CHIMERIC ANTIGEN RECEPTOR FOR SOLID CANCER AND T CELLS EXPRESSING CHIMERIC ANTIGEN RECEPTOR

Abstract

Disclosed is a chimeric antigen receptor with improved persistency.

Claims

1. A chimeric antigen receptor, which comprises IL-7Rα or a part thereof.

2. The chimeric antigen receptor of claim 1, wherein the IL-7Rα or a part thereof is interposed between a costimulatory domain and a signaling domain.

3. The chimeric antigen receptor of claim 2, wherein the signaling domain is CD3ζ domain.

4. The chimeric antigen receptor of claim 1, wherein the part of IL-7Rα is cleaved cytoplasmic domain of IL-7Rα.

5. The chimeric antigen receptor of claim 1, wherein a part of IL-7Rα is a sequence of SEQ ID NO: 15.

6. The chimeric antigen receptor of claim 3, which comprises three immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3ζ domain, wherein in a third ITAM region, a first motif, YxxL, is substituted with YxxQ; and a second motif region, YxxLHM, is substituted with YxYVTM, wherein x is any amino acid.

7. The chimeric antigen receptor of claim 3, which comprises three immunoreceptor tyrosine-based activation motifs (ITAMs) within the CD3ζ domain, wherein a third ITAM region is mutated to a sequence of SEQ ID NO: 31.

8. The chimeric antigen receptor of claim 3, wherein the CD3ζ domain is a sequence represented by SEQ ID NO: 18.

9. The chimeric antigen receptor of claim 1, wherein the antigen binding domain of the chimeric antigen receptor binds to an antigen selected from the group consisting of IL13Rα2, an antigen associated with an angiogenesis activity, EGFRvIII, EphA2, αVβ3, mesothelin, and glypican.

10. The chimeric antigen receptor of claim 2, wherein the costimulatory domain comprises one or more selected from the group consisting of 4-1BB and a CD28 domain.

11. The chimeric antigen receptor of claim 1, for the treatment of solid cancer or blood cancer.

12. A nucleic acid encoding the chimeric antigen receptor of claim 1.

13. A CAR expression vector comprising a nucleic acid of claim 12.

14. A CAR-T cell, wherein the chimeric antigen receptor of claim 1 is expressed.

15. An anticancer agent comprising the CAR-T cell of claim 13.

16. A polypeptide comprising a chimeric antigen receptor of claim 1, which further comprises an antigen binding domain; a hinge region; a transmembrane domain; a costimulatory domain; and a signaling domain, wherein the signaling domain comprises a CD3ζ domain.

17. A nucleic acid encoding the polypeptide of claim 16.

18. A CAR expression vector comprising a nucleic acid of claim 17.

19. A CAR-T cell, wherein the polypeptide of claim 16 is expressed.

20. An anticancer agent comprising the CAR-T cell of claim 19.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0059] FIG. 1 shows a diagram illustrating a process of effectively treating solid cancer by a CAR-containing polypeptide according to the present invention.

[0060] FIG. 2 shows a diagram and a table illustrating an antigen binding domain which can be used for a CAR-containing polypeptide platform according to the present invention.

[0061] FIG. 3 shows diagrams illustrating the introduction of a cytokine signaling domain into CAR-T cells so as to control CAR tonic signaling in the CAR-containing polypeptide according to the present invention.

[0062] FIG. 4 shows diagrams illustrating the introduction of a chimeric switch receptor so as to convert an immune suppressive signal in a hostile tumor microenvironment into an activation signal in CAR-T cells.

[0063] FIG. 5 shows diagrams illustrating the release of cytokine IL-21 to the outside of CAR-T cells so as to aid the activation of innate immune-related cells and to increase the content of less-differentiated memory CAR-T cells.

[0064] FIG. 6 shows diagrams illustrating the structures of CAR-containing polypeptides according to the present invention.

[0065] FIG. 7 shows diagrams illustrating the self-cleaving peptides used in the CAR-containing polypeptides according to the present invention.

[0066] FIG. 8 shows a graph and tables illustrating the growth rate and viability of CAR-T cells, which are transformed with the CAR of the present invention.

[0067] FIG. 9 shows graphs and a table illustrating the analysis results of the CAR expression rate of CAR-T cells, which were transformed with the CAR of the present invention, by flow cytometry analysis.

[0068] FIG. 10 shows a graph and a table illustrating the analysis results of the chimeric switch receptor expression rate of TGF-βR2 exodomain and transmembrane domain and an endodomain of IL18R of CAR-T cells, which were transformed with the CAR of the present invention, by flow cytometry analysis.

[0069] FIG. 11 shows the results of Day 10 phenotypes of CAR-T cells, which were transformed with the CAR of the present invention, by flow cytometry analysis.

[0070] FIG. 12 shows a graph, which illustrates the cytotoxicity of CAR-T cells, which were transformed with the CAR of the present invention, confirmed through LDH assay after 24 hours of co-culture with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells); and a table, which illustrates the purity and viability of CAR-T cells used in a CAR-T cytotoxicity test.

[0071] FIG. 13 shows graphs illustrating the analysis results of spontaneous toxicity of CAR-T cells, which were transformed with the CAR of the present invention, after 24 hours or 96 hours.

[0072] FIGS. 14 to 16 each show graphs and a table illustrating the analysis results of changes in a CAR expression rate of CAR-T cells, which were transformed with the CAR of the present invention, after 24 hours (FIG. 14), 48 hours (FIG. 15), and 96 hours (FIG. 16) of co-culture of the CAR-T cells with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells), by flow cytometry analysis.

[0073] FIG. 17 shows graphs and a table illustrating the analysis results of changes in the chimeric switch receptor expression rate of TGF-βR2 exodomain and transmembrane domain and an endodomain of IL-18R of CAR-T cells, which were transformed with the CAR of the present invention, after 24 hours of co-culture of the CAR-T cells, which were transformed with the CAR of the present invention, with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells), by flow cytometry analysis.

[0074] FIG. 18 shows graphs illustrating the comparison results of IFN-γ cytokine production after 24 hours and 48 hours of co-culture of CAR-T cells, which were transformed with the CAR of the present invention, with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells).

[0075] FIG. 19 shows graphs illustrating the comparison results of IL-21 cytokine production after 24 hours and 48 hours of co-culture of CAR-T cells, which were transformed with the CAR of the present invention, with U87 cells (human brain cancer cell line) and 293FT target cells (which were used as a control group for normal cells).

[0076] FIG. 20 shows a graph and tables illustrating the growth rate and viability of CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is EGFRvIII, in which the CAR expression rate and persistence in CAR-T cells were optimized, while simultaneously changing the target sequence so as to minimize the binding affinity for EGFR wild-type, where the specificity for EGFRvIII is maintained so as to reduce the side effects of CAR-T cells that appear through non-specific binding (YYB105, #13, #14) (see SEQ ID NOS: 39, 34, 35); and a graph and tables illustrating the growth rate and viability of CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is αVβ3, in which the CAR expression rate in CAR-T cells was optimized (YYB107, #15, #16) (see SEQ ID NOS: 38, 36, 37).

[0077] FIG. 21 shows a graph illustrating the CAR expression rate in CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is EGFRvIII, in which the CAR expression rate in CAR-T cells was optimized, while simultaneously changing the target sequence so as to minimize the binding affinity for EGFR wild-type, where the specificity for EGFRvIII is maintained so as to reduce the side effects of CAR-T cells that appear through non-specific binding (YYB105, #13, #14) (see SEQ ID NOS: 39, 34, 35); and a graph illustrating the CAR expression rate in CAR-T cells, which were transformed with a chimeric antigen receptor (CAR), when the cancer antigen is αVβ3, in which the CAR expression rate in CAR-T cells was optimized (YYB107, #15, #16) (see SEQ ID NOS: 38, 36, 37).

BEST MODE FOR CARRYING OUT THE INVENTION

[0078] Hereinafter, the present invention will be described in detail through examples. However, it should be noted that the following Examples are only for illustrating the present invention, and the spirit and technical scope of the present invention are not limited in any sense. Further, it should be understood that the present invention is not limited to the precise arrangement and means of the embodiments shown in the drawings, which are cited as reference.

Example 1: Preparation of Novel Chimeric Antigen Receptor (CAR) Platform for Effective Treatment by Specifically Binding to IL13Rα2 Overexpressed in Solid Cancer Cells

[0079] With regard to human IL13 (P35225.1), human CD3 (P20963-1), human CD8A (P01732), human CD28 (P10747), human CD3ζ (P20963), human 41BB (Q07011), IL7RA (P16871), IL2RB (P14784), TGFR2 (P37173), IL18R (Q13478), IL21 (Q9HBE4), IL2 (P60568), T2A, P2A, and human kappa light chain signal sequence (HuVHCAMP), CAR-containing polypeptides were prepared using a chimeric antigen-containing polypeptide (see SEQ ID NOS: 23 to 30 and 34 to 37; and #1, #2, #5, #6, #7, #8, #11, and #12 of FIG. 6) expression vector consisting of codon-optimized synthetic DNA, using scientific literature and publicly available databases (see FIGS. 6 and 7).

[0080] Specifically, T cells, in which a CAR-containing polypeptide is expressed, can be prepared by preparing a vector that expresses a CAR-containing polypeptide via conjugation of synthetic DNA to an MFG retrovirus expression vector digested with XhoI/NotI, followed by transduction of the vector into the T cells.

[0081] The completed structure of polypeptide #1 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human IL13.E11K.R64D.S67D.R107K mature protein sequence (see YYB-103 of PCT International Publication No. WO 2017/023138), three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein (see YYB-103 of PCT International Publication No. WO 2017/023138), a hinge region of human CD8α, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of cytokine IL-7RA (amino acid positions of 265 to 328; SEQ ID NO: 15), a mutant CD3ζ (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 31), T2A, a CD8A signal sequence (leader sequence), a chimeric switch receptor which comprises TGF-βR2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R (i.e., a cytokine receptor) (amino acid positions of 323 to 541; SEQ ID NO: 20), P2A, a human IL2 signal sequence (leader sequence), an IL-21 (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an XhoI/NotI cleavage site (see CAR-containing polypeptide #1 of FIG. 6; and FIG. 7).

[0082] The completed structure of CAR-containing polypeptide #2 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human IL13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8α, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL-7RA (amino acid positions of 265 to 328; SEQ ID NO: 15), a mutant CD3ζ (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 31), T2A, a CD8A signal sequence (leader sequence), a chimeric switch receptor which comprises TGF-βR2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R (i.e., a cytokine receptor) (amino acid positions of 323 to 541; SEQ ID NO: 20), and an XhoI/NotI cleavage site (see CAR-containing polypeptide #2 of FIG. 6; and FIG. 7).

[0083] The completed structure of CAR-containing polypeptide #5 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human IL13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8α, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL-7RA (amino acid positions of 265 to 328; SEQ ID NO: 15), a mutant CD3ζ (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 31), T2A, a human IL2 signal sequence, an IL-21 (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an XhoI/NotI cleavage site (see CAR-containing polypeptide #5 of FIG. 6; and FIG. 7).

[0084] The completed structure of CAR-containing polypeptide #6 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human IL13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8α, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cytoplasmic domain of a cytokine IL-7RA (amino acid positions of 265 to 328; SEQ ID NO: 15), a mutant CD3ζ (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 31), and an XhoI/NotI cleavage site (see CAR-containing polypeptide #6 of FIG. 6; and FIG. 7).

[0085] The completed structure of polypeptide CAR-containing #7 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human IL13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8α, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL2Rβ (amino acid positions of 266 to 369; SEQ ID NO: 17), a mutant CD3ζ (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), T2A, a CD8A signal sequence, a chimeric switch receptor which comprises a TGF-βR2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R (i.e., a cytokine receptor) (amino acid positions of 323 to 541; SEQ ID NO: 20), P2A, a human IL2 signal sequence (leader sequence), an IL-21 (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an XhoI/NotI cleavage site (see #7 of FIG. 6; and FIG. 7).

[0086] The completed structure of CAR-containing polypeptide #8 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human IL13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8α, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL2Rβ (amino acid positions of 266 to 369; SEQ ID NO: 19), a mutant CD3ζ (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), T2A, a CD8A signal sequence, a chimeric switch receptor which comprises a TGF-βR2 exodomain (amino acid positions of 23 to 166; SEQ ID NO: 19) and a transmembrane domain and an endodomain of IL-18R (i.e., a cytokine receptor) (amino acid positions of 323 to 541; SEQ ID NO: 20), and an XhoI/NotI cleavage site (see CAR-containing polypeptide #8 of FIG. 6; and FIG. 7).

[0087] The completed structure of CAR-containing polypeptide #11 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human IL13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8α, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL2Rβ (amino acid positions of 266 to 369; SEQ ID NO: 17), a mutant CD3ζ (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), T2A, a human IL2 signal sequence, an IL-21 (amino acid positions of 23 to 155; SEQ ID NO: 21) sequence, and an XhoI/NotI cleavage site (see CAR-containing polypeptide #11 of FIG. 6; and FIG. 7).

[0088] The completed structure of polypeptide CAR-containing #12 of FIG. 6 includes a Kozak consensus ribosome-binding sequence, a human kappa light chain signal sequence (HuVHCAMP), human IL13.E11K.R64D.S67D.R107K mature protein sequence, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a human hinge region of CD8α, a human CD8 transmembrane domain, a costimulatory signal domain of cytoplasmic 4-1BB, a cleaved cytoplasmic domain of a cytokine IL212β (amino acid positions of 266 to 369; SEQ ID NO: 17), a mutant CD3ζ (positions 91 to 113 of SEQ ID NO: 18 are mutated to a sequence of SEQ ID NO: 32), and an XhoI/NotI cleavage site (see #12 of FIG. 6; and FIG. 7).

[0089] The entire sequences of CAR-containing polypeptides #1, #2, #5, #6, #7, #8, #11, and #12 are shown in SEQ ID NOS: 23 to 30.

[0090] The finally prepared CAR gene fragments were conjugated to the MFG retrovirus expression vector digested with XhoI/NotI (Emtage P C et al., Clin Cancer Res, 2008, 14:8112-8122). In this Example, in order to compare the activity of chimeric antigen receptors, YYB103 (SEQ ID NO: 22) was further prepared.

Example 2: Preparation of CAR-T Cells Transformed with Novel Chimeric Antigen Receptors

[0091] High-titer CAR-expressing PG13 clones were prepared such that Phoenix-Ampho and Phoenix-Eco cells were transiently transfected with the retroviral expression vector prepared in Example 1, and then, cell-free vector stocks were prepared from the transfected Phoenix-Ampho and Phoenix-Eco cells by transfecting PG13 cells.

[0092] For high-titer monoclones, PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 cells were stained using an anti-IL-13 monoclonal antibody (BD Pharmingen), and these single clones were isolated using a flow cytometer. The PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 clones were prepared by the second subcloning according to the limiting dilution method. These subclones stably showed high CAR expression and were selected for the efficient transduction ability in the peripheral blood.

[0093] The transduction degree of PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 cells, which were transduced using an anti-IL-13 monoclonal antibody (BD Pharmingen), was analyzed using a flow cytometer. The supernatants of the transduced PG13/#1, PG13/#2, PG13/#5, PG13/#6, PG13/#7, PG13/#8, PG13/#11, or PG13/#12 cells contained retrovirus, and the supernatants were collected for genetic modification of T cells.

[0094] The peripheral blood mononuclear cells (PBMCs) were separated using centrifugation by adding the whole blood obtained from a healthy human donor into Ficoll Paque (GE Healthcare). The separated PBMCs were cultured by adding an anti-CD3 monoclonal antibody (eBioscience) at a concentration of 100 ng/mL under the condition of human IL-2 (Novartis) at a concentration of 100 IU/mL to activate the T cell fraction (BL Levine, Cancer Gene Therapy, 2015, 22:79-84). Two to three days after the cultivation, most of the cells were T cells and included natural killer cells at a percentage of 0% to 2%. Two to three days after the activation step, the T cells were subjected to transduction two times over two days using the retroviral supernatant and washed, and then proliferated for four to seven days in a flask. The cells were cultured in a stirring platform device (a WAVE bio-reactor system) for 12 to 28 days. IL-2 was maintained at a concentration of 100 IU/mL. The T cells modified as such were used for an analysis experiment.

Experimental Example 1: Checking of Growth Rate and Viability of CAR-T Cells Transformed with Novel Chimeric Antigen Receptors

[0095] Experimental Results

[0096] For the T cells prepared in Example 2, the number of cells was counted to confirm the growth rate and viability of CAR-T, and the results are shown in FIG. 8.

[0097] The number of cells and growth rate of all of the groups (#1, #2, #5, #6, #7, #8, #11, or #12) were shown to be higher than those of the control group (i.e., YYB103) depending on the day of the week, the cells were shown to grow rapidly from Day 12 of culture, and the viability was also shown to be 90% or higher (see FIG. 8).

Experimental Example 2: Checking of CAR Expression Rate on Surface of CAR-T Cells Transformed with Novel Chimeric Antigen Receptors

[0098] Experimental Method (Flow Cytometric Analysis)

[0099] For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a PE-conjugated anti-human IL-13 monoclonal antibody (BD Pharmingen) thereto. After washing, the cells were reacted with each antibody at 4° C. for 30 minutes in a state where light was blocked, and washed once, and thereafter, the expression rate of CAR on the surface of transduced T cells was checked.

[0100] Experimental Results

[0101] In order to confirm whether the 8 kinds of IL13Rα2-specific, CAR-containing polypeptides prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) were expressed on the T cell surface, T cell culture was performed for 28 days according to Example 2, and then flow cytometric analysis was performed according to the experimental method.

[0102] As a result of the analysis, as shown in FIG. 9, the expression rate of the chimeric antigen receptors expressed on the cell surface of live T cells was shown to be in the range of 24.5% to 84.2%, and the expression of IL13Rα2-specific chimeric antigen receptors was stably maintained for 4 weeks, without additional activation or transduction of T cells (see FIG. 9).

[0103] Experimental Example 3: Checking of chimeric switch receptor expression rates of TGF-βR2 exodomain and transmembrane domain and an endodomain of IL-18R on the cell surface of CAR-T cells transformed with novel chimeric antigen receptors

[0104] Experimental Methods (Flow Cytometric Analysis)

[0105] For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a human TGF-βR2 fluorescein-conjugated antibody (FAB2411F) (BD Pharmingen) thereto. After washing, the cells were reacted with each antibody at 4° C. for 30 minutes in a state where light was blocked and washed once, and thereafter, the chimeric switch receptor expression rates of TGF-βR2 exodomain and transmembrane domain and an endodomain of IL-18R on the cell surface of transduced T cells were checked.

[0106] Experimental Results

[0107] In order to confirm whether the chimeric switch receptors of TGFβR2 exodomain and transmembrane domain and an endodomain of IL-18R were expressed on the cell surface of CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), T cell cultivation was performed for 14 days according to Example 2, and then flow cytometric analysis was performed according to the experimental method. As a result of the analysis, the expression rates of the chimeric switch receptors of TGFβR2 exodomain and transmembrane domain and an endodomain of IL-18R on the cell surface of live T cells were shown to be about 85% (see FIG. 10).

Experimental Example 4: Checking of Phenotypes on the Cell Surface of CAR-T Cells Transformed with Novel Chimeric Antigen Receptors

[0108] Experimental Methods (Flow Cytometric Analysis)

[0109] For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and a BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding an FITC-conjugated CD45RA Ab (HI100) (Biolegend), a PE-conjugated CCR7Ab (G043H7) (Biolegend), and a PE-Cy7-conjugated CD62L Ab (DREG-56) (Biolegend) thereto. After washing, the cells were reacted with each antibody at 4° C. for 30 minutes in a state where light was blocked and washed once, and thereafter, the content of less-differentiated memory CAR-T cells and CCR7.sup.+CD45RA.sup.+CD62L.sup.+ phenotypes on the surface of transduced T cells were checked.

[0110] Experimental Results

[0111] In order to confirm the content of the less-differentiated memory CAR-T cells on the cell surface of CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), T cell cultivation was performed for 10 days according to Example 2, and then flow cytometric analysis was performed according to the experimental method. As a result of the analysis, the content of less-differentiated memory CAR-T cells showing CCR7.sup.+CD45RA.sup.+CD62L.sup.+ phenotypes, which were expressed on live T cells, compared to a non-transduced sample (i.e., a control group) by 5% or higher, and compared to YYB103 (i.e., the transduced sample, CAR-T control group) by 10% or higher, on the surface of all of the groups of CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), was confirmed (see FIG. 11).

Experimental Example 5: Checking of Cytotoxicity of CAR-T Cells Transformed with Novel Chimeric Antigen Receptors Against Glioma Cell Line in which IL13Rα2 is Overexpressed

[0112] Experimental Methods

[0113] In order to measure the cytotoxicity of IL13Rα2-specific CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12), cytotoxicity assay was performed using an LDH (Promega) kit. Specifically, CAR-T cells (effector cells) were used 10 days after activation of the cells with an anti-CD3 Ab, and the CAR-T cells (having a CAR expression rate of 20% to 40%) were added to a 6-well plate at a ratio 1:2, in which effector:target was 1×10.sup.6 cells:2×10.sup.6 cells and reacted at 37° C. for 24 hours. As the used target cells, U87 cells (human brain cancer cell line expressing IL13Rα2) and 293FT cells (the control group for normal cells) were used.

[0114] Experimental Results

[0115] An analysis was performed to examine whether IL13Rα2-specific CAR-T cells prepared in the present invention can effectively kill the target cancer cells (U87 expressing IL13Rα2). The method used was such that where target cancer cells (U87 expressing IL13Rα2) and normal cells (293FT) described above were cultured together with each of the activated CAR-T cells and cytotoxicity was compared and analyzed. As shown in FIG. 12, all of the CAR-T cells prepared according to the present invention showed a higher effect by 50% to 70% of inducing the death of the target cancer cell (U87) compared to non-transduced activated T cells. In particular, CAR-T #5, #6, and #11 of the present invention showed high cytotoxicity. In addition, CAR-T cells (#1, #2, #5, and #6) containing IL-7Rα exhibited higher overall cytotoxicity compared to CAR-T cells (#7, #8, #11, and #12) containing IL-2Rβ.

[0116] From the experimental result where 293FT cells, which do not express IL13Rα2, were used as the control group for normal cells, as an object to be compared with the target cells, it was confirmed that the CAR-T cells specific to IL13Rα2 showed very weak cytotoxicity (2% to 4%). This result shows that the chimeric antigen receptors used in this experiment bind specifically to IL13Rα2.

[0117] Through this Experimental Example, it was found that IL13Rα2-specific chimeric antigen receptor T cells do not show cytotoxicity to a normal cell (293FT) and significantly kill target cancer cells (U87), which express IL13Rα2.

[0118] FIG. 13 shows the results in which the cytotoxicity of the CAR-T cells, which were transformed with the chimeric antigen receptor (CAR) of the present invention, was confirmed through LDH assay after 24 hours or 96 hours without co-culture with target cells. Since the spontaneous toxicity of YYB103 is higher than that of other groups, the 8 groups are expected to show higher cytotoxicity compared to YYB103 in the co-culture with U87 cells, and the CAR-T cells (#1, #2, #5, #6, #7, #8, #11, or #12), which were transformed with the chimeric antigen receptor (CAR) of the present invention, are expected to have a comparative advantage in terms of safety and stability.

Experimental Example 6: Checking of Changes in CAR Expression Rate when CAR-T Cells, which were Transformed with Novel Chimeric Antigen Receptors, are Co-Cultured with Human Glioma Cell Line in which IL13Rα2 is Overexpressed

[0119] Experimental Methods

[0120] In order to check the changes in CAR expression rate when the CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) are co-cultured with human glioma cell line in which IL13Rα2 is overexpressed, the CAR-T cells (effector cells) having a CAR expression rate of 20% to 40% were used 10 days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio 1:2, in which effector:target was 1×10.sup.6 cells:2×10.sup.6 cells and reacted at 37° C. for 24, 48, and 96 hours. To the sample, which was reacted for 96 hours, were added 2×10.sup.6 cells of the target cells after 48 hours, and a BD LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used for flow cytometry (>30,000 events). Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a PE-conjugated anti-human IL-13 monoclonal antibody (BD Pharmingen) thereto. After washing, the cells were reacted with each antibody at 4° C. for 30 minutes in a state where light was blocked and washed once, and thereafter, the changes in the CAR expression rate were checked.

[0121] Experimental Results

[0122] In order to confirm the changes in the CAR expression rate when the 8 kinds of IL13Rα2-specific CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) are co-cultured with human glioma cell line in which IL13Rα2 is overexpressed, the T cells were cultured for 10 days, and then flow cytometric analysis was performed according to the experimental method according to Example 2. As a result of the analysis, as shown in FIG. 14, the CAR-T cells which were co-cultured with U87 (human glioma cell line cells for 24 hours showed a smaller change in CAR expression in all of the 8 groups compared to that of YYB103 (FIG. 14).

[0123] In co-culture with U87 (human glioma cell line) cells for 48 hours, the change in CAR expression was shown to be smaller than that of YYB103 in all of the 8 groups (FIG. 15).

[0124] In co-culture with U87 (human glioma cell line) cells for 96 hours, the change in CAR expression was shown to be smaller than that of YYB103 in 5 groups (i.e., #1, #2, #5, #6, and #7) (FIG. 16). The CAR expression of YYB103 after co-culture with U87 (human glioma cell line) cells for 96 hours was shown to be insignificant (FIG. 16).

[0125] The changes in CAR expression during the co-culture with human glioma cell line, in which IL13Rα2 is overexpressed, the changes in the CAR expression rate without co-culture with target cells or during the co-culture with 293FT cells (which were used as the control group for normal cells) can confirm that the chimeric antigen receptor (CAR) according to the present invention is expected to show excellent in vivo persistence and anti-tumor effects of CAR-T cells in a hostile tumor microenvironment, while simultaneously reducing toxicity.

Experimental Example 7: Checking of Changes in Chimeric Switch Receptor Expression Rate of TGFβR2 Exodomain and Transmembrane Domain and an Endodomain of IL-18R when CAR-T Cells which were Transformed with Novel Chimeric Antigen Receptors are Co-Cultured with Human Glioma Cell Line, in which IL13Rα2 is Overexpressed

[0126] Experimental Methods

[0127] In order to check the changes in the chimeric switch receptor expression rate of TGFβR2 exodomain and transmembrane domain and an endodomain of IL-18R when CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) are co-cultured with glioma cell line, in which IL13Rα2 is overexpressed, the CAR-T cells (effector cells) having a CAR expression rate of 20% to 40% were used 10 days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio 1:2, in which effector:target was 1×10.sup.6 cells:2×10.sup.6 cells and reacted at 37° C. for 24, 48, and 96 hours. To the sample, which was reacted for 96 hours, were added 2×10.sup.6 cells of the target cells after 48 hours, and a BD LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used for flow cytometry (>30,000 events). Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding an anti-human TGF-βR2 fluorescein-conjugated antibody (FAB2411F) (BD Pharmingen) thereto. After washing, the cells were reacted with each antibody at 4° C. for 30 minutes in a state where light was blocked and washed once, and thereafter, the changes in the chimeric switch receptor expression rate of TGFβR2 exodomain and transmembrane domain and an endodomain of IL-18R were checked.

[0128] Experimental Results

[0129] In order to confirm the changes in the chimeric switch receptor expression rate of TGFβR2 exodomain and transmembrane domain and an endodomain of IL-18R when the 8 kinds of IL13Rα2-specific CAR prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) are co-cultured with human glioma cell line in which IL13Rα2 is overexpressed, the T cells were cultured for 10 days according to Example 2, and then flow cytometric analysis was performed according to the experimental method.

[0130] As a result of the analysis, as shown in FIG. 17, the expression of the chimeric switch receptors of TGFβR2 exodomain and transmembrane domain and an endodomain of IL-18R in the 4 groups (#1, #2, #7, and #8) after 24 hours of co-culture showed a tendency of a decrease in the groups co-cultured with human glioma cell line, U87 cells compared to those cultured with CAR-T cells only, but the average expression rate was 30% (FIG. 17). The expression of the chimeric switch receptors of TGFβR2 exodomain and transmembrane domain and an endodomain of IL-18R in the 4 groups (#1, #2, #7, and #8) after 48 hours of co-culture showed a tendency of a decrease in the groups co-cultured with U87 cells compared to those cultured with CAR-T cells only, but the average expression rate was 25%. The expression of the chimeric switch receptors of TGFβR2 exodomain and transmembrane domain and an endodomain of IL-18R in the 4 groups (#1, #2, #7, and #8) after 72 hours of co-culture showed a tendency of a decrease in the groups co-cultured with human glioma cell line, U87 cells compared to those cultured with CAR-T cells only, but the average expression rate was 15%. Although the changes in the expression rate were greater compared to one without co-culture with target cells or the co-culture with 293FT cells (which were used as the control group for normal cells), by the confirmation of the results of the chimeric switch receptor expression rate of TGFβR2 exodomain and transmembrane domain and an endodomain of IL-18R against human glioma cell line, U87 in which IL13Rα2 is overexpressed, the chimeric antigen receptor (CAR) according to the present invention is expected to show excellent in vivo persistence and anti-tumor effects of CAR-T cells in a hostile tumor microenvironment.

[0131] Experimental Example 8: Checking of cytokine (IFN-γ) production by T cells, which were transformed with novel chimeric antigen receptors, against target cells

[0132] Experimental Methods

[0133] In order to confirm the cytokine (IFN-γ) production after 24 hours and 48 hours of co-culture between the CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) and human glioma cell line (U87 cells) and 293FT target cells (which were used as a control group for normal cells), the CAR-T cells (effector cells) in which the CAR expression rate is in the range of 20% to 40% were used 10 days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio in which effector:target was 1×10.sup.6 cells:2×10.sup.6 cells, 6 mL of a culture medium was added per well, and the cells reacted at 37° C. for 24 hours. 100 μL of the supernatant was collected and transferred to a 1.5 mL tube, the cells were cultured further up to 48 hours, and 100 μL of the supernatant was collected from the culture in the same manner.

[0134] The IFN-γ analysis experiment was performed as follows according to the manufacturer's instructions of the ELISA analyzer (R&D systems). 3 mL of the calibrator diluent RD6-21 was added to an IFN-γ standard bottle and mixed in a shaker for 15 minutes, dispensed in an amount of 1 mL per 1.5 mL tube to prepare two “standard 1”s. 500 μL was taken from 1 mL of the “standard 1” dispensed and was subjected to serial dilution to prepare up to “standard 7”. In order to prepare blanks, 500 μL of the culture medium and 500 μL of the calibrator diluent RD6-21 were mixed.

[0135] In order to dilute the samples by 1/20, 190 μL of the calibrator diluent RD6-21 was added into each of new 1.5 mL tubes as many as the number of samples. The assay samples were each prepared to a total volume of 200 μL by collecting 10 μL of the supernatant of each sample. To prepare a wash buffer, 500 mL of distilled water and 20 mL of wash buffer concentrate were mixed well in a 500 mL storage bottle.

[0136] Assay diluent RD1-51 (blue dye) in an amount of 100 μL each was added to the IFN-γ microplate for each well of “standard 1” to “standard 7”, blank, and samples. After adding 100 μL each of the blank, standards, and samples prepared above, a plate sealer was attached thereto and reacted at room temperature for 2 hours. After the reaction, the reaction solution was discarded, and 400 μL of the prepared wash buffer was added thereto, and the wells were washed 4 times. 200 μL of IFN-γ conjugate was added to each well, a plate sealer was attached thereto, and the reaction was performed at room temperature for 2 hours. The reaction solution was discarded, and the wells were washed 4 times using 400 μL of the wash buffer. After mixing the color reagent A: B in a 1:1 ratio, the mixture was added in an amount of 200 μL per well, a plate sealer was attached thereto, the light was blocked with foil, and the reaction was performed at room temperature for 30 minutes. After the reaction, 50 μL of the stop solution was added to each well and measured at 450 nm within 30 minutes.

[0137] Experimental Results

[0138] YYB103 was shown to secrete a relatively high amount of IFN-γ compared to other samples. When CAR-T cells were cultured in the presence of human glioma U87 cells, CAR-T (#1, #2, #5, and #6) containing an IL-7Rα signaling domain were shown to contain more IFN-γ compared to CAR-T (#7, #8, #11, and #12) containing an IL-2Rβ signaling domain. Results between samples after 24 hours and 48 hours of incubation showed similar patterns (FIG. 18).

[0139] As shown in FIG. 12, when the cytotoxicity was confirmed through LDH assay after 24 hours of co-culture between the CAR-T (which were transformed with the chimeric antigen receptor (CAR) of the present invention), U87 cells (human glioma cell line), and 293FT target cells (which are used as a control group for normal cells), the 8 groups showed similar abilities of killing target cells. However, considering that the production of IFN-γ was small and thus target cells could be killed without adverse effects caused by cytokines (cytokine release syndrome, CRS), in terms of safety, the effect of the CAR-T cell therapeutic is expected to have a fewer side effects while having excellent therapeutic effects.

Experimental Example 9: Checking of Cytokine (IL-21) Production by CAR-T Cells, which were Transformed with Novel Chimeric Antigen Receptors

[0140] Experimental Methods

[0141] In order to confirm the cytokine (IL-21) production after 24 hours and 48 hours of co-culture between the CAR-T cells prepared in Example 2 (#1, #2, #5, #6, #7, #8, #11, or #12) and human glioma U87 cells and 293FT target cells (which were used as a control group for normal cells), the CAR-T cells (effector cells) in which the CAR expression rate is in the range of 20% to 40% were used 10 days after activation of the cells with an anti-CD3 Ab. The CAR-T cells were added to a 6-well plate at a ratio in which effector:target was 1×10.sup.6 cells:2×10.sup.6 cells, 6 mL of a culture medium was added per well, and the cells were reacted at 37° C. for 24 hours. Then, 100 μL of the supernatant was collected and transferred to a 1.5 mL tube, the cells were cultured further up to 48 hours, and 100 μL of the supernatant was collected from the culture in the same manner.

[0142] The IL-21 analysis experiment was performed as follows according to the manufacturer's instructions of the ELISA analyzer (Invitrogen). 100 μL of a capture antibody was added to each well of a 96-well ELISA plate and reacted at 4° C. overnight. After the reaction, the wells were washed 3 times using 250 μL of wash buffer. ELISA/ELISASPOT diluent was added in an amount of 200 μL per well and reacted at room temperature for one hour.

[0143] After the reaction, 100 μL each of the prepared blank, cytokine IL-21 standard, and samples were added thereto, and ELISA/ELISASPOT diluent was added in an amount of 100 μL per well, and a plate sealer was attached thereto and the reaction was performed at room temperature for two hours. After the reaction, the wells were washed 3 times using 250 μL of wash buffer. 100 μL of a detection antibody was added to each well, and a plate sealer was attached thereto and the reaction was performed at room temperature for one hour. After the reaction, the wells were washed 3 times using 250 μL of wash buffer. 100 μL of Avidin-HRP was added to each well, a plate sealer was attached thereto, and the reaction was performed at room temperature for 30 minutes. After the reaction, the wells were washed 5 times using 250 μL of wash buffer. 100 μL of 1×TMB solution was added to each well, a plate sealer was attached thereto, and the reaction was performed at room temperature for 30 minutes. After the reaction, 50 μL of the stop solution was added to each well and measured at 450 nm within 30 minutes.

[0144] Experimental Results

[0145] It was confirmed that IL-21 was secreted in IL-21 gene-containing CAR-T cells (#1, #5, #7, and #11). When the CAR-T cells were co-cultured with human glioma U87 cells, a greater amount of IL-21 was secreted, and considering that the overall experimental results at 24 hours and 48 hours were similar, no significant difference according to the incubation time was observed. As the result of the experiment at 48 hours showed a higher IL-21 concentration than that at 24 hours, the secretion of IL-21 appeared to proceed steadily (FIG. 19).

[0146] The secretion of IL-21 in an environment of cancer cells is speculated to increase the ability to kill cancer cells by assisting the activation of innate immune-related cells.

Example 3: Preparation of Cancer Antigen EGFRvIII Targeting CAR Vector and αVβ3 Targeting CAR Vector

[0147] In the case of EGFRvIII, which is a major antigen for tumors such as glioblastoma and lung cancer, in order to reduce the side effects of CAR-T cells through non-specific binding while minimizing the binding affinity for EGFR wild-type in which the specificity for EGFRvIII is maintained, in SEQ ID NO: 33, positions 52 to 57 of SEQ ID NO: 3 were first changed from STGGYN to DPENDE (a CDR2 part of a heavy chain), position 101 of SEQ ID NO: 3 was changed from S to G (a CDR3 part of a heavy chain), and position 229 of SEQ ID NO: 3 was changed from V to G (a CDR3 part of a light chain).

[0148] Human CD3 (P20963-1), human CD8A (P01732), human 4-1BB (Q07011), human CD3Z (P20963), a Gaussia princeps luciferase, and human kappa light chain signal sequence (HuVHCAMP) were optimized using scientific literature and publicly available databases, and thereby chimeric antigen receptor-containing polypeptides consisting of codon-optimized synthetic DNA (#13, #14, #15, and #16 of SEQ ID NOS: 34 to 37) were prepared. The completed structure of CAR-containing polypeptide #13 includes a Kozak consensus ribosome-binding sequence, a CD8A signal sequence, an antigen binding domain which binds to EGFRv111 of SEQ ID NO: 3, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8A, a human CD8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3ζ cytoplasmic domain (SEQ ID NO: 18), and an XhoI/NotI cleavage site.

[0149] The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with XhoI/NotI (Emtage P C et al., Clin Cancer Res, 2008, 14:8112-8122). In this Example, in order to compare the activity of chimeric antigen receptors, YYB105 (SEQ ID NO: 39; see PCT International Publication No. WO 2017/023138) was additionally prepared.

[0150] The completed structure of CAR-containing polypeptide #14 includes a Kozak consensus ribosome-binding sequence, a CD8A signal sequence, an antigen binding domain which binds to EGFRv111 of SEQ ID NO: 33, three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8A, a human CD8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3ζ cytoplasmic domain (SEQ ID NO: 18), and an XhoI/NotI cleavage site. The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with XhoI/NotI (Emtage P C et al., Clin Cancer Res, 2008, 14:8112-8122).

[0151] In the case of αVβ3 anti-angiogenic, the CAR expression rate and persistence in CAR-T cells targeting these were optimized. In the case of αVβ3, the CAR expression rate and persistence in CAR-T cells targeting these were optimized using Gaussia princeps luciferase signal sequence or CD8 signal sequence (SEQ ID NO: 36 and SEQ ID NO: 37).

[0152] The completed structure of CAR-containing polypeptide #15 includes a Kozak consensus ribosome-binding sequence, a Gaussia princeps luciferase signal sequence, SEQ ID NO: 5 (an antigen binding domain which binds to αVβ3), three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8A, a human CD8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3ζ cytoplasmic domain (SEQ ID NO: 18), and an XhoI/NotI cleavage site. The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with XhoI/NotI (Emtage P C et al., Clin Cancer Res, 2008, 14:8112-8122). In this Example, in order to compare the activity of chimeric antigen receptors, YYB107 (SEQ ID NO: 38; see PCT International Publication No. WO 2017/023138) was additionally prepared.

[0153] The completed structure of CAR-containing polypeptide #16 includes a Kozak consensus ribosome-binding sequence, a Gaussia princeps luciferase signal sequence, SEQ ID NO: 5 (an antigen binding domain which binds to αVβ3), three glycines (GGG) which are introduced between an antigen binding domain and a hinge region so as to increase the expression of a chimeric antigen receptor by increasing the solubility of a CAR protein, a hinge region of human CD8A, a human CD8 transmembrane domain, a cytoplasmic 4-1BB costimulatory signal domain, a CD3ζ cytoplasmic domain (SEQ ID NO: 18), and an XhoI/NotI cleavage site. The finally prepared CAR gene fragment was conjugated to the MFG retrovirus expression vector digested with XhoI/NotI (Emtage P C et al., Clin Cancer Res, 2008, 14:8112-8122).

Example 4: Preparation of CAR-T Cells Transformed with Chimeric Antigen Receptors

[0154] High-titer CAR-expressing PG13 clones were prepared such that Phoenix-Ampho and Phoenix-Eco cells were transiently transfected with the retroviral expression vector prepared in Example 1, and then, cell-free vector stocks were prepared from the transfected Phoenix-Ampho and Phoenix-Eco cells by transfecting PG13 cells. For high-titer monoclones, PG13/#13, PG13/#14, PG13/#15, and PG13/#16 cells were stained using an anti-myc Ab (BD Pharmingen), and these monoclones were isolated using a flow cytometer. The transduction degree of PG13/#13, PG13/#14, PG13/#15, and PG13/#16 cells, which were transduced using an anti-myc Ab, was analyzed using a flow cytometer. The supernatants of the transduced PG13/#13, PG13/#14, PG13/#15, and PG13/#16 cells contained retrovirus, and the supernatants were collected for genetic modification of T cells. The peripheral blood mononuclear cells (PBMCs) were separated using centrifugation by adding the whole blood obtained from a healthy human donor into Ficoll Paque (GE Healthcare). The separated PBMCs were cultured by adding an anti-CD3 monoclonal antibody (eBioscience) at a concentration of 100 ng/mL under the condition of human IL-2 (Novartis) at a concentration of 100 IU/mL to activate the T cell fraction (BL Levine, Cancer Gene Therapy, 2015, 22:79-84). Two to three days after the cultivation, most of the cells were T cells and included natural killer cells at a percentage of 0% to 2%. Two to three days after the activation step, the T cells were subjected to transduction two times over two days using the retroviral supernatant and washed, and then proliferated for 14 days in a flask. IL-2 was maintained at a concentration of 100 IU/mL. The T cells modified as such were used for an analysis experiment.

[0155] Experimental Example 1: Checking of Growth Rate and Viability of CAR-T Cells Transformed with Chimeric Antigen Receptors

[0156] Experimental Results

[0157] For the T cells prepared in Example 4 above, the number of cells was counted to confirm the growth rate and viability rate of CAR-T, and the results are shown in FIG. 20. The number of cells and growth rate of all of the groups (#13, #14, #15, and #16) were shown to be similar to those of the control group (i.e., YYB105) depending on the day of the week, the cells were shown to grow rapidly from Day 12 of culture, and the viability was also shown to be 90% or higher (see FIG. 20).

[0158] Experimental Example 2: Checking of CAR Expression Rate on the Cell Surface of CAR-T Cells Transformed with Chimeric Antigen Receptors

[0159] Experimental Methods (Flow Cytometric Analysis)

[0160] For flow cytometry (>30,000 events), a BD LSRII device (Becton Dickinson) and BD FACSDiva software (Becton Dickinson) were used. Specifically, the cells were washed once with PBS containing 2% bovine serum albumin before adding a PE-conjugated anti-myc antibody (BD Pharmingen) thereto. After washing, the cells were reacted with each antibody at 4° C. for 30 minutes in a state where light was blocked and washed once, and thereafter, the expression rate of CAR on the surface of transduced T cells was checked.

[0161] Experimental Results

[0162] In order to confirm whether the 4 kinds of CAR prepared in Example 2 (#13, #14, #15, and #16) were expressed on the T cell surface, T cell cultivation was performed for 14 days according to Example 4, and then flow cytometric analysis was performed according to the experimental method.

[0163] As a result of the analysis, as shown in FIG. 21, the expression rate of the chimeric antigen receptors expressed on the surface of live T cells was shown to increase in #13 (43.0%) and #14 (50.8%) compared to YYB105 (37.4%) (i.e., a control group) (FIG. 21); and was shown to increase in #15 (45.9%) and #16 (40.0%) compared to YYB107 (24.6%) (i.e., a control group) (FIG. 21).

INDUSTRIAL APPLICABILITY

[0164] The present invention relates to rapidly developing a CAR-T cell in the field of cancer treatment. The CAR-T cell according to the present invention has a remarkably excellent expression rate and persistence, and thus has an improved therapeutic effect for solid cancer, etc., and can be effectively used in the field of customized cancer treatment.

TABLE-US-00001 [SEQUENCE LISTING] SEQ ID NO: 1 {antigen binding wild type IL13 domain binding to IL13Ra2] Length: 112 Type: ligand protein Organism: human Sequence: GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMY CAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRD TKIEVAQFVKDLLLHLKKLFREGQFN SEQ ID NO: 2 {antigen binding domain capable of binding to an antigen associated with an angiogenic activity} Length: 92 Type: ligand protein Organism: human Sequence: EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVLQ PPSTATISGLKPGVDYTITVYAVVERNGRELNTPPISINYRTHH HHHH SEQ ID NO: 3 {antigen binding domain binding to EGFRvIII} Length: 252 Type: scFv protein Organism: human Sequence: QVQLQESGGGLVKPGGSLKLSCAASGFTFSKFGMSWVRQTPD KRLEWVASISTGGYNTFYSDNVKGRFTISRDNAKNTLYLQMSS LKSEDTAMYYCARGYSSTSFAMDYWGQGTMVTVSSGSTSGSG KPGSGEGSDIQMTQSPSSLSASVGDRVTITCMTSTDIDDDMNW YQQKPGKTPKLLIYEGNTLRPGVPSRFSGSGSGTDFIFTISSLQP EDIATYYCLQSFNVPLTFGGGTKVEIKEQKLISEEDL SEQ ID NO: 4 {antigen binding domain binding to EphA2} Length: 141 Type: ligand protein Organism: human Sequence: DRHTVFWNSSNPKFRNEDYTIHVQLNDYVDIICPHYEDHSVAD AAMEQYILYLVEHEEYQLCQPQSKDQVRWQCNRPSAKHGPEK LSEKFQRFTAFALAKEFKAGHSYYYISKPIHQHEDRCLRLKVT VSGEQKLISEEDL SEQ ID NO: 5 {antigen binding domain binding to αVβ3} Length: 104 Type: ligand protein Organism: human Sequence: VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNS PVQEFTVPGSKSTATISGLKPGVDYTITVYAVTPRGDWNEGSK PISINYRTEQKLISEEDL SEQ ID NO: 6 {antigen binding domain binding to glypican1} Length: 418 Type: ligand protein Organism: human Sequence: TSPCDNFDCQNGAQCIVRINEPICQCLPGYQGEKCEKLVSVNFI NKESYLQIPSAKVRPQTNITLQIATDEDSGILLYKGDKDHIAVE LYRGRVRASYDTGSHPASAIYSVETINDGNFHIVELLALDQSLS LSVDGGNPKIITNLSKQSTLNFDSPLYVGGMPGKSNVASLRQA PGQNGTSFHGCIRNLYINSELQDFQKVPMQTGILPGCEPCHKK VCAHGTCQPSSQAGFTCECQEGWMGPLCDQRTNDPCLGNKCV HGTCLPINAFSYSCKCLEGHGGVLCDEEEDLFNPCQAIKCKHG KCRLSGLGQPYCECSSGYTGDSCDREISCRGERIRDYYQKQQG YAACQTTKKVSRLECRGGCAGGQCCGPLRSKRRKYSFECTDG SSFVDEVEKVVKCGCTRCVSEQKLISEEDL SEQ ID NO: 7 {antigen binding domain binding to mesothelin} Length: 262 Type: scFv protein Organism: human Sequence: QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTN YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGKNGAFDIWGQGTMVTVSS GSTSGSGKPGSGEGSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSR GLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTP EDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGS SEQ ID NO: 8 {hinge region sequence-1} Length: 47 Type: protein Organism: human Sequence: KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACD SEQ ID NO: 9 {hinge region sequence-2} Length: 45 Type: protein Organism: human Sequence: KPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDF A SEQ ID NO: 10 {transmembrane domain sequence-1} Length: 21 Type: protein Organism: human Sequence: IYIWAPLAGTCGVLLLSLVIT SEQ ID NO: 11 {transmembrane domain sequence-2} Length: 23 Type: protein Organism: human Sequence: LAYLLDGILFIYGVILTALFLRV SEQ ID NO: 12 (4-1BB) Length: 42 Type: protein Organism: human Sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 13 {Wild type CD28} Length: 41 Type: protein Organism: human Sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 14 {IL-7Rα} Length: 459 Type: protein Organism: human Sequence: MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTCAF EDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLTCKKI DLTTIVKPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENKW THVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSSGEM DPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNP ESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESF GRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQ SGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ SEQ ID NO: 15 {part of IL-7Rα} Length: 64 Type: protein Organism: human Sequence: KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFL QDTF SEQ ID NO: 16 {IL-2Rβ} Length: 551 Type: protein Organism: human Sequence: MAAPALSWRLPLLILLLPLATSWASAAVNGTSQFTCFYNSRANISCVWSQDGALQDTSC QVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWR VMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHT WEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAAL GKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEH GGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTS CFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDA YCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPG VPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTD AYLSLQELQGQDPTHLV SEQ ID NO: 17 {part of IL-2Rβ} Length: 104 Type: protein Organism: human Sequence: NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLE VLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHL SEQ ID NO: 18 (CD3ζ) Length: 113 Type: protein Organism: human Sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD PEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 19 (TGF-βR2 exodomain} Length: 137 Type: protein Organism: human Sequence: TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQE VCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPD SEQ ID NO: 20 {transmembrane domain and endodomain of IL-18R} Length: 219 Type: protein Organism: human Sequence: PGHVFTRGMIIAVLILVAVVCLVTVCVIYRVDLVLFYRHLTRRDETLTDGKTYDAFVSY LKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVL SKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFL PQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES SEQ ID NO: 21 {IL-21} Length: 133 Type: protein Organism: human Sequence: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSA NTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPK EFLERFKSLLQKMIHQHLSSRTHGSEDS SEQ ID NO: 22 {YYB 103} Length: 359 Type: protein Organism: human Sequence: MGWSCIILFLVATATGVHSGPVPPSTALRKLIEELVNITQNQKA PLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQDMLDG FCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFKEGQF NGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQP FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR SEQ ID NO: 23 {#1} Length: 998 Type: protein Organism: human Sequence: MGWSCIILFLVATATGVHSGPVPPSTALRKLIEELVNITQNQKAPLCNGSMVWSINLTAG MYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL HLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQAR DEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTATKDTYD YVTMQALPPREGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPTIPPHVQKSV NNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE EYNTSNPDPGHVFTRGMIIAVLILVAVVCLVTVCVIYRVDLVLFYRHLTRRDETLTDGKT YDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIE KSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRV LKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSESRRKRSGSGATNFSL LKQAGDVEENPGPMYRMQLLSCIALSLALVTNSQGQDRHMIRMRQLIDIVDQLKNYVN DLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGR RQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS SEQ ID NO: 24 {#2} Length: 818 Type: protein Organism: human Sequence: MGWSCIILFLVATATGVHSGPVPPSTALRKLIEELVNITQNQKAPLCNGSMVWSINLTAG MYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL HLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQAR DEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTATKDTYD YVTMQALPPREGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPTIPPHVQKSV NNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSE EYNTSNPDPGHVFTRGMIIAVLILVAVVCLVTVCVIYRVDLVLFYRHLTRRDETLTDGKT YDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIE KSRRLIIVLSKSYMSNEVRYELESGLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRV LKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVLPVLSES SEQ ID NO: 25 {#5} Length: 594 Type: protein Organism: human Sequence: MGWSCIILFLVATATGVHSGPVPPSTALRKLIEELVNITQNQKAPLCNGSMVWSINLTAG MYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL HLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQAR DEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTATKDTYD YVTMQALPPREGRGSLLTCGDVEENPGPMYRMQLLSCIALSLALVTNSQGQDRHMIRM RQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVS IKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQH LSSRTHGSEDS SEQ ID NO: 26 {#6} Length: 423 Type: protein Organism: human Sequence: MGWSCIILFLVATATGVHSGPVPPSTALRKLIEELVNITQNQKAPLCNGSMVWSINLTAG MYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL HLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQAR DEVEGFLQDTFRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYLPQSTATKDTYD YVTMQALPPR SEQ ID NO: 27 {#7} Length: 1038 Type: protein Organism: human Sequence: MGWSCIILFLVATATGVHSGPVPPSTALRKLIEELVNITQNQKAPLCNGSMVWSINLTAG MYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL HLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGL APEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYLSLSTATKDTYLPQHMQALPPREGRGSLLTCGDV EENPGPMALPVTALLLPLALLLHAARPTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFC DVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE DAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDPGHVFTRGMIIAVLIL VAVVCLVTVCVIYRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFA VEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELES GLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYL MPAKTVKPGRDEPEVLPVLSESRRKRSGSGATNFSLLKQAGDVEENPGPMYRMQLLSCI ALSLALVTNSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSC FQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLE RFKSLLQKMIHQHLSSRTHGSEDS SEQ ID NO: 28 {#8} Length: 858 Type: protein Organism: human Sequence: MGWSCIILFLVATATGVHSGPVPPSTALRKLIEELVNITQNQKAPLCNGSMVWSINLTAG MYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL HLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGL APEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYLSLSTATKDTYLPQHMQALPPREGRGSLLTCGDV EENPGPMALPVTALLLPLALLLHAARPTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFC DVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILE DAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDPGHVFTRGMIIAVLIL VAVVCLVTVCVIYRVDLVLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFA VEILPRVLEKHFGYKLCIFERDVVPGGAVVDEIHSLIEKSRRLIIVLSKSYMSNEVRYELES GLHEALVERKIKIILIEFTPVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYL MPAKTVKPGRDEPEVLPVLSES SEQ ID NO: 29 {#11} Length: 634 Type: protein Organism: human Sequence: MGWSCIILFLVATATGVHSGPVPPSTALRKLIEELVNITQNQKAPLCNGSMVWSINLTAG MYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL HLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGL APEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYLSLSTATKDTYLPQHMQALPPREGRGSLLTCGDV EENPGPMYRMQLLSCIALSLALVTNSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLP APEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS SEQ ID NO: 30 {#12} Length: 463 Type: protein Organism: human Sequence: MGWSCIILFLVATATGVHSGPVPPSTALRKLIEELVNITQNQKAPLCNGSMVWSINLTAG MYCAALESLINVSGCSAIEKTQDMLDGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL HLKKLFKEGQFNGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGL APEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYLSLSTATKDTYLPQHMQALPPR SEQ ID NO: 31 {mutated 3rd ITAM-1} Length: 23 Type: protein Organism: human Sequence: YLPQSTATKDTYDYVTMQALPPR SEQ ID NO: 32 {mutated 3rd ITAM-2} Length: 23 Type: protein Organism: human Sequence: YLSLSTATKDTYLPQHMQALPPR SEQ ID NO: 33 {antigen binding domain binding to EGFRvIII} Length: 252 Type: scFv protein Organism: human Sequence: QVQLQESGGGLVKPGGSLKLSCAASGFTFSKFGMSWVRQTPD KRLEWVASIDPENDETFYSDNVKGRFTISRDNAKNTLYLQMSS LKSEDTAMYYCARGYGSTSFAMDYWGQGTMVTVSSGSTSGSG KPGSGEGSDIQMTQSPSSLSASVGDRVTITCMTSTDIDDDMNW YQQKPGKTPKLLIYEGNTLRPGVPSRFSGSGSGTDFIFTISSLQP EDIATYYCLQSFNGPLTFGGGTKVEIKEQKLISEEDL SEQ ID NO: 34 {#13} Length: 499 Type: protein Organism: human Sequence: MALPVTALLLPLALLLHAARPQVQLQESGGGLVKPGGSLKLSC AASGFTFSKFGMSWVRQTPDKRLEWVASISTGGYNTFYSDNV KGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGYSSTSFA MDYWGQGTMVTVSSGSTSGSGKPGSGEGSDIQMTQSPSSLSAS VGDRVTITCMTSTDIDDDMNWYQQKPGKTPKLLIYEGNTLRPG VPSRFSGSGSGTDFIFTISSLQPEDIATYYCLQSFNVPLTFGGGT KVEIKEQKLISEEDLGGGPRKPTTTPAPRPPTPAPTIASQPLSLR PEAARPAAGGAVHTRGLDFALAYLLDGILFIYGVILTALFLRV KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 35 {#14} Length: 499 Type: protein Organism: human Sequence: MALPVTALLLPLALLLHAARPQVQLQESGGGLVKPGGSLKLSC AASGFTFSKFGMSWVRQTPDKRLEWVASIDPENDETFYSDNV KGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGYGSTSFA MDYWGQGTMVTVSSGSTSGSGKPGSGEGSDIQMTQSPSSLSAS VGDRVTITCMTSTDIDDDMNWYQQKPGKTPKLLIYEGNTLRPG VPSRFSGSGSGTDFIFTISSLQPEDIATYYCLQSFNGPLTFGGGT KVEIKEQKLISEEDLGGGPRKPTTTPAPRPPTPAPTIASQPLSLR PEAARPAAGGAVHTRGLDFALAYLLDGILFIYGVILTALFLRV KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 36 {#15} Length: 349 Type: protein Organism: human Sequence: MGVKVLFALICIAVAEAVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTI TVYAVTPRGDWNEGSKPISINYRTEQKLISEEDLGGGPRKPTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY IWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQE EDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELN LGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR SEQ ID NO: 37 {#16} Length: 353 Type: protein Organism: human Sequence: MALPVTALLLPLALLLHAARPVSDVPRDLEVVAATPTSLLISW DAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPG VDYTITVYAVTPRGDWNEGSKPISINYRTEQKLISEEDLGGGPR KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNE LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR SEQ ID NO: 38 {YYB 107} Length: 351 Type: protein Organism: human Sequence: MGWSCIILFLVATATGVHSVSDVPRDLEVVAATPTSLLISWDA PAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVD YTITVYAVTPRGDWNEGSKPISINYRTEQKLISEEDLGGGPRKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNE LNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR SEQ ID NO: 39 {YYB 105} Length: 497 Type: protein Organism: human Sequence: MGWSCIILFLVATATGVHSQVQLQESGGGLVKPGGSLKLSCAA SGFTFSKFGMSWVRQTPDKRLEWVASISTGGYNTFYSDNVKG RFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGYSSTSFAMD YWGQGTMVTVSSGSTSGSGKPGSGEGSDIQMTQSPSSLSASVG DRVTITCMTSTDIDDDMNWYQQKPGKTPKLLIYEGNTLRPGVP SRFSGSGSGTDFIFTISSLQPEDIATYYCLQSFNVPLTFGGGTKV EIKEQKLISEEDLGGGPRKPTTTPAPRPPTPAPTIASQPLSLRPE AARPAAGGAVHTRGLDFALAYLLDGILFIYGVILTALFLRVKR GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 40 {T2A peptide} Length: 21 Type: protein Organism: human Sequence: GSGEGRGSLLTCGDVEENPGP SEQ ID NO: 41 {P2A peptide} Length: 22 Type: protein Organism: human Sequence: GSGATNFSLLKQAGDVEENPGP SEQ ID NO: 42 {E2A peptide} Length: 23 Type: protein Organism: human Sequence: GSGQCTNYALLKLAGDVESNPGP SEQ ID NO: 43 {F2A peptide} Length: 25 Type: protein Organism: human Sequence: GSGVKQTLNFDLLKLAGDVESNPGP