaactg gatattctga aaaccgtaaa ggaaatcaca 1800 gggtttttgc tgattcaggc ttggcctgaa aacaggacgg acctccatgc ctttgagaac 1860 ctagaaatca tacgcggcag gaccaagcaa catggtcagt tttctcttgc agtcgtcagc 1920 ctgaacataa catccttggg attacgctcc ctcaaggaga taagtgatgg agatgtgata 1980 atttcaggaa acaaaaattt gtgctatgca aatacaataa actggaaaaa actgtttggg 2040 acctccggtc agaaaaccaa aattataagc aacagaggtg aaaacagctg caaggccaca 2100 ggccaggtct gccatgcctt gtgctccccc gagggctgct ggggcccgga gcccagggac 2160 tgcgtctctt gccggaatgt cagccgaggc agggaatgcg tggacaagtg caaccttctg 2220 gagggtgagc caagggagtt tgtggagaac tctgagtgca tacagtgcca cccagagtgc 2280 ctgcctcagg ccatgaacat cacctgcaca ggacggggac cagacaactg tatccagtgt 2340 gcccactaca ttgacggccc ccactgcgtc aagacctgcc cggcaggagt catgggagaa 2400 aacaacaccc tggtctggaa gtacgcagac gccggccatg tgtgccacct gtgccatcca 2460 aactgcacct acggatgcac tgggccaggt cttgaaggct gtccaacgaa tgggcctaag 2520 atcccgtcca tcgccactgg gatggtgggg gccctcctct tgctgctggt ggtggccctg 2580 gggatcggcc tcttcatgtg a 2601 <210> 16 <211> 866 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide (hYP7-CAR) <400> 16 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro His Met Glu Val Gln Leu Val Glu Ser Gly 20 25 30 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 35 40 45 Ser Gly Phe Thr Phe Asn Lys Asn Ala Met Asn Trp Val Arg Gln Ala 50 55 60 Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Arg Asn Lys Thr Asn 65 70 75 80 Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Ala Arg Phe Thr Ile 85 90 95 Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu 100 105 110 Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala Gly Asn Ser Phe 115 120 125 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly 130 135 140 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met 145 150 155 160 Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr 165 170 175 Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys 180 185 190 Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 195 200 205 Leu Ile Tyr Trp Ala Ser Ser Arg Glu Ser Gly Val Pro Asp Arg Phe 210 215 220 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 225 230 235 240 Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr 245 250 255 Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Ser Thr 260 265 270 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 275 280 285 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 290 295 300 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 305 310 315 320 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 325 330 335 Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 340 345 350 Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg 355 360 365 Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser 370 375 380 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 385 390 395 400 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 405 410 415 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 420 425 430 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 435 440 445 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 450 455 460 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 465 470 475 480 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Glu Gly Arg Gly Ser 485 490 495 Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu Leu 500 505 510 Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu 515 520 525 Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys 530 535 540 Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys 545 550 555 560 Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly 565 570 575 Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile 580 585 590 Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp 595 600 605 Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile 610 615 620 Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser 625 630 635 640 Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp 645 650 655 Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr 660 665 670 Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile 675 680 685 Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys 690 695 700 His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp 705 710 715 720 Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys 725 730 735 Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu 740 745 750 Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr 755 760 765 Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile 770 775 780 Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu 785 790 795 800 Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His 805 810 815 Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu 820 825 830 Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met 835 840 845 Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu 850 855 860 Phe Met 865 <210> 17 <211> 2214 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct (pMH288) <400> 17 atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60 atcccacata tgcaggtgca gctggtgcag tctgggggag gcttggtaca gcctggaggg 120 tccctgagac tctcctgtgc agcctcttat ttcgatttcg attcttatga aatgagctgg 180 gtccgccagg ctccagggaa gggcctagag tggattggga gtatctatca tagtgggagc 240 acctactaca acccgtccct caagagtcga gtcaccatct ccagagacaa ttccaagaac 300 acgctgtatc tgcaaatgaa caccctgaga gccgaggaca cagccacgta ttactgtgcg 360 agagtaaata tggaccgatt tgactactgg ggccagggaa ccctggtcac cgtctcctca 420 actagtacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 480 cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 540 gggctggact tcgcctgtga catctacatc tgggcgccct tggccgggac ttgtggggtc 600 cttctcctgt cactggttat caccaaacgg ggcagaaaga aactcctgta tatattcaaa 660 caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 720 ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag cgcagacgcc 780 cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 840 gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 900 aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 960 tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1020 cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1080 cctcgcgagg gcagaggag tcttctaaca tgcggtgacg tggaggaga tcccggccct atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg atcccacgca aagtgtgtaa cggaatagt attggtgaat ttaaagactc actctccata 1320. attack cttcaaaaac tgcacctcca tcagtggcga tctccacatc ctgccggtgg catttagggg tgactccttc acacatactc ctcctctgga tccacagga ctggatattc tgaaaaccgt aaaggaatc acagggtttt tgctgattca ggcttggcct 1440 gaaaacagga cggacctcca tgcctttgag aacctagaaa tcatacgcgg caggaccaag caacatggtc agttttctct tgcagtcgtc agcctgaaca taacatcctt gggattacgc tccctcaagg agataagtga tggagatgtg ataatttcag gaaacaaaaa tttgtgctat gcaataca taaactggaa aaaactgttt gggacctccg gtcagaaac caaattata agcaacagag gtgaaaacag ctgcaaggcc acaggccagg tctgccatgc cttgtgctcc cccgagggct gctggggccc ggagcccagg gactgcgtct cttgccgga tgtcagccga ggcagggaat gcgtggacaa gtgcaacctt ctggagggtg agccaaggga gtttgtggag 1860 aactctgagt gcatacagtg ccacccagag tgcctgcctc aggccatgaa catcacctgc 1920 acaggacggg gaccagacaa ctgtatccag tgtgcccact acattgacgg cccccactgc 1980 gtcaagacct gcccggcagg agtcatggga gaaaacaaca ccctggtctg gaagtacgca 2040 gacgccggcc atgtgtgcca cctgtgccat ccaaactgca cctacggatg cactgggcca 2100 ggtcttgaag gctgtccaac gaatgggcct aagatcccgt ccatcgccac tgggatggtg 2160 ggggccctcc tcttgctgct ggtggtggcc ctggggatcg gcctcttcat gtga 2214 <210> 18 <211> 737 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide (HN3-CAR) <400> 18 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro His Met Gln Val Gln Leu Val Gln Ser Gly 20 25 30 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 35 40 45 Ser Tyr Phe Asp Phe Asp Ser Tyr Glu Met Ser Trp Val Arg Gln Ala 50 55 60 Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Tyr His Ser Gly Ser 65 70 75 80 Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp 85 90 95 Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu 100 105 110 Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Val Asn Met Asp Arg Phe Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Ser Thr Thr 130 135 140 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 145 150 155 160 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 165 170 175 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 180 185 190 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 195 200 205 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 210 215 220 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 225 230 235 240 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg 245 250 255 Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 260 265 270 Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 275 280 285 Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 290 295 300 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 305 310 315 320 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 325 330 335 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 340 345 350 Ala Leu His Met Gln Ala Leu Pro Pro Arg Glu Gly Arg Gly Ser Leu 355 360 365 Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Leu Leu Leu 370 375 380 Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu 385 390 395 400 Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp 405 410 415 Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr 420 425 430 Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp 435 440 445 Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu 450 455 460 Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro 465 470 475 480 Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg 485 490 495 Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu 500 505 510 Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly 515 520 525 Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile 530 535 540 Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile 545 550 555 560 Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His 565 570 575 Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys 580 585 590 Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys 595 600 605 Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys 610 615 620 Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys 625 630 635 640 Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp 645 650 655 Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn 660 665 670 Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu 675 680 685 Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly 690 695 700 Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val 705 710 715 720 Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe 725 730 735 Met <210> 19 <211> 2226 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct (pMH290) <400> 19 atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60 atcccacata tgcaggtgca gctggtgcag tctgggggag gcttggtaca gcctggaggg 120 tccctgagac tctcctgtgc agcctctgat ttctatttct atgattatga aatgagctgg 180 gtccgccagg ctccagggaa gggtctggag tggattggga ctgtctccta tagtgggagc 240 acctactaca acccgtccct caagagtcga gtcaccatct ccagagacaa ttccaagaac 300 acgctgtatc tgcaatgaa caccctaaga gccgaggaca cagccatgta ttactgtgcg 360 agaggttaca gctatgatga ctcccgatat tttgactact ggggccaggg aaccctggtc 420 accgtctcct caactac cacgacgcca gcgccgcgac caccacacc ggcgcccacc 480 atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 540 gtgcacacga gggggctgga cttcgcctgt gatactaca cttggccggg 600 acttgtgggg tccttcct gtcactggtt atcaccaac ggggcagaaa gaaactcctg 660 tatatattca aaaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 720 agctgccgat ttccagaga agagaagga ggatgtgaac tgagagtgaa gttcagcagg 780 agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 840 ggacgaagg aggagtacga tgttttggac agagacgtg gccgggaccc tgagatgggg 900 ggaaagccga gaaggagaa ccctcaggaa ggcctgtaca atgaactgca gaagataag 960 atggcggagg cctacagtga gattgggatg aaggcgagc gccggagggg caggggcac 1020 gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1080 caggccctgc cccctcgcga gggcagagga agtcttctaa catgcggtga cgtggaggag 1140 aatcccggcc ctatgcttct cctggtgaca agccttctgc tctgtgagtt accacaccca 1200 gcattcctcc tgatcccacg caaagtgtgt aacggaatag gtattggtga atttaaagac 1260 tcactctcca taaatgctac gaatattaaa cacttcaaaa actgcacctc catcagtggc 1320 gatctccaca tcctgccggt ggcatttagg ggtgactcct tcacacatac tcctcctctg 1380 gatccacagg aactggatat tctgaaaacc gtaaaggaaa tcacagggtt tttgctgatt 1440 caggcttggc ctgaaaacag gacggacctc catgcctttg agaacctaga aatcatacgc 1500 ggcaggacca agcaacatgg tcagttttct cttgcagtcg tcagcctgaa cataacatcc 1560 ttgggattac gctccctcaa ggagataagt gatggagatg tgataatttc aggaaacaaa 1620 aatttgtgct atgcaaatac aataaactgg aaaaaactgt ttgggacctc cggtcagaaa 1680 accaaaatta taagcaacag aggtgaaaac agctgcaagg ccacaggcca ggtctgccat 1740 gccttgtgct cccccgaggg ctgctggggc ccggagccca gggactgcgt ctcttgccgg 1800 aatgtcagcc gaggcaggga atgcgtggac aagtgcaacc ttctggaggg tgagccaagg 1860 gagtttgtgg agaactctga gtgcatacag tgccacccag agtgcctgcc tcaggccatg 1920 aacatcacct gcacaggacg gggaccagac aactgtatcc agtgtgccca ctacattgac 1980 ggcccccact gcgtcaagac ctgcccggca ggagtcatgg gagaaaacaa caccctggtc 2040 tggaagtacg cagacgccgg ccatgtgtgc cacctgtgcc atccaaactg cacctacgga 2100 tgcactgggc caggtcttga aggctgtcca acgaatgggc ctaagatccc gtccatcgcc 2160 actgggatgg tgggggccct cctcttgctg ctggtggtgg ccctggggat cggcctcttc 2220 atgtga 2226 <210> 20 <211> 741 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide (LH7-CAR) <400> 20 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro His Met Gln Val Gln Leu Val Gln Ser Gly 20 25 30 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 35 40 45 Ser Asp Phe Tyr Phe Tyr Asp Tyr Glu Met Ser Trp Val Arg Gln Ala 50 55 60 Pro Gly Lys Gly Leu Glu Trp Ile Gly Thr Val Ser Tyr Ser Gly Ser 65 70 75 80 Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp 85 90 95 Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu 100 105 110 Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gly Tyr Ser Tyr Asp Asp Ser 115 120 125 Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140 Thr Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 145 150 155 160 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala 165 170 175 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile 180 185 190 Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser 195 200 205 Leu Val Ile Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 210 215 220 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 225 230 235 240 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 245 250 255 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 260 265 270 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 275 280 285 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg 290 295 300 Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys 305 310 315 320 Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg 325 330 335 Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 340 345 350 Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Glu Gly 355 360 365 Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro 370 375 380 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 385 390 395 400 Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly 405 410 415 Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe 420 425 430 Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala 435 440 445 Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu 450 455 460 Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile 465 470 475 480 Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu 485 490 495 Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala 500 505 510 Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu 515 520 525 Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr 530 535 540 Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys 545 550 555 560 Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly 565 570 575 Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu 580 585 590 Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys 595 600 605 Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu 610 615 620 Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met 625 630 635 640 Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala 645 650 655 His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val 660 665 670 Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His 675 680 685 Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro 690 695 700 Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala 705 710 715 720 Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly 725 730 735 Ile Gly Leu Phe Met 740 <210> 21 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide (YP7 VH Domain) <400> 21 gaggtgcagc ttgttgagac tggtggagga atggtgcagc ctgaagggtc attgaaactc 60 tcatgtgcag cctctggatt caccttcaat aagaatgcca tgaattgggt ccgccaggct 120 ccaggaaagg gtttggaatg ggttgctcgc ataagaaata aaactaataa ttatgcaaca 180 tattatgccg attcagtgaa agccaggttt accatctcca gagatgattc acaaagcatg 240 ctctatctgc aaatgaacaa cttgaaaatt gaggacacag ccatgtacta ttgtgtggct 300 ggtaactcgt ttgcttactg gggccaaggg actctggtca ctgtctctgc a 351 <210> 22 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide (YP7 VH domain) <400> 22 Glu Val Gln Leu Val Glu Thr Gly Gly Gly Met Val Gln Pro Glu Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Asn 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Asn Lys Thr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Ala Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Ile Glu Asp Thr Ala Met Tyr 85 90 95 Tyr Cys Val Ala Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> twenty three <211> 339 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (YP7 VL domain) <400> twenty three gacattgtga tgtcacagtc tccatcctcc ctagttgtgt caattggaga gaaggttatact 60 atgacctgca agtccagtca gagcctttta tatagcagca atcaaaagaa ctacttggcc 120 tggtaccaac agaaaccagg gcagtctcct aaactgctga tttactgggc atccagtagg 180 gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240 atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttataactat 300 ccgctcacgt tcggtgctgg gaccaagttg gagctgaaa 339 <210> twenty four <211> 113 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide (YP7 VL domain) <400> twenty four Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Val Val Ser Ile Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ser Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 25 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Polynucleotide (hYP7 VH Domain) <400> 25 gaggtgcagc ttgttgagtc tggtggagga ttggtgcagc ctggagggtc attgagactc 60 tcatgtgcag cctctggatt caccttcaat aagaatgcca tgaattgggt ccgccaggct 120 ccaggaaagg gtttggaatg ggttggccgc ataagaaata aaactaataa ttatgcaaca 180 tattatgccg attcagtgaa agccaggttt accatctcca gagatgattc aaagaactca 240 ctctatctgc aaatgaacag cttgaaaacc gaggacacag ccgtgtacta ttgtgtggct 300 ggtaactcgt ttgcttactg gggccaaggg actctggtca ctgtctctgc a 351 <210> 26 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide (hYP7 VH domain) <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Asn 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Asn Lys Thr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Ala Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Ala Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <210> 27 <211> 339 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (hYP7 VL domain) <400> 27 gacattgtga tgacccagtc tccagactcc ctagctgtgt cactgggaga gagggccact 60 atcaactgca agtccagtca gagcctttta tatagcagca atcaaaagaa ctacttggcc 120 tggtaccaac agaaaccagg gcagcctcct aaactgctga tttactgggc atccagtagg 180 gaatctgggg tccctgatcg cttcagtggc agtggatctg ggacagattt cactctcacc 240 atcagcagtc tgcaggctga agacgtggca gtttattact gtcagcaata ttataactat 300 ccgctcacgt tcggtcaggg gaccaagttg gagatcaaa 339 <210> 28 <211> 113 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide (hYP7 VL domain) <400> 28 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ser Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 29 <211> 351 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (HN3) <400> 29 caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly tcctgtgcag cctcttattt cgatttcgat tcttatgaaa tgagctgggt ccgccaggct 120 Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Phe Asp Phe Asp Ser Tyr Glu Met Ser Trp Val Arg Gln Ala ccagggaagg gcctagagtg gattgggagt atctatcata gtgggagcac ctactacaac 180 Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn ccgtccctca agagtcgagt caccatctcc agagacaatt ccaagaacac gctgtatctg 240 Pro Ser Leu Lys Ser Glu Ser Thr Ile Ser Arg Asp Ile Pro Lys Asn Thr Leu Tyr Leu caaatgaaca ccctgagagc cgaggacaca gccacgtatt actgtgcgag agtaaatatg 300 Gln Met Asn Thr Leu Glu Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Glu Ser Asn Tyr gaccgatttg actactgggg ccagggaacc ctggtcaccg tctcctcaag t 351 Asp Asp Phe Asp Thr Trp Gly Pro Gly Asn Pro Gly Thr Arg Ser Leu Lys <210> 30<210> 30 <211> 117<211> 117 <212> PRT<212> PRT <213> 人工序列<213> Artificial sequence <220><220> <223> 合成多肽 (HN3) <223> Synthetic polypeptide (HN3) <400> 30 <400> 30 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Phe Asp Phe Asp Ser Tyr Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Phe Asp Phe Asp Ser Tyr 20 25 30 20 25 30 Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 35 40 45 Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Val Asn Met Asp Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ser 115 <210> 31 <211> 360 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide (LH7) <400> 31 caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60 tcctgtgcag cctctgattt ctatttctat gattatgaaa tgagctgggt ccgccaggct 120 ccagggaagg gtctggagtg gattgggact gtctcctata gtgggagcac ctactacaac 180 ccgtccctca agagtcgagt caccatctcc agagacaatt ccaagaacac gctgtatctg 240 caaatgaaca ccctaagagc cgaggacaca gccatgtatt actgtgcgag aggttacagc 300 tatgatgact cccgatattt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360 <210> 32 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide (LH7) <400> 32 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Phe Tyr Phe Tyr Asp Tyr 20 25 30 Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Thr Val Ser Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Gly Tyr Ser Tyr Asp Asp Ser Arg Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 33 <211> 360 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (LH4) <400> 33 caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60 tcctgtgcag cctcttcttt ctatttcgat gattatgaaa tgagctgggt ccgccaggct 120 ccagggaagg ccctggagtg gattgggcgt atctatacca gtggggagcac caactacaac 180 ccctccctca agagtcgagt caccatctcc agagacaatt ccaagaacac gctgtatctg 240 caaatgaaca ccctgagagc cgaggacaca gccacgtatt actgtgcgag gggatattgt 300 agtggtggta gctgctactt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360 <210> 34 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide (LH4) <400> 34 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Ser Ser Phe Tyr Phe Asp Asp Tyr 20 25 30 Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Gly Tyr Cys Ser Gly Gly Ser Cys Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 35 <211> 363 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (LH6) <400> 35 caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60 tcctgtgcag cctctgattt ctatttcgat gattatgaaa tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaact attagtggta gtggtggtgg cacatactac 180 gcagactcag tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acaccctgag agccgaggac acagccacat attactgtgc gagaggttac 300 agttatgacg actcccgata ttttgactac tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 36 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide (LH6) <400> 36 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Phe Tyr Phe Asp Asp Tyr 20 25 30 Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Ser Tyr Asp Asp Ser Arg Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Free Mp3 Download 115 120 <210> 37 <211> 369 <212> DNA <213> The snowstorm <220> <223> Switchgear (YP218 VH switch) <400> 37 60. cgcagcagc tggaggagc cgggggaggc ctggtcaagc ctgagggatc cctgacactc acctgcaaag cctctggatt cgacctcggt ttctactttt acgcctgttg ggtccgccag 120 gctccaggga agggcctgga gtggatcgca tgcatttata ctgctggtag tggtagcacg 180 240. tactacgcga gctgggcga aggccgattc accatctcca aagcctcgtc gaccacggtg actctgcaaa tgaccagtct ggcagccgcg gacacggcca cctatttctg tgcgagatct actgctaata ctagaagtac ttattatctt aacttgtggg gcccaggcac cctggtcacc gtctcctca 369 <210> 38 <211> 123 <212> PRT <213> The snowstorm <220> <223> Container (YP218 VH Container) <400> 38 Gln Gln Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Glu Gly 1 5 10 15 Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Asp Leu Gly Phe Tyr 20 25 30 Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Ala Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr Ala Ser 50 55 60 Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Ala Ser Ser Thr Thr Val 65 70 75 80 Thr Leu Gln Met Thr Ser Leu Ala Ala Ala Asp Thr Ala Thr Tyr Phe 85 90 95 Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu Asn Leu 100 105 110 Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 39 <211> 330 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (YP218 VL domain) <400> 39 gacgtcgtga tgacccagac tccagcctcc gtgtctgaac ctgtgggagg cacagtcacc 60 atcaagtgcc aggccagtca gaggattagt agttacttat cctggtatca gcagaaacca 120 gggcagcgtc ccaagctcct gatctttggt gcatccactc tggcatctgg ggtcccctcg 180 cggttcaaag gcagtggatc tgggacagaa tacactctca ccatcagcga cctggagtgt 240 gccgatgctg ccacttacta ctgtcagagt tatgcttatt ttgatagtaa taattggcat 300 gctttcggcg gagggaccga ggtggtggtc 330 <210> 40 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide (YP218 VL domain) <400> 40 Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Ser Glu Pro Val Gly 1 5 10 15 Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln Arg Ile Ser Ser Tyr 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45 Phe Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Ala Tyr Phe Asp Ser 85 90 95 Asn Asn Trp His Ala Phe Gly Gly Gly Thr Glu Val Val Val 100 105 110 <210> 41 <211> 339 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide (SD1) <400> 41 caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60 tcctgtgcag cctctgattt cgatttcgct gcttatgaaa tgagctgggt ccgccaggct 120 ccaggacaag gccttgagtg ggtggcaatt atatcacatg atggaatcga taaatactac 180 acagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acaccctgag agccgaggac acagccacgt attactgttt aaggcttggt 300 gctgtaggcc agggaaccct ggtcaccgtc tcctcaagt 339 <210> 42 <211> 113 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide (SD1) <400> 42 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Asp Phe Asp Phe Ala Ala Tyr 20 25 30 Glu Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Ser His Asp Gly Ile Asp Lys Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Leu Arg Leu Gly Ala Val Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Ser <210> 43 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 43 gcagtctctg gaagaaggag 20 <210> 44 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 44 tggtgacagg tggcgtccgg 20 <210> 45 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 45 cggttttcca aggtgagttc 20 <210> 46 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 46 ggtcacgtct tgctcctcgg 20 <210> 47 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 47 gacatcaatg agtgcctccg 20 <210> 48 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 48 gataataagc agatctatat 20 <210> 49 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 49 cgttttccgc cacagggcta 20 <210> 50 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 50 agggtgtcgt tttccgccac 20 <210> 51 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 51 gaggcagagc aggtagtcag 20

Claims

1. A nucleic acid molecule encoding a chimeric antigen receptor (CAR), comprising, in a 5' to 3' direction: a nucleic acid encoding the first granulocyte-macrophage colony stimulating factor receptor signal sequence (GMCSFRss); A nucleic acid encoding a glypican-3 (GPC3)-specific antibody or an antigen-binding fragment thereof, wherein the nucleic acid encoding the GPC3-specific antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain complementarity determining region 1 (CDR1), CDR2 and CDR3 nucleic acid sequence of SEQ ID NO: 25 and a light chain variable (VL) domain CDR1, CDR2 and CDR3 nucleic acid sequence of SEQ ID NO: 27, wherein the VH domain CDR1, CDR2 and CDR3 and the VL domain CDR1, CDR2 and CDR3 are determined by the Kabat numbering scheme or by the IMGT numbering scheme; 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-cleaved 2A peptide; a nucleic acid encoding a second GMCSFRss; and Nucleic acid encoding truncated human epidermal growth factor receptor (huEGFRt).

2. The nucleic acid molecule of claim 1, wherein the extracellular hinge region comprises a CD8α hinge region or a CD28 hinge region.

3. The nucleic acid molecule of claim 1, wherein the transmembrane domain comprises a CD8α transmembrane domain or a CD28 transmembrane domain.

4. The nucleic acid molecule of claim 1, wherein the intracellular co-stimulatory domain comprises a 4-1BB, CD28, ICOS, OX40, CD27 or DAP10 co-stimulatory domain.

5. The nucleic acid molecule of claim 1, wherein the intracellular signaling domain comprises a CD3ζ or FcεRIγ signaling domain.

6. The nucleic acid molecule of claim 1, wherein the extracellular hinge region comprises a CD8α hinge region, the transmembrane domain comprises a CD8α transmembrane domain, the intracellular co-stimulatory domain comprises a 4-1BB co-stimulatory domain, and the intracellular signaling domain comprises a CD3ζ signaling domain.

7. The nucleic acid molecule of claim 6, wherein the nucleic acid encoding the CD8α hinge comprises the sequence of SEQ ID NO:

3.

8. The nucleic acid molecule of claim 6, wherein the nucleic acid encoding the CD8α transmembrane domain comprises the sequence of SEQ ID NO:

5.

9. The nucleic acid molecule of claim 6, wherein the nucleic acid encoding the 4-1BB co-stimulatory domain comprises the sequence of SEQ ID NO:

7.

10. The nucleic acid molecule of claim 6, wherein the nucleic acid encoding the CD3 zeta signaling domain comprises the sequence of SEQ ID NO:

9.

11. The nucleic acid molecule of any one of claims 1-10, 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. 12 . The nucleic acid molecule of claim 1 , wherein 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.

13. The nucleic acid molecule of any one of claims 1-10, wherein the nucleic acid encoding the huEGFRt comprises the sequence of SEQ ID NO:

13.

14. The nucleic acid molecule of any one of claims 1 to 10, further comprising a human elongation factor 1 alpha (EF1 alpha) promoter sequence 5' to the nucleic acid encoding the first GMCSFRss.

15. The nucleic acid molecule of any one of claims 1-10, wherein the antigen binding fragment is a single chain variable fragment (scFv) or a single domain antibody.

16. The nucleic acid molecule of any one of claims 1-10, wherein the nucleic acid encoding the antibody binding fragment comprises the sequence of nucleotides 73-807 of SEQ ID NO:

15.

17. A vector comprising the nucleic acid molecule of any one of claims 1 to 16.

18. The vector of claim 17, wherein the vector is a viral vector.

19. The vector of claim 18, wherein the viral vector is a lentiviral vector.

20. An isolated host cell comprising the nucleic acid molecule of any one of claims 1-16 or the vector of any one of claims 17-19.

21. An isolated host cell co-expressing a chimeric antigen receptor (CAR) and a truncated human epidermal growth factor receptor (huEGFRt), wherein: The CAR comprises a glypican-3 (GPC3)-specific antibody or an antigen-binding fragment thereof, an extracellular hinge region, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain, wherein the amino acid sequence of the antigen-binding fragment comprises a heavy chain variable (VH) domain complementarity determining region 1 (CDR1), CDR2, and CDR3 sequence of SEQ ID NO: 26 and a light chain variable (VL) domain CDR1, CDR2, and CDR3 sequence of SEQ ID NO: 28, wherein the VH domain CDR1, CDR2, and CDR3 and the VL domain CDR1, CDR2, and CDR3 are determined by the Kabat numbering scheme or by the IMGT numbering scheme; 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.

22. The isolated host cell of claim 21, wherein the extracellular hinge region comprises a CD8α hinge region or a CD28 hinge region.

23. The isolated host cell of claim 21, wherein the transmembrane domain comprises a CD8α transmembrane domain or a CD28 transmembrane domain.

24. The isolated host cell of claim 21, wherein the intracellular co-stimulatory domain comprises a 4-1BB, CD28, ICOS, OX40, CD27, or DAP10 co-stimulatory domain.

25. The isolated host cell of claim 21, wherein the intracellular signaling domain comprises a CD3ζ or FcεRIγ signaling domain.

26. The isolated host cell of claim 21, wherein the extracellular hinge region comprises a CD8α hinge region, the transmembrane domain comprises a CD8α transmembrane domain, the intracellular co-stimulatory domain comprises a 4-1BB co-stimulatory domain, and the intracellular signaling domain comprises a CD3ζ signaling domain.

27. The isolated host cell of claim 26, wherein the CD8α hinge region comprises the amino acid sequence of SEQ ID NO:

4.

28. The isolated host cell of claim 26, wherein the CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO:

6.

29. The isolated host cell of claim 26, wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:

8.

30. The isolated host cell of claim 26, wherein the CD3zeta signaling domain comprises the amino acid sequence of SEQ ID NO:

10.

31. The isolated host cell of any one of claims 21-30, wherein the huEGFRt comprises the amino acid sequence of SEQ ID NO:

14.

32. The isolated host cell of any one of claims 21-30, wherein the antigen binding fragment is a single chain variable fragment (scFv) or a single domain antibody.

33. The isolated host cell of any one of claims 21-30, wherein the amino acid sequence of the antibody binding fragment comprises residues 25-269 of SEQ ID NO:

16.

34. The isolated host cell of any one of claims 20-30, wherein the cell is a T lymphocyte.

35. The isolated host cell of claim 34, wherein the T lymphocytes are autologous T lymphocytes or allogeneic T lymphocytes.

36. A composition comprising the isolated host cell of any one of claims 20-35 and a pharmaceutically acceptable carrier.

37. Use of a therapeutically effective amount of an isolated host cell comprising a nucleic acid molecule according to any one of claims 1 to 16, or a therapeutically effective amount of an isolated host cell according to any one of claims 20 to 35, in the preparation of a medicament for treating a GPC3-positive cancer in a subject, wherein the GPC3-positive cancer is hepatocellular carcinoma (HCC) or epidermoid carcinoma.

38. The use of claim 37, wherein the isolated host cell is a T lymphocyte.

39. The use of claim 38, wherein the T lymphocytes are autologous T lymphocytes or allogeneic T lymphocytes.

40. 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 a glypican-3 (GPC3)-specific antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to the tumor antigen glypican-3 (GPC3), wherein the nucleic acid encoding the antibody or antigen-binding fragment comprises a heavy chain variable (VH) domain complementarity determining region 1 (CDR1), CDR2 and CDR3 nucleic acid sequence of SEQ ID NO: 25 and a light chain variable (VL) domain CDR1, CDR2 and CDR3 nucleic acid sequence of SEQ ID NO: 27, wherein the VH domain CDR1, CDR2 and CDR3 and the VL domain CDR1, CDR2 and CDR3 are determined by the Kabat numbering scheme or by the IMGT numbering scheme; Nucleic acid encoding the CD8α hinge region; A nucleic acid encoding a CD8α transmembrane domain; A nucleic acid encoding a 4-1BB co-stimulatory domain; a nucleic acid encoding a CD3ζ signaling domain; a nucleic acid encoding a self-cleaved 2A peptide; a nucleic acid encoding a second GMCSFRss; and Nucleic acid encoding truncated human epidermal growth factor receptor (huEGFRt).

41. The nucleic acid molecule of claim 40, wherein the nucleic acid encoding the antibody binding fragment comprises the sequence of nucleotides 73-807 of SEQ ID NO:

15.

42. The nucleic acid molecule of claim 40 or claim 41 comprising the nucleotide sequence of SEQ ID NO:

15.

43. An isolated host cell co-expressing a chimeric antigen receptor (CAR) and a truncated human epidermal growth factor receptor (huEGFRt), wherein: The CAR comprises a glypican-3 (GPC3)-specific antibody or an antigen-binding fragment thereof, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain, wherein the amino acid sequence of the antigen-binding fragment comprises a heavy chain variable (VH) domain complementarity determining region 1 (CDR1), CDR2, and CDR3 sequence of SEQ ID NO: 26 and a light chain variable (VL) domain CDR1, CDR2, and CDR3 sequence of SEQ ID NO: 28, wherein the VH domain CDR1, CDR2, and CDR3 and the VL domain CDR1, CDR2, and CDR3 are determined by the Kabat numbering scheme or by the IMGT numbering scheme; and The huEGFRt includes domain III, domain IV and transmembrane domain from human EGFR, but lacks the EGF-binding domain and cytoplasmic domain.

44. The isolated host cell of claim 43, wherein the amino acid sequence of the antigen binding fragment comprises residues 25-269 of SEQ ID NO:

16.

45. The isolated host cell of claim 43 or claim 44, wherein the amino acid sequence of the CAR comprises residues 25-491 of SEQ ID NO: 16 and the amino acid sequence of the huEGFRt comprises SEQ ID NO: 14.

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