Anti-L1CAM antibody or antigen-binding fragment thereof and chimeric antigen receptor comprising same

Abstract

The present invention relates to an anti-L1CAM antibody specifically binding to L1CAM antigen or an antigen-binding fragment thereof, a chimeric antigen receptor comprising same, and uses thereof. The anti-L1CAM antibody or the antigen-binding fragment of the present invention is excellent in specificity and affinity to L1CAM and thus may be used in the treatment and diagnosis of cancers related to high expression of L1CAM and diseases related to inflammatory disorders. In particular, when the chimeric antigen receptor comprising the anti-L1CAM antibody of the present invention is expressed in effector cells such as T lymphocytes, the chimeric antigen receptor may be effectively used as immunotherapy for cancers related to L1CAM and inflammatory disorders.

Claims

1. A chimeric antigen receptor polypeptide comprising: (a) an L1CAM binding domain; (b) a transmembrane domain (TM); (c) a costimulatory domain; and (d) an intracellular signaling domain (ICD), wherein the L1CAM binding domain comprises the anti-L1CAM antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising CDRH1 (complementarity determining region 1 of heavy chain), CDRH2, and CDRH3 below and a light chain variable region (VL) comprising CDRL1, CDRL2, and CDRL3 below: i) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 1, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 8, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 15; and CDRL1 consisting of the amino acid sequence of SEQ ID NO: 32, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 37, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 43; ii) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 2, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 9, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 16; and CDRL1 consisting of the amino acid sequence of SEQ ID NO: 33, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 44; iii) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 3, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 10, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 17; and CDRL1 consisting of the amino acid sequence of SEQ ID NO: 34, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 44; iv) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 4, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 11, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 18; and CDRL1 consisting of the amino acid sequence of SEQ ID NO: 34, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 39, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 45; v) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 3, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 9, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 19; and CDRL1 consisting of the amino acid sequence of SEQ ID NO: 35, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 39, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 46; vi) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 12, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 20; and CDRL1 consisting of the amino acid sequence of SEQ ID NO: 33, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 40, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 47; vii) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 6, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 13, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 21; and CDRL1 consisting of the amino acid sequence of SEQ ID NO: 34, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 41, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 44; viii) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 7, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 9, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 22; and CDRL1 consisting of the amino acid sequence of SEQ ID NO: 36, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 42, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 43; or ix) CDRH1 consisting of the amino acid sequence of SEQ ID NO: 3, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 14, and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 23; and CDRL1 consisting of the amino acid sequence of SEQ ID NO: 34, CDRL2 consisting of the amino acid sequence of SEQ ID NO: 37, and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 44.

2. The chimeric antigen receptor polypeptide of claim 1, wherein the transmembrane domain includes a transmembrane domain of a protein selected from the group consisting of a T-cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

3. The chimeric antigen receptor polypeptide of claim 1, wherein the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of TNF receptor proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAMs), B and T lymphocyte attenuators (BTLAs), Toll-like ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and CD83.

4. The chimeric antigen receptor polypeptide of claim 1, wherein the intracellular signaling domain includes a functional signaling domain of 4-1BB, CD28, OX40, or CD3 zeta, or a combination thereof.

5. The chimeric antigen receptor polypeptide of claim 1, wherein the anti-L1CAM antibody or antigen-binding fragment thereof comprising VH and VL below: i) VH consisting of the amino acid sequence of SEQ ID NO: 52, and VL consisting of the amino acid sequence of SEQ ID NO: 56; ii) VH consisting of the amino acid sequence of SEQ ID NO: 53, and VL consisting of the amino acid sequence of SEQ ID NO: 57; iii) VH consisting of the amino acid sequence of SEQ ID NO: 54, and VL consisting of the amino acid sequence of SEQ ID NO: 58; or iv) VH consisting of the amino acid sequence of SEQ ID NO: 55, and VL consisting of the amino acid sequence of SEQ ID NO: 59.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 schematically shows a phage display library panning procedure.

(2) FIG. 2 shows graphs depicting the degree of enrichment of phages to the antigen mL1CAM according to the phage panning round (top: phage output titer, bottom: elution titer ratio).

(3) FIGS. 3A, 3B and 3C show the results of performing monoclonal phage ELISA to select phage clones specifically binding to the antigen mL1CAM for each phage panning round

(4) FIG. 4 shows the selection frequency of nine types of scFv clones selected in the present disclosure.

(5) FIG. 5 shows the results of performing monoclonal clone phage ELISA for hL1CAM on nine types of unique anti-mL1CAM scFv clones cross-reactive to mouse L1CAM, which were selected in the present disclosure, in order to discover antibodies cross-reactive to human L1CAM and mouse L1CAM.

(6) FIG. 6 shows SDS-PAGE analysis results of purified anti-mL1CAM scFv clones.

(7) FIGS. 7A, 7B and 7C show affinity to mL1CAM and hL1CAM antigens in four types of anti-L1CAM scFv antibodies of the present disclosure according to the soluble ELISA results in FIG. 5.

(8) FIGS. 7D and 7E show affinity to mL1CAM and hL1CAM antigens in four types of anti-L1CAM scFv antibodies of the present disclosure according to the octet system results.

(9) FIG. 8 shows a vector map of the pMT-CART plasmid used to manufacture a CAR-construct comprising the anti-L1CAM scFv selected in the present disclosure.

(10) FIG. 9 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising anti-L1CAM scFv of the present disclosure.

(11) FIGS. 10A and 10B show structures of CAR-constructs comprising anti-L1CAM scFv (L1-CAR-001, L1-CAR-002, L1-CAR-003, and L1-CAR-004) constructed in the example of the present disclosure.

(12) FIGS. 11A and 11B show retroviral vectors into which four types of CAR-constructs comprising anti-L1CAM scFv (L1-CAR-001, L1-CAR-002, L1-CAR-003, and L1-CAR-004) of the present disclosure were introduced.

(13) FIG. 12 shows expression rates of L1CAM in SKOV3 cells and 293T cells.

(14) FIGS. 13A and 13B show anticancer activity of the anti-L1CAM-CAR-expressing T cells of the present disclosure on SKOV3 cells (high expression of L1CAM, FIG. 13A) and 293T cells (low expression of L1CAM, FIG. 13B).

(15) FIG. 14 shows in-vivo anticancer activity of the anti-L1CAM CAR (anti-L1-CAR)-expressing T cells of the present disclosure.

(16) FIG. 15 shows a vector map of the pMT-CAR-002 plasmid used to manufacture a CAR-construct comprising the anti-L1CAM scFv selected in the present disclosure.

(17) FIG. 16 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising the anti-L1CAM scFv of the present disclosure.

(18) FIG. 17 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-002) constructed in the example of the present disclosure.

(19) FIG. 18 shows a vector map of the pMT-CART-003 plasmid used to manufacture a CAR-construct comprising the anti-L1CAM scFv selected in the present disclosure.

(20) FIG. 19 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising the anti-L1CAM scFv of the present disclosure.

(21) FIG. 20 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-003) constructed in the example of the present disclosure.

(22) FIG. 21 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising the anti-L1CAM scFv of the present disclosure.

(23) FIG. 22 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-004) constructed in the example of the present disclosure.

(24) FIGS. 23A, 23B, 23C to 23D show retroviral vectors into which four types of CAR-constructs comprising the anti-L1CAM scFv (L1-H8-CAR-001, L1-H8-CAR-002, L1-H8-CAR-003, and L1-H8-CAR-004) of the present disclosure were introduced.

(25) FIGS. 24A, 24B, 24C, 24D, 24E, 24F to 24G show the expression rates of L1CAM in SKOV3 cells, Hela cells, SH-SY5Y cells, and 293T cells.

(26) FIG. 25 shows anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on SKOV3 cells (high expression of L1CAM).

(27) FIG. 26 shows anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on 293T cells (low expression of L1CAM).

(28) FIGS. 27A and 27B show anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on SH-SY5Y cells (high expression of L1CAM).

(29) FIGS. 28A and 28B show anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on HeLa cells (high expression of L1CAM).

(30) FIG. 29 shows in-vivo anticancer activity of anti-L1CAM-CAR (anti-L1-CAR)-expressing T cells of the present disclosure.

(31) FIG. 30 shows a vector map of the pMT-CART-004 plasmid used to manufacture a CAR-construct comprising the selected anti-L1CAM scFv.

(32) FIG. 31 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising anti-L1CAM scFv.

(33) FIG. 32 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-001-28BB) constructed in the example of the present disclosure.

(34) FIG. 33 shows a vector map of the pBHA-ICOS TM+ICD plasmid used to manufacture a CAR-construct comprising the selected anti-L1CAM scFv.

(35) FIG. 34 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising anti-L1CAM scFv.

(36) FIG. 35 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-001-28ICOS) constructed in the example of the present disclosure.

(37) FIG. 36 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising anti-L1CAM scFv.

(38) FIG. 37 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-001-28) constructed in the example of the present disclosure.

(39) FIG. 38 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising anti-L1CAM scFv.

(40) FIG. 39 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-001-OX) constructed in the example of the present disclosure.

(41) FIG. 40 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising anti-L1CAM scFv.

(42) FIG. 41 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-001-BB) constructed in the example of the present disclosure.

(43) FIG. 42 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising anti-L1CAM scFv.

(44) FIG. 43 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-001-ICOS) constructed in the example of the present disclosure.

(45) FIGS. 44A, 44B, 44C, 44D, 44E to 44F show retroviral vectors into which six types of CAR-constructs comprising the anti-L1 CAM scFv (L1-H8-CAR-001-28BB, L1-H8-CAR-001-28ICOS, L1-H8-CAR-001-28, L1-H8-CAR-001-OX, L1-H8-CAR-001-BB, and L1-H8-CAR-001-ICOS) of the present disclosure were introduced.

(46) FIGS. 45A, 45B, 45C, 45D, 45E, 45F, 45G, 45H to 45I show the expression rates of L1CAM in SKOV3 cells, SH-SY5Y cells, HeLa cells, and 293T cells.

(47) FIG. 46 shows anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on SKOV3 cells (high expression of L1CAM).

(48) FIG. 47 shows anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on 293T cells (low expression of L1CAM).

(49) FIGS. 48A, 48B to 48C show anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on SH-SY5Y cells (high expression of L1CAM).

(50) FIGS. 49A, 49B to 49C show anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on HeLa cells (high expression of L1CAM).

(51) FIG. 50 shows in-vivo anticancer activity of anti-L1CAM-CAR (anti-L1-CAR)-expressing T cells of the present disclosure.

(52) FIG. 51 shows a vector map of the pBHA-3E8LS-H8Rev plasmid used to manufacture a CAR-construct comprising the anti-L1CAM scFv selected in the present disclosure.

(53) FIG. 52 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising the anti-L1CAM scFv of the present disclosure.

(54) FIG. 53 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-005) constructed in the example of the present disclosure.

(55) FIG. 54 shows a vector map of the pMT-CART-005 plasmid used to manufacture a CAR-construct comprising the anti-L1CAM scFv selected in the present disclosure.

(56) FIG. 55 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising the anti-L1CAM scFv of the present disclosure.

(57) FIG. 56 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-006) constructed in the example of the present disclosure.

(58) FIG. 57 shows a vector map of the pMT-CART-006 plasmid used to manufacture a CAR-construct comprising the anti-L1CAM scFv selected in the present disclosure.

(59) FIG. 58 is a schematic diagram showing a series of PCR amplification procedures in order to manufacture a CAR-construct comprising the anti-L1CAM scFv of the present disclosure.

(60) FIG. 59 shows a structure of the CAR-construct comprising anti-L1CAM scFv (L1-H8-CAR-007) constructed in the example of the present disclosure.

(61) FIGS. 60A, 60B to 60C show retroviral vectors into which three types of CAR-constructs comprising anti-L1CAM scFv (L1-H8-CAR-005, L1-H8-CAR-006, and L1-H8-CAR-007) of the present disclosure were introduced.

(62) FIGS. 61A, 61B, 61C, 61D, 61E to 61F show the expression rates of L1CAM in SKOV3 cells, SH-SY5Y cells, HeLa cells, and 293T cells.

(63) FIG. 62 shows anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on SKOV3 cells (high expression of L1CAM).

(64) FIG. 63 shows anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on SH-SY5Y cells (high expression of L1CAM).

(65) FIG. 64 shows anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on HeLa cells (high expression of L1CAM).

(66) FIG. 65 shows anticancer activity of anti-L1CAM-CAR-expressing T cells of the present disclosure on 293T cells (low expression of L1CAM).

DETAILED DESCRIPTION

(67) Hereinafter, the present disclosure will be described in more detail with reference to examples. These examples are provided only for the purpose of illustrating the present disclosure in more detail, and therefore, according to the purpose of the present disclosure, it would be apparent to a person skilled in the art that these examples are not construed to limit the scope of the present disclosure.

EXAMPLES

(68) Throughout the present specification, the % used to express the concentration of a specific material, unless otherwise particularly stated, refers to (wt/wt) % for solid/solid, (wt/vol) % for solid/liquid, and (vol/vol) % for liquid/liquid.

Example 1: Selection of scFv Antibodies for L1CAM Antigen

1.1. Human Synthetic scFv Phage Display Antibody Library Panning

(69) To select anti-mL1CAM scFv antibodies binding to mouse L1CAM (mL1CAM) antigen, phage panning for the antigen mL1CAM protein was performed up to 4 rounds by using the human synthetic scFv phage display library (KscFv-1, KBIO HEALTH) (FIG. 1). The antigen mL1CAM protein (R&D system, Cat No. 5674-NC) was added to immunotubes, incubated at 4 C. overnight, and then blocked by incubation with PBS (MPBS) comprising 5% skim milk at room temperature for 1 hour. MPBS was added to KscFv-1, followed by incubation at room temperature for 1 hour, thereby preparing blocked phages. The blocked phages were added to immunotubes coated with the antigen mL1CAM protein, followed by incubation at 37 C. for 90 minutes. After the phages were washed with PBS comprising 0.05% Tween20, 100 mM trimethylamine was added to harvest (elution) phages adhering to the immunotubes. The harvested phages were neutralized by addition of 1 M Tris-HCl, and then TG1 E. coli (Lucigen, Cat No. 60502-2) cultured in the mid-log phase (OD.sub.600=0.5-1.0) was added, followed by incubation at 37 C. for 1 hour. After incubation, cell pellets were collected, and inoculated on TB medium plates comprising ampicillin and 2% glucose. The cultured colonies were collected, and then stored at 80 C. after the addition of 50% glycerol. Since the antigen mL1CAM protein (R&D system, Cat No. 5674-NC) was fused with the Fc domain, Fc control panning for Fc depletion was also performed in the panning step, and the enrichment of phages was monitored through the elution titer ratio by comparing respective output titer values at each round. The elution titer ratio is the value obtained by dividing the phage output titer value (antigen mL1CAM) by the Fc control output titer value (no antigen mL1CAM). As shown in FIG. 2, mL1CAM-Fc showed a large difference in output titer from Fc control from the 2nd round of phage panning. The enrichment was initiated from the 2nd round of phage panning, and for the antigen mL1CAM, mL1CAM-Fc showed a difference by about 23.9 times compared to the control in the second round of phage panning, a difference by 66.1 times in the third round of phage panning, and a difference by 141.4 times in the fourth round of phage panning.

1.2 Phage ELISA Screening

(70) To select clones specifically adhering to the antigen mL1CAM protein among the phages obtained by phage panning, monoclonal phage ELISA was performed on 95 clones obtained in the 2nd, 3rd, and 4th rounds of panning.

(71) Specifically, the antigen mL1CAM protein was added to 96-well plates, incubated at 4 C. overnight, and then blocked with 2% MPBS at 37 C. for 2 hours. Since the antigen mL1CAM protein was fused with the Fc domain, Fc as an Fc control was also added to the 96-well plates, incubated at 4 C. overnight, and blocked with 2% MPBS at 37 C. for 2 hours. Then, the phages (up to 10.sup.11 cfu) were added to the 96-well plates. After incubation at room temperature for 90 minutes, HRP-anti-M13 (Sino Biological, Cat No. 11973-MM05) was diluted in PBS to 1:5000, and added to 96-well plates. After incubation at room temperature for 1 hour, TMB substrate (Sigma, Cat No. T0440) and 2N H2504 (Merck, Cat No. 100731) were sequentially added, and the absorbance (OD) at 450 nm was measured. As a result, when the absorbance (A450 nm) cut-off for the antigen mL1CAM was set to at least 0.4 for selection, one clone in the 2nd round, a total of 26 clones in the 3rd round, and a total of 9 clones in the 4th round specifically bound (positive) to the antigen mL1CAM in ELISA (FIG. 3).

1.3 Sequencing of Unique scFv Clones for mL1CAM Antigen of the Present Disclosure

(72) 36 types of scFv clones for the antigen mL1CAM, which showed a positive response in the monoclonal phage ELISA, were sequenced, and the sequences were grouped by alignment through Kabat numbering, and as a result, a total of 9 types of unique anti-mL1CAM scFv clones were obtained (Tables 1 and 2). Considering the selection frequency of the scFv clones obtained for the antigen mL1CAM, the 3rd round clones (mL1CAM-3R-H8, mL1CAM-3R-E1, and mL1CAM-3R-C9) were selected as major clones by accounting for 33%, 26%, and 16%, respectively, and the remaining clones were selected as minor clones by accounting for a range of 3-10% (FIG. 4).

(73) TABLE-US-00008 TABLE1 Aminoacidsequencesofheavychainvariableregionsandlinkerof9types ofanti-mL1CAMscFvclonesselectedinpresentdisclosure(Kabat) ID FR1_VH CDR1_VH FR2_VH CDR2_VH FR3_VH CDR3_VH FR4_VH VH_Vk_linker 1 EVQLVESGGG DYAMN WVRQAPG AISSTGSTIYY RFTISRDNSKN QSTYFYS WGQGTL GGGGSGGG LVQPGGSLRL KGLEWVS ADSVKG TLYLQMNSLRA YFDV VTVSS GSGGGGS SCAASGFTFS EDTAVYYCAK 2 EVQLVESGGG SYAMH WVRQAPG AISSSGGSTY RFTISRDNSKN DEGSGLG WGQGTL GGGGSGGG LVQPGGSLRL KGLEWVS YADSVKG TLYLQMNSLRA AFDI VTVSS GSGGGGS SCAASGFTFS EDTAVYYCAK 3 EVQLVESGGG SYAMS WVRQAPG AISSSGSSTYY RFTISRDNSKN DESTGLG WGQGTL GGGGSGGG LVQPGGSLRL KGLEWVS ADSVKG TLYLQMNSLRA AFDY VTVSS GSGGGGS SCAASGFTFS EDTAVYYCAK 4 EVQLVESGGG SYAMH WVRQAPG AISSSGSSKYY RFTISRDNSKN DESYGW WGQGTL GGGGSGGG LVQPGGSLRL KGLEWVS ADSVKG TLYLQINSLRA LYAFDL VTVSS GSGGGGS SCAASGFTFS EDTAVYYCAK 5 EVQLVESGGG SYAMS WVRQAPG AISSSGGSTY RFTISRDNSKN VLELWEG WGQGTL GGGGSGGG LVQPGGSLRL KGLEWVS YADSVKG TLYLQMNSLRA LDY VTVSS GSGGGGS SCAASGFTFS EDTAVYYCAK 6 EVQLVESGGG NYAMH WVRQAPG AIYQSGGDTY RFTISRDNSKN VRGTYYG WGQGTL GGGGSGGG LLQPGGSLRL KGLEWVS YADSVKG TLYLQMNSLRA SYLDY VTVSS GSGGGGS SCAASGFTFS EDTAVYYCAK 7 EVQLVESGGG SYAMN WVRQAPG RISSSGTTFYA RFTISRDNSKN VEEGRYV WGQGTL GGGGSGGG LVQPGGSLRL KGLEWVS DSVKG TLYLQMNSLRA QAFDY VTVSS GSGGGGS SCAASGFTFS EDTAVYYCAK 8 EVQLVESGGG DYAMH WVRQAPG AISSSGGSTY RFTISRDNSKN HGGTWW WGQGTL GGGGSGGG LVQPGGSLRL KGLEWVS YADSVKG TLYLQMNSLRA GRAFDY VTVSS GSGGGGS SCAASGFTFS EDTAVYYCAK 9 EVQLVESGGG SYAMS WVRQAPG AISSSGGTKY RFTISRDNSKN HGSYAFV WGQGTL GGGGSGGG LAQPGGSLRL KGLEWVS YADSVKG TLYLQMNSLRA FDY VTVSS GSGGGGS SCAASGFTFS EDTAVYYCAK 1: mL1CAM-3R-H8_: mL1CAM-3R-E6_: mL1CAM-3R-G10_: mL1CAM-3R-B5_: mL1CAM-3R-H3_: mL1CAM-3R-G5_: mL1CAM-3R-C12_: mL1CAM-3R-G8_: mL1CAM-3R-G4_: mL1CAM-4R-D5_ 2: mL1CAM-3R-C9_: mL1CAM-3R-B8_: mL1CAM-3R-B4_: mL1CAM-3R-B6_: mL1CAM-3R-D6_ 3: mL1CAM-3R-E1_: mL1CAM-3R-C6_: mL1CAM-3R-C11_: mL1CAM-3R-H6_: mL1CAM-3R-F3_: mL1CAM-4R-E11_: mL1CAM-4R-F4_: mL1CAM-4R-H6_ 4: mL1CAM-3R-F6_: mL1CAM-3R-G2_: mL1CAM-3R-E7_ 5: mL1CAM-3R-F1_ 6: mL1CAM-3R-G6_ 7: mL1CAM-3R-A2_ 8: mL1CAM-3R-E9_ 9: mL1CAM-2R-F8_

(74) The clone IDs expressed in bold mean the clone IDs representing respective groups.

(75) TABLE-US-00009 TABLE2 Aminoacidsequencesoflightchainvariableregionsof9typesofanti- mL1CAMscFvclonesselectedinpresentdisclosure(Kabat) ID FR1_Vk CDR1_Vk FR2_Vk CDR2_Vk FR3_Vk CDR3_Vk FR4_Vk Frequency 1 DIQMTQSPSSLSA RASQSISR WYQQKPGK AASSL GVPSRFSGSG QQSYS FGQGT 10 SVGDRVTITC DLN APKLLIY QS SGTDFTLTISSL TPYT KVEIK QPEDFATYYCL 2 DIQMTQSPSSLSA RASQSISR WYQQKPGK AASNL GVPSRFSGSG QQSYS FGQGT 5 SVGDRVTITC YLN APKLLIY QS SGTDFTLTISSL FPWT KVEIK QPEDFATYYC 3 DIQMTQSPSSLSA RASQSISN WYQQKPGK AASNL GVPSRFSGSG QQSYS FGQGT 8 SVGDRVTITC YLN APKLLIY QS SGTDFTLTISSL FPWT KVEIK QPEDFATYYC 4 DIQMTQSPSSLSA RASQSISN WYQQKPGK AASRL GVPSRFSGSG QQSYS FGQGT 3 SVGDRVTITC YLN APKLLIY QS SGTDFTLTISSL FPLT KVEIK QPEDFATYYC 5 DIQMTQSPSSLSA RASQSISS WYQQKPGK AASRL GVPSRFSGSG QQSES FGQGT 1 SVGDRVTITC YLN APKLLIY QS SGTDFTLTISSL FPYT KVEIK QPEDFATYYC 6 DIQMTQSPSSLSA RASQSISR WYQQKPGK AASTL GVPSRFSGSG QQSYS FGQGT 1 SVGDRVTITC YLN APKLLIY QS SGTDFTLTISSL YPFT KVEIK QPEDFATYYC 7 DIQMTQSPSSLSA RASQSISN WYQQKPGK ATSRL GVPSRFSGSG QQSYS FGQGT 1 SVGDRVTITC YLN APKLLIY QS SGTDFTLTISSL FPWT KVEIK QPEDFATYYC 8 DIQMTQSPSSLSA RASQSIGS WYQQKPGK ATSSL GVPSRFSGSG QQSYS FGQGT 1 SVGDRVTITC YLN APKLLIY QS SGTDFTLTISSL TPYT KVEIK QPEDFATYYC 9 DIQMTQSPSSLSA RASQSISN WYQQKPGK AASSL GVPSRFSGSG QQSYS FGQGT 1 SVGDRVTITC YLN APKLLIY QS SGTDFTLTISSL FPWT KVEIK QPEDFATYYC 1: mL1CAM-3R-H8_: mL1CAM-3R-E6_: mL1CAM-3R-G10_: mL1CAM-3R-B5_: mL1CAM-3R-H3_: mL1CAM-3R-G5_: mL1CAM-3R-C12_: mL1CAM-3R-G8_: mL1CAM-3R-G4_: mL1CAM-4R-D5_ 2: mL1CAM-3R-C9_: mL1CAM-3R-B8_: mL1CAM-3R-B4_: mL1CAM-3R-B6_: mL1CAM-3R-D6_ 3: mL1CAM-3R-E1_: mL1CAM-3R-C6_: mL1CAM-3R-C11_: mL1CAM-3R-H6_: mL1CAM-3R-F3_: mL1CAM-4R-E11_: mL1CAM-4R-F4_: mL1CAM-4R-H6_ 4: mL1CAM-3R-F6_: mL1CAM-3R-G2_: mL1CAM-3R-E7_ 5: mL1CAM-3R-F1_ 6: mL1CAM-3R-G6_ 7: mL1CAM-3R-A2_ 8: mL1CAM-3R-E9_ 9: mL1CAM-2R-F8_

(76) The clone IDs expressed in bold mean the clone IDs representing respective groups.

1.4. Discovery of scFv Antibodies Cross-Reactive to Human L1CAM (hL1CAM) and Mouse L1CAM (mL1CAM)

(77) To discover antibodies cross-reactive to human L1CAM (hL1CAM, R&D system, Cat No. 777-NC) and mouse L1CAM, monoclonal phage ELISA was performed on a total of 9 types of unique anti-mL1CAM scFv clones for the antigen hL1CAM. As a result, when the absorbance (A450 nm) cut-off for the antigen hL1CAM was set to at least 0.4 for selection, a total of four clones (mL1CAM-3R-H8, mL1CAM-3R-C9, mL1CAM-3R-E1, and mL1CAM-3R-E9) were cross-reactive to the antigen hL1CAM (FIG. 5 and Tables 3 and 4).

(78) TABLE-US-00010 TABLE3 Aminoacidsequencesofheavychainvariableregionsandlinkeroffour typesofanti-L1CAMscFvclonesfinallyselectedinthepresentdisclosure(Kabat) ID FR1_VH CDR1_VH FR2_VH CDR2_VH FR3_VH CDR3_VH FR4_VH VH_Vk_linker 1 EVQLVESGGGLV DYAMN WVRQAPGK AISSTGSTIYYA RFTISRDNSKNT QSTYFYSY WGQGTL GGGGS QPGGSLRLSCAA GLEWVS DSVKG LYLQMNSLRAE FDV VTVSS GGGGS SGFTFS DTAVYYCAK GGGGS 2 EVQLVESGGGLV SYAMH WVRQAPGK AISSSGGSTYY RFTISRDNSKNT DEGSGLG WGQGTL GGGGS QPGGSLRLSCAA GLEWVS ADSVKG LYLQMNSLRAE AFDI VTVSS GGGGS SGFTFS DTAVYYCAK GGGGS 3 EVQLVESGGGLV SYAMS WVRQAPGK AISSSGSSTYY RFTISRDNSKNT DESTGLG WGQGTL GGGGS QPGGSLRLSCAA GLEWVS ADSVKG LYLQMNSLRAE AFDY VTVSS GGGGS SGFTFS DTAVYYCAK GGGGS 8 EVQLVESGGGLV DYAMH WVRQAPGK AISSSGGSTYY RFTISRDNSKNT HGGTWW WGQGTL GGGGS QPGGSLRLSCAA GLEWVS ADSVKG LYLQMNSLRAE GRAFDY VTVSS GGGGS SGFTFS DTAVYYCAK GGGGS 1: mL1CAM-3R-H8_: mL1CAM-3R-E6_: mL1CAM-3R-G10_: mL1CAM-3R-B5_: mL1CAM-3R-H3_: mL1CAM-3R-G5_: mL1CAM-3R-C12_: mL1CAM-3R-G8_: mL1CAM-3R-G4_: mL1CAM-4R-D5_ 2: mL1CAM-3R-C9_: mL1CAM-3R-B8_: mL1CAM-3R-B4_: mL1CAM-3R-B6_: mL1CAM-3R-D6_ 3: mL1CAM-3R-E1_: mL1CAM-3R-C6_: mL1CAM-3R-C11_: mL1CAM-3R-H6_: mL1CAM-3R-F3_: mL1CAM-4R-E11_: mL1CAM-4R-F4_: mL1CAM-4R-H6_ 8: mL1CAM-3R-E9_

(79) The clone IDs expressed in bold mean the clone IDs representing respective groups.

(80) TABLE-US-00011 TABLE4 Aminoacidsequencesoflightchainvariableregionsoffourtypesofanti- L1CAMscFvclonesfinallyselectedinthepresentdisclosure(Kabat) ID FR1_Vk CDR1_Vk FR2_Vk CDR2_Vk FR3_Vk CDR3_Vk FR4_Vk Frequency 1 DIQMTQSPSSLSA RASQSISR WYQQKPGKA AASSL GVPSRFSGSGS QQSYST FGQGTK 10 SVGDRVTITC DLN PKLLIY QS GTDFTLTISSLQP PYT VEIK EDFATYYC 2 DIQMTQSPSSLSA RASQSISR WYQQKPGKA AASNL GVPSRFSGSGS QQSYSF FGQGTK 5 SVGDRVTITC YLN PKLLIY QS GTDFTLTISSLQP PWT VEIK EDFATYYC 3 DIQMTQSPSSLSA RASQSISN WYQQKPGKA AASNL GVPSRFSGSGS QQSYSF FGQGTK 8 SVGDRVTITC YLN PKLLIY QS GTDFTLTISSLQP PWT VEIK EDFATYYC 8 DIQMTQSPSSLSA RASQSIGS WYQQKPGKA ATSSL GVPSRFSGSGS QQSYST FGQGTK 1 SVGDRVTITC YLN PKLLIY QS GTDFTLTISSLQP PYT VEIK EDFATYYC 1: mL1CAM-3R-H8_: mL1CAM-3R-E6_: mL1CAM-3R-G10_: mL1CAM-3R-B5_: mL1CAM-3R-H3_: mL1CAM-3R-G5_: mL1CAM-3R-C12_: mL1CAM-3R-G8_: mL1CAM-3R-G4_: mL1CAM-4R-D5_ 2: mL1CAM-3R-C9_: mL1CAM-3R-B8_: mL1CAM-3R-B4_: mL1CAM-3R-B6_: mL1CAM-3R-D6_ 3: mL1CAM-3R-E1_: mL1CAM-3R-C6_: mL1CAM-3R-C11_: mL1CAM-3R-H6_: mL1CAM-3R-F3_: mL1CAM-4R-E11_: mL1CAM-4R-F4_: mL1CAM-4R-H6_ 8: mL1CAM-3R-E9_

(81) The clone IDs expressed in bold mean the clone IDs representing respective groups.

1.5. E. coli Expression and Purification of Four Types of Unique scFv Clones Cross-Reactive to Human L1CAM and Mouse L1CAM

(82) A total of four types of unique anti-mL1CAM scFv clones obtained through cross-reactivity evaluation and monoclonal phage ELISA were cloned into E. coli expression vectors (pKFAB, KBIO HEALTH), induced to be expressed through 0.5 M IPTG in 200 mL of TB media, and incubated at 30 C. overnight. The soluble proteins were obtained through periplasmic protein extraction, and then purified through affinity chromatography using a strep tag II column. The expression of each purified clone was confirmed through SDS-PAGE analysis (FIG. 6).

1.6. Affinity Analysis

(83) The affinity of each clone binding to the L1CAM protein was compared and analyzed through soluble ELISA using the anti-L1CAM scFv (4 types) antibody proteins that were selected and purified.

(84) Specifically, the antigen mL1CAM protein or antigen hL1CAM protein was added to 96-well plates, incubated at 4 C. overnight, and then blocked with 2% MPBS at room temperature for 1 hour. Then, the purified anti-L1CAM scFv antibody protein was added. After incubation at room temperature for 90 minutes, HRP-anti-StrepMAB (IBA, Cat No. 2-1509-001) was diluted in 2% MPBS to 1:5000 and added. After incubation at room temperature for 1 hour, TMB substrate (Sigma, Cat No. T0440) and 2N H.sub.2SO.sub.4 (Merck, Cat No. 100731) were sequentially added, and the absorbance (OD) at 450 nm was measured. As a result, each clone bound to the antigen mL1CAM with an affinity ranging from 5 nM (mL1CAM-3R-C9) to 50 nM (mL1CAM-3R-E1). As a result of comparing and analyzing affinity for the hL1CAM protein, each clone bound to the antigen hL1CAM with an affinity ranging from 2 nM (mL1CAM-3R-H8) to 20.87 M (mL1CAM-3R-E9) (FIGS. 7A to 7C). When the binding affinity of four types of cross-reactive clones was synthetically compared for each L1CAM, the binding affinity was high in the order of H8>E1>C9>E9.

(85) The clone 3R-H8 showing the highest binding affinity among four types of anti-L1CAM scFv was subjected to a conversion procedure, thereby securing the whole IgG1 antibody. Through the Octet system (Forte Bio, Model No. QK384) using the purified whole IgG1 antibody, the antigen-antibody affinity was analyzed for the antigen hL1CAM (Sino biological, Cat No. 10140-H08H) protein or mL1CAM (R&D, Cat No. 5674-NC) protein. The result verified that the corresponding antibody had a binding affinity of 4.14E-09 KD(M) with the antigen hL1CAM protein and a binding affinity of 2.05E-08 KD(M) with the antigen mL1CAM (FIGS. 7D to 7E).

Example 2: Fabrication of Anti-L1CAM-CAR Gene-Expressing T Cells and Verification of Activity Thereof

2.1. Obtainment of Anti-L1CAM-CAR Gene

2.1.1. Obtainment of Anti-mL1CAM scFv Antibody Gene

(86) The nucleotide sequences of the anti-L1CAM scFv clones were obtained through sequencing using Lac promoter-forward primers from the phagemids comprising the anti-L1CAM scFv clones selected in the present disclosure. (Table 5). Forward and reverse primers were prepared based on the analyzed nucleotide sequences, and PCR products were obtained by amplifying the phagemids as templates by PCR method. The obtained PCR products of the anti-L1CAM scFv antibodies as templates were amplified by PCR using the primer of SEQ ID NO: 68 (Table 6) and the primer of SEQ ID NO: 69 (Table 6). The primer binding to the 5 site of the anti-L1CAM scFv antibody variable heavy chain (VH) has the 12-nucleotide sequence of the leader sequence (LS) of the 3E8 antibody, which is a mouse monoclonal IgG, and the primer binding to the 3 site of the anti-L1CAM scFv antibody variable light chain (VL) has the 12-nucleotide sequence of the IgD hinge. Therefore, the PCR product amplified by the primers has the hinge nucleotide sequence of 3E8 LS-scFv-IgD. The amplified PCR product was used in the next PCR amplification process.

(87) TABLE-US-00012 TABLE5 Nucleotidesequencesencodingfourtypesofanti-L1CAMscFvclones finallyselectedinpresentdisclosure(Kabat) ID Nucleotidesequence 1 GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGC TGCGCCGCCTCGGGTTTTACTTTCTCTGATTATGCAATGAATTGGGTTCGTCAGGCGCCGGGCAA GGGTCTCGAATGGGTTTCAGCAATCTCTTCTACTGGTTCTACTATCTACTATGCCGATTCAGTGAA GGGTCGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCG TGCCGAAGACACGGCCGTCTATTATTGCGCCAAACAGTCTACTTACTTTTACTCTTACTTTGATGTT TGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGCGGTTCAGGCGGAGGTGGATCC GGCGGTGGCGGATCGGACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGC GATCGTGTGACAATTACTTGTCGCGCTAGCCAGTCTATCTCTCGTGATCTGAACTGGTATCAGCAG AAACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCATCCTCTCTGCAGTCTGGTGTACCGT CCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAA GATTTTGCCACCTATTATTGTCAGCAATCTTACTCTACTCCGTACACGTTCGGGCAGGGAACTAAA GTGGAAATTAAA 2 GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGC TGCGCCGCCTCGGGTTTTACTTTCTCTTCTTATGCAATGCACTGGGTTCGTCAGGCGCCGGGCA AGGGTCTCGAATGGGTTTCAGCAATCTCTTCTTCTGGTGGTTCTACTTACTATGCCGATTCAGTGA AGGGTCGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGC GTGCCGAAGACACGGCCGTCTATTATTGCGCCAAAGATGAAGGTTCTGGTCTGGGTGCATTTGAT ATCTGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGCGGTTCAGGCGGAGGTGGA TCCGGCGGTGGCGGATCGGACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTG GGCGATCGTGTGACAATTACTTGTCGCGCTAGCCAGTCTATCTCTCGTTACCTGAACTGGTATCA GCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCATCCAATCTGCAGTCTGGTGTA CCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGC CTGAAGATTTTGCCACCTATTATTGTCAGCAATCTTACTCTTTTCCGTGGACGTTCGGGCAGGGAA CTAAAGTGGAAATTAAA 3 GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGC TGCGCCGCCTCGGGTTTTACTTTCTCTTCTTATGCAATGTCTTGGGTTCGTCAGGCGCCGGGCAA GGGTCTCGAATGGGTTTCAGCAATCTCTTCTTCTGGTTCTTCTACTTACTATGCCGATTCAGTGAA GGGTCGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCG TGCCGAAGACACGGCCGTCTATTATTGCGCCAAAGATGAATCTACTGGTCTGGGTGCATTTGATTA CTGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGCGGTTCAGGCGGAGGTGGATC CGGCGGTGGCGGATCGGACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGG CGATCGTGTGACAATTACTTGTCGCGCTAGCCAGTCTATCTCTAATTACCTGAACTGGTATCAGCA GAAACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCATCCAATCTGCAGTCTGGTGTACCG TCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGA AGATTTTGCCACCTATTATTGTCAGCAATCTTACTCTTTTCCGTGGACGTTCGGGCAGGGAACTAA AGTGGAAATTAAA 8 GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGC TGCGCCGCCTCGGGTTTTACTTTCTCTGATTATGCAATGCACTGGGTTCGTCAGGCGCCGGGCA AGGGTCTCGAATGGGTTTCAGCAATCTCTTCTTCTGGTGGTTCTACTTACTATGCCGATTCAGTGA AGGGTCGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGC GTGCCGAAGACACGGCCGTCTATTATTGCGCCAAACATGGTGGTACTTGGTGGGGTCGTGCATT CGATTACTGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGTGGAGGCGGTTCAGGCGGAGG TGGATCCGGCGGTGGCGGATCGGACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGC GTGGGCGATCGTGTGACAATTACTTGTCGCGCTAGCCAGTCTATCGGTTCTTACCTGAACTGGTA TCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAACTTCCTCTCTGCAGTCTGGT GTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCA GCCTGAAGATTTTGCCACCTATTATTGTCAGCAATCTTACTCTACTCCGTACACGTTCGGGCAGGG AACTAAAGTGGAAATTAAA

(88) TABLE-US-00013 TABLE6 SEQIDNO Primername Nucleotidesequence 68 3E8VH_LS+L1ScFv(F) GGTGTCCACTCCGAAGTACAGTTGGTC 69 L1ScFv+hIgDhinge(R) ACCTGGCCAGCGTTTAATTTCCACTTT 70 Mlu1+3E8VH(F) ACGCGTATGGAATGGAGCTGGGTC 71 3E8VH+L1ScFv(R) CAACTGTACTTCGGAGTGGACACCTGT 72 L1ScFv+hIgDhinge(F) GTGGAAATTAAACGCTGGCCAGGTTCT 73 XhoI+CD3zeta(R) CCGCTCGAGTTAGCGAGGGGGCAGGGC 74 T7(F) TATACGACTCACTATAGGG 75 SP6(R) ATTTAGGTGACACTATAG

2.1.2. Obtainment of 3E8 Antibody Leader Sequence Gene

(89) The pMT-CAR plasmid comprising the 3E8 antibody leader sequence (FIG. 8) as a template was amplified by PCR using the primer of SEQ ID NO: 70 (Table 6) and the primer of SEQ ID NO: 71 (Table 6) before use. The primer binding to the 5 site of the 3E8 leader sequence (LS) has the nucleotide sequence of the Mlu I restriction enzyme, and the primer binding to the 3 site of the 3E8 leader sequence (LS) has the 12-nucleotide sequence of the heavy chain variable region of the anti-L1CAM scFv antibody. Therefore, the amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-scFv. The amplified PCR product was used in the next PCR amplification process.

2.1.3. Obtainment of Human IgD Hinge Region, Transmembrane Domain, Intracellular Signaling Domain, Costimulatory Domain, and CD3 Gene

(90) To manufacture the CAR-constructs of the present disclosure, the gene of human IgD hinge region, CD28 transmembrane domain (TM), intracellular signaling domain (ICD), costimulatory domain OX40, and CD3 was obtained by the following methods.

(91) First, the pMT-CAR plasmid (FIG. 8) as a template was amplified by PCR using the primer of SEQ ID NO: 72 (Table 6) and the primer of SEQ ID NO: 73 (Table 6). The primer binding to the 5 site of the human IgD hinge region includes the 12-nucleotide sequence of the anti-L1CAM scFv antibody light chain variable region, and the primer binding to the 3 site of CD3 includes the nucleotide sequence of XhoI restriction enzyme. Therefore, the PCR product amplified by the primers has the nucleotide sequence of scFv-IgD hinge-CD28 TM-ICD-OX40-CD3-Xho I (Table 7). The amplified PCR product was used in the next PCR amplification process.

(92) TABLE-US-00014 TABLE7 Leadersequence,hinge,transmembranedomain(TM),intracellular domain(ICD),costimulatorydomain,andCD3genesequencesused inconstructionofCARconstructsofpresentdisclosure ID Nucleotidesequence MluI-start ACGCGTATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACTACAGG codon-3E8 TGTCCACTCC LS ScFv (SeeTable5) IgDhinge CGCTGGCCAGGTTCTCCAAAGGCACAGGCCTCCTCCGTGCCCACTGCACAA CCCCAAGCAGAGGGCAGCCTCGCCAAGGCAACCACAGCCCCAGCCACCAC CCGTAACACAGGTAGAGGAGGAGAAGAGAAGAAGAAGGAGAAGGAGAAAGA GGAACAAGAAGAGAGAGAGACAAAGACACCAGGTTGTCCG CD28TM TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAG TAACAGTGGCCTTTATTATTTTCTGGGTG CD28ICD AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCC GCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCG ACTTCGCAGCCTATCGCTCC OX40 GCCCTGTACCTGCTCCGGAGGGACCAGAGGCTGCCCCCCGATGCCCACAA GCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCCG ACGCCCACTCCACCCTGGCCAAGATC CD3-stop AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA codon-XhoI GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTT TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGA AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGG CGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAG GGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC GACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAACTCGAG

2.1.4. Preparation of pGemT-L1CAM-CAR Vectors

(93) Mlu I-3E8 LS-scFv, which is the PCR product amplified in 2.1.2, and the 3E8 LS-scFv-IgD hinge, which is the PCR product amplified in 2.1.1, as templates, were amplified by overlap extension PCR (OE-PCR) using the primer of SEQ ID NO: 70 (Table 6) and the primer of SEQ ID NO: 69 (Table 6).

(94) The resulting amplified Mlu I-3E8 LS-scFv-IgD hinge, and scFv-IgD hinge-CD28 TM-ICD-OX40-CD3-Xho I, which is the PCR product amplified in 2.1.3, as templates, were amplified by OE-PCR using the primer of SEQ ID NO: 70 (Table 6) and the primer of SEQ ID NO: 73 (Table 6) (FIG. 9). The resulting amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-scFv-IgD hinge-CD28 TM-ICD-OX40-CD3-Xho I. The amplified PCR product was ligated to pGemT EASY vector (Promega, WI, USA) having the multiple T sequences at both ends of linear DNA to give the CAR constructs, pGemT-L1-CAR-001, pGemT-L1-CAR-002, pGemT-L1-CAR-003, and pGemT-L1-CAR-004. The obtained CAR constructs were confirmed to be the same as the original sequence through sequencing (FIGS. 10A and 10B). A pair of primers of SEQ ID NOs: 74 and 75 (Table 6) was used for the sequencing.

2.1.5. Preparation of pMIN-L1-CAR Retroviral Vectors

(95) Four types of pGemT-L1-CAR vectors were treated with Mlu I and Xho I restriction enzymes to obtain DNA fragments. The obtained DNA fragments were ligated to the pMT retroviral vectors (U.S. Pat. No. 7,049,143) previously treated with Mlu I and Xho I restriction enzymes to construct four types of pMT-L1-CAR retroviral vectors (FIG. 11). The pMT-L1-CAR retroviral vectors thus constructed include sequences encoding anti-L1-CAR under the control of the MLV LTR promoter.

2.2. Preparation of Anti-L1-CAR Gene-Expressing T Cells

2.2.1. Preparation of Anti-L1-CAR Gene-Expressing Retroviruses (anti-L1-CAR Retroviruses)

(96) The retroviruses for anti-L1-CAR gene delivery were prepared using plasmid DNA transformation (Soneoka Y et al., 1995). The TransiT 293 transformation system (Mirus Bio LLC, Wis., USA) was used and operated according to the manufacturer's protocol. The previous day, pMT-L1-CAR retroviral vectors (pMT-L1-CAR-001, pMT-L1-CAR-002, pMT-L1-CAR-003, and pMT-L1-CAR-004) constructed in 2.1 above, the gag-pol expression vector, and the RD114 env expression vector were transformed into 293T cell lines seeded at 110.sup.6 on 60 mm dishes, and then the cells were cultured for about 48 hours. Upon completion of the culture, the cell cultures were all harvested, and then filtered through a 0.45-m filter. The four types of anti-L1-CAR retroviruses thus produced were measured for titer by real-time PCR using a retrovirus titer set (TaKaRa, JAPAN), and then stored frozen at 80 C. before use.

2.2.2 Preparation of Anti-L1-CAR Gene-Expressing T Cells

(97) Mononuclear cells were obtained from the blood of a donee by using SepMate-50 (STEMCELL) and Ficoll-Paque PLUS (GE healthcare, Sweden). The mononuclear cells were dispensed at 110.sup.7 in 100-mm dishes while AIMV medium (Invitrogen) comprising 5% human serum was used as a culture medium, and then the anti-CD3 (OKT3, eBioscience) antibody was added at 50 ng per mL, thereby activating T cells. For the growth of T cells, human IL-2 (R&D) was added to the culture medium at 300 U per mL, and cultured. After 48-hour incubation, the activated T cells were harvested, and used for delivery of four types of anti-L1-CAR retroviruses.

(98) Retronectin (TaKaRa, Japan) prepared at a concentration of 10 g/mL was added to 6-well plates at 2 mL per well, and then coated on the plates by incubation at room temperature for 2 hours. After the incubation, the residual Retronectin was removed, and then phosphate-buffered saline (PBS) comprising 2.5% bovine serum albumin (BSA) was added at 2 mL per well, and blocked by incubation at room temperature for 30 minutes. After the incubation, the solution used for blocking was removed, and the cells were washed by addition of HBSS comprising 2.5% of 1 M HEPES at 3 mL per well. Anti-L1-CAR retroviruses were diluted to 310.sup.10 copies per well with AIMV media comprising 5% human serum, and 4 mL of the dilution was added, followed by centrifugation under conditions of 2000 g and 32 C. for 2 hours, thereby immobilizing the retroviruses on Retronectin. The same amount of the medium used for retrovirus dilution was added to the wells to be used as a control. After the incubation, the residual retroviruses were removed, and activated T cells were added at 210.sup.6 per well, followed by incubation at 1000 g for 15 minutes, thereby delivering anti-L1-CAR retroviruses to T cells. To increase the delivery efficiency, the delivery procedure was repeated once more the next day, and thus a total of 2 times of delivery was performed. After 24 hours of delivery, T cells were all harvested, and subcultured in T flasks at 510.sup.5 cells per mL with AIMV media comprising 300 U/mL of 5% human serum and human IL-2. The cells were subcultured at 510.sup.5 per mL every 3-4 days, and maintained so as not to exceed 210.sup.6 per mL.

(99) It was investigated whether anti-L1-CAR was expressed in the activated T cells (anti-L1-CAR-expressing T cells) delivering anti-L1-CAR retroviruses. On days 8 and 20 of the incubation, 110.sup.6 cells were prepared, and incubated with biotinylated protein L (Genescript, Cat No. M00097) at 4 C. for 45 minutes. After the incubation, the cells were incubated with phycoerythrin-conjugated streptavidin (BD, Cat No. 554061) at 4 C. for 30 minutes, and the expression rate of anti-L1-CAR was checked by flow cytometry. The results verified that although there is a difference depending on the donor, the expression rate of anti-L1-CAR was about 19.9% to 67.2% on day 8 of the incubation and about 34.5% to 94.9% on day 20 of the incubation (Table 8).

(100) TABLE-US-00015 TABLE 8 Expression rates of anti-L1-CAR on surface of anti-L1-CAR-expressing T cells L1- L1- L1- L1- Donor Days of CAR- CAR- CAR- CAR- No incubation Control 001 002 003 004 30 8 Days 1.1% 51.6% 43.1% 24.7% 26.3% 20 Days 2.0% 65.7% 59.7% 58.4% 36.2% 32 8 Days 3.4% 67.2% 46.8% 63.7% 59.7% 20 Days 4.6% 84.6% 73.1% 94.9% 61.9% 34 8 Days 1.3% 36.6% 40.1% 20.9% 19.9% 20 Days 2.0% 53.9% 54.9% 40.8% 34.5%

2.3. Verification of Anticancer Activity of Anti-L1-CAR Gene-Expressing T Cells

2.3.1. Verification of Expression Rates of L1CAM in Target Cells

(101) The human ovarian adenocarcinoma cell line SKOV3 is known to highly express L1CAM, which is an antigen in the present disclosure, and thus is a cell line suitable for investigating the anticancer activity of the anti-L1CAM-CAR-expressing T cells of the present disclosure. To check this, the SKOV3 cell line was prepared at 510.sup.5 cells in 100 L of PBS, and 0.25 g of the anti-hCD171-PE (5G3 clone) (eBioscience, Cat No. 12-1719-42) antibody was added, followed by incubation at 4 C. for 30 minutes. After the incubation, the cells were washed twice with PBS, and the expression rate of L1CAM was checked by flow cytometry. The results verified that the L1CAM expression rate was about 74% in SKOV3 cancer cells. Meanwhile, as a result of investigating the expression of L1CAM in the human embryonic kidney cell line 293T by the same method, an expression rate of about 3% was confirmed (FIG. 12).

2.3.2. Verification of Anticancer Activity of L1CAM-Expressing T Cells on Target Cells

(102) To investigate the anticancer activity of the anti-L1CAM-CAR (anti-L1-CAR)-expressing T cells (effector cells, E) of the present disclosure on target cells (T), the xCELLigence Real-Time Cell Analysis (RTCA) method was used. According to the xCELLigence RTCA method, the electron flow is displayed numerically as an index value when an electroconductive solution (e.g., culture media) is included on a plate coated with a gold microelectrode biosensor, and the electron flow is disturbed to result in changed index values when target cells adhere to the plate. Upon the addition of CAR-expressing T cells (CAR-T), the adhering target cells are separated from the plate due to cytotoxicity of the T cells, and the anticancer activity (cytotoxicity) can be checked by analyzing the change in index value. Target cells were prepared at 110.sup.4 cells in 50 L of a culture medium, and added to a plate for analysis. After 21 hours, anti-L1-CAR-expressing T cells were prepared at 110.sup.4, 510.sup.4, and 110.sup.5 (E:T ratio=1, 5, and 10) in 50 uL of AIMV media comprising human serum and human IL-2, and added to wells comprising target cells, to check the cell index value in real time for 50 hours. In addition, wells comprising only target cells were prepared, and the anticancer activity of anti-L1-CAR-expressing T cells was calculated as follows.
Cytotoxicity (%)={(index value of target cell well)(index value of target cell and T cell incubation well)}/(index value of target cell well)100Equation

(103) The results verified that among the four types of anti-L1-CAR-expressing T cells of the present disclosure, L1-CAR-004 showed higher cytotoxicity in SKOV3 cells than CAR-non-expressing T cells (control). Although there is a difference depending on the donor, L1-CAR-001 showed cytotoxicity in SKOV3 cells compared with the control (FIG. 13A). All the four types showed lower cytotoxicity than the control in 293T cells showing a low expression rate of L1CMA (FIG. 13B). Therefore, the anti-L1-CAR-expressing T cells of the present disclosure exhibited anticancer activity in target cancer cells highly expressing L1CAM antigens, and thus can be advantageously used as a cell therapeutic agent for anti-cancer use.

Example 3: Verification of Anti-L1 CAM-CAR Gene-Expressing T Cells in Vivo

(104) To investigate anticancer activity of anti-L1CAM-CAR gene-expressing T cells in vivo, cancer-induced animal models were used. SKOV3 cancer cells (Target, T) mixed with Matrigel at 1:1 were subcutaneously (SC) administered at 310.sup.6 to the right flank of NOD/SCID mice (7 weeks old, female) lacking T cells, B cells, and natural killer cells (NK cells), to thereby induce cancer. L1-CAR-002 and L1-CAR-004, which are two types of anti-L1CAM-CAR-expressing T cells confirmed to have efficacy in vitro, and control T cells were administered to each NOD/SCID mouse 3 days after cancer cell administration, once a day, a total of 3 times. T cells were administered through the tail vein (intravenous, IV) at 210.sup.7 per dose, and the cancer size was measured up to day 25. The results verified that both two types of anti-L1CAM-CAR-expressing T cells inhibited the cancer growth rate compared with the control T cell administration group (FIG. 14). Through the fact that L1-CAR-004 greatly inhibited the cancer growth rate compared with L1-CAR-002, it was verified that the efficacy of L1-CAR-004 was better in vivo.

Example 4: Fabrication of T Cells Expressing Anti-L1CAM-CAR Genes with Various Spacer Domain Structures and Verification of Activity Thereof

4.1. Obtainment of L1-H8-CAR Genes with Various Spacer Domain Structures

4.1.1. Selection of Anti-mL1CAM scFv Antibody

(105) It was verified through the anticancer activity test conducted in Example 3 that the cancer growth rate-inhibitory effect of L1-CAR-004 was best. The nucleotide sequences of polynucleotides encoding the heavy chain and light chain variable regions of the L1CAM-specific antibody of L1-CAR-004 (FIG. 10B) were obtained, and used to prepare the next gene. Hereinafter, pMT-L1-CAR-004 was expressed as pMT-L1-H8-CAR-001.

4.1.2. Obtainment of L1-H8-CAR-002 Gene

4.1.2.1. Obtainment of 3E8 Antibody Leader Sequence (LS) and Anti-mL1CAM scFv Antibody Gene

(106) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 70 (Table 9) and SEQ ID NO: 69 (Table 9). The primer binding to the 5 end of the 3E8 leader sequence (LS) has the nucleotide sequence of Mlu I restriction enzyme and the 18-nucleotide sequence of the 3E8 leader sequence (LS), and the primer binding to the 3 end of L1-H8 scFv has the 12-nucleotide sequence of hIgD, and thus the amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge (Table 10). The amplified PCR product was used in the next PCR amplification process.

(107) TABLE-US-00016 TABLE9 Nucleotidesequenceinformationofusedprimers SEQID NO Primername Nucleotidesequence 70 Mlu1+3E8VH(F) ACGCGTATGGAATGGAGCTGGGTC 69 L1ScFv+hIgDhinge(R) ACCTGGCCAGCGTTTAATTTCCACTTT 72 L1ScFv+hIgDhinge(F) GTGGAAATTAAACGCTGGCCAGGTTCT 73 XhoI+CD3zeta(R) CCGCTCGAGTTAGCGAGGGGGCAGGGC 83 L1-H8scFv+IgG1hinge(R) AGATTTGGGCTCTTTAATTTCCACTTT 84 L1-H8scFv+IgG1hinge(F) GTGGAAATTAAAGAGCCCAAATCTTGT

(108) TABLE-US-00017 TABLE10 LS,L1-H8scFv,Hinge,CH3,TM,ICD,costimulatorydomain, andCD3genesequences ID Nucleotidesequence MluI-start ACGCGTATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACTACAG codon-3E8 GTGTCCACTCC LS L1-H8scFv GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTC (L1CAM-3R- ACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCTGATTATGCAATG H8) AATTGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCAGCAATC TCTTCTACTGGTTCTACTATCTACTATGCCGATTCAGTGAAGGGTCGCTTTAC CATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGC GTGCCGAAGACACGGCCGTCTATTATTGCGCCAAACAGTCTACTTACTTTTA CTCTTACTTTGATGTTTGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGT GGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATTCA AATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGAC AATTACTTGTCGCGCTAGCCAGTCTATCTCTCGTGATCTGAACTGGTATCAGC AGAAACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCATCCTCTCTGC AGTCTGGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTAC CCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAG CAATCTTACTCTACTCCGTACACGTTCGGGCAGGGAACTAAAGTGGAAATTA AA IgDhinge CGCTGGCCAGGTTCTCCAAAGGCACAGGCCTCCTCCGTGCCCACTGCACA ACCCCAAGCAGAGGGCAGCCTCGCCAAGGCAACCACAGCCCCAGCCACCA CCCGTAACACAGGTAGAGGAGGAGAAGAGAAGAAGAAGGAGAAGGAGAAA GAGGAACAAGAAGAGAGAGAGACAAAGACACCAGGTTGTCCG IgG1hinge GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA IgG1CH3 GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGA GCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCC CAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA TGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAA CD28TM TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTA GTAACAGTGGCCTTTATTATTTTCTGGGTG CD28ICD AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCC CGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACG CGACTTCGCAGCCTATCGCTCC OX40 GCCCTGTACCTGCTCCGGAGGGACCAGAGGCTGCCCCCCGATGCCCACAA GCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCC GACGCCCACTCCACCCTGGCCAAGATC CD3-iso1- AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA stopcodon- GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTT XhoI TTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGA GAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGA TGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACAC CTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAACTCGAG

4.1.2.2. Obtainment of Hinge, CH3, TM, ICD, Costimulatory Domain, and CD3 Gene

(109) The pMT-CAR-002 plasmid (FIG. 15), comprising the human IgD hinge and IgG1 hinge, CH3, CD28 TM and ICD, costimulatory domain OX40, and CD3-iso1, as a template was amplified by PCR using the primers of SEQ ID NO: 72 (Table 9) and SEQ ID NO: 73 (Table 9) before use. The primer binding to the 5 end of the hIgD hinge has the 12-nucleotide sequence of the light chain variable region (VL) of L1-H8 scFv antibody, and the primer binding to the 3 end of CD3-iso1 has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of L1-H8 scFv-IgD hinge-IgG1 hinge-CH3-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I (Table 10). The amplified PCR product was used in the next PCR amplification process.

4.1.2.3. Obtainment of 3E8 LS, L1-H8 scFv, Hinge, CH3, TM, ICD, Costimulatory Domain, and CD3 Gene

(110) Mlu I-3E8 LS-L1-H8 scFv-IgD hinge and L1-H8-scFv-IgD hinge-IgG1 hinge-CH3-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I, which were the amplified PCR products, as templates, were amplified by the overlap extension PCR (OE-PCR) method using the primers of SEQ ID NO: 70 (Table 9) and SEQ ID NO: 73 (Table 9) (FIG. 16). The amplified PCR product has the nucleotide sequence of Mlu I-3E8-L1-H8 scFv-IgD hinge-IgG1 hinge-CH3-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I, and has a structure of L1-H8-CAR-002 (FIG. 17).

4.1.3. Obtainment of L1-H8-CAR-003 Gene

4.1.3.1. Obtainment of 3E8 Antibody Leader Sequence (LS) and Anti-mL1CAM scFv Antibody Gene

(111) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 70 (Table 9) and SEQ ID NO: 69 (Table 9). The primer binding to the 5 end of the 3E8 leader sequence (LS) has the nucleotide sequence of Mlu I restriction enzyme and the 18-nucleotide sequence of the 3E8 leader sequence (LS), and the primer binding to the 3 end of L1-H8 scFv has the 12-nucleotide sequence of hIgD hinge, and thus the amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge (Table 10) The amplified PCR product was used in the next PCR amplification process.

4.1.3.2. Obtainment of Hinge, CH3, TM, ICD, Costimulatory Domain, and CD3 Gene

(112) The pMT-CAR-003 plasmid (FIG. 18), comprising human IgD hinge and IgG1 hinge, CD 28 TM and ICD, costimulatory domain OX40, and CD3-iso1, as a template was amplified by PCR using the primers of SEQ ID NO: 72 (Table 9) and SEQ ID NO: 73 (Table 9) before use. The primer binding to the 5 end of the hIgD hinge has the 12-nucleotide sequence of the light chain variable region (VL) of L1-H8 scFv antibody, and the primer binding to the 3 end of CD3-iso1 has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of L1-H8 scFv-IgD hinge-IgG1 hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I (Table 10). The amplified PCR product was used in the next PCR amplification process.

4.1.3.3. Obtainment of 3E8 LS, L1-H8 scFv, Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene

(113) Mlu I-3E8 LS-L1-H8 scFv-IgD hinge and L1-H8-scFv-IgD hinge-IgG1 hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 70 (Table 9) and SEQ ID NO: 73 (Table 9) (FIG. 19). The amplified PCR product has the nucleotide sequence of Mlu I-3E8-L1-H8 scFv-IgD hinge-IgG1 hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I, and has a structure of L1-H8-CAR-003 (FIG. 20).

4.1.4. Obtainment of L1-H8-CAR-004 Gene

4.1.4.1. Obtainment of 3E8 Antibody Leader Sequence (LS) and Anti-mL1CAM scFv Antibody Gene

(114) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 70 (Table 9) and SEQ ID NO: 83 (Table 9). The primer binding to the 5 end of the 3E8 leader sequence (LS) has the nucleotide sequence of Mlu I restriction enzyme and the 18-nucleotide sequence of the 3E8 leader sequence (LS), and the primer binding to the 3 end of L1-H8 scFv has the 12-nucleotide sequence of hIgG1 hinge, and thus the amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgG1 hinge (Table 10). The amplified PCR product was used in the next PCR amplification process.

4.1.4.2. Obtainment of Hinge, CH3, TM, ICD, Costimulatory Domain, and CD3 Gene

(115) The pMT-CAR-002 plasmid (FIG. 15), comprising IgG1 hinge, CH3, CD28 TM and ICD, costimulatory domain OX40, and CD3-iso1, as a template, was amplified by PCR using the primers of SEQ ID NO: 84 (Table 9) and SEQ ID NO: 73 (Table 9). The primer binding to the 5 end of the hIgG1 hinge has the 12-nucleotide sequence of the light chain variable region (VL) of L1-H8 scFv antibody, and the primer binding to the 3 end of CD3-iso1 has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of L1-H8 scFv-IgG1 hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I (Table 10). The amplified PCR product was used in the next PCR amplification process.

4.1.4.3. Obtainment of 3E8 LS, L1-H8 scFv, Hinge, CH3, TM, ICD, Costimulatory Domain, and CD3 Gene

(116) Mlu I-3E8 LS-L1-H8 scFv-IgG1 hinge and L1-H8 scFv-IgG1 hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 70 (Table 9) and SEQ ID NO: 73 (Table 9) (FIG. 21). The amplified PCR product has the nucleotide sequence of Mlu I-3E8-L1-H8 scFv-IgG1 hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I, and has a structure of L1-H8-CAR-004 (FIG. 22).

4.1.5. Preparation of pMT-L1-H8-CAR Retroviral Vectors

(117) Three types of the amplified PCR products were treated with Mlu I and Xho I restriction enzymes to obtain DNA fragments. The obtained DNA fragments were ligated to the pMT retroviral vectors (U.S. Pat. No. 6,451,595) previously treated with Mlu I and Xho I restriction enzymes to prepare three types of pMT-L1-H8-CAR retroviral vectors (FIG. 23). The pMT-L1-H8-CAR retroviral vectors thus prepared include a sequence encoding anti-L1-CAR under the control of the MLV LTR promoter.

4.2. Preparation of Retroviruses Expressing L1-H8-CAR Genes with Various Spacer Domain Structures (L1-H8-CAR Retroviruses)

(118) The retroviruses for L1-H8-CAR gene delivery were prepared using plasmid DNA transformation (Soneoka Y et al., 1995). The TransiT 293 transformation system (Mirus Bio LLC, Wis., USA) was used and operated according to the manufacturer's protocol. The previous day, four types of pMT-L1-H8-CAR retroviral vectors, the gag-pol expression vector, and the RD114 env expression vector were transformed into 293T cell lines seeded at 110.sup.6 on 60 mm dishes, and then the cells were cultured for about 48 hours. Upon completion of the culture, the cell cultures were all harvested, and then filtered through a 0.45-m filter. The four types of L1-H8-CAR retroviruses thus produced were measured for titer by real-time PCR using a retrovirus titer set (TaKaRa, JAPAN), and then stored frozen at 80 C. before use.

4.3. Preparation of T Cells Expressing L1-H8-CAR Genes with Various Spacer Domain Structures

(119) Mononuclear cells were obtained from the blood of a donee using SepMate-50 (STEMCELL) and Ficoll-Paque PLUS (GE healthcare, Sweden). The mononuclear cells were dispensed at 110.sup.7 in 100-mm dishes while AIMV medium (Invitrogen) comprising 5% human serum was used as a culture medium, and then the anti-CD3 (OKT3, eBioscience) antibody was added at 50 ng per mL, thereby activating T cells. For the growth of T cells, human IL-2 (R&D) was added to the culture medium at 300 U per mL, and cultured. After 48-hour incubation, the activated T cells were harvested, and used for delivery of four types of anti-L1-H8-CAR retroviruses.

(120) Retronectin (TaKaRa, Japan) prepared at a concentration of 10 g/mL was added to 6-well plates at 2 mL per well, and then coated on the plates by incubation at room temperature for 2 hours. After the incubation, the Retronectin was removed, and then phosphate-buffered saline (PBS) comprising 2.5% human albumin was added at 2 mL per well, and blocked by incubation at room temperature for 30 minutes. After the incubation, the solution used for blocking was removed, and washed by addition of HBSS comprising 2.5% of 1 M HEPES at 3 mL per well. L1-H8-CAR retroviruses were diluted to 310.sup.10 copies per well with AIMV media comprising 5% human serum, and 4 mL of the dilution was added, followed by centrifugation under conditions of 2000 g and 32 C. for 2 hours, thereby immobilizing the retroviruses on Retronectin. The same amount of the medium used for retrovirus dilution was added to the wells to be used as a control. After culture, the retroviruses were removed, and activated T cells were added at 210.sup.6 per well, followed by incubation at 1000 g for 15 minutes, thereby delivering L1-H8-CAR retroviruses to T cells. To increase the delivery efficiency, the delivery procedure was repeated once more the next day, and thus a total of 2 times of delivery was performed. After 24 hours of delivery, T cells were all harvested, and subcultured in T flasks at 510.sup.5 cells per mL with AIMV media comprising 300 U/mL of 5% human serum and human IL-2. The cells were subcultured at 510.sup.5 per mL every 3-4 days, and maintained so as not to exceed 210.sup.6 per mL.

(121) It was investigated whether L1-H8-CAR was expressed in the activated T cells (L1-H8-CAR-expressing T cells) delivering L1-H8-CAR retroviruses. At the first and second weeks of culture, 110.sup.6 cells were prepared, and incubated with FITC-conjugated protein L (ACROBiosystems, Cat No. RPL-PF141) at 4 C. for 30 minutes, and the expression rate of L1-H8-CAR was checked by flow cytometry. The results verified that although there is a difference depending on the donor, the expression rate of L1-H8-CAR was about 16.4% to 52.4% on day 8 of incubation and about 29.6% to 69.2% on day 15 or day 18 of incubation (Table 11).

(122) TABLE-US-00018 TABLE 11 Expression rates of L1-H8-CAR on surface of L1-H8-CAR-expressing T cells L1-H8- L1-H8- L1-H8- L1-H8- Donor Days of CAR- CAR- CAR- CAR- NO. incubation Control 001 002 003 004 45 8 Days 1.64% 33.6% 40.5% 34.1% 16.4% 18 Days 0.37% 52.4% 64.7% 54.5% 30.0% 36 8 Days 1.26% 33.6% 44.8% 42.2% 21.6% 15 Days 1.00% 50.5% 69.2% 63.1% 29.6% 43 8 Days 1.84% 42.7% 52.4% 50.6% 27.6% 15 Days 0.64% 52.6% 60.0% 63.8% 33.2%

4.4. Verification of Anticancer Activity of T Cells Expressing L1-H8-CAR Genes with Various Spacer Domain Structures (In Vitro)

4.4.1. Verification of Expression Rates of L1CAM in Target Cells

(123) The human ovarian adenocarcinoma cell line SKOV3 is known to highly express L1CAM, and thus is a cell line suitable for investigating the anticancer activity of the anti-L1CAM-CAR-expressing T cells. To check this, the SKOV3 cell line was prepared at 510.sup.5 cells in 100 L of PBS, and 0.25 g of the anti-hCD171-PE (5G3 clone) (eBioscience, Cat No. 12-1719-42) antibody was added, followed by incubation at 4 C. for 30 minutes. After the incubation, the cells were washed with PBS twice, and then the expression of L1CAM was investigated by flow cytometry. The results verified that the L1CAM expression rate was about 93.4 to 99.2% in SKOV3 cancer cells (FIGS. 24A to 24C). As a result of investigating the expression of L1CAM in the human cervical cancer cell line HeLa, the human neuroblastoma cell line SH-SY5Y, and the human embryonic kidney cell line 293T, the expression rate was about 99.9% in HeLa (FIG. 24F), about 89.6% in SH-SY5Y (FIG. 24A), and about 0.57 to 0.61% in 293T (FIGS. 24D and 24E).

4.4.2. Verification of Anticancer Activity of L1CAM-Expressing T Cells on Target Cells (In Vitro)

4.4.2.1. Verification of Anticancer Activity Using xCelligence Assay

(124) To investigate the anticancer activity of the anti-L1CAM-CAR (L1-H8-CAR)-expressing T cells (effector cells, E) on target cells (T), xCELLigence Real-Time Cell Analysis (RTCA) was used. According to the xCELLigence RTCA method, the electron flow is displayed numerically as an index value when an electroconductive solution (e.g., culture media) is included on a plate coated with a gold microelectrode biosensor, and the electron flow is disturbed to result in changed index values when target cells adhere to the plate. Upon the addition of CAR-expressing T cells, the adhering target cells are separated from the plate due to cytotoxicity of the T cells, and the anticancer activity (cytotoxicity) can be checked by analyzing the change in index value. Target cells were prepared at 110.sup.4 cells in 50 L of a culture medium, and added to a plate for analysis. After about 21 hours, anti-L1-H8-CAR-expressing T cells were prepared at 110.sup.4, 510.sup.4, and 110.sup.5 (E:T ratio=1, 5, 10) in 50 uL of AIMV media comprising human serum and human IL-2, and added to wells comprising target cells, to check the cell index value in real time for 30 hours. In addition, wells comprising only target cells were prepared, and the anticancer activity of L1-H8-CAR-expressing T cells was calculated as follows.
Cytotoxicity (%)={(index value of target cell well)(index value of target cell and T cell incubation well)}/(index value of target cell well)100Equation

(125) As a result, four types of T cells expressing L1-H8-CAR-001, -002, -003, and -004 showed high cytotoxicity on SKOV3 cells compared with T cells not expressing 1-H8-CAR (control) (FIG. 25).

(126) The cytotoxicity on 293T cells were checked by the same method. The target cells were added at 2.510.sup.4 to 50 uL of culture media, and after about 21 hours, L1-H8-CAR-expressing T cells were prepared at 2.510.sup.4, 1.2510.sup.5, and 2.510.sup.5 (E:T ratio=1, 5, and 10) in 50 uL of AIMV media comprising human serum and human IL-2, and added to wells comprising target cells, to check the cell index value in real time for 30 hours. In addition, wells comprising only target cells were prepared, and the anticancer activity of L1-H8-CAR-expressing T cells was calculated in the same manner as in the above tests. As a result, all the four types showed similar cytotoxicity to the control in 293T cells showing a low expression rate of L1CMA (FIG. 26).

4.4.2.2. Verification of Anticancer Activity Using CellTox Green Dye

(127) To investigate the anticancer activity of the anti-L1 CAM-CAR (L1-H8-CAR)-expressing T cells (effector cells, E) on target cells (T), CellTox Green dye was used. CellTox Green dye is a dye that attaches to DNA released from dead cells to exhibit fluorescence, and is used to investigate anticancer activity (cytotoxicity). The target cells were prepared at 110.sup.4 in 50 uL of culture media, and 0.2 uL of CellTox Green dye was added, and the mix was added to 96-well black plates. The L1-H8-CAR-expressing T cells were prepared at 510.sup.3, 110.sup.4, 510.sup.4, and 110.sup.5 (E:T ratio=0.5, 1, 5, and 10) in 50 uL of AIMV media comprising human serum and human IL-2, and added to wells comprising target cells, followed by incubation in a CO.sub.2 incubator at 37 C. for 24 hours. The group added with only L1-H8-CAR-expressing T cells was prepared in the wells comprising the culture media of CellTox Green dye and target cells, and the reaction value of the dye, occurring by attachment to DNA released from dead L1-H8-CAR-expressing T cells during the incubation was excluded. The wells comprising only target cells were prepared to correct the low control (spontaneous DNA release) value, and a lysis solution was added to the well comprising only the target cells to correct the high control (maximum DNA release) value. The cytotoxicity on the target cells was calculated by the following method.
Cytotoxicity (%)={(reaction value of Target cells and Effector cells)(reaction value of Effector cells)}(Low control)/(High controlLow control)100Equation 2

(128) As a result, four types of T cells expressing L1-H8-CAR-001, -002, -003, and -004 showed high cytotoxicity on SH-SY5Y cells compared with T cells not expressing 1-H8-CAR (control) (FIGS. 27A and 27B).

(129) The cytotoxicity on HeLa cells were checked by the same test method. The target cells were prepared at 3.510.sup.3 in 50 uL of culture media, and 0.2 uL of CellTox Green dye was added, and the mix was added to 96-well black plates. The L1-H8-CAR-expressing T cells were prepared at 1.7510.sup.3, 3.510.sup.3, 1.7510.sup.4, and 3.510.sup.4 (E:T ratio=0.5, 1, 5, and 10) in 50 uL of AIMV media comprising human serum and human IL-2, and added to wells comprising target cells, followed by incubation in a CO.sub.2 incubator at 37 C. for 24 hours. The cytotoxicity on the target cells was corrected and calculated by the same method. As a result, four types of T cells expressing L1-H8-CAR-001, -002, -003, and -004 showed high cytotoxicity on HeLa cells compared with T cells not expressing 1-H8-CAR (control) (FIGS. 28A and 28B).

4.5. Verification of Anticancer Activity of T Cells Expressing L1-H8-CAR Genes with Various Spacer Domain Structures (In Vitro)

(130) To investigate anticancer activity of anti-L1CAM-CAR (L1CAM-CAR) gene-expressing T cells in vivo, cancer-induced animal models were used. SKOV3 cancer cells (Target, T) mixed with Matrigel at 1:1 were subcutaneously (SC) administered at 310.sup.6 to the right flank of NOD/SCID mice (7 weeks old, female) lacking T cells, B cells, and natural killer cells (NK cells), to thereby induce cancer. Four types of L1-H8-CAR-expressing T cells confirmed to have efficacy in vitro and control T cells were administered to each NOD/SCID mouse 3 days and 5 days after cancer cell administration, once a day, a total of 2 times. T cells were administered through the tail vein (intravenous, IV) at 210.sup.7 per dose, and the cancer size was measured up to day 25. The results verified that both two types of anti-L1CAM-CAR-expressing T cells inhibited the cancer growth rate compared with the control T cell administration group. It was especially verified that the cancer growth inhibitory effect of L1-H8-CAR-003 was the best (FIG. 29).

Example 5: Fabrication of T Cells Expressing Anti-L1CAM-CAR With Various Costimulatory Domain Structures and Verification of Activity Thereof

5.1. Obtainment of L1CMA-CAR Genes with Various Costimulatory Domain Structures

5.1.1. Obtainment of L1-H8-CAR-001-28BB Gene

5.1.1.1. Obtainment of 3E8 Antibody Leader Sequence (LS), L1-H8 scFv, Hinge, TM, and ICD Gene

(131) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 87 (Table 12). The primer binding to the 5 end of the 3E8 leader sequence (LS) has the nucleotide sequence of Mlu I restriction enzyme and the 18-nucleotide sequence of the 3E8 leader sequence (LS), and the primer binding to the 3 end of CD28 ICD has the 12-nucleotide sequence of 4-1BB, and thus the amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-CD28 ICD-4-1BB (Table 13) The amplified PCR product was used in the next PCR amplification process.

(132) TABLE-US-00019 TABLE12 Nucleotidesequenceinformationofusedprimers SEQID NO Primername Nucleotidesequence 70 Mlu1+3E8VH(F) ACGCGTATGGAATGGAGCTGGGTC 87 CD28ICD+41BB(R) TCTGCCCCGTTTGGAGCGATAGGCTGC 88 CD28ICD+41BB(F) GCCTATCGCTCCAAACGGGGCAGAAAG 73 XhoI+CD3zeta(R) CCGCTCGAGTTAGCGAGGGGGCAGGGC 89 CD28ICD+ICOSICD(R) GGATGAATACTTGGAGCGATAGGCTGC 90 CD28ICD+ICOSICD(F) GCCTATCGCTCCAAGTATTCATCCAGT 91 ICOSICD+CD3zeta(R) GAACTTCACTCTGGTCACATCTGTGAG 92 ICOSICD+CD3zeta(F) ACAGATGTGACCAGAGTGAAGTTCAGC 93 CD28ICD+CD3zeta(R) GAACTTCACTCTGGAGCGATAGGCTGC 94 CD28ICD+CD3zeta(F) GCCTATCGCTCCAGAGTGAAGTTCAGC 95 CD28TM+CX40(R) CAGGTACAGGGCCACCCAGAAAATAAT 96 CD28TM+CX40(F) ATTTTCTGGGTGGCCCTGTACCTGCTC 97 CD28TM+41BB(R) TCTGCCCCGTTTCACCCAGAAAATAAT 98 CD28TM+41BB(F) ATTTTCTGGGTGAAACGGGGCAGAAAG 99 CD28TM+ICOSICD(R) TGGATGAATACTTCACCCAGAAAATAATA 100 CD28TM+ICOSICD(F) ATTTTCTGGGTGAAGTATTCATCCAGT

(133) TABLE-US-00020 TABLE13 LS,L1-H8scFv,Hinge,TM,ICD,costimulatorydomain, andCD3genesequences ID Nucleotidesequence MluI-start ACGCGTATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACTAC codon-3E8LS AGGTGTCCACTCC L1-H8scFv GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGT (L1CAM-3R- TCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCTGATTATGC H8) AATGAATTGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTC AGCAATCTCTTCTACTGGTTCTACTATCTACTATGCCGATTCAGTGAAGGG TCGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGAT GAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAACAG TCTACTTACTTTTACTCTTACTTTGATGTTTGGGGTCAGGGCACTTTAGTG ACCGTCTCATCGGGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGCGG TGGCGGATCGGACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCT AGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTAGCCAGTCTATCT CTCGTGATCTGAACTGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATT GCTGATTTACGCAGCATCCTCTCTGCAGTCTGGTGTACCGTCCCGTTTCT CTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCA GCCTGAAGATTTTGCCACCTATTATTGTCAGCAATCTTACTCTACTCCGTA CACGTTCGGGCAGGGAACTAAAGTGGAAATTAAA IgDhinge CGCTGGCCAGGTTCTCCAAAGGCACAGGCCTCCTCCGTGCCCACTGCA CAACCCCAAGCAGAGGGCAGCCTCGCCAAGGCAACCACAGCCCCAGC CACCACCCGTAACACAGGTAGAGGAGGAGAAGAGAAGAAGAAGGAGAA GGAGAAAGAGGAACAAGAAGAGAGAGAGACAAAGACACCAGGTTGTCC G CD28TM TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGC TAGTAACAGTGGCCTTTATTATTTTCTGGGTG CD28ICD AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTC CCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCAC CACGCGACTTCGCAGCCTATCGCTCC OX40 GCCCTGTACCTGCTCCGGAGGGACCAGAGGCTGCCCCCCGATGCCCA CAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGC AGGCCGACGCCCACTCCACCCTGGCCAAGATC 4-1BB AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGA CCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAG AAGAAGAAGAAGGAGGATGTGAACTG ICOS AAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAG AGCAGTGAACACAGCCAAAAAATCTAGACTCACAGATGTGACC CD3-iso1-stop AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGG codon-XhoI CCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTAC GATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAG CCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAG CGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACA GCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGCTAACTCGAG

5.1.1.2. Obtainment of Costimulatory Domain and CD3 Gene

(134) The pMT-CAR-004 plasmid (FIG. 30), comprising the costimulatory domain 4-1BB, and CD3-iso1, as a template, was amplified by PCR using the primers of SEQ ID NO: 88 (Table 12) and SEQ ID NO: 73 (Table 12). The primer binding to the 5 end of 4-1BB has the 12-nucleotide sequence of CD28 ICD, and the primer binding to the 3 end of CD3-iso1 has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of ICD-4-1BB-CD3-iso1-Xho I (Table 13). The amplified PCR product was used in the next PCR amplification process.

5.1.1.3. Obtainment of 3E8 LS, L1-H8 scFv, Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene

(135) Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-CD28 ICD-4-1BB and CD28 ICD-4-1BB-CD3-iso1-Xho I, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 73 (Table 12) (FIG. 31). The amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-CD28 ICD-4-1BB-CD3-iso1-Xho I, and has a structure of L1-H8-CAR-001-28BB (FIG. 32).

5.1.2. Obtainment of L1-H8-CAR-001-28ICOS Gene

5.1.2.1. Obtainment of 3E8 Antibody Leader Sequence (LS), L1-H8 scFv, Hinge, TM, and ICD Gene

(136) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 89 (Table 12). The primer binding to the 5 end of the 3E8 leader sequence (LS) has the nucleotide sequence of Mlu I restriction enzyme and the 18-nucleotide sequence of the 3E8 leader sequence (LS), and the primer binding to the 3 end of CD28 ICD has the 12-nucleotide sequence of ICOS, and thus the amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-CD28 ICD-ICOS ICD (Table 13) The amplified PCR product was used in the next PCR amplification process.

5.1.2.2. Obtainment of Costimulatory Domain ICOS Gene

(137) TM and ICD structures of the costimulatory domain ICOS gene were synthesized. The pBHA-ICOS TM+ICD (FIG. 33) secured through gene synthesis as a template was amplified by PCR using the primers of SEQ ID NO: 90 (Table 12) and SEQ ID NO: 91 (Table 12). The primer binding to the 5 end of ICOS ICD has the 12-nucleotide sequence of CD28 ICD, and the primer binding to the 3 end of ICOS ICD has the nucleotide sequence of CD3-iso1, and thus the amplified PCR product has the nucleotide sequence of CD28 ICD-ICOS ICD-CD3-iso1 (Table 13). The amplified PCR product was used in the next PCR amplification process.

5.1.2.3. Obtainment of CD3 Gene

(138) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 92 (Table 12) and SEQ ID NO: 73 (Table 12). The primer binding to the 5 end of CD3-iso1 has the 12-nucleotide sequence of ICOS ICD, and the primer binding to the 3 end of CD3-iso1 has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of ICOS ICD-CD3-iso1-Xho I (Table 13). The amplified PC R product was used in the next PCR amplification process.

5.1.2.4. Obtainment of 3E8 LS, L1-H8 scFv, Hinge, TM, ICD, and Costimulatory Domain Gene

(139) Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-CD28 ICD-ICOS ICD and CD28 ICD-ICOS ICD-CD3-iso1, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 91 (Table 12) (FIG. 34). The amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-CD28 ICD-ICOS ICD-CD3-iso1 (Table 13). The amplified PCR product was used in the next PCR amplification process.

5.1.2.5. Obtainment of 3E8 LS, L1-H8 scFv, Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene

(140) Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-CD28 ICD-ICOS ICD-CD3-iso1 and ICOS ICD-CD3-iso1-Xho I, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 73 (Table 12) (FIG. 34). The amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-CD28 ICD-ICOS ICD-CD3-iso1-Xho I and a structure of L1-H8-CAR-001-28ICOS (FIG. 35).

5.1.3. Obtainment of L1-H8-CAR-001-28 Gene

5.1.3.1. Obtainment of 3E8 Antibody Leader Sequence (LS), L1-H8 scFv, Hinge, TM, and ICD Gene

(141) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 93 (Table 12). The primer binding to the 5 end of the 3E8 leader sequence (LS) has the nucleotide sequence of Mlu I restriction enzyme and the 18-nucleotide sequence of the 3E8 leader sequence (LS), and the primer binding to the 3 end of CD28 ICD has the 12-nucleotide sequence of CD3-iso1, and thus the amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-CD28 ICD-CD3-iso1 (Table 13). The amplified PCR product was used in the next PCR amplification process.

5.1.3.2. Obtainment of CD3 Gene

(142) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 94 (Table 12) and SEQ ID NO: 73 (Table 12). The primer binding to the 5 end of CD3-iso1 has the 12-nucleotide sequence of CD28 ICD, and the primer binding to the 3 end of CD3-iso1 has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of ICD28 ICD-CD3-iso1-Xho I (Table 13). The amplified PCR product was used in the next PCR amplification process.

5.1.3.3. Obtainment of 3E8 LS, L1-H8 scFv, Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene

(143) Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-CD28 ICD-CD3-iso1 and CD28 ICD-CD3-iso1-Xho I, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 73 (Table 12) (FIG. 36). The amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-CD28 ICD-CD3-iso1-Xho I and a structure of L1-H8-CAR-001-28 (FIG. 37).

5.1.4. Obtainment of L1-H8-CAR-001-OX Gene

5.1.4.1. Obtainment of 3E8 Antibody Leader Sequence (LS), L1-H8 scFv, Hinge, and TM Gene

(144) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 95 (Table 12). The primer binding to the 5 end of the 3E8 leader sequence (LS) has the nucleotide sequence of Mlu I restriction enzyme and the 18-nucleotide sequence of the 3E8 leader sequence (LS), and the primer binding to the 3 end of CD28 TM has the 12-nucleotide sequence of OX40, and thus the amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-OX40 (Table 13). The amplified product was used in the next PCR amplification process.

5.1.4.2. Obtainment of Costimulatory Domain and CD3 Gene

(145) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 96 (Table 12) and SEQ ID NO: 73 (Table 12). The primer binding to the 5 end of OX40 has the 12-nucleotide sequence of CD28 TM, and the primer binding to the 3 end of CD3-iso1 has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of CD28 TM-OX40-CD3-iso1-Xho I (Table 13). The amplified PCR product was used in the next PCR amplification process.

5.1.4.3. Obtainment of 3E8 LS, L1-H8 scFv, Hinge, TM, Costimulatory Domain, and CD3 Gene

(146) Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-OX40 and CD28 TM-OX40-CD3-iso1-Xho I, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 73 (Table 12) (FIG. 38). The amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-OX40-CD3-iso1-Xho I and a structure of L1-H8-CAR-001-OX (FIG. 39).

5.1.5. Obtainment of L1-H8-CAR-001-BB Gene

5.1.5.1. Obtainment of 3E8 Antibody Leader Sequence (LS), L1-H8 scFv, Hinge, and TM Gene

(147) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 97 (Table 12). The primer binding to the 5 end of the 3E8 leader sequence (LS) has the nucleotide sequence of Mlu I restriction enzyme and the 18-nucleotide sequence of the 3E8 leader sequence (LS), and the primer binding to the 3 end of CD28 TM has the 12-nucleotide sequence of 4-1BB, and thus the amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-4-1BB (Table 13) The amplified product was used in the next PCR amplification process.

5.1.5.2. Obtainment of Costimulatory Domain and CD3 Gene

(148) pMT-L1-H8-CAR-004 (FIG. 30) as a template was amplified by PCR using the primers of SEQ ID NO: 98 (Table 12) and SEQ ID NO: 73 (Table 12). The primer binding to the 5 end of 4-1BB has the 12-nucleotide sequence of CD28 TM, and the primer binding to the 3 end of CD3-iso1 has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of CD28 TM-4-1BB-CD3-iso1-Xho I (Table 13). The amplified PCR product was used in the next PCR amplification process.

5.1.5.3. Obtainment of 3E8 LS, L1-H8 scFv, Hinge, TM, Costimulatory Domain, and CD3 Gene

(149) Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-4-1BB and CD28 TM-4-1BB-CD3-iso1-Xho I, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 73 (Table 12) (FIG. 40). The amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-4-1BB-CD3-iso1-Xho I and a structure of L1-H8-CAR-001-BB (FIG. 41).

5.1.6. Obtainment of L1-H8-CAR-001-ICOS Gene

5.1.6.1. Obtainment of 3E8 Antibody Leader Sequence (LS), L1-H8 scFv, Hinge, and TM Gene

(150) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 99 (Table 12). The primer binding to the 5 end of the 3E8 leader sequence (LS) has the nucleotide sequence of Mlu I restriction enzyme and the 18-nucleotide sequence of the 3E8 leader sequence (LS), and the primer binding to the 3 end of CD28 TM has the 13-nucleotide sequence of ICOS-ICD, and thus the amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-ICOS ICD (Table 13). The amplified product was used in the next PCR amplification process.

5.1.6.2. Obtainment of Costimulatory Domain ICOS Gene

(151) The pBHA-ICOS TM+ICD (FIG. 33) as a template was amplified by PCR using the primers of SEQ ID NO: 100 (Table 12) and SEQ ID NO: 91 (Table 12). The primer binding to the 5 end of ICOS ICD has the 12-nucleotide sequence of CD28 TM, and the primer binding to the 3 end of ICOS ICD has the nucleotide sequence of CD3-iso1, and thus the amplified PCR product has the nucleotide sequence of CD28 TM-ICOS ICD-CD3-iso1 (Table 13). The amplified PCR product was used in the next PCR amplification process.

5.1.6.3. Obtainment of CD3 Gene

(152) pMT-L1-H8-CAR-001 as a template was amplified by PCR using the primers of SEQ ID NO: 92 (Table 12) and SEQ ID NO: 73 (Table 12). The primer binding to the 5 end of CD3-iso1 has the 12-nucleotide sequence of ICOS ICD, and the primer binding to the 3 end of CD3-iso1 has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of ICOS ICD-CD3-iso1-Xho I (Table 13). The amplified product was used in the next PCR amplification process.

5.1.6.4. Obtainment of 3E8 LS, L1-H8 scFv, Hinge, TM, and Costimulatory Domain Gene

(153) Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-ICOS ICD and CD28 TM-ICOS ICD-CD3-iso1, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 91 (Table 12) (FIG. 42). The amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-ICOS ICD-CD3-iso1 (Table 13). The amplified PCR product was used in the next PCR amplification process.

5.1.6.5. Obtainment of 3E8 LS, L1-H8 scFv, Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene

(154) Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-ICOS ICD-CD3-iso1 and ICOS ICD-CD3-iso1-Xho I, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 70 (Table 12) and SEQ ID NO: 73 (Table 12) (FIG. 42). The amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-hIgD hinge-CD28 TM-ICOS ICD-CD3-iso1-Xho I and a structure of L1-H8-CAR-001-ICOS (FIG. 43).

5.1.7. Preparation of pMT-L1-H8-CAR Retroviral Vectors

(155) Six types of the amplified PCR products were treated with Mlu I and Xho I restriction enzymes to obtain DNA fragments. The obtained DNA fragments were ligated to the pMT retroviral vectors (U.S. Pat. No. 6,451,595) previously treated with Mlu I and Xho I restriction enzymes to prepare six types of pMT-L1-H8-CAR retroviral vectors (FIG. 44). The pMT-L1-H8-CAR retroviral vectors thus prepared include sequences encoding L1-H8-CAR under the control of the MLV LTR promoter.

5.2. Preparation of Retroviruses Expressing L1-H8-CAR Genes with Various Costimulatory Domain Structures

(156) Seven types of retroviruses expressing L1-H8-CAR-001 and L1-H8-CAR-001-28BB, -28ICOS, -28, -OX, -BB, and -ICOS were prepared by the same method as in Example 4.2.

5.3. Preparation of T Cells Expressing L1-H8-CAR Genes with Various Costimulatory Domain Structures

(157) Seven types of L1-H8-CAR-T were prepared by the same method as in Example 4.3. The results verified that although there is a difference depending on the donor, the expression rate of L1-H8-CAR was about 7.7% to 88.4% on day 7 or day 8 of incubation, about 9.0% to 82.4% on day 11 of incubation, and about 6.7% to 89.8% on day 15 or day 17 of incubation (Table 14).

(158) TABLE-US-00021 TABLE 14 Expression rates of L1-H8-CAR on surfaces of L1-H8-CAR-expressing T cells L1-H8- L1-H8- L1-H8- L1-H8- L1-H8- L1-H8- Donor Days of L1-H8- CAR-001- CAR-001- CAR-001- CAR-001- CAR-001- CAR-001- NO. incubation Control CAR-001 28BB 28ICOS 28 OX BB ICOS 39 8 Days 1.14% 76.8% 68.7% 75.4% 31.9% 88.4% 63.6% 67.4% 15 Days 0.96% 79.0% 72.4% 74.6% 33.3% 89.8% 59.3% 65.6% 37 7 Days 1.87% 66.1% 65.2% 68.8% 76.7% 7.7% 52.7% 62.5% 11 Days 0.39% 64.8% 46.4% 58.6% 74.0% 9.0% 31.8% 52.6% 17 Days 0.41% 62.4% 58.3% 62.5% 83.2% 6.7% 35.2% 50.5% 40 7 Days 2.21% 60.0% 59.8% 64.3% 76.3% 11.3% 50.5% 55.3% 11 Days 0.90% 70.7% 53.2% 67.1% 82.4% 12.2% 39.9% 59.2% 17 Days 0.32% 86.3% 82.4% 80.6% 88.5% 33.9% 65.5% 67.4%

5.4. Verification of Anticancer Activity of T Cells Expressing L1-H8-CAR Genes with Various Spacer Domain Structures (In Vitro)

5.4.1. Verification of Expression Rates of L1CAM in Target Cells

(159) The expression rate of L1CAM in target cells was investigated by the same method as in Example 4.4.1. The results verified that the L1CAM expression rate was about 80.4 to 98.5% in SKOV3 cancer cells (FIGS. 45A to 45C). As a result of investigating the expression of L1CAM in the human cervical cancer cell line HeLa, the human neuroblastoma cell line SH-SY5Y, and the human embryonic kidney cell line 293T by the same method, the expression rate was about 99.6 to 99.9% in HeLa (FIGS. 45F and 45G), about 52.1 to 98.1% in SH-SY5Y (FIGS. 45H and 45I), and about 0.023 to 4.72% in 293T (FIGS. 45D and 45E).

5.4.2. Verification of Anticancer Activity of L1CAM-Expressing T Cells on Target Cells (In Vitro)

5.4.2.1. Verification of Anticancer Activity Using xCelligence Assay

(160) The activity of seven types of L1-H8-CAR on SKOV3 was investigated by the same method as in Example 4.4.2.1. As a result, seven types of T cells expressing L1-H8-CAR-001 and L1-H8-CAR-001-28BB, -28ICOS, -28, -OX, -BB, and -ICOS showed high cytotoxicity on SKOV3 cells compared with T cells not expressing L1-H8-CAR (control) (FIG. 46).

(161) The cytotoxicity on 293T cells were investigated by the same method as in Example 4.4.2.1. As a result, all the seven types showed similar cytotoxicity to the control in 293T cells showing a low expression rate of L1CMA (FIG. 47).

5.4.2.2. Verification of Anticancer Activity Using CellTox Green Dye

(162) The cytotoxicity on SH-SY5Y cells were investigated by the same method as in Example 4.4.2.2. As a result, seven types of T cells expressing L1-H8-CAR-001 and L1-H8-CAR-001-28BB, -28ICOS, -28, -OX, -BB, and -ICOS showed high cytotoxicity on SH-SY5Y cells compared with T cells not expressing L1-H8-CAR (control) (FIG. 48A to 48C).

(163) The cytotoxicity on HeLa cells were investigated by the same method as in Example 4.4.2.2. As a result, seven types of T cells expressing L1-H8-CAR-001 and L1-H8-CAR-001-28BB, -28ICOS, -28, -OX, -BB, and -ICOS showed high cytotoxicity on HeLa cells compared with T cells not expressing L1-H8-CAR (control) (FIG. 49A to 49C).

5.5. Verification of Anticancer Activity of T Cells Expressing L1-H8-CAR Genes with Various Spacer Domain Structures (In Vivo)

(164) To investigate anticancer activity of anti-L1CAM-CAR(L1-H8-CAR) gene-expressing T cells in vivo, cancer-induced animal models were used. SKOV3 cancer cells (Target, T) mixed with Matrigel at 1:1 were subcutaneously (SC) administered at 310.sup.6 to the right flank of NOD/SCID mice (7 weeks old, female) lacking T cells, B cells, and natural killer cells (NK cells), to thereby induce cancer. Seven types of L1-H8-CAR-expressing T cells confirmed to have efficacy in vitro and control T cells were administered to each NOD/SCID mouse 3 days and 5 days after cancer cell administration, once a day, a total of 2 times. T cells were administered through the tail vein (intravenous, IV) at 210.sup.7 per dose, and the cancer size was measured up to day 25. The results verified that all the seven types of anti-L1CAM-CAR-expressing T cells inhibited the cancer growth rate compared with the control T cell administration group. It was especially verified that the cancer growth inhibitory effect of L1-H8-CAR-001-28ICOS was best (FIG. 50).

Example 6: Fabrication of Anti-L1CAM-CAR-Expressing T Cells With Various Structures and Verification of Activity Thereof

6.1. Obtainment of L1CMA-CAR Genes with Various Structures

6.1.1. Obtainment of L1-H8-CAR-005 Gene

6.1.1.1. Obtainment of 3E8 Antibody Leader Sequence (LS) and L1-H8 scFv_Reverse Gene

(165) The structure of 3E8 LS, L1-H8 scFv antibody light chain variable region (VL), linker, and L1-H8 scFv antibody heavy chain variable region (VH) was synthesized. The pBHA-3E8-H8Rev (FIG. 51) obtained through gene synthesis as a template was amplified by PCR using the primers of SEQ ID NO: 70 (Table 15) and SEQ ID NO: 103 (Table 15). The primer binding to the 5 end of the 3E8 leader sequence (LS) has the nucleotide sequence of Mlu I restriction enzyme and the 18-nucleotide sequence of the 3E8 leader sequence (LS), and the primer binding to the 3 end of L1-H8 scFv-Reverse has the 12-nucleotide sequence of hIgD hinge, and thus the amplified PCR product has the nucleotide sequence of Mlu I-3E8 LS-L1-H8 scFv-Rev-IgD hinge (Table 16) The amplified product was used in the next PCR amplification process.

(166) TABLE-US-00022 TABLE15 Nucleotidesequenceinformationofusedprimers SEQID NO. Primername Nucleotidesequence 70 Mlu1+3E8VH(F) ACGCGTATGGAATGGAGCTGGGTC 103 L1-H8HC+IgDhinge(R) ACCTGGCCAGCGCGATGAGACGGTCAC 104 L1-H8HC+IgDhinge(F) ACCGTCTCATCGCGCTGGCCAGGTTCT 73 XhoI+CD3zeta(R) CCGCTCGAGTTAGCGAGGGGGCAGGGC 105 AS+MluI+2173-CD8a_LS(F) CGACGCGTATGGCCCTCCCTGTCACCG 106 2173-CD8a_LS+C9ScFv(R) CAACTGTACTTCGGGCCGAGCGGCGTG 107 2173-CD8a_LS+C9ScFv(F) GCCGCTCGGCCCGAAGTACAGTTGGTC 108 C9ScFv+hCD8a_Hinge(R) TGGGGTAGTGGTTTTAATTTCCACTTT 109 C9ScFv+hCD8a_Hinge(F) GTGGAAATTAAAACCACTACCCCAGCA 110 AS+XhoI+2173-0D3zeta(R) CCGCTCGAGTTACCGAGGCGGCAGGGC 111 AS+MluI+GMCSFrec.aLS(F) CGACGCGTATGCTTCTCCTGGTGACAA 112 GMCSFrec.aLS+L1-H8 CAACTGTACTTCTGGGATCAGGAGGAA scFv(R) 113 GMCSFrec.aLS+L1-H8 CTCCTGATCCCAGAAGTACAGTTGGTC scFv(F) 114 L1-H8scFv+hinge+hCD28(R) AATTGCGGCCGCTTTAATTTCCACTTT 115 L1-H8scFv+hinge+hCD28(F) GTGGAAATTAAAGCGGCCGCAATTGAA 116 AS+XhoI+CD3-(R) CCGCTCGAGTTATTAGCGAGGGGGCAGG

(167) TABLE-US-00023 TABLE16 LS,L1-H8scFv,Hinge,TM,ICD,costimulatorydomain, andCD3genesequences ID Nucleotidesequence MluI-start ACGCGTATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACTACA codon-3E8LS GGTGTCCACTCC L1-H8scFv- GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCG Rev ATCGTGTGACAATTACTTGTCGCGCTAGCCAGTCTATCTCTCGTGATCTGA (L1CAM-3R- ACTGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTACGCA H8Rev) GCATCCTCTCTGCAGTCTGGTGTACCGTCCCGTTTCTCTGGCAGCGGTTC TGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGC CACCTATTATTGTCAGCAATCTTACTCTACTCCGTACACGTTCGGGCAGGG AACTAAAGTGGAAATTAAAGGTGGAGGCGGTTCAGGCGGAGGTGGATCC GGCGGTGGCGGATCGGAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTC GTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTA CTTTCTCTGATTATGCAATGAATTGGGTTCGTCAGGCGCCGGGCAAGGGT CTCGAATGGGTTTCAGCAATCTCTTCTACTGGTTCTACTATCTACTATGCCG ATTCAGTGAAGGGTCGCTTTACCATTTCCCGTGACAACTCTAAGAATACTC TGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTAT TGCGCCAAACAGTCTACTTACTTTTACTCTTACTTTGATGTTTGGGGTCAG GGCACTTTAGTGACCGTCTCATCG IgDhinge CGCTGGCCAGGTTCTCCAAAGGCACAGGCCTCCTCCGTGCCCACTGCAC AACCCCAAGCAGAGGGCAGCCTCGCCAAGGCAACCACAGCCCCAGCCA CCACCCGTAACACAGGTAGAGGAGGAGAAGAGAAGAAGAAGGAGAAGGA GAAAGAGGAACAAGAAGAGAGAGAGACAAAGACACCAGGTTGTCCG CD28TM TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCT AGTAACAGTGGCCTTTATTATTTTCTGGGTG CD28ICD AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCC CCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCA CGCGACTTCGCAGCCTATCGCTCC OX40 GCCCTGTACCTGCTCCGGAGGGACCAGAGGCTGCCCCCCGATGCCCAC AAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAG GCCGACGCCCACTCCACCCTGGCCAAGATC CD3-iso1- AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGC stopcodon- CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGA XhoI TGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCC GCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAA GATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCC GGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCAC CAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA CTCGAG

6.1.1.2. Obtainment of Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene

(168) The pMT-L1-H8-CAR-003 plasmid (FIG. 23), comprising human IgD hinge and IgG1 hinge, CD28 TM and ICD, the costimulatory domain OX40, and CD3-iso1, as a template, was amplified by PCR using the primers of SEQ ID NO: 104 (Table 15) and SEQ ID NO: 73 (Table 15) before use. The primer binding to the 5 end of the hIgD hinge has the 12-nucleotide sequence of the heavy chain variable region (VH) of L1-H8 scFv antibody, and the primer binding to the 3 end of CD3-iso1 has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of L1-H8 scFv-Rev-IgD hinge-IgG1 hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I (Table 16). The amplified PCR product was used in the next PCR amplification process.

6.1.1.3. Obtainment of 3E8 LS, L1-H8 scFv-Rev, Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene

(169) Mlu I-3E8 LS-L1-H8 scFv-Rev-IgD hinge and L1-H8 scFv-Rev-IgD hinge-IgG1 hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 70 (Table 15) and SEQ ID NO: 73 (Table 15) (FIG. 52). The amplified PCR product has the nucleotide sequence of Mlu I-3E8-L1-H8 scFv-Rev-IgD hinge-IgG1 hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I, and has a structure of L1-H8-CAR-005 (FIG. 53).

6.1.2. Obtainment of L1-H8-CAR-006 Gene

6.1.2.1. Obtainment of CD8 Alpha Leader Sequence (LS) Gene

(170) pMT-CAR-005 (FIG. 54) plasmid, comprising CD8 alpha LS, as a template, was amplified by PCR using the primers of SEQ ID NO: 105 (Table 15) and SEQ ID NO: 106 (Table 15). The primer binding to the 5 end of the CD8 alpha leader sequence (LS) has the nucleotide sequence of Mlu I restriction enzyme and the 18-nucleotide sequence of the CD8 alpha leader sequence (LS), and the primer binding to the 3 end of CD8 alpha leader sequence (LS) has the 12-nucleotide sequence of L1-H8 scFv antibody heavy chain variable region (VH), and thus the amplified PCR product has the nucleotide sequence of Mlu I-hCD8 LS-L1-H8 scFv (Table 17) The amplified product was used in the next PCR amplification process.

(171) TABLE-US-00024 TABLE17 LS,L1-H8scFv,Hinge,TM,ICD,costimulatorydomain, andCD3genesequences ID Nucleotidesequence MluI-startcodon- ACGCGTATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACTAC hCD8LS AGGTGTCCACTCC L1-H8scFv GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGT (L1CAM-3R-H8) TCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCTGATTATG CAATGAATTGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTT CAGCAATCTCTTCTACTGGTTCTACTATCTACTATGCCGATTCAGTGAAGG GTCGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAG ATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAAC AGTCTACTTACTTTTACTCTTACTTTGATGTTTGGGGTCAGGGCACTTTAG TGACCGTCTCATCGGGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGC GGTGGCGGATCGGACATTCAAATGACGCAGAGTCCCTCCTCACTGAGT GCTAGCGTGGGCGATCGTGTGACAATTACTTGTCGCGCTAGCCAGTCTA TCTCTCGTGATCTGAACTGGTATCAGCAGAAACCGGGCAAGGCGCCAAA ATTGCTGATTTACGCAGCATCCTCTCTGCAGTCTGGTGTACCGTCCCGTT TCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCT CCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCAATCTTACTCTACTCC GTACACGTTCGGGCAGGGAACTAAAGTGGAAATTAAA hCD8ahinge ACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCC TCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGT GGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGAT hCD8aTM ATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTT CACTCGTGATCACTCTTTACTGT 4-1BB AAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGA GGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCC CAGAGGAGGAGGAAGGCGGCTGCGAACTG CD3-iso2M-stop CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGG codon-XhoI CAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACG ACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAG CCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAG GATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCA GAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCC ACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG TAACTCGAG

6.1.2.2. Obtainment of L1-H8 scFv Gene

(172) pMT-L1-H8-CAR-001 (FIG. 23) plasmid, comprising L1-H8 scFv, as a template, was amplified by PCR using the primers of SEQ ID NO: 107 (Table 15) and SEQ ID NO: 108 (Table 15) before use. The primer binding to the 5 end of L1-H8 scFv has the 12-nucleotide sequence of CD8 alpha LS, and the primer binding to the 3 end of L1-H8 scFv 3 has the 12-nucleotide sequence of hCD8 alpha Hinge, and thus the amplified PCR product has the nucleotide sequence of hCD8 LS-L1-H8 scFv-hCD8 hinge (Table 17). The amplified PCR product was used in the next PCR amplification process.

6.1.2.3. Obtainment of Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene

(173) The pMT-CAR-005 plasmid (FIG. 54), comprising human CD8 alpha hinge, TM, the costimulatory domain 4-1BB, and CD3-iso2M, as a template, was amplified by PCR using the primers of SEQ ID NO: 109 (Table 15) and SEQ ID NO: 110 (Table 5) before use. The primer binding to the 5 end of the hCD8 hinge has the 12-nucleotide sequence of the light chain variable region (VL) of L1-H8 scFv antibody, and the primer binding to the 3 end of CD3-iso2M has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of L1-H8 scFv-hCD8 hinge-hCD8 TM-4-1BB-CD3-iso2M-Xho I (Table 17). The amplified PCR product was used in the next PCR amplification process.

6.1.2.4. Obtainment of CD8 LS and L1-H8 scFv Gene

(174) Mlu I-hCD8 LS-L1-H8 scFv and hCD8 LS-L1-H8 scFv-hCD8 hinge, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 105 (Table 15) and SEQ ID NO: 108 (Table 15) (FIG. 55). The amplified PCR product has the nucleotide sequence of Mlu I-hCD8 LS-L1-H8 scFv-CD28 hinge. The amplified PCR product was used in the next PCR amplification process.

6.1.2.5. Obtainment of CD8 LS, L1-H8 scFv, Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene

(175) Mlu I-hCD8 LS-L1-H8 scFv-hCD8hinge and L1-H8 scFv-hCD8 hinge-hCD8 TM-4-1BB-CD3-iso2M-Xho I, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 105 (Table 15) and SEQ ID NO: 110 (Table 15) (FIG. 55). The amplified PCR product has the nucleotide sequence of Mlu I-hCD8LS-L1-H8 scFv-hCD8 hinge-hCD8 TM-4-1BB-CD3-iso2M-Xho I, and has a structure of L1-H8-CAR-006 (FIG. 56).

6.1.3. Obtainment of L1-H8-CAR-007 Gene

6.1.3.1. Obtainment of hGM-CSF Receptor Alpha-Chain Signal Sequence Gene

(176) The pMT-CAR-006 (FIG. 57) plasmid, comprising the hGM-CSF rec. signal sequence, as a template, was amplified by PCR using the primers of SEQ ID NO: 111 (Table 15) and SEQ ID NO: 112 (Table 15). The primer binding to the 5 end of the hGM-CSF rec. has the nucleotide sequence of Mlu I restriction enzyme, and the primer binding to the 3 end of hGM-CSF rec. has the 12-nucleotide sequence of L1-H8 scFv heavy chain variable region (VH), and thus the amplified PCR product has the nucleotide sequence of Mlu I-hGM-CSF rec.-L1-H8 scFv (Table 18). The amplified product was used in the next PCR amplification process.

(177) TABLE-US-00025 TABLE18 LS,L1-H8scFv,Hinge,TM,ICD,costimulatorydomain, andCD3genesequences ID Nucleotidesequence MluI-start ACGCGTATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACTACAG codon-hGM- GTGTCCACTCC CSFrec.aLS L1-H8scFv GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTC (L1CAM-3R- ACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCTGATTATGCAATG H8) AATTGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCAGCAATC TCTTCTACTGGTTCTACTATCTACTATGCCGATTCAGTGAAGGGTCGCTTTAC CATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGC GTGCCGAAGACACGGCCGTCTATTATTGCGCCAAACAGTCTACTTACTTTTAC TCTTACTTTGATGTTTGGGGTCAGGGCACTTTAGTGACCGTCTCATCGGGTG GAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATTCAA ATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACA ATTACTTGTCGCGCTAGCCAGTCTATCTCTCGTGATCTGAACTGGTATCAGCA GAAACCGGGCAAGGCGCCAAAATTGCTGATTTACGCAGCATCCTCTCTGCA GTCTGGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACC CTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGTCAGCA ATCTTACTCTACTCCGTACACGTTCGGGCAGGGAACTAAAGTGGAAATTAAA hinge GCGGCCGCA hCD28pECD ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAAC CATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGAC CTTCTAAGCCC hCD28TM TTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATAGCTTGCTA GTAACAGTGGCCTTTATTATTTTCTGGGTG hCD28ICD AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCC CGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACG CGACTTCGCAGCCTATCGCTCC CD3-iso2- AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCA stopcodon- GAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTT XhoI TTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGC GGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGG GGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACG ACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAATAATAACTCGAG

6.1.3.2. Obtainment of L1-H8 scFv Gene

(178) The pMT-L1-H8-CAR-001 (FIG. 23) plasmid, comprising L1-H8 scFv, as a template, was amplified by PCR using the primers of SEQ ID NO: 113 (Table 15) and SEQ ID NO: 114 (Table 15) before use. The primer binding to the 5 end of L1-H8 scFv has the 12-nucleotide sequence of hGM-CSF rec. LS, and the primer binding to the 3 end of L1-H8 scFv has the 9-nucleotide sequence of Hinge and the 3-nucleotide sequence of hCD28 pECD, and thus the amplified PCR product has the nucleotide sequence of hGM-CSF rec. LS-L1-H8 scFv-hinge-hCD28 pECD (Table 18). The amplified PCR product was used in the next PCR amplification process.

6.1.3.3. Obtainment of Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene

(179) The pMT-CAR-006 plasmid (FIG. 57), comprising Hinge, hCD28 pECD, TM, ICD, and hCD3-iso2, as a template, was amplified by PCR using the primers of SEQ ID NO: 115 (Table 13) and SEQ ID NO: 116 (Table 13). The primer binding to the 5 end of Hinge has the 12-nucleotide sequence of the light chain variable region (VL) of L1-H8 scFv, and the primer binding to the 3 end of CD3-iso2 has the nucleotide sequence of Xho I restriction enzyme, and thus the amplified PCR product has the nucleotide sequence of L1-H8 scFv-Hinge-hCD28 pECD-hCD28 TM-hCD28 ICD-CD3-iso2-Xho I (Table 16). The amplified product was used in the next PCR amplification process.

6.1.3.4. Obtainment of hGM-CSF Receptor Alpha-Chain Signal Sequence and L1-H8 scFv Gene

(180) Mlu I-hGM-CSF rec.-L1-H8 scFv and hGM-CSF rec. LS-L1-H8 scFv-hinge-hCD28 pECD, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 111 (Table 15) and SEQ ID NO: 114 (Table 15) (FIG. 58). The amplified PCR product has the nucleotide sequence of Mlu I-hGM-CSF rec.-L1-H8 scFv-hinge-hCD28 pECD. The amplified PCR product was used in the next PCR amplification process.

6.1.3.5. Obtainment of hGM-CSF Receptor Alpha-Chain Signal Sequence, L1-H8 scFv, Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene

(181) Mlu I-hGM-CSF rec.-L1-H8 scFv-hinge-hCD28 pECD and L1-H8 scFv-Hinge-hCD28 pECD-hCD28 TM-hCD28 ICD-CD3-iso2-Xho I, which were the amplified PCR products, as templates, were amplified by OE-PCR using the primers of SEQ ID NO: 111 (Table 15) and SEQ ID NO: 116 (Table 15) (FIG. 58). The amplified PCR product has the nucleotide sequence of Mlu I-hGM-CSF rec.-L1-H8 scFv-hinge-hCD28 pECD-hCD28 TM-hCD28 ICD-CD3-iso2-Xho I and the structure of L1-H8-CAR-007 (FIG. 59).

6.1.4. Preparation of pMT-L1-H8-CAR Retroviral Vectors

(182) Three types of the amplified PCR products were treated with Mlu I and Xho I restriction enzymes to obtain DNA fragments. The obtained DNA fragments were ligated to the pMT retroviral vectors (U.S. Pat. No. 6,451,595) previously treated with Mlu I and Xho I restriction enzymes to prepare three types of pMT-L1-H8-CAR retroviral vectors (FIG. 60). The pMT-L1-H8-CAR retroviral vectors thus prepared include a sequence encoding L1-H8-CAR under the control of the MLV LTR promoter.

6.2. Preparation of Retroviruses Expressing L1-H8-CAR Genes with Various Structures (L1-H8-CAR Retroviruses)

(183) Four types of retroviruses expressing L1-H8-CAR-003, -005, -006, and -007 genes were prepared by the same method as in Example 4.2.

6.3. Preparation of T Cells Expressing L1-H8-CAR Genes with Various Structures

(184) Four types of L1-H8-CAR-T were prepared by the same method as in Example 4.3. The results verified that although there is a difference depending on the donor, the expression rate of L1-H8-CAR was about 22.1% to 74.1% on day 8 of incubation, about 27.1% to 77.1% on day 11 of incubation, about 24.6% to 76.6% on day 14 of incubation, and about 29.8% to 81.9% on day 16 of incubation (Table 19).

(185) TABLE-US-00026 TABLE 19 Expression rates of L1-H8-CAR on surfaces of L1-H8-CAR-expressing T cells L1-H8- L1-H8- L1-H8- L1-H8- Donor Days of CAR- CAR- CAR- CAR- NO. culture Control 003 005 006 007 37 8 Days 1.07% 67.1% 22.1% 71.4% 63.4% 11 Days 1.75% 65.7% 27.1% 72.8% 61.2% 14 Days 1.01% 60.0% 24.6% 64.5% 52.7% 16 Days 0.81% 69.5% 29.8% 78.3% 70.7% 50 8 Days 1.36% 73.2% 34.5% 74.1% 63.3% 11 Days 1.73% 75.6% 39.0% 77.1% 68.3% 14 Days 0.87% 76.6% 35.4% 69.8% 62.5% 16 Days 0.59% 81.9% 43.9% 81.8% 74.3%

6.4. Verification of Anticancer Activity of T Cells Expressing L1-H8-CAR Genes with Various Structures (In Vitro)

6.4.1. Verification of Expression Rates of L1CAM in Target Cells

(186) The expression rate of L1CAM in target cells was investigated by the same method as in Example 4.4.1. The results verified that the L1CAM expression rate was about 67.1% to 87.0% in SKOV3 cancer cells. As a result of investigating the expression of L1CAM in the human cervical cancer cell line HeLa, the human neuroblastoma cell line SH-SY5Y, and the human embryonic kidney cell line 293T, the expression rate was about 98.4% in HeLa, about 65.0 to 70.9% in SH-SY5Y, and about 0.082% in 293T (FIGS. 61A to 61F).

6.4.2. Verification of Anticancer Activity of L1CAM-Expressing T Cells on Target Cells (In Vitro)

6.4.2.1. Verification of Anticancer Activity Using xCelligence Assay

(187) The ability of four types of L1-H8-CAR on SKOV3 were investigated by the same method as in Example 4.4.2.1. As a result, four types of T cells expressing L1-H8-CAR-003 and L1-H8-CAR-005, -006, and -007 showed high cytotoxicity on SKOV3 cells compared with T cells not expressing L1-H8-CAR (control) (FIG. 62).

(188) The cytotoxicity on SH-SY5Y cells were investigated by the same method as in Example 4.4.2.1. The target cells were added at 1.010.sup.5 to 50 uL of culture media, and after about 21 hours, L1-H8-CAR-expressing T cells were prepared at 5.010.sup.4, 1.010.sup.5, and 5.010.sup.5 (E:T ratio=0.5, 1, and 5) in 50 uL of AIMV media comprising human serum and human IL-2, and added to wells comprising target cells, to check the cell index value in real time for 30 hours. In addition, wells comprising only target cells were prepared, and the anticancer activity of L1-H8-CAR-expressing T cells was calculated in the same manner as in the above tests. As a result, four types of T cells expressing L1-H8-CAR-003 and L1-H8-CAR-005, -006, and -007 showed high cytotoxicity on SH-SY5Y cells compared with T cells not expressing L1-H8-CAR (control) (FIG. 63).

6.4.2.2. Verification of Anticancer Activity Using CellTox Green Dye

(189) The activity of four types of L1-H8-CAR on HeLa cells were investigated by the same method as in Example 4.4.2.2. As a result, four types of T cells expressing L1-H8-CAR-003 and L1-H8-CAR-005, -006, and -007 showed high cytotoxicity on HeLa cells compared with T cells not expressing L1-H8-CAR (control) (FIG. 64).

(190) The cytotoxicity on 293T cancer cells were investigated by the same method as in Example 4.4.2.2. The target cells were prepared at 1.010.sup.4 in 50 uL of culture media, and 0.2 uL of CellTox Green dye was added, and the mix was added to 96-well black plates. The L1-H8-CAR-expressing T cells were prepared at 5.010.sup.3, 1.010.sup.4, 5.010.sup.4, and 1.010.sup.5 (E:T ratio=0.5, 1, 5, and 10) in 50 uL of AIMV media comprising human serum and human IL-2, and added to wells comprising target cells, followed by incubation in a CO.sub.2 incubator at 37 C. for 24 hours. The cytotoxicity on the target cells was corrected and calculated by the same method.

(191) As a result, all the four types showed cytotoxicity similar to or lower than that of the control in 293T cells showing a low expression rate of L1CMA (FIG. 65).