Chimeric antigen receptor comprising anti c-met antibody or antigen binding fragment thereof, and use thereof

Abstract

The present invention relates to a chimeric antigen receptor comprising a c-Met binding domain, and a use thereof. The chimeric antigen receptor comprising a c-Met domain, of the present invention, can be effectively usable as an agent for treating various diseases associated with c-Met expression.

Claims

1. A nucleic acid molecule coding for an anti-c-Met chimeric antigen receptor, comprising: a c-Met-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the c-Met-binding domain is an antibody or an antigen binding fragment thereof binding specifically to c-Met, wherein the antibody or the antigen binding fragment thereof comprises a heavy chain variable region (VH) consisting of the amino acid sequence of SEQ ID NO: 21; and a light chain variable region (VL) consisting of the amino acid sequence of SEQ ID NO: 22.

2. The nucleic acid molecule of claim 1, wherein the chimeric antigen receptor further comprises a leader sequence (LS).

3. The nucleic acid molecule of claim 2, wherein the leader sequence is a leader sequence of hCD8 alpha, a leader sequence of hGM-CSF receptor alpha-chain, or a leader sequence of 3E8 antibody.

4. The nucleic acid molecule of claim 2, wherein the leader sequence is a leader sequence comprising the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 26, 28, or 30.

5. The nucleic acid molecule of claim 1, wherein the c-Met-binding domain is linked to the transmembrane domain by a hinge region, a spacer region, or a combination thereof.

6. The nucleic acid molecule of claim 5, wherein the hinge region and the spacer region is a hinge of IgG1, a hinge of IgG4, a hinge of IgD, a hinge of CD8 alpha, IgG1 CH3, an extracellular domain of CD28, or a combination thereof.

7. The nucleic acid molecule of claim 5, wherein the hinge region or the spacer region comprises the amino acid sequence encoded by the nucleotide sequence of 32, 34, 36, 38, 40, or 44.

8. The nucleic acid molecule of claim 1, wherein the chimeric antigen receptor comprises a transmembrane domain of a protein selected from the group consisting of a T-cell receptor, CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

9. The nucleic acid molecule of claim 8, wherein the transmembrane domain comprises the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 42, 46, or 47.

10. The nucleic acid molecule of claim 1, wherein the intracellular signaling domain comprises a signaling domain of CD3 zeta.

11. The nucleic acid molecule of claim 10, wherein the signaling domain of CD3 zeta comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 55, 57, or 59.

12. The nucleic acid molecule of claim 1, wherein the intracellular signaling domain further comprises as a costimulatory domain a signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137).

13. The nucleic acid molecule of claim 12, wherein the costimulatory domain comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 49, 51, or 53.

14. An anti-c-Met chimeric antigen receptor molecule comprising a polypeptide encoded by the nucleic acid molecule of claim 1.

15. An effector cell, having the anti-c-Met chimeric antigen receptor molecule of claim 14 expressed on a surface thereof.

16. The effector cell of claim 15, wherein the effector cell is selected from the group consisting of dendritic cells, killer dendritic cells, mast cells, natural killer cells, B lymphocytes, T lymphocytes, macrophages, and progenitor cells thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram illustrating the structure of c-Met-CAR-001 construct according to the present disclosure.

(2) FIG. 2 is a map illustrating the structure of pBHA-c-Met-CAR-001 plasmid according to the present disclosure.

(3) FIG. 3 is a map illustrating the structure of pMT-CAR-001 plasmid according to the present disclosure.

(4) FIG. 4 is a map illustrating the structure of pMT-CAR-002 plasmid according to the present disclosure.

(5) FIG. 5 is a map illustrating the structure of pMT-CAR-003 plasmid according to the present disclosure.

(6) FIG. 6 is a schematic diagram illustrating a PCR amplification procedure for c-Met-CAR-002 according to the present disclosure.

(7) FIG. 7 is a schematic diagram illustrating the structure of c-Met-CAR-002 construct according to the present disclosure.

(8) FIG. 8 is a schematic diagram illustrating a PCR amplification procedure for c-Met-CAR-003 according to the present disclosure.

(9) FIG. 9 is a schematic diagram illustrating the structure of c-Met-CAR-003 construct according to the present disclosure.

(10) FIG. 10 is a schematic diagram illustrating a PCR amplification procedure for c-Met-CAR-004 according to the present disclosure.

(11) FIG. 11 is a schematic diagram illustrating the structure of c-Met-CAR-004 construct according to the present disclosure.

(12) FIGS. 12a, 12b, 12c and 12d are maps illustrating the structures of pMT-c-Met-CAR-001, pMT-c-Met-CAR-002, pMT-c-Met-CAR-003, and pMT-c-Met-CAR-004 plasm ids according to the present disclosure.

(13) FIGS. 13a, 13b and 13c are views illustrating the CAR expression as confirmed by the expression of CD3 zeta in the anti-c-Met-CAR-expressing T cells according to the present disclosure.

(14) FIG. 14 are views illustrating the CAR expression on the surface of the anti-c-Met-CAR-expressing T cells according to the present disclosure.

(15) FIGS. 15a, 15b, 15c, 15d, 15e and 15f are views illustrating c-Met expression levels in the cancer cell lines A549, PC-3, MCF-7, SKOV3, SK-HEP-1, and Jurkat.

(16) FIGS. 16a, 16b, 16c and 16d are plots illustrating anticancer activity of the anti-c-Met CAR-expressing T cells according to the present disclosure against A549 cancer cell line.

(17) FIGS. 17a, 17b, 17c and 17d are plots illustrating anticancer activity of the anti-c-Met CAR-expressing T cells according to the present disclosure against PC-3 cancer cell line.

(18) FIGS. 18a, 18b, 18c and 18d are plots illustrating anticancer activity of the anti-c-Met CAR-expressing T cells according to the present disclosure against MCF-7 cancer cell line.

(19) FIGS. 19a. 19b, 19c and 19d are plots illustrating anticancer activity of the anti-c-Met CAR-expressing T cells according to the present disclosure against SKOV3 cancer cell line.

(20) FIG. 20 is a plot illustrating anticancer activity of the anti-c-Met CAR-expressing T cells according to the present disclosure against SK-HEP-1 cancer cell line.

(21) FIGS. 21a, 21b and 21c are plots illustrating anticancer activity of the anti-c-Met CAR-expressing T cells according to the present disclosure against Jurkat cell line.

DETAILED DESCRIPTION

(22) A better understanding of the present disclosure may be obtained via the following examples which are set forth to illustrate, but are not construed to limit the present disclosure.

EXAMPLES

(23) 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 List

(24) Examples 1 to 4: Preparation of Anti-c-Met-Chimeric Antigen Receptor Gene Example 5. Construction of pGemT-c-Met-CAR Vector Example 6. Construction of pMT-c-Met-CAR Retroviral Vector Example 7. Preparation of Anti-c-Met-CAR Gene-Expressing T Cell Example 8. In Vitro Assay for Anticancer Activity of Anti-c-Met-CAR-Expressing T Cell

Example 1. Preparation of c-Met-CAR-001 Gene

Example 1-1. Preparation of Anti-c-Met scFv Antibody Gene

(25) The nucleotide sequence of the polynucleotide coding for the heavy-chain variable region and light-chain variable region of the c-Met-specific antibody of the present disclosure was obtained through the previous application (Korean Patent Application No. 10-2018-0140196) (Table 1).

(26) TABLE-US-00001 TABLE1 Nucleotidesequenceoffinallyselectedunique anti-c-MetscFvclones SEQ ID ID Nucleotidesequence NO 1E4- CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAGCC 24 H4k2 CGGCTCCTCCGTGAAGGTCTCCTGCCAGGGCTCCGGCTACTC CTTCCCCACCCACTGGATCACCTGGGTGCGACAGGCCCCCGG CCAAGGCCTGGAATGGATGGGCACCATCGACCCCACCGACTC CTACAACTTCTACGGCCCCAGCTTCCAGGGCAGAGTGACCAT CACCGCCGACTCCTCCACGTCCACCGCCTACATGGAGCTGTC CTCCCTGAGATCTGAGGACACCGCCATGTACTACTGCGCCAG GGACGGCAACTACTACGACTCCCGGGGCTACTACTACGATAC CTTCGACATGTGGGGCCAGGGCACCCTGGTCACCGTCTCCTC AGGCGGTGGAGGATCTGGAGGAGGCGGCTCTGGGGGGGGC GGCTCTGACATCCAGATGACCCAGTCCCCCAGCTCCCTGTCC GCCTCCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCTCC CAGGGCATCTCCACCTACCTGGCCTGGTATCAGCAGAAGCCC GGCAAAGCCCCCAAGCTGCTGATCTACTCCGCCTCCACCCTG GAATCCGGCGTGCCCTCCAGATTCTCCGGCTCCGGCTCTGGC ACCGACTTCACCCTGACCATCTCCAGCCTGCAGCCCGAGGAC TTTGCCACCTACTACTGCCAGCAGGCCGACTCCTTCCCCCTG ACCTTCGGCGGAGGCACCAAGGTGGAAATCAAACGT

Example 1-2. Preparation of c-Met-CAR-001 Gene

(27) A nucleotide sequence for the anti-c-Met scFv antibody of the present disclosure was designed to include the BamH I restriction site and the leader sequence of CD8 alpha in the 5 region of the variable heavy chain (VH) and the hinge and TM of hCD8 alpha, the costimulatory domains 4-1BB, CD3-iso2M (modified CD3-iso2), and the Xho I restriction site in the 3 region of the variable light chain (VL). The nucleotide sequence thus obtained has the structure of BamH I-hCD8LS-scFv-hCD8 hinge-hCD8TM-41BB-CD3-iso2M-Xho I (Table 2). Based on this structure, c-Met-CAR-001 construct of SEQ ID NO: 60 (FIG. 1) was synthesized. The synthesized pBHA-c-Met-CAR-001 (FIG. 2) was used to establish other c-Met-CAR constructs.

(28) TABLE-US-00002 TABLE2 LS,Hinge,TM,ICD,CostimulatoryDomain,and CD3GeneSequences ID Nucleotidesequence BamHI-start GGATCCATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGC codon- TCTTCTGCTCCACGCCGCTCGGCCC hCD8aLS ScFv (Table1) hCD8ahinge ACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCAT CGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCG CAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTG CGAT hCD8aTM ATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCT GCTTTCACTCGTGATCACTCTTTACTGT 4-1BB AAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTT CATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCAT GCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG CD3-iso2M- CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCA stopcodon- GGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAG XhoI AGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGA AATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTG TACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGA GATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGAC GGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGA CGCTCTTCACATGCAGGCCCTGCCGCCTCGGTAACTCGAG

Example 2. Preparation of c-Met-CAR-002 Gene

Example 2-1. Preparation of Anti-c-Met scFv Antibody Gene

(29) Amplification was performed by PCR using the primers of SEQ ID NOS: 1 and 2 (Table 3) on the template pBHA-c-Met-CAR-001 (FIG. 2) obtained through gene synthesis. In this regard, the primer designed to bind to the 5 region of the variable heavy chain (VH) of the anti-c-Met scFv antibody had 12 nucleotide sequences of hGM-CSF rec. (human GM-CSF receptor alpha-chain) while the primer designed to bind to the 3 region of the variable light chain (VL) of the anti-c-Met scFv antibody had nine nucleotide sequences of hinges and three nucleotide sequences of hCD28 pECD. The PCR product thus obtained had the nucleotide sequence of hGM-CSF rec.-scFv-hinge-hCD28 pECD (Table 4). The PCR product was used in subsequent PCR procedures.

(30) TABLE-US-00003 TABLE3 InformationonNucleotideSequenceofPrimer SEQID NO Primer Nucleotidesequence 1 GMCSFrec.aLS+1E4-H4k2 CTCCTGATCCCACAGGTGCAGCTGGTG scFv(F) 2 1E4-H4k2scFv+ AATTGCGGCCGCACGTTTGATTTCCAC hinge+hCD28pECD(R) 3 AS+BamHI+GMCSFrec.a CGGGATCCATGCTTCTCCTGGTGACAA LS(F) 4 GMCSFrec.aLS+1E4-H4k2 CAGCTGCACCTGTGGGATCAGGAGGAA scFv(R) 5 1E4-H4k2scFv+hinge+ GAAATCAAACGTGCGGCCGCAATTGAA CD28pECD(F) 6 AS+Xho1+CD3-(R) CCGCTCGAGTTATTAGCGAGGGGGCAGG 7 3E8LS+1E4-H4k2scFv(F) GGTGTCCACTCCCAGGTGCAGCTGGTG 8 1E4-H4k2scFv+hIgD ACCTGGCCAGCGACGTTTGATTTCCAC hinge(R) 9 BamHI+3E8VH(F) GGATCCATGGAATGGAGCTGGGTC 10 1E4-H4k2scFv+3E8LS(R) CAGCTGCACCTGGGAGTGGACACCTGT 11 1E4-H4k2scFv+hIgD GAAATCAAACGTCGCTGGCCAGGTTCT hinge(F) 12 XhoI+CD3zeta(R) CCGCTCGAGTTAGCGAGGGGGCAGGGC 13 T7(F) TATACGACTCACTATAGGG 14 SP6(R) ATTTAGGTGACACTATAG

Example 2-2. Preparation of Signal Sequence Gene of hGM-CSF Receptor Alpha-Chain

(31) Amplification was performed by PCR using the primers of SEQ ID NOS: 3 and 4 (Table 3) on the template pMT-CAR-001 plasmid carrying the signal sequence of hGM-CSF rec.. In this regard, the primer designed to bind to a 5 region of hGM-CSF rec. had the BamH I restriction site while the primer designed to bind to a 3 region of hGM-CSF rec. had 12 nucleotide sequences of the variable heavy chain (VH) of the anti-c-Met antibody. The PCR product thus obtained had the nucleotide sequence of BamHI-hGM-CSF rec.-scFv (Table 4). The PCR product was used in subsequent PCR procedures.

(32) TABLE-US-00004 TABLE4 LS,Hinge,TM,ICD,CostimulatoryDomain,andCD3Gene Sequences ID Nucleotidesequence BamHI-start GGATCCATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTT codon-hGM- ACCACACCCAGCATTCCTCCTGATCCCA CSFrec.aLS ScFv (Table1) hinge GCGGCCGCA hCD28pECD ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGC AATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAG TCCCCTATTTCCCGGACCTTCTAAGCCC hCD28TM TTTTGGGTGCTGGTGGTGGTTGGGGGAGTCCTGGCTTGCTATA GCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG hCD28ICD AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACA TGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCC CTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC CD3-iso2-stop AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGC codon-XhoI AGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAG AGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCT GAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGC CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGC ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACAC CTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAATAA CTCGAG

Example 2-3. Preparation of Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene Sequences

(33) PCR was performed using the primers of SEQ ID NOS: 5 and 6 (Table 3) on the template pMT-CAR-001 plasmid including a hinge, pECD and TM of hCD28, ICD, and hCD3-iso2 (FIG. 3). In this regard, the primers designed to bind to 5 regions of the hinge had 12 nucleotide sequences of the variable light chain (VL) of anti-c-Met antibody while the primer designed to bind to a 3 region of CD3-iso2 had a Xho I restriction site. The PCR product thus obtained had the nucleotide sequence of scFv-Hinge-hCD28 pECD-hCD28 TM-hCD28 ICD-CD3-iso2-XhoI (Table 4). The PCR product was used in subsequent PCR procedures.

Example 3. Preparation of c-Met-CAR-003 Gene

Example 3-1. Preparation of Anti-c-Met scFv Antibody Gene

(34) Amplification was performed by PCR using the primers of SEQ ID NOS: 7 and 8 (Table 3) on the template pBHA-c-Met-CAR-001 (FIG. 2) obtained through gene synthesis. In this regard, the primer designed to bind to a 5 region of the variable heavy chain (VH) of the anti-c-Met scFv antibody had 12 nucleotide sequences of 3E8 leader sequence (LS) while the primer designed to bind to a 3 region of the variable light chain (VL) of the anti-c-Met scFv antibody had 12 nucleotide sequences of hIgD hinge. The PCR product thus obtained had the nucleotide sequence of 3E8 LS-scFv-hIgD hinge (Table 5). The PCR product was used in subsequent PCR procedures.

(35) TABLE-US-00005 TABLE5 LS,Hinge,TM,ICD,CostimulatoryDomain,andCD3Gene Sequence ID Nucleotidesequence BamHI- GGATCCATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAAC startcodon- TACAGGTGTCCACTCC 3E8LS ScFv (Table1) IgDhinge CGCTGGCCAGGTTCTCCAAAGGCACAGGCCTCCTCCGTGCCCACTG CACAACCCCAAGCAGAGGGCAGCCTCGCCAAGGCAACCACAGCCC CAGCCACCACCCGTAACACAGGTAGAGGAGGAGAAGAGAAGAAGAA GGAGAAGGAGAAAGAGGAACAAGAAGAGAGAGAGACAAAGACACCA GGTTGTCCG IgG1hinge GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA IgG1CH3 GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG GATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA CD28TM TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTT GCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG CD28ICD AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGC CCCACCACGCGACTTCGCAGCCTATCGCTCC OX40 GCCCTGTACCTGCTCCGGAGGGACCAGAGGCTGCCCCCCGATGCC CACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAG GAGCAGGCCGACGCCCACTCCACCCTGGCCAAGATC CD3-iso1- AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAG stopcodon- GGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG XhoI AGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGG GGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAAT GAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGA TGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACC AGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACAT GCAGGCCCTGCCCCCTCGCTAACTCGAG

Example 3-2. Preparation of Leader Sequence (LS) Gene of 3E8 Antibody

(36) Amplification was performed by PCR using the primers of SEQ ID NOS: 9 and 10 (Table 3) on the template pMT-CAR-002 plasmid carrying the leader sequence (LS) of 3E8 antibody (FIG. 4). In this regard, the primer designed to bind to a 5 region of 3E8 leader sequence (LS) had the BamH I restriction site while the primer designed to bind to a 3 region of 3E8 leader sequence (LS) had 12 nucleotide sequences of the variable heavy chain (VH) of the anti-c-Met antibody. The PCR product thus obtained had the nucleotide sequence of BamH I-3E8 LS-scFv (Table 5). The PCR product was used in subsequent PCR procedures.

Example 3-3. Preparation of Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene Sequences

(37) PCR was performed using the primers of SEQ ID NOS: 11 and 12 (Table 3) on the template pMT-CAR-002 plasmid including a hinge of human IgD, a hinge and CH3 of IgG1, TM and ICD of CD28, and the costimulatory domains OX40 and CD3-iso1 (FIG. 4). In this regard, the primer designed to bind to a 5 region of the hIgD hinge had 12 nucleotide sequences of the variable light chain (VL) of anti-c-Met antibody while the primer designed to bind to a 3 region of CD3-iso1 had a Xho I restriction site. The PCR product thus obtained had the nucleotide sequence of scFv-IgD hinge-IgG1 hinge-CH3-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I (Table 5). The PCR product was used in subsequent PCR procedures.

Example 4. Preparation of c-Met-CAR-004 Gene

Example 4-1. Preparation of Anti-c-Met scFv Antibody Gene

(38) Amplification was performed by PCR using the primers of SEQ ID NOS: 7 and 8 (Table 3) on the template pBHA-c-Met-CAR-001 (FIG. 2) obtained through gene synthesis. In this regard, the primer designed to bind to a 5 region of the variable heavy chain (VH) of the anti-c-Met scFv antibody had 12 nucleotide sequences of 3E8 leader sequence (LS) while the primer designed to bind to a 3 region of the variable light chain (VL) of the anti-c-Met scFv antibody had 12 nucleotide sequences of hIgD hinge. The PCR product thus obtained had the nucleotide sequence of 3E8 LS-scFv-hinge-hIgD hinge (Table 6). The PCR product was used in subsequent PCR procedures.

(39) TABLE-US-00006 TABLE6 LS,Hinge,TM,ICD,CostimulatoryDomain,andCD3 GeneSequence ID Nucleotidesequence BamHI- GGATCCATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAA startcodon- CTACAGGTGTCCACTCC 3E8LS ScFv (Table1) IgDhinge CGCTGGCCAGGTTCTCCAAAGGCACAGGCCTCCTCCGTGCCCACT GCACAACCCCAAGCAGAGGGCAGCCTCGCCAAGGCAACCACAGC CCCAGCCACCACCCGTAACACAGGTAGAGGAGGAGAAGAGAAGA AGAAGGAGAAGGAGAAAGAGGAACAAGAAGAGAGAGAGACAAAG ACACCAGGTTGTCCG CD28TM TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGC TTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG CD28ICD AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATG ACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTAT GCCCCACCACGCGACTTCGCAGCCTATCGCTCC OX40 GCCCTGTACCTGCTCCGGAGGGACCAGAGGCTGCCCCCCGATGC CCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAG AGGAGCAGGCCGACGCCCACTCCACCCTGGCCAAGATC CD3-iso1- AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCA stopcodon- GGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA XhoI GGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGAT GGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGT ACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGA TTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGG CCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC CCTTCACATGCAGGCCCTGCCCCCTCGCTAACTCGAG

Example 4-2. Preparation of Leader Sequence (LS) Gene of 3E8 Antibody

(40) Amplification was performed by PCR using the primers of SEQ ID NOS: 9 and 10 (Table 3) on the template pMT-CAR-003 plasmid carrying the leader sequence (LS) of 3E8 antibody (FIG. 5). In this regard, the primer designed to bind to a 5 region of 3E8 leader sequence (LS) had the BamH I restriction site while the primer designed to bind to a 3 region of 3E8 leader sequence (LS) had 12 nucleotide sequences of the variable heavy chain (VH) of the anti-c-Met antibody. The PCR product thus obtained had the nucleotide sequence of BamH I-3E8 LS-scFv (Table 6). The PCR product was used in subsequent PCR procedures.

Example 4-3. Preparation of Hinge, TM, ICD, Costimulatory Domain, and CD3 Gene Sequences

(41) PCR was performed using the primers of SEQ ID NOS: 11 and 12 (Table 3) on the template pMT-CAR-003 plasmid including the hinge of human IgD, TM and ICD of CD28, and the costimulatory domains OX40 and CD3-iso1 (FIG. 5). In this regard, the primer designed to bind to a 5 region of the hIgD hinge had 12 nucleotide sequences of the variable light chain (VL) of anti-c-Met antibody while the primer designed to bind to a 3 region of CD3-iso1 had a Xho I restriction site. The PCR product thus obtained had the nucleotide sequence of scFv-hIgD hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I (Table 6). The PCR product was used in subsequent PCR procedures.

Example 5. Construction of pGemT-c-Met-CAR Vector

Example 5-1. Construction of pGemT-c-Met-CAR-002 Vector

(42) Amplification was performed by OE-PCR (overlap extension PCR) using the primers of SEQ ID NOS: 2 and 3 (Table 3), with the PCR products BamH I-hGM-CSF rec.-scFv and hGM-CSF rec.-scFv-hinge-hCD28 pECD serving as templates. OE-PCR was performed using the primers of SEQ ID NOS: 3 and 6 (Table 3) while the amplicons BamH I-hGM-CSF rec.-scFv-hinge-hCD28 pECD and scFv-Hinge-hCD28 pECD-hCD28 TM-hCD28 ICD-CD3-iso2-Xho I were used as templates (FIG. 6). The PCR product thus obtained had the nucleotide sequence of SEQ ID NO: 61 and was named c-Met-CAR-002 which was structured to include BamH I-hGM-CSF rec.-scFv-Hinge-hCD28 pECD-hCD28 TM-hCD28 ICD-CD3-iso2-Xho I (FIG. 7). This PCR product was ligated to pGemT ESAY vector (Promega, WI, USA) containing poly-T sequences at both ends of the linear DNA to attain pGemT-c-Met-CAR-002 construct. Sequencing analysis with the aid of primers of SEQ ID NOS: 13 and 14 (Table 3) confirmed that the amplicon has the same original sequence.

Example 5-2. Construction of pGemT-c-Met-CAR-003 Vector

(43) Amplification was performed by OE-PCR (overlap extension PCR) using the primers of SEQ ID NOS: 9 and 8 (Table 3), with the PCR products BamH I-3E8 LS-scFv and 3E8 LS-scFv-hIgD hinge serving as templates. OE-PCR was performed using the primers of SEQ ID NOS: 9 and 12 (Table 3) while the amplicons BamH I-3E8 LS-scFv-hIgD hinge and scFv-IgD hinge-IgG1 hinge-CH3-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I were used as templates (FIG. 8). The PCR product thus obtained had the nucleotide sequence of SEQ ID NO: 62 and was named c-Met-CAR-003 which was structured to include BamH I-3E8 LS-scFv-hIgD hinge-IgG1 hinge-CH3-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I (FIG. 9). This PCR product was ligated to pGemT ESAY vector containing poly-T sequences at both ends of the linear DNA to attain pGemT-c-Met-CAR-003 construct. Sequencing analysis with the aid of primers of SEQ ID NOS: 13 and 14 (Table 3) confirmed that the amplicon has the same original sequence.

Example 5-3. Construction of pGemT-c-Met-CAR-004 Vector

(44) Amplification was performed by OE-PCR (overlap extension PCR) using the primers of SEQ ID NOS: 9 and 8 (Table 3), with the PCR products BamH I-3E8 LS-scFv and 3E8 LS-scFv-hIgD hinge serving as templates. OE-PCR was performed using the primers of SEQ ID NOS: 9 and 12 (Table 3) while the amplicons BamH I-3E8 LS-scFv-hIgD hinge and scFv-IgD hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I were used as templates (FIG. 10). The PCR product thus obtained had the nucleotide sequence of SEQ ID NO: 63 and was named c-Met-CAR-004 which was structured to include BamH I-3E8 LS-scFv-IgD hinge-CD28 TM-CD28 ICD-OX40-CD3-iso1-Xho I (FIG. 11). This PCR product was ligated to pGemT ESAY vector containing poly-T sequences at both ends of the linear DNA to attain pGemT-c-Met-CAR-004 construct. Sequencing analysis with the aid of primers of SEQ ID NOS: 13 and 14 (Table 3) confirmed that the amplicon has the same original sequence.

Example 6. Construction of pMT-c-Met-CAR Retroviral Vector

(45) A single type of pBHA-c-Met-CAR-001 and three types of pGemT-c-Met-CAR vectors (pGemT-c-Met-CAR-002, pGemT-c-Met-CAR-003, pGemT-c-Met-CAR-004) were treated with BamH I and Xho I restriction enzymes to obtain DNA fragments. These DNA fragments were ligated to the pMT retroviral vector (U.S. Pat. No. 6,451,595) previously cut with BamH I and Xho I restriction enzymes to construct four types of pMT-c-Met-CAR retroviral vectors (pMT-c-Met-CAR-001, -002, -003, and -004) (FIGS. 12a to 12d). The pMT-c-Met-CAR retroviral vectors each include a sequence encoding c-Met-CAR under the control of the MLV LTR promoter.

Example 7. Preparation of Anti-c-Met-CAR Gene-Expressing T Cell

Example 7-1. Construction of Anti-c-Met-CAR Gene-Expressing Retrovirus

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

Example 7-2. Preparation of Anti-c-Met-CAR Gene-Expressing T Cell

(47) Mononuclear cells were obtained from the blood of a donor by using SepMate-50 (STEMCELL) and Ficoll-Paque PLUS (GE healthcare, Sweden). The mononuclear cells were dispensed at 110.sup.7 cells in 100-mm dishes while AIMV medium (Invitrogen) supplemented with 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-c-Met-CAR retroviruses.

(48) 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) containing 2.5% human serum 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 the wells were washed by addition of HBSS containing 2.5% of 1 M HEPES at 3 mL per well. The anti-c-Met-CAR retroviruses were diluted to 310.sup.10 copies per well with AIMV media containing 5% human serum, and 4 mL of the dilution was added, followed by centrifugation under conditions of 2000g 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. Activated T cells were added at 210.sup.6 cells per well, followed by centrifugation at 1000g for 15 minutes, thereby delivering anti-c-Met-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 rounds of delivery was performed. After 24 hours of delivery, the T cells were all harvested, and subcultured in T flasks at 510.sup.5 cells per mL with AIMV media containing 5% human serum and 300 U/mL human IL-2. The cells were passaged at 510.sup.5 cells per mL every 3-4 days, and maintained so as not to exceed 210.sup.6 cells per mL.

(49) Next, it was investigated whether the anti-c-Met-CAR was expressed in the activated T cells (anti-c-Met-CAR-expressing T cells) to which the anti-c-Met-CAR retroviruses had been delivered. Proteins were extracted from 210.sup.6 cells harvested, and quantitatively analyzed by the Bradford assay. The proteins were mixed with a 4 sample buffer (Invitrogen) and dithiothreitol and then boiled at 95 for 5 minutes for reduction. The expression of anti-c-Met-CAR was confirmed by western blotting. To this end, mouse anti-human CD247 (BD, CA, USA) was used as a primary antibody against CD3 while goat anti-mouse IgG(H+L)-HRP (Thermo, USA) was used as a secondary antibody. The western blotting analysis for the four types of anti-c-Met-CAR-expressing T cells detected a band at 50-80 KDa in each cell, demonstrating the expression of anti-c-Met-CAR (FIGS. 13a to 13c).

(50) It was investigated whether the anti-c-Met-CAR-expressing T cells expressed anti-c-Met-CAR on the surface thereof. After being harvested, 510.sup.5 cells were washed twice with PBS (phosphate buffered saline) and incubated at 4 C. for 30 minutes with 2.5 g of FITC-conjugated Protein L (AcroBiosystem, RPL-PF141), which binds specifically to the single-chain variable fragment of CAR. After incubation, the cells were washed twice with PBS and analyzed for the expression of the anti-c-Met-CAR by flow cytometry. As a result, about 57.7% of the T cells to which MT-c-Met-CAR-004 retrovirus had been transfected were observed to have the anti-c-Met-CAR expressed on the surface thereof (FIG. 14).

Example 8. In Vitro Assay For Anticancer Activity of Anti-c-Met-CAR-Expressing T Cell

Example 8-1. Assay For Expression Rate of c-Met on Target Cell

(51) The lung cancer cell line A549 is known to express c-Met at a high level and as such, exhibits adequacy for examining the anticancer activity of the anti-c-Met-CAR-expressing T cells. In this regard, 510.sup.5 A549 cells in 100 L of PBS was incubated with 1 g of 1E4-H4k2 antibody at 4 C. for 30 minutes. After incubation, the cells were washed twice with PBS and reacted with 2 L of goat anti-human IgG-PE (Southern Biotech) at 4 C. for 30 minutes. Thereafter, the cells were washed twice with PBS and analyzed for c-Met expression by flow cytometry. As a result, the A549 cancer cells were observed to exhibit a c-Met expression rate of about 83.9%. In the same manner, c-Met was measured to be expressed at a rate of about 48.5% in the human prostate cancer cell line PC-3, about 66.0% in the human breast cancer cell line MCF-7, about 58.0% in the ovarian cancer cell line SKOV3, about 99.9% in the human liver adenocarcinoma cell line SK-HEP-1, and about 2.37% in the human acute T cell leukemia cell line Jurkat.

Example 8-2. Assay For Anticancer Activity Using CellTox Green Dye

(52) To investigate the anticancer activity of the anti-c-Met-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 a density of 110.sup.4 cells per 50 L of a culture medium, and added with 0.2 L of CellTox Green dye, and the mix was seeded to 96-well black plates. The anti-c-Met-CAR-expressing T cells were prepared at a density of 510.sup.3, 110.sup.4, or 510.sup.4 cells (E:T ratio=0.5, 1, and 5) in 50 L of AIMV medium containing human serum and human IL-2, and added to wells containing the target cells, followed by incubation in a CO.sub.2 incubator at 37 C. for 24 hours. The group added with only anti-c-Met-CAR-expressing T cells was prepared in the wells containing CellTox Green dye and the target cell culture medium, and the reaction value of the dye, occurring by attachment to DNA released from dead anti-c-Met-CAR-expressing T cells during the incubation was excluded. The wells containing only target cells were prepared to correct the low control (spontaneous DNA release) value, and a lysis solution was added to the well containing only the target cells to correct the high control (maximum DNA release) value. The cytotoxicity on the target cells was calculated by the following equation.
Cytotoxicity %={(Reaction value of Target cells and Effector cells)(Reaction value of Effector cells)}(Low control)/(High controlLow control)100Equation

(53) As a result, four types of T cells expressing c-Met-CAR-001, -002, -003, and -004 showed high cytotoxicity against A549 cancer cells, compared with T cells expressing no anti-c-Met-CAR (FIG. 16).

(54) In the same assay manner, cytotoxicity against PC-3 cancer cells was confirmed. T cells expressing c-Met-CAR-001, -002, -003, and -004 were observed to exhibit higher anticancer activity against PC-3 cells, compared with T cells expressing no anti-c-Met-CAR (FIG. 17). Particularly, c-Met-CAR-001 was inferior to the other CAR structures in terms of cytotoxic effect.

(55) In the same assay manner, cytotoxicity against MCF-7 cancer cells was confirmed. T cells expressing c-Met-CAR-001, -002, -003, and -004 were observed to exhibit higher anticancer activity against MCF-7 cells, compared with T cells expressing no anti-c-Met-CAR (FIG. 18).

(56) In the same assay manner, cytotoxicity against SKOV3 cancer cells was confirmed. T cells expressing c-Met-CAR-001, -002, -003, and -004 were observed to exhibit higher anticancer activity against SKOV3 cells, compared with T cells expressing no anti-c-Met-CAR (FIG. 19).

(57) In the same assay manner, cytotoxicity against SK-HEP-1 was confirmed. T cells expressing c-Met-CAR-001, -002, -003, and -004 were observed to exhibit higher anticancer activity against SK-HEP-1 cancer cells, compared with T cells expressing no anti-c-Met-CAR (FIG. 20).

(58) In the same assay manner, cytotoxicity against Jurkat was examined. T cells expressing c-Met-CAR-001, -002, -003, and -004 were observed to have almost no cytotoxicity against Jurkat, which expresses c-Met at a low level. Taken together, the data imply that anti-c-Met-CAR-expressing T cells exhibit specific cytotoxicity only for cells expressing high levels of c-Met (FIG. 21).

(59) Although the preferred embodiments of the present disclosure have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

(60) This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as the sequence listing text file entitled 000302usnp_SequenceListing. TXT, file size 37 kilobytes (KB), created on 24 Mar. 2022. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. 1.52 (e) (5).